The purpose of this project is to characterize rodent and avian TGF-betas in terms of chemistry, biology, and molecular biology of each of the component members and to understand their mechanisms of expression. The emphasis has been on continuing our investigation of the expression of the TGF-beta isoforms in the rat and chicken using rat and chicken TGF-beta cDNA homologs that have been previously cloned. Rat TGF-beta1 cDNAs corresponding to the 2.4 and 1.9 Kb TGF-beta1 mRNAs have been cloned by polymerase chain reaction (PCR) amplification of reverse-transcribed mRNAs extracted from adult rat liver and adult rat heart following experimental myocardial infarction. respectively. The 1.9 Kb transcript level has been shown to be significantly higher in infarcted heart tissue than in normal heart tissue, suggesting an important role for this mRNA species in response to injury. In addition, cDNA probes and antibodies for TGF-betas 1, 2 and 3 were used to study expression of these different isoforms in normal liver and during rat liver regeneration following partial hepatectomy. Expression of all three TGF-beta mRNAs increased following hepatectomy, and all three isoforms were shown to inhibit DNA synthesis in cultured hepatocytes, suggesting that the different TGF-beta isoforms may function in an inhibitory mechanism that is activated following liver injury. In addition, cDNA probes and antibodies for TGF- betas 1, 2, 3 and 4 were used to study expression of the different TGF-beta isoforms in cultured chicken embryo chondrocytes and myocytes, as well as in developing cartilage and heart tissues and also in the developing chicken embryonic nervous system. RNA Northern blot analysis using TGF-beta cDNA probes and immunohistochemical staining using TGF-beta antibodies suggest that chicken TGF-betas 2, 3 and 4 mRNAs and proteins are co-expressed in chicken cartilage and heart. Similarly, RNA analysis and immunohistochemical staining has shown expression of TGF-betas 2, 3 and 4 in the chicken embryonic nervous system. In situ hybridization is being used to identify cell-specific localization of chicken TGF-beta mRNAs in the developing chicken nervous system.
