Effect of MC1R variant allele status on MSH-ligand induction of dopachrome tautomerase in melanocytes co-cultured with keratinocytes

Abstract

epidermal components. Immunohistochemically, the regenerated hairs were positive for loricrin, filaggrin, keratin 10, p63 and Ki67 in a similar pattern to normal skin . There remained a possibility that the difference in media between the two-dimensional and three-dimensional culture accounted for the difference in the hair-inducing capacity of transplanted cells. However, in our preliminary experiments (n = 12), we could not observe hair regeneration in the co-transplantation of epidermal cells and twodimensionally cultured dermal cells maintained in the medium used for the three-dimensional culture condition (DMEM: F12 + B27 + EGF + bFGF).Our data described previously raise a possibility that sphere formation may be sufficient to give mesenchymal cells hair-inducing capacity regardless of the cell identity as dermal cells. To test this, we selected lungs as the origin of mesenchymal cells outside the dermis. Immunohistochemistry showed that some cells stained positive for both Sca-1 and nestin in adult lungs ( Fig. 2a-d). We cultured dissociated lung cells in two-dimensional culture condition and thereafter transferred them into the sphere-forming condition. They formed spheroids (Fig. 2e) in which some cells expressed Sca-1, nestin and fibronectin ( Fig. 2f-i). Each cell attached directly to one another by adherens junctions (Fig. 2j). Of the genes for Wnt ligand members, wnt5a and wnt5b, b-catenin and lef-1 were significantly upregulated during cultivation in the sphere-forming condition ( Fig. 2k-n). Co-transplantation of epidermal cells and sphere-forming lung cells regenerated black hairs in the transplanted area (9 ⁄ 13) ( Fig. 2o, q). Occasionally, epidermal cysts, presumably formed by transplanted epidermal cells that failed to participate in hair follicles, were observed (Fig. 2q). On the contrary, co-transplantation of epidermal cells and two-dimensionally lung cells did not regenerate hairs (0 ⁄ 9) (Fig. 2p, r). We traced the transplanted lung cells derived from CAG-EGFP mice and found that GFP + cells are localized in the dermal papilla and in the sub-epidermal lesion (Fig. 2s, t). The regenerated hairs showed normal expression pattern for loricrin, filaggrin, keratin 10, p63 and Ki67 (Fig. S3k-t). ConclusionIn summary, mesenchymal cells obtain hair-inducing capacity through sphere formation, even if the cell does not originally possess dermal cell identity. Our data provide a new methodology to successfully expand the hair follicle niche cells for regenerative medicine of hair follicles. Two-dimensionally expanded mesenchymal cells that have once lost their potential as hair-inducing niche cells can recover hair-inducing capacity in sphere-forming culture. AcknowledgementsThis work was supported by Grants-in-Aid for Specially Promoted Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan. Conflict of interestsThe authors state no conflict of interest. Supporting InformationAdditional Supporting Information may be found in the online version of this article: Fig...

MC1R Variant Allele Effects on UVR-Induced Phosphorylation of p38, p53, and DDB2 Repair Protein Responses in Melanocytic Cells in Culture

“…The work presented supports the hypothesis that MC1R is required to mediate the NER process through p38 activation and the postulation that persons with RHC skin phenotype are compromised in their ability to activate p38 and so mediate DDB2 degradation. Dysfunctional DNA repair ability might pose a significantly high risk for melanoma in addition to the reduction of DCT expression and pigmentation responses that we have previously reported (Roberts et al, 2008;Ainger et al, 2011). Improper execution of DNA repair in individuals with the RHC phenotype might be a major contributor to the pathological phenomenon seen in melanoma development.…”

MC1R, the cAMP pathway, and the response to solar UV: extending the horizon beyond pigmentation

“…This moderate functional impairment can be accounted for by misstrafficking with significant intracellular retention and reduced cell surface levels. However, the residual activity of the V60L variant is apparently sufficient to sustain normal dendricity and dopachrome tautomerase responses in homozygous NHMs, upon activation of MC1R (Ainger et al., ). For V92M, slightly impaired (Herraiz et al., ; Ringholm et al., ) or WT coupling (Beaumont et al., ) has been reported, and both the dose‐response curves for agonist‐induced cAMP and the maximal stimulation appear normal for R163Q (Nakayama et al., ; Ringholm et al., ).…”

Melanoma mutations modify melanocyte dynamics in co-culture with keratinocytes or fibroblasts

“…Cultured neonatal foreskin melanoblasts are a well-established in vitro model for human primary melanocytic cells and have been extensively characterised in clonal strains genotyped for melanomarisk-conferring loci (Ainger et al, 2011;Cook et al, 2003;Jagirdar et al, 2014;Praetorius et al, 2013;Wong et al, 2012). Since neonatal melanoblasts differ from adult melanocytes in some features (Kim et al, 2000), we assessed both cell types for their response to lentiviral vector-driven expression of NRAS.Q61K and CDK4.R24C.…”

“…Melanocytes have been shown to deliver both pigmented (melanosomes) and nonpigmented vesicles to keratinocytes, while keratinocyte-excreted factors could influence both melanocyte proliferation and dendricity in culture (Ando et al, 2012;Hirobe, 2005;Lo Cicero et al, 2015). The MC1R allelic variant melanocytes also vary in the level of this response to keratinocytes, with R/R variants showing lower induced dendricity compared to wild-type variants with reduced melanoma risk (Ainger et al, 2011;Roberts et al, 2008).…”