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• W1999560320 abstract "Both the diversity and the precisely regulated tissue- and differentiation-specific expression patterns of keratins suggest that these proteins have specific functions in epithelia besides their well known maintenance of cell integrity. In the search for these specific functions, our previous results have demonstrated that the expression of K10, a keratin expressed in postmitotic suprabasal cells of the epidermis, prevents cell proliferation through the inhibition of Akt kinase activity. Given the roles of Akt in NF-κB signaling and the importance of these processes in the epidermis, a study was made into the possible alterations of the NF-κB pathway in transgenic mice expressing K10 in the proliferative basal layer. It was found that the inhibition of Akt, mediated by K10 expression, leads to impaired NF-κB activity. This appears to occur through the decreased expression of IKKβ and IKKγ. Remarkably, increased production of tumor necrosis factor α and concomitant JNK activation was observed in the epidermis of these transgenic mice. These results confirm that keratin K10 functions in vivo include the control of many aspects of epithelial physiology, which affect the cells not only in a cell autonomous manner but also influence tissue homeostasis. Both the diversity and the precisely regulated tissue- and differentiation-specific expression patterns of keratins suggest that these proteins have specific functions in epithelia besides their well known maintenance of cell integrity. In the search for these specific functions, our previous results have demonstrated that the expression of K10, a keratin expressed in postmitotic suprabasal cells of the epidermis, prevents cell proliferation through the inhibition of Akt kinase activity. Given the roles of Akt in NF-κB signaling and the importance of these processes in the epidermis, a study was made into the possible alterations of the NF-κB pathway in transgenic mice expressing K10 in the proliferative basal layer. It was found that the inhibition of Akt, mediated by K10 expression, leads to impaired NF-κB activity. This appears to occur through the decreased expression of IKKβ and IKKγ. Remarkably, increased production of tumor necrosis factor α and concomitant JNK activation was observed in the epidermis of these transgenic mice. These results confirm that keratin K10 functions in vivo include the control of many aspects of epithelial physiology, which affect the cells not only in a cell autonomous manner but also influence tissue homeostasis. IκB kinase tumor necrosis factor a c-Jun terminal kinase interleukin-6 phosphate-buffered saline enzyme-linked immunosorbent assay wild type c-Jun NH2-terminal kinase TNF receptor-associated factor 2 electrophoretic mobility shift analysis glutathione S-transferase bromodeoxyuridine receptor-interacting protein tumor necrosis factor-associated death domain The keratins are the main components of the intermediate filament cytoskeleton in epithelial cells. The functions of these proteins were clarified when human epithelial fragility syndromes were attributed to mutations of epidermal keratin genes (for reviews see Refs. 1Fuchs E.V. Weber K. Annu. Rev. Biochem. 1994; 63: 345-382Crossref PubMed Scopus (1284) Google Scholar, 2McLean W.H.I. Lane E.B. Curr. Opin. Cell Biol. 1995; 7: 118-125Crossref PubMed Scopus (219) Google Scholar, 3Fuchs E.V. Mol. Biol. Cell. 1997; 69: 899-902Google Scholar, 4Irvine A.D. McLean W.H.I. Br. J. Dermatol. 