Biman C. Paria

Impairment of Store-Operated Ca2+ Entry in TRPC4−/− Mice Interferes With Increase in Lung Microvascular Permeability

We investigated the possibility that the TRPC gene family of putative store-operated Ca2+ entry channels contributes to the increase in microvascular endothelial permeability by prolonging the rise in intracellular Ca2+ signaling. Studies were made in wild-type (wt) and TRPC4 knockout (TRPC4−/−) mice and lung vascular endothelial cells (LECs) isolated from these animals. RT-PCR showed expression of TRPC1, TRPC3, TRPC4, and TRPC6 mRNA in wt LECs, but TRPC4 mRNA expression was not detected in TRPC4−/− LECs. We studied the response to thrombin because it is known to increase endothelial permeability by the activation of G protein-coupled proteinase-activated receptor-1 (PAR-1). In wt LECs, thrombin or PAR-1 agonist peptide (TFLLRNPNDK-NH2) resulted in a prolonged Ca2+ transient secondary to influx of Ca2+. Ca2+ influx activated by thrombin was blocked by La3+ (1 &mgr;mol/L). In TRPC4−/− LECs, thrombin or TFLLRNPNDK-NH2 produced a similar initial increase of intracellular Ca2+ secondary to Ca2+ store depletion, but Ca2+ influx induced by these agonists was drastically reduced. The defect in Ca2+ influx in TRPC4−/− endothelial cells was associated with lack of thrombin-induced actin-stress fiber formation and a reduced endothelial cell retraction response. In isolated-perfused mouse lungs, the PAR-1 agonist peptide increased microvessel filtration coefficient (Kf,c), a measure of vascular permeability, by a factor of 2.8 in wt and 1.4 in TRPC4−/−; La3+ (1 &mgr;mol/L) addition to wt lung perfusate reduced the agonist effect to that observed in TRPC4−/−. These results show that TRPC4-dependent Ca2+ entry in mouse LECs is a key determinant of increased microvascular permeability.

Role of Ca2+ signaling in the regulation of endothelial permeability

The vascular endothelial cell forms a semipermeable barrier between blood and interstitium. Inflammatory mediators such as thrombin and histamine induce vascular leakage defined as increased endothelial permeability to plasma proteins and other solutes. Increased endothelial permeability is the hallmark of inflammatory vascular edema. Inflammatory mediators that bind to heptahelical G protein-coupled receptors (GPCR) trigger increased endothelial permeability by increasing the intracellular Ca(2+) concentration ([Ca(2+)](i)). The rise in [Ca(2+)](i) activates key signaling pathways, which mediate cytoskeletal reorganization (through myosin light chain (MLC)-dependent contraction) and disassembly of VE-cadherin at the adherens junctions. The Ca(2+)-dependent protein kinase C (PKC) isoform, PKC-alpha, plays a critical role in initiating endothelial cell contraction and disassembly of VE-cadherin junctions. The increase in [Ca(2+)](i) induced by a variety of agonists is achieved by the generation of inositol 1,4,5-trisphosphate (IP3), activation of IP3 receptors (IP3R), release of stored intracellular Ca(2+), and Ca(2+) entry through plasma membrane channels. Recent findings demonstrate that IP3-sensitive Ca(2+) store depletion activates plasma membrane cation channels (i.e., store-operated cation channels (SOC) or Ca(2+) release activated channels) to cause Ca(2+) influx in endothelial cells. This mode of Ca(2+) influx is also known as capacitative Ca(2+) entry (CCE). Store-operated Ca(2+) influx signals increase in permeability and nitric oxide (NO) production and provokes changes in gene expression in endothelial cells. Recent studies have established that the Drosophila transient receptor potential (TRP) gene family of channels expressed in endothelial cells can function as SOC. Deletion of one of the TRP homologues, TRPC4, in mouse caused impairment in store-operated Ca(2+) current and Ca(2+) store release activated Ca(2+) influx in aortic and lung endothelial cells (LEC). In TRPC4 knockout (TRPC4(-/-)) mice, acetylcholine-induced endothelium-dependent smooth muscle relaxation was drastically reduced. In addition, TRPC4(-/-) mice LEC exhibited lack of actin stress fiber formation and cell retraction in response to thrombin activation of proteinase-activated receptor-1 (PAR-1) in endothelial cells. The increase in lung microvascular permeability in response to thrombin receptor activation was inhibited in TRPC4(-/-) mice. These results indicate that endothelial TRP channels such as TRPC1 and TRPC4 play an important role in signaling the increase in endothelial permeability.

RhoA Interaction with Inositol 1,4,5-Trisphosphate Receptor and Transient Receptor Potential Channel-1 Regulates Ca2+ Entry ROLE IN SIGNALING INCREASED ENDOTHELIAL PERMEABILITY

We tested the hypothesis that RhoA, a monomeric GTP-binding protein, induces association of inositol trisphosphate receptor (IP3R) with transient receptor potential channel (TRPC1), and thereby activates store depletion-induced Ca2+ entry in endothelial cells. We showed that RhoA upon activation with thrombin associated with both IP3R and TRPC1. Thrombin also induced translocation of a complex consisting of Rho, IP3R, and TRPC1 to the plasma membrane. IP3R and TRPC1 translocation and association required Rho activation because the response was not seen in C3 transferase (C3)-treated cells. Rho function inhibition using Rho dominant-negative mutant or C3 dampened Ca2+ entry regardless of whether Ca2+ stores were emptied by thrombin, thapsigargin, or inositol trisphosphate. Rho-induced association of IP3R with TRPC1 was dependent on actin filament polymerization because latrunculin (which inhibits actin polymerization) prevented both the association and Ca2+ entry. We also showed that thrombin produced a sustained Rho-dependent increase in cytosolic Ca2+ concentration [Ca2+]i in endothelial cells overexpressing TRPC1. We further showed that Rho-activated Ca2+ entry via TRPC1 is important in the mechanism of the thrombin-induced increase in endothelial permeability. In summary, Rho activation signals interaction of IP3R with TRPC1 at the plasma membrane of endothelial cells, and triggers Ca2+ entry following store depletion and the resultant increase in endothelial permeability.

