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.

ery and, thus, is important in the control of vascular endothelial barrier function (1)(2)(3). An increase in [Ca 2ϩ ] i induces barrier dysfunction leading to leakiness of the endothelial monolayer to plasma proteins (3). Mediators such as thrombin, histamine, and reactive oxygen species increase vascular permeability by activating Ca 2ϩ -sensitive signaling pathways (3). We showed that Ca 2ϩ entry through plasma membrane cation channels activated by Ca 2ϩ store depletion is a critical determinant of increased endothelial permeability (4 -6). We have also shown that activation of endothelial cell surface protease-activated receptor-1 (PAR-1) 3 by thrombin caused a rapid and transient increase in cytosolic Ca 2ϩ concentration ([Ca 2ϩ ] i ) secondary to the release of stored Ca 2ϩ and subsequent longer-lived Ca 2ϩ entry triggered by store depletion (4,6). In endothelial cells, the plasma membrane cation channels, known as store-operated cation channels (SOCs), mediate the entry of Ca 2ϩ (7)(8)(9). Several studies have shown that influx of Ca 2ϩ in vascular endothelial cells occurs via SOCs (6,8,10,11). Studies have also identified that the mammalian homologues of the transient receptor potential (TRP) gene family of channels function as SOCs (6,8,12,13). These TRP genes encode a superfamily of proteins with six transmembrane helices that are divided into seven subfamilies: TRPC (canonical or classical), TRPV (vaniloid), TRPM (melastatin), TRPA (ankyrin), TRPML (mucolipin), TRPP (polycystin), and the TRPN (no mechanoreceptor potential C (NOMPC) (10,14). Members of the TRPC subfamily contain 700 -1000 amino acids, and 7 isoforms (TRPC1-7) are expressed in mammalian cells. Primary endothelial cells express TRPC1-7 (6,12,15). TRPC4 is the predominant isoform expressed in mouse endothelial cells (6,12). Deletion of TRPC4 in mice caused impairment in SOC current in mouse aortic and lung vascular endothelial cells (6,12). We showed that SOC-mediated Ca 2ϩ influx induced by thrombin causing increased microvascular permeability was impaired in TRPC4 knock-out mice (6); thus, TRPC4 is an essential SOC in endothelial cells mediating increased vascular permeability. Moore et al. (11) showed that antisense oligonucleotide-induced inhibition of TRPC1 expression, a predominant isoform expressed in human vascular endothelial cells (15), reduced the thapsigargin-induced Ca 2ϩ entry (11). We showed that overexpression of TRPC1 augmented the thrombin-induced Ca 2ϩ influx in human endothelial cells (15), and this response resulted in markedly increased endothelial permeability (15). The cloning of human TRPC1 5Ј-regulatory region revealed that it contains multiple nuclear factor-B (NF-B) binding sites (16). Tumor necrosis factor-␣-activated TRPC1 expression in human endothelial cells was shown to occur via NF-B signaling (16). In addition, tumor necrosis factor-␣ stimulation of human endothelial cells resulted in increased TRPC1 expression (15,16).
Thrombin mediates the expression of genes such as ICAM-1 and VCAM-1 by activating NF-B signaling in endothelial cells (17)(18)(19). In the present study we examined the role of Ca 2ϩ influx induced by the depletion of Ca 2ϩ stores in signaling TRPC1 expression in endothelial cells and its consequences in augmenting Ca 2ϩ signaling. Utilizing TRPC1 promoter deletion mutant construct expression studies, we identified that NF-B binding sites upstream of TRPC1 5Ј-regulatory region were critical for thrombin-induced TRPC1 expression. We showed that store-operated Ca 2ϩ influx signaled NF-B activation and induced TRPC1 expression and thereby augmented Ca 2ϩ influx in response to Ca 2ϩ store depletion. Thus, augmented Ca 2ϩ influx signal may play an important role in the pathogenesis of vascular inflammation and injury and provides a novel therapeutic target against vascular inflammatory diseases.
Reverse Transcription (RT)-PCR-Confluent HUVEC monolayers were washed with serum-free medium and incubated with 1% FBS-containing medium for 2 h. After this period, the endothelial cells were treated with 50 nM thrombin in the presence or absence of actinomycin D (0.5 M) in 1% FBS-containing medium for different time intervals. After thrombin treatment, total RNA was isolated using TRIzol reagent. RT-PCR was performed as described (16). Human TRPC1 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were amplified using the following primer sets: TRPC1 (forward, 5Ј-GATTT-TGGAAAATTTCTTGGGATGT-3Ј, and reverse, 5Ј-TTTGT-CTTCATGATTTGCTATCA-3Ј); GAPDH (forward, 5Ј-TAT-CGTGGAAGGACTCATGACC-3Ј, and reverse, 5Ј-TACAT-GGCAACTGTG AGGGG-3Ј). RT product (2 l) was amplified in a 50-l volume containing 100 pmol of primers and 2.5 units of TaqDNA polymerase. Reaction conditions are as follows: 95°C for 30 s, 55°C for 30 s, 72°C for 1 min for 35 cycles, and then 72°C for 7 min. Amplification of GAPDH was also performed following the protocol described above. PCR products were separated on 1.2% agarose gel and identified by ethidium bromide staining. The band intensity of scanned gel photograph was determined by using Scion imaging software (from the National Institutes of Health). Normalization of TRPC1 expression was achieved by comparing the expression of GAPDH for the corresponding sample.
Immunoblotting-TRPC1 expression in response to PAR-1 activation in HUVEC was determined by immunoblotting cell lysate proteins with anti-TRPC1 antibody (16).
Cytosolic Ca 2ϩ Measurement-The cytoplasmic Ca 2ϩ concentration ([Ca 2ϩ ] i ) in single endothelial cells was measured by Fura-2 fluorescence imaging (15,16). Cells grown on 25-mm diameter glass coverslips were washed twice in Hanks' balanced salt solution (HBSS) and loaded with 3 M Fura-2 AM for 20 min at 37°C. Cells were then washed twice in HBSS and imaged by using an Attofluor RatioVision digital fluorescence microscopy system (Atto Instruments, Rockville, MD) equipped with a Zeiss Axiovert S100 inverted microscope and F-Fluar ϫ40, 1.3 NA oil immersion objective. Regions of interest in individual cells were marked and excited at 334 and 380 nm with emission at 520 nm at 5-s intervals. The 334/380 nm fluorescence ratio has been used to represent changes in [Ca 2ϩ ] i .
