BACKGROUND: beta2 microglobulin (beta2m) amyloidosis is a destructive articular disease affecting dialysis patients. We have demonstrated that beta2m increases the expression of vascular cell adhesion molecule (VCAM-1) and cyclooxygenase-2 (COX-2) in human osteoarthritic synovial fibroblasts (SFLs). METHODS: To determine the cell signaling pathways, SFLs were incubated with beta2m in the presence or absence of various inhibitors for 24 hours. Intracellular calcium ([Ca2+]i) was measured by fluorometric techniques and vascular cell adhesion molecule-1 (VCAM-1) and cyclooxygenase-2 (COX-2) expression was determined by immunohistochemistry and Western blotting. RESULTS: beta2m increased [Ca2+]i levels in a dose dependent manner (P < 0.05) in SFLs. BAPTA-AM, a [Ca2+]i chelator, completely inhibited beta2m-induced expression of VCAM-1 and COX-2. U73122 [phospholipase C (PLC) inhibitor] or 2-APB [specific inhibitor of inositol 1,4,5-trisphosphate (IP3)-induced [Ca2+]i release] completely blocked the beta2m-induced increase in [Ca2+]i and the up-regulation of VCAM-1 and COX-2. However, pretreatment with staurosporin, a protein kinase C inhibitor, had no effect. Disruption of the actin cytoskeleton by treatment with cytochalasin D or latrunculin A blocked beta2m up-regulation of VCAM-1 and COX-2. Finally, cells treated with phosphatidylinositol-3 kinase (PI-3 kinase) inhibitors wortmannin or LY294002 also failed to express VCAM-1 and COX-2. CONCLUSIONS: These results demonstrate that IP3-mediated [Ca2+]i release, PI-3 kinase, and actin cytoskeleton reorganization are involved in beta2m-induced expression of VCAM-1 and COX-2 in human SFLs. Understanding the potential pathways by which beta2m exerts its inflammatory-like effects may lead to the development of future therapies.
BACKGROUND:beta2 microglobulin (beta2m) amyloidosis is a destructive articular disease affecting dialysis patients. We have demonstrated that beta2m increases the expression of vascular cell adhesion molecule (VCAM-1) and cyclooxygenase-2 (COX-2) in humanosteoarthritic synovial fibroblasts (SFLs). METHODS: To determine the cell signaling pathways, SFLs were incubated with beta2m in the presence or absence of various inhibitors for 24 hours. Intracellular calcium ([Ca2+]i) was measured by fluorometric techniques and vascular cell adhesion molecule-1 (VCAM-1) and cyclooxygenase-2 (COX-2) expression was determined by immunohistochemistry and Western blotting. RESULTS:beta2m increased [Ca2+]i levels in a dose dependent manner (P < 0.05) in SFLs. BAPTA-AM, a [Ca2+]i chelator, completely inhibited beta2m-induced expression of VCAM-1 and COX-2. U73122 [phospholipase C (PLC) inhibitor] or 2-APB [specific inhibitor of inositol 1,4,5-trisphosphate (IP3)-induced [Ca2+]i release] completely blocked the beta2m-induced increase in [Ca2+]i and the up-regulation of VCAM-1 and COX-2. However, pretreatment with staurosporin, a protein kinase C inhibitor, had no effect. Disruption of the actin cytoskeleton by treatment with cytochalasin D or latrunculin A blocked beta2m up-regulation of VCAM-1 and COX-2. Finally, cells treated with phosphatidylinositol-3 kinase (PI-3 kinase) inhibitors wortmannin or LY294002 also failed to express VCAM-1 and COX-2. CONCLUSIONS: These results demonstrate that IP3-mediated [Ca2+]i release, PI-3 kinase, and actin cytoskeleton reorganization are involved in beta2m-induced expression of VCAM-1 and COX-2 in human SFLs. Understanding the potential pathways by which beta2m exerts its inflammatory-like effects may lead to the development of future therapies.