Previous studies by the investigative group have provided evidence for a cation-sensing mechanism in osteoblasts that regulates osteoblast-mediated bone formation. Initial in vivo investigations of aluminum-induced neo- osteogenesis led to the discovery that cations stimulate de novo bone formation through activation of osteoblastic precursors residing in the bone marrow. More recently, in vitro investigations in the prototypic MC3T3-E1 osteoblast model indicate that aluminum and other polyvalent cations directly stimulate pre-osteoblast proliferation through G-protein coupled receptor activation and protein kinase C (PKC)-dependent signaling pathways. The cation specificity and relative affinities for osteoblast stimulation resembles that of the recently cloned parathyroid calcium receptor (PCaR1). However, definite knowledge of a similar cation sensing receptor in osteoblasts is lacking. The investigators indicate that they have successfully employed a PCaR1 homology-based polymerase chain reaction screening strategy to isolate and clone, from osteoblasts, a novel and putative G-protein coupled receptor, ObCaRs (for Osteoblast Cation-sensing Receptor-related Sequence), that is highly expressed in MC3T3-E1 osteoblasts. ObCaRs is highly homologous to PCaR1, but is a separate, closely-related gene located on a different chromosome. In the proposed investigations, plans are advanced to continue studies of the mechanisms by which aluminum and other cations stimulate osteoblast function. Efforts will be directed towards advancing the hypothesis that a cation-sensing PCaR1-like receptor plays an important regulatory role in osteoblast-mediated bone formation. Specifically, the investigators will complete their functional characterization of the PCaR1-like cation-sensing mechanism in osteoblasts by evaluating the ability of cations and calcimimetics to stimulate the endogenous receptor in vitro, by examining cation ligand- receptor interactions in osteoblast membranes and by defining the cation receptor coupling to its intracellular effectors. In addition, they will screen osteoblast cDNA libraries to isolate the full length ObCaRs cDNA. Further studies will examine the tissue distribution and intraosseous expression of the ObCaRs using Northern and in situ hybridization approaches. To evaluate the cation-sensing properties of ObCaRs, the investigators will functionally express in host cells this putative cation-sensing receptor and use antisense cDNA to disrupt the expression of the endogenous ObCaRs receptor in MC3T3-E1 osteoblasts. It is suggested that information derived from these studies will provide vital data regarding the cation-sensing mechanism in osteoblasts. It is hoped that such kn owledge will permit a better understanding of physiologic and pharmacologic control of de novo bone formation by cations that may lead to new therapies for osteopenic disorders.
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