Molecular Characterization of Transforming Growth Factor Type β3a

Abstract

Normal tissue homeostasis is controlled by a critical balance of positive and negative modulators. Chapter 2 gives an overview of the molecular aspects of growth control, in particular the role of growth factors and oncogene and anti-oncogene products. Uncontrolled growth of cancer cells may result from either an abrogation of growth stimulatory or a deficiency of growth inhibitory pathways. Mediators of growth inhibition include secretory polypeptide growth inhibitors, like transforming growth factor β(TGF-β) and nuclear proteins, like the retinoblastoma gene product. Early studies on organogenesis suggested the presence of growth inhibitors (challones) to regulate the growth of organs. Postulating that growth inhibitory proteins might have potential in cancer therapy, we began to analyze human tissues for the presence of novel tumor inhibitory factors. Purification of these activities and physico-chemical characterization suggested a relatedness to TGF-β. The biochemistry and cell biology of (TGF-β) will be reviewed in Chapter 3. At the start of the investigation, only one (TGF-β) had been identified. Our subsequent results indicated that a family of TGF-βproteins exists. Conventional purification of these TGF-βlike activities provided only limited quantities of material for analysis. We therefore adopted an alternative strategy which included the isolation of the cDNAs for TGF-β-like factors using TGF-β1 as a probe, assuming that related molecules might possess sufficient sequence similarity to cross-hybridize to a TGF-β1 probe. Differential hybridization of a Southern blot with human genomic DNA probed with TGF-β1 cDNA suggested the presence of a related gene, which we termed TGF-β3. The research described in this thesis includes the molecular cloning and expression of TGF-β3. Furthermore, experiments were carried out to gain insight into the effects of TGF-β3 on cell growth and differentiation and its mechanism of action, including initial studies to gauge the potential therapeutic uses of this factor. In Chapter 4, we report the cloning of the human TGF-β3 cDNA and the encoded TGF-β3 protein is compared with other members of the TGF-βfamily. In Chapter 5 the interspecies conservation of TGF-B3 is examined and the chromosomal location of the human TGF-β3 gone is determined. In Chapter 6, the recombinant expression and purification of TGF-β3 is described. The purified TGF-β3 protein has potent growth modulating effects on a number of normal as well as tumor cells. The studies in Chapter 7 were performed to assess the effect of TGF-β3 on osteoblasts and to characterize the specific binding of TGF-β3 to bone cells. TGF-β3 appears to be a potent regulator of functions associated with bone formation. Crosslinking studies showed that TGF-β3 and TGF-β1 associate in a similar fashion with three cell surface binding proteins, which have been characterized as putative receptor types I and II and a membrane-bound proteoglycan, termed betaglycan. The different (TGF-β) isoforms appear to have different potencies on Mv1Lu mink lung epithelia] and fetal bovine heart endothelial cells. In Chapter 8, we investigate the role of TGF-β. receptors and serum factors as determinants of the cell-specific responsiveness to the three homodimeric isoforms. The induction of mesoderm in Xenopus laevis animal cap explants by TGF-β3 is discussed in Chapter 9. Finally, in Chapter 10 we review the therapeutic applications of growth factors for wound healing.