Melanocyte Precursors in the Hair Follicle Bulge of Repigmented Vitiligo Skin Are Controlled by RHO-GTPase, KCTD10, and CTNNB1 Signaling

In repigmentation of human vitiligo, the melanocyte (MC) precursors in hair follicle bulge proliferate, migrate, and differentiate to repopulate depigmented epidermis. Here, we present a comprehensive characterization pathways signals that control process. Using biopsies from patients with have selectively harvested, by laser capture microdissection, MC keratinocyte untreated vitiligo skin treated narrow-band UVB. The captured material was subjected whole transcriptome RNA-sequencing. With this strategy, found is driven KCTD10, signal unknown roles skin, CTNNB1 (encoding ?-catenin) RHO guanosine triphosphatase [RHO GTPase, RHO], two signaling previously shown be involved pigmentation biology. Knockdown studies cultured MCs RHOJ, upmost differentially expressed family component, corroborated our findings identified RHOJ involvement UV response melanization, confirmed melanocytic cell migration apoptosis. A better understanding mechanisms govern will enable discovery molecules induce robust phenotypes vitiligo. Vitiligo progressive autoimmune disease characterized white spots resulting loss epidermal melanocytes (MCs) because attack destruction CD8+ T cells (Eby et al., 2014Eby J.M. Kang H.K. Klarquist J. Chatterjee S. Mosenson J.A. Nishimura M.I. al.Immune responses mouse model spontaneous de- repigmentation.Pigment Cell Melanoma Res. 2014; 27: 1075-1085Crossref PubMed Scopus (15) Google Scholar; Rashighi Harris, 2017Rashighi M. Harris J.E. pathogenesis emerging treatments.Dermatol Clin. 2017; 35: 257-265Abstract Full Text PDF (63) Roberts 2019Roberts G.H.L. Paul Yorgov D. Santorico S.A. Spritz R.A. Family clustering results principally polygenic inheritance common risk alleles.Am J Hum Genet. 2019; 105: 364-372Abstract Andersen, 2017Spritz Andersen G.H. Genetics vitiligo.Dermatol 245-255Abstract (77) Scholar). depigmentation reversed process, classic example regenerative medicine (Birlea 2017aBirlea Costin G.E. Roop D.R. Norris D.A. Trends medicine: through stem mobilization.Med Res Rev. 37: 907-935Crossref (33) Scholar, Birlea 2017bBirlea Goldstein N.B. Repigmentation regeneration 205-218Abstract (16) Scholar), which (HF), are stimulated differentiate, migrate epidermis (Goldstein 2015Goldstein Koster Hoaglin L.G. Spoelstra N.S. Kechris K.J. Robinson S.E. al.Narrow band ultraviolet B treatment for associated proliferation, migration, differentiation precursors.J Invest Dermatol. 2015; 135: 2068-2076Abstract (60) Common treatments function immunomodulation stimulation HF include UVB (NBUVB), topical bimatoprost, JAK inhibitors (Barbulescu 2020Barbulescu C.C. Harnessing power therapy alopecia areata.J 2020; 140: 29-37Abstract (9) NBUVB one most effective therapies stimulate repigmentation; however, molecular changes occur interfollicular during not well-understood. To advance knowledge, developed system combines immunostaining, fluorescent microdissection (F-LCM) separately harvest keratinocytes (KCs) NBUVB-treated RNA-sequencing (RNA-seq), identifying important modulated repopulated 2018Goldstein Jones K.L. Gao B. al.Repigmentation controlled transcription GLI1 activation ?-catenin pathway cells.J 2018; 138: 657-668Abstract study, harvested F-LCM KCs performed RNA-seq. genes were cellular proliferation and/or but represent unreported NBUVB: upregulation KCTD10 (RHO) pathway, being top component gene. Stable knockdown impacted differentiation, cycle function, DNA damage UV, apoptosis, expression key Understanding interactions between RHO, CTNNB1, other repopulation disease. For precursors, used frozen obtained We mapping, rapid immunostaining (to label MCs), separately, RNA-seq, confirmatory quantitative real-time RT–PCR (qRT-PCR), functional using MCs. RNA-seq collected generalized (N = 12) (Caucasians or Latinos types II–IV) trunk limbs. Six six (Supplementary Table S1). This study focused on laser-captured bulge–outer root sheath. Separately KC samples also sheath validate method identify canonical NBUVB. total RNA isolated By principal analysis, segregation gene values both Scholar) Figure S1a) versus S1b). Additionally, significant enrichment MC-specific compared KC-specific S1c–e). These indicate accurate isolating distinct populations adjacent skin. repigmented after 3–6 months (n 6 unpaired samples). dataset composed 54,008 transcripts, 280 (0.52%) protein-coding (P ? 