1999; 140: 815-828Crossref PubMed Scopus (329) Google Scholar). However, this shared function does not clearly explain the great diversity of these proteins, which suggests they may have additional family member-specific functions. In the search for possible specific keratin functions, a study was made of keratin K10. This protein is expressed in postmitotic differentiating keratinocytes in epidermis in vivo (5Fuchs E.V. Green H. Cell. 1980; 19: 1033-1042Abstract Full Text PDF PubMed Scopus (822) Google Scholar), and its expression is severely reduced in hyperproliferative situations, including skin tumors (6Toftgard R. Yuspa S.H. Roop D.R. Cancer Res. 1985; 45: 5845-5850PubMed Google Scholar, 7Roop D.R. Krieg T.M. Mehrel T. Cheng C.K. Yuspa S.H. Cancer Res. 1988; 48: 3245-3252PubMed Google Scholar). It has been shown that the expression of this keratin inhibits cell proliferation in cultured cells and in transgenic mice (8Paramio J.M. Casanova M.Ll. Segrelles C. Mittnacht S. Lane E.B. Jorcano J.L. Mol. Cell. Biol. 1999; 19: 3086-3094Crossref PubMed Scopus (148) Google Scholar, 9Paramio J.M. Segrelles C. Ruiz S. Jorcano J.L. Mol. Cell. Biol. 2001; 21: 7449-7459Crossref PubMed Scopus (106) Google Scholar, 10Santos M. Paramio J.M. Bravo A. Ramirez A. Jorcano J.L. J. Biol. Chem. 2002; 277: 19122-19130Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). The modulation of cell growth by keratin K10 is linked to the retinoblastoma (pRB) protein and the molecular machinery controlling cell cycle progression during G1, in particular cyclin D1 expression (8Paramio J.M. Casanova M.Ll. Segrelles C. Mittnacht S. Lane E.B. Jorcano J.L. Mol. Cell. Biol. 1999; 19: 3086-3094Crossref PubMed Scopus (148) Google Scholar, 9Paramio J.M. Segrelles C. Ruiz S. Jorcano J.L. Mol. Cell. Biol. 2001; 21: 7449-7459Crossref PubMed Scopus (106) Google Scholar). This activity appears to be promoted by the sequestration of Akt to the keratin cytoskeleton, mediated by keratin K10 through its amino terminus. This leads to decreased Akt kinase activity (9Paramio J.M. Segrelles C. Ruiz S. Jorcano J.L. Mol. Cell. Biol. 2001; 21: 7449-7459Crossref PubMed Scopus (106) Google Scholar). More recently these results have been amplified to include in vivo situations (10Santos M. Paramio J.M. Bravo A. Ramirez A. Jorcano J.L. J. Biol. Chem. 2002; 277: 19122-19130Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). Transgenic mice were generated in which human keratin K10 gene expression was targeted to the basal layer of the epidermis by using the bovine keratin K5 promoter (11Ramirez A. Bravo A. Jorcano J.L. Vidal M. Differentiation. 1994; 58: 53-64PubMed Google Scholar). These animals displayed severe alterations in the epidermis, including decreased proliferation, which results in hypoplastic epidermis, associated with impaired activation of Akt kinase activity in the skin. Finally, by using chemical skin carcinogenesis protocols, it was demonstrated that K10 expression reduces the formation of tumorsin vivo (10Santos M. Paramio J.M. Bravo A. Ramirez A. Jorcano J.L. J. Biol. Chem. 2002; 277: 19122-19130Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). These results are in agreement with the recently described fundamental roles of Akt kinase in mouse skin carcinogenesis (12Segrelles C. Ruiz S. Perez P. Murga C. Santos M. Budunova I.V. Martinez J. Larcher F. Slaga T.J. Gutkind J.S. Jorcano J.L. Paramio J.M. Oncogene. 2002; 21: 53-64Crossref PubMed Scopus (159) Google Scholar). Akt is a serine/threonine kinase that plays an important role not only in tumorigenesis but also in many aspects of cell physiology (reviewed in Refs. 13Datta S.R. Brunet A. Greenberg M.E. Genes Dev. 1999; 13: 2905-2927Crossref PubMed Scopus (3729) Google Scholar, 14Testa J.R. Bellacosa A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 10983-10985Crossref PubMed Scopus (774) Google Scholar, 15Scheid M.P. Woodgett J.R. Nat. Rev. Mol. Cell. Biol. 2001; 2: 760-768Crossref PubMed Scopus (540) Google Scholar, 16Lawlor M.A. Alessi D.R. J. Cell Sci. 2001; 114: 2903-2910Crossref PubMed Google Scholar). This kinase phosphorylates many cellular substrates involved in the control of cell proliferation, apoptosis, and metabolism. Its role in activating Akt in NF-κB signaling has recently been demonstrated, which may be an essential antiapoptotic event. However, the mechanism responsible for the activation of NF-κB by Akt remains uncertain and may depend on the cell type analyzed (17Factor V. Oliver A.L. Panta G.R. Thorgeirsson S.S. Sonenshein G.E. Arsura M. Hepatology. 