Transendothelial Electrical Resistance-The real-time change in endothelial monolayer resistance was measured to assess endothelial barrier function as described by us (20). In brief, HUVEC were grown to confluence on a small gold electrode (4.9 ϫ 10 Ϫ4 cm 2 ). The small electrode and the larger counter electrode were connected to a phase-sensitive lock-in amplifier. An approximate constant current of 1 A was supplied by a 1-V, 4000-Hz AC signal connected serially to 1-megaohm resistor between the small electrode and the larger counter electrode. The voltage between small electrode and large electrode was monitored by lock-in amplifier, stored, and processed by a personal computer. The same computer controlled the output of the amplifier and switched the measurement to different electrodes in the course of an experiment. Before the experiment, confluent endothelial monolayer was kept in 1% FBS-containing medium for 2 h, and then cells were either stimulated with thrombin or not stimulated with thrombin for 20 h. After this treatment, thrombin-induced change in resistance of endothelial monolayer was measured. The data are presented in resistance normalized to its value at time zero as described (20,22).
Expression of Reporter Constructs-HMEC grown to 50% confluency in 6-well culture plates were used for reporter constructs transfection (16,21). Plasmid DNA mixtures containing 1 g of hTRPC1 promoter-luciferase (hTRPC1-Pro-Luc) in pGL2 vector and 0.035 g of pRL/TK (Promega) were transfected using Lipofectamine. Lipofectamine-DNA complexes were diluted with 0.8 ml of Opti-MEM I before being added to HMEC and prewashed two times with Opti-MEM I for 2-4 h. To end transfection, 2 ml of MCBD131 medium supplemented with 10% FBS was added to each well.
Dual Luciferase Reporter Assay-At 48 h after transfection, the cells were incubated in MCBD131 medium containing 1% FBS for 2 h and then stimulated with thrombin or PAR-1 agonist peptide.
In some experiments cells were pretreated with either 2-aminoethoxydiphenyl borate (2-APB) (75 M) for 30 min before thrombin treatment. After stimulation, cells were lysed, and 20 l of lysate was used to measure reporter gene expression (16,21). Firefly (P. pyralis) and sea pansy (R. reniformis) luciferase activity were assayed by the dual luciferase reagent assay system (Promega). Protein concentrations were determined using Bio-Rad reagents.
Nuclear Protein Extraction-Nuclear extracts were prepared from HUVEC after thrombin treatment as described (18). Cells grown in 100-mm cell culture dishes were washed twice with ice-cold Tris-buffered saline, scraped, and resuspended in 400 l of buffer A (10 mM KCl, 10 mM HEPES, pH 7.9, 0.1 mM EDTA, pH 8.0, 0.1 mM EGTA, 1 mM dithiothreitol, and 0.5 mM phenylmethylsulfonyl fluoride). The suspension was homogenized for 10 strokes using a Glass Dounce homogenizer and centrifuging at 3000 ϫ g for 30 s. Nuclear pellets were then resuspended in 100 l of solution B (20 mM HEPES, 1 mM EDTA, 0.4 M NaCl, 1 mM EGTA, 1 mM dithiothreitol, and 0.5 mM phenylmethylsulfonyl fluoride) and incubated on ice for 20 min. The nuclei were then pelleted by centrifugation at FIGURE 1. PAR-1 activation increases TRPC1 expression in endothelial cells. HUVEC grown to confluence were washed and incubated in serum-free medium for 2 h at 37°C and then stimulated with 50 nM thrombin (A) or 40 M PAR-1 agonist peptide (TFLLRNPNDK) (B) for 0, 2, 4, and 6 h. HUVEC were also exposed to thrombin in the presence of actinomycin D (0.5 M) for 0, 2, 4, and 6 h (C ). After this incubation period, RT-PCR was performed to determine TRPC1 mRNA expression (see "Experimental Procedures"). TRPC1 induction -fold was calculated by measuring the ratio of TRPC1 to GAPDH (D). The results are the mean Ϯ S.E. from five separate experiments shown. *, p Ͻ 0.05, significantly different from the control. 25,000 ϫ g for 1 min. Supernatants containing nuclear proteins were used for electrophoretic mobility shift assay.
Electrophoretic Mobility Shift Assay (EMSA)-EMSAs were performed as described (18,23). The probes used for EMSAs were 21-bp double-stranded TRPC1 promoter-specific NF-B binding sequences that were listed in Fig. 4A. End labeling was performed by T4 kinase in the presence of [␣-32 P]ATP. Labeled oligonucleotides were purified on a Sephadex G-50 column. An aliquot of 10 g of nuclear protein extract was incubated with the labeled double-stranded probe (ϳ80,000 cpm) in the presence of 2.5 l of binding buffer (Promega). The binding reactions were incubated at 25°C for 20 min. After adding non-denaturing sample buffer, the DNA-protein complexes were resolved by 6% native PAGE in low ionic strength buffer (0.5ϫ Tris borate-EDTA). To study the effect of antibodies on DNA-protein binding, nuclear extracts were first incubated with NF-B protein-specific antibodies (2 g/assay) for 30 min at 25°C, and then labeled double-stranded probe was added, and the incubation was continued for additional 20 min. After this incubation, non-denaturing sample buffer was added, and the DNA-protein complexes were separated as described above.
PKC-␣-specific siRNA Transfection-Validated human PKC␣specific siRNA targeted to exon 4 was obtained from Ambion (Austin, TX). HUVEC grown to ϳ70 confluence on gelatin-coated culture dishes were transfected with either 100 nM PKC␣-siRNA or scrambled sequence using GeneEraser siRNA transfection reagent (Stratagene) according to the manufacture's protocol. At 48 h after transfection, cells were used for experiments.
Statistical Analysis-Statistical comparisons were made using two-tailed Student's t test. Experimental values were reported as the means Ϯ S.E. Differences in mean values were considered significant at p Ͻ 0.05.

PAR-1 Activation Induces TRPC1 Expression in Endothelial
Cells-We addressed whether thrombin induces TRPC1 expression in endothelial cells. We exposed HUVEC to thrombin and determined TRPC1 mRNA expression by RT-PCR. We observed that thrombin increased TRPC1 mRNA expression in a time-dependent manner ( Fig. 1A) without affecting housekeeping gene GAPDH expression (Fig. 1A). At 2 h after thrombin treatment, TRPC1 transcript level increased ϳ2-fold, at 4 h the expression level was increased 3-fold, and at 6 h it was increased ϳ4-fold over basal ( Fig. 1, B and D). To address whether the increase in TRPC1 mRNA was due to increased transcription, we incubated HUVEC with actinomycin D and exposed the cells to thrombin. Thrombin-induced TRPC1 expression was blocked by actinomycin D (Fig. 1C). Thrombin activates PAR-1 by proteolytic cleavage of PAR-1, which couples to multiple heterotrimeric G-proteins to elicit cellular responses including NF-B activation (17,18,21,24,25,27). We also used PAR-1-specific agonist peptide (TFLLRN-PNDK) (24,26) to mimic the effects of thrombin in inducing TRPC1 expression. Results showed that PAR-1 agonist peptide, like thrombin, activated TRPC1 expression in HUVEC ( Fig. 1, B and D).
