JP6460646B2 - Screening method for skin barrier function promoter - Google Patents

Description

   The present invention relates to a method for screening a skin barrier function promoter and a skin disease therapeutic agent, and a method for producing acylceramide.

  The skin is exposed to various stimuli from the external environment. The skin has a barrier function (barrier function) that prevents irritation from the outside, foreign substances, and microorganisms, and retains moisture to prevent transpiration. When this barrier function is lowered, the moisture of the skin evaporates, leading to dryness and being susceptible to external irritation, which causes skin diseases such as atopic dermatitis. Severe skin barrier dysfunction causes ichthyosis, leading to premature death due to water loss and infection.

  The epidermis of the skin has a four-layer structure of the basal layer, spiny layer, granule layer, and stratum corneum in order from the inside, and most of the skin barrier function is carried by the outermost stratum corneum. In particular, a special structure called “keratin intercellular lipid” having a lamellar structure (a multi-layer structure with a short-period phase of about 6 nm and a long-period layer of about 13 nm) in which a lipid layer and an aqueous layer are alternately repeated, It is essential to prevent evaporation of moisture in the skin. Epidermal keratinocytes proliferated in the basal layer differentiate and enucleate before reaching the stratum corneum, and have a "peripheral zone" that is a physically and chemically very stable insoluble structure, and keratin filaggrin aggregates inside It becomes keratinocytes clogged with. When keratinized cells are likened to bricks, a strong barrier is formed by filling the interstitial lipids like mortar (Fig. 1A).

  This stratum corneum intercellular lipid is a lamellar granule that is biosynthesized by cells in the spinous layer and granule layer. Is. The stratum corneum intercellular lipid is composed of ceramide, cholesterol, fatty acid, phospholipid, and the like, and acylceramide is the most important among various ceramide molecular species for the formation of a lamellar structure of stratum corneum lipid.

  Acylceramide present in the epidermis has a structure in which linoleic acid (C18: 2) is ester-bonded to the ω position of a very long chain fatty acid (28 to 34 carbon atoms) (FIG. 1C).

  The keratinocytes are covered with a keratinized insoluble membrane (peripheral zone) in which a backing protein such as involucrin and loricrin is cross-linked and thickened. Furthermore, ω-hydroxyceramide (also referred to as ω-OH ceramide) produced by the metabolism of acylceramide is covalently bonded to glutamic acid and glutamine residues located on the intercellular space side of the peripheral zone, and the cornified extracellular lipid (cornified lipid) By forming a -bound envelope (CLE), the keratinocytes and stratum corneum lipids are tightly linked to create a robust structure.

  Therefore, the acylceramide that exists uniquely in the skin is an essential component for the formation of a stratum corneum permeation barrier realized by the lamellar structure of stratum corneum lipids and the presence of CLE (Non-patent Document 1: Breiden B, Sandhoff K. The role of sphingolipid metabolism in cutaneous permeability barrier formation. Biochim Biophys Acta 1841: 441-452, 2014).

  The acyl ceramide present in the skin has a unique structure and function as compared with other known ceramides. Acylceramide acts as a “molecular rivet” because it has a very long hydrocarbon chain in which linoleic acid is further bonded via an ester bond at the ω position of a very long chain fatty acid having 18 to 42 carbon atoms. Two or more lipid layers of intercellular lipids are connected to each other, and as a result, the movement of moisture and electrolytes is restricted to maintain a barrier function peculiar to the skin.

Acylceramide secreted between the corneocytes is hydrolyzed at the ω position to release linoleic acid, and then becomes a glutamic acid residue of the protein constituting the peripheral zone or ω-hydroxyceramide that forms an ester bond with a glutamine residue. Furthermore, when it is hydrolyzed by acid ceramidase and the sphingosine skeleton is liberated, it becomes ω-hydroxy very long chain fatty acid ester-linked at the ω position. Ω-OH ceramide and ω-OH fatty acid covalently bonded to these peripheral bands by ester bonds constitute CLE on the keratinocyte surface, and strongly bind keratinocytes and interkeratin lipids.

  In essential fatty acid-deficient animals that have been fed diets lacking essential fatty acids for a long time, other ceramides in the stratum corneum have almost no quantitative abnormalities, but the amount of water transpiration increases and the skin barrier function is increased. It is known that the skin (and hair) can no longer be kept healthy due to a decrease in the skin, resulting in exfoliation, dryness and thickening of the skin. From the detailed analysis of the ceramide composition, it was discovered that most of the linoleic acid ester-bonded to the ω-position of ω-O-acylceramide was changed to oleic acid (Non-Patent Document 2: Wertz PW, Downig DT: Glysolipids in mammalian epidermis; Structure and function of the water barrier. Science 21, 1261-1262, 1982). That is, when linoleic acid, which is one of the essential fatty acids, is insufficient, the acyl moiety at the ω position of acylceramide is replaced from linoleic acid to oleic acid, suggesting that such an acylceramide cannot exert a skin barrier function. Therefore, it is considered that acyl ceramides in which linoleic acid is ester-bonded at the ω position play an important role that cannot be replaced by other ceramides in the skin barrier function.

  There are at least types of ceramides 1 to 12 depending on the combination of the sphingosine skeleton, which is a constituent, and the structure of a fatty acid. Examples of sphingoid bases include three types: sphingosine (D-erythro-dihydrosphingosine, SPH), dihydrosphingosine (D-erythro-dihydrosphingosine, DHS), and phytosphigosine (PHS). Among various ceramide molecular species, ceramide 1, 4, 9, 12 or EOS, EOH, EOP, EODS are acylceramide components present in the stratum corneum of skin. The existence ratio is in the order of EOS, EOH, EOP, EODS.

  As indicated above, ceramide is an important component of the skin and is said to contribute greatly to the skin barrier function. For this reason, blending ceramide into a topical skin preparation such as cosmetics and administering it to the skin is effective in improving the skin barrier function of the skin whose skin barrier function has been reduced due to the decrease in ceramide, and reduces water loss. It has been shown to recover and exert a wrinkle prevention effect. Furthermore, ceramide is used as a pharmaceutical for skin diseases, for example, for the treatment of atopic eczema. It has been found that the amount of ceramide in the corneum of the skin examination area and the non-rash area of atopic dermatitis patients is significantly reduced compared with that in healthy subjects, and among the ceramide species, acylceramides that are deeply related to barrier function Is the cause of the decrease in water retention and barrier function in the skin of patients with atopic dermatitis (Non-patent Document 3: Imokawa G. et al. A decrease level of ceramides in stratum corneum of atopic dermatitis: An etiologic factor in atopic dry skin? J Invest Dermatol, 96, 523-526, 1991).

J Invest Dermatol. 96: 523-526, 1991 (Non-Patent Document 4) and Arch Dermatol Res. 283: 219-223,1991 (Non-Patent Document 5) are “ceramides in atopic dermatitis and senile xeroderma” Regarding the “decrease in the amount”, J Dermatol Sci. 1: 79-83, 1990 (Non-patent document 6) and Acta Derm Venereol. 74: 337-340, 1994 (Non-patent document 7) described “Decrease in ceramide amount and lipids”. Regarding “metabolic enzyme abnormalities”, Contact Dermatitis. 45: 280-285, 2001 (Non-Patent Document 8) and J Eur Acad Dermatol Venereol. 16: 587-594, 2002 (Non-Patent Document 9) "Recovery" is disclosed.

  Studies have also been made to prevent or cure rough skin. Rough skin refers to dry skin in which exfoliation of keratinocytes is generally observed. Such rough skin is caused by elution of interstitial lipids such as cholesterol, ceramide, fatty acids, etc., and formation of a stratum corneum permeation barrier due to degeneration of keratinocytes caused by ultraviolet rays, detergents, etc., and disruption of proliferation / keratinization balance of epidermal cells. Etc. For the purpose of preventing or healing this rough skin, studies have been made on supplying a keratinocyte lipid component or a synthetic keratinocyte lipid similar thereto.

  In recent years, it has been attracting attention as a therapeutic agent for skin diseases associated with reduced skin barrier function and dry skin, or as a material for cosmetics and beauty health foods. In fact, many products containing ceramide are already on the market as cosmetics, foods and supplements, etc., and the scale of the ceramide raw material market continues to expand.

  Currently, known ceramides (natural ceramides, synthetic ceramides, synthetic pseudo-ceramides are used as ceramides blended in cosmetics and skin external preparations. Examples of commercially available ceramides include cosmoferm. "Ceramide 1" (ceramide 1), "Ceramide 2" (ceramide 2), "Ceramide III" (ceramide 3), "Ceramide IIIB" (ceramide 3) from Cosmoferm and Evonik () , “Ceramide VI” (Ceramide 6) and “Ceramide TTI-001” (Ceramide 2), CERAMIDE II (Quest International), DS-Ceramide VI, DS-CLA-Phytoceramide, C6-Phytoceramide, manufactured by Takasago International Corporation DS-ceramide Y3S (DOOSAN), CERAMIDE2 (Cedera), and the like.

  “Ceramide 1” (ceramide 1) manufactured by Cosmoferm is stearoyloxyheptacosanoylphytosphingosine, not ceramide 1 itself, but an analog of ceramide 1, that is, pseudo-ceramide and not acylceramide. The sphingosine skeleton of ceramide 1 is replaced with phytosphingosine (thus more precisely an analogue of ceramide 9), and the fatty acid is not ester-bonded at the ω position, but instead stearic acid is bound by an ether bond. doing. This ceramide 1 analog is a substance obtained by organic synthesis and does not exist in nature. More importantly, since it has structural characteristics different from acylceramide, it cannot be metabolized in the same manner as acylceramide. Therefore, it cannot function as a CLE to bind keratinocyte lipids and keratinocytes. Therefore, the effect of promoting the skin barrier function is considered to be limited.

  So far, ceramides have been derived from animals such as cattle, but problems with infectious diseases have been pointed out, and plant ceramides such as rice, wheat, soybeans and straw are the mainstream. Recent basic research (J. Clin. Invest. 112: 1372-1382, 2003 (Non-Patent Document 10)) has revealed that the structure of ceramide is extremely important for the skin moisturizing and barrier functions. The question remains whether plant ceramides with different structures are highly functional lipids, and ceramides present in animals and plants are difficult to extract and purify in minute amounts, and are poor in productivity and high in cost. Development of new production technology that can overcome this is strongly desired.

Currently, most of ceramides used for blending cosmetics and skin external preparations are ceramide 2 (also referred to as NS or simply ceramide). Ceramide 2 is a precursor of ω-OH ceramide (ω-hydroxyceramide), and as described above, the presence of acylceramide is indispensable for the recovery and enhancement of skin barrier function, so ceramides are simply administered. However, it is necessary that the administered ceramide 2 is efficiently metabolized via ω-OH ceramide to the keratinocyte lipid and acylceramide which is the most important component of CLE.

  Many enzymes and transporters that make up the ceramide metabolic pathway of the skin have been reported (Fig. 1B). Interestingly, many of the molecular mutations and defects involved in the skin-specific ceramide metabolic pathway cause ichthyosis. There are some ceramide metabolism molecules whose molecular entities have not yet been clarified, but acyl ceramides (ω-O-acyl ceramides), which are considered to be most important for layered stratum corneum lipids and CLE formation, are known. ) Has not been identified. It has been reported that ω-OH ceramide can be synthesized enzymatically by allowing cytochrome P450 4F (CYP4F22) to act on ceramide (Non-patent Document 11: Ohno Y, Ohkuni A., Kamiyama N., Kihara A; The cytochrome P450 4F subfamily protein CYP4F22 catalyzes ω-hydroxylation of ultra long-chain-containing ceramides in the epidermis.FASEB Summer Research conferences (Lysophospholipid and Other Related Mediators-From Bench to Clinic), Niseko, 2013. 8. 5-6. Non-Patent Document 12: Ohno Y, Ohkuni A, Kamiyama N, Kihara A; Epidermal-specific ω-hydroxylated ceramide is synthesized by the cytochrome P450 4F subfamily protein CYP4F22. 11th International Symposium on Future Drug Discovery and Medical Innovation, Sapporo , 2013. 8. 2.). In addition, from MATREYA (Pennsylvania, USA), ω-OH ceramide (ω-hydroxytriacontanoyl-sphingosine, N-ω-Hydroxytriacontanoyl-D-erythro-sphingosine) containing very long chain fatty acids (30 carbon atoms) Is available. Therefore, by identifying and using an acylceramide synthase that has the activity of transferring fatty acids, more preferably linoleic acid, to the ω-position of ω-OH ceramide, industrial mass production of acylceramide can be realized. .

It has been reported that PNPLA1 gene belonging to the Ca ²⁺ -independent phospholipase A ₂ / PNPLA (Patatin-like phospholipase or Pattain-like phospholipase domain containing) family causes ichthyosis in humans and dogs (non- Patent Document 3), the mechanism of ichthyosis development due to the physiological function and mutation of this molecule was unknown. There are nine molecular species in the PNPLA family, which are characterized by having a common catalytic domain called a patatin domain (FIG. 2, Non-Patent Document 13). Molecules belonging to this family have been reported to be involved in various lipid metabolisms such as hydrolysis of phospholipids, lysophospholipids, triglycerides and retinoid esters, and fatty acid transfer reactions to retinol and lysophosphatidic acid.

  The human PNPLA1 gene has its nucleotide sequence described in NCBI Reference Sequence: NM_173676.2 and the protein amino acid sequence encoded thereby, and the mouse Pnpla1 gene is described in NCBI Reference Sequence: NM_001034885.3.