2001; 34: 32-41Crossref PubMed Scopus (72) Google Scholar, 18Gustin J.A. Maehama T. Dixon J.E. Donner D.B. J. Biol. Chem. 2001; 276: 27740-27744Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 19Kane L.P. Shapiro V.S. Stokoe D. Weiss A. Curr. Biol. 1999; 9: 601-604Abstract Full Text Full Text PDF PubMed Scopus (742) Google Scholar). The canonical activation of NF-κB requires the phosphorylation of the inhibitory IκB protein by a high molecular weight complex that includes IKKα, IKKβ, and IKKγ1 proteins. This allows the NF-κB to move to the nucleus and activate target genes. In this context, it has been shown that Akt may increase the transactivation of the p65 subunit of NF-κB, without interfering with its nuclear localization (20Madrid L.V. Mayo M.W. Reuther J.Y. Baldwin Jr., A.S. J. Biol. Chem. 2001; 276: 18934-18940Abstract Full Text Full Text PDF PubMed Scopus (700) Google Scholar, 21Madrid L.V. Wang C.Y. Guttridge D.C. Schottelius A.J. Baldwin Jr., A.S. Mayo M.W. Mol. Cell. Biol. 2000; 20: 1626-1638Crossref PubMed Scopus (589) Google Scholar, 22Sizemore N. Leung S. Stark G.R. Mol. Cell. Biol. 1999; 19: 4798-4805Crossref PubMed Google Scholar). Similarly, the increased transactivation of p50 subunit by Akt phosphorylation has been reported (23Koul D. Yao Y. Abbruzzese J.L. Yung W.K. Reddy S.A. J. Biol. Chem. 2001; 276: 11402-11408Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). Furthermore, the phosphorylation of IKK proteins by Akt, leading to increased phosphorylation and thus degradation of IκB proteins, has been widely reported (24Romashkova J.A. Makarov S.S. Nature. 1999; 401: 86-90Crossref PubMed Scopus (1670) Google Scholar, 25Jones R.G. Parsons M. Bonnard M. Chan V.S. Yeh W.C. Woodgett J.R. Ohashi P.S. J. Exp. Med. 2000; 191: 1721-1734Crossref PubMed Scopus (286) Google Scholar, 26Ozes O.N. Mayo L.D. Gustin J.A. Pfeffer S.R. Pfeffer L.M. Donner D.B. Nature. 1999; 401: 82-85Crossref PubMed Scopus (1904) Google Scholar, 27Zhou B.P. Hu M.C. Miller S.A. Yu Z. Xia W. Lin S.Y. Hung M.C. J. Biol. Chem. 2000; 275: 8027-8031Abstract Full Text Full Text PDF PubMed Scopus (342) Google Scholar). Finally, in breast cancer lines, it has been suggested that Akt mediates the calpain degradation of IκB protein instead of the consensus ubiquitin pathway (28Pianetti S. Arsura M. Romieu-Mourez R. Coffey R.J. Sonenshein G.E. Oncogene. 2001; 20: 1287-1299Crossref PubMed Scopus (242) Google Scholar). Collectively, all these studies demonstrate the importance of Akt in activating NF-κB signaling, but also emphasize the controversy in identifying the molecular mechanisms responsible for such activation. The importance of NF-κB signaling in epidermis has been highlighted in recent years (reviewed in Ref. 29Kaufman C.K. Fuchs E. J. Cell Biol. 2000; 149: 999-1004Crossref PubMed Scopus (104) Google Scholar). This stems from findings obtained in transgenic mice ectopically expressing a non-degradable IκBα protein (IκBαM), or which overexpress p50 and/or p65 subunits in the epidermis (30Seitz C.S. Lin Q. Deng H. Khavari P.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2307-2312Crossref PubMed Scopus (382) Google Scholar). In these animals, increased NFκB activity leads to epidermal hypoplasia and growth inhibition, probably in association with p21waf/cip1 expression (30Seitz C.S. Lin Q. Deng H. Khavari P.