To address the functional relevance of TRPC1 expression, we measured TRPC1 protein expression after exposing HUVEC to thrombin or PAR-1 agonist peptide. Cells were exposed to PAR-1 control peptide (scrambled sequence, FTLLRNPNDK), PAR-1 agonist peptide (TFLLRNPNDK), or thrombin for 20 h, and then TRPC1 protein expression was determined by immunoblotting cell lysates with anti-TRPC1 antibody. TRPC1 protein expression was increased ϳ3-fold after PAR-1 activation compared with control ( Fig. 2, A and B). We measured the thrombin-induced increase in intracellular Ca 2ϩ in control and thrombin-or TFLLRNPNDK-exposed cells. In the presence of extracellular Ca 2ϩ (1.26 mM), thrombin produced an increase in intracellular Ca 2ϩ ([Ca 2ϩ ] i ) followed by a gradual decline to base-line value after thrombin stimulation (Fig. 2C). In the thrombin or TFLLRNPNDK prestimulated cells, the increase in [Ca 2ϩ ] i elicited by thrombin was significantly greater compared with control (Fig. 2C). We also compared thrombin-induced Ca 2ϩ influx in control and thrombin-stimulated cells. Cells were first challenged with thrombin for endoplasmic reticulum (ER)-store Ca 2ϩ depletion, and then Ca 2ϩ was re-applied to the extracellular medium to assess Ca 2ϩ influx. In both control and experimental group cells (i.e. thrombin-or TFLLRNPNDKpretreated cells), the thrombin-induced increase in the initial peak was similar; however, Ca 2ϩ re-application produced more than a 2-fold increase in Ca 2ϩ influx in the thrombin-or TFLL-RNPNDK-pretreated cells compared with controls (Fig. 2D). These results suggest that the increased TRPC1 protein expression induced by PAR-1 activation contributes to augmented Ca 2ϩ influx in response to ER store Ca 2ϩ depletion.
To address the functional relevance of increased Ca 2ϩ influx associated with TRPC1 expression, we measured thrombin-induced permeability increase in endothelial permeability in control cells and cells exposed to thrombin for 20 h. Transendothelial monolayer resistance (TER) was measured to assess endothelial permeability increase as previously described (20). HUVEC grown to confluence on gold electrodes were incubated with 1% FBS containing medium and exposed to thrombin for 20 h. At 20 h after thrombin, cells were re-challenged with thrombin to assess changes in TER. In control cells (not pretreated with thrombin for 20 h), thrombin produced an ϳ50% decrease in TER and TER return to normal value within 5 h (Fig. 2E). In the thrombin prestimulated cells, thrombin produced an ϳ55% decrease in TER, but the TER failed to return to normal level after thrombin challenge (Fig. 2E), suggesting that increased Ca 2ϩ influx via TRPC1 may augment the permeability increase.
Thrombin Induces TRPC1 Expression via NF-B Activation-PAR-1 activation in endothelial cells is known to increase the expression of inflammatory genes by activating NF-B signaling pathways (17,18). In recent studies we cloned the 5Ј-reg-FIGURE 2. Thrombin exposure induces TRPC1 protein expression and augments store-operated Ca 2؉ influx and increase in endothelial permeability. A, HUVEC grown to confluence were incubated in 1% FBS containing medium for 2 h, and then cells were exposed to 40 M TFLLRNPNDK (PAR-1 agonist peptide), FTLLRNPNDK (PAR-1 control peptide), or thrombin (50 nM) for 20 h. After this treatment cells were washed and lysed using lysis buffer. Total cell lysate proteins (50 g) from each sample were separated on SDS-PAGE and immunoblotted with anti-TRPC1 antibody. The membrane was stripped and immunoblotted with anti-tubulin antibody (bottom panel). Results were representative of four experiments. B, TRPC1 induction -fold was calculated by measuring the ratio of TRPC1 to tubulin. *, p Ͻ 0.05, significantly different from the control. C, thrombin-induced increase in [Ca 2ϩ ] i was measured. HUVEC grown on glass coverslips were stimulated with thrombin (50 nM) or TFLLRNPNDK (40 M) for 20 h as described above. Cells were then loaded with 3 M Fura-2 AM for 30 min at 37°C (15,16). After Fura-2 AM loading, cells were used to measure the thrombin (T)-induced increase in cytosolic Ca 2ϩ ([Ca 2ϩ ] i ). During this measurement, the extracellular medium contained a nominal Ca 2ϩ concentration (1.26 mM). The arrow indicates the time at which cells were stimulated with thrombin (50 nM). D, Ca 2ϩ entry was measured after thrombin-induced ER-stored Ca 2ϩ depletion. The experiment was carried out as described above. Cells exposed to either thrombin or TFLLRNPNDK for 20 h as described above were washed 2 times, placed in Ca 2ϩ -and Mg 2ϩ -free HBSS (Ca 2ϩ free medium), and then stimulated with thrombin (50 nM). After the return of [Ca 2ϩ ] i to base-line levels, 1.5 mM CaCl 2 was re-applied to the extracellular medium to induce Ca 2ϩ influx. Arrows, times at which thrombin (T ) or Ca 2ϩ was added. In this figure bottom tracing shows the Ca 2ϩ entry in response to extracellular Ca 2ϩ addition in the absence of thrombin stimulation in control cells. The experiments were repeated four times, and the data obtained were similar. The results show in C and D are from representative experiments. E, thrombin-induced decrease in TER was measured to assess endothelial permeability. HUVEC were grown to confluence on gold electrode (20) and then treated with or without thrombin (50 nM) in 1% FBS containing medium for 20 h. After this treatment cells were washed, incubated with 1% FBS containing medium for 2 h, and then challenged with thrombin (50 nM) to measure changes in TER (top panel). The arrow indicates the time thrombin (50 nM) or medium was added. In the thrombin-pretreated control (top line) only medium was added. Data from this experiment presented as a maximal decrease in TER at 0.5, 2, and 4 h after thrombin stimulation (bottom panel). Values are the means Ϯ S.E. from four experiments. *, p Ͻ 0.05 compared with 20-h thrombin-pretreated cells with control.

