Gene knockout studies unexpectedly reveal a pivotal role for IkB kinase alpha (IKKa) in mouse embryonic skin development. Skin carcinogenesis experiments show that Ikk a heterozygous mice are highly susceptible to chemical carcinogen or ultraviolet B light (UVB) induced benign and malignant skin tumors in comparison to wild-type mice. IKKa deletion mediated by keratin 5 (K5).Cre or K15.Cre in keratinocytes induces epidermal hyperplasia and spontaneous skin squamous cell carcinomas (SCCs) in Ikk a floxed mice. On the other hand, transgenic mice overexpressing IKKa in the epidermis, under the control of a truncated loricrin promoter or K5 promoter, develop normal skin and show no defects in the formation of the epidermis and other epithelial organs, and the transgenic IKKa represses chemical carcinogen or UVB induced skin carcinogenesis. Moreover, IKKa deletion mediated by a mutation, which generates a stop codon in the Ikk a gene, has been reported in a human autosomal recessive lethal syndrome. Downregulated IKKa and Ikk a mutations and deletions are found in human skin SCCs. The collective evidence not only highlights the importance of IKKa in skin development, maintaining skin homeostasis, and preventing skin carcinogenesis, but also demonstrates that mouse models are extremely valuable tools for revealing the mechanisms underlying these biological events, leading our studies from bench side to bedside.
References
[1]
Connelly, M.A.; Marcu, K.B. CHUK, a new member of the helix-loop-helix and leucine zipper families of interacting proteins, contains a serine-threonine kinase catalytic domain. Cell. Mol. Biol. Res. 1995, 41, 537–549.
[2]
Mock, B.A.; Connelly, M.A.; McBride, O.W.; Kozak, C.A.; Marcu, K.B. CHUK, a conserved helix-loop-helix ubiquitous kinase, maps to human chromosome 10 and mouse chromosome 19. Genomics 1995, 27, 348–351, doi:10.1006/geno.1995.1054.
[3]
Balkhi, M.Y.; Willette-Brown, J.; Zhu, F.; Chen, Z.; Liu, S.; Guttridge, D.C.; Karin, M.; Hu, Y. IKKalpha-mediated signaling circuitry regulates early B lymphopoiesis during hematopoiesis. Blood 2012, 119, 5467–5477, doi:10.1182/blood-2012-01-401547.
[4]
Ghosh, S.; Karin, M. Missing pieces in the NF-kappaB puzzle. Cell 2002, 109, S81–S96, doi:10.1016/S0092-8674(02)00703-1.
[5]
Liu, B.; Xia, X.; Zhu, F.; Park, E.; Carbajal, S.; Kiguchi, K.; DiGiovanni, J.; Fischer, S.M.; Hu, Y. IKKalpha is required to maintain skin homeostasis and prevent skin cancer. Cancer Cell 2008, 14, 212–225, doi:10.1016/j.ccr.2008.07.017.
[6]
Descargues, P.; Sil, A.K.; Sano, Y.; Korchynskyi, O.; Han, G.; Owens, P.; Wang, X.J.; Karin, M. IKKalpha is a critical coregulator of a Smad4-independent TGFbeta-Smad2/3 signaling pathway that controls keratinocyte differentiation. Proc. Natl. Acad. Sci. USA 2008, 105, 2487–2492.
[7]
Marinari, B.; Moretti, F.; Botti, E.; Giustizieri, M.L.; Descargues, P.; Giunta, A.; Stolfi, C.; Ballaro, C.; Papoutsaki, M.; Alema, S.; et al. The tumor suppressor activity of IKKalpha in stratified epithelia is exerted in part via the TGF-beta antiproliferative pathway. Proc. Natl. Acad. Sci. USA 2008, 105, 17091–17096.
[8]
Maeda, G.; Chiba, T.; Kawashiri, S.; Satoh, T.; Imai, K. Epigenetic inactivation of IkappaB Kinase-alpha in oral carcinomas and tumor progression. Clin. Cancer Res. 2007, 13, 5041–5047, doi:10.1158/1078-0432.CCR-07-0463.