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2307-2312Crossref PubMed Scopus (382) Google Scholar, 31Seitz C.S. Deng H. Hinata K. Lin Q. Khavari P.A. Cancer Res. 2000; 60: 4085-4092PubMed Google Scholar). The repression of NF-κB activity, on the other hand, produces epidermal hyperplasia (30Seitz C.S. Lin Q. Deng H. Khavari P.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2307-2312Crossref PubMed Scopus (382) Google Scholar) and may cause spontaneous squamous cell carcinomas in transgenic mice (32van Hogerlinden M. Rozell B.L. Ahrlund-Richter L. Toftgard R. Cancer Res. 1999; 59: 3299-3303PubMed Google Scholar). Nevertheless, we have observed increased endogenous NF-κB activity during chemically induced mouse skin tumorigenesis or upon UV treatment of mouse skin (33Budunova I.V. Perez P. Vaden V.R. Spiegelman V.S. Slaga T.J. Jorcano J.L. Oncogene. 1999; 18: 7423-7431Crossref PubMed Scopus (112) Google Scholar, 34Perez P. Page A. Jorcano J.L. Mol. Carcinog. 2000; 27: 272-279Crossref PubMed Scopus (19) Google Scholar). This apparent discrepancy might be explained in that the overexpression of regulatory molecules used in earlier transgenic studies may differ from those involved in endogenous activity. In this regard, one has to take into account that dominant negative IκBα expression does not generate a true NF-κB null phenotype and that nuclear accumulation of Bcl-3 and its interaction with other NF-κB dimers is independent of inhibition by other IκB proteins and IKK stimulation. In agreement with this, neither tumors nor phenotypic alterations were observed in the absence of IκBM overexpression in epidermis (32van Hogerlinden M. Rozell B.L. Ahrlund-Richter L. Toftgard R. Cancer Res. 1999; 59: 3299-3303PubMed Google Scholar). This suggests that a threshold in the level of expression of this protein is required to produce the phenotype. More recently, using a knock inapproach, which does not produce massive overexpression, it has been shown that the repression of NF-κB promotes severe epidermal defects affecting hair follicle development and increased apoptosis but not increased proliferation or abnormalities in differentiation (35Schmidt-Ullrich R. Aebischer T. Hulsken J. Birchmeier W. Klemm U. Scheidereit C. Development. 2001; 128: 3843-3853PubMed Google Scholar). Finally, the epidermal-specific ablation of IKKβ, which is accompanied by a deficiency in NF-κB activation, results in a TNF-mediated inflammatory skin disease but does not led to hyperproliferation or impaired keratinocyte differentiation (36Pasparakis M. Courtois G. Hafner M. Schmidt-Supprian M. Nenci A. Oksoy A. Krampert M. Goebeler M. Gillitzer R. Israel A. Kireg T. Rajewsky K. Haase I. Nature. 2002; 417: 861-866Crossref PubMed Scopus (404) Google Scholar). Another line of information on the roles of NF-κB signaling in keratinocytes comes from the strong phenotypes observed in the epidermis of mice lacking the IKKγ or IKKα subunits of the IKK complex (37Makris C. Godfrey V.L. Krahn-Senftleben G. Takahashi T. Roberts J.L. Schwarz T. Feng L. Johnson R.S. Karin M. Mol. Cell. 2000; 5: 969-979Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar, 38Schmidt-Supprian M. Bloch W. Courtois G. Addicks K. Israel A. Rajewsky K. Pasparakis M. Mol. Cell. 2000; 5: 981-992Abstract Full Text Full Text PDF PubMed Scopus (374) Google Scholar, 39Takeda K. Takeuchi O. Tsujimura T. Itami S. Adachi O. Kawai T. Sanjo H. Yoshikawa K. Terada N. Akira S. Science. 1999; 284: 313-316Crossref PubMed Scopus (538) Google Scholar, 40Hu Y. Baud V. Delhase M. Zhang P. Deerinck T. Ellisman M. Johnson R. Karin M. Science. 