Store-operated Ca 2؉ Influx Induces TRPC1 Expression
ulatory region of hTRPC1 gene (16) and showed that the hTRPC1 regulatory region contains multiple binding sites for the transcription factors NF-B (Fig. 3A). As thrombin activates NF-B signaling in endothelial cells, we addressed whether thrombin-induced TRPC1 expression is dependent on NF-B activation. In the classical NF-B activation signaling pathway, IB phosphorylation and subsequent degradation promotes NF-B translocation to the nucleus and initiates gene transcription (28 -30). To address the role of NF-B signaling, first we transfected TRPC1-Pro-Luc construct with non-phosphorylatable IB␣ dominant negative mutant (IB␣-DN) and measured thrombin-induced TRPC1 expression (16). At 48 h after transfection, cells were stimulated with thrombin. In control cells (transfected only wild type TRPC1-Pro-Luc), thrombin-induced reporter expression was time-dependent. The reporter activity was at maximal level (ϳ6-fold over basal) at 4 h after thrombin stimulation (Fig. 3B). Thrombin-induced TRPC1 expression was prevented by co-expression of IB␣-DN with TRPC1-Pro-Luc in HMEC (Fig. 3B). To address the role of IKK activation, we co-transfected kinase-defective dominant negative IKK␤ mutant (IKK␤-DN) construct with a TRPC1-promoter-luciferase (TRPC1-Pro-Luc) construct in HMEC and measured thrombin-induced TRPC1 expression. We observed that IKK␤-DN mutant expression in a dose-dependent manner inhibited thrombin-induced TRPC1 expression (Fig. 3C), indicating that NF-B activation is important in mediating TRPC1 expression.
Previous studies have shown that interaction of IKK␥/ NEMO with the IKK complex is critical for the activation of the IKK complex and the subsequent activation of NF-B (30). A cell-permeable synthetic peptide (NBD peptide) corresponding to the NEMO N-terminal ␣-helical region was shown to block tumor necrosis factor-␣-induced NF-B activation (30). To further validate the role of IKK activation in the signaling mechanism of thrombin-induced TRPC1 expression, we pretreated HUVEC with WT-NBD peptide or MT-NBD peptide, and we measured thrombin-induced TRPC1 expression. In MT-NBD peptide-treated cells, thrombin-induced TRPC1 mRNA expression was not significantly altered compared with controls (i.e. in the absence of peptide) (Fig. 3D); however, in WT-NBD peptide-treated cells, thrombin-induced TRPC1 mRNA expression was markedly reduced (Fig. 3D). These results collectively suggest that IKK activation is critical in the signaling mechanism of NF-B-dependent TRPC1 expression in response to thrombin in endothelial cells.
Because NF-B activation is necessary for thrombin-induced TRPC1 expression, we constructed TRPC1 promoter mutants to identify NF-B binding sites in the TRPC1 promoter. The schematics of the expression constructs are shown in Fig. 3E. The promoter mutants in pGL2-basic vector were transiently expressed into HMEC. At 48 h cells were treated with thrombin for 4 h, and reporter activity was measured to assess TRPC1 expression as described above. Transfection of the wild type (Ϫ1685 to ϩ40) TRPC1-pro-Luc in HMEC showed ϳ6-fold increase in reporter expression over basal (Fig. 3E). Expression of the construct in which distal NF-B site at Ϫ1623 (⌬1623NF-B) is deleted showed basal Luc activity, indicating that this NF-B site is important for TRPC1 expression. Expression of the construct in which the NF-B site at Ϫ880 (⌬880NF-B) is deleted markedly reduced thrombin-induced Luc expression (Fig. 3E). Deletion of the most proximal NF-B site at Ϫ708 (⌬NF-B708) showed an ϳ25% inhibition on thrombin-induced reporter gene expression (Fig. 3E). These results demonstrate that two NF-B sites located at position Ϫ1623 and Ϫ880 are essential in the mechanism of thrombin-induced TRPC1 expression.
To address further the mechanism of TRPC1 expression, we designed oligonucleotide probes for the three NF-B consensus sites in hTRPC1 promoter to examine by EMSA the in vitro binding of NF-B to the oligonucleotides. The nuclear extracts obtained from thrombin-stimulated HUVEC showed markedly increased DNA binding activity with NF-B (Ϫ1623) oligos compared with control (not stimulated with thrombin) (Fig. 4,  A and B). DNA binding activity increased within 30 min of thrombin treatment and remained elevated for up to 2 h with the Ϫ1623 NF-B oligos (see details under "Experimental Procedures"). The DNA binding of the NF-B oligos at position Ϫ880 was also elevated over basal (Fig. 4, A and B). We failed to detect DNA binding activity of NF-B oligos at position Ϫ708 in nuclear extracts obtained from control and thrombin-stimulated cells (Fig. 4, A and B). Thus, we observed utilizing both promoter deletion analysis and electrophoretic mobility shift HMEC were transfected into hTRPC1-Pro-Luc (wild type promoter, hTRPC1-Pro-Luc (Ϫ1686 to ϩ40)) construct together with pRL/TK plasmid as described under "Experimental Procedures." In addition, the wild type hTRPC1 promoter (1 g/ml), pRL/TK (0.035 g/ml) plasmid, was co-transfected with IB␣-DN (1 g/ml). At 48 h after transfection cells were stimulated with thrombin (50 nM) for 4 h. After this treatment cells were lysed, and reporter activity was measured as described (16,21). Luciferase reporter activity (relative light unit ratio (RLU)/mg of protein) was measured, and relative activity was expressed after subtracting the basal activity at each time point. The mean Ϯ S.E. from four experiments is repeated in triplicate is shown. The asterisks indicate the difference from thrombin-stimulated control (*, p Ͻ 0.005). C, co-expression of dominant negative IKK␤ (IKK␤-DN) inhibits thrombin-induced TRPC1 expression. The wild type hTRPC1 promoter (1 g/ml), pRL/TK (0.035 g/ml) plasmid, was co-transfected with varying concentrations of IKK␤-DN expression constructs. At 48 h after transfection cells were stimulated with thrombin (50 nM) for 4 h. After this treatment cells were lysed, and the reporter activity was measured as described above. The mean Ϯ S.E. from four experiments repeated in triplicate is shown. The asterisks indicate the difference from thrombin-stimulated control (*, p Ͻ 0.005). Cell lysates (25 g of protein) were immunoblotted with anti-IKK␤ antibody to assess the expression of IKK␤-DN (top panel). D, NEMO-binding domain peptide inhibits thrombin-induced TRPC1 expression in endothelial cells. HUVEC grown to confluence were incubated with cell-permeable wild type NBD (Wt-NBD) or mutant NBD (Mt-NBD) peptides (150 M) for 2 h in serum-free medium. Cells were then challenged with thrombin (50 nM) for the indicated times. Total RNA was isolated, and RT-PCR was performed to determine the expression of TRPC1 and GAPDH (see details under "Experimental Procedures"). The experiment was repeated three times with similar results. E, localization of thrombin-responsive NF-B sites in the hTRPC1 promoter. Deletion mutant analysis was utilized to identify the NF-B binding sites in the 5Ј-regulatory region of the hTRPC1 gene. PCR-based method was used to prepare the NF-B deletion constructs from the wild type hTRPC1-Pro-Luc described under "Experimental Procedures." The wild type and the NF-B site-deleted (⌬NF-B Ϫ1623, ⌬NF-B Ϫ880, and ⌬NF-B Ϫ708) constructs were transfected into HMEC to assess TRPC1 promoter-driven reporter expression. At 48 h after transfection, cells were stimulated with thrombin (50 nM) for 4 h. After this treatment cells were lysed, and the reporter expression was measured (16,21). The mean Ϯ S.E. from four experiments repeated in triplicate is shown. The asterisk indicates the difference from the (Ϫ1685 to ϩ40) TRPC1-Pro-Luc construct (*, p Ͻ 0.001).