Breiden B, Sandhoff K. The role of sphingolipid metabolism in cutaneous permeability barrier formation. Biochim Biophys Acta 1841: 441-452, 2014 Wertz PW, Downig DT: Glysolipids in mammalian epidermis; Structure and function of the water barrier.Science 21, 1261-1262, 1982 Imokawa G. et al. A decrease level of ceramides in stratum corneum of atopic dermatitis: An etiologic factor in atopic dry skin? J Invest Dermatol, 96, 523-526, 1991 J Invest Dermatol. 96: 523-526, 1991 Arch Dermatol Res. 283: 219-223,1991 J Dermatol Sci. 1: 79-83, 1990 Acta Derm Venereol. 74: 337-340, 1994 Contact Dermatitis. 45: 280-285, 2001 J Eur Acad Dermatol Venereol. 16: 587-594, 2002 J. Clin. Invest. 112: 1372-1382, 2003 Ohno Y, Ohkuni A., Kamiyama N., Kihara A; The cytochrome P450 4F subfamily protein CYP4F22 catalyzes ω-hydroxylation of ultra long-chain-containing ceramides in the epidermis.FASEB Summer Research conferences (Lysophospholipid and Other Related Mediators-From Bench to Clinic), Niseko, 2013. 8. 5-6. Ohno Y, Ohkuni A, Kamiyama N, Kihara A; Epidermal-specific ω-hydroxylated ceramide is synthesized by the cytochrome P450 4F subfamily protein CYP4F22. 11th International Symposium on Future Drug Discovery and Medical Innovation, Sapporo, 2013. 8. 2. Feingold KR, Jiang YJ. The mechanisms by which lipids coordinately regulate the formation of the protein and lipid domains of the stratum corneum: Role of fatty acids, oxysterols, cholesterol sulfate and ceramides as signaling molecules.Dermatoendocrinol 3: 113-118, 2011

  An object of the present invention is to provide a method for screening a skin barrier function promoter and a skin disease therapeutic agent using PNPLA1, and a method for producing acylceramide.

  As a result of intensive studies to solve the above problems, the present inventor has found that PNPLA1 is involved in the synthesis of acylceramide, and has completed the present invention.

That is, the present invention is as follows.
(1) When a test substance is administered to a non-human animal deficient in the PNPLA1 gene and a skin barrier function promoting effect is obtained in the non-human animal after administration of the test substance, the test substance is removed from the skin. The screening method of a skin barrier function promoter including the process selected as a barrier function promoter.
(2) When a test substance is administered to a non-human animal having the PNPLA1 gene, the expression level of PNPLA1 in the non-human animal after administration of the test substance, or the amount of acylceramide in which linoleic acid is ester-linked at the ω position A screening method for a skin barrier function promoter, comprising a step of selecting the test substance as a skin barrier function promoter when increased.
(3) Obtained by detecting acyl ceramide with linoleic acid ester-bonded at the ω position after contacting the test substance with a biomaterial collected from a cell having PNPLA1 gene or a non-human animal having PNPLA1 gene A screening method for a skin barrier function promoter, comprising a step of selecting a skin barrier function promoter using a detection result as an index.
(4) When acylceramide having linoleic acid ester-bonded at the ω position is increased compared to control cells or biomaterials that do not contact the test substance, the test substance is selected as a skin barrier function promoter. The method according to (3).
(5) Measure the expression level of PNPLA1 or the expression level of ceramide metabolism-related gene after contacting the test substance with the biomaterial collected from the cell having PNPLA1 gene or the non-human animal having PNPLA1 gene A method for screening a skin barrier function promoter, comprising a step of selecting a substance that promotes skin barrier function using the measured result as an index.
(6) When the expression level of PNPLA1 is increased compared to control cells or biomaterials that are not brought into contact with the test substance, the test substance is selected as a skin barrier function promoter, (5) Method.
(7) The method according to (5), wherein the ceramide metabolism-related gene is at least one selected from the group consisting of Elovl4, Lass3, Cyp4f39, Ugcg, Abca12, Gba, Abhd5, Aloxe3 and Alox12b.
(8) When the expression level of the ceramide metabolism-related gene is increased as compared with a control cell or biomaterial that is not brought into contact with the test substance, the test substance is selected as a skin barrier function promoter, (7) The method described in 1.
(9) A skin barrier function comprising a step of detecting acylceramide after contacting a test substance with a sample containing PNPLA1 and ω-hydroxyceramide, and selecting a substance that promotes the skin barrier function using the obtained detection result as an index. Accelerator screening method.
(10) The method according to (9), wherein the test substance is selected as a skin barrier function promoter when the acylceramide is increased as compared with a control sample that is not brought into contact with the test substance.
(11) After contacting the test substance with a microarray carrying the PNPLA1 gene and / or ceramide metabolism-related gene, the expression of the PNPLA1 gene and / or ceramide metabolism-related gene is detected, and the skin barrier function using the obtained detection result as an index A method for screening a skin barrier function promoter, comprising the step of selecting a promoter.
(12) When the expression level of the PNPLA1 gene is increased as compared with the PNPLA1 gene mounted on the control microarray that does not contact the test substance, the test substance is selected as a skin barrier function promoter. ) Method.
(13) The method according to (11), wherein the ceramide metabolism-related gene is at least one selected from the group consisting of Elovl4, Lass3, Cyp4f39, Ugcg, Abca12, Gba, Abhd5, Aloxe3 and Alox12b.
(14) When the expression level of a ceramide metabolism-related gene is increased as compared with a ceramide metabolism-related gene mounted on a control microarray that does not contact the test substance, the test substance is selected as a skin barrier function promoter. The method according to (13).
(15) When a test substance is administered to a non-human animal deficient in the PNPLA1 gene and a skin barrier function promoting effect is obtained in the non-human animal after administration of the test substance, the test substance is A screening method for a skin disease therapeutic agent, comprising a step of selecting as a disease therapeutic agent.
(16) A test substance is administered to a non-human animal having the PNPLA1 gene, and the expression level of PNPLA1 in the non-human animal after administration of the test substance, or the amount of acylceramide in which linoleic acid is ester-linked at the ω position A screening method for a therapeutic agent for skin disease, comprising a step of selecting the test substance as a therapeutic agent for skin disease when increased.
(17) After contacting a test substance with a cell material having a PNPLA1 gene or a biological material collected from a non-human animal having a PNPLA1 gene, an acylceramide in which linoleic acid is ester-bonded at the ω position is detected and obtained. A screening method for a skin disease therapeutic drug, comprising a step of selecting a skin disease therapeutic substance using a detection result as an index.
(18) When the acyl ceramide in which linoleic acid is ester-bonded at the ω position is increased as compared with a control cell or biomaterial that does not contact the test substance, the test substance is selected as a skin disease therapeutic agent. The method according to (17).
(19) Measuring the expression level of PNPLA1 or the expression level of a gene related to ceramide metabolism after contacting a test substance with a biomaterial collected from a cell having a PNPLA1 gene or a non-human animal having a PNPLA1 gene A method for screening a therapeutic agent for skin diseases, comprising a step of selecting a therapeutic agent for skin diseases using an obtained measurement result as an index.
(20) The method according to (19), wherein when the expression level of PNPLA1 is increased as compared with a control sample not contacted with the test substance, the test substance is selected as a therapeutic agent for skin diseases.
(21) The method according to (19), wherein the ceramide metabolism-related gene is at least one selected from the group consisting of Elovl4, Lass3, Cyp4f39, Ugcg, Abca12, Gba, Abhd5, Aloxe3 and Alox12b.
(22) When the expression level of a ceramide metabolism-related gene is increased as compared with a control cell or biomaterial that is not brought into contact with the test substance, the test substance is selected as a skin barrier function promoter, (21) The method described in 1.
(23) After contacting a test substance with a sample containing PNPLA1 and ω-hydroxyceramide, acyl ceramide in which linoleic acid is ester-bonded at the ω position is detected, and a skin disease therapeutic substance is selected using the obtained detection result as an index. A method for screening a skin disease therapeutic agent, comprising a step.
(24) When the acyl ceramide in which linoleic acid is ester-bonded at the ω position is increased as compared with a control sample that does not contact the test substance, the test substance is selected as a therapeutic agent for skin disease. (23) The method described in 1.
(25) After contacting a test substance with a microarray equipped with the PNPLA1 gene and / or ceramide metabolism-related gene, the expression of the PNPLA1 gene and / or ceramide metabolism-related gene is detected, and the treatment result is used as an index to treat skin diseases. A screening method for a skin disease therapeutic drug, comprising a step of selecting a substance.
(26) When the expression level of the PNPLA1 gene is increased as compared to the PNPLA1 gene mounted on the control microarray that does not contact the test substance, the test substance is selected as a therapeutic agent for skin disease. (25) The method described in 1.
(27) The method according to (25), wherein the ceramide metabolism-related gene is at least one selected from the group consisting of Elovl4, Lass3, Cyp4f39, Ugcg, Abca12, Gba, Abhd5, Aloxe3 and Alox12b.
(28) When the expression level of a ceramide metabolism-related gene is increased as compared with a ceramide metabolism-related gene mounted on a control microarray that does not contact the test substance, the test substance is selected as a skin barrier function promoter. The method according to (27).
(29) The skin disease is at least one selected from the group consisting of ichthyosis, atopic dermatitis, psoriasis, rough skin, dry skin, keratosis, dry skin, eczema and dry dermatitis (15) to (28) The method according to any one of (28).
(30) A method for producing an acylceramide, characterized in that acylceramide is collected after PNPLA1 is allowed to act on ω-hydroxyceramide.
(31) PNPLA1 gene and / or ceramide metabolism-related after contacting a test substance with a cell material having a PNPLA1 gene or ceramide metabolism-related gene or a biomaterial collected from a non-human animal having a PNPLA1 gene or ceramide metabolism-related gene A method for testing a test substance, comprising measuring a gene expression level and evaluating the property of the test substance using the obtained measurement result as an index.

(32) Measure the expression level of the PNPLA1 gene and / or ceramide metabolism-related gene after contacting the test substance with the microarray carrying the PNPLA1 gene and / or ceramide metabolism-related gene, and test using the obtained measurement result as an index A method for inspecting a test substance, characterized by evaluating a property of the substance.
(33) After bringing a test substance into contact with a biomaterial collected from a cell having a PNPLA1 gene or a ceramide metabolism-related gene or a non-human animal having a PNPLA1 gene or a ceramide metabolism-related gene, linoleic acid is esterified at the ω position. A method for testing a test substance, comprising detecting bound acylceramide and evaluating the property of the test substance using the obtained detection result as an index.
(34) When the expression level of the PNPLA1 gene is decreased as compared to a control cell or biomaterial that does not contact the test substance, the test substance is determined to be a substance that reduces the skin barrier function. 31) The method.
(35) The ceramide metabolism-related gene is at least one selected from the group consisting of Elovl4, Lass3, Cyp4f39, Ugcg, Abca12, Gba, Abhd5, Aloxe3 and Alox12b, in any one of (31) to (33) The method described.
(36) When the expression level of a ceramide metabolism-related gene is lower than that of a ceramide metabolism-related gene mounted on a control microarray that does not contact the test substance, the test substance is a substance that reduces the skin barrier function The method according to (35), wherein the method is determined.
(37) When the acyl ceramide in which linoleic acid is ester-bonded at the ω position is lower than that of a control cell or biomaterial that does not contact the test substance, the test substance is a substance that lowers the skin barrier function. The method according to (33), which is determined to be present.

  The present invention provides a method for synthesizing acyl ceramides using PNPLA1, and a screening method for skin barrier function promoters and skin disease therapeutic agents.

  According to the present invention, PNPLA1 was shown to be an enzyme necessary for the biosynthesis of acylceramide in epidermal keratinocytes. Acylceramide has a complex and special structure and is a major component of intercellular corneocyte lipids that are important for the formation of a skin barrier, but no metabolic enzyme has been identified. Since ω-hydroxyceramide can already be synthesized, by using PNPLA1, it is possible to biochemically synthesize acylceramide that could not be industrially mass-produced until now. In addition, drugs that increase the expression level of PNPLA1 in the skin can increase the ability to synthesize endogenous acylceramide, making them useful as therapeutic agents for dermatologic diseases such as ichthyosis and atopic dermatitis with abnormal skin barriers. .

It is a figure which shows the ichthyosis resulting from formation of a skin barrier and abnormality of ceramide metabolism. PNPLA is a diagram showing a (Patatin-like PLA) / iPLA 2 family. It is a figure which shows that Pnpla1 is expressed with the differentiation of an epidermal keratinocyte. It is a figure explaining establishment of a systemic Pnpla1 gene-deficient mouse. It is a figure which shows abnormality of the skin barrier function and epidermis form of Pnpla1 ^{− / −} mouse. It is a figure which shows that acyl ceramide decreases sharply in the epidermis of Pnpla1 ^{− / −} mouse. It is a figure which shows the comprehensive analysis of epidermis ceramide molecular species by a liquid chromatograph mass spectrometer (LC-MS). It is a figure which shows that the various acyl ceramides which have linoleic acid in the ω position decrease in the epidermis of Pnpla1 ^{− / −} mice. It is a figure which shows the summary of the change of the ceramide molecular species in the epidermis of Pnpla1 ^{− / −} mouse. It is a figure which shows that the expression level of a ceramide metabolism related gene increases in Pnpla1 ^{− / −} mouse. It is a figure which shows that the expression of keratin structure protein falls in Pnpla1 ^{− / −} mouse. It is a figure which shows that a skin specific Pnpla1 deficient mouse also dies by skin abnormality. It is a figure which shows that a skin barrier function falls in the skin-specific Pnpla1 deficient mouse several days after birth. It is a figure which shows the expression level increase of the ceramide metabolism related gene in a skin specific Pnpla1 deficient mouse. It is a figure which shows that skin barrier formation falls in the three-dimensional culture system of a keratinocyte derived from Pnpla1 ^{− / −} mouse. It is a figure which shows the synthesis | combination of the acyl ceramide from the omega-hydroxyceramide by PNPLA1.

