The embryogenesis and regeneration of the skeleton are complex developmental events dependent on the successful induction of endochondral osteogenesis. Little is known, however, about the genetic control of bone induction. Bone morphogenetic proteins (BMPs) are members of a highly conserved family of molecules involved in the regulation of embryonic pattern formation and osteogenesis. Recombinant human BMP-4 can induce the entire developmental program of endochondral osteogenesis in an ectopic site in a manner identical to that seen in the autosomal dominant genetic disorder, fibrodysplasia ossificans progressive (FOP). FOP is a progressively disabling condition characterized by congenital malformations of the toes and disordered temporal and spatial induction of endochondral osteogenesis at ectopic sites. BMP-4 messenger RNA and proteins uniquely over-expressed inlymphocytes from patients who have FOP. These data indicate that over expression of BMP-4, a potent bone-inducing morphogen, is associated with disabling ectopic osteogenesis in man. Tow related hypotheses provide the focus for our longterm goals; First, the molecular structure and function of the human BMP-4 gene provides fundamental insight into the genetic regulation of endochondral bone induction and pattern formation in humans; second, BMP-4 is the prime candidate gene for FOP; and the molecular structure and/or regulatory control of that gene is abnormal in patients who have FOP. To address these hypothesis, we intend to: 1. Define the regulatory regions of the human BMP-4 gene by reporter gene expression assays. 2. Identify DNA-binding proteins for the BMP-4 gene in normal and FOP cells by gel mobility shift and footprinting assays using nuclear protein extracts from FOP and non-FOP cells. 3. Examine the rate of BMP-4 transcript initiation and mRNA stability in FOP cells relative to control cells by nuclear run-on and transcription inhibition experiments. 4. Examine the expression of BMP type I and type II receptors in FOP cells by RT-PCR and immunohistochemistry. 5. Identify genetic markers that are closely linked to the BMP-4 gene locus and utilize these markers to establish or exclude genetic linkage of the BMP-4 gene with FOP. Analysis of the regulatory control of the human BPM-4 gene will foster that longterm goal of elucidating basic mechanisms of normal and disordered bone induction, and of designing rational molecular diagnostic and treatment strategies for a wide range of developmental disorders of the skeleton in humans.
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