Tumor necrosis factor-α induces nuclear factor-κB-dependent TRPC1 expression in endothelial cells

We investigated the role of tumor necrosis factor-α (TNF-α) in activating the store-operated Ca2+ channels in endothelial cells via the expression of transient receptor potential channel (TRPC) isoforms. We observed that TNF-α 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-κB (NF-κB)-binding sites in the 5′-regulatory region. To address the contribution of NF-κB in the mechanism of TRPC1 expression, we determined the effects of TNF-α 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-α challenge. TNF-α-induced increase in reporter activity was markedly reduced by co-expression of either kinase-defective IKKβ kinase mutant or non-phosphorylatable IκB mutant. Treatment with NEMO-binding domain peptide, which prevents NF-κB activation by selectively inhibiting IKKγ interaction with IKK complex, also blocked the TNF-α-induced TRPC1 expression. Thus, TNF-α induces TRPC1 expression through an NF-κB-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-α.

Ca2+ Influx Induced by Protease-activated Receptor-1 Activates a Feed-forward Mechanism of TRPC1 Expression via Nuclear Factor-κB Activation in Endothelial Cells

Thrombin activation of protease-activated receptor-1 induces Ca2+ influx through store-operated cation channel TRPC1 in endothelial cells. We examined the role of Ca2+ influx induced by the depletion of Ca2+ stores in signaling TRPC1 expression in endothelial cells. Both thrombin and a protease-activated receptor-1-specific agonist peptide induced TRPC1 expression in human umbilical vein endothelial cells, which was coupled to an augmented store-operated Ca2+ influx and increase in endothelial permeability. To delineate the mechanisms of thrombin-induced TRPC1 expression, we transfected in endothelial cells TRPC1-promoter-luciferase (TRPC1-Pro-Luc) construct containing multiple nuclear factor-κB (NF-κB) binding sites. Co-expression of dominant negative IκBα mutant prevented the thrombin-induced increase in TRPC1 expression, indicating the key role of NF-κB activation in mediating the response. Using TRPC1 promoter-deletion mutant constructs, we showed that NF-κB binding sites located between –1623 and –871 in the TRPC1 5′-regulatory region were required for thrombin-induced TRPC1 expression. Electrophoretic mobility shift assay utilizing TRPC1 promoter-specific oligonucleotides identified that the DNA binding activities of NF-κB to NF-κB consensus sites were located in this domain. Supershift assays using NF-κB protein-specific antibodies demonstrated the binding of p65 homodimer to the TRPC1 promoter. Inhibition of store Ca2+ depletion, buffering of intracellular Ca2+, or down-regulation of protein kinase Cα downstream of Ca2+ influx all blocked thrombin-induced NF-κB activation and the resultant TRPC1 expression in endothelial cells. Thus, Ca2+ influx via TRPC1 is a critical feed-forward pathway responsible for TRPC1 expression. The NF-κB-regulated TRPC1 expression may be an essential mechanism of vascular inflammation and, hence, a novel therapeutic target.

TRPC3 Controls Agonist-stimulated Intracellular Ca2+ Release by Mediating the Interaction between Inositol 1,4,5-Trisphosphate Receptor and RACK1

Activation of TRPC3 channels is concurrent with inositol 1,4,5-trisphosphate (IP3) receptor (IP3R)-mediated intracellular Ca2+ release and associated with phosphatidylinositol 4,5-bisphosphate hydrolysis and recruitment to the plasma membrane. Here we report that interaction of TRPC3 with receptor for activated C-kinase-1 (RACK1) not only determines plasma membrane localization of the channel but also the interaction of IP3R with RACK1 and IP3-dependent intracellular Ca2+ release. We show that TRPC3 interacts with RACK1 via N-terminal residues Glu-232, Asp-233, Glu-240, and Glu-244. Carbachol (CCh) stimulation of HEK293 cells expressing wild type TRPC3 induced recruitment of a ternary TRPC3-RACK1-IP3R complex and increased surface expression of TRPC3 and Ca2+ entry. Mutation of the putative RACK1 binding sequence in TRPC3 disrupted plasma membrane localization of the channel. CCh-stimulated recruitment of TRPC3-RACK1-IP3R complex as well as increased surface expression of TRPC3 and receptor-operated Ca2+ entry were also attenuated. Importantly, CCh-induced intracellular Ca2+ release was significantly reduced as was RACK1-IP3R association without any change in thapsigargin-stimulated Ca2+ release and entry. Knockdown of endogenous TRPC3 also decreased RACK1-IP3R association and decreased CCh-stimulated Ca2+ entry. Furthermore, an oscillatory pattern of CCh-stimulated intracellular Ca2+ release was seen in these cells compared with the more sustained pattern seen in control cells. Similar oscillatory pattern of Ca2+ release was seen after CCh stimulation of cells expressing the TRPC3 mutant. Together these data demonstrate a novel role for TRPC3 in regulation of IP3R function. We suggest TRPC3 controls agonist-stimulated intracellular Ca2+ release by mediating interaction between IP3R and RACK1.