Hereinafter, the present invention will be described in detail.
1. Overview
PNPLA1 (patatin-like phospholipase domain containing 1) is a lipid metabolic enzyme that is expressed with the differentiation of epidermal keratinocytes. As a result of producing a mouse deficient in the Pnpla1 gene and conducting phenotypic analysis, the present inventor suffered severe transdermal water loss and weight loss due to severe skin barrier function deterioration, and died soon after birth. I found out. Lipid analysis of the epidermis reveals that PNPLA1 is essential for the reaction of biosynthesis of acylceramide from ceramide. If this process does not proceed normally, not only does the formation of the stratum corneum lipid layer fail, but also the stratum corneum It was found that the expression of the constituent proteins filaggrin and loricrin was decreased and the skin barrier function was lowered.

  Providing a method of synthesizing acylceramide using PNPLA1 is expected to be an effective means for developing therapeutic agents and cosmetics that promote enhancement of skin barrier function.

  The findings found in the present invention are described in detail below.

  In the present specification, “PNPLA1” represents a protein (enzyme), and “Pnpla1” represents a gene, but for convenience of explanation, a gene encoding PNPLA1 is referred to as “PNPLA1 gene”. Sometimes written. “Pnpla1” and “PNPLA1 gene” are synonymous.

  Moreover, the abbreviations of ceramides used in the present specification are as follows.

EOdS: N- (ω-OH-acyl) acyl-dihydrosphingosine
EOS: N- (ω-OH-acyl) acyl-sphingosine,
EOP: N- (ω-OH-acyl) acyl-phytosphingosine
EOH: N- (ω-OH-acyl) acyl-6-OH-sphingosine
NdS: N-acyl-dihydrosphingosine
NS: N-acyl-sphingosine
NP: N-acyl-phytosphingosine
NH: N-acyl-6-OH-sphingosine
AdS: N- (α-OH-acyl) -dihydrosphingosine
AS: N- (α-OH-acyl) -sphingosine
AP: N- (α-OH-acyl) -phytosphingosine
AH: N- (α-OH-acyl) -6-OH-sphingosine

(1) Pnpla1 mRNA induces expression with keratinocyte differentiation
When quantitative expression of Pnpla1 mRNA in each organ of C57BL / 6 mice was analyzed by quantitative PCR, it was found to be specifically expressed in the skin (Fig. 3A) and distributed in the epidermis from the results of in situ hybridization. (Figure 3B). Furthermore, when MPEK cells, which are mouse epidermal keratinocyte progenitor cells, were cultured in the presence of 1 mM CaCl ₂ to induce differentiation, the expression of Pnpla1 mRNA was induced from the second day of differentiation induction as in Krt10 and Lor (FIG. 3C). . Therefore, PNPLA1 expression was found to be closely related to keratinocyte differentiation.

(2) Failure of skin barrier function in Pnpla1-deficient mice
A homologue was introduced by introducing a targeting vector with a structure in which the FRT-LacZ-loxP-Neo-FRT-loxP sequence was inserted upstream of exon 2 of the Pnpla1 gene and the loxP sequence was inserted downstream of exon 3 into the RENKA strain of C57BL / 6-derived ES cells. Recombinant ES cells were selected (FIG. 4A). After producing chimeric mice and F1 mice, mice lacking exons 2 and 3 were mated in the order of Flippase overexpressing mice and Cre recombinase overexpressing mice, and then heterozygous mice were mated. Pnpla1 homo-deficient mice were born according to Mendel's law (FIGS. 4B and C), but dryness and hardening of the skin, low body weight were observed (FIGS. 4D and E), and died within one day after birth. When cesarean section was performed on day 18.5, the weight of homo-deficient mice was the same as that of wild-type mice, but decreased with time (Fig. 5A), and skin drying and hardening progressed. The skin barrier function was evaluated by transepidermal water loss (TEWL) and skin permeability test using toluidine blue staining solution, and severe transdermal water loss and staining solution penetration were observed ( FIG. 5B, C). Further, when the skin section was stained with HE, a striking decrease in intercellular corneocyte lipids consisting of ceramide and cholesterol was observed (FIG. 5D).

  From the above results, in Pnpla1-deficient mice, the skin barrier function accompanied by dysplasia of keratinocyte lipids was markedly decreased, and it was thought that death occurred early due to water loss.

(3) Abnormal biosynthesis of acyl ceramide in Pnpla1-deficient mice Keratinocytes produce lamellar bodies containing abundant lipids such as ceramide in the granule layer, and when further differentiation proceeds to the stratum corneum, It is secreted extracellularly and forms keratin intercellular lipids that are important for skin barrier function (Fig. 1A). When frozen sections of skin were stained with Nile Red, keratinocyte lipids were regularly stained in sheet form in wild-type mice, but many granular structures were observed in the stratum corneum in Pnpla1 homo-deficient mice (Fig. 5E), an abnormal secretion of lamina was suspected. Furthermore, when lipid composition analysis was performed by developing lipids extracted from the neonatal mouse epidermis using thin layer chromatography (TLC), there were significant changes in phospholipids such as phosphatidylcholine (PC) and phosphatidylethanolamine (PE). In contrast, acylceramide (EOS) and acylglucosylceramide (ω-O-AcylGlcCer) were drastically decreased in Pnpla1 homo-deficient mice, and ω-hydroxyceramide (ω-OH-) located immediately upstream of the metabolic pathway. Cer), glucosylceramide (GlcCer) and 1-O-acylceramide (1-O-AcylCer) derived from ceramide (NS) were increased (FIG. 6B). This result shows that in the absence of PNPLA1, the reaction to biosynthesize acylceramide EOS from ω-OH-Cer (and acylglucosylceramide (ω-O-AcylGlcCe) from ω-hydroxyglucoside ceramide (ω-OH-GlcCer) is produced. (Reaction to synthesize) does not proceed, and instead, metabolites that flowed to other pathways were thought to increase (FIG. 6A).

  Furthermore, through comprehensive analysis of epidermal ceramide molecular species using LC / MS, detailed analysis to detect differences in sphingosine skeleton, fatty acid carbon number, degree of unsaturation, or modification by hydroxy group, it is in the ω position. Various acylceramide molecular species (EOH, EOP, EOS, EODS, etc.) to which linoleic acid is ester-linked, acylglucoside ceramide (Glc-EOS, Glc-EOH), and ultra-long length covalently bonded to keratinocytes at the ω position The chain fatty acid was remarkably decreased (FIGS. 7, 8, and 9). On the other hand, non-linoleic acid ester type ceramides such as NS, NdS, AP and NP before being metabolized to acylceramides showed an increasing tendency in Pnpla1 homo-deficient mice (FIGS. 7 and 9). Therefore, it was confirmed that in the absence of Pnpla1, synthesis of not only EOS but also various other acylceramide molecular species would not proceed.

(4) Cernamide synthesis pathway and keratin structure protein expression change in Pnpla1-deficient mice
We examined gene expression changes in newborn mouse epidermis by DNA microarray analysis, and found that many of the gene expressions of enzymes and transporters related to the ceramide synthesis pathway with ultra-long chain fatty acids unique to the skin known so far Was found to increase more than twice in Pnpla1-deficient mice. Quantitative PCR confirmed that the expression of Elovl4, Lass3, Cyp4f39, Ugcg, Abca12, Abhd5, Tgm1, etc. increased several fold (FIG. 10). Since the synthesis of acylceramide is stopped in Pnpla1-deficient mice, the expression of these genes is thought to increase due to a feedback mechanism that increases the supply of raw materials necessary for acylceramide synthesis. This also means that even if a metabolic pathway upstream of acylceramide synthesis is promoted, the absence of Pnpla1 does not lead to acylceramide synthesis or enhancement of the skin barrier function.

  Furthermore, when the expression of the differentiation marker gene group corresponding to the four-layer structure of the epidermis was analyzed, the expression of the basal layer marker Krt5 and the spiny layer marker Krt1 was not significantly different between the wild-type and Pnpla1-deficient mice. In addition, among the main components of the peripheral zone, which is an insoluble structure that lines the cell membrane of keratinocytes, the expression level of involucrin (Ivl) produced in spinous cells did not change, whereas in granule cells The amount of loricrin (Lor) produced was drastically reduced (Fig. 11A). In addition, the expression of filaggrin (Flg), which is an important factor for the synthesis of natural moisturizing factor and keratin fiber aggregation and the main component of granule layer keratohyaline granules, was also significantly decreased in Pnpla1-deficient mice (Fig. 11A). ).

  Also in immunohistochemical staining using skin sections of newborn mice, it was confirmed that the expression of loricrin and filaggrin at the protein level was significantly reduced (FIG. 11B). On the other hand, the expression of S100a9, an inflammatory marker, increased dramatically (FIG. 11A).

  Therefore, in Pnpla1-deficient mice, not only the inhibition of acylceramide synthesis and abnormal formation of the lipid layer between keratinocytes, but also the decrease in loricrin and filaggrin synthesized in the outermost layer of the granule layer are factors in reducing the skin barrier function. Furthermore, it was revealed that inflammation was secondarily increased by the breakdown of the skin barrier function.

(5) Decreased skin barrier function and abnormal acylceramide synthesis in skin-specific Pnpla1-deficient mice
Repeated mating of Pnpla1 ^{f / +} mice with loxP sequences upstream of exon 2 and downstream of exon 3 of the Pnpla1 gene and K14-Cre mice expressing Cre recombinase specifically in the skin, resulting in skin-specific Pnpla1 deficiency Mice (Pnpla1 ^{f / f} K14-Cre mice) were prepared (FIG. 12A). Although the expression of Pnpla1 in the skin of this mouse was not completely lost, it was significantly reduced (FIG. 12B). From 0 to 2 days after birth, no abnormal appearance was observed (FIG. 13A), but from around 3 days after birth, an increase in body weight (FIG. 12C) and skin peeling (FIG. 12D) were observed. Died by day 6 after birth (FIG. 12E). The transcutaneous water transpiration of this skin-specific Pnpla1-deficient mouse showed a normal value at the non-peeling site of the skin, but a markedly high value at the peeling site (FIG. 13B). Therefore, although skin-specific Pnpla1 deficiency has a slightly longer survival time than systemic Pnpla1 deficiency, it was considered that water loss was caused by a marked decrease in skin barrier function.

  Analysis of the composition of lipids extracted from the epidermis of skin-specific Pnpla1-deficient mice on the fifth day of life (P5) by TLC revealed that acylceramide EOS and acylglucosylceramide were found in the same way as in systemic Pnpla1-deficient newborns. The content was drastically reduced, and ω-OH ceramide and 1-O-acylceramide accumulated instead (FIG. 13C). Changes in the expression of other ceramide-metabolizing enzymes in the skin of skin-specific Pnpla1-deficient mice showed the same tendency as in systemic Pnpla1-deficient mice (FIG. 14). These results suggest that the decrease in skin barrier function and acylceramide synthesis abnormalities due to Pnpla1 deficiency are not the effects of Pnpla1 that can be expressed in small amounts in tissues and cells other than skin keratinocytes, and the role of Pnpla1 expressed in epidermal keratinocytes Is directly indicated.

(6) Role of Pnpla1 in skin barrier formation in 3D cultured skin model Keratinocytes prepared from wild-type mice formed a stratum corneum with basket wave appearance after 2 weeks of 3D culture, but derived from Pnpla1 homo-deficient mice In keratinocytes, the stratum corneum with stratum corneum lipids was not formed even after three-dimensional culture (FIG. 15A). Furthermore, from the permeability test of the fluorescent dye Lucifer Yellow into the tissues, the wild type keratinocytes acquired the skin barrier function, but the keratinocytes derived from the Pnpla1 homo-deficient mice showed that the fluorescent dye penetrated into the tissues (Fig. 15B), it has been clarified that Pnpla1 is also necessary for obtaining the skin barrier function in an in vitro skin model.

(7) Synthesis of acylceramides using recombinant PNPLA1 protein
In the epidermis of Pnpla1-deficient mice, acylceramides required for the skin barrier function have been drastically reduced, and ω-OH ceramide and metabolites generated in the reaction pathway branched from the upstream have increased, so PNPLA1 has been The possibility of being an acylceramide synthase that has not been discovered is assumed. Therefore, recombinant PNPLA1 protein was prepared using a baculovirus-insect cell expression system suitable for mass production of proteins, and acylceramide synthesis activity was examined using ω-hydroxyceramide as a substrate. By adding linoleyl CoA labeled with radioactive isotope ¹⁴ C to the reaction system as a linoleic acid source, synthesis of acylceramide (EOS) in which linoleic acid was bonded to the ω position was promoted (FIG. 16).