0.05) (165 [0.31%] upregulated 115 [0.21%] downregulated). None these surpassed false rate significance threshold (false rate–adjusted Q-value 0.05). ranked basis P-values (significant P fold change ? 2 10 summarized 1, several different types. next selected qRT-PCR confirmation five bulge. included CDR1 2.2E?04, +3.0-fold), LY6E 7.4E?04, +8.1-fold), GMCL1 1.1E?03, +4.6-fold), 1.2E?03, +9.7-fold), upregulated, RIOK2 6.3E?04, ?8.01-fold), downregulated. qRT-PCR, seven (three four new samples) (four samples), named Confirmation Group, rounds F-LCM. three (Benjamini-Hochberg adjusted 0.05 2) including 4.1E-03, Padjusted 2.0E?02, +3.0-fold) (Figure 1a), 2.1E?02, 3.4E?02, +51.5-fold), downregulation 1.8E?02, 4.6E?02, ?3.0-fold) S1f). As exhibited modulation NBUVB, second showed its transcript RNAscope technology (Advanced Diagnostics, Newark, CA), utilizes an situ hybridization 1b c). Although multiple testing corrections > 0.05), (LY6E GMCL1) followed trend.Table 1Top Genes Differentially Expressed RNA-Seq DataGeneFold ChangeP-ValueQ-ValueFunctionReferenceCDR13.032.20E?040.47Involved progression promotes mitosisOpalko Moseley, 2017Opalko H.E. Moseley J.B. Dynamic regulation Cdr1 kinase localization phosphorylation osmotic stress.J Biol Chem. 292: 18457-18468Abstract (8) ScholarRIOK2?8.016.30E?040.50Pro-proliferative role inhibits p53 glioblastomaRead 2013Read R.D. Fenton T.R. Gomez G.G. Wykosky Vandenberg S.R. Babic I. al.A kinome-wide RNAi screen Drosophila glia reveals RIO kinases mediate survival TORC2-Akt glioblastoma.PLoS 2013; 9e1003253Crossref (75) ScholarLY6E8.077.40E?040.50Cellular differentiationUpadhyay, 2019Upadhyay G. Emerging lymphocyte antigen-6 cancer immune cells.Front. Immunol. 10: 819Crossref (32) ScholarGMCL14.641.15E?030.50Regulator tumor suppressor MDM2-P53Masuhara al 2003Masuhara Nagao K. Nishikawa Kimura T. Nakano Enhanced degradation MDM2 nuclear envelope germ cell-less.Biochem Biophys Commun. 2003; 308: 927-932Crossref (7) ScholarKCTD109.691.19E?030.50Impacts interacts proliferating antigenWang 2009Wang Y. Zheng Luo F. Fan X. Chen Zhang C. al.KCTD10 antigen down-regulation could inhibit proliferation.J Biochem. 2009; 106: 409-413Crossref (24) ScholarMFAP45.741.27E?030.50Overexpression protected UVB-induced photodamageKasamatsu 2011Kasamatsu Hachiya A. Fujimura Sriwiriyanont P. Haketa Visscher M.O. al.Essential microfibrillar-associated protein 4 cutaneous homeostasis photoprotection.Sci Rep. 2011; 1: 164Crossref (44) ScholarPHAX5.21.30E?030.50Pluripotency pluripotent cellsShao 2017Shao Taniguchi Gurdziel Townshend R.F. Xue Yong K.M.A. al.Self-organized amniogenesis biomimetic implantation-like niche.Nat Mater. 16: 419-425Crossref ScholarGNG122.861.30E?030.50Modulator transducer transmembrane systemsTan 2014Tan Gong Y.Z. Wu Liao D.F. X.L. Mesenchymal cell-derived microparticles: promising therapeutic strategy.Int Mol Sci. 15: 14348-14363Crossref (22) ScholarSYNPO3.831.59E?030.50Induces via RHOA signalingAsanuma 2006Asanuma Yanagida-Asanuma E. Faul Tomino Kim Mundel Synaptopodin orchestrates actin organization motility RhoA signalling.Nat Biol. 2006; 8: 485-491Crossref (314) ScholarFAM127BRTL8A6.491.77E?030.50Unknown functionN/AAbbreviations: N/A, applicable; UVB; RNA-sequencing.Top encoding Fold changes, P-values, (Q-values), functions provided. Open table tab Abbreviations: Top describe signature examined 193 published panel process (nCounter Virtual Stem Gene Set, NanoString Technologies, Seattle, WA). There bulge: NOTCH3 3.3E?03, ?4.0-fold), COL1A1 8.1E?03, +6.55-fold), TLE1 1.5E?02, ?2.4-fold), +2.0-fold), FGF1 4.0E?02, +12.4-fold), APC 4.5E?02, +18.1-fold) 2a). attempted confirm Group described previously. only 