1999; 284: 316-320Crossref PubMed Scopus (713) Google Scholar, 41Li Q. Lu Q. Hwang J.Y. Buscher D. Lee K.F. Izpisua-Belmonte J.C. Verma I.M. Genes Dev. 1999; 13: 1322-1328Crossref PubMed Scopus (418) Google Scholar). In the case of IKKα, the abnormalities are not only due to the possible alterations of NF-κB signaling but also to the decreased production of a yet-unidentified soluble factor capable of inducing keratinocyte differentiation (42Hu Y. Baud V. Oga T. Kim K.I. Yoshida K. Karin M. Nature. 2001; 410: 710-714Crossref PubMed Scopus (300) Google Scholar). Given the above importance of Akt modulation of NF-κB activity in a range of systems, and the importance of NF-κB in epidermal physiology, an investigation was made into the possible alterations in this pathway that might arise as a consequence of keratin K10 ectopic expression in the basal layer of the epidermis. A dramatic decrease was seen in NF-κB activity in both transgenic skin and cultured mouse keratinocytes. This decrease is attributed to the inhibition of IKK activity associated with decreased expression of IKKβ and IKKγ. Remarkably, the transgenic animals showed aberrant overproduction of TNFα, and concomitant increased JNK activity, which may account for some of the phenotypic characteristics observed in bK5hK10 transgenic mice. The plasmid bK5hK10 was used to generate transgenic mice in a (C57 BL/10 × BALB/c) F1 and (C57BL/10 × DBA/2) F1 genetic background as previously described (10Santos M. Paramio J.M. Bravo A. Ramirez A. Jorcano J.L. J. Biol. Chem. 2002; 277: 19122-19130Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). The presence of the transgene was analyzed by Southern blots. Homozygous and heterozygous mice were identified using a PhosphorImager scanner (Bio-Rad) as reported (10Santos M. Paramio J.M. Bravo A. Ramirez A. Jorcano J.L. J. Biol. Chem. 2002; 277: 19122-19130Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). Primary keratinocyte cultures were established isolating keratinocytes from newborn mice cultured in Eagle's minimal medium containing 8% Chelex-treated serum and 0.03 mm Ca2+ as previously described (43Hennings H. Michael D. Cheng C. Steinert P. Holbrook K. Yuspa S.H. Cell. 1980; 19: 245-254Abstract Full Text PDF PubMed Scopus (1507) Google Scholar). Dorsal skin samples and tumors were fixed either in formalin or ethanol and embedded in paraffin prior to sectioning. 4-μm sections were cut and stained with hematoxylin-eosin. Immunodetection of dermal inflammatory cells including T lymphocytes (anti-CD3e), granulocytes (anti-Ly-6G/Gr-1), and macrophages (anti-CD11b/Mac1) was performed in frozen sections using fluorescein isothiocyanate-labeled specific rat anti-mouse monoclonal antibodies (145-2C11, RB6–8C5, and M1/70 clones, respectively; Amersham Biosciences). Sections were also stained for the expression of K10 as previously described (10Santos M. Paramio J.M. Bravo A. Ramirez A. Jorcano J.L. J. Biol. Chem. 2002; 277: 19122-19130Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). The localization of TNFα and IL-6 was carried out in formalin-fixed, paraffin-embedded sections from transgenic and non-transgenic littermates using specific rabbit polyclonal antibodies purchased from Calbiochem and diluted 1/100, followed by horseradish-peroxidase-labeled anti-rabbit antibody (Jackson ImmunoResearch Laboratory, diluted 1:2000). Positive staining was determined using diaminobenzidine as a substrate (diaminobenzidine kit, Vector, Burlingame, CA) following the manufacturer's recommendations. Sections were then counterstained with hematoxylin and mounted. For ultrastructural analysis, skin samples were fixed in 2.5% glutaraldehyde in PBS and postfixed in 1% osmium tetroxide prior to dehydration and embedding in Epon 812 resin. Ultrathin sections were stained with uranyl acetate and lead citrate. The 8xκB-Tk-CAT plasmid reporter gene used for transfection assays contained eight copies of the Igκ enhancer