Store-operated Ca 2؉ Influx Induces TRPC1 Expression
assays for which NF-B consensus sites located at position Ϫ1623 and Ϫ880 in TRPC1 promoter are critical for thrombininduced TRPC1 expression.
We next carried out supershift assays with specific antibodies to p65, p52, p50, RelB, and c-Rel to identify the NF-B protein binding to the TRPC1 promoter in response to thrombin. Nuclear extracts prepared from HUVEC treated with thrombin for 60 min were incubated with the NF-B protein-specific antibodies, and then labeled NF-B1 double-stranded probe was added to determine the supershift of NF-B complex (see details under "Experimental Procedures"). The incubation of anti-p65 antibody resulted in a supershift of the DNA-protein complexes (Fig. 4C), whereas the addition of antibodies to p52, anti-p50, RelB, and c-Rel had no effect (Fig. 4C). We also performed supershift assay using NF-B2 double-stranded probe. We observed a supershift of the DNA-protein complex only with anti-p65 antibody (data not shown). These results suggest that p65 homodimer binds to the TRPC1 promoter in response to thrombin to initiate TRPC1 transcription in endothelial cells.
Ca 2ϩ Signaling Induces TRPC1 Expression-Because thrombin-induced TRPC1 expression was dependent on NF-B activation, we addressed the role of Ca 2ϩ signaling in the mechanism of thrombin-induced TRPC1 expression. In this experiment we first measured the effect of cell-permeable Ca 2ϩ chelator BAPTA/AM on the thrombin-induced increase in TRPC1 mRNA expression. HUVEC were incubated for 30 min with 20 M BAPTA/AM and challenged with thrombin for the indicated periods. BAPTA/AM prevented a thrombin-induced rise in cytosolic Ca 2ϩ in HUVEC (data not shown). In control cells, PAR-1 signaling increased TRPC1 expression without altering GAPDH expression (Fig. 5A); however, pretreatment of cells with BAPTA/AM failed to increase TRPC1 mRNA expression in response to PAR-1 activation (Fig. 5B). Next we determined the effect of other G protein-coupled receptor agonists such as bradykinin and histamine (known to increase cytosolic Ca 2ϩ level in endothelial cells (34 -36)) and measured TRPC1 transcript expression. We observed that these agonists challenge increased TRPC1 mRNA expression in a time-dependent manner (Fig. 5, C and D). We treated HUVEC with thapsigargin (Ca 2ϩ -ATPase inhibitor) to increase intracellular Ca 2ϩ levels and measured TRPC1 expression. Thapsigargin also increased TRPC1 transcript expression (Fig. 5E). To further address the role of Ca 2ϩ signaling in the mechanism of TRPC1 expression, we treated HUVEC with the inositol 1,4,5trisphosphate receptor antagonist 2-APB to prevent an increase in intracellular Ca 2ϩ and measured PAR-1 activationmediated TRPC1 mRNA expression. 2-APB (75 M) inhibited the thrombin-induced rise in intracellular Ca 2ϩ in HUVEC (data not shown). We also observed that 2-APB treatment prevented thrombin-induced TRPC1 transcript expression in HUVEC (Fig. 5F), whereas 2-APB treatment had no effect on GAPDH expression (Fig. 5F). In addition, PAR-1 signaling-induced reporter expression in the presence and absence of 2-APB was measured in HMEC-transfected wild type hTRPC1-Pro-Luc plasmid. We observed that either thrombin-or PAR-1-specific agonist peptide (TFLLRNPNDK) stimulation increased reporter expression ϳ6-fold over basal (Fig. 5G); however, in 2-APB-treated cells PAR-1 activation-induced reporter expression was markedly reduced (Fig. 5G), indicating that Ca 2ϩ signaling is critical in the mechanism of thrombininduced TRPC1 expression in endothelial cells.
Because 2-APB prevented the thrombin-induced TRPC1 expression, we addressed whether store-operated Ca 2ϩ influx (i.e. Ca 2ϩ influx through TRPC1) signaling is necessary for FIGURE 4. Identification of thrombin-responsive DNA binding activity of NF-B in hTRPC1 promoter. In A, the hTRPC1 promoter-specific NF-B oligonucleotide probes used for EMSA were shown. The underlined sequence represents the consensus NF-B sites in hTRPC1 promoter. In B, HUVEC grown to confluence were stimulated with 50 nM thrombin for 0.5, 1, and 2 h in serum-free medium. After this treatment nuclear extracts were prepared, and EMSA was performed using 32 P-labeled double-stranded oligonucleotide containing the TRPC1 promoter-specific three NF-B sequences and assayed for NF-B-DNA binding. The two upstream NF-B binding sites showed NF-B-DNA complex formation in response to thrombin. The experiment was repeated four times. The results from representative experiments are shown in this figure. In C, nuclear extracts prepared from HUVEC stimulated for 1 h with thrombin were incubated with antibodies specific to NF-B proteins for 30 min at room temperature before the addition of radiolabeled NF-B1 probe. The experiment was repeated four times. The results from representative experiments are shown in this figure.