2.3E?02, +5.8-fold) 2b). genes, although trend. explore relationship among data, Ingenuity Pathway Analysis tool, comparing 323 485 (significance, activated (highest Z-score, lowest P-value) integrin (Z-score 3.2; 3.5E?05) S2) 2.4; 1.2E?02) S3). Most data 3a) S4). Of validated 3.5E?03, +2.6-fold) similar trend VIM 2.7E?02, CDH3 0.11, +1.8-fold), CDH11 0.08, +1.7-fold), RELA +1.5-fold) corrections) 3b). highest upstream transcriptional regulators AHR ?2.1; P=5.6E?06) S5) TP53 protein) 2.0, 5.8E?15) S6). Furthermore, MCs, growth 2.6; 2.9E?14) movement 3.1; 1.8E?13) S7). understand RHOJ’s induced neonatal moderately pigmented (HEMn-MPs). immature cells, excellent experimental precursors’ behavior they proliferate many passages without terminal differentiation. created stable HEMn-MP pools transducing them SMARTvector Inducible Lentiviral short hairpin (shRNA) particles (GE Healthcare Dharmacon, Lafayette, CO), pool transduced shRNA (shRHOJ) (shControl). shRHOJ had 70% decrease steady-state (vs. shControl), as S2a) 80% depletion protein, western blot S2b), indicating efficient knockdown. Next, studied how impacts viability, light melanoma invasion (Ho 2013Ho H. Soto Hopkin Kapadia R. Vasudeva Schilling Ganesan A.K. RhoJ modulates altering cytoskeletal dynamics.Pigment 26: 218-225Crossref (19) melanogenesis, finding RHOJ-depleted smaller, elongated spindle-like shape S2c), than shControl polygonal shape, 30% reduced normal medium, analyzed scratch wound assay < 4a). effect even more pronounced (45% reduction) when ?-MC–stimulating hormone, known mitogenic melanogenic (Scott 1997Scott Cassidy L. Abdel-Malek Z. Alpha-melanocyte-stimulating hormone endothelin-1 opposing effects adhesion, pp125FAK phosphorylation.Exp 1997; 237: 19-28Crossref 0.01) 4b No difference viability observed first 72-hour incubation addition tested Vision Blue Assay (BioVision, Inc, Milpitas, CA; shown); two-fold CCND1 cyclin D1, progression) 4.0E?04) 16-hour serum starvation subsequent 24-hour transcripts TYR, TYRP1, PMEL decreased 50%, 31%, respectively, shControl, 48 hours doxycycline induction, quantified 4d). following:1.UV-induced expression. Because increase wanted see whether cells. Preliminary experiments single dose 90 mJ/cm2 resulted death 24 eye, indicated rounded floating chose lower 30 UV. +2.2-fold post-irradiation 3.0E?03) 4e). contrast, same 52% reduction shControl; 4.0E?03), persisted treatment, 4e).2.Cell plays apoptosis following previous lines 2012Ho Aruri Mehr White M.A. regulates chemoresistance suppressing sense damage.Cancer 2012; 72: 5516-5528Crossref (39) exposed increased ?-H2AX foci count (2.9-fold higher; 1.0E-03) area calculation (3.1-fold 4g), parameters examining immunostained images 4f); cleaved PARP (pro-apoptotic signals) (+2.3-fold +1.6-fold, at hours); phospho-BAD (prosurvival signal) (?4.8-fold hours), S4a b). sum, low amounts induction anti-apoptotic modulation. apoptotic RHOJ. explored levels SOX10 effector ?1.6-fold, ?1.7-fold, respectively 1.0E?02) S5a), whereas exposure all +1.3-fold, 1E?02) unexposed S5b). 5) bulge, building signals, knowledge unreported, repigmentation: (RNA-seq, hybridization) significantly CDR1, qRT-PCR). Our inability highest-ranked result low-powered analysis between-patient variation some limitations appears (Opalko has attributed glioblastoma inhibition (Read repair, control, interaction (Olaisen 2015Olaisen Müller Nedal Otterlei PCNA-interacting peptides reduce Akt TLR-mediated cytokine secretion suggesting PCNA signaling.Cell Signal. 1478-1487Crossref (29) Ren 2014Ren Yuan Yang Ding Zhou cardiovascular Notch early embryonic development.PLoS One. 9e112275Crossref (18) Wang 5). Proliferating antigen, complexes can transiently crucial (Xu Morris 1999Xu G.F. p53-mediated ionizing radiation.Mol 1999; 19: 12-20Crossref (78) regulator Together, suggest possible orchestrating consistent hypermelanosis (Murase 2009Murase Amano Ohuchi Kitahara Takema essential hyperpigmentation paracrine receptor signaling.J 284: 4343-4353Abstract induces release GFs KCs, activate their corresponding receptors 2017bBirl