Transforming growth factor beta mediates the angiotensin-II-induced stimulation of collagen type IV synthesis in cultured murine proximal tubular cells

Background. Angiotensin II (Ang II) stimulates synthesis of type IV collagen in a cultured murine proximal tubular cell line (MCT cells). In addition, Ang II also induces the expression of TGF-beta(1) in these cells. Since TGF-beta has well-known stimulatory effects on the transcription of various collagens, we tested whether the Ang-II-mediated stimulation of type IV collagen is due to induction of endogenous TGF-beta(1) synthesis in MCT cells. Results. A neutralizing monoclonal anti-TGF-beta(1-3) antibody abolished the Ang II-stimulated release of type IV collagen in culture supernatants. The anti-TGF-beta(1-3) antibody also partly blocked Ang-II-mediated incorporation of (3)[H]proline into de novo synthesized collagens. Moreover, 5 mu M TGF-beta(1) antisense oligonucleotides, but not the same concentration of sense oligonucleotides, completely blocked Ang-II-stimulated (3)[H]proline incorporation. MCT cells incubated with TGF-beta(1) antisense phosphorothioate-modified oligonucleotides failed to synthesize TGF-beta(1) protein after Ang II treatment as measured by a sandwich ELISA in culture supernatants. SDS-polyacrylamide electrophoresis of (3)[H] proline-labelled collagens and comparison with standard collagens also demonstrated that the neutralizing anti-TGF-beta(1-3) antibody abolished the Ang-II-mediated stimulation in type IV collagen. Semiquantitative cDNA amplification for collagen type alpha 1(IV) transcripts revealed that the anti-TGF-beta(1-3) antibody abrogates the increase in mRNA after Ang II treatment. Transient transfection studies in MCT cells using murine collagen alpha 1(IV) enhancer/promoter promoter constructs also demonstrated the suppressive effect of the neutralizing antibody on Ang-II-stimulated gene transcription. Conclusions. Our data collectively suggest that the Ang-II-mediated increase in type IV collagen in MCT cells is mediated by endogenous synthesis and autocrine action of TGF-beta(1). These findings may be important in changes of the tubulointerstitial architecture during the progression of renal disease.