Abstract
Introduction
A catalytic subunit of NADPH oxidase 1 (Nox1) is implicated to be involved in neoplastic progression in human epithelial cancers. We had previously demonstrated that Nox1 overexpression of immortalized epithelial cells was able to induce the generation of progenitor cells that expressed fetal-type cytokeratins 8 and 18.
Purpose
We aimed to investigate the direct effects and underlying mechanisms of Nox1 on expression of cytokeratin 18 (CK18).
Methods
Immortalized human epithelial GM16 cells with low CK18 were used in Nox1 overexpression experiments. NuB2 cells with high CK18 were used in Nox1 knockdown experiments. Protein expression of CK18, phosphorylated and ubiquitinated CK18 were analyzed by Western blot.
Results
With no effects on the mRNA levels, CK18 protein was increased upon Nox1 overexpression and decreased upon Nox1 knockdown. Treatment with proteasome inhibitor MG132 prevented CK18 degradation and increased CK18 protein indicating translational regulation of CK18. Treatment for NuB2 cells with N-acetyl-l-cysteine, diphenyleneiodonium, or apocynin decreased CK18 protein levels indicating its regulation involving reactive oxygen species and flavoprotein Nox. It has been known that phosphorylation of CK18 regulates CK18 turnover by ubiquination. Consistently, Nox1 modulated CK18 phosphorylation at ser52. Nox1 knockdown and treatment with diphenyleneiodonium accumulated the levels of ubiquinated CK18 enhancing degradation causing decreased CK18 protein.
Conclusion
We demonstrated that Nox1 was able to induce CK18 stabilization by inhibiting CK18 protein degradation in a phosphorylation-dependent manner. CK18 accumulation induced by Nox1 is consistent with the persistence of fetal-type CK18 protein in many epithelial carcinomas.
Similar content being viewed by others
Abbreviations
- CK:
-
Cytokeratin
- DPI:
-
Diphenyleneiodonium
- EGF:
-
Epidermal growth factor
- GM16:
-
Human gingival mucosal epithelial cells immortalized by HPV16
- HPV16:
-
Human papilloma virus type 16
- KGM:
-
Keratinocyte growth medium
- NAC:
-
N-acetyl-l-cysteine
- Nox1:
-
NADPH oxidase 1
- qPCR:
-
Quantitative real-time PCR
- ROS:
-
Reactive oxygen species
- siRNA:
-
Small interfering RNA
References
Arbiser JL, Petros J, Klafter R, Govindajaran B, McLaughlin ER, Brown LF, Cohen C, Moses M, Kilroy S, Arnold RS, Lambeth JD (2002) Reactive oxygen generated by Nox1 triggers the angiogenic switch. Proc Nat Acad Sci USA 99:715–720
Arnold RS, He J, Remo A, Ritsick D, Yin-Goen Q, Lambeth JD, Datta MW, Young AN, Petros JA (2007) Nox1 expression determines cellular reactive oxygen and modulates c-fos-induced growth factor, interleukin-8, and Cav-1. Am J Pathol 171:2021–2032
Bánfi B, Maturana A, Jaconi S, Arnaudeau S, Laforge T, Sinha B, Ligeti E, Demaurex N, Krause KH (2000) A mammalian H+ channel generated through alternative splicing of the NADPH oxidase homolog NOH-1. Science 287:138–142
Busso N, Masur SK, Lazega D, Waxman S, Ossowski L (1994) Induction of cell migration by pro-urokinase binding to its receptor: possible mechanism for signal transduction in human epithelial cells. J Cell Biol 126:259–270
Byun HS, Won M, Park KA, Kim YR, Choi BL, Lee H, Hong JH, Piao L, Park J, Kim JM, Kweon GR, Kang SH, Han J, Hur GM (2008) Prevention of TNF-induced necrotic cell death by rottlerin through a Nox1 NADPH oxidase. Exp Mol Med 40:186–195
Chamulitrat W, Schmidt R, Tomakidi P, Stremmel W, Chunglok W, Kawahara T, Rokutan K (2003) Association of gp91phox homolog Nox1 with anchorage-independent growth and MAP kinase-activation of transformed human keratinocytes. Oncogene 22:6045–6053