NF-κB binding site. The expression vectors for IKKα, IKKβ, and IKKγ containing the open reading frame of the indicated genes, and cloned in pCDNA3 under the cytomegalovirus promoter/enhancer were a generous gift of Dr. A. Israël. The plasmid coding for hK10 has been described previously (8Paramio J.M. Casanova M.Ll. Segrelles C. Mittnacht S. Lane E.B. Jorcano J.L. Mol. Cell. Biol. 1999; 19: 3086-3094Crossref PubMed Scopus (148) Google Scholar, 9Paramio J.M. Segrelles C. Ruiz S. Jorcano J.L. Mol. Cell. Biol. 2001; 21: 7449-7459Crossref PubMed Scopus (106) Google Scholar). Plasmids coding for wt and dominant-negative Akt were a generous gift of Dr. J. S. Gutkind. Chloramphenicol acetyltransferase assays were performed using an ELISA kit (Roche Molecular Biochemicals) following the manufacturer's instructions. Retrovirus coding for wt Akt or dominant negative Akt were generated by subcloning the corresponding cDNAs into pStMCS vector (kindly provided by Dr. J. C. Segovia, CIEMAT). These plasmids were transfected into Nxe cells and the supernatants were collected 48 h after transfection. For infections, exponentially growing PB keratinocytes were cultured in the presence of retroviral supernatants for 3 h, and afterward fresh medium was added and the cultures were further incubated overnight. Protein extracts were collected after 48 h and used in Western blotting analyses as commented below. The antibodies used included rabbit polyclonal antibodies to p50 (sc-114), p65/RelA (sc-372), IκBα (sc-371), IKKα (sc-7182), IKKγ (sc-8032) (Santa Cruz Biotechnology, Santa Cruz, CA), and IKKβ (Imgenex IMG-19). Antibodies against JNK, RIP, TRAF2, and TRADD were obtained from Transduction Laboratories. Antibodies against phosphorylated ATF2 and JNK were purchased from Cell Signaling. An anti-rabbit immunoglobulin G and anti-mouse peroxidase-conjugated (Amersham Biosciences, Aylesbury, UK) were used for immunoblotting. IL-1α, was purchased from Endogen and IL-1β and TNFα from Sigma. All cytokines were used at 10 ng/ml. Protein extracts (20 μg) were boiled in Laemmli buffer and separated on 10% SDS-PAGE and transferred to nitrocellulose filters (Hybond ECL, Amersham Biosciences, Aylesbury, UK). Filters were blocked with 5% nonfat dry milk in PBS/0.1% Tween 20 at 4 °C overnight, washed three times in PBS/0.1% Tween 20, and incubated with the indicated antibodies. After washing, membranes were incubated with a peroxidase-conjugated secondary antibody, washed again, and analyzed using the enhanced chemiluminescence method (West Picosignal, Pierce), according to the manufacturer's instructions. Membranes were stripped by incubation with 62.5 mm Tris-HCl (pH 6.7)/2% SDS/100 mm β-mercaptoethanol at 55 °C for 30 min and reprobed with different antibodies. No cross-reactivity with other NF-κB/IκB/IKK proteins was detected. Electrophoretic mobility shift assays (EMSA) were performed by incubating whole cell extracts from mouse skin with a labeled oligonucleotide corresponding to a palindromic κB site as previously described (44Perez P. Lira S.A. Bravo R. Mol. Cell. Biol. 1995; 15: 3523-3530Crossref PubMed Google Scholar). The sequence of the κB oligonucleotide coding strand was: 5′-GATCCAACGGCAGGGGAATTCCCCTCTCCTTA-3′ (44Perez P. Lira S.A. Bravo R. Mol. Cell. Biol. 1995; 15: 3523-3530Crossref PubMed Google Scholar). Complexes were separated on 5.5% native polyacrylamide gels in 0.25× Tris borate-EDTA buffer, dried, and exposed to Hyperfilm-MP (Amersham Biosciences) at −70 °C. The composition of the κB complexes in newborn mouse skin has been previously described (34Perez P. Page A. Jorcano J.L. Mol. Carcinog. 