The p63 protein plays a key role in regulating human keratinocyte proliferation and differentiation. Although some p63-regulating microRNAs (miRNAs) have been identified in the control of epidermal homeostasis, little is known about miRNAs acting downstream of p63. In this paper, we characterized multiple p63-regulated miRNAs (miR-17, miR-20b, miR-30a, miR-106a, miR-143 and miR-455-3p) and elucidated their roles in the onset of keratinocyte differentiation. We identified RB, p21 and multiple MAPKs as targets of these p63-controlled miRNAs. Upon inhibition of most of these miRNAs, we observed defects in commitment to differentiation that could be reversed by siRNA-mediated silencing of their targets. Furthermore, knockdown of MAPK8 and MAPK9 efficiently restored expression of the early differentiation markers keratin 1 and keratin 10 in p63-silenced primary human keratinocytes. These results highlight new mechanistic roles of multiple miRNAs, particularly the miR-17 family (miR-17, miR-20b and miR-106a), as regulatory intermediates for coordinating p63 with MAPK signaling in the commitment of human mature keratinocytes to early differentiation.
References
[1]
Fuchs E, Raghavan S (2002) Getting under the skin of epidermal morphogenesis. Nat Rev Genet 3: 199–209.
[2]
Muller EJ, Williamson L, Kolly C, Suter MM (2008) Outside-in signaling through integrins and cadherins: a central mechanism to control epidermal growth and differentiation? J Invest Dermatol 128: 501–516.
[3]
Blanpain C, Fuchs E (2009) Epidermal homeostasis: a balancing act of stem cells in the skin. Nat Rev Mol Cell Biol 10: 207–217.
[4]
Mills AA, Zheng B, Wang XJ, Vogel H, Roop DR, et al. (1999) p63 is a p53 homologue required for limb and epidermal morphogenesis. Nature 398: 708–713.
[5]
Yang A, Schweitzer R, Sun D, Kaghad M, Walker N, et al. (1999) p63 is essential for regenerative proliferation in limb, craniofacial and epithelial development. Nature 398: 714–718.
[6]
Truong AB, Kretz M, Ridky TW, Kimmel R, Khavari PA (2006) p63 regulates proliferation and differentiation of developmentally mature keratinocytes. Genes Dev 20: 3185–3197.
[7]
Wu N, Rollin J, Masse I, Lamartine J, Gidrol X (2012) p63 Regulates Human Keratinocyte Proliferation via MYC-regulated Gene Network and Differentiation Commitment through Cell Adhesion-related Gene Network. J Biol Chem 287: 5627–5638.
[8]
Pasquinelli AE, Reinhart BJ, Slack F, Martindale MQ, Kuroda MI, et al. (2000) Conservation of the sequence and temporal expression of let-7 heterochronic regulatory RNA. Nature 408: 86–89.
Kim VN, Han J, Siomi MC (2009) Biogenesis of small RNAs in animals. Nat Rev Mol Cell Biol 10: 126–139.
[11]
Valencia-Sanchez MA, Liu J, Hannon GJ, Parker R (2006) Control of translation and mRNA degradation by miRNAs and siRNAs. Genes Dev 20: 515–524.
[12]
Ghildiyal M, Zamore PD (2009) Small silencing RNAs: an expanding universe. Nat Rev Genet 10: 94–108.
[13]
Ambros V (2004) The functions of animal microRNAs. Nature 431: 350–355.
[14]
Bushati N, Cohen SM (2007) microRNA functions. Annu Rev Cell Dev Biol 23: 175–205.
[15]
Farh KK, Grimson A, Jan C, Lewis BP, Johnston WK, et al. (2005) The widespread impact of mammalian MicroRNAs on mRNA repression and evolution. Science 310: 1817–1821.
[16]
Lu J, Getz G, Miska EA, Alvarez-Saavedra E, Lamb J, et al. (2005) MicroRNA expression profiles classify human cancers. Nature 435: 834–838.
[17]
Kanellopoulou C, Muljo SA, Kung AL, Ganesan S, Drapkin R, et al. (2005) Dicer-deficient mouse embryonic stem cells are defective in differentiation and centromeric silencing. Genes Dev 19: 489–501.
[18]
Yi R, O’Carroll D, Pasolli HA, Zhang Z, Dietrich FS, et al. (2006) Morphogenesis in skin is governed by discrete sets of differentially expressed microRNAs. Nat Genet 38: 356–362.
[19]
Aberdam D, Candi E, Knight RA, Melino G (2008) miRNAs, ‘stemness’ and skin. Trends Biochem Sci 33: 583–591.
[20]
Yi R, Poy MN, Stoffel M, Fuchs E (2008) A skin microRNA promotes differentiation by repressing ‘stemness’. Nature 452: 225–229.
[21]
Yi R, Fuchs E (2010) MicroRNA-mediated control in the skin. Cell Death Differ 17: 229–235.
[22]
Lena AM, Shalom-Feuerstein R, Rivetti di Val Cervo P, Aberdam D, Knight RA, et al. (2008) miR-203 represses ‘stemness’ by repressing DeltaNp63. Cell Death Differ 15: 1187–1195.
[23]
Scheel AH, Beyer U, Agami R, Dobbelstein M (2009) Immunofluorescence-based screening identifies germ cell associated microRNA 302 as an antagonist to p63 expression. Cell Cycle 8: 1426–1432.
[24]
Manni I, Artuso S, Careccia S, Rizzo MG, Baserga R, et al.. (2009) The microRNA miR-92 increases proliferation of myeloid cells and by targeting p63 modulates the abundance of its isoforms. FASEB J.
