Human gingival mucosal keratinocytes exhibiting anchorage-independent growth express increased inducible nitric oxide synthase: regulation by MAP kinases.
Inducible nitric oxide synthase (iNOS) has been implicated in cancer formation because of its vast presence cancer tissues. Studies to support such a role during transformation of human cells are very limited. We have developed a cell culture system, which renders a more transformed epithelial phenotype. The model cells generated from immortalized human gingival mucosal (GM) keratinocytes are consisted of less transformed epithelial-like (EPI) cells and more transformed fibroblast-like (FIB) cells. The latter exhibit anchorage independent growth (AIG). Our data showed that iNOS at mRNA and protein levels was up-regulated in more transformed FIB cells in comparison with less transformed EPI cells. FIB cells at low passages (p<22) were unstable being able to morphologically and functionally revert back to EPI phenotype, while no reversion was observed in FIB cells at high passages (p>43). The morphological reversion of FIB cells was associated with the reversal of vimentin expression as well as AIG. More importantly, these revertants showed reduced levels of iNOS mRNA as well as MAP kinase ERK and phospho-ERK protein expression, while FIB cells without reversion maintained the expression. Furthermore, the MEK1/2 inhibitor U0126 could reduce detectable iNOS mRNA levels suggesting that MAP kinases were upstream regulators of iNOS transcription. U0126 caused both morphological and functional reversion of FIB cells indicating involvement of MAP kinases in these functions. Taken together, we provide evidence for an up-regulation of iNOS in cultured human keratinocytes which exhibit AIG. This up-regulation may reflect progressive transformation which still requires further changes to reach tumorigenic conversion.
Chunglok
Warangkana
W
Department of Internal Medicine IV, University Hospital Heidelberg, Im Neuenheimer Feld 410, 69120 Heidelberg, Germany.
Ittarat
Wanida
W
Tomakidi
Pascal
P
Schmidt
Rainer
R
Stremmel
Wolfgang
W
Chamulitrat
Walee
W
D002070:Butadienes / Q000494:pharmacology; D002455:Cell Division; D002478:Cells, Cultured; D003260:Contact Inhibition; D004847:Epithelial Cells / Q000166:cytology / Q000201:enzymology; D048049:Extracellular Signal-Regulated MAP Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D005347:Fibroblasts; D015971:Gene Expression Regulation, Enzymologic; D005881:Gingiva / Q000166:cytology* / Q000201:enzymology; D006801:Humans; D015603:Keratinocytes / Q000166:cytology* / Q000201:enzymology*; D009092:Mucous Membrane / Q000166:cytology* / Q000201:enzymology; D019001:Nitric Oxide Synthase / Q000235:genetics / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II; D009570:Nitriles / Q000494:pharmacology; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism
D002070:Butadienes; D009570:Nitriles; D012333:RNA, Messenger; C113580:U 0126; C496319:NOS2 protein, human; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; D048049:Extracellular Signal-Regulated MAP Kinases