A Side-by-Side Comparison of Wildtype and Variant Melanocortin 1 Receptor Signaling with Emphasis on Protection against Oxidative Damage to DNA
<p>Stable expression of WT and variant MC1R in melanoma cells of identical genetic background. (<b>A</b>) Strategy for the generation of HBL human melanoma cell-derived clones expressing a single and defined variant of the MC1R. See text and <a href="#app1-ijms-24-14381" class="html-app">Supplementary Figure S1</a> for details. (<b>B</b>) Expression and intracellular stability of the WT, R151C and D294H forms of MC1R. Cells were treated with 0.1 mM cycloheximide for the indicated times, detergent-solubilized, electrophoresed, and analyzed for MC1R with anti-flag. (<b>C</b>) Steady-state level of expression of WT and variant MC1R. Detergent-solubilized cell extracts were analyzed for MC1R expression via Western blot. The intensity of the MC1R band was corrected for protein loading using β-actin (ACTB) as the loading control. Results are normalized to the expression of WT MC1R and are the mean ± sem for 7 independent experiments. (<b>D</b>) Semi-logarithmic plots for the estimation of the rate of decay of WT or variant MC1R in live HBL cells. The intracellular half-lives of the different receptor forms, estimated from the slopes of the adjusted linear plots, are indicated (mean ± sem, n ≥ 3). (<b>E</b>) Confocal micrographs of HBL cells expressing defined MC1R variants. MC1R was immunostained with anti-FLAG, with or without a 15 min treatment with 0.4% Triton X-100 in PBS for permeabilization of the cell membrane. The graphs on the right show the MC1R staining intensity normalized to the cells expressing the WT receptor. **, <span class="html-italic">p</span> < 0.01; ***, <span class="html-italic">p</span> < 0.001; ****, <span class="html-italic">p</span> < 0.0001. Scale bar 50 µm.</p> "> Figure 2
<p>Functional coupling of WT and variant MC1R. Cells expressing the indicated MC1R variants were stimulated with 100 nM NDP-MSH for the times shown and analyzed for (<b>A</b>) intracellular cAMP and (<b>B</b>) ERK activation. The upper Western blots are representative of three independent experiments, and the lower bar graphs represent the quantification of the active ERK intensity, normalized to the non-stimulated control (0 min timepoint, mean ± sem, n = 3). (<b>C</b>) Steady-state levels of MITF in unstimulated cells expressing the indicated MC1R forms. The image is representative of five independent Western blots, whose quantification is shown below as a bar graph (values normalized to the MC1R-KO expression, results given as mean ± sem, n = 5). (<b>D</b>) Changes in MITF expression upon stimulation of cells expressing the indicated MC1R form with 100 nM NDP-MSH for 48 h. Representative blots are shown at the top, and the fold change in band intensity, normalized to each non-stimulated control, is shown below (mean ± sem, n = 3).</p> "> Figure 3
<p>MC1R activation modulates proliferation and cell cycle progression. (<b>A</b>) Growth curves of MC1R clones cultured in 2D and complete medium (DMEM + 10% FCS) determined by manual cell counting. Equal numbers of cells were seeded, cells were allowed to attach for 24 h (time 0) and counted every 24 h. Growth was represented as fold increase in cell number relative to the initial number in the 0-time point. The statistical significance of the cell numbers in MC1R-expressing cells compared with MC1R-KO cells at 72 h is shown. (<b>B</b>) FACS analysis of MC1R clones cultured in total medium (DMEM + 10% FCS) for three days. Results are given as mean ± sd (n = 5). Stars indicate the statistical significance of each phase compared with the same phase in MC1R-KO cells. (<b>C</b>) FACS analysis of MC1R clones cultured under FCS-starved conditions (DMEM and no FCS) for two days in the presence or absence of 100 nM NDP-MSH. For each receptor form, the stars within the bars of the NDP-MSH condition indicate the statistical significance of each phase compared with the same phase in the corresponding untreated control (results given as mean ± sd, n ≥ 3; ns, not significant). *, <span class="html-italic">p</span> < 0.05; **, <span class="html-italic">p</span> < 0.01; ***, <span class="html-italic">p</span> < 0.001.</p> "> Figure 4
