The Critical Role Played by Mitochondrial MITF Serine 73 Phosphorylation in Immunologically Activated Mast Cells
"> Figure 1
<p>IgE-Ag stimulation affects mitochondrial MITF Serine 73 phosphorylation. (<b>A</b>), RBL cells were sensitized with 100 ng/mL IgE for 2 h followed by a 0–60 min DNP challenge. Non-sensitized cells are presented as Control (Q: Quiescent). The protein levels of MITF in the whole cell lysates were determined by Western blot analysis using anti-pS73-MITF, anti-MITF and anti-GAPDH antibodies. (<b>B</b>), Densitometry of MITF-pS73/MITF levels in RBL cells that were activated for 0–60 min. (<b>C</b>), RBL and BMMCs were treated with 100 ng/mL IgE for 2 h followed by a 15 min DNP challenge and were fractionated into cytosol and mitochondria. The protein levels of MITF in both fractions were determined by Western blot analysis using anti-pS73-MITF, anti-MITF, anti-GAPDH (cytosolic marker) and anti-VDAC (mitochondrial marker) antibodies. (<b>D</b>), Densitometry of MITF-pS73/GAPDH and MITF-pS73/VDAC levels in RBL cells that were activated for 15 min followed by cytosolic and mitochondrial fractionation. A two-tailed Student <span class="html-italic">t</span>-test was performed for all the western blots. Results represent means ± SEMs (n = 4, **** <span class="html-italic">p</span> ≤ 0.0001; *** <span class="html-italic">p</span> ≤ 0.001; ** <span class="html-italic">p</span> ≤ 0.01; * <span class="html-italic">p</span> ≤ 0.05; ns <span class="html-italic">p</span> > 0.05).</p> "> Figure 2
<p>ERK1/2-dependent mitochondrial MITF Serine 73 phosphorylation in immunologically activated mast cells. (<b>A</b>), RBL cells were sensitized with 100 ng/mL IgE for 2 h, followed by a 15 min DNP challenge. Thirty minutes before the addition of DNP, 20 µmol/L U0126 was added to the medium. The protein levels of MITF and ERK in the whole cell lysates were determined by Western blot analysis using anti-pS73-MITF, anti-MITF, anti-pERK, anti-ERK and anti-GAPDH antibodies. (<b>B</b>), RBL cells were treated as in (<b>A</b>). The cells were fractionated into cytosol and mitochondria. The protein levels of MITF in both fractions were determined by Western blot analysis using anti-pS73-MITF, anti-MITF, anti-pERK, anti-ERK, anti-GAPDH (cytosolic marker) and anti-VDAC (mitochondrial marker) antibodies. (<b>C</b>,<b>D</b>), Densitometry of MITF-pS73/total MITF levels in RBL cells wholecell lysate (<b>C</b>) and mitochondrial fractions (<b>D</b>) that were treated with U0126 and DMSO as control as in (<b>A</b>). A two-tailed Student <span class="html-italic">t</span>-test was performed for all the western blots. Results represent means ± SEMs (n = 4, *** <span class="html-italic">p</span> ≤ 0.001; ** <span class="html-italic">p</span> ≤ 0.01; * <span class="html-italic">p</span> ≤ 0.05; ns <span class="html-italic">p</span> > 0.05).</p> "> Figure 3
<p>Serine 73 phosphorylation of MITF affects MITF-PDH interaction during mast cell stimulation. (<b>A</b>), IP of MITF in IgE-DNP activated RBL cell lysates in the presence and absence of ERK1/2 inhibitor. Cell lysates were pre-treated with U0126 as described in <a href="#cells-11-00589-f002" class="html-fig">Figure 2</a>. Non-sensitized cells are presented as Control (Q: Quiescent). For IP anti-MITF antibody was used and for Western blot analysis, anti-pS73-MITF, anti-MITF, anti-PDHA antibodies were used. (<b>B</b>), Densitometry of PDHA/MITF levels in RBL cells after IP experiment. A two-tailed Student <span class="html-italic">t</span>-test was performed for all the western blots. Results represent means ± SEMs (n = 4, ** <span class="html-italic">p</span> ≤ 0.01; * <span class="html-italic">p</span> ≤ 0.05; ns <span class="html-italic">p</span> > 0.05).</p> "> Figure 4
