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Regulation of type II transforming-growth-factor-beta receptors by protein kinase C iota.

ABSTRACT: TGF-beta (transforming growth factor-beta) is implicated in the pathogenesis of diabetic nephropathy. We previously demonstrated that up-regulation of type II TGF-beta receptor (TbetaRII) induced by high glucose might contribute to distal tubular hypertrophy [Yang, Guh, Yang, Lai, Tsai, Hung, Chang and Chuang (1998) J. Am. Soc. Nephrol. 9, 182-193]. We have elucidated the mechanism by using cultured Madin-Darby canine kidney cells. Enhancer assay and electrophoretic-mobility-shift assay were used to estimate the involvement of transcription factors. Western blotting and an in vitro kinase assay were used to evaluate the level and activity of protein kinase. We showed that glucose (100-900 mg/dl) induced an increase in mRNA level and promoter activity of TbetaRII (note: 'mg/dl' are the units commonly used in diabetes studies). The promoter region -209 to -177 appeared to contribute to positive transactivation of TbetaRII promoter by comparing five TbetaRII-promoter-CAT (chloramphenicol acetyl-transferase) plasmids. Moreover, the transcription factor AP-1 (activator protein 1) was significantly activated and specifically binds to TbetaRII promoter (-209 to -177). More importantly, we found that atypical PKC iota might be pivotal for high glucose-induced increase in both AP-1 binding and TbetaRII promoter activity. First, high glucose induced cytosolic translocation, activation and autophosphorylation of PKC iota. Secondly, antisense PKC iota expression plasmids attenuated high-glucose-induced increase in AP-1 binding and TbetaRII promoter activity; moreover, sense PKC iota expression plasmids enhanced these instead. Finally, we showed that antisense PKC iota expression plasmids might partly attenuate a high-glucose/TGF-beta1-induced increase in fibronectin. We conclude that PKC iota might mediate high-glucose-induced increase in TbetaRII promoter activity. In addition, antisense PKC iota expression plasmid effectively suppressed up-regulation of TbetaRII and fibronectin in hyperglycaemic distal-tubule cells.

SUBMITTER: Chuang LY

PROVIDER: S-EPMC1223681 | biostudies-other | 2003 Oct

REPOSITORIES: biostudies-other

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Project description:BACKGROUND: Tumors expressing a transforming growth factor-beta type I receptor (T beta RI) mutant with sequence deletions in a nine-alanine (9A) stretch of the signal peptide are reported to be highly associated with disease progression. Expression of this mutant could interfere with endogenous TGFbeta signaling in the cell. However, little is known about the importance of the remaining part of the signal peptide on the cellular function of T beta RI. RESULTS: We cloned and identified four new in-frame deletion variants of T beta RI, designated DM1 to DM4, in pleural effusion-derived tumor cells. Intriguingly, DM1 and DM2, with a small region truncated in the putative signal peptide of T beta RI, had a serious defect in their protein expression compared with that of the wild-type receptor. Using serial deletion mutagenesis, we characterized a region encoded by nucleotides 16-51 as a key element controlling T beta RI protein expression. Consistently, both DM1 and DM2 have this peptide deleted. Experiments using cycloheximde and MG132 further confirmed its indispensable role for the protein stability of T beta RI. In contrast, truncation of the 9A-stretch itself or a region downstream to the stretch barely affected T beta RI expression. However, variants lacking a region C-terminal to the stretch completely lost their capability to conduct TGFbeta-induced transcriptional activation. Intriguingly, expression of DM3 in a cell sensitive to TGFbeta made it significantly refractory to TGFbeta-mediated growth inhibition. The effect of DM3 was to ablate the apoptotic event induced by TGFbeta. CONCLUSION: We identified four new transcript variants of T beta RI in malignant effusion tumor cells and characterized two key elements controlling its protein stability and transcriptional activation. Expression of one of variants bestowed cancer cells with a growth advantage in the presence of TGFbeta. These results highlight the potential roles of some naturally occurring T beta RI variants on the promotion of tumor malignancy.

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Regulation of type II transforming-growth-factor-beta receptors by protein kinase C iota.

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