TRPC Is Involved In Hypoxia-induced VEGF Expression In U-87 MG Cells

BackgroundGlioblastoma multiforme(GBM) is the most common malignant brain tumor. The patients of GBM usually have a bad prognosis,representing a leading cause of cancer-related death.Usually,the growth of these tumors is highly angiogenesis-dependent,while higher grade malignant astrocytoma show higher degree of vascularity.Many growth factors can regulate angiogenesis including: Fibroblast growth factors(FGF),Vascular endothelia growth factors(VEGF), Endothelial growth factors(EGF) and Transforming growth factors(TGF-β).Of these factors,VEGF is a most potent angiogenic factor implicated in solid tumor angiogenesis.VEGF can promote vascular endothelial cell(VEC) proliferation, leading to Angiogenesis.Hypoxia is an important characteristic of glioblastomas, and a well-known stimulus for inducing VEGF expression.It has been found that in GBM,pseudopalisading cells in areas adjacent to areas of necrosis are severely hypoxic and express high levels of VEGF.Regulation of VEGF is complex and occurs at many different levels,including induction of VEGF transcription,stabilization of VEGF mRNA,and secretion and diffusion of mature protein.In most cells,Hypoxia inducible factor(HIF-1α) is the key transcription factor accounting for hypoxia-induced VEGF expression.Under hypoxic condition,protein levels and transcription activity of HIF-1αincrease. HIF-1αtranslocates into cell nucleus due to hypoxia and form heterodimer with HIF-1β.Then,HIF-1 binds to a specific consensus sequence,5'-RCGTG-3',which is found within the HRE in the VEGF promoter and induces VEGF gene expression.Usually,transcription factors directly regulate expression of downstream target gene.Meanwhile,ion channels also play an important role in regulating gene expression.Pheochromocytoma(PC12),an generally accepted excitable oxygen-sensing cell line,depolarized during hypoxia as the result of inhibition of hypoxia sensitive potassium ehannel(Kvl.2),which coupled to expression of HIF-1 target gene for tyrosine hydroxylase,in addition,non-selective cation channels are also found to be involved in hypoxia signal pathway.Severe hypoxia combined with glucose deprivation caused neuronal death by Ca2+ overload via TRPM7-ROS positive feedback pathway.Under moderate hypoxia(1%O2),non-selective cation channels have been reported to participate in the regulation of gene expression in endothelial cells.Ca2+ entry via non-selective cation channels raised[Ca²⁺]_i in astrocytes exposed to hypoxia,while Ca²⁺ was an important regulator for gene expression.Non-selective cation channels were formed by transient receptor potential(TRP) channels,calcium activated non-selective channels,hyperpolarization activated cation currents,acid-sensitive cationic channels(ASIC),etc.Recently,TRP channels become hot spot in this research filed.TRP channels have at least three subfamilies: TRPC(TRP-canonical),TRPV(TRP-vanilloid) and TRPM(TRP-melastatin). Although previous reports showed that non-selective cation channels participate in hypoxic response using channel blockers such as La³⁺ or Gd³⁺,these blockers are not specific and can not be used to identify specific member molecule.We focus on TRPC channels which can sense diverse stimulus in local environments.Human express six isoforms(TRPC1-7) except TRPC2(a pseudogene for human).By combined use of pharmacological and molecular biology methods,we identify that a specific molecule of TRPC channels,TRPC1,is involved in hypoxia-induced VEGF expression in U-87 MG cells.ObjectiveBy combined use of several cell biology and molecular biology methods:MTT, hoechst staining,real-time RT-PCR,western blot,immunofluorescence technique, RNA interference,overexpression,calcium concentration assay,etc,Effect of TRPC channels on hypoxia-induced VEGF expression in U-87 MG cells is investigated.Methods1.To establish a model of hypoxia-induced VEGF expression in vitro in U-87 MG cells,cell apoptosis,proliferation,expression of HIF-1αand VEGF was examined in cells treated with hypoxia(1%O2) for different time.Cell apoptosis,proliferation, expression of HIF-1αand VEGF was examined using Hoechst,MTT,western blot and real-time RT-PCR.2.U-87 MG cells were exposed to normoxia or hypoxia for 10h in the presence of 0, 25 or 40μM SKF96365.Total RNA was extracted and real-time RT-PCR was performed to determine whether TRPC channels participate in VEGF