Abstract
Proliferation in the epidermis is a tightly controlled process. During skin development, epidermis formation and hair follicle morphogenesis crucially depend on the regulated balance between proliferation and differentiation. Here we deleted all three Ras loci (H-Ras, N-Ras and K-Ras) from keratinocytes in vitro as well as specifically from the epidermis in mice using a K5Cre strain. Upon Ras elimination, keratinocytes ceased proliferation and entered into senescence without any signs of apoptosis induction. Constitutive activation of the mitogen-activated protein kinase pathway was able to partially rescue the proliferative defects. In mice, Ras signaling was essential for proper development of the epidermis and hair follicles. Deletion of the three Ras loci during epidermis formation in mouse embryos caused a dramatic decrease in proliferation, resulting in a substantially thinner epidermis and delayed appearance of differentiation markers. We could not detect apoptotic or senescent cells in these embryos suggesting that loss of Ras protein expression only leads to severe hypoproliferation. These observations provide genetic evidence for an essential role of Ras proteins in the control of keratinocyte and epidermal proliferation.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 50 print issues and online access
$259.00 per year
only $5.18 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Simpson CL, Patel DM, Green KJ . Deconstructing the skin: cytoarchitectural determinants of epidermal morphogenesis. Nat Rev Mol Cell Biol 2011; 12: 565–580.
Blanpain C, Fuchs E . Epidermal homeostasis: a balancing act of stem cells in the skin. Nat Rev Mol Cell Biol 2009; 10: 207–217.
Koster MI, Roop DR . Mechanisms regulating epithelial stratification. Annu Rev Cell Dev Biol 2007; 23: 93–113.
Koster MI . Making an epidermis. Ann N Y Acad Sci 2009; 1170: 7–10.
Byrne C, Tainsky M, Fuchs E . Programming gene expression in developing epidermis. Development 1994; 120: 2369–2383.
Lechler T, Fuchs E . Asymmetric cell divisions promote stratification and differentiation of mammalian skin. Nature 2005; 437: 275–280.
Fuchs E . Epidermal differentiation: the bare essentials. J Cell Biol 1990; 111: 2807–2814.
Candi E, Schmidt R, Melino G . The cornified envelope: a model of cell death in the skin. Nat Rev Mol Cell Biol 2005; 6: 328–340.
Millar SE . Molecular mechanisms regulating hair follicle development. J Invest Dermatol 2002; 118: 216–225.
Lee J, Tumbar T . Hairy tale of signaling in hair follicle development and cycling. Semin Cell Dev Biol 2012; 23: 906–916.
Fuchs E . Scratching the surface of skin development. Nature 2007; 445: 834–842.
Malumbres M, Barbacid M . RAS oncogenes: the first 30 years. Nat Rev Cancer 2003; 3: 459–465.
Pylayeva-Gupta Y, Gradocka E, Bar-Sagi D . Ras oncogenes: weaving a tumorigenic web. Nat Rev Cancer 2011; 11: 761–774.
Rojas AM, Fuentes G, Raussell A, Valencia A . The Ras protein superfamily: evolutionary tree and role of conserved amino acids. J Cell Biol 2012; 196: 189–201.
Johnson L, Greenbaum D, Cichowski K, Mercer K, Murphy E, Schmitt E et al. K-ras is an essential gene in the mouse with partial functional overlap with N-ras. Genes Dev 1997; 11: 2468–2481.
Drosten M, Dhawahir A, Sum EYM, Urosevic J, Lechuga CG, Esteban LM et al. Genetic analysis of Ras signaling pathways in cell proliferation, migration and survival. EMBO J 2010; 29: 1091–1104.
Dajee M, Tarutani M, Deng H, Cai T, Khavari PA . Epidermal Ras blockade demonstrates spatially localized Ras promotion of proliferation and inhibition of differentiation. Oncogene 2002; 21: 1527–1538.
Luetteke NC, Phillips HK, Qiu TH, Copeland NG, Earp HS, Jenkins NA et al. The mouse waved-2 phenotype results from a point mutation in the EGF receptor tyrosine kinase. Genes Dev 1994; 8: 399–413.
Sibilia M, Wagner EF . Strain-dependent epithelial defects in mice lacking the EGF receptor. Science 1995; 269: 234–238.
