Synovial fluid basic calcium phosphate (BCP) crystals are markers of severe joint degeneration in osteoarthritis. BCP crystals cause mitogenesis of articular cells and stimulate matrix metalloprotease production, thus promoting degradation of articular tissues. Previous work suggested that BCP crystal-induced cell activation required intracellular crystal dissolution, induction of proto-oncogene expression, and activation of signal transduction pathways involving protein kinase C and mitogen-activated protein kinases. Here we further elucidate the mechanisms of BCP crystal-induced cell activation as BCP crystals activate transcription factors nuclear factor B and activator protein 1 in human fibroblasts. We confirm the role of protein kinase C in BCP crystal-induced mitogenesis in human fibroblasts.In contrast, we demonstrate that BCP crystals do not activate signal transduction pathways involving protein tyrosine kinases or phosphatidylinositol 3-kinase. These data further define the mechanism of cell activation by BCP crystals and confirm its selectivity, an observation that may have therapeutic implications. Synovial fluid basic calcium phosphate (BCP)1 (hydroxyapatite, octacalcium phosphate, and tricalcium phosphate) crystals are common in osteoarthritis (OA) and are associated with severe degenerative arthropathies (1). The prevalence of BCP crystals in synovial fluid from patients with knee OA is between 30 and 60%, and their presence correlates strongly with radiographic evidence of cartilage degeneration (2). Larger joint effusions are seen in affected joints when compared with joint fluid from OA knees without BCP crystals (3).Clinical and pathological studies have demonstrated that synovial lining proliferation of varying degrees is associated with BCP crystals in osteoarthritis (4). BCP crystals themselves are at least partly responsible for such proliferation since they stimulate cell replication in vitro (5). Increased cellularity in the synovial lining enhances the capacity for secretion of cytokines, which may promote chondrolysis. Noninflammatory destruction of matrix-rich articular structures including cartilage, ligament, and tendon is also characteristic of BCP crystal deposition (1). BCP crystals promote tissue damage by induction of matrix metalloprotease (MMP) synthesis and secretion. Since there are no available drugs to inhibit deposition or affect reabsorption of these crystals, prevention of the biological consequences of the destructive processes initiated by BCP crystals is an attractive therapeutic strategy.The in vitro effects of BCP crystals emphasize their pathogenic potential. BCP crystals induce mitogenesis in cultured fibroblasts (5, 6). However, mechanisms by which BCP crystals induce mitogenesis have been incompletely studied. Endocytosis and intracellular dissolution of the crystals, producing an elevated cytoplasmic calcium concentration, are important (7). BCP crystals activate a protein kinase signal transduction pathway involving p42 and p44 mitogen-activated protein (MA...

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Human PF4 is a heparin-binding chemokine known to be capable of inhibiting endothelial cell proliferation and angiogenesis. To explore the biological mechanisms responsible for this action, we investigated the effect of PF4 on epidermal growth factor (EGF)-stimulated human umbilical vein endothelial cells (HUVEC), a model system in which stimulation is essentially independent of interaction with cell-surface glycosaminoglycans. Based on previous findings that PF4 blocks endothelial cell cycle entry and progression into S phase, we studied the molecular mechanism(s) of PF4 interference with cell cycle machinery. PF4 treatment of EGF-stimulated HUVEC caused a decrease in cyclin E–cyclin-dependent kinase 2 (cdk2) activity with resulting attenuation of retinoblastoma protein phosphorylation. PF4-dependent downregulation of cyclin E-cdk2 activity was associated with increased binding of the cyclin-dependent kinase inhibitor, p21Cip1/WAF1, to the cyclin E-cdk2 complex. Analysis of total cellular p21Cip1/WAF1 showed that in the presence of PF4, p21Cip1/WAF1 levels were sustained at time points when p21Cip1/WAF1 was no longer detectable in cells stimulated by EGF in the absence of PF4. These findings indicate that PF4 inhibition of HUVEC proliferation in response to EGF is associated with impaired downregulation of p21Cip1/WAF1 and provide the first evidence for interference with cell cycle mechanisms by a chemokine.