[9]
DiDonato, J.A.; Hayakawa, M.; Rothwarf, D.M.; Zandi, E.; Karin, M. A cytokine-responsive IκB kinase that activates the transcription factor NF-κB. Nature 1997, 388, 548–554, doi:10.1038/41493.
[10]
Mercurio, F.; Zhu, H.; Murray, B.W.; Shevchenko, A.; Bennett, B.L.; Li, J.; Young, D.B.; Barbosa, M.; Mann, M.; Manning, A.; et al. A. KK-1 and IKK-2: Cytokine-activated IκB kinases essential for NF-κB activation. Science 1997, 278, 860–866, doi:10.1126/science.278.5339.860.
[11]
Beg, A.A.; Sha, W.C.; Bronson, R.T.; Ghosh, S.; Baltimore, D. Embryonic lethality and liver degeneration in mice lacking the RelA component of NF-κB. Nature 1995, 376, 167–170.
[12]
Li, Z.W.; Chu, W.; Hu, Y.; Delhase, M.; Deerinck, T.; Ellisman, M.; Johnson, R.; Karin, M. The IKKβ subunit of IκB kinase (IKK) is essential for nuclear factor κB activation and prevention of apoptosis. J. Exp. Med. 1999, 189, 1839–1845, doi:10.1084/jem.189.11.1839.
[13]
Rudolph, D.; Yeh, W.C.; Wakeham, A.; Rudolph, B.; Nallainathan, D.; Potter, J.; Elia, A.J.; Mak, T.W. Severe liver degeneration and lack of NF-κB activation in NEMO/IKKgamma-deficient mice. Genes Dev. 2000, 14, 854–862.
[14]
Makris, C.; Godfrey, V.L.; Krahn-Senftleben, G.; Takahashi, T.; Roberts, J.L.; Schwarz, T.; Feng, L.; Johnson, R.S.; Karin, M. Female mice heterozygous for IKK gamma/NEMO deficiencies develop a dermatopathy similar to the human X-linked disorder incontinentia pigmenti. Mol. Cell 2000, 5, 969–979, doi:10.1016/S1097-2765(00)80262-2.
[15]
Li, Q.; Lu, Q.; Hwang, J.Y.; Buscher, D.; Lee, K.F.; Izpisua-Belmonte, J.C.; Verma, I.M. IKK1-deficient mice exhibit abnormal development of skin and skeleton. Genes Dev. 1999, 13, 1322–1328, doi:10.1101/gad.13.10.1322.
[16]
Li, Q.; van Antwerp, D.; Mercurio, F.; Lee, K.F.; Verma, I.M. Severe liver degeneration in mice lacking the IkappaB kinase 2 gene. Science 1999, 284, 321–325, doi:10.1126/science.284.5412.321.
[17]
Schmidt-Supprian, M.; Bloch, W.; Courtois, G.; Addicks, K.; Israel, A.; Rajewsky, K.; Pasparakis, M. NEMO/IKK gamma-deficient mice model incontinentia pigmenti. Mol. Cell 2000, 5, 981–992, doi:10.1016/S1097-2765(00)80263-4.
[18]
Doi, T.S.; Marino, M.W.; Takahashi, T.; Yoshida, T.; Sakakura, T.; Old, L.J.; Obata, Y. Absence of tumor necrosis factor rescues RelA-deficient mice from embryonic lethality. Proc. Natl. Acad. Sci. USA 1999, 96, 2994–2999.
[19]
Alcamo, E.; Mizgerd, J.P.; Horwitz, B.H.; Bronson, R.; Beg, A.A.; Scott, M.; Doerschuk, C.M.; Hynes, R.O.; Baltimore, D. Targeted mutation of TNF receptor I rescues the RelA-deficient mouse and reveals a critical role for NF-kappa B in leukocyte recruitment. J. Immunol. 2001, 167, 1592–1600.