(8) Discussion
According to the present invention, PNPLA1 was shown to be an enzyme necessary for the biosynthesis of acylceramide in epidermal keratinocytes. Acylceramide has a complex and special structure and is a major component of intercellular corneocyte lipids that are important for the formation of a skin barrier, but no metabolic enzyme has been identified. Since ω-hydroxyceramide can be synthesized at the cellular level, it is possible to biochemically synthesize acylceramide that could not be industrially mass-produced until now by using PNPLA1. In addition, a drug that increases the expression level of PNPLA1 in the skin may have a medicinal effect on skin diseases such as ichthyosis and atopic dermatitis with abnormal skin barrier by increasing the ability to synthesize endogenous acylceramide.

  In Pnpla1-deficient mice, not only metabolites downstream from acylceramide were produced, but the expression levels of loricrin and filaggrin were reduced. It has been pointed out that lipid metabolites in the stratum corneum act as ligands for nuclear receptors such as PPAR and LXR to regulate keratinocyte differentiation and stratum corneum structural protein expression (Reference 5). It is also assumed that such transcriptional regulation may be affected by acylceramide or a metabolite located in the metabolic pathway downstream thereof.

2. Screening method According to the present invention, PNPLA1 was shown to be an enzyme necessary for the biosynthesis of acylceramide in epidermal keratinocytes, so that expression of PNPLA1 or acylceramide (acylceramide in which linoleic acid was ester-linked to the ω position) A skin barrier function promoter or a skin disease therapeutic agent can be screened using synthesis as an index.

  Therefore, in the screening method of the present invention, when a test substance is administered to a non-human animal deficient in the PNPLA1 gene, and a skin barrier function promoting effect is obtained in the non-human animal after administration of the test substance, A step of selecting the test substance as a skin barrier function promoter.

  The present invention also provides an acyl ceramide in which a test substance is administered to a non-human animal having a PNPLA1 gene, and the expression level of PNPLA1 in the non-human animal after administration of the test substance, or linoleic acid is ester-linked to the ω position. A step of selecting the test substance as a skin barrier function promoter.

  In another embodiment, the screening method of the present invention is a method in which a test substance is brought into contact with a biological material collected from a cell having a PNPLA1 gene or a non-human animal having a PNPLA1 gene, and then linoleic acid is ester-linked to the ω position. And a step of selecting a substance that promotes the skin barrier function using the obtained detection result as an index.

  In addition, the screening method of the present invention includes the expression level of PNPLA1 or the expression of a gene related to ceramide metabolism after contacting a test substance with a biomaterial collected from a cell having a PNPLA1 gene or a non-human animal having a PNPLA1 gene. A step of measuring the amount and selecting a substance that promotes the skin barrier function using the obtained measurement result as an index.

  In still another embodiment, the screening method of the present invention is a method for detecting acylceramide after contacting a test substance with a sample containing PNPLA1 and ω-hydroxyceramide, and using the obtained detection result as an index, a substance that promotes skin barrier function The step of selecting is included.

  In still another embodiment, the screening method of the present invention detects the expression of a PNPLA1 gene and / or a ceramide metabolism-related gene after contacting a test substance with a microarray equipped with the PNPLA1 gene and / or a ceramide metabolism-related gene, The method includes a step of selecting a substance that promotes the skin barrier function using the obtained detection result as an index.

  In the present invention, a test substance (candidate substance) that is a candidate for a skin barrier function promoter and a skin disease treatment drug is not particularly limited, and examples thereof include peptides, proteins, DNA, non-peptide compounds, and synthetic compounds. , Fermentation products, cell extracts, plant extracts, and the like. These compounds may be novel compounds or known compounds. These test substances may form a salt. As the salt of the test substance, a salt with a physiologically acceptable acid (for example, an inorganic acid) or a base (for example, an organic acid) is used. Physiologically acceptable acid addition salts are preferred. Examples of such salts include salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid, etc.), or organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, And salts with succinic acid, tartaric acid, citric acid, malic acid, succinic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid, and the like. The test substance may be tested on a single substance independently or on a mixture (including a library, etc.). Examples of the library containing a plurality of test substances include a synthetic compound library (such as a combinatorial library) and a peptide library (such as a combinatorial library).

  The method of administering the test substance to the non-human animal is not particularly limited. For example, oral administration, parenteral administration (injection, application, etc.), etc. are used, depending on the type of animal and the nature of the test substance. Can be selected as appropriate. Further, the dose of the test substance can be appropriately selected according to the administration method, the nature of the test substance, and the like. In order to bring the test substance into contact with the non-human animal itself, there are modes in which the test substance is inoculated into the animal by injection, applied to the skin of the test substance, spread, sprayed, or the like.

  The non-human animal (test animal) to be administered with the test substance and the control animal are not particularly limited, but usually the same kind of non-human animal is used. Moreover, it is preferable to use animals of the same litter as the test animal and the control animal, and it is more preferable to use animals of the same sex and the same age. Examples of non-human animals include rodents such as mice, rats, and guinea pigs, as well as rabbits, birds, goats, cows, horses, pigs, and the like.

  In the present invention, a test substance is administered to a non-human animal to examine the regeneration, repair or enhancement effect of the skin barrier function.

  A test substance having these effects can be selected as a skin barrier function promoter. The promoter thus selected can be selected as a drug or cosmetic for skin diseases, for example, a therapeutic agent or cosmetic for ichthyosis, atopic dermatitis and the like.

  The “skin barrier function” means a function of the skin that prevents foreign substances from entering from the outside of the body, or prevents evaporation of body water or leakage of body fluids. The epidermis of the skin has a four-layer structure consisting of the basal layer, spiny layer, granule layer, and stratum corneum in order from the inside. The outermost stratum corneum is responsible for most of the skin barrier function, and it has a “peripheral zone” which is an insoluble structure that is physically and chemically very stable. The epidermis is filled with keratin and filaggrin aggregates. It consists of keratinocytes and a multi-layered “interkeratinic lipid” composed mainly of ceramide, cholesterol, and fatty acids that fills them (Fig. 1A). In analogy to a brick block, a keratinocyte lipid equivalent to mortar (cement) fills the space between the epidermal keratinocytes equivalent to bricks to form a strong wall, and promotes the skin barrier function.

  “Promotion of skin barrier function” means regenerating, repairing, or enhancing keratinocyte lipids or CLE having a barrier function in the skin. The “skin barrier function promoting substance” means a substance having an effect of regenerating, repairing or enhancing the skin barrier function or an effect of preventing a decrease in the skin barrier function.

  “Regeneration” means to re-create the lost skin barrier function, “Repair” means to restore and restore the partially lost skin barrier function, and “Enhancement” means that the existing barrier function is further increased. Enhancing and preventing means preventing deterioration of the barrier function already possessed.

The items for evaluating whether the test substance promoted the skin barrier function include, for example, PNPLA1 expression level, ceramide metabolism-related gene expression level, acyl ceramide level, and keratinocyte detachment phenomenon. The presence or absence of skin in the state, the presence or absence or degree of epidermal tissue reduction, epidermal cell differentiation and proliferation marker, transdermal moisture transpiration, filaggrin expression and the like. These evaluations can be performed using methods such as appearance observation of animals, histological analysis, genetic engineering analysis, biochemical analysis, and the like. And, regarding the skin barrier function promoting effect, at least one of effects such as suppression of onset of skin disease, reduction of skin disease, increase in the amount of acylceramide, increase in the expression level of PNPLA1, and the appearance or disappearance of epidermal cell differentiation / proliferation markers If observed, it is judged that the skin barrier function has been regenerated, repaired, enhanced or reduced, and the test substance is selected as a substance having a skin barrier function promoting action (skin barrier function promoter). . The ceramide metabolism-related gene includes at least one selected from the group consisting of Elovl4, Lass3, Cyp4f39, Ugcg, Abca12, Gba, Abhd5, Aloxe3 and Alox12b. Cyp4f39 is CYP4F22 in humans. In PNPLA1 gene knockout mice, the expression of these ceramide metabolism-related genes increases, but this works as a feedback mechanism to compensate for the lack of acylceramide and promotes the upstream ceramide metabolic pathway that supplies acylceramide raw materials it is conceivable that. Therefore, when the expression level of the ceramide metabolism-related gene is increased compared to the control, the test substance is selected as a skin barrier function promoter. In other words, when the expression level of the ceramide metabolism-related gene changes compared to the control, the test substance is selected as a skin barrier function modifier.

The non-human animal deficient in the PNPLA1 gene used in the present invention may be an animal in which the PNPLA1 gene is homoknocked out or heterozygously knocked out, but a systemic homoknockout animal is very early after birth as described above. Because of death, it is preferable to use skin-specific homo knockout animals or hetero knockout animals.

  In the present invention, in addition to non-human animals deficient in the PNPLA1 gene, non-human animals having the PNPLA1 gene but mutated in this gene can be used. Mutation refers to a gene containing a sequence that does not encode a protein having PNPLA1 activity, such as deletion, substitution, addition, etc. in part of the full-length sequence of PNPLA1 gene, that is, the original function of PNPLA1 gene. It means a gene that you do not have.

  In the present invention, in order to evaluate the effect of promoting skin barrier function, a non-human animal having the PNPLA1 gene itself can be used. However, the present invention is not limited to this, and the cell having the PNPLA1 gene, or the non-human animal Biomaterials collected from can also be used. In this case, an acylceramide, for example, a ceramide esterified with linoleic acid at the ω position is detected after contacting the test substance with a cell having the PNPLA1 gene or a biological material collected from a non-human animal having the PNPLA1 gene. . When using a hetero knockout non-human animal or a non-human animal having a mutant PNPLA1 gene, the expression of PNPLA1 can also be used as an index. When acylceramide or PNPLA1 increases, or when the expression level of the PNPLA1 gene increases, the test substance is selected as a skin barrier function promoter.

Furthermore, in the present invention, when using a non-human animal deficient or mutated in the PNPLA1 gene or a biomaterial obtained from a non-human animal deficient or mutated in the PNPLA1 gene, the PNPLA1 gene is knocked out or mutated as a comparative control. It is preferable to use a wild type non-human animal that has not been knocked out or a cell in which the PNPLA1 gene has not been knocked out or mutated. For example, when the amount of acylceramide in which linoleic acid is ester-bonded at the ω position is increased compared to control cells or biomaterials that do not contact the test substance, the test substance is selected as a skin barrier function promoter.

  Furthermore, in the present invention, not a non-human animal deficient in the PNPLA1 gene but a non-human animal having the PNPLA1 gene, for example, a wild type non-human animal, or a biomaterial collected from the non-human animal, or a cell having the PNPLA1 gene Can also be used. In this case, a non-human animal containing the PNPLA1 gene, a biomaterial collected from the animal, or a cell containing the PNPLA1 gene is brought into contact with the test substance, and the amount of acylceramide in which linoleic acid is ester-linked to the ω position after contact. A skin barrier function promoter can also be selected using the expression level of PNPLA1, the PNPLA1 enzyme activity or the expression of epidermal cell differentiation / proliferation marker as an index. The selection method and the determination method when the control is used are the same as described above.

Examples of biomaterials collected from non-human animals include non-human animal-derived skin sections (skin tissues), cells, body fluids, blood, lymph fluids, and the like. The “cell containing PNPLA1 gene” means a cell other than a biomaterial collected from a non-human animal, and its type is not limited. Examples include established cultured cells, transformed cells in which the PNPLA1 gene has been introduced by genetic engineering (including transformed E. coli, yeast, plant cells, etc.).

  “Contact” means an aspect in which a test substance is administered to a non-human animal, an aspect in which the test substance is added to a biological material, an aspect in which the test substance is cultured in the presence of the test substance, and the like. In order to administer a test substance to a non-human animal itself, there are modes in which the test substance is inoculated into these animals by injection or the like, applied to the skin of the test substance, sprayed, sprayed, or the like. Examples of the mode of adding the test substance to the biological material include adding the test substance to a cell culture, adding the test substance to a body fluid, blood, lymph, or the like. “Culture” means any of cells, culture media, and cell extracts. “Culturing in the presence of a test substance” means culturing under conditions where the cell and the test substance are in contact with each other, and the contact of the test substance with the cells or the like is, for example, a cell culture medium Or by adding a test substance into various buffer solutions (for example, HEPES buffer solution, phosphate buffer solution, phosphate buffered saline, etc.) and incubating the cells for a certain period of time.

  The concentration of the test substance added to the culture varies depending on the characteristics (type of compound, solubility, toxicity, etc.), but is appropriately selected within the range of 1 nM to 1,000 μM, for example. Examples of the incubation time include 24-72 hours.

  “Epithelial cell differentiation / proliferation marker” means a molecule whose expression is specifically induced when the basal cells in the lowermost layer of the epidermis proliferate and differentiate into epidermal spiny cells, granule cells, and corneocytes. Examples include Keratin 1, Keratin 5, loricrin, and filaggrin. The measurement method for measuring epidermal cell differentiation / proliferation markers is not limited, and mRNA expression level is analyzed by quantitative PCR, or protein expression level is analyzed by Western blotting or immuno-antibody staining. Regarding the selection criteria when the epidermal cell differentiation / proliferation marker is used, when the expression of Keratin 1, Keratin 5, loricrin, filaggrin or the like is increased, it is determined that the proliferation / differentiation of epidermis cells has progressed.

  Furthermore, in the present invention, it is possible to biochemically measure the enzyme activity of PNPLA1 without using the animals and cells, and to use the measurement result as an index. It is also possible to detect the expression of the PNPLA1 gene using PCR or DNA microarray.