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Phosphatidylinositol 3-kinase (PI3K) is a heterodimer lipid kinase consisting of an 85-kD subunit bound to a 110-kD catalytic subunit that also possesses intrinsic, Mn2+-dependent protein serine kinase activity capable of phosphorylating the 85-kD subunit. Here, we examine the Mn2+-dependent protein kinase activity of PI3Kα immunoprecipitated from normal resting or thrombin-stimulated platelets, and characterize p85/p110 phosphorylation, in vitro. Phosphoamino acid analysis of phosphorylated PI3Kα showed p85 and p110 were phosphorylated on serine, but in contrast to previous results, were also phosphorylated on threonine and tyrosine. Wortmannin and LY294002 inhibited p85 phosphorylation; however, p110 phosphorylation was also inhibited suggesting p110 autophosphorylation on serine/threonine. The protein tyrosine kinase inhibitor, erbstatin analog, partially inhibited p85 and p110 phosphorylation but did not appear to affect PI3K lipid kinase activity. The in vitro phosphorylation of p85α or p110α derived from thrombin-stimulated platelets was no different than that of resting platelets, but we confirm that in thrombin receptor-stimulated platelets enhanced levels of p85α and PI3K lipid kinase activity were recovered in antiphosphotyrosine antibody immunoprecipitates. These results suggest PI3Kα can autophosphorylate on serine and threonine, and both p85α and p110α are substrates for a constitutively-associated protein tyrosine kinase in platelets.

No abstract

Human PF4 is a heparin-binding chemokine known to be capable of inhibiting endothelial cell proliferation and angiogenesis. To explore the biological mechanisms responsible for this action, we investigated the effect of PF4 on epidermal growth factor (EGF)-stimulated human umbilical vein endothelial cells (HUVEC), a model system in which stimulation is essentially independent of interaction with cell-surface glycosaminoglycans. Based on previous findings that PF4 blocks endothelial cell cycle entry and progression into S phase, we studied the molecular mechanism(s) of PF4 interference with cell cycle machinery. PF4 treatment of EGF-stimulated HUVEC caused a decrease in cyclin E–cyclin-dependent kinase 2 (cdk2) activity with resulting attenuation of retinoblastoma protein phosphorylation. PF4-dependent downregulation of cyclin E-cdk2 activity was associated with increased binding of the cyclin-dependent kinase inhibitor, p21Cip1/WAF1, to the cyclin E-cdk2 complex. Analysis of total cellular p21Cip1/WAF1 showed that in the presence of PF4, p21Cip1/WAF1 levels were sustained at time points when p21Cip1/WAF1 was no longer detectable in cells stimulated by EGF in the absence of PF4. These findings indicate that PF4 inhibition of HUVEC proliferation in response to EGF is associated with impaired downregulation of p21Cip1/WAF1 and provide the first evidence for interference with cell cycle mechanisms by a chemokine.

Phosphatidylinositol 3-kinase (PI3K) is a heterodimer lipid kinase consisting of an 85-kD subunit bound to a 110-kD catalytic subunit that also possesses intrinsic, Mn2+-dependent protein serine kinase activity capable of phosphorylating the 85-kD subunit. Here, we examine the Mn2+-dependent protein kinase activity of PI3Kα immunoprecipitated from normal resting or thrombin-stimulated platelets, and characterize p85/p110 phosphorylation, in vitro. Phosphoamino acid analysis of phosphorylated PI3Kα showed p85 and p110 were phosphorylated on serine, but in contrast to previous results, were also phosphorylated on threonine and tyrosine. Wortmannin and LY294002 inhibited p85 phosphorylation; however, p110 phosphorylation was also inhibited suggesting p110 autophosphorylation on serine/threonine. The protein tyrosine kinase inhibitor, erbstatin analog, partially inhibited p85 and p110 phosphorylation but did not appear to affect PI3K lipid kinase activity. The in vitro phosphorylation of p85α or p110α derived from thrombin-stimulated platelets was no different than that of resting platelets, but we confirm that in thrombin receptor-stimulated platelets enhanced levels of p85α and PI3K lipid kinase activity were recovered in antiphosphotyrosine antibody immunoprecipitates. These results suggest PI3Kα can autophosphorylate on serine and threonine, and both p85α and p110α are substrates for a constitutively-associated protein tyrosine kinase in platelets.