Chamulitrat W, Stremmel W, Kawahara T, Rokutan K, Fujii H, Wingler K, Schmidt HH, Schmidt RA (2004) A constitutive NADPH oxidase-like system containing gp91phox homologs in human keratinocytes. J Invest Dermatol 122:1000–1009
Chamulitrat W, Huber A, Riedel HD, Stremmel W (2007) Nox1 induces differentiation resistance in immortalized human keratinocytes generating cells that express simple epithelial keratins. J Invest Dermatol 127:2171–2183
Chiera F, Meccia E, Degan P, Aquilina G, Pietraforte D, Minetti M, Lambeth D, Bignami M (2008) Overexpression of human NOX1 complex induces genome instability in mammalian cells. Free Radic Biol Med 44:332–342
Chu YW, Runyan RB, Oshima RG, Hendrix MJ (1993) Expression of complete keratin filaments in mouse L cells augments cell migration and invasion. Proc Natl Acad Sci USA 90:4261–4265
Fillies T, Werkmeister R, Packeisen J, Brandt B, Morin P, Weingart D, Joos U, Buerger H (2006) Cytokeratin 8/18 expression indicates a poor prognosis in squamous cell carcinomas of the oral cavity. BMC Cancer 6:10
Fukuyama M, Rokutan K, Sano T, Miyake H, Shimada M, Tashiro S (2005) Overexpression of a novel superoxide-producing enzyme, NADPH oxidase 1, in adenoma and well differentiated adenocarcinoma of the human colon. Cancer Lett 221:97–104
Geiszt M, Lekstrom K, Leto TL (2004) Analysis of mRNA transcripts from the NAD(P)H oxidase 1 (Nox1) gene. Evidence against production of the NADPH oxidase homolog-1 short (NOH-1S) transcript variant. J Biol Chem 279:51661–51668
Ii M, Yoshida H, Aramaki Y, Masuya H, Hada T, Terada M, Hatanaka M, Ichimori Y (1993) Improved enzyme immunoassay for human basic fibroblast growth factor using a new enhanced chemiluminescence system. Biochem Biophys Res Commun 193:540–545
Ku NO, Omary MB (1994) Identification of the major physiologic phosphorylation site of human keratin 18: potential kinases and a role in filament reorganization. J Cell Biol 127:161–171
Ku NO, Omary MB (2000) Keratins turn over by ubiquitination in a phosphorylation-modulated fashion. J Cell Biol 149:547–552
Ku NO, Omary MB (2001) Effect of mutation and phosphorylation of type I keratins on their caspase-mediated degradation. J Biol Chem 276:26792–26798
Kulesh DA, Ceceña G, Darmon YM, Vasseur M, Oshima RG (1989) Posttranslational regulation of keratins: degradation of mouse and human keratins 18 and 8. Mol Cell Biol 9:1553–1565
Lambeth JD, Kawahara T, Diebold B (2007) Regulation of Nox and Duox enzymatic activity and expression. Free Radic Biol Med 43:319–331
Laurent E, McCoy JW III, Macina RA, Liu W, Cheng G, Robine S, Papkoff J, Lambeth JD (2008) Nox1 is over-expressed in human colon cancers and correlates with activating mutations in K-Ras. Int J Cancer 123:100–107
Liao J, Lowthert LA, Ku NO, Fernandez R, Omary MB (1995) Dynamics of human keratin 18 phosphorylation: polarized distribution of phosphorylated keratins in simple epithelial tissues. J Cell Biol 131:1291–1301
Lim SD, Sun C, Lambeth JD, Marshall F, Amin M, Chung L, Petros JA, Arnold RS (2005) Increased Nox1 and hydrogen peroxide in prostate cancer. Prostate 62:200–207
Marceau N, Loranger A, Gilbert S, Daigle N, Champetier S (2001) Keratin-mediated resistance to stress and apoptosis in simple epithelial cells in relation to health and disease. Biochem Cell Biol 79:543–555
Mitsushita J, Lambeth JD, Kamata T (2004) The superoxide-generating oxidase Nox1 is functionally required for Ras oncogene transformation. Cancer Res 64:3580–3585
Oshima RG, Baribault H, Caulín C (1996) Oncogenic regulation and function of keratins 8 and 18. Cancer Metastasis Rev 15:445–471