2000; 27: 272-279Crossref PubMed Scopus (19) Google Scholar). Whole skin extracts from newborn mice were obtained in buffer A (1% Triton X-100, 10% glycerol, 137 mm NaCl, 20 mm Tris-HCl, pH 7.5, 1 μg/ml aprotinin and leupeptin, 1 mm phenylmethylsulfonyl fluoride, 20 mm NaF, 1 mm disodium pyrophosphate, and 1 mm Na3VO4). The kinase activity for Akt and IKK complexes was determined by immunoprecipitation of the endogenous kinase proteins using anti Akt1/2 antibody or a mixture of different IKKα antibodies (Santa Cruz Biotechnology). Histone H2B was used as a substrate for Akt, and full-length IκBα (Santa Cruz Biotechnology) for IKK in in vitro kinase assays (essentially as described in Refs. 12Segrelles C. Ruiz S. Perez P. Murga C. Santos M. Budunova I.V. Martinez J. Larcher F. Slaga T.J. Gutkind J.S. Jorcano J.L. Paramio J.M. Oncogene. 2002; 21: 53-64Crossref PubMed Scopus (159) Google Scholar and 45Tanaka M. Fuentes M.E. Yamaguchi K. Durnin M.H. Dalrymple S.A. Hardy K.L. Goeddel D.V. Immunity. 1999; 10: 421-429Abstract Full Text Full Text PDF PubMed Scopus (495) Google Scholar). Jun kinase assay was performed essentially as described (46Okubo Y. Blakesley V.A. Stannard B. Gutkind S. Le Roith D. J. Biol. Chem. 1998; 273: 25961-25966Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar) upon immunoprecipitation of JNK using GST-c-Jun (kindly provided by Dr. J. S. Gutkind) as substrate. Total RNA from freshly harvested mouse epidermis and frozen tumors was isolated by guanidine isothiocyanate-phenol-chloroform extraction. Northern blots containing total RNA (15 μg/lane) were probed for expression of TNFα, IL-6, and IL-1 employing DNA probes prepared by random primed reactions using the complete sequences. The membranes were also hybridized with a keratin K14 cDNA probe to verify that equal amounts of mRNA were loaded and transferred. The quantification of TNFα in mouse serum and in the culture supernatant from primary keratinocytes was performed using an ELISA kit (R&D) following the manufacturer's recommendations. Transgenic mice ectopically expressing K10 in the basal layer of the epidermis display severe epidermal abnormalities associated with decreased proliferation in close conjunction with Akt kinase activity inhibition (10Santos M. Paramio J.M. Bravo A. Ramirez A. Jorcano J.L. J. Biol. Chem. 2002; 277: 19122-19130Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). Given the importance of Akt in the activation of NF-κB (see the introduction), the present study was designed to investigate NF-κB binding activity in the epidermis of transgenic mice through electrophoretic mobility shift analysis using a κB-labeled oligonucleotide. In non-transgenic samples, two retarded complexes were observed (Fig.1A, lanes 1 and 2) that were identified as p50-p50 homodimers and p50-p65 heterodimers by supershift experiments using specific antibodies (not shown; see also Ref. 34Perez P. Page A. Jorcano J.L. Mol. Carcinog. 2000; 27: 272-279Crossref PubMed Scopus (19) Google Scholar). Severely decreased DNA binding activity was observed in heterozygous transgenic mice (Fig. 1A,lanes 3 and 4). The κB binding activity in homozygous animals was barely detectable (Fig. 1A,lanes 5 and 6). Interestingly, the inhibition of Akt kinase activity runs in parallel with the increased K10 expression observed in heterozygous and homozygous transgenic mice (Fig.1A′) (not shown, see also Ref. 10Santos M. Paramio J.M. Bravo A. Ramirez A. Jorcano J.L. J. Biol. Chem. 2002; 277: 19122-19130Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). Given that we used whole skin extracts for these experiments, and to rule out the possibility that other cell types were actually responsible for the observed effect, similar analysis were performed using primary keratinocytes derived from wt and bK5hK10 transgenic mice. In addition, we also tested whether stimulation with IL-1α could induce increased DNA binding in these cells. IL-1α treatment led to increased NF-κB