c-Fos-driven transcriptional activation of transforming growth factor β-1: inhibition of high glucose-induced promoter activity by thiazolidinediones

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Abstract

The peroxisome proliferator-activated receptor γ activating compounds thiazolidinedione (TZD) have been shown to inhibit diabetes-induced glomerular transforming growth factor-β1 (TGF-β1) expression, thereby ameliorating diabetic nephropathy. Here we examined the hypothesis that TZDs block high glucose-induced TGF-β1 gene activation by interaction with the activated protein kinase C-c-Fos-TGF-β1 promoter cascade in mesangial cells. The TZD compounds troglitazone and rosiglitazone completely prevented the high glucose induction of both TGF-β1 promoter activity and elevation in nuclear c-Fos protein levels. The scavenging properties of troglitazone were shown not to be responsible for this inhibitory action, because hydrogen peroxide-mediated stimulation of TGF-β1 promoter activity was not blocked. TZD-treatment did not interfere with the transcriptional activity of c-Fos responsible for stimulation of the TGF-β1 promoter. The findings suggest a molecular mechanism by which TZD-treatment reduces specifically high glucose-induced, c-Fos-mediated gene activation, since phorbol ester-induced c-Fos mRNA and protein expression and subsequent elevation of TGF-β1 mRNA expression were not prevented by TZDs.

Section snippets

Methods

Materials. Troglitazone was kindly provided by Sankyo Pharmaceuticals (Tokyo, Japan). Rosiglitazone was a generous gift of Glaxo SmithKline (Muenchen, Germany). Mouse mesangial cell line SV40 MES [26] was obtained from ATCC (Manassas, USA). Oligonucleotides were synthesized by Life Technologies (Karlsruhe, Germany). Cell culture media, supplements, Ultroser, and fetal calf serum (FCS) were from Gibco (Eggenstein, Germany); Klenow enzyme and poly[d(I–C)] were from Boehringer (Mannheim, Germany);

Effect of TZDs on c-Fos-induced TGF-β1 promoter activation

First we studied the impact of the transcription factor c-Fos on TGF-β1 promoter activity. Transient transfection of SV40 MES, which we used to obtain higher transfection efficiencies and expression levels compared to transfection of porcine mesangial cells, with the c-Fos expression vector resulted in clearly elevated nuclear levels of c-Fos protein (Fig. 1B). In co-transfection experiments the overexpression of c-Fos induced the activity of the TGF-β1 promoter construct −453/+11 (Fig. 1A)

Discussion

We investigated the interaction of TZDs, namely troglitazone and rosiglitazone, with the c-Fos-driven transcriptional activation of the prosclerotic cytokine TGF-β1 in mesangial cells. First, we demonstrated that increases in nuclear c-Fos protein levels are sufficient to induce TGF-β1 promoter activity. Second, we found that treatment with TZDs did not reduce nuclear translocation or transcriptional activity of c-Fos or prevent phorbol ester-stimulated c-Fos expression and activity. Third, we

Acknowledgements

The work was supported by the Deutsche Forschungsgemeinschaft (Schl 239-7) to E.S. We gratefully acknowledge the kind support of Dr. H. Horikoshi (Sankyo Pharmaceuticals) and Dr. Seidel (Glaxo SmithKline) and we thank F. Machicao for critical discussion.

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    Abbreviations: AP-1, activating protein-1; DAG, diacylglycerol; PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate; PPARγ, peroxisome proliferator activator receptor; ROS, reactive oxygen species; TZD, thiazolidinedione; VSMCs, vascular smooth muscle cells.

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