[20]
Zandi, E.; Rothwarf, D.M.; Delhase, M.; Hayakawa, M.; Karin, M. The IκB kinase complex (IKK) contains two kinase subunits, IKKα and IKKβ, necessary for IκB phosphorylation and NF-κB activation. Cell 1997, 91, 243–252, doi:10.1016/S0092-8674(00)80406-7.
[21]
Hu, Y.; Baud, V.; Delhase, M.; Zhang, P.; Deerinck, T.; Ellisman, M.; Johnson, R.; Karin, M. Abnormal morphogenesis but intact IKK activation in mice lacking the IKKα subunit of IκB kinase. Science 1999, 284, 316–320, doi:10.1126/science.284.5412.316.
Fuchs, E.; Byrne, C. The epidermis: Rising to the surface. Curr. Opin. Genet. Dev. 1994, 4, 725–736, doi:10.1016/0959-437X(94)90140-X.
[24]
Hu, Y.; Baud, V.; Oga, T.; Kim, K.I.; Yoshida, K.; Karin, M. IKKα controls formation of the epidermis independently of NF-κB. Nature 2001, 410, 710–714, doi:10.1038/35070605.
[25]
Richardson, R.J.; Dixon, J.; Malhotra, S.; Hardman, M.J.; Knowles, L.; Boot-Handford, R.P.; Shore, P.; Whitmarsh, A.; Dixon, M.J. IRF6 is a key determinant of the keratinocyte proliferation-differentiation switch. Nat. Genet. 2006, 38, 1329–1334.
[26]
Herron, B.J.; Liddell, R.A.; Parker, A.; Grant, S.; Kinne, J.; Fisher, J.K.; Siracusa, L.D. A mutation in stratifin is responsible for the repeated epilation (Er) phenotype in mice. Nat. Genet. 2005, 37, 1210–1212, doi:10.1038/ng1652.
[27]
Li, Q.; Lu, Q.; Estepa, G.; Verma, I.M. Identification of 14-3-3{sigma} mutation causing cutaneous abnormality in repeated-epilation mutant mouse. Proc. Natl. Acad. Sci. USA 2005, 102, 15977–15982.
[28]
Kondo, S.; Schutte, B.C.; Richardson, R.J.; Bjork, B.C.; Knight, A.S.; Watanabe, Y.; Howard, E.; de Lima, R.L.; Daack-Hirsch, S.; Sander, A.; et al. Mutations in IRF6 cause Van der Woude and popliteal pterygium syndromes. Nat. Genet. 2002, 32, 285–289, doi:10.1038/ng985.
[29]
Blackburn, J.; Ohazama, A.; Kawasaki, K.; Otsuka-Tanaka, Y.; Liu, B.; Honda, K.; Rountree, R.B.; Hu, Y.; Kawasaki, M.; Birchmeier, W.; et al. The role of Irf6 in tooth epithelial invagination. Dev. Biol. 2012, 365, 61–70, doi:10.1016/j.ydbio.2012.02.009.
[30]
Urano, T.; Saito, T.; Tsukui, T.; Fujita, M.; Hosoi, T.; Muramatsu, M.; Ouchi, Y.; Inoue, S. EFP targets 14-3-3σ for proteolysis and promotes breast tumour growth. Nature 2002, 417, 871–875, doi:10.1038/nature00826.
[31]
Zhu, F.; Xia, X.; Liu, B.; Shen, J.; Hu, Y.; Person, M.; Hu, Y. IKKalpha Shields 14-3-3sigma, a G(2)/M Cell Cycle Checkpoint Gene, from Hypermethylation, Preventing Its Silencing. Mol. Cell 2007, 27, 214–227, doi:10.1016/j.molcel.2007.05.042.
[32]
Hermeking, H.; Lengauer, C.; Polyak, K.; He, T.C.; Zhang, L.; Thiagalingam, S.; Kinzler, K.W.; Vogelstein, B. 14-3-3σ is a p53-regulated inhibitor of G2/M progression. Mol. Cell 1997, 1, 3–11, doi:10.1016/S1097-2765(00)80002-7.
