The synthesis of type I collagen in osteoblastic cells is under the complex regulation of systemic and locally-produced hormones and growth factors and it is likely that other influences such as components of the extracellular matrix (ECM) are also involved in this regulation. The long-term goals of this project are investigate the regulation of alpha1(l) collagen gene expression in osteoblasts by parathyroid hormone (PTH) and insulin-like growth factors (IGFs) and to study the role of the ECM in modulating the activity of alpha1(l) collagen promoter in osteoblasts. For many of these studies, transgenic animals which harbor alpha1(l) collagen promoter DNA constructs will be used. Transgenic mice represent a unique model system in which to appreciate the hormonal regulation of collagen gene expression in a physiologic context. The inhibitory effect of PTH on alpha1(l) collagen promoter activity will be characterized in cultured calvariae from transgenic mice and the role of cyclic AMP, interleukin-6 and prostaglandins as possible mediators of this Inhibitory action will be explored. PTH-responsive loci in the alpha1(l) collagen gene will be identified by deletion mapping using stably transfected osteoblastic ROS 17/2.8 cells. To examine protein-DNA Interactions, mobility shift assays and DNase footprinting using nuclear extracts from PTH-treated cells and oligodeoxynucleotides spanning the alpha1(l) collagen promoter region of interest will be performed. Once DNA loci are identified, these regions will be altered using oligonucleotide-directed mutagenesis. Selected mutant DNA sequences will be tested for functional activity in cultured osteoblastic cells and used to generate new transgenic mouse lines for in vivo validation. The effect of IGFs on alpha1(l) collagen promoter activity in cultured calvariae from transgenic animals will be determined and the possible autocrine/paracrine role of IGFs in regulating alpha1(I) collagen promoter activity will be explored using purified inhibitory insulin-like binding protein-2, IGF neutralizing antibodies and IGF oligodeoxynucleotides. A variety experimental systems will be used to study mechanisms for the downregulation of an alpha1(l) collagen promoter construct in primary cell cultures derived from transgenic mouse calvariae and to determine the role of the ECM in regulating promoter expression. Primary cell digests from transgenic mouse calvariae will be plated on ECM substrates and mixed calvarial cells digests will be cultured in medium which promotes bone nodule formation. These studies will help elucidate molecular mechanisms by which PTH, IGF-I and ECM modulate collagen gene expression thereby providing experimental findings which will be useful in planning rationale treatment protocols for metabolic diseases of bone.
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