TRPC1 expression in endothelial cells. To study whether Ca 2ϩ influx signaling is necessary for thrombin-induced NF-B activation-dependent TRPC1 expression, we performed EMSAs to determine the DNA binding activities of NF-B. In this experiment, HUVEC were incubated in the presence and absence of Ca 2ϩ in the extracellular medium, and cells were stimulated with thrombin. Nuclear extracts prepared from these cells were used for NF-B-DNA binding activities. We used TRPC1 promoter NF-B sites (NF-B1 (Ϫ1623 to Ϫ1614))and NF-B2 (Ϫ880 to Ϫ871))-specific oligos as probes for EMSAs (Fig. 4A). Thrombin increased NF-B-DNA binding activities in the presence of Ca 2ϩ influx in a time-dependent manner (Fig. 6, A and B), whereas thrombin failed to increase NF-B-DNA activities in the absence of Ca 2ϩ influx (Fig. 6, A and B). To address the role of Ca 2ϩ influx in this response, we measured thrombin-induced TRPC1 mRNA expression in the presence and absence of store-operated Ca 2ϩ influx. We observed thrombin-induced TRPC1 mRNA expression in the presence of Ca 2ϩ influx (Fig. 6C), whereas this response was abrogated in the absence of Ca 2ϩ influx (Fig. 6C). These results collectively show that Ca 2ϩ influx plays a critical role in the signaling mechanism of thrombin-induced TRPC1 expression.
Ca 2ϩ Influx-induced PKC␣ Activation Induces TRPC1 Expression-We next addressed the possible role of PKC activation in the signaling mechanism of thrombin-induced TRPC1 expression. In this experiment, HUVEC were incubated with PKC inhibitor calphostin C (a pan-PKC inhibitor) and measured thrombin-induced NF-B activation by EMSA. We used NF-B1 (Fig. 4A) site-specific oligos as probes. In control cells thrombin increased NF-B-DNA complex formation in a time-dependent manner, whereas thrombin-induced increased NF-B-DNA complex formation was markedly reduced in calphostin C-treated cells (data not shown). Because PKC signaling was required for NF-B activation in response to thrombin, we addressed the possible role of the Ca 2ϩ -dependent PKC␣ isoform in the signaling of TRPC1 expression. In this experiment HUVEC were exposed to PKC␣-specific inhibitor Gö6976, and DNA-NF-B binding using EMSA was determined. The thrombin-induced DNA-NF-B binding activity was markedly reduced in Gö6976-treated cells (Fig. 7A) compared with control (not treated with Gö6976). Also the thrombin-induced TRPC1 transcript expression was determined in FIGURE 5. PAR-1-activated Ca 2؉ signaling is required for TRPC1 expression in endothelial cells. HUVEC grown to confluence on culture dishes were washed and incubated in serum-free medium (Ca 2ϩ containing HBSS) for 2 h at 37°C. After this treatment cells were incubated in the absence (A) or presence (B) of 20 M BAPTA/AM for 30 min and washed, and then cells were challenged with thrombin (50 nM) for 0, 2, 4, and 6 h. After thrombin chal-lenge, total RNA was isolated using Trizol reagent, and RT-PCR was performed to determine TRPC1 mRNA expression. PCR amplification of GAPDH was also performed for each sample. HUVEC incubated with serum-free medium for 2 h were challenged with 1 M bradykinin (C), 10 M histamine (D), or 1 M thapsigargin (E) for 0, 2, and 4 h. After this, total RNA was isolated, and RT-PCR was performed to determine TRPC1 and GAPDH expression. In F, HUVEC were incubated with 75 M 2-APB for 30 min, and then cells were challenged with thrombin (50 nM) for 0, 2, 4, and 6 h. The experiments in A, B, C, D, E, and F were repeated three times, and the results from representative experiment are shown. In G, wild type hTRPC1-Pro-Luc construct was transfected into HMEC as described under "Experimental Procedures." At 48 h after transfection cells were first incubated in the presence and absence of 75 M 2-APB for 30 min and then stimulated with thrombin (50 nM) or TFLLRNPN (40 M) for 4 h. After this treatment cells were lysed, and reporter activity was measured (16,21). The results are the mean Ϯ S.E. from four separate experiments shown. *, p Ͻ 0.005, significantly different from thrombin-or TFLLRNPNDK-challenged control. RLU, relative light units.
control and Gö6976-treated cells. In control cells thrombin increased TRPC1 mRNA expression in a time-dependent manner (Fig. 1A); however, in Gö6976-treated cells, thrombin-induced TRPC1 expression was significantly reduced (Fig. 7B). We performed additional experiments to address the role of PKC␣ signaling in the mechanism of TRPC1 expression. First, dominant negative PKC␣ (PKC␣-DN) mutant expression construct with TRPC1-Pro-Luc were co-transfected in HMEC, and thrombin-induced reporter expression was recorded. The thrombin-induced TRPC1 expression was prevented by co-expression of PKC␣-DN mutant with TRPC1-Pro-Luc (Fig. 7C). Second, PKC␣-specific siRNA was transfected to down-regulate endogenous PKC␣ expression. In PKC␣-siRNA trans-fected cells, PKC␣ protein expression was inhibited Ͼ80% compared with control (scrambled sequence-transfected cells) (Fig. 7D). PKC␦ is known to be involved in the signaling mechanism of NF-B activation-dependent ICAM-1, and VCAM-1 expression in endothelial cells (17,37) was not altered by transfecting PKC␣-siRNA (Fig. 7D). We measured TRPC1 transcript expression in control and the PKC␣-siRNA transfected cells. Thrombin-induced TRPC1 expression was prevented in after PKC␣-siRNA transfection as compared with cells transfected with scrambled siRNA (Fig. 7E). We also measured ICAM-1 transcript expression in this experiment. The thrombin-induced ICAM-1 transcript expression was not significantly altered in PKC␣-siRNA-transfected cells compared with control (data not shown). These results collectively show that Ca 2ϩ influx induced by ER store Ca 2ϩ depletion activates the PKC␣ to increase TRPC1 expression signaling via NF-B in endothelial cells.

