Osteoporosis is a major cause of morbidity, most commonly in older women, and represents the end result of unbalanced bone turnover.Intracellular signals including Ca+2, cell attachment, and intercellular signals affect bone turnover, but coupling of these diverse elements to cellular activity is enigmatic. On the other hand, it is established that osteoclastic acid secretion plays a central role in bone turnover, but how osteoclast secretion is controlled is poorly understood. By studying changes in osteoclastic acid secretion as a function of cellular environment, the investigators have determined that cellular signals and the osteoclast ion transport mediating bone degradation are coupled through calmodulin. Further, previously undescribed and major changes in the calmodulin regulatory system and related intracellular calcium activity occur in osteoclasts as a result of cell-bone binding. Understanding how calmodulin controls the activity of bone-bound osteoclasts is the focus of this application. Preliminary studies show that the calmodulin in osteoclasts increases 10 fold with bone attachment, is concentrated at the acid secreting ruffled border, and modulates acid transport in isolated ruffled membrane vesicles. The principal investigator hypothesizes that calmodulin expression is induced by bone attachment and controls acid transport activity by regulating, via phosphorylation, HCI transport in concert with Ca+2i.
Specific aim 1 will study osteoclast-attachment induced polarized expression of the calmodulin as a function of time and substrate binding. A highly specific antibody, a cDNA probe, and intracellular calcium measurements will assay calmodulin concentration, subcellular localization, intracellular [Ca+2], and mRNA level in bone-bound and non-bone exposed cells. Measurements on cell fractions, calmodulin fluorescent-antibody localization by scanning confocal microscopy and colloidal gold antibody localization by electron microscopy will determine how calmodulin is distributed in osteoclasts and how this distribution varies in bone- degrading and non-bone attached cells.
Specific aim 2 will identify and characterize calmodulin binding proteins by affinity chromatography with [Ca+2] specific elution, and study calmodulin dependent kinase and phosphatase effects on acid transport using normal and calmodulin- depleted cell membranes, assaying ruffled membrane H+-ATPase and C1- channel activities. These results will tell what factors affect calmodulin accumulation, indicate relative concentration in subcellular components, and show how they are related to cellular activity.The proposed work will primarily use avian osteoclasts to characterize the osteoclast's calmodulin regulatory pathway because they are practically obtainable in requisite quantity for direct biochemical study. The highly conserved nature of bone turnover suggests that this pathway will be similar in humans. However, specific aim 3 will compare calmodulin- dependent processes in mammalian and avian osteoclasts. These studies will be performed using antibody probes, in situ hybridization, pit resorption assays, and fluorescent intracellular probes.
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