Threadgill DW, Dlugosz AA, Hansen LA, Tennenbaum T, Lichti U, Yee D et al. Targeted disruption of mouse EGF receptor: effect of genetic background on mutant phenotype. Science 1995; 269: 230–234.
Petiot A, Conti FJA, Grose R, Revest JM, Hodivala-Dilke KM, Dickson C . A crucial role for Fgfr2-IIIb signaling in epidermal development and hair follicle patterning. Development 2003; 130: 5493–5501.
Kolch W, Pitt A . Functional proteomics to dissect tyrosine kinase signaling pathways in cancer. Nat Rev Cancer 2010; 10: 618–629.
Scholl FA, Dumesic PA, Barragan DI, Harada K, Bissonauth V, Charron J et al. Mek1/2 MAPK kinases are essential for mammalian development, homeostasis, and Raf-induced hyperplasia. Dev Cell 2007; 12: 615–629.
Dumesic PA, Scholl FA, Barragan DI, Khavari PA . Erk1/2 MAP kinases are required for epidermal G2/M progression. J Cell Biol 2009; 185: 409–422.
Khavari TA, Rinn J . Ras/Erk MAPK signaling in epidermal homeostasis and neoplasia. Cell Cycle 2007; 6: 2928–2931.
Kern F, Niault T, Baccarini M . Ras and Raf pathways in epidermis development. Br J Cancer 2011; 104: 229–234.
Guerra C, Mijimolle N, Dhawahir A, Dubus P, Barradas M, Serrano M et al. Tumor induction by an endogenous K-ras oncogene is highly dependent on cellular context. Cancer Cell 2003; 4: 111–120.
Truong AB, Kretz M, Ridky TW, Kimmel R, Khavari PA . p63 regulates proliferation and differentiation of developmentally mature keratinocytes. Genes Dev 2006; 20: 8723–8762.
Oskarsson T, Essers MAG, Dubois N, Offner S, Dubey C, Roger C et al. Skin epidermis lacking the c-myc gene is resistant to Ras-driven tumorigenesis but can reacquire sensitivity upon additional loss of the p21Cip1 gene. Genes Dev 2006; 20: 2024–2029.
Lichti U, Anders J, Yuspa SH . Isolation and short-term culture of primary keratinocytes, hair follicle populations and dermal cells from newborn mice and keratinocytes from adult mice for in vitro analysis and for grafting to immunodeficient mice. Nat Protocols 2008; 3: 799–810.
Buse P, Tran SH, Luther E, Phu PT, Aponte GW, Firestone GL . Cell cycle and hormonal control of nuclear-cytoplasmic localization of the serum- and glucocorticoid-inducible protein kinase, Sgk, in mammary tumor cells. J Biol Chem 1999; 274: 7253–7363.
Kim WY, Sharpless NE . The regulation of INK4/ARF in cancer and aging. Cell 2006; 127: 265–275.
Abbas T, Dutta A . p21 in cancer: intricate networks and multiple activities. Nat Rev Cancer 2009; 9: 400–414.
Candi E, Cipollone R, Rivetti de Val Cervo P, Gonfloni S, Melino G, Knight R . p63 in epithelial development. Cell Mol Life Sci 2008; 65: 3126–3133.
Ramirez A, Page A, Gandarillas A, Zanet J, Pibre S, Vidal M et al. A keratin K5Cre transgenic line appropriate for tissue-specific or generalized Cre-mediated recombination. Genesis 2004; 39: 52–57.
Di Cunto F, Topley G, Calautti E, Hsiao J, Ong L, Seth PK et al. Inhibitory function of p21Cip1/WAF1 in differentiation of primary mouse keratinocytes independent of cell cycle control. Science 1998; 280: 1069–1072.
Paus R, Müller-Röver S, van der Veen C, Maurer M, Eichmüller S, Ling G et al. A comprehensive guide for the recognition and classification of distinct stages of hair follicle morphogenesis. J Invest Dermatol 1999; 113: 523–532.
Fuchs E, Horsley V . More than one way to the skin. Genes Dev 2008; 22: 976–985.