[33]
Liu, B.; Zhu, F.; Xia, X.; Park, E.; Hu, Y. A tale of terminal differentiation: IKKalpha, the master keratinocyte regulator. Cell Cycle 2009, 8, 527–531.
[34]
Mills, A.A.; Zheng, B.; Wang, X.J.; Vogel, H.; Roop, D.R.; Bradley, A. p63 is a p53 homologue required for limb and epidermal morphogenesis. Nature 1999, 398, 708–713, doi:10.1038/19531.
[35]
Candi, E.; Terrinoni, A.; Rufini, A.; Chikh, A.; Lena, A.M.; Suzuki, Y.; Sayan, B.S.; Knight, R.A.; Melino, G. p63 is upstream of IKK alpha in epidermal development. J. Cell Sci. 2006, 119, 4617–4622, doi:10.1242/jcs.03265.
[36]
Koster, M.I.; Dai, D.; Marinari, B.; Sano, Y.; Costanzo, A.; Karin, M.; Roop, D.R. p63 induces key target genes required for epidermal morphogenesis. Proc. Natl. Acad. Sci. USA 2007, 104, 3255–3260.
[37]
Marinari, B.; Ballaro, C.; Koster, M.I.; Giustizieri, M.L.; Moretti, F.; Crosti, F.; Papoutsaki, M.; Karin, M.; Alema, S.; Chimenti, S.; et al. IKKalpha is a p63 transcriptional target involved in the pathogenesis of ectodermal dysplasias. J. Invest. Dermatol. 2009, 129, 60–69, doi:10.1038/jid.2008.202.
[38]
Liu, B.; Willette-Brown, J.; Liu, S.; Chen, X.; Fischer, S.M.; Hu, Y. IKKalpha represses a network of inflammation and proliferation pathways and elevates c-Myc antagonists and differentiation in a dose-dependent manner in the skin. Cell Death Differ. 2011, 18, 1854–1864, doi:10.1038/cdd.2011.56.
[39]
Lahtela, J.; Nousiainen, H.O.; Stefanovic, V.; Tallila, J.; Viskari, H.; Karikoski, R.; Gentile, M.; Saloranta, C.; Varilo, T.; Salonen, R.; et al. Mutant CHUK and severe fetal encasement malformation. N. Engl. J. Med. 2010, 363, 1631–1637, doi:10.1056/NEJMoa0911698.
[40]
Sil, A.K.; Maeda, S.; Sano, Y.; Roop, D.R.; Karin, M. IKKα acts in the epidermis to control skeletal and craniofacial morphogenesis. Nature 2004, 428, 660–664.
[41]
Park, E.; Zhu, F.; Liu, B.; Xia, X.; Shen, J.; Bustos, T.; Fischer, S.M.; Hu, Y. Reduction in IkappaB kinase alpha expression promotes the development of skin papillomas and carcinomas. Cancer Res. 2007, 67, 9158–9168, doi:10.1158/0008-5472.CAN-07-0590.
[42]
Quintanilla, M.; Brown, K.; Ramsden, M.; Balmain, A. Carcinogen-specific mutation and amplification of Ha-ras during mouse skin carcinogenesis. Nature 1986, 322, 78–80, doi:10.1038/322078a0.
Ziegler, A.; Jonason, A.S.; Leffell, D.J.; Simon, J.A.; Sharma, H.W.; Kimmelman, J.; Remington, L.; Jacks, T.; Brash, D.E. Sunburn and p53 in the onset of skin cancer. Nature 1994, 372, 773–776, doi:10.1038/372773a0.
[45]
Melnikova, V.O.; Ananthaswamy, H.N. Cellular and molecular events leading to the development of skin cancer. Mutat. Res. 2005, 571, 91–106, doi:10.1016/j.mrfmmm.2004.11.015.