  In the case where the animal or cell is not used biochemically, it includes the step of treating PNPLA1 with a test substance to detect the enzyme activity of PNPLA1, and selecting a skin barrier function promoter using this detection result as an index. . “Treatment with a test substance” means that both are mixed in an assay system in which PNPLA1 and the test substance are in contact with each other. For example, mixing PNPLA1 and a test substance in a test tube, adding a test substance to a test tube containing PNPLA1, adding PNPLA1 to a test tube containing a test substance, and reacting for a predetermined time if necessary It means that Specifically, a step of detecting acylceramide after contacting a test substance with a sample containing PNPLA1 and a substrate (ω-hydroxyceramide), and selecting a substance that promotes skin barrier function using the obtained detection result as an index. Including.

  Enzyme activity can be measured by adding PNPLA1 recombinant enzyme preparation to ω-hydroxyceramide as a substrate and linoleic acid-containing lipid and quantifying the resulting acylceramide.

  As an apparatus for measuring enzyme activity, for example, TLC, LC / MS (mass spectrometry), LSC-6100 liquid scintillation counter (ALOKA), ARVO mx multi-label counter (Perkin Elmer) and the like can be used.

  When the measurement result measured in cells cultured in the presence of the test substance or the measurement result of the activity of PNPLA1 treated with the test substance is increased compared to the control, the test substance is removed from the skin barrier. Select as a function promoter.

  When using a DNA microarray, contact the test substance with the microarray equipped with the PNPLA1 gene and / or ceramide metabolism-related gene, and then detect the expression of the PNPLA1 gene and / or ceramide metabolism-related gene. Selecting a substance that promotes skin barrier function. A “DNA microarray” is an array of a plurality of nucleic acids (probes) that bind to a test substance on a microsubstrate. The specimen is brought into contact with the probe arranged on the microarray substrate, the test substance is reacted with the probe, and then the presence or expression level of the test substance present in the specimen is examined by detecting the conjugate. Can do. The microarray that can be used in the present invention is not particularly limited, and various types of microarrays can be used. For example, Whole Mouse Genome Oligo DNA Microarray Kit Ver2.0 (4x44K) (Agilent Technologies), SurePrint G3 Human GE Microarray 8x60K ver.2.0 (Agilent Technologies), GeneChip (registered trademark) Human Transcriptome Array 2.0 (Affymetrix) Manufactured microarray). When the PNPLA1 gene (probe) is not mounted on these microarrays, they can be mounted by well-known methods, for example, by photolithography on a substrate, or by spotting a pre-synthesized probe on the substrate. it can. The PNPLA1 gene may be full length or partial length.

  In order to detect the expression of the PNPLA1 gene using a DNA microarray, a predetermined microarray scanner (SureScan; Agilent Technologies) or the like can be used.

  Since the substance selected as a skin barrier function promoter as described above increases the expression of PNPLA1 or promotes the synthesis of acylceramide, the effect of regenerating, repairing or enhancing the skin barrier function can be obtained. Therefore, such a substance can be selected as a blended product such as a skin disease therapeutic drug or cosmetic.

  The skin disease is not particularly limited. For example, ichthyosis, atopic dermatitis, rough skin, psoriasis, psoriasis, contact dermatitis, keratosis, eczema, dry dermatitis, rough skin, dryness Although skin etc. are mentioned, it is not limited to these diseases.

3. Method for evaluating safety of test substance In the present invention, it is considered that a substance that decreases the expression of the PNPLA1 gene decreases the skin barrier function.

  Therefore, the present invention can be used as an inspection method for confirming the safety of a test substance.

  First, a test substance is brought into contact with a biomaterial collected from a cell having a PNPLA1 gene or a ceramide metabolism-related gene or a non-human animal having a PNPLA1 gene or a ceramide metabolism-related gene. Next, the expression level of the PNPLA1 gene and / or ceramide metabolism-related gene is measured. The obtained measurement result is an index for evaluating the property of the test substance.

  In the present invention, the amount of expression of the PNPLA1 gene and / or ceramide metabolism-related gene is measured after contacting the test substance with a microarray equipped with the PNPLA1 gene and / or ceramide metabolism-related gene, and the same evaluation as above is performed. can do.

  Furthermore, in the present invention, after contacting the test substance with a biological material collected from a cell having a PNPLA1 gene or a ceramide metabolism-related gene or a non-human animal having a PNPLA1 gene or a ceramide metabolism-related gene, the ω position An acylceramide in which linoleic acid is ester-bonded to can be detected and evaluated in the same manner as described above. For example, when the expression level of the PNPLA1 gene after treatment with the test substance decreases compared to the control, the test substance is determined to be a substance that reduces the skin barrier function. In addition, when the acyl ceramide in which linoleic acid is ester-bonded at the ω position is lower than that of a control cell or biomaterial that does not contact the test substance, the test substance is a substance that reduces the skin barrier function. Is determined.

  On the other hand, at least one ceramide metabolism-related gene selected from the group consisting of Elovl4, Lass3, Cyp4f39, Ugcg, Abca12, Gba, Abhd5, Aloxe3 and Alox12b was confirmed to increase in PNPLA1 gene knockout mice as described above. It is thought that feedback to increase the raw materials necessary for ceramide synthesis works. Therefore, it can be determined that a substance that increases these ceramide metabolism-related genes is a substance that increases the skin barrier function.

4). Biosynthesis of acylceramides using PNPLA1 The present invention provides a method for producing acylceramides, wherein PNPLA1 is allowed to act on ω-hydroxyceramides.

  ω-Hydroxyceramide can be prepared, for example, by allowing CYP4F22 (human) or Cyp4f39 (mouse) to act on ultra-super ceramide (Ohno Y, Ohkuni A, Kamiyama N, Kihara A; Epidermal-specific ω- hydroxylated ceramide is synthesized by the cytochrome P450 4F subfamily protein CYP4F22. 11th International Symposium on Future Drug Discovery and Medical Innovation, Sapporo, 2013. 8. 2.). Alternatively, commercially available ω-hydroxyceramide (N-ω-Hydroxytriacontanoyl-D-erythro-sphingosine; MATERYA) can be used.

  PNPLA1 is allowed to act on the above ω-hydroxyceramide.

  “Activate” means that the substrate ω-hydroxyceramide is made to catalyze the enzyme PNPLA1, and for example, a linoleic acid donor and PNPLA1 are added to a solution containing ω-hydroxyceramide. What is necessary is just to make it react for a predetermined time.

<Structure and definition of ω-hydroxyceramide and acylceramide>
The structural formulas of ω-hydroxyceramide and acylceramide are as follows.

In which R ₁ and R ₂ are straight or branched hydrocarbon chains having up to 50 carbon atoms, which may optionally be interrupted by oxygen atoms, optionally one Or may contain more double bonds and optionally be substituted with one or more hydroxyl groups, etc., and R ₃ is a carbohydrate such as H or a hexose or pentose moiety (optional And may be further linked to a carbohydrate moiety).

In a more preferred embodiment of the invention, R ₁ is a linear or branched fatty acid having 14 to 24 carbon atoms, optionally containing one or more double bonds, and optionally Optionally substituted with one or more hydroxyl groups, etc., and R ₂ is a straight or branched fatty acid having 20 to 40 carbon atoms, optionally one or more double bonds And optionally substituted with one or more hydroxyl groups and the like, R ₃ is H or a glucosyl or galactosyl group.

In which R ₁ , R ₂ and R ₄ are linear or branched hydrocarbon chains having up to 50 carbon atoms, which may optionally be interrupted by oxygen atoms, optionally May contain one or more double bonds and optionally be substituted with one or more hydroxyl groups, etc., and R ₃ is a carbohydrate such as H or a hexose or pentose moiety. Yes (optionally further linked to a carbohydrate moiety).

In a more preferred embodiment of the invention, R ₁ is a linear or branched fatty acid having 14 to 24 carbon atoms, optionally containing one or more double bonds, and optionally Optionally substituted with one or more hydroxyl groups, etc., and R ₂ is a straight or branched fatty acid having 20 to 40 carbon atoms, optionally one or more double bonds And optionally substituted with one or more hydroxyl groups and the like, R ₃ is H or a glucosyl or galactosyl group, and R ₄ is linoleic acid.

As the acylceramide synthase PNPLA1, those contained in cells or microorganism homogenates expressing the Pnpla1 gene can be used. Alternatively, a recombinant protein having a mutation in Pnpla1 can be prepared using genetic engineering techniques based on the amino acid sequence and DNA base sequence information of Pnpla1.

  The amount (concentration) of PNPLA1 added to the ω-hydroxyceramide-containing solution is 1 μM to 1 mM, preferably 50 μM to 300 μM, and the reaction temperature is 20 to 40 ° C., preferably 35 to 37 ° C. The reaction time is 10 minutes to 6 hours, preferably 30 minutes to 2 hours. As the linoleic acid donor, Linoely CoA, Trilinolein and the like can be used.

  After the reaction, the produced acylceramide is collected. As the collection method, HPLC or TLC can be used.

  Confirmation that the collected substance is acylceramide can be determined by measuring a known method, that is, a gas chromatograph mass spectrometer (GC / MS), TLC, LC / MS, etc. alone or in combination. .

Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to these examples.

Figure 3
Analysis of Pnpla1 gene expression level in mouse tissues:
After anesthetizing an 8-week-old male mouse with isoflurane, the back skin and other tissues were collected, rapidly frozen in liquid nitrogen and stored at -80 ° C. 1 mL of TRIZOL (Invitrogen) for RNA preparation was added to 20 mg of frozen mouse tissue, and crushed using Polytron PT10-35 GT (KINEMATICA). After adding 1/5 volume of chloroform to this solution and stirring vigorously, it was centrifuged at 15,000 rpm for 10 minutes at room temperature, and the supernatant was collected. The same volume of 2-propanol was added to the supernatant, mixed by inversion, allowed to stand for 15 minutes, and then centrifuged at 5,000 rpm at 4 ° C. for 15 minutes to precipitate RNA. The supernatant was removed, 70% (v / v) ethanol was added, and RNA was washed by centrifugation at 15,000 rpm for 5 minutes at 4 ° C. An appropriate amount of sterilized water was added to the precipitate from which the supernatant was removed to redissolve it, and the obtained RNA was quantified by measuring the absorbance at 260 nm. A reverse transcription reaction was performed using 1 μg of RNA as a template and ReverTraAce (Toyobo) to prepare cDNA. The RNA expression level was evaluated by quantitative PCR (TaqMan method qPCR) using a fluorescently labeled probe. Using ThunderBird qPCR Master Mix (Toyobo), add 2 x qPCR Master Mix 10 μL, reference gene Hprt1 and primer (Pnpla1) of the target gene to be detected, and fluorescently labeled UPL probe (Roche) to 1 μL of cDNA sample and sterilize The total volume was adjusted to 20 μL with water. For qPCR reaction, 96-well plate (LightCycler (registered trademark) 480 Multiwell Plate 96, 04729692001; Roche) was used, and LightCycler (registered trademark) 480 Instrument II (Roche) was used. 50 ° C, 2 min → 95 ° C , 10 min → (95 ℃, 15 sec → 60 ℃, 1 min) and reacted at x 40 cycles conditions, gene expression of interest by using comparison from the change curve of the fluorescence intensity C _T method (DDC _T method) Was calculated.

  The combinations of primers and UPL probe numbers used for quantifying the gene expression levels are shown below.

Hprt1: TGATAGATCCATTCCTATGACTGTAGA (SEQ ID NO: 1), AAGACATTCTTTCCAGTTAAAGTTGAG (SEQ ID NO: 2), UPL # 22
Pnpla1: TGCGGGATTGAGATGGAG (SEQ ID NO: 3), CGCATCATCTGTACCATCTTG (SEQ ID NO: 4), UPL # 94

In situ hybridization:
Entrusted to Geno Staff, embryonic day 17.5 (E17.5) fetal mice were anesthetized with isoflurane, immersed and fixed with 4% paraformaldehyde-phosphate buffer (pH 7.4), and sliced to about 4 μm Sections were used. The detection probe used was an antisense RNA against Pnpla1 mRNA, and a sense RNA complementary to this sequence was used as a control. A digoxigenin-labeled (DIG RNA Labeling Mix; Roche) probe was prepared by in vitro transcription from a plasmid encoding a partial sequence of Pnpla1 shown below. For detection, an alkaline phosphatase-labeled anti-dicoxygenin antibody (Roche) and a chromogenic substrate NBT / BCIP (Sigma) were used, and counterstaining was performed with Kernechtrot staining solution (Mudo Chemical).

A partial sequence of Pnpla1 cDNA used for preparation of the expression detection probe is shown below (SEQ ID NO: 41).


Gene expression analysis associated with differentiation induction of epidermal progenitor cells:
MPEK-BL6 cells (Mouse Epidermal Keratinocyte Progenitors Cell, C57BL / 6; CELnTEC) were seeded in a 12-well plate at a cell density of 3.8 × 10 ⁵ cells / well, and 37 ° C. with epidermal cell culture medium CnT-07 (CELnTEC) Culturing was performed under conditions of 5% CO ₂ . Three days later, cell differentiation was initiated by exchanging the medium with epidermal cell culture solution CnT-02 (CELnTEC) supplemented with 1 mM CaCl ₂ . The cell lysate was collected using TRIZOL solution at 0, 2, 4, 6, and 8 days after differentiation induction. CDNA was synthesized from RNA prepared by the same method as described above, and the expression levels of Keratin10 (Krt10), Loricrin (Lor), and Pnpla1 were quantified by qPCR. Hprt1 was used as a reference gene.

  The combinations of primers and UPL probe numbers used for quantifying the gene expression levels are shown below.