up-regulation in hypoxic U-87 MG cells.3.Total RNA was isolated from U-87 MG cells in normoxia.The cDNAs were amplified for 35 cycles and PCR products(5μl) were subjected to 2%agarose gel electrophoresis to analyze expression profile of TRPC in normoxic U-87 cells.4.Confocal microscopy was used to observe changes of calcium concentration when agonist or antagonist of TRPC channels were added.5.U-87 MG cells were incubated in normoxia or hypoxia for 16h.Then,TRPCs mRNA levels were determined by real-time RT-PCR.6.TRPC1 was down-regulated by RNA interference.Real-time RT-PCR and ELISA assay were used to determine whether knockdown of TRPC1 affected VEGF expression in mRNA level and protein level induced by hypoxia.7.TRPC3 and TRPC5 were overexpressed by transiently transfecting corresponding plasmids.After overexpression,real-time RT-PCR was used to evaluate effects of TRPC3 and TRPC5 on expression of VEGF.8.By immunofluorescence technique,location of TRPC1 was observed after hypoxic exposure in U-87 MG cells.Results1.Results from Hoechst staining show that hypoxia(1%O2) for 24h or 48h do not cause cell apoptosis;Hypoxia for 24h slightly inhibits cell proliferation(decrease by 3.23%) and for 48h inhibits cell proliferation by 22.35%(P<0.001).Hypoxia(1%O2) for 2h and 4h increase expression of HIF-1α.Also,VEGF was substantially induced by hypoxia for 10h(P<0.01).2.The up-regulation of VEGF mRNA during hypoxia was inhibited by 25μM SKF96365(P<0.01) and further suppressed by a increased dose of SKF96365.3.Agarose gel electrophoresis of the product from RT-PCR detected four single bands of 168bp,112bp,119bp,159bp as expected size,corresponding to TRPC1, TRPC3,TRPC4,TRPC5 in normoxic U-87 MG cells.4.OAG(agonist of TRPC) can increase calcium concentration of U-87 cells,while 2-APB(antagonist of TRPC) can attenuate OAG-induced calcium concentration rise.5.Under hypoxia stress,TRPC3 and TRPC4 expression decreased by 67.7%,68.2% (P<0.05),respectively.The mRNA of TRPC5 can not be detected after hypoxia; On the other hand,No change was found in TRPC1 mRNA level between normoxia and hypoxia.6.siRNAs delivery efficiency was over 90%.Real-time RT-PCR showed the mRNA of TRPC1 in U-87 cells transfected with three different siRNAs against TRPC1,si-1, si-2 and si-3,were reduced to 63%,39%and 36%,respectively,of that found in cultures transfected with the NC siRNA(P<0.01).Furthermore,si-1,si-2 and si-3 reduced the amount of TRPC1 protein to 48%,29%and 33%,respectively.Hypoxia enhanced VEGF gene expression in NC siRNA-transfected U-87 cells.However, transfection with either si-2 or si-3 greatly inhibited up-regulation of VEGF gene expression by hypoxia(P<0.01).Moreover,the protein levels of secreted VEGF in medium were in accordance with mRNA results.7.Transient transfection of TRPC3 or TRPC5 increase expression of these genes under hypoxic conditions.However,overexpression of TRPC3 or TRPC5 neither promote VEGF expression in hypoxic U-87 MG cells,nor prevent VEGF expression (P>0.05).8.Under normoxic conditions,TRPC1 protein located in cytoplasm and cell membrane.After hypoxia,TRPC1 mainly located in cell membrane,rather than cytoplasm.Conclusion1.Hypoxic conditions(1%O2) exists in microenvironment of most solid tumors. Experiments in vitro demonstrate that hypoxic conditions(1%O2) can induce expression of HIF-1αand VEGF.2.TRPC channels participate in hypoxia-induced VEGF expression in U-87 MG cells.3.Under normoxic conditions,U-87 MG cells express four TRPC isoforms:TRPC1, TRPC3,TRPC4 and TRPC54.Effective response of TRPC channels to agonist and antagonist demonstrates TRPC in U-87 MG cells is functional channels.5.Hypoxia has different effects on TRPCs gene expression,and imply TRPC1 and other TRPCs might play different roles in hypoxia signal pathway. 6.Knockdown of TRPC1 largely suppressed hypoxia-induced VEGF gene expression.Moreover,secreted VEGF protein was also inhibited.These data suggest TRPC1 is critical for hypoxia-induced VEGF expression and therefore it's expression is stable during hypoxia.7.Overexpression of TRPC3 or TRPC5 neither promote VEGF expression in hypoxic U-87 MG cells,nor prevent VEGF expression.These results suggest that these TRPCs do not contribute to VEGF up-regulation induced by hypoxia in U-87 MG cells8.Hypoxia can induce translocation of TRPC1 into cell membrane,which imply TRPC1 was activated by translocation and play a role in VEGF expression induced by hypoxia.