[25]
Antonini D, Russo MT, De Rosa L, Gorrese M, Del Vecchio L, et al. (2010) Transcriptional repression of miR-34 family contributes to p63-mediated cell cycle progression in epidermal cells. J Invest Dermatol 130: 1249–1257.
[26]
Orom UA, Kauppinen S, Lund AH (2006) LNA-modified oligonucleotides mediate specific inhibition of microRNA function. Gene 372: 137–141.
[27]
Koster MI, Kim S, Mills AA, DeMayo FJ, Roop DR (2004) p63 is the molecular switch for initiation of an epithelial stratification program. Genes Dev 18: 126–131.
[28]
Sonkoly E, Wei T, Pavez Lorie E, Suzuki H, Kato M, et al. (2010) Protein kinase C-dependent upregulation of miR-203 induces the differentiation of human keratinocytes. J Invest Dermatol 130: 124–134.
[29]
Kouwenhoven EN, van Heeringen SJ, Tena JJ, Oti M, Dutilh BE, et al. (2010) Genome-wide profiling of p63 DNA-binding sites identifies an element that regulates gene expression during limb development in the 7q21 SHFM1 locus. PLoS Genet 6: e1001065.
[30]
Pozzi S, Zambelli F, Merico D, Pavesi G, Robert A, et al. (2009) Transcriptional network of p63 in human keratinocytes. PLoS ONE 4: e5008.
[31]
Vigano MA, Lamartine J, Testoni B, Merico D, Alotto D, et al. (2006) New p63 targets in keratinocytes identified by a genome-wide approach. EMBO J 25: 5105–5116.
[32]
O’Donnell KA, Wentzel EA, Zeller KI, Dang CV, Mendell JT (2005) c-Myc-regulated microRNAs modulate E2F1 expression. Nature 435: 839–843.
[33]
Dews M, Homayouni A, Yu D, Murphy D, Sevignani C, et al. (2006) Augmentation of tumor angiogenesis by a Myc-activated microRNA cluster. Nat Genet 38: 1060–1065.
[34]
Chang TC, Yu D, Lee YS, Wentzel EA, Arking DE, et al. (2008) Widespread microRNA repression by Myc contributes to tumorigenesis. Nat Genet 40: 43–50.
[35]
Paramio JM, Segrelles C, Casanova ML, Jorcano JL (2000) Opposite functions for E2F1 and E2F4 in human epidermal keratinocyte differentiation. J Biol Chem 275: 41219–41226.
[36]
Ruiz S, Santos M, Segrelles C, Leis H, Jorcano JL, et al. (2004) Unique and overlapping functions of pRb and p107 in the control of proliferation and differentiation in epidermis. Development 131: 2737–2748.
[37]
He L, Thomson JM, Hemann MT, Hernando-Monge E, Mu D, et al. (2005) A microRNA polycistron as a potential human oncogene. Nature 435: 828–833.
[38]
Cordes KR, Sheehy NT, White MP, Berry EC, Morton SU, et al. (2009) miR-145 and miR-143 regulate smooth muscle cell fate and plasticity. Nature 460: 705–710.
[39]
Foshay KM, Gallicano GI (2009) miR-17 family miRNAs are expressed during early mammalian development and regulate stem cell differentiation. Dev Biol 326: 431–443.
[40]
Wang Q, Li YC, Wang J, Kong J, Qi Y, et al. (2008) miR-17–92 cluster accelerates adipocyte differentiation by negatively regulating tumor-suppressor Rb2/p130. Proc Natl Acad Sci U S A 105: 2889–2894.
[41]
Weston CR, Wong A, Hall JP, Goad ME, Flavell RA, et al. (2004) The c-Jun NH2-terminal kinase is essential for epidermal growth factor expression during epidermal morphogenesis. Proc Natl Acad Sci U S A 101: 14114–14119.
[42]
Gazel A, Banno T, Walsh R, Blumenberg M (2006) Inhibition of JNK promotes differentiation of epidermal keratinocytes. J Biol Chem 281: 20530–20541.
[43]
Alameda JP, Fernandez-Acenero MJ, Moreno-Maldonado R, Navarro M, Quintana R, et al. (2011) CYLD regulates keratinocyte differentiation and skin cancer progression in humans. Cell Death Dis 2: e208.
[44]
Hara T, Miyazaki M, Hakuno F, Takahashi S, Chida K (2011) PKCeta promotes a proliferation to differentiation switch in keratinocytes via upregulation of p27Kip1 mRNA through suppression of JNK/c-Jun signaling under stress conditions. Cell Death Dis 2: e157.
[45]
Li G, Gustafson-Brown C, Hanks SK, Nason K, Arbeit JM, et al. (2003) c-Jun is essential for organization of the epidermal leading edge. Dev Cell 4: 865–877.
[46]
Zenz R, Scheuch H, Martin P, Frank C, Eferl R, et al. (2003) c-Jun regulates eyelid closure and skin tumor development through EGFR signaling. Dev Cell 4: 879–889.
[47]
Mehic D, Bakiri L, Ghannadan M, Wagner EF, Tschachler E (2005) Fos and jun proteins are specifically expressed during differentiation of human keratinocytes. J Invest Dermatol 124: 212–220.
[48]
Sabapathy K, Hochedlinger K, Nam SY, Bauer A, Karin M, et al. (2004) Distinct roles for JNK1 and JNK2 in regulating JNK activity and c-Jun-dependent cell proliferation. Mol Cell 15: 713–725.
[49]
Boukamp P, Petrussevska RT, Breitkreutz D, Hornung J, Markham A, et al. (1988) Normal keratinization in a spontaneously immortalized aneuploid human keratinocyte cell line. J Cell Biol 106: 761–771.