DISCUSSION
Thrombin, a serine protease, catalyzes the conversion of fibrinogen to fibrin (38,39). Thrombin also activates a variety of cell types, including endothelial cells, smooth muscle cells, and leukocytes (40,41); for example, thrombin induces leukocyte adhesion to endothelial cell surface by increasing the cell surface expression of adhesion molecules ICAM-1, VCAM-1, P-selectin, and E-selectin (18,42,43). Thrombin promotes the expression of ICAM-1 and VCAM-1 via the activation of the transcription factor NF-B in endothelial cells (18,37,42). We and others have shown that thrombin also causes endothelial cell shape change and thereby increases endothelial permeability via opening of interendothelial junctions (3). Thrombin mediates these cellular responses by activating the G proteincoupled PAR-1 on the endothelial cell surface (24,25,40). We showed that ligation of PAR-1 increased endothelial permeability by depletion of ER Ca 2ϩ stores and the subsequent activation of Ca 2ϩ influx via SOC (6,15,16). The TRPC isoform TRPC1 is a prominent SOC present in human vascular endothelial cells (15,16). In the present study we observed that thrombin induces TRPC1 mRNA and protein expression via NF-B activation such that the resultant TRPC1 expression augmented the store-operated Ca 2ϩ influx and increased endothelial permeability.
NF-B is composed of homodimers and heterodimers of five different proteins (p50, p52, p65 (RelA), RelB, cRel) (44). These dimers exist in the cytoplasm in an inactive form bound to the inhibitory protein I-B (IB) (28 -31). Agonistinduced signals activate IB kinases ␣ and ␤ (28 -30, 44), which in turn phosphorylate serine residues 32 and 36 of IB␣ and serine residues 19 and 23 of IB␤, respectively (28 -31). These phosphorylation events lead to proteolytic degradation of IB and dissociation of NF-B, and the released NF-B via its nuclear localization signal translocates to the nucleus to induce gene transcription (28 -31). Because the hTRPC1 promoter contains multiple binding sites for NF-B (16), we examined the possibility that the thrombin-induced TRPC1 expression was the result of activation of NF-B. We first addressed the role of NF-B in the mechanism of TRPC1 expression. We showed that co-expres-FIGURE 6. Thrombin-induced Ca 2؉ influx is required for NF-B binding to TRPC1 promoter and transcription. HUVEC grown to confluence were incubated with serum-free medium (HBSS) for 2 h. Cells were then washed twice with HBSS (ϩCa 2ϩ ) or Ca 2ϩ -free HBSS (ϪCa 2ϩ ). After washing, cells were incubated with either ϩCa 2ϩ medium or ϪCa 2ϩ medium and then stimulated with thrombin (50 nM) for the indicated periods. We have shown previously that the absence of Ca 2ϩ in the extracellular medium prevented thrombin-induced Ca 2ϩ influx in endothelial cells (6,15,16). Cells incubated with ϩCa 2ϩ medium or ϪCa 2ϩ medium were stimulated with thrombin (50 nM) for 0, 0.5, 1, and 2 h at 37°C. After this treatment, nuclear extracts were prepared, and EMSA was performed using 32 P-labeled double-stranded oligos containing the TRPC1-specific NF-B1 (A) and NF-B2 (B) sequences (see Fig. 4A). The experiment was repeated three times, and results shown are from representative experiments. In C, the experiment was carried out as described above except that after thrombin stimulation, total RNA was isolated, and RT-PCR was performed to determine TRPC1 and GAPDH mRNA expression. The experiment was repeated three times with similar results.
sion of either non-phosphorylatable IB␣-DN mutant or kinase-defective IKK␤-DN mutant with wild type hTRPC1-Pro-Luc reporter prevented thrombin-induced TRPC1 expression in endothelial cells. Furthermore, we showed that thrombin-induced TRPC1 mRNA expression was markedly reduced by treating endothelial cells with NEMO-binding domain peptide. These findings suggest that NF-B signaling is a key requirement for thrombin-induced TRPC1 expression.
To determine the promoter NF-B binding sites responsible for TRPC1 transcription, we made NF-B deletion mutant constructs utilizing the wild type hTRPC1-Pro-Luc construct. We transfected these constructs in endothelial cells to identify the thrombin-induced TRPC1 promoter-driven reporter expression. Deletion of the upstream NF-B site at position Ϫ1623 or at Ϫ880 from the transcription initiation site prevented the thrombininduced reporter expression, whereas deletion of downstream site at Ϫ708 had only a minimal effect. Thus, thrombin appears to induce TRPC1 expression via the formation NF-B-DNA protein complex upstream of these two TRPC1 promoter NF-B consensus sites. To address further the specificity of NF-B binding to TRPC1 5Ј-regulatory region, we performed EMSAs utilizing the NF-B consensus sequence present in TRPC1 5Ј-regulatory region. Thrombin increased DNA-NF-B complex formation with the TRPC1 promoter consensus NF-B sites at position Ϫ1623 (NF-B1) and Ϫ880 (NF-B2); however, thrombin failed to induce DNA-NF-B complex with consensus NF-B site at Ϫ703 (NF-B3). These results, in agreement with the mutant TRPC1-Pro-Luc expression studies described above, indicate the importance of NF-B binding sites at position Ϫ1623 (NF-B1) and Ϫ880 (NF-B2) in regulating the transcription of TRPC1.
