The preservation of bone mass relies on adequate proliferation, differentiation, secretion of matrix proteins and rate of apoptosis of the bone-forming osteoblasts. Although growing body of evidence indicates that the transient receptor potential (TRP) channels play important roles in numerous cellular functions, limited information is available about the TRP channels in osteoblasts. Here, we inventoried the gene expression and addressed some roles of the TRP channels in various osteoblast-like cells. The transcripts of canonical TRP (TRPC) channels were revealed for TRPC1, TRPC3, TRPC4 and TRPC6 in human MG-63, SaOS and U2 OS osteoblasts while transcripts for TRPC2, TRPC4, TRPC6 and TRPC7 were observed in the murine MC3T3 osteoblasts. PCR products were shown for the melastatin-related TRP (TRPM) channels TRPM4, TRPM6, TRPM7 and TRPM8 in all cell lines. The TRPM1 was specifically expressed by murine MC3T3 cells while the TRPM3 transcripts were revealed solely in human osteoblast-like cells. Transcripts for TRPV2 and TRPV4 were shown in osteoblastic cells. By interfering RNA approaches, the TRPC1 channels in osteoblasts were shown to be responsible for the capacitative calcium entry (CCE) and for the stimulation of cell proliferation by platelet-derived growth factor. On the other hand, interfering RNA-mediated abrogation of the expression of TRPM7, known as calcium and magnesium channels, resulted in the reduction of both basal and growth factor-stimulated osteoblastic cell proliferation. Our results provide the first complete reference for the gene expression of TRP channels in osteoblasts and point to their importance in cell proliferation.
Our results indicate that CCE, likely attributed to the activation of TRPCs, might be the main route for Ca2+ influx involved in osteoblast proliferation.
The contributions of membrane type-1 matrix metalloproteinase (MT1-MMP) and of the glucose-6-phosphate transporter (G6PT) in sphingosine-1-phosphate (S1P)-mediated Ca 2+ mobilization were assessed in glioblastoma cells. We show that gene silencing of MT1-MMP or G6PT decreased the extent of S1P-induced Ca 2+ mobilization, chemotaxis, and extracellular signal-related kinase phosphorylation. Chlorogenic acid and (À)-epigallocatechin-3-gallate, two diet-derived inhibitors of G6PT and of MT1-MMP, respectively, reduced S1P-mediated Ca 2+ mobilization. An intact MT1-MMP/G6PT signaling axis is thus required for efficient Ca 2+ mobilization in response to bioactive lipids such as S1P. Targeted inhibition of either MT1-MMP or G6PT may lead to reduced infiltrative and invasive properties of brain tumor cells.
The plasminogen activator/plasmin system represents a key component of the proteolytic machinery underlying angiogenesis. In this work, we investigated the effect of Neovastat (AE-941), a naturally occurring multifunctional antiangiogenic agent that is currently in Phase III clinical trials, on tissue and urokinase plasminogen activator activities. We found that in vitro, Neovastat at 100 microg/ml markedly stimulates t-PA-mediated plasmin generation, while it slightly inhibits the generation of plasmin mediated by uPA. The stimulatory effect of Neovastat on t-PA activity was markedly increased by a heat treatment, resulting in a 15-fold increase in the rate of activation of plasminogen. Neovastat did not directly stimulate the activity of t-PA or plasmin towards exogenous substrates, suggesting that its effect requires the presence of plasminogen. Accordingly, kinetic analysis showed that Neovastat increases both the k(cat) of t-PA as well as its affinity for plasminogen by 10-fold. The stimulation of t-PA activity by Neovastat was also correlated with a direct interaction of Neovastat with plasminogen as monitored by the surface plasmon resonance technology. Overall, these results identify Neovastat as a potent stimulator of t-PA-dependent activation of plasminogen, further emphasizing its pleiotropic mechanism of action on several molecular events involved in angiogenesis.
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