<p>Effect of the <span class="html-italic">MC1R</span> genotype on cell shape and motility. (<b>A</b>) Effect of WT MC1R on shape and dendricity. MC1R-KO cells or cells expressing WT MC1R were grown in complete medium with 10% FCS. Micrographs were taken using an Eclipse TS2 microscope with 20x objective lenses, scale bar 50 µm. A quantitative analysis of the number and length of dendrites per cell in randomly selected images is shown below the micrographs (at least 100 cells per condition were analyzed, and the results are given as median ± SEM for length or mean ± SEM for number, n = 3). (<b>B</b>) Effect of NDP-MSH on shape of cells expressing WT or variant MC1R. When required, cells were treated for 48 h with 100 nM NDP-MSH before acquisition and analysis of the micrographs (100 cells quantified per condition; the scale bar and results are formatted as in (<b>A</b>)). (<b>C</b>) Basal and MC1R-agonist induced migration of cells expressing the different MC1R forms. Cells were seeded on Oris™ 96-well plates with silicon stoppers in serum-reduced medium, and if required were treated with 100 nM NDP-MSH for 24 h. The stoppers were removed, and images were taken at 24 h or 48 h after removal of the stoppers. Representative images are shown, along with the quantification of wound healing at 24 h or 48 h (results are given as mean ± SEM, n = 3). *, <span class="html-italic">p</span> < 0.05; **, <span class="html-italic">p</span> < 0.01; ****, <span class="html-italic">p</span> < 0.0001.</p> "> Figure 5
<p>Genoprotective action against oxidative stress of the different MC1R forms. (<b>A</b>) γH2AX immunostaining of control cells and cells challenged with Luperox (150 µM, 30 min) with or without a previous treatment with NDP-MSH (100 nM, 48 h). The confocal images correspond to one of two independent experiments with comparable results. Scale bar 50 µm. For each experiment, at least 100 cells were randomly selected and analyzed for staining intensity. The plots below show the median of the staining intensity of cells, normalized to the control condition (no treatment with NDP-MSH or Luperox). (<b>B</b>) Neutral comet assay. In this case, cells pretreated or not with NDP-MSH were challenged with 100 µM Luperox for 20 min. Two independent experiments were performed with consistent results. *, <span class="html-italic">p</span> < 0.05; **, <span class="html-italic">p</span> < 0.01; ***, <span class="html-italic">p</span> < 0.001; ****, <span class="html-italic">p</span> < 0.0001. Scale bar 25 µm.</p> ">
Abstract
:1. Introduction
2. Results
2.1. Generation of MC1R Knockout Cells (MC1R-KO) and Reconstitution with Defined MC1R Variants
2.2. Signaling Downstream of WT and Variant MC1R
2.3. Effects of MC1R Genotype on Melanoma Cell Proliferation
2.4. Effects of MC1R Genotype on Melanoma Cell Shape and Motility
2.5. Protection of DNA from Oxidative Stress
3. Discussion
4. Materials and Methods
4.1. Reagents
4.2. Generation of CRISPR/Cas9-Based MC1R-KO Cells and Reconstitution with Defined MC1R Variants
4.3. Cell Culture and Analysis of Proliferation and Cell Cycle Progression
4.4. Analysis of Cell Morphology and Migration
4.5. Analysis of DNA Integrity and Detection of DSBs
4.6. Immunoblotting and Immunofluorescence
4.7. Functional Assays
4.8. Statistical Analysis
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Abbreviation
DSB | Double-strand break |
ERK | Extracellular signal-regulated protein kinase |
FCS | Fetal calf serum |
GPCR | G protein-coupled receptor |
MC1R | Melanocortin 1 receptor |
αMSH | α-melanocyte-stimulating hormone |
MITF | Microphthalmia-associated transcription factor |
NDP-MSH | [Nle4, dphe7]-α-melanocyte-stimulating hormone |
NER | Nucleotide excision repair |
PPARγ | Peroxisome proliferator-activated receptor |
γH2AX | Phosphorylated histone h2ax |
ROS | Reactive oxygen species |
SSB | Single-strand break |
UVR | Ultraviolet solar radiation |
WT | Wildtype |
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Culture Conditions | Doubling Time (h) 1 | |||
---|---|---|---|---|
MC1R-KO | WT | R151C | D294H | |
10% FCS | 21.5 ± 0.7 | 19.8 ± 1.5 | 26.5 ± 1.4 | 26.7 ± 0.6 |
1% FCS | 64.6 ± 1.6 * | 45.3 ± 2.8 * | 56.0 ± 3.8 * | 42.7 ± 2.4 * |
1% FCS + 100 nM NDP-MSH | 55.8 ± 6.4 * | >100 | 59.1 ± 8.3 | 41.3 ± 3.0 * |
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Cerdido, S.; Sánchez-Beltrán, J.; Lambertos, A.; Abrisqueta, M.; Padilla, L.; Herraiz, C.; Olivares, C.; Jiménez-Cervantes, C.; García-Borrón, J.C. A Side-by-Side Comparison of Wildtype and Variant Melanocortin 1 Receptor Signaling with Emphasis on Protection against Oxidative Damage to DNA. Int. J. Mol. Sci. 2023, 24, 14381. https://doi.org/10.3390/ijms241814381
Cerdido S, Sánchez-Beltrán J, Lambertos A, Abrisqueta M, Padilla L, Herraiz C, Olivares C, Jiménez-Cervantes C, García-Borrón JC. A Side-by-Side Comparison of Wildtype and Variant Melanocortin 1 Receptor Signaling with Emphasis on Protection against Oxidative Damage to DNA. International Journal of Molecular Sciences. 2023; 24(18):14381. https://doi.org/10.3390/ijms241814381
Chicago/Turabian StyleCerdido, Sonia, José Sánchez-Beltrán, Ana Lambertos, Marta Abrisqueta, Lidia Padilla, Cecilia Herraiz, Conchi Olivares, Celia Jiménez-Cervantes, and José C. García-Borrón. 2023. "A Side-by-Side Comparison of Wildtype and Variant Melanocortin 1 Receptor Signaling with Emphasis on Protection against Oxidative Damage to DNA" International Journal of Molecular Sciences 24, no. 18: 14381. https://doi.org/10.3390/ijms241814381