activity in non-transgenic keratinocytes but not in keratinocytes derived from transgenic animals (Fig. 1B). Western blot analysis against keratin K14 (Fig. 1B′) also demonstrated that this effect was not due to different amounts of keratinocyte protein in the assays. These data show that the expression of K10 leads to a dramatic reduction in NF-κB activity in keratinocytes and impedes the activation of this complex on stimulation. Experiments were then performed to see whether this decreased NF-κB activity was due to K10 expression and K10-mediated Akt inhibition. PB mouse keratinocytes were transfected with a NFκB reporter element (8xκBCAT) plus empty vector, K10, or K10 plus an expression vector for wt Akt (Fig. 1C). It has previously been shown that co-expression of wt Akt is sufficient to override K10-induced cell cycle arrest in keratinocytes (10Santos M. Paramio J.M. Bravo A. Ramirez A. Jorcano J.L. J. Biol. Chem. 2002; 277: 19122-19130Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). The expression of K10 produced a severe inhibition of NF-κB transcriptional activity induced either by IL-1β or TNFα (Fig. 1, C and C′, respectively), whereas co-expression of Akt almost completely abolished such inhibition (Fig. 1, C and C′). Finally, to determine whether the inhibition of Akt activity might be sufficient to account for the decreased NF-κB activity, the reporter plasmid was co-transfected with a dominant negative form of Akt, resulting in almost complete inhibition of NF-κB activity in response to TNFα or IL-1β (Fig. 1, C and C′). Together, these results demonstrate that K10-mediated Akt inhibition leads to impaired NF-κB basal activity and NF-κB activation in cultured keratinocytes and in epidermis in vivo. A study was then undertaken to determine whether the decreased NF-κB activity found in the transgenic mouse epidermis was due to altered expression of different NF-κB family members (Fig. 2A). No major alterations in p65 (RelA) or IκBα protein levels were detected by Western blotting (Fig. 2A), and a decrease in only p50 was observed in whole skin extracts from homozygous transgenic mice (Fig.2A). Because the NF-κB activity is dependent on the activity of the IKK complex, the expression of IKKα, IKKβ, and IKKγ was analyzed. Surprisingly, in homozygous transgenic mice there was a significant decrease in IKKβ and IKKγ levels (Fig.2A′). Because these two components of the IKK complex are essential for IκB phosphorylation and for NF-κB activation (47Li Z.W. Chu W. Hu Y. Delhase M. Deerinck T. Ellisman M. Johnson R. Karin M. J. Exp. Med. 1999; 189: 1839-1845Crossref PubMed Scopus (824) Google Scholar,48Rothwarf D.M. Zandi E. Natoli G. Karin M. Nature. 1998; 395: 297-300Crossref PubMed Scopus (853) Google Scholar), IκB kinase activity was evaluated in the transgenic mouse extracts (45Tanaka M. Fuentes M.E. Yamaguchi K. Durnin M.H. Dalrymple S.A. Hardy K.L. Goeddel D.V. Immunity. 1999; 10: 421-429Abstract Full Text Full Text PDF PubMed Scopus (495) Google Scholar). Almost complete inhibition of the IκB kinase activity was seen in homozygous mouse extracts (Fig. 2A“), and, surprisingly, a significant inhibition of" @default.
• W1999560320 created "2016-06-24" @default.
• W1999560320 creator A5000001579 @default.
• W1999560320 creator A5002672600 @default.
• W1999560320 creator A5026925465 @default.
• W1999560320 creator A5051333971 @default.
• W1999560320 creator A5064016844 @default.
• W1999560320 creator A5070674443 @default.
• W1999560320 date "2003-04-01" @default.
• W1999560320 modified "2023-10-03" @default.
• W1999560320 title "Impaired NF-κB Activation and Increased Production of Tumor Necrosis Factor α in Transgenic Mice Expressing Keratin K10 in the Basal Layer of the Epidermis" @default.
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