Vitale-Cross L, Amornphimoltham P, Fisher G, Molinolo AA, Gutkind JS . Conditional expression of K-ras in an epithelial compartment that includes the stem cells is sufficient to promote squamous cell carcinogenesis. Cancer Res 2004; 64: 8804–8807.
Stewart S, Guan KL . The dominant-negative Ras mutant, N17Ras, can inhibit signaling independently of blocking Ras activation. J Biol Chem 2000; 275: 8854–8862.
Potenza N, Vecchione C, Notte A, De Rienzo A, Rosica A, Bauer L et al. Replacement of K-Ras with H-Ras supports normal embryonic development despite inducing cardiovascular pathology in adult mice. EMBO Rep 2005; 6: 432–437.
Tarutani M, Cai T, Dajee M, Khavari PA . Inducible activation of Ras and Raf in adult epidermis. Cancer Res 2003; 63: 319–323.
Scholl FA, Dumesic PA, Khavari PA . Mek1 alters epidermal growth and differentiation. Cancer Res 2004; 64: 6035–6040.
He J, Kallin EM, Tsukada Y, Zhang Y . The H3K36 demethylase Jhdm1b/Kdm2b regulates cell proliferation and senescence through p15(Ink4b). Nat Struct Mol Biol 2008; 15: 1169–1175.
Weinberg WC, Denning MF . P21Waf1 control of epithelial cell cycle and cell fate. Crit Rev Oral Biol Med 2002; 13: 453–463.
Keyes WM, Wu Y, Vogel H, Guo X, Lows SW, Mills AA . p63 deficiency activates a program of cellular senescence and leads to accelerated aging. Genes Dev 2005; 19: 1986–1999.
Trigg MJ . Hair growth in mouse mutants affecting coat texture. J Zool 1972; 168: 165–198.
Esteban LM, Vicario-Abejón C, Fernández-Salguero P, Fernández-Medarde A, Swaminathan N, Yienger K et al. Targeted genomic disruption of H-ras and N-ras, individually or in combination, reveals the dispensability of both loci for mouse growth and development. Mol Cell Biol 2001; 21: 1444–1452.
Navas C, Hernández-Porras I, Schuhmacher AJ, Sibilia M, Guerra C, Barbacid M . EGF receptor signaling is essential for K-Ras oncogene-driven pancreatic ductal adenocarcinoma. Cancer Cell 2012; 22: 318–330.
Yuan JS, Reed A, Chen F, Stewart CN . Statistical analysis of real-time PCR data. BMC Bioinformatics 2006; 7: 85.
Itahana K, Campisi J, Dimri GP . Methods to detect biomarkers of cellular senescence: the senescence-associated β-galactosidase assay. Methods Mol Biol 2007; 371: 21–31.
Guerra C, Collado M, Navas C, Schuhmacher AJ, Hernández-Porras I, Cañamero M et al. Pancreatitis-induced inflammation contributes to pancreatic cancer by inhibiting oncogene-induced senescence. Cancer Cell 2011; 19: 728–739.
Acknowledgements
We thank the Histopatholoy and Confocal Microscopy Units at the CNIO for their help. This work was supported by grants from the European Research Council (ERC-AG/250297-RAS AHEAD), EU-Framework Programme (LSHG-CT-2007-037665/CHEMORES, HEALTH-F2-2010-259770/LUNGTARGET and HEALTH-2010-260791/EUROCANPLATFORM), Spanish Ministry of Economy and Competitiveness (SAF2011-30173) and Autonomous Community of Madrid (S2011/BDM-2470/ONCOCYCLE) to MB.
Author information
Authors and Affiliations
Corresponding authors
Ethics declarations
Competing interests
The authors declare no conflict of interest.
Additional information
Supplementary Information accompanies this paper on the Oncogene website
Supplementary information
Rights and permissions
About this article
Cite this article
Drosten, M., Lechuga, C. & Barbacid, M. Ras signaling is essential for skin development. Oncogene 33, 2857–2865 (2014). https://doi.org/10.1038/onc.2013.254
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/onc.2013.254
Keywords
This article is cited by
-
Cutis verticis gyrata and Noonan syndrome: report of two cases with pathogenetic variant in SOS1 gene
Italian Journal of Pediatrics (2022)
-
RAS-targeted therapies: is the undruggable drugged?
Nature Reviews Drug Discovery (2020)