[46]
Halliday, G.M. Inflammation, gene mutation and photoimmunosuppression in response to UVR-induced oxidative damage contributes to photocarcinogenesis. Mutat. Res. 2005, 571, 107–120, doi:10.1016/j.mrfmmm.2004.09.013.
[47]
Liu, B.; Park, E.; Zhu, F.; Bustos, T.; Liu, J.; Shen, J.; Fischer, S.M.; Hu, Y. A critical role for I{kappa}B kinase {alpha} in the development of human and mouse squamous cell carcinomas. Proc. Natl. Acad. Sci. USA 2006, 103, 17202–17207.
[48]
Jonason, A.S.; Kunala, S.; Price, G.J.; Restifo, R.J.; Spinelli, H.M.; Persing, J.A.; Leffell, D.J.; Tarone, R.E.; Brash, D.E. Frequent clones of p53-mutated keratinocytes in normal human skin. Proc. Natl. Acad. Sci. USA 1996, 93, 14025–14029.
[49]
Xia, X.; Park, E.; Liu, B.; Willette-Brown, J.; Gong, W.; Wang, J.; Mitchell, D.; Fischer, S.M.; Hu, Y. Reduction of IKKalpha expression promotes chronic ultraviolet B exposure-induced skin inflammation and carcinogenesis. Am. J. Pathol. 2010, 176, 2500–2508, doi:10.2353/ajpath.2010.091041.
[50]
Ouhtit, A.; Gorny, A.; Muller, H.K.; Hill, L.L.; Owen-Schaub, L.; Ananthaswamy, H.N. Loss of Fas-ligand expression in mouse keratinocytes during UV carcinogenesis. Am. J. Pathol. 2000, 157, 1975–1981, doi:10.1016/S0002-9440(10)64836-5.
[51]
Li, Q.; Sambandam, S.A.; Lu, H.J.; Thomson, A.; Kim, S.H.; Lu, H.; Xin, Y.; Lu, Q. 14-3-3sigma and p63 play opposing roles in epidermal tumorigenesis. Carcinogenesis 2011, 32, 1782–1788.
[52]
Restivo, G.; Nguyen, B.C.; Dziunycz, P.; Ristorcelli, E.; Ryan, R.J.; Ozuysal, O.Y.; di Piazza, M.; Radtke, F.; Dixon, M.J.; Hofbauer, G.F.; et al. IRF6 is a mediator of Notch pro-differentiation and tumour suppressive function in keratinocytes. EMBO J. 2011, 30, 4571–4585, doi:10.1038/emboj.2011.325.
[53]
Botti, E.; Spallone, G.; Moretti, F.; Marinari, B.; Pinetti, V.; Galanti, S.; de Meo, P.D.; de Nicola, F.; Ganci, F.; Castrignano, T.; et al. Developmental factor IRF6 exhibits tumor suppressor activity in squamous cell carcinomas. Proc. Natl. Acad. Sci. USA 2011, 108, 13710–13715.
[54]
Pasparakis, M.; Courtois, G.; Hafner, M.; Schmidt-Supprian, M.; Nenci, A.; Toksoy, A.; Krampert, M.; Goebeler, M.; Gillitzer, R.; Israel, A.; et al. TNF-mediated inflammatory skin disease in mice with epidermis-specific deletion of IKK2. Nature 2002, 417, 861–866, doi:10.1038/nature00820.
[55]
Nenci, A.; Huth, M.; Funteh, A.; Schmidt-Supprian, M.; Bloch, W.; Metzger, D.; Chambon, P.; Rajewsky, K.; Krieg, T.; Haase, I.; et al. Skin lesion development in a mouse model of incontinentia pigmenti is triggered by NEMO deficiency in epidermal keratinocytes and requires TNF signaling. Hum. Mol. Genet. 2006, 15, 531–542, doi:10.1093/hmg/ddi470.
