| Summary: | 碩士 === 國立成功大學 === 生理學研究所 === 101 === Previously we demonstrated that collagen gel-induced apoptosis mediated by Ca2+ homeostasis disturbance which was resulted from STIM1 coupled to Orai1, a essential pore subunit of store-operated Ca2+ channels in normal epithelial cells. On the other hand, TRPC1 have been proposed as mechanosensitive ion channels, assembly of STIM1-Orai1-TRPC1 complex have reported to elevate store-operated Ca2+ entry (SOCE). However, whether TRPC1 coordinates with STIM-Orai1 and contributes to collagen gel-induced elevation of SOCE is still unclear. To test this hypothesis, we established LLC-PK1 cells stably expressed two types of dominant negative STIM1 construct : STIM1-ΔCAD, which was reported a mutant prevented STIM-Orai1 interaction ; STIM1-ΔERM, which blocked Orai1 and TRPC1 coupled to STIM1. In immunofluoresence results, LLC-PK1 cells cultured on collagen gel enhanced the colocalization of STIM-Orai1 and STIM1-TRPC1, and this phenomenon was inhibited in cells overexpressed STIM-ΔCAD or STIM-ΔERM. Furthermore, SOCE was increased when cells cultured on collagen gel, but inhibited by STIM1-ΔCAD for 70% and totally blocked by STIM1-ΔERM. Besides, knockdown of TRPC1 or STIM1 also inhibited collagen gel-induced Ca2+ influx by 30% and 80% respectively. In addition to SOC channel molecules, the actin filament and microtubule were disorganized when LLC-PK1 cells seeded on collagen gel. Previous studies indicated that disruption of actin filament or microtubule enhanced SOCE resulted from STIM1-Orai1-TRPC1 interaction. To test this hypothesis, we applied cytochalasin D and nocodazole to disrupt actin filament and microtubule, the result showed that either cytochalasin D or nocodazole were up-regulated SOCE of LLC-PK1 cells. Finally, we analyzed apoptosis ratio in four methods. In low molecular DNA electrophoresis result, DNA ladder obviously in LLC-PK1 cells cultured on collagen gel, but this phenomenon were not prevented from STIM-ΔCAD or STIM-ΔERM overexpressed cells. In contrast, western blot results showed that cells overexpressed wild-type STIM1 prevented cleavage caspase-3 expression under collagen gel stimulation. In Hoechst 33258 staining and flow cytometry results, overexpression of STIM1-ΔCAD or STIM-ΔERM were slightly decreased apoptosis ratio and nucleus fragmentation, but dramatically decreased in wild-type STIM1 overexpressed cells. Moreover, we also applied SOC channel inhibitors or siRNA for STIM1 and TRPC1 to analyzed the apoptosis ratio by flow cytometry, results showed that collagen gel-induced Ca2+ influx was prevented by knockdown of STIM1 or TRPC1 as well as SOC channel inhibitors.
Taken together, we concluded that coordination of STIM1, Orai1 and TRPC1 contributed to collagen gel-induced elevation of store-operated Ca2+ entry and apoptosis in LLC-PK1 cells. The disorganization of cytoskeleton may involved in the regulation of this process.
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