Krt10: GAACAACTTGCAGAAAAGAATCG (SEQ ID NO: 5), TGTGGTGAGTTCCTTGCTCTT (SEQ ID NO: 6), UPL # 89
Lor: GGTTGCAACGGAGACAACA (SEQ ID NO: 7), CATGAGAAAGTTAAGCCCATCG (SEQ ID NO: 8), UPL # 11

Figure 4
Generation of Pnpla1 gene-deficient mice:
A mouse Pnpla1 gene targeting vector (Clone name HTGRS6008_A_A04) was obtained from BACPAC Resources (Children's Hospital Oakland Research Institute, Oakland, Calif., USA). As shown in the schematic diagram of a part of this targeting vector in FIG. 4A, this targeting vector has exon 2 to 6 of genomic DNA of mouse Pnpla1, and is an autonomous promoter sandwiched between two FRT sequences upstream of exon 2. / Neomycin resistance gene cassette (hBactP-neo) and loxP sequence, loxP sequence between exon 3 and exon 4, phosphoglycerate kinase promoter / diphtheria toxin A fragment gene (PGK-DTA) downstream of exon 6. This targeting vector was cleaved with the restriction enzyme Sal I to be linearized, and the gene was introduced into RENKA strain (obtained from Professor Kenji Sakimura, Niigata University Brain Research Institute) by electroporation. The transfected ES cells were cultured at 37 ° C. in 17.5% KSR (knockout serum replacement; Lifetechnologies) -DMEM medium containing antibiotic G418 (100 μg / mL), and homologous recombination clones were selected by Southern blotting. Mouse embryonic stem cells that have undergone appropriate homologous recombination are co-cultured with ICR 8-cell stage embryo (Arc Resource) to produce an aggregation chimera embryo, and transplanted to the uterus of pseudopregnant ICR mouse (Charles River Japan) Heterogeneous Pnpla1 gene modified chimeric mice were generated. The selected male individuals are mated with female C57BL / 6 (CLEA Japan), and PCR using primers that detect GoTaq polymerase (Promega) and transgene using the genomic DNA extracted from the tail of the resulting offspring as a template. Genotyping was performed and it was confirmed that the transgene was transmitted to the offspring. In this state, the Pnpla1 gene is still active, and the Pnpla1 gene can be knocked out by removing exons 2 and 3 sandwiched between two loxP sequences by the Cre-loxP system.

First, as a first step, Pnpla1 ^{f / +} mice were produced by mating with flippase-expressing mice to remove the hBactP-neo cassette sandwiched between two FRT sequences from the genomic DNA. As a second step, this Pnpla1 ^{f / +} mouse was further mated with a CAG-Cre mouse expressing Cre recombinase throughout the body, and PCR genotyping was performed on the genomic DNA extracted from the tail tip of the resulting litter, An individual lacking exons 2 and 3 of the Pnpla1 gene was obtained and used as a Pnpla1 hetero-deficient (Pnpla1 ^+/− ) mouse. As shown in Fig.4C, wild-type (Pnpla1 ^{+ / +} ), Pnpla1 hetero-deficiency (Pnpla1 ^+/- ), Pnpla1 homo-deficiency (Pnpla1 ^-/-) ) Neonates were obtained in a ratio of approximately 1: 2: 1. The genotype was determined by extracting genomic DNA again from the tail tip of the resulting litter and performing PCR genotyping. In addition, RNA was extracted from the skin and qPCR analysis was performed, and as shown in FIG. 4B, it was confirmed that Pnpla1 mRNA was not expressed in the skin of Pnpla1 homo-deficient newborns.

FIG.
Weight measurement of fetus obtained by cesarean section:
Heterogeneous Pnpla1-deficient mice were mated with each other and female mice that became pregnant on day 18.5 were anesthetized with isoflurane, and then laparotomized to remove the fetus from the uterus. The E18.5 fetus was marked with a magic marker, and the time course of body weight was measured using an electronic balance ScoutPro (OHAUS, New Jersey, USA) every hour for up to 16 hours. After completion of the measurement, the tip of the tail was cut, and the genotype was determined by PCR using the extracted genomic DNA.

Evaluation of skin barrier function Measurement of transepidermal water transpiration:
An open chamber portion at the tip of a probe of Tewameter (registered trademark) TM300 (Courage + Khazaka, Germany) was pressed against the skin of a newborn mouse, and transepidermal water transpiration was measured.

Skin permeability test with toluidine blue staining:
A newborn immediately after birth was dehydrated with 100% methanol, and then the whole body was immersed in a 0.1% toluidine blue solution for 5 minutes. The skin barrier function was determined by the degree of penetration of the blue pigment into the body.

Hematoxylin and eosin staining (HE staining):
Newborn mice on the day of birth (PO) were anesthetized with isoflurane and the dorsal skin was collected. Tissue fixed overnight with 10% (w / v) neutral formalin buffer solution (Wako Pure Chemical Industries) is molded to an appropriate size, placed in an embedding cassette (Unicassette; Sakura Finetech), and 70% ethanol It was immersed in it for 1 hour. Using a vacuum automatic fixed embedding device (Sakura Vacuum Rotary VRX-23; Sakura Finetech), increase the ethanol concentration in the tissue as follows, and then replace with paraffin and embed (70% ethanol → 80% Ethanol → 90% ethanol → 100% ethanol (5 times) → 100% xylene (3 times) → 100% xylene (4 times)) (Tissue Enbedding Medium; Mc Cormick Sientific) → 100% paraffin (60 ° C) → 100 % Paraffin (60 ° C) → 100% paraffin (60 ° C), immersed for 3 hours each). The embedded paraffin block was cut with a sliding microtome (REM710; Daiwa Koki Kogyo Co., Ltd.) at a thickness of about 5 μm in the direction perpendicular to the skin surface. The obtained sections were floated on a 42 ° C water bath, flattened, attached to a slide glass (Platinum Pro White PRO-01; Matsunami Glass), and then a 42 ° C Tissue Tech Paraffin Extender (PS-53; Sakura Fine). (Tech Co.) overnight. Place the slide of the paraffin-embedded section in the staining basket and deparaffinize by immersing in each solution in the following order (xylene 5 minutes (4 times) → 99% ethanol 30 seconds → 95% ethanol 30 seconds → 90% Ethanol 30 seconds → 80% ethanol 30 seconds → 70% ethanol 30 seconds → 50% ethanol 30 seconds). It was immersed in Mayer's hematoxylin solution (Wako Pure Chemical Industries) for about 1 minute and washed with tap water for 3 minutes. Then, it was immersed in eosin (0.5% Eosin Y ethanol solution; Wako Pure Chemical Industries, Ltd.) for about 1 minute, and washed again with tap water for 3 minutes. After dehydration and penetration, it was sealed with a cover glass (NEO MICRO COVER GLASS) using a soft mount (Wako Pure Chemicals). The prepared tissue sections were observed using a microscope (BX61; Olympus) and image analysis software (DP2-BSW, Olympus).

Nile Red staining:
On the day of birth (P0), mice were anesthetized with isoflurane, the skin on the back was collected and fixed overnight using a neutral formalin buffer solution. This was immersed in a 10% sucrose / PBS solution at 4 ° C. overnight, and further replaced with a 30% sucrose / PBS solution and allowed to stand for another half day. Freezing embedding (Tissue-Tek®) OCT compound (Sakura Finetech) / 30% sucrose = 3: 1 was used to prepare a block in which the skin specimen was frozen. Using a cryostat (Leica CM3050), a 5 μm-thick section was sliced, attached to a slide glass (Super Frost White S9441, Matsunami Glass), and air-dried. An appropriate amount of Nile Red staining solution (5 mg / ml in 75% glycerol, Wako Pure Chemical Industries, Ltd.) was dropped onto the obtained skin section and sealed with a cover glass. Using a confocal laser microscope (LSM710; Carl Zeiss), observation was performed at an excitation wavelength of 557 nm and a fluorescence wavelength of 572 nm.

Analysis of skin lipids by thin layer chromatography (TLC):
The skin specimen was immersed in a 1.5 mg / ml dispase (Invitrogen) / PBS solution at 4 ° C. overnight, and the epidermis layer was peeled off using tweezers. After lyophilization, the dry weight was weighed, 1 ml of methanol was added, and the mixture was crushed using Polytron PT10-35 GT (KINEMATICA). Extract lipids according to the Folch method (FOLCH J, LEES M, SLOANE STANLEY GH.A simple method for the isolation and purification of total lipides from animal tissues.J Biol Chem 226: 497-509, 1957). After spraying to evaporate the organic solvent, the lipid sample was dissolved by adding chloroform to 1 μL per 1 mg of dry skin weight. Spot a 5 μL lipid sample on a silica gel 60 TLC glass plate (1.05721.0001, 20 cm × 20 cm; Merck), 2 cm in chloroform / methanol / water (40: 10: 1), chloroform / methanol / water (40: 10: 1) was developed in the order of 5 cm, chloroform / methanol / acetic acid (47: 2: 0.5) was 8.5 cm, and hexane / diethyl ether / acetic acid (65: 35: 1) was developed in the order of 18 cm. Bands developed with iodine vapor and separated by TLC were visualized, and images were acquired using LAS4000 (GE Healthcare).
7 to 9
Analysis of skin ceramide molecular species by mass spectrometry:
Lipids were repeatedly extracted from mouse epidermis three times, and butylated hydroxytoluene (BHT) was added to 50 μg / mL in order to suppress lipid oxidation. The covalently bonded lipid was treated in 2 mL of 1M NaOH, 90% methanol solution for 60 minutes, adjusted to pH 6 with 3M hydrochloric acid, and added with 2.5 mL of chloroform. The organic layer obtained by centrifugation at 3,000 × g for 15 minutes was collected, and the liquid layer was further mixed with 1 mL of chloroform and centrifuged at 3,000 × g for 10 minutes. The obtained organic layer was combined with the previous organic layer, washed twice with sterilized water, then blown with nitrogen gas to evaporate the organic solvent, and dissolved in 1 μl of chloroform per 1 mg of dry weight. Reverse phase HPLC was used to separate free sphingolipids. 5 μL of the lipid extract was injected into an HPLC column (Phnomenex Luna C18, 100 mm × 3 mm, 2.6 μm particle size, 100 mm pore size: Phenomenex, CA, USA). 95: 5), 100% ethyl acetate was used for the mobile layer B. Set to a flow rate of 650 μL / min until 14.15 minutes, maintain moving bed B to 0% for 2 minutes, then linearly increase the rate of moving bed B to 55% by 10.5 minutes and then increase to 100 until 14.15 minutes. Increased to%. Subsequently, all lipids were eluted from the column by maintaining at 100% for 4 minutes. For separation of covalently bound lipids, 5 μl of sample was injected into a normal phase TLC Advantage Silica column (250 mm x 4.6 mm, 5 μm particle size, 150 mm pore size: Thomson Instrument, CA, USA) and 1 mL / Elution with hexane / inopropanol / acetic acid (90: 10: 0.1) at a flow rate of min.

A ThemoFinniganDSQ instrument was used in positive ion mode with an APCI ion source for detection of free ceramide species separated by reverse phase HPLC with a mass spectrometer. Capillary temperature 200 ° C, ion transfer voltage 2000 V, vaporizer temperature 450 ° C, electron energy 70 eV. For detection of covalently bonded lipids, conditions of a capillary temperature of 275 ° C., an ion transfer voltage of 2000 V, a vaporizer temperature of 450 ° C., and an electron energy of 70 eV were used. In both cases, scanning was performed at 400 to 1400 amu (± 0.4 amu). In order to perform relative quantification of the detected lipid molecular species, the peak area of the ion chromatogram was calculated, and comparison was made between samples by correcting with the dry weight of each epidermal lipid sample.

FIG.
Expression level analysis of ceramide metabolism related genes:
The newborn mouse on the day of birth (P0) was anesthetized with isoflurane, the back skin was collected, rapidly frozen with liquid nitrogen, and stored at -80 ° C. 1 mL of TRIZOL (Invitrogen) for RNA preparation was added to frozen mouse skin pieces, and crushed using Polytron PT10-35 GT (KINEMATICA). CDNA was synthesized from RNA prepared in the same manner as described above, and the expression levels of Elovl4, Lass3, Cyp4f39, Ugcg, Abca12, Abhd5, Aloxe3, Alox12b, and Tgm1 were quantified by qPCR.

The combinations of primers and UPL probe numbers used are shown below.
Elovl4: GCGGCCAGTCTGCTACAC (SEQ ID NO: 9), CTTTGGTCCTGATGGCAAC (SEQ ID NO: 10), UPL # 85
Lass3: CATGATATATCTGACATTTGGCTAGAG (SEQ ID NO: 11), GTGAAGATGAAGAACAGGGTGTTA (SEQ ID NO: 12), UPL # 38
Cyp4f39: AAAGAAGAAAGCCAAGAGAATTGA (SEQ ID NO: 13), TGCAACAGGTAGTCGGTGAG (SEQ ID NO: 14), UPL # 89
Ugcg: AGTTTCAATCCAGAATGATCAGG (SEQ ID NO: 15), CATTCTGAAATTGGCTCACAAAT (SEQ ID NO: 16), UPL # 4
Abca12: CCTGCTAAACCAGACGATCC (SEQ ID NO: 17), ACTTGCACAAAGGGGTTCC (SEQ ID NO: 18), UPL # 50
Abhd5: ATCTTTGGAGCCCGATCCT (SEQ ID NO: 19), CTTCTGGCTGATCTGCATACAC (SEQ ID NO: 20), UPL # 89
Aloxe3: GGCCTCACTGATCTTCAACG (SEQ ID NO: 21), GTCCAGGAGACCTCGAATCTT (SEQ ID NO: 22), UPL # 4
Alox12b: CTTTGGTCCTGATGGCAAC (SEQ ID NO: 23), GACAATCAGGCCCAGGAGT (SEQ ID NO: 24), UPL # 105
Tgm1: CAGAAAGGACCGATCTCCAG (SEQ ID NO: 25), TCCGGTGACGGTGTTGTAG (SEQ ID NO: 26), UPL # 51

FIG.
Expression analysis of epidermal keratinocyte differentiation marker gene group: Filaggrin (Flg), Loricrin (Lor), Involucrin (Inv), Keratin 1 by qPCR using the cDNA prepared from newborn mouse skin on the day of birth (P0) as a template. The expression levels of (Krt1), Keratin 10 (Krt10), Keratin 5 (Krt5), Keratin 14 (Krt14), and S100a9 were quantified.

