Cardiac fibroblasts are responsible for synthesis and deposition of fibrillar collagen types I and III. Transforming growth factor-beta-1 (TGF-beta-1) has been proved to increase collagen biosynthesis in various systems, both in vivo and in vitro. We have investigated the effect of TGF-beta-1 on collagen gene expression in cultured cardiac fibroblasts and have compared this effect with that of a mitogenic agent, phorbol myristate acetate (PMA). The regulation of collagen types I and III gene expression was examined by using cDNA probes to rat alpha-2 (I) and mouse alpha-1 (III) procollagens. Quiescent cultured cardiac fibroblasts from rabbit heart were treated with TGF-beta-1 (10-15 ng/ml) and PMA (200 ng/ml). After 24 hours of treatment with TGF-beta-1, the abundance of mRNA for pro-alpha-2 (I) and pro-alpha-1 (III) collagens was increased by 112% (p < 0.001) and 97% (p = 0.05), respectively, in treated fibroblasts compared with untreated cells. However, PMA-treated cells showed an opposite response: a 42% (p = 0.01) decrease in mRNA levels for pro-alpha-2 (I) collagen was observed. Immunofluorescent staining of cardiac fibroblasts in culture with anti-type I collagen antibody showed that alterations in mRNA levels led to altered collagen synthesis: cellular collagen was relatively increased in TGF-beta-1-treated cells and significantly diminished in PMA-treated cells. The abundance of mRNA for pro-alpha-1 (III) collagen was not affected by PMA treatment. Increased collagen gene expression by TGF-beta-1 was abolished in the presence of cycloheximide, whereas the inhibitory effect of PMA on collagen type I mRNA did not change after addition of cycloheximide to the culture medium. After 3 hours of treatment, only PMA led to the induction of mRNAs for early growth response gene (Egr-1) and proto-oncogenes c-fos and c-jun in cardiac fibroblasts. Using a rabbit anti-mouse Egr-1 antibody and immunofluorescent light microscopy, this protein was clearly localized to the nuclei after 4-5 hours of treatment with PMA. TGF-beta-1 did not induce the expression of Egr-1, c-fos, or c-jun in cardiac fibroblasts. [H-3]Thymidine incorporation into the cell nuclei was increased 1.5-fold (p < 0.001) in TGF-beta-1-treated cells and 1.8-fold (p < 0.001) in PMA-treated cells compared with untreated cells. These findings suggest that TGF-beta-1 in the myocardium may play an important role in the regulation of collagen biosynthesis and, hence, myocardial fibrosis. They may further imply that increase in collagen biosynthesis in the heart may be independent of the fibroblast response to immediate growth-enhancing events, such as induction of transcriptional regulator Egr-1 and proto-oncogenes c-fos and c-jun, that accompany cardiac myocyte hypertrophy.