<p>Inhibition of mitochondrial MITF Serine 73 phosphorylation using U0126 affects mast cell degranulation and mitochondrial functions. Mast cells were stimulated with IgE-DNP and incubated either with 20 µmol/L U0126 or DMSO as control and degranulation activity was assessed by measuring β-hexosaminidase release and cytokine (TNF-α, granzyme B) secretion using the ELISA assays in RBL cells (<b>A</b>–<b>C</b>) and BMMCs (<b>D</b>–<b>F</b>). Mitochondrial activity assay results are shown in (<b>G</b>) mitochondrial ATP, (<b>H</b>) Oxygen consumption rate with the increased pyruvate levels (<b>I</b>) and the decreased pyruvate dehydrogenase activity (<b>J</b>) in RBL cells. A two-tailed Student’s <span class="html-italic">t</span>-test was performed for all the assays. Results represent means ± SEMs (n = 4, **** <span class="html-italic">p</span> ≤ 0.0001; *** <span class="html-italic">p</span> ≤ 0.001; ** <span class="html-italic">p</span> ≤ 0.01; ns <span class="html-italic">p</span> > 0.05).</p> "> Figure 5
<p>Effect of overexpression of mitochondrial MITF Serine 73 mutants (<b>D</b> or <b>A</b>) on mast cell degranulation and mitochondrial functions. Transfections were made in RBL cells either with EV or mitochondrial MITF-S73D or mitochondrial MITF-S73A. After 48 h, cells were stimulated with IgE-DNP and degranulation activity was assessed by measuring β-hexosaminidase release (<b>A</b>). Relative oxygen consumption rate at basal respiration was compared in transfected RBL cells (<b>B</b>). One (out of 3 experimental repeats from <a href="#cells-11-00589-f005" class="html-fig">Figure 5</a>B) of the Seahorse XF cell Mito Stress profile is shown to assess the effect of mitochondrial MITF-S73D or S73A on mitochondrial respiration, where each dot represents means ± SDs (n = 12) (<b>C</b>). Mitochondrial ATP (<b>D</b>) and pyruvate levels (<b>E</b>) 48 h after transfection were measured and compared with mitochondrial MITF-WT. Results represent means ± SEMs (n = 3 or 4, ** <span class="html-italic">p</span> ≤ 0.01; * <span class="html-italic">p</span> ≤ 0.05; ns <span class="html-italic">p</span> > 0.05).</p> ">
Abstract
:1. Introduction
2. Materials and Methods
2.1. Antibodies
2.2. Chemical Inhibitor Treatment
2.3. Cell Culture
2.4. Bone Marrow-Derived Mast Cells (BMMCs) from Mice
2.5. Cytosol and Mitochondrial Fractionation
2.6. Gel Electrophoresis and Western Blotting
2.7. Mitochondrial Targeted Plasmids
2.8. Transfection
2.9. β-Hexosaminidase Release Assay (Degranulation Assay)
2.10. ATP Determination
2.11. Oxygen Consumption
2.12. Measurement of Cytokines, Pyruvate Levels and PDH Activity
2.13. Coimmunoprecipitation
2.14. Statistical Analysis
3. Results
3.1. IgE-Ag Mast Cell Activation Induces Phosphorylation of MITF in Mitochondria
3.2. Mitochondrial Serine 73-MITF Is ERK1/2-Dependently Phosphorylated during Mast Cell IgE-Ag Stimulation
3.3. Dissociation of Mitochondrial Serine 73-Phosphorylated MITF from PDH after Immunological Activation
3.4. Inhibiting Serine 73 Phosphorylation of Mitochondrial MITF during IgE-Ag Stimulation Reduces the Mast Cell Reactivity
3.5. Checking the Overexpression of Phosphorylated and Dephosphorylated Mimicking Mitochondrial MITF Serine 73 on Mitochondrial Function and Mast Cell Activity
4. Discussion
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
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Paruchuru, L.B.; Govindaraj, S.; Razin, E. The Critical Role Played by Mitochondrial MITF Serine 73 Phosphorylation in Immunologically Activated Mast Cells. Cells 2022, 11, 589. https://doi.org/10.3390/cells11030589
Paruchuru LB, Govindaraj S, Razin E. The Critical Role Played by Mitochondrial MITF Serine 73 Phosphorylation in Immunologically Activated Mast Cells. Cells. 2022; 11(3):589. https://doi.org/10.3390/cells11030589
Chicago/Turabian StyleParuchuru, Lakshmi Bhargavi, Sharmila Govindaraj, and Ehud Razin. 2022. "The Critical Role Played by Mitochondrial MITF Serine 73 Phosphorylation in Immunologically Activated Mast Cells" Cells 11, no. 3: 589. https://doi.org/10.3390/cells11030589