CONTROL OF TGF-BETA RECEPTOR EXPRESSION IN BONE

Michael Centrella, Changhua Ji, Thomas L. McCarthy

Department of Surgery, Plastic Surgery Section, Yale University School of Medicine, 333 Cedar Street, PO Box 208041, New Haven, CT 06520-8041,

Received 12/1/97 Accepted 12/5/97

9. TGF-BETA RECEPTOR I PROMOTER

Consequently, variations in TGF-beta receptors can define the extent or the nature of TGF-beta activity for bone cells. Initial studies to understand how this occurs suggested a very short half-life of 2-6 h for cell surface TGF-beta receptor proteins in untreated bone cell cultures. In some instances, there is a rapid recovery after hormone or growth factor treatments. Measurements of transcript half-lives of 6-20 h for TGF-beta receptor mRNAs show reasonable turnover rates, and predict that constitutive expression is needed to maintain an adequate supply of cell surface TGF-beta receptor protein (73). Again, TGF-betaRI is essential for TGF-beta activity. Its mRNA half-life is short (6-7 h) by comparison to TGF-betaRII and betaglycan (17-20 h), and its expression varies in appropriate ways on bone cells with differentiation and in response to specific bone growth regulators. To examine transcriptional control of TGF-betaRI in better detail, its promoter was cloned in our lab from a rat genomic library (64). Consistent with the widespread importance of TGF-beta in most tissues, the organization of the TGF-betaRI promoter is like many constitutively expressed genes. The rat TGF-betaRI promoter is very similar to the human TGF-betaRI promoter that was cloned by others simultaneously (95). However, reporter constructs used to analyze the human TGF-betaRI promoter failed to include important upstream and downstream control elements that our lab established by sequence, reporter, gel shift, and mutation analyses. Deletions and point mutations showed that two downstream CCAAT boxes in the TGF-betaRI promoter do not contribute to its expression in osteoblasts. In contrast, the promoter contains multiple binding sequences for transcription factor Sp1, a condition that is often associated with constitutive gene expression (96,97). The many Sp1 binding sequences may contribute in part to multiple transcription start sites (64). Also, at least one Sp1 site is essential for basal TGF-betaRI promoter activity (96). Even so, and consistent with initial observations of TGF-betaRI mRNA and protein (21), the TGF-betaRI promoter is significantly more active in more differentiated bone cells (64). This predicted the presence of tissue specific cis-acting elements within the TGF-betaRI promoter itself, and specific trans-acting factors within osteoblast nuclei (96,98). Consistent with this possibility, there are four separate binding sequences for members of the CBFa transcription factor family within the maximally active 1.0 kb region of the TGF-betaRI promoter, and two others within the next 5' 0.1 kb (98).