Previous studies have shown that NF-B p65 homodimer binds to VCAM-1 and ICAM-1 promoters in thrombin-stimulated endothelial cells (17,18). To identify the NF-B proteins interacting with TRPC1 promoter, we carried out supershift assays utilizing antibodies specific to NF-B proteins. We observed a supershift of NF-B-DNA complex with anti-p65 antibody in thrombinstimulated endothelial cells, suggesting that p65 homodimer FIGURE 7. A, pharmacological inhibition of PKC␣ prevents thrombin-induced NF-B binding to TRPC1 promoter. HUVEC grown to confluence were incubated in serum-free medium for 2 h at 37°C. After this incubation cells were incubated in the presence and absence of PKC␣ inhibitor Gö 6976 at the indicated concentrations for 30 min. Cells were then stimulated with 50 nM thrombin for 60 min. After this treatment nuclear extracts were prepared, and EMSA was performed using 32 P-labeled double-stranded oligonucleotide containing the TRPC1-specific NF-B1 sequences (see Fig. 4A) and assayed for NF-B-DNA binding. The experiment was repeated three times with similar results. B, PKC␣ inhibitor prevents thrombin-induced TRPC1 transcript expression; HUVEC grown to confluence were incubated in serum-free medium for 2 h at 37°C. After this incubation cells were incubated in the presence and absence of PKC␣-specific inhibitor Gö 6976 (1 M) for 30 min at 37°C. Cells were then stimulated with thrombin (50 nM) for 0, 2, 4, and 6 h. After thrombin treatment, total RNA isolated, and RT-PCR was performed to determine TRPC1 and GADH expression as described under "Experimental Procedures." Results are representative of three experiments. C, PKC␣ dominant negative (PKC␣-DN) mutant expression prevents thrombin-induced TRPC1 expression. HMEC were transfected with hTRPC1-Pro-Luc and pRL/TK or hTRPC1-Pro-Luc and pRL/TK plasmids co-transfected with PKC␣-DN mutant construct with varying concentrations as described under "Experimental Procedures." At 48 h after transfection cells were stimulated with thrombin (50 nM) for 4 h. After this treatment cells were lysed, and the reporter activity was measured. Also, the cell lysates were immunoblotted with anti-PKC␣ antibody to determine PKC␣-DN protein expression (upper panel). The mean Ϯ S.E. from four experiments repeated in triplicate is shown. The asterisks indicate the difference from thrombin-stimulated control (*, p Ͻ 0.001). RLU, relative light units. D and E, down-regulation of endogenous PKC␣ inhibits thrombin-induced TRPC1 expression. HUVEC were transfected with either scrambled siRNA sequence (Scr) or PKC␣-siRNA as described under "Experimental Procedures." In D, 48 h after transfection, cells lysed, and cell lysate proteins (50 g) were immunoblotted with the indicated antibodies. The experiment was repeated three times. The results from representative experiments are shown in this figure. In E, 48 h after transfection cells were stimulated with thrombin (50 nM) for 0, 2, 4, and 6 h. After thrombin treatment, total RNA was isolated, and RT-PCR was performed to determine TRPC1 and GADH expression as described under "Experimental Procedures." The results from representative experiments are shown in this figure (upper panel). TRPC1 induction -fold was calculated by measuring the ratio of TRPC1 to GAPDH (lower panel). The results are the mean Ϯ S.E. from four separate experiments shown. *, p Ͻ 0.05, significantly different from the control (scrambled siRNA-transfected). JULY 28, 2006 • VOLUME 281 • NUMBER 30 binds to TRPC1 promoter in response to thrombin to initiate TRPC1 transcription in endothelial cells.

Store-operated Ca 2؉ Influx Induces TRPC1 Expression
Because TRPC1 promoter sequence has multiple NF-B binding sites and thrombin-induced TRPC1 expression requires NF-B activation, we addressed the possible role of thrombin-induced increase in cytosolic Ca 2ϩ in signaling TRPC1 expression. These studies were based on the premise that a rise in cytosolic Ca 2ϩ would be involved in signaling NF-B-dependent gene expression (31)(32)(33). The activation of PAR-1 caused a rapid and transient increase cytosolic Ca 2ϩ concentration (([Ca 2ϩ ] i ) (phase I) secondary to the release of ER-stored Ca 2ϩ and subsequent Ca 2ϩ entry occurring via TRPC1 activated by store depletion (phase II) (6,15,16). The inositol 1,4,5-trisphosphate receptor antagonist 2-APB was shown to prevent both the phase I and phase II increases in [Ca 2ϩ ] i (45,46). Strikingly, we also observed that exposure of endothelial cells to 2-APB prevented the PAR-1 activation-mediated TRPC1 expression. In other studies, we transfected TRPC1-Pro-Luc construct in endothelial cells and measured PAR-1-activated reporter expression. In this experiment, 2-APB treatment also significantly reduced thrombin-induced reporter expression, indicating that Ca 2ϩ is a critical signal mediating TRPC1 expression.
We have shown previously that the thrombin-induced Ca 2ϩ entry signal is a requirement for increased endothelial perme-ability (3,6,15). Thus, to address the role of Ca 2ϩ entry in the mechanism of TRPC1 expression, Ca 2ϩ was omitted from the extracellular medium such that there is no Ca 2ϩ influx after ER-stored Ca 2ϩ depletion. Both thrombin-induced TRPC1 mRNA expression and NF-B interaction with the TRPC1 promoter were markedly reduced in the absence of Ca 2ϩ influx, indicating that store-operated Ca 2ϩ influx via TRPC1 plays an important role in the signaling TRPC1 expression in endothelial cells.
PKC-induced activation of NF-B pathways can also signal the expression of inflammatory genes such as ICAM-1 and VCAM-1 in endothelial cells (17,19,37). Three different families of PKC isoforms (conventional, novel, and atypical) have been identified based on their domain structure and their ability to respond to Ca 2ϩ and diacylglycerol (DAG) (47). The "conventional" PKC isoforms (␣, ␤I, ␤II, and ␥) require DAG and Ca 2ϩ for activation (47). The "novel" PKC isoforms (␦, ⑀, , and ) require only DAG for activity (47). The "atypical" PKC isoforms (, /, and ) are activated independently of Ca 2ϩ or DAG (47). Human vascular endothelial cells in culture express PKC␣, PKC␦, PKC⑀, and PKC (4, 48). Rahman et al. (37) showed that PKC␦ activation is required for thrombin-induced NF-B activation and ICAM-1 gene expression in human vascular endothelial cells. We have shown that PAR-1 activation increases Ca 2ϩ -dependent PKC␣ activity and thereby contributes to signaling the increase endothelial permeability (4). In this study we addressed the possibility that PKC signaling was involved in the mechanism of thrombin-induced TRPC1 expression. We showed that the pan-PKC inhibitor calphostin C prevented the thrombin-induced NF-B binding to TRPC1 promoter, indicating that PKC signaling is crucial in activating TRPC1 transcription. Because the Ca 2ϩ influx signal is required for NF-B activation and TRPC1 gene expression, we addressed the possible role of Ca 2ϩ -dependent PKC␣ activation in the mechanism of thrombin-induced TRPC1 expression. The PKC␣-specific inhibitor Gö6976 prevented the thrombin-induced TRPC1 transcript expression. Moreover, co-transfection of PKC␣-DN expression construct with wild type TRPC1-Pro-Luc plasmid in endothelial cells prevented the thrombin-induced TRPC1 promoter-driven reporter expression. Also down-regulation of endogenous PKC␣ expression by transfecting PKC␣-siRNA prevented the thrombin-induced TRPC1 expression. Taken together, these results demonstrate the essential role of Ca 2ϩ -dependent PKC␣ activation in signaling thrombin-induced TRPC1 expression in endothelial cells.
In summary, we show herein that PAR-1 activation of storeoperated Ca 2ϩ influx signals PKC␣-dependent NF-B activation, which in turn induces TRPC1 gene transcription in endothelial cells (Fig. 8). This feed-forward mechanism of TRPC1 expression is important in augmenting Ca 2ϩ influx in response to Ca 2ϩ store depletion and, thus, in signaling increased endothelial permeability. The present results demonstrating the role of Ca 2ϩ signaling in inducing TRPC1 expression may inform a novel therapeutic target in inflammatory diseases.