[56]
Moore, R.J.; Owens, D.M.; Stamp, G.; Arnott, C.; Burke, F.; East, N.; Holdsworth, H.; Turner, L.; Rollins, B.; Pasparakis, M.; et al. Mice deficient in tumor necrosis factor-alpha are resistant to skin carcinogenesis. Nat. Med. 1999, 5, 828–831, doi:10.1038/10552.
[57]
Ramirez, A.; Page, A.; Gandarillas, A.; Zanet, J.; Pibre, S.; Vidal, M.; Tusell, L.; Genesca, A.; Whitaker, D.A.; Melton, D.W.; et al. A keratin K5Cre transgenic line appropriate for tissue-specific or generalized Cre-mediated recombination. Genesis 2004, 39, 52–57, doi:10.1002/gene.20025.
[58]
Hafner, M.; Wenk, J.; Nenci, A.; Pasparakis, M.; Scharffetter-Kochanek, K.; Smyth, N.; Peters, T.; Kess, D.; Holtkotter, O.; Shephard, P.; et al. Keratin 14 Cre transgenic mice authenticate keratin 14 as an oocyte-expressed protein. Genesis 2004, 38, 176–181, doi:10.1002/gene.20016.
[59]
Gareus, R.; Huth, M.; Breiden, B.; Nenci, A.; Rosch, N.; Haase, I.; Bloch, W.; Sandhoff, K.; Pasparakis, M. Normal epidermal differentiation but impaired skin-barrier formation upon keratinocyte-restricted IKK1 ablation. Nat. Cell Biol. 2007, 9, 461–469, doi:10.1038/ncb1560.
[60]
Zenz, R.; Eferl, R.; Scheinecker, C.; Redlich, K.; Smolen, J.; Schonthaler, H.B.; Kenner, L.; Tschachler, E.; Wagner, E.F. Activator protein 1 (Fos/Jun) functions in inflammatory bone and skin disease. Arthritis Res. Ther. 2008, 10, 201.
[61]
Yu, H.; Pardoll, D.; Jove, R. STATs in cancer inflammation and immunity: A leading role for STAT3. Nat. Rev. Cancer 2009, 9, 798–809, doi:10.1038/nrc2734.
[62]
Page, A.; Navarro, M.; Garin, M.; Perez, P.; Casanova, M.L.; Moreno, R.; Jorcano, J.L.; Cascallana, J.L.; Bravo, A.; Ramirez, A. IKKbeta leads to an inflammatory skin disease resembling interface dermatitis. J. Invest. Dermatol. 2010, 130, 1598–1610, doi:10.1038/jid.2010.28.
[63]
Alameda, J.P.; Moreno-Maldonado, R.; Fernandez-Acenero, M.J.; Navarro, M.; Page, A.; Jorcano, J.L.; Bravo, A.; Ramirez, A.; Casanova, M.L. Increased IKKalpha expression in the basal layer of the epidermis of transgenic mice enhances the malignant potential of skin tumors. PLoS One 2011, 6, e21984.
[64]
Senftleben, U.; Cao, Y.; Xiao, G.; Greten, F.R.; Krahn, G.; Bonizzi, G.; Chen, Y.; Hu, Y.; Fong, A.; Sun, S.C.; et al. Activation by IKKα of a second, evolutionary conserved, NF-κB signaling pathway. Science 2001, 293, 1495–1499, doi:10.1126/science.1062677.
[65]
Moreno-Maldonado, R.; Ramirez, A.; Navarro, M.; Fernandez-Acenero, M.J.; Villanueva, C.; Page, A.; Jorcano, J.L.; Bravo, A.; Llanos Casanova, M. IKKalpha enhances human keratinocyte differentiation and determines the histological variant of epidermal squamous cell carcinomas. Cell Cycle 2008, 7, 2021–2029, doi:10.4161/cc.7.13.6147.
[66]
Descargues, P.; Sil, A.K.; Karin, M. IKKalpha, a critical regulator of epidermal differentiation and a suppressor of skin cancer. EMBO J. 2008, 27, 2639–2647, doi:10.1038/emboj.2008.196.