The combinations of primers and UPL probe numbers used are shown below (those already described are omitted).
Krt1: TTTGCCTCCTTCATCGACA (SEQ ID NO: 27), GTTTTGGGTCCGGGTTGT (SEQ ID NO: 28), UPL # 62
Krt5: CAGAGCTGAGGAACATGCAG (SEQ ID NO: 29), CATTCTCAGCCGTGGTACG (SEQ ID NO: 30), UPL # 22
Krt14: ATCGAGGACCTGAAGAGCAA (SEQ ID NO: 31), TCGATCTGCAGGAGGACATT (SEQ ID NO: 32), UPL # 83
Inv: AGCAGCTGCAGGTGAAAAAG (SEQ ID NO: 33), TCTCCAGATGCAGTTCCTGTT (SEQ ID NO: 34), UPL # 18
Flg: GGAGGAAGAAACACTGAGCAA (SEQ ID NO: 35), CGATGTCTTGGTCATCTGGA (SEQ ID NO: 36), UPL # 96
S100a9: GACACCCTGACACCCTGAG (SEQ ID NO: 37), TGAGGGCTTCATTTCTCTTCTC (SEQ ID NO: 38), UPL # 31

Detection of expression of keratinocyte differentiation marker by immunohistochemical staining: A section attached to a slide glass was prepared in the same manner as HE staining. After deparaffinization, wash with phosphate buffer (PBS) twice (3 minutes each) and block for 30 minutes at room temperature with Block Ace (Dainippon Pharmaceutical) to block nonspecific proteins. went. Thereafter, the cells were washed once with PBS (3 minutes), once with T-PBS (0.05% Tween 20-containing PBS) (each for 3 minutes), and rabbit anti-Keratin 5 antibody (1,000-fold dilution; Covance) as a primary antibody, Anti-Keratin 1 antibody (1,000-fold dilution; Covance), anti-Loricrin antibody (1,000-fold dilution; Covance), and anti-Filaggrin antibody (1,000-fold dilution; Covance) were reacted overnight at 4 ° C. Wash twice with PBS (3 minutes each) and once with T-PBS (3 minutes), and react with Alexa546-labeled anti-rabbit IgG antibody (1,000-fold dilution; Invitrogen) as the secondary antibody for 1 hour at room temperature. It was. After washing twice with PBS (3 minutes each), encapsulated with VECTASHIELD Mounting Medium with DAPI (Vector laboratories) and cover glass, and observed fluorescence signal with confocal laser microscope (LSM710; Carl Zeiss)

The results obtained in Example 1 are shown in FIGS.

FIG. 3 shows that Pnpla1 is specifically expressed in epidermal keratinocytes of the skin. (Panel A)
In 8-week-old mice, Pnpla1 was expressed in the skin, but was hardly detected in the liver, white adipose tissue (WAT), and skeletal muscle. (Panel B) As a result of in situ hybridization using a slice of E17.5 mouse fetal skin, Pnpla1 expression was expressed locally in the epidermis. (Panel C) When MPEK cells, which are cultured keratinocyte progenitor cells, were induced to differentiate by the addition of 1 mM CaCl ₂ , the mRNA expression level of Pnpla1 increased. It was confirmed that the mRNA expression of keratinocyte differentiation marker genes such as Keratin10 and Loricrin also increased under this differentiation-inducing condition.

In FIG. 4, panel A shows a schematic diagram of the Pnpla1 gene site of the mouse genome used in the Examples, and panel B shows that the expression level of Pnpla1 mRNA differs in the dorsal skin of P0 neonate corresponding to the genotype. . As shown in Panel C, wild-type (Pnpla1 ^{+ / +} ), Pnpla1 hetero-deficient (Pnpla1 ^+/- ), and Pnpla1 homo-deficient (Pnpla1 ^-/- ) neonates were obtained at a ratio of approximately 1: 2: 1. Therefore, it can be said that Pnpla1 homo deficiency does not show embryonic lethality.

The characteristics of heterozygous Pnpla1 gene-deficient mice and homozygous Pnpla1 gene-deficient mice when compared with C57BL / 6 mouse (wild-type) newborn mice are summarized as follows.

Heterotype Pnpla1 gene-deficient mice showed no difference in appearance, weight, life span, and optical microscopic pathological findings of skin structure compared to wild-type mice.

All individuals died within 24 hours after birth of homozygous Pnpla1 gene-deficient mice. There were also homozygous Pnpla1 gene-deficient mice with firm and inflexible skin and necrosis at the tip of the tail (Fig. 4D). The average body weight of wild-type mice was about 1.4 g, whereas homo-deficient mice averaged about 1.0 g (FIG. 4E). As shown in Fig.5A, the weight change of Pnpla1 ^-/- mice obtained by cesarean section of pregnant mice at 18.5 days of gestation is the Since the body weight decreased rapidly over time, the cause of the body weight loss was considered not to be poor growth but to body water loss due to water evaporation from the skin (FIG. 5A). In fact, when measuring the transdermal water transpiration (TEWL) of the newborn (P0) immediately after birth, wild type mice were 1.22 ± 0.08 g / h / m ² (19 mice), heterozygous mice were 1.56 ± 0.11 g / While it was h / m ² (45 mice), that of Pnpla1-deficient mice was 6.34 ± 0.29 g / h / m ² (19 mice), about 5 times higher than that of the wild type (FIG. 5B). In addition, when a skin permeability test using a toluidine blue staining solution was performed, the pigment penetrated into the body and stained blue in the homo-deficient mouse (FIG. 5C). Therefore, if Pnpla1 is not expressed, a serious reduction in skin barrier function occurs.

The results of histological analysis using skin tissue sections are shown in FIG. 5D. In HE-stained wild type mice, a basket weave appearance corresponding to lipids in the stratum corneum was clearly observed in the stratum corneum, but this structure disappeared in homozygous mice (intracellular corneum lipids). Disappearance), and in addition, thickening of the epidermis was observed. When the lipid part of a frozen section of skin was stained with Nile Red, the stratum corneum was regularly stained in the wild-type mouse, but in the homo-Pnpla1-deficient mouse, a large number of granular structures were observed in the stratum corneum as indicated by the arrows. It was suggested that the process of lamelabotti secretion and spreading in layers did not proceed normally (FIG. 5E).

FIG. 6 shows TLC analysis (panel B) of the ceramide metabolic pathway (panel A) and the major lipid components of the epidermis. When lipid extracted from the epidermis of a newborn mouse is developed by thin layer chromatography (TLC), blue-colored EOS and acylglucosylceramide (ω-O-AcylGlcCer) almost disappeared in homo-deficient mice and shown in red ω-OH-Ceramide, glucosylceramide (GlcCer), and 1-O-AcylCer were significantly increased. This result indicates that the reaction of producing AcylCer (EOS) from ω-OH-Ceramide is almost completely inhibited in Pnpla1 homo-deficient mice. In addition, since the production pathway of acylceramide does not function, ω-OH-Ceramide, an upstream metabolite, accumulates, and one type of Cermide (NS) from the other upstream via other metabolic pathways to other types of ceramide This suggests that the production of metabolites is increasing.

7 and 8
FIG. 7 shows the result of comprehensive analysis of epidermis ceramide molecular species by separating a lipid sample prepared from a neonatal epidermis by liquid chromatography and ionizing and detecting it with a mass spectrometer. The horizontal axis is a multistage mass chromatogram in which the retention time by the HPLC column is plotted, and the mass number / charge (m / z) is plotted on the vertical axis. Each ceramide molecular species class was detected in the circled position, and EOH, EOP, EOS, Glc-EOH, and Glc-EOS circled in blue circles were decreased in Pnpla1 ^{− / −} mice. All of these molecules are ceramide molecular species in which linoleic acid is ester-bonded to the ω position. On the other hand, ceramides such as NS, NdS, AP, and NP surrounded by red dotted circles showed an increasing tendency in Pnpla1 ^{− / −} mice than in Pnpla1 ^{+ / +} mice.

FIG. 8 shows a graph obtained by comparing and quantifying the data obtained by the method of FIG. 7 using Pnpla1 ^{+ / +} mice and Pnpla1 ^+/− mice Pnpla1 ^{− / −} mice. In addition, the results of quantification of ultra-superchain fatty acids covalently bound to keratinocytes released by alkali treatment are also shown. These results show that in Pnpla1 homo-deficient mice, various acylceramide molecular species (EOH, EOP, EOS, EODS, etc.) with linoleic acid ester-linked to the ω position of the ultralong chain, acylglucoside ceramide, and keratinocytes This means that the extremely long chain fatty acid covalently bonded to the ω position is significantly reduced.

FIG.
The figure summarizes the ceramide biosynthetic pathway in the skin and changes in the abundance of various ceramide molecular species in Pnpla1 ^{− / −} mice. As can be seen from FIGS. 7 and 8, acyl ceramides esterified with linoleic acid at the ω position are decreased, and the non-esterified ceramide molecular species located immediately upstream is increased. Thus, PNPLA1 has been shown to be required not only for EOS but also for the biosynthesis of other types of acylceramides.

FIG. 10 shows that expression of many genes of metabolic enzymes and transporters involved in epidermis-specific ceramide metabolism is increased in Pnpla1 homo-deficient mice. Panel A shows in which process each gene shown in panel B works in the ceramide metabolic pathway. When the expression of these known genes related to the metabolic pathway of skin ceramide is decreased, the supply of ω-hydroxyceramide and the like is decreased, and as a result, synthesis of acylceramides may be insufficient. However, when the expression of known ceramide metabolism-related genes in the epidermis of Pnpla1 gene-deficient mice was analyzed by qPCR, the expression levels of Elovol4, Lass3, Cypf39, Ugcg, Abc12, Abhd5, etc. increased more than twice compared to the wild type Was. Therefore, the decrease in the amount of acylceramide in Pnpla1-deficient mice is not due to decreased gene expression in the upstream metabolic pathway or activation of the downstream metabolic pathway, but is a direct result of the lack of function of the defective Pnpla1. To support. In addition, even if the ceramide metabolic pathway upstream of acylceramide synthesis is promoted, acylceramide is not synthesized without Pnpla1, meaning that the decrease in skin barrier function cannot be compensated.

FIG.
In Pnpla1 homo-deficient mice, the expression of basal layer markers Krt5 and Krt14 and spinous layer markers Krt1 and Krt10 was equivalent to the wild type, but granule layer marker Lor and stratum corneum marker Flg expression were at the mRNA level. And decreased at the protein level. Both were important as stratum corneum constituent proteins, and it was shown that not only the amount of acylceramide was decreased by Pnpla1 deficiency, but also gene expression after the granular layer of epidermal keratinocytes was affected. Furthermore, S100a9 is an inflammatory marker that increases in the epidermis of patients suffering from atopic dermatitis and psoriasis, and the increased expression of S100a9 in the epidermis of Pnpla1 homo-deficient mice results in an inflammatory response due to disruption of the skin barrier function Suggest that

FIG.
Generation of skin-specific Pnpla1-deficient mice:
Pnpla1 ^{f / +} mice were crossed to obtain Pnpla1 ^{f / f} mice. On the other hand, Pnpla1 ^{f / +} mice were mated with skin-specific K14-Cre mice expressing Cre recombinase to obtain Pnpla1 ^{f / +} K14-Cre mice. By further crossing the obtained Pnpla1 ^{f / f} mouse and Pnpla1 ^{f / +} K14-Cre mouse, Pnpla1 ^{f / f} mouse, Pnpla1 ^{f / f} K14-Cre mouse, Pnpla1 ^{f / +} mouse, Pnpla1 ^{f / +} K14 Four pups of -Cre mice are obtained. Pnpla1 ^{f / f} K14-Cre mice are skin-specific Pnpla1-deficient mice, and littermate Pnpla1 ^{f / f} mice were used as controls with normal Pnpla1 expression.

When the expression level of Pnpla1 mRNA in each tissue was quantified by qPCR in the same manner as in Example 1, Pnpla1 ^{f / f} K14-Cre mice, which are skin-specific Pnpla1-deficient mice, had markedly decreased expression of Pnpla1 in the skin ( FIG. 12B). However, no abnormal appearance was observed from day 0 to day 2 after birth (FIG. 13A), weight loss (FIG. 12C), and increase in transdermal water transpiration (FIG. 13C) were not observed. Pnpla1 ^{f / f} K14-Cre mice were observed from the 3rd day of life to individuals whose weight gain stopped (Fig. 12C) and skin detachment of the legs and back (Figs. 12D and 13A). All individuals died by 6 days (FIG. 12E). The transdermal moisture transpiration of the control Pnpla1 ^{f / f} mice was 1.43 ± 0.10 g / h / m ² (10 animals). In contrast, the transcutaneous water transpiration of Pnpla1 ^{f / f} K14-Cre mice showed a normal value of 1.46 ± 0.04 g / h / m ² (19 mice) at the non-exfoliated site of the skin, but at the exfoliated site. The value was extremely high at 12.48 ± 0.79 g / h / m ² (19 animals).