Pankov R, Umezawa A, Maki R, Der CJ, Hauser CA, Oshima RG (1994) Oncogene activation of human keratin 18 transcription via the Ras signal transduction pathway. Proc Natl Acad Sci USA 91:873–877
Park HS, Lee SH, Park D, Lee JS, Ryu SH, Lee WJ, Rhee SG, Bae YS (2004) Sequential activation of phosphatidylinositol 3-kinase, beta Pix, Rac1, and Nox1 in growth factor-induced production of H2O2. Mol Cell Biol 24:4384–4394
Pei XF, Leigh IM, Watt FM (1992) Changes in type I keratin expression associated with HPV16 transformation of human epidermal keratinocytes. Epithelial Cell Biol 1:84–89
Perner A, Andresen L, Pedersen G, Rask-Madsen J (2003) Superoxide production and expression of NAD(P)H oxidases by transformed and primary human colonic epithelial cells. Gut 52:231–236
Puca R, Nardinocchi L, Starace G, Rechavi G, Sacchi A, Givol D, D’Orazi G (2010) Nox1 is involved in p53 deacetylation and suppression of its transcriptional activity and apoptosis. Free Radic Biol Med 48:1338–1346
Ranjan P, Anathy V, Burch PM, Weirather K, Lambeth JD, Heintz NH (2006) Redox-dependent expression of cyclin D1 and cell proliferation by Nox1 in mouse lung epithelial cells. Antioxid Redox Signal 8:1447–1459
Raul U, Sawant S, Dange P, Kalraiya R, Ingle A, Vaidya M (2004) Implications of cytokeratin 8/18 filament formation in stratified epithelial cells: induction of transformed phenotype. Int J Cancer 111:662–668
Sancho P, Bertran E, Caja L, Carmona-Cuenca I, Murillo MM, Fabregat I (2009) The inhibition of the epidermal growth factor (EGF) pathway enhances TGF-beta-induced apoptosis in rat hepatoma cells through inducing oxidative stress coincident with a change in the expression pattern of the NADPH oxidases (NOX) isoforms. Biochim Biophys Acta 1793:253–263
Schaafsma HE, Ramaekers FC, van Muijen GN, Lane EB, Leigh IM, Robben H, Huijsmans A, Ooms EC, Ruiter DJ (1990) Distribution of cytokeratin polypeptides in human transitional cell carcinomas, with special emphasis on changing expression patterns during tumor progression. Am J Pathol 136:329–343
Shinohara M, Shang WH, Kubodera M, Harada S, Mitsushita J, Kato M, Miyazaki H, Sumimoto H, Kamata T (2007) Nox1 redox signaling mediates oncogenic Ras-induced disruption of stress fibers and focal adhesions by down-regulating Rho. J Biol Chem 282:17640–17648
Shinohara M, Adachi Y, Mitsushita J, Kuwabara M, Nagasawa A, Harada S, Furuta S, Zhang Y, Seheli K, Miyazaki H, Kamata T (2010) Reactive oxygen generated by NADPH oxidase 1 (Nox1) contributes to cell invasion by regulating matrix metalloprotease-9 production and cell migration. J Biol Chem 285:4481–4498
Suh YA, Arnold RS, Lassegue B, Shi JX, Sorescu D, Chung A, Griendling KK, Lambeth JD (1999) Cell transformation by the superoxide-generating oxidase Mox1. Nature 40:79–82
Yamaura M, Mitsushita J, Furuta S, Kiniwa Y, Ashida A, Goto Y, Shang WH, Kubodera M, Kato M, Takata M, Saida T, Kamata T (2009) NADPH oxidase 4 contributes to transformation phenotype of melanoma cells by regulating G2-M cell cycle progression. Cancer Res 69:2647–2654
Acknowledgments
This study was supported by Deustche Forschungsgemeinschaft to WC (CH 288/3-3), and Thailand Research Fund/Deutscher Akademischer Austausch Dienst for PhD studentship of AS.
Conflict of interest
There is no conflict of interests to declare.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Sattayakhom, A., Ittiwat, W., Stremmel, W. et al. Redox regulation of cytokeratin 18 protein by NADPH oxidase 1 in preneoplastic human epithelial cells. J Cancer Res Clin Oncol 137, 1669–1678 (2011). https://doi.org/10.1007/s00432-011-1041-x
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00432-011-1041-x