Ca2+ Entry via TRPC Channels Is Necessary for Thrombin-induced NF-κB Activation in Endothelial Cells through AMP-activated Protein Kinase and Protein Kinase Cδ*

The transient receptor potential canonical (TRPC) family channels are proposed to be essential for store-operated Ca2+ entry in endothelial cells. Ca2+ signaling is involved in NF-κB activation, but the role of store-operated Ca2+ entry is unclear. Here we show that thrombin-induced Ca2+ entry and the resultant AMP-activated protein kinase (AMPK) activation targets the Ca2+-independent protein kinase Cδ (PKCδ) to mediate NF-κB activation in endothelial cells. We observed that thrombin-induced p65/RelA, AMPK, and PKCδ activation were markedly reduced by knockdown of the TRPC isoform TRPC1 expressed in human endothelial cells and in endothelial cells obtained from Trpc4 knock-out mice. Inhibition of Ca2+/calmodulin-dependent protein kinase kinase β downstream of the Ca2+ influx or knockdown of the downstream Ca2+/calmodulin-dependent protein kinase kinase β target kinase, AMPK, also prevented NF-κB activation. Further, we observed that AMPK interacted with PKCδ and phosphorylated Thr505 in the activation loop of PKCδ in thrombin-stimulated endothelial cells. Expression of a PKCδ-T505A mutant suppressed the thrombin-induced but not the TNF-α-induced NF-κB activation. These findings demonstrate a novel mechanism for TRPC channels to mediate NF-κB activation in endothelial cells that involves the convergence of the TRPC-regulated signaling at AMPK and PKCδ and that may be a target of interference of the inappropriate activation of NF-κB associated with thrombosis.

important role both in the mechanism of host defense as well as inthepathogenesisofinflammationandtissueinjury (1).NF-Bdependent expression of intercellular adhesion molecule-1 (ICAM-1; CD54) on the surface of endothelial cells mediates stable polymorphonuclear leukocyte adhesion and transendothelial migration of polymorphonuclear leukocyte (1). Mediators as diverse as TNF-␣, lipopolysaccharide, and thrombin induce ICAM-1 expression by activating NF-B signaling in endothelial cells (1)(2)(3)(4). There are, however, differences in the activation mechanism. Thrombin, in contrast to TNF-␣ and lipopolysaccharide, signals NF-B activity by activating its G protein-coupled receptor, PAR-1 (protease-activated receptor-1) (3,4). Also, thrombin may be crucial in linking the activation of the coagulation cascade to the innate immune and inflammatory responses regulated by NF-B (5,6).
A rise in [Ca 2ϩ ] i was found to signal the activation of NF-B (16,17). However, the targets of Ca 2ϩ signaling that mediate NF-B activation are unknown. PAR-1 activation in endothelial cells mediates increase in [Ca 2ϩ ] i by activating the G q/11 -phospholipase C pathway that results in depletion of endoplasmic reticulum (ER) Ca 2ϩ stores and the subsequent store depletiondependent Ca 2ϩ entry across the plasma membrane (18). This accounts for the sustained rise in [Ca 2ϩ ] i required for the activation of NF-B. We (13,19,20) and others (21)(22)(23) have identified members of the transient receptor potential canonical (TRPC) family of channels that are essential for Ca 2ϩ entry induced by PAR-1 agonist. The TRPC family contains seven isoforms (TRPC1 to -7). Recent studies have shown that TRPC1 forms a complex with STIM1 and ORAI1 and is involved in store-operated Ca 2ϩ entry (24,25). Primary endothelial cells in culture express multiple TRPC isoforms, TRPC1 to -7 (18,19). We have shown that human endothelial cells predominantly express TRPC1 and contribute to thrombin-induced Ca 2ϩ entry (18,19). Mouse lung endothelial cells predominantly express Trpc4 (20,21). Further, we have shown that in Trpc4 knock-out (Trpc4 Ϫ/Ϫ ) mouse lung endothelial cells, the thrombin-induced Ca 2ϩ entry was markedly reduced (20). Here, we show that thrombin-induced Ca 2ϩ entry via TRPC channels activates the calmodulin (CaM) kinase kinase ␤ (CaMKK␤), AMPK, and PKC␦ signaling pathway to induce p65/RelA nuclear translocation and transactivation. Further, we show that AMPK activation is required for PKC␦ phosphorylation at Thr 505 in its activation loop to mediate NF-B activation. Thus, Ca 2ϩ entry via TRPC channels is an essential signal responsible for activating NF-B in response to thrombin and thereby serves as a crucial link between activation of the coagulation cascade and innate immunity and inflammation.
Cell Culture-Primary human pulmonary arterial endothelial cells (HPAECs) obtained from Cambrex Bio Science (Walkersville, MD) were grown in endothelial growth medium-2 supplemented with 10% FBS as described (13,19). HPAECs between passages 4 and 7 were used for all described experiments. Lung endothelial cells from 129SvJ (wild type) and Trpc4 Ϫ / Ϫ mice were isolated and cultured as described by us (20). Cells passaged between 3 and 4 times were used in experiments. Mouse lung endothelial cells were characterized by their cobblestone morphology, PECAM-1 (platelet/endothelial cell adhesion molecule-1) (or CD31) expression, and Dil-Ac-LDL uptake (20).
Nuclear Protein Extraction-HPAECs grown to confluence were incubated with 1% FBS-containing medium for 2 h prior exposure to different pharmacological inhibitors for 30 min. Nuclear extracts were prepared from endothelial cells and mouse lung tissue using the method described before (13). After thrombin exposure, cells were scraped and washed twice with ice-cold Tris-buffered saline. Cells were then homogenized with 400 l of solution A (10 mM KCl, 10 mM Hepes, pH 7.9, 0.1 mM EDTA, pH 8.0, 0.1 mM EGTA, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 5 g/ml aprotinin, and 5 g/ml leupeptin). The cells were lysed with 10 strokes using a Dounce homogenizer and centrifuging at 14,000 rpm for 1 min. