PLASMA MEMBRANE CALCIUM PUMP IN OSTEOBLASTS

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The overall objective of this grant is to isolate and characterize the plasma membrane calcium pump (Ca2+ -pump, Ca2+ -Pumping-Ca2+-Mg-ATPase) from human osteoblast-like cells and to examine its physiology in these cells. We hypothesize that the plasma membrane Ca2+-pump plays a vital role in the control of intracellular calcium within human osteoblasts. Several investigators have shown that intracellular Ca2+ concentration is important in determining the functional state of the osteoblast. Therefore, an understanding of the structure and physiology of this pump is of importance. Our specific aims are to: 1.Isolate and characterize the plasma membrane Ca 2+- pump from human osteoblast-like cells. These experiments will include the demonstration of Ca 2+ transport by the isolated plasma membrane Ca 2+_Mg2+ -ATPase in reconstitution studies. 2.Isolate cDNA clones for the human osteoblast plasma membrane Ca 2+- pump. 3.Determine whether the activity of the plasma membrane Ca 2+- pump is altered by exposure of human osteoblast-like cells to hormonal agents such as parathyroid hormone analogs, 1,25-dihydroxyvitamin [1,25(OH)2 D3] and other vitamin D metabolites. Both short-term and long-term experiments will be performed. Changes in Ca 2+- pump mRNA will be quantitated using Northern blot analysis. If changes are noted, nuclear run-off experiments will be performed. 4. Use a series of expression systems to assess the possible physiological role of the plasma membrane Ca 2+- pump in osteoblast-like cell lines. An understanding of the physiology and biochemistry of the osteoblast is central to our understanding of important diseases of bone. One such disease, of significant public health importance, is osteoporosis. Premenopausal and senile osteoporosis account for at least $10 billion of health care expenditures per year. It is clear from the studies of many laboratories, including our own, that abnormalities in various hormonal factors nd in calcium intake are important in the genesis of this disease. Our studies will add greatly to the understanding of osteoblast biology which in turn can be utilized to devise rational therapeutic principles for the treatment of osteoporosis.