Knockout of STIM1 enhances PDGF-mediated Ca2+ signaling through the PDGFR-PLCγ-STIM2 cascade in MEF cells

Abstract

STIM1 (stromal interaction molecule 1) has been reported as a key molecule in store-operated calcium entry (SOCE) which is one of the major mechanisms that allows extracellular Ca2+ refill into cytoplasm to regulate many cell functions Platelet-derived growth factor-BB (PDGF-BB) induces cell migration and proliferation in MEF (murine embryo fibroblast) cells Recent studies have suggested that PDGF-BB induces endo-plasm reticulum (ER) Ca2+ release and SOCE through the STIM1-Orai1 complex In this study we investigated the role of STIM1 in PDGF-BB-induced Ca2+ oscillation and its functions in MEF cells PDGF-BB activates Ca2+ elevation by activating the PLCγ sig-naling pathway and the PIP2 hydrolysis into IP3 and DAG resulted in ER Ca2+ release The depletion of ER Ca2+ induces SOCE through forming a SOC channel (STIM-TRPC-Orai complex) Also there are several signaling pathways co-activated by PDGF-BB stimulation including AKT JNK ERK and STAT3 According to data collect-ed from the FlexStation3 microplate reader unexpectedly we found PDGF-BB (100 ng/ml) induced manifest Ca2+ elevation in STIM1 knockout MEF cells dramatically with different doses and immunoblots data also indicated that PDGFR (PDGF? and PDGFRβ) overexpression induced PLCγ activity time-dependently with PDGF-BB stimulation in STIM1 knockout MEF cells Furthermore we applied SOCE PLC PDGFR inhibitors to examine the signaling transduction pathway 2-APB SKF96365 YM58483 Gd3+ La3+ were applied to inhibit PDGF-BB-induced Ca2+ elevation U73122 and D609 were applied to inhibit PLC phosphorylation and the AG-1295 was applied to inhibit PDGFR activa-tion after PDGF-BB triggering These data show that PDGFR protein levels are enhanced in STIM1 knockout MEF cells and then raise the PDGF-PLCγ-STIM2 signaling cascade to increase the [Ca2+]i with PDGF-BB stimulation

Date of Award	2016 Aug 2
Original language	English
Supervisor	Wen-Tai Chiu (Supervisor)