Caveolin-1 scaffold domain interacts with TRPC1 and IP3R3 to regulate Ca2+ store release-induced Ca2+ entry in endothelial cells.

Caveolin-1 (Cav-1) regulates agonist-induced Ca(2+) entry in endothelial cells; however, how Cav-1 regulates this process is poorly understood. Here, we describe that Cav-1 scaffold domain (NH(2)-terminal residues 82-101; CSD) interacts with transient receptor potential canonical channel 1 (TRPC1) and inositol 1,4,5-trisphosphate receptor 3 (IP(3)R3) to regulate Ca(2+) entry. We have shown previously that the TRPC1 COOH-terminal residues 781-789 bind to CSD. In the present study, we show that the TRPC1 COOH-terminal residues 781-789 truncated (TRPC1-CDelta781-789) mutant expression abolished Ca(2+) store release-induced Ca(2+) influx in human dermal microvascular endothelial cell line (HMEC) and human embryonic kidney (HEK-293) cells. To understand the basis of loss of Ca(2+) influx, we determined TRPC1 binding to IP(3)R3. We observed that the wild-type (WT)-TRPC1 but not TRPC1-CDelta781-789 effectively interacted with IP(3)R3. Similarly, WT-TRPC1 interacted with Cav-1, whereas TRPC1-CDelta781-789 binding to Cav-1 was markedly suppressed. We also assessed the direct binding of Cav-1 with TRPC1 and observed that the WT-Cav-1 but not the Cav-1DeltaCSD effectively interacted with TRPC1. Since the interaction between TRPC1 and Cav-1DeltaCSD was reduced, we measured Ca(2+) store release-induced Ca(2+) influx in Cav-1DeltaCSD-transfected cells. Surprisingly, Cav-1DeltaCSD expression showed a gain-of-function in Ca(2+) entry in HMEC and HEK-293 cells. We observed a similar gain-of-function in Ca(2+) entry when Cav-1DeltaCSD was expressed in lung endothelial cells of Cav-1 knockout mice. Immunoprecipitation results revealed that WT-Cav-1 but not Cav-1DeltaCSD interacted with IP(3)R3. Furthermore, we observed using confocal imaging the colocalization of IP(3)R3 with WT-Cav-1 but not with Cav-1DeltaCSD on Ca(2+) store release in endothelial cells. These findings suggest that CSD interacts with TRPC1 and IP(3)R3 and thereby regulates Ca(2+) store release-induced Ca(2+) entry in endothelial cells.

Caveolin-1 regulates store-operated Ca2+ influx by binding of its scaffolding domain to transient receptor potential channel-1 in endothelial cells.

Caveolin-1 associates with store-operated cation channels (SOC) in endothelial cells. We examined the role of the caveolin-1 scaffolding domain (CSD) in regulating the SOC [i.e., transient receptor potential channel-1 (TRPC1)] in human pulmonary artery endothelial cells (HPAECs). We used the cell-permeant antennapedia (AP)-conjugated CSD peptide, which competes for protein binding partners with caveolin-1, to assess the interactions of caveolin-1 with TRPC1 and its consequences on thrombin-induced Ca2+ influx. We observed that AP-CSD peptide markedly reduced thrombin-induced Ca2+ influx via SOC in HPAECs in contrast to control peptide. AP-CSD also suppressed thapsigargin-induced Ca2+ influx. Streptavidin-bead pull-down assay indicated strong binding of biotin-labeled AP-CSD peptide to TRPC1. Immunoprecipitation studies demonstrated an interaction between endogenous TRPC1 and ectopically expressed hemagglutinin-tagged CSD. Analysis of the deduced TRPC1 amino acid sequence revealed the presence of CSD binding consensus sequence in the TRPC1 C terminus. We also observed that an AP-TRPC1 peptide containing the CSD binding sequence markedly reduced the thrombin-induced Ca2+ influx. We identified the interaction between biotin-labeled AP-TRPC1 C terminus peptide and caveolin-1. Thus, these results demonstrate a crucial role of caveolin-1 scaffolding domain interaction with TRPC1 in regulating Ca2+ influx via SOC.

Tumor necrosis factor-alpha induces nuclear factor-kappaB-dependent TRPC1 expression in endothelial cells.

We investigated the role of tumor necrosis factor-alpha (TNF-alpha) in activating the store-operated Ca2+ channels in endothelial cells via the expression of transient receptor potential channel (TRPC) isoforms. We observed that TNF-alpha exposure of human umbilical vein endothelial cells resulted in TRPC1 mRNA and protein expression, whereas it had no effect on TRPC3, TRPC4, or TRPC5 expression. The TRPC1 expression was associated with increased Ca2+ influx after intracellular Ca2+ store depletion with either thrombin or thapsigargin. We cloned the 5'-regulatory region of the human TRPC1 (hTRPC1) gene which contained a TATA box and CCAAT sequence close to the transcription initiation site. We also identified four nuclear factor-kappaB (NF-kappaB)-binding sites in the 5'-regulatory region. To address the contribution of NF-kappaB in the mechanism of TRPC1 expression, we determined the effects of TNF-alpha on expression of the reporter luciferase after transfection of hTRPC1 promoter-luciferase (hTRPC1-Pro-Luc) construct in the human dermal microvascular endothelial cell line. Reporter activity increased >4-fold at 4 h after TNF-alpha challenge. TNF-alpha-induced increase in reporter activity was markedly reduced by co-expression of either kinase-defective IKKbeta kinase mutant or non-phosphorylatable IkappaB mutant. Treatment with NEMO-binding domain peptide, which prevents NF-kappaB activation by selectively inhibiting IKKgamma interaction with IKK complex, also blocked the TNF-alpha-induced TRPC1 expression. Thus, TNF-alpha induces TRPC1 expression through an NF-kappaB-dependent pathway in endothelial cells, which can trigger augmented Ca2+ entry following Ca2+ store depletion. The augmented Ca2+ entry secondary to TRPC1 expression may be an important mechanism of endothelial injury induced by TNF-alpha.