MSU alters OB functions Mineralization MSU present in the culture medium of human OBs affects parameters implicated in bone mineralization, kinase inhibitor Tubacin such as alkaline phosphatase activity and osteocalcin content. To assess the mineralization function of OBs in the presence of MSU or vehicle in vitro, OB cultures were stained with alizarin red S, a marker of matrix calcium that allows a quantitative evaluation of mineralization. OBs incubated with MSU showed a reduced ARS staining of the newly Inhibitors,Modulators,Libraries calcified matrix. The quantities of ARS in cultures of MSU Inhibitors,Modulators,Libraries activated OBs were dose dependently de creased by 1. 6 and 2. 1 fold compared with those ob served in vehicle treated OBs. Moreover, the addition of MSU suppressed in a time dependent manner the expression of the mRNA of procollagen 1, a typical bone matrix constituent, with a sixfold decrease at 48 hours in the presence of 1 mg MSU.

These data indicate that MSU affects the formation of certain matrix Inhibitors,Modulators,Libraries components and in fine bone matrix mineralization. MMP activity Bone matrix degradation depends, among other factors, on enzymes such as matrix metalloproteinases that are known to be Inhibitors,Modulators,Libraries implicated in pathophysiological processes. Although bone matrix degradation is re lated mainly to osteoclasts, OBs can also be involved in bone resorption through their production of several MMPs. The activity of generic MMPs, as evaluated in supernatants of OBs cultured with MSU, was increased by 120% over that of unstimulated cells. These results indicate that MSU stimulated OBs may be directly implicated in matrix degradation of bone with MSU deposits.

Phagocytosis of MSU by OBs is tightly regulated Inhibitors,Modulators,Libraries Signaling pathways affected by MSU These data document profound effects of MSU on the behavior of OBs. These data indicate that the pathways regulating OB functions are likely to be affected by the presence of MSU. By using a protein kinase array that detects specific phosphorylation of 46 kinase phos phorylation sites, certain effector signaling proteins were investigated in MSU stimulated OBs. cytoskeletons. Therefore, the effects of cytochalasin D, an inhibitor of actin polymerization, and colchicine, an inhibitor of microtubule polymerization, were examined on MSU internalization by OBs. Cytochalasin D pretreatment abrogated the formation of vacuoles as sociated with MSU phagocytosis. In contrast, colchicine did not inhibit the appearance of vacuoles containing MSU.

Mechanisms underlying phagocytosis also impli cate several intracellular things signaling pathways that lead to cytoskeleton reorganization and ingestion of particles. From that point of view, pharmacological inhibitors can help decipher signaling pathways associated with MSU phagocytosis by OBs. The phosphoinositide 3 kinases that control cytoskeleton dynamics, signal trans duction, and membrane trafficking were targeted by two pan PI3K inhibitors, wortmannin and LY294002.