Lipids were extracted from the back of the mice 5 days after birth (P5) in the same manner as in Example 1 and analyzed for lipids using silica gel 60 TLC. As a result, as in Example 1, EOS indicated by blue letters, acylglucosylceramide (ω-O-AcylGlcCer) almost disappeared, ω-OH-Ceramide indicated by red letters, glucosylceramide (GlcCer), 1-O- AcylCer was significantly increased (FIG. 13E). From the results of Example 2, it was shown that Pnpla1 expressed in the skin is essential for the production of acylceramide.

Figure 15
Fluorescent dye permeability test using 3D culture of primary cultured epidermal cells:
Mice on the day of birth (PO) were left on ice for 30 minutes to suppress activity, back skin was collected, and the tail tip of the same mouse was used for genotype determination. The skin was treated with a 5 mg / ml dispase (Invitrogen) solution / epidermal cell culture solution (CnT-57; CELnTEC) at 4 ° C. overnight.

The epidermis was peeled off with tweezers and treated with TrypLE Select (Invitrogen) at room temperature for 20 minutes to disperse epidermal cells. After suspending the cells by adding an epidermal culture solution, the cells were collected by centrifugation at 800 × g for 5 minutes. Resuspend the cells in epidermal cell culture medium, seed 1 × 10 ⁵ cells per Millicell® Cell Culture Insert (12 mm, 0.4 μm; Merck), and leave it in a 6 cm culture dish. And cultured in an epidermal cell culture solution. Since CaCl ₂ is not contained in the epidermal cell culture, cells other than epidermal keratinocytes die at this stage. Six days later, one Cell Culture Insert was taken out, immersed in 100% methanol and fixed at room temperature for 5 minutes, and the cell density was confirmed using a cell staining reagent (CnT-ST-100; CELnTEC). After confirming that the cell density was 100%, the medium was replaced with a three-dimensional culture medium (CnT-02-3DP1; CELnTEC).

  After overnight culture, the medium in Cell Culture Insert was removed and 3D differentiation induction was started. Thereafter, the medium in the 6 cm culture dish was changed every 3 days and cultured for 14 days. The Cell Culture Insert was transferred to a 24-well plate, 50 μL of 1 mM Lucifer Yellow solution (Invitrogen) was dropped, and the plate was allowed to stand at 37 ° C. for 1 hour. After removing the Lucifer Yellow solution, the solution was fixed in a neutral formalin buffer solution (Wako Pure Chemical Industries, Ltd.) at 4 ° C. overnight. The membrane portion of Cell Culture Insert was cut out and replaced with paraffin. Using a microtome, it was cut to a thickness of about 5 μm in a direction perpendicular to the film surface and attached to a slide glass. After deparaffinization, the sample was sealed with VECTASHIELD Mounting Medium with DAPI (Vector laboratories) and observed with a confocal laser microscope (LSM710; Carl Zeiss). The penetration of Lucifer Yellow into the three-dimensional cultured epidermal tissue was detected at an excitation wavelength of 553 nm and a fluorescence wavelength of 637 nm.

A stratum corneum with basket wave appearance (corresponding to corneum intercellular lipid) was formed in a wild type (Pnpla1 ^{+ / +} ) three-dimensional cultured tissue piece, and almost no infiltration of the fluorescent dye Lucifer Yellow into the tissue was observed. . On the other hand, in the Pnpla1 homo-deficient (Pnpla1 ^{− / −} ) three-dimensional cultured tissue piece, the stratum corneum with keratin intercellular lipid was not sufficiently formed, and the inside of the tissue was stained with Lucifer Yellow. Therefore, it was suggested that Pnpla1 is necessary for the skin barrier function in an in vitro skin model by three-dimensional culture.

Preparation of recombinant PNPLA1 protein:
Transform pFastBac1 vector with mouse Pnpla1 cDNA into Max Efficiency DH10BAC competent cells (10361-012 Invitrogen), 10 μg / ml Tetracycline, 100 μg / ml X-Gal, 7 μg / ml Gentamycin, 40 μg / ml We spread on LB plate to which IPTG was added. Blue colonies that had undergone recombination were selected and amplified, and Bacmid DNA was purified. Using a primer set having the sequences of CCCAGTCACGACGTTGTAAAACG (SEQ ID NO: 39) and AGCGGATAACAATTTCACACAGG (SEQ ID NO: 40), using GoTaq polymerase (M5005; Promega), 93 ° C., 45 seconds → (94 ° C., 45 seconds → 55 ° C. 45 seconds → 72 ° C., 5 minutes) × 30 cycles → 72 ° C., 3 minutes of PCR reaction was performed to confirm that site-specific transposition occurred.

Grace's Insect medium containing 10% FBS, 100 Unit / ml Penicillin, 100 Unit / ml Streptomycin, 1% CD Lipid Concentrate (Invitorgen), seeded in a 6-well plate at a cell density of 1 × 10 ⁶ cells / well (11605-102; Invitorgen) at 27 ° C. for 30 minutes. After confirmation of cell adhesion, 2 μg of Bacmid DNA was introduced using Cellfectin (Invitrogen). After culturing at 27 ° C. for 5 days, the medium was collected and centrifuged at 2,000 rpm for 10 minutes, and the supernatant was collected to obtain a baculovirus suspension.

Sf9 cells were seeded in a 12-well plate at a cell density of 5 × 10 ⁵ cells / well, 1 μl of a baculovirus suspension encoding PNPLA1 was added, and cultured at 27 ° C. for 3 days. The cells were removed by cell scraper and collected, and 100 μl of lysis buffer (20 mL Tris, pH 7.4, 50 mM NaCl, 1 mM DTT, 100 μg / mL leupeptin, 10 μg / mL pepstatin) was added and sonicated. Centrifugation was performed at 1,000 xg for 5 minutes, and the supernatant was used as an enzyme source. As a control, a protein solution was prepared in the same manner from Sf9 cells not infected with virus.

FIG.
Synthesis of acylceramide:
Linoleoyl Coenzyme A as a reaction substrate, [linoleoyl 1- ¹⁴ C] (final concentration 20 μM, ARV1195; American Radiolabeled Chemicals, Inc.), N-ω-Hydroxytriacontanoyl- D-erythro-sphingosine ( final concentration 266 [mu] M, Matreya Inc.) Glass Dissolve in a 100 μl reaction solution (100 mM Tris, pH 7.0, 100 mM NaCl, 0.1% BSA, 0.03% TritonX-100) in a test tube and use 1 μg of protein solution prepared from Sf9 as the enzyme source. In addition, the reaction was carried out with stirring at 37 ° C. for 30 minutes. The alkali treatment for confirming the formation of the fatty acid ester was carried out at 37 ° C. for 60 minutes by adding 1 mL of an alkali treatment solution (chloroform / methanol / 10 M NaOH = 2: 7: 1) immediately after the enzyme reaction. To the resulting reaction solution, 2 ml of chloroform, 2 ml of methanol and 1.6 ml of acetate buffer were added, and after stirring, the mixture was centrifuged at 3,000 rpm for 10 minutes, and the lower layer was recovered. Nitrogen gas was blown to evaporate the organic solvent, and 5 μl chloroform was added to dissolve the lipid sample. The whole amount was spotted on a silica gel 60 TLC plate and developed in the same manner as the lipid analysis by TLC described above. Radiation was detected using an imaging plate (BAS IP MS2040; Fuji Film) and visualized using an imaging analyzer (BAS-2500; Fuji Film).

In the sample to which PNPLA1 was added as an enzyme source, an isotope-labeled band was observed at the position of acylceramide (EOS), and this band disappeared by alkali treatment, so it was esterified with ¹⁴ C-labeled linoleic acid. It was shown that This result indicates that PNPLA1 has an activity of transferring linoleic acid to the ω-position of ω-OH ceramide and synthesizing an acylceramide essential for the skin barrier function. Therefore, it is suggested that a mass production system of acylceramide can be constructed by using PNPLA1.

  According to the present invention, acylceramide can be synthesized in large quantities biochemically and industrially. In addition, compounds that increase the expression level of PNPLA1 can be used as therapeutic agents for skin diseases such as ichthyosis with abnormal skin barriers, atopic dermatitis, and as cosmetics for maintaining and restoring skin barrier functions. .

  SEQ ID NOs: 1 to 40: synthetic DNA

Claims (14)

  When a test substance is administered to a non-human animal deficient in the PNPLA1 gene and the skin barrier function is promoted in the non-human animal after administration of the test substance, the test substance is promoted to promote the skin barrier function. A method for screening a skin barrier function promoter, comprising a step of selecting as an agent.   When a test substance is administered to a non-human animal having the PNPLA1 gene, and the expression level of PNPLA1 or the amount of acylceramide in which linoleic acid is ester-linked in the non-human animal after administration of the test substance is increased The screening method of a skin barrier function promoter including the process of selecting the said test substance as a skin barrier function promoter.   After contacting a test substance with a biomaterial collected from a cell having a PNPLA1 gene or a non-human animal having a PNPLA1 gene, the expression level of PNPLA1 or an acylceramide in which linoleic acid is ester-linked at the ω position is detected. A skin barrier function promoter comprising a step of selecting the test substance as a skin barrier function promoter when the expression level of the PNPLA1 or the amount of acylceramide in which linoleic acid is ester-bonded to the ω position is increased. Screening method.   The acyl ceramide esterified with linoleic acid at the ω position is selected as a skin barrier function promoter when the test ceramide is increased compared to a control cell or biomaterial that does not contact the test substance. 3. The method according to 3.   When acylceramide is detected after contacting a test substance with a sample containing PNPLA1 and ω-hydroxyceramide, and the acylceramide is increased as compared with a control sample not contacting the test substance, A screening method for a skin barrier function promoter, comprising a step of selecting a substance as a skin barrier function promoter.   When a test substance is administered to a non-human animal deficient in the PNPLA1 gene and a skin barrier function promoting effect is obtained in the non-human animal after administration of the test substance, the test substance is treated with acylceramide. A screening method for a therapeutic agent for skin diseases caused by abnormal synthesis of acylceramide, comprising a step of selecting the therapeutic agent for skin diseases caused by synthetic abnormalities.   When a test substance is administered to a non-human animal having the PNPLA1 gene, and the expression level of PNPLA1 or the amount of acylceramide in which linoleic acid is ester-linked in the non-human animal after administration of the test substance is increased A screening method for a therapeutic agent for skin diseases caused by abnormal synthesis of acylceramide, comprising the step of selecting the test substance as a therapeutic agent for skin diseases caused by abnormal synthesis of acylceramide.   After contacting a test substance with a cell material having a PNPLA1 gene or a biomaterial collected from a non-human animal having a PNPLA1 gene, an acylceramide having an ester bond of linoleic acid at the ω position is detected, and linole is detected at the ω position. A step of selecting the test substance as a therapeutic agent for skin diseases caused by abnormal synthesis of acylceramide when the acid-ceramic acylceramide is increased as compared with a control cell or biomaterial that does not contact the test substance. A screening method for a therapeutic agent for skin diseases caused by abnormal acylceramide synthesis.   Measure the expression level of PNPLA1 after contacting the test substance to cells with PNPLA1 gene or biomaterial collected from non-human animals with PNPLA1 gene, and the expression level of PNPLA1 does not contact the test substance A method of screening for a therapeutic agent for skin diseases caused by abnormal synthesis of acylceramide, comprising the step of selecting the test substance as a therapeutic agent for skin diseases caused by abnormal synthesis of acylceramide when increased compared to a control sample.   After contacting a test substance with a sample containing PNPLA1 and ω-hydroxyceramide, acyl ceramide with linoleic acid ester-bonded at the ω position is detected, and the acyl ceramide with linoleic acid ester-bonded at the ω position is the test substance. Screening for a therapeutic agent for skin diseases caused by abnormal synthesis of acylceramide, comprising the step of selecting the test substance as a therapeutic agent for skin diseases caused by abnormal synthesis of acylceramide. Method. The skin disease due to acylceramide synthesis abnormality is at least one selected from the group consisting of ichthyosis, atopic dermatitis, psoriasis, rough skin, psoriasis, keratosis, dry skin, eczema and dry dermatitis Item 11. The method according to any one of Items 6 to 10 .   A method for producing an acylceramide, characterized by collecting acylceramide after allowing PNPLA1 to act on ω-hydroxyceramide.   Measure the expression level of the PNPLA1 gene after contacting the test substance with a biomaterial collected from a cell having the PNPLA1 gene or a non-human animal having the PNPLA1 gene, and the expression level of the PNPLA1 gene When the test substance is increased or decreased as compared with a control cell or biomaterial that is not contacted, the test substance is determined to be a substance that increases or decreases the skin barrier function. Inspection method of skin barrier function.   After contacting a test substance with a cell material having a PNPLA1 gene or a biomaterial collected from a non-human animal having a PNPLA1 gene, an acylceramide having an ester bond of linoleic acid at the ω position is detected, and linole is detected at the ω position. When the acyl ceramide esterified with an acid is increased or decreased compared to a control cell or biomaterial that does not contact the test substance, the test substance is a substance that increases or decreases the skin barrier function, respectively. A test method for the skin barrier function of a test substance, characterized in that