Involvement of calcium-sensing receptor in oxLDL-induced MMP-2 production in vascular smooth muscle cells via PI3K/Akt pathway.

Matrix metalloproteinase-2 (MMP-2) is constitutively expressed in vascular smooth muscle cells (VSMCs) and up-regulated in atherosclerotic lesion by various stimuli, such as oxidized low-density lipoprotein (oxLDL). Calcium-sensing receptor (CaSR) is also expressed in VSMCs, but it remains unclear whether CaSR is associated with overproduction of MMP-2 in VSMCs. In this study, the expression of MMP-2 was detected by real-time PCR and Western blot analysis, and the gelatinolytic activity of MMP-2 was measured using gelatin zymography. Our results showed that oxLDL enhanced MMP-2 expression and activity in rat aortic VSMCs in a time- and dose-dependent manner. In addition, CaSR expression was up-regulated by oxLDL. Manipulating CaSR function in these cells by NPS2390 (an antagonist of CaSR) or GdCl(3) (an agonist of CaSR) affected the oxLDL-induced MMP-2 production. In VSMCs, oxLDL stimulated the rapid activation of phosphatidylinositol 3-kinase (PI3K)/Akt signal pathway, as determined by Western blot analysis. Phosphorylation of Akt and MMP-2 production stimulated by oxLDL were attenuated by LY294002 (a specific inhibitor of PI3K). Activation of Akt was suppressed by NPS2390 but enhanced by GdCl(3). In contrast, oxLDL had no stimulatory effect on the phosphorylation of JNK, and pretreatment with SP600125 (an inhibitor of JNK) produced no significant effect on oxLDL-induced MMP-2 production. These results suggest that CaSR mediates oxLDL-induced MMP-2 production in VSMCs via PI3K/Akt signal pathway.

Calcium-sensing receptors induce apoptosis during simulated ischaemia-reperfusion in Buffalo rat liver cells.

1. Calcium-sensing receptors (CaSR) exist in a variety of tissues. In 2010, we first identified its functional expression in Buffalo rat liver (BRL) cells and demonstrated that the activation of CaSR was involved in an increased intracellular calcium through the Gq subunit-phospholipase C-inositol triphosphate pathway. However, its role and related mechanism in hepatic ischaemia/reperfusion (I/R) injury is still unclear. 2. Therefore, in the present study, BRL cells were incubated in ischaemia-mimetic solution for 4 h, then reincubated in the normal culture medium for 10 h to establish a simulated I/R model. We assayed the apoptotic ratio of BRL cells by flow cytometry and Hoechst 33342 staining; analyzed the expression of CaSR, cytochrome c (Cyt-c), caspase-3, Bcl-2, Bax, extracellular signal-regulated protein kinase (ERK), and p38 by Western blotting; and measured the concentration of intracellular calcium by laser-scanning confocal microscopy. 3. The results showed that simulated I/R increased the expression of CaSR and induced apoptosis in BRL cells. GdCl(3), a specific activator of CaSR, further increased CaSR expression, intracellular calcium, and apoptosis in BRL cells during I/R. The activation of CaSR downregulated Bcl-2 expression, upregulated Cyt-c, caspase-3, and Bax expressions, and promoted p38 and ERK-1/2 phosphorylation. 4. In conclusion, increased CaSR expression plays a vital role in apoptosis induced by I/R injury, in which its mechanism is related with calcium overload and the activation of the mitochondrial and mitogen-activated protein kinase apoptotic pathways. The regulation of CaSR activity might serve as a novel pharmacological target to prevent and treat liver disease.