Management of osteoporosis is limited by poor understanding of how bone mass is regulated. Osteoclasts are the acid-secreting cells that degrade bone. We found that Ca+2/calmodulin at the acid-secreting membrane of osteoclasts is required for HCI production. In contrast, inactivation of acid secretion is calmodulin independent, but is mediated by the cGMP- dependent enzymes function in a coordinated counter-regulatory processes that mediates the reversible phosphorylation of proteins in the acid secreting membrane to regulate osteoclastic activity. We will study this hypothesis using human osteoclasts in vitro.
Aim 1 will determine the role of local intracellular Ca+2 increases (calcium puffs or sparks) that occur near the acid-secreting cell membrane. H+- ATPase or acid secretion will be modified, and effects on intracellular calcium puffs will be measured using fluorescent indicators. The effects of calcium transport antagonists will be studied to identify the mechanism(s) that cause the calcium puffs, and the effects of calcium puffs on membrane protein phosphorylation will be determined.
Aim 2 will use adenovirus infection to modify osteoclastic expression of the cGMP- dependent protein kinase and calcineurin (the calmodulin-dependent phosphatase). Eliminating the kinase should promote acid transport and abolish cGMP effects in osteoclasts; constitutive kinase should inactivate acid secretion. In cells without calcineurin, phosphorylation of membrane regulatory proteins should increase, decreasing acid secretion.
Aim 3 will characterize the interaction of cGMP and Ca+2 signals in osteoclasts. How calmodulin-dependent cGMP phosphodiesterase activity varies with cGMP, acid secretion, and calcium puff activity will be assessed, using Western analysis and immune localization, to establish the role of this enzyme in balancing cGMP and Ca+2/calmodulin effects. Interactions of cGMP- and calmodulin-dependent kinases/phosphatases in the acid-secreting cell membrane will also be studied, using kinase, membrane activity, and immune localization assays. This will show whether Ca+2/calmodulin and cGMP counter- regulation occurs via common target proteins, and how distribution of regulatory proteins varies with cGMP and calcium signals. These studies will characterize an intracellular regulatory system that controls osteoclastic activity. This knowledge will be useful in understanding how osteoporosis develops, and may assist in the design of treatments to reduce bone resorption and prevent osteoporosis.
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