Roles Of STIM1and STIM2in The CaR Mediated Ca²⁺ Entry And NO Generation And Its Mechanisms In Human Umbilical Vein Endothelial Cells

Objective: To study the role of calcium sensor stromal interaction molecules （STIM1、2）in extracellularCa²⁺-sensing receptor （CaR）-induced extracellular Ca²⁺influx and the production of nitric oxide （NO）, andits mechanisms in human umbilical vein endothelial cells （HUVEC）.Methods:（1） HUVEC were cultured using trypsinzation method and identified by growth pattern andimmunofluorescent staining.（2） The second to fifth passage of HUVEC were randomly divided into:①Control group;②CaR agonist group (spermine+Ca²⁺);③SOC inhibitor groups(MRS1845/Gd³⁺/2-APB/SKF-9636+spermine+Ca²⁺);④ROC inhibitor groups(Ro31-8220/Go6976+spermine+Ca²⁺);⑤ROC inhibitor+ROC agonist groups(Ro31-8220/Go6976+TPA+spermine+Ca²⁺);⑥ROC+SOC inhibitor groups(MRS1845+Ro31-8220/Go6976+spermine+Ca²⁺);⑦STIM1shRNA groups: the untransfected group（control group）; interference groups: shSTIM1-002group, shSTIM1-003group, shSTIM1-004group;vehicle group;⑧STIM2shRNA groups: the untransfected group （control group）; interference groups:shSTIM2-001group, shSTIM2-002group, shSTIM2-003group; vehicle group. STIM1/STIM2shRNAgroups were incubated with CaR agonist spermine （activating ROC and SOC）, ROC analog TPA andnegative allosteric modulator of CaR Calhex231（activating ROC, blocking SOC）, PKC inhibitorRo31-8220and classic-type PKCs and PKCμ inhibitor Go6976（activating SOC, blocking ROC）,respectively.（3） The shRNA specifically targeted to STIM1/STIM2gene were designed and synthesizedaccording to the cDNA sequence of human STIM1/STIM2gene in Genbank, shRNA were transfected into2-5HUVEC by liposome method. The transfection efficiency of STIM1/STIM2shRNA was detected bylaser scanning confocal microscopy in each group. The STIM1/STIM2proteins expression and interferenceefficiency were determined by Western blot in each group. The interference efficiencys of STIM1/STIM2mRNA were determined by Real time RT-PCR respectively.（4） The co-localization of STIM1and STIM2were determined using the immunofluorescence analysis.（5） Intracellular Ca²⁺concentration ([Ca²⁺]i) wasmeasured by Fura-2/AM loading, the production of NO were determined by the DAF-FM diacetate（DAF-FM DA）.（6） The SPSS17.0was used. Data are reported as means±SE. Statistical comparisons weremade using t-test or chi-square test for the paired and the unpaired groups. An analysis of variance wasused when multiple comparisons were performed. A difference was considered significant at p<0.05.Results:（1） HUVEC were confirmed by cell morphology and immunocytochemistry measuring Ⅷ factorantigen.（2） The green/red fluorescence by fluorescence microscopy could show the successful transfectionof shRNA in HUVEC.（3） Immunocytochemical results showed that STIM1and STIM2co-localized in thecytoplasm.（4） Western blot results showed that: Compared with control group, the STIM1and STIM2protein levels in each group were not considered significant,respectively （P>0.05）.（5） In STIM1/STIM2shRNA groups, Western blot results demonstrated that: Compared with control group, Vehicle group wasnot considered significant （P>0.05）; shRNA targeted to the STIM1specifically decreased the expression ofSTIM1protein in shSTIM1-002, shSTIM1-003and shSTIM1-004group （the interference efficiency was 43%,50%,74%, respectively）48h after transfection （P<0.05）, especially the shSTIM1-004group（P<0.05）;shRNA targeted to the STIM2specifically decreased the expression of STIM2protein in shSTIM2-001,shSTIM2-002and shSTIM2-003group （the interference efficiency was44%,80%,30%, respectively）48hafter transfection （P<0.05）, especially the ShSTIM2-002group （P<0.05）.（6） Real time RT-PCR resultsshowed that: Compared with control group, Vehicle group was not considered significant （P>0.05）;shSTIM1-002, shSTIM1-003and shSTIM1-004（the interference efficiency was56%,73%,91%,respectively） targeted to the STIM1specifically decreased the gene of STIM1（P<0.05）, especially theshSTIM1-004group （P<0.05）; shSTIM2-001, shSTIM2-002and shSTIM2-003（the interference efficiencywas80%,88%,41%, respectively） targeted to the STIM2specifically decreased the gene of STIM2（P<0.05）, especially the shSTIM2-002（P<0.05）.（7） The results of dynamic changes of intracellular Ca²⁺fluorescence intensity ratio in HUVEC by Fura-2/AM showed that: Compared with the control group (CaRagonist group, spermine+Ca²⁺group)（Δratio=5.84±0.10） and vehicle group （Δratio=5.80±0.05）, the [Ca²⁺]iof shSTIM1-004Group （Δratio=4.60±0.18） was decreased （p<0.05）, the [Ca²⁺]iof shSTIM2-002group（Δratio=5.75±0.13） was not changed （p>0.05）, compared with the control group, vehicle group was nosignificant difference （p>0.05）. Compared with the control group (Calhex231+OAG+spermine+Ca²⁺group)（Δratio=2.79±0.04） and vehicle group （Δratio=2.91±0.18）, the [Ca²⁺]iof shSTIM1-004Group（Δratio=1.09±0.02） was decreased （p<0.05）, compared with the control group, vehicle group was nosignificant difference （p>0.05）. Compared with the control group (Ro31-8220+spermine+Ca²⁺)（Δratio=3.15±0.10） and vehicle group （Δratio=2.90±0.08）, the [Ca²⁺]iof shSTIM1-004group（Δratio=1.15±0.02） was decreased （p<0.05）, compared with the control group, vehicle group was nosignificant difference （p>0.05）. Compared with the control group (Go6976+spermine+Ca²⁺group)（Δratio=4.26±0.04） and vehicle group （Δratio=4.17±0.14）, the [Ca²⁺]iof shSTIM1-004Group（Δratio=1.21±0.07） was decreased （p<0.05）, compared with the control group, vehicle group was nosignificant difference （p>0.05）.（8） The results of net NO fluorescence intensity by DAF-FM DA showedthat: Compared with the control group (CaR agonist group, spermine+Ca²⁺group)（1105.18±31.97） andvehicle group （983.33±30.33）, the net NO fluorescence intensity of shSTIM1-004group （160.08±11.12）was diminished （p<0.05）, the net NO fluorescence intensity of shSTIM2-002group （1035.80±27.78） wasno significant difference （p>0.05）, compared with the control group, vehicle group was no significantdifference （p>0.05）. Compared with the control group (Calhex231+OAG+spermine+Ca²⁺group)（177.17±20.09） and vehicle group （179.92±16.82）, the net NO fluorescence intensity of shSTIM1-004group （59.83±7.43） was diminished （p<0.05）, compared with the control group, vehicle group was nosignificant difference （p>0.05）. Compared with the control group (Ro31-8220+spermine+Ca²⁺)（182.66±11.93） and Vehicle group （175.52±31.64）, the net NO fluorescence intensity of shSTIM1-004group （56.27±5.71） was diminished （p<0.05）, compared with the control group, vehicle group was nosignificant difference （p>0.05）. Compared with the control group (Go6976+spermine+Ca²⁺group)（201.43±7.48） and vehicle group （188.19±19.77）, the net NO fluorescence intensity of shSTIM1-004group（56.06±7.71） was diminished （p<0.05）, compared with the control group, vehicle group was no significant difference （p>0.05）.Conclusion:（1） STIM1plays an important role in CaR-mediating Ca²⁺influx and NO production inHUVEC.（2） STIM1is a key component in CaR-mediated Ca²⁺influx and NO production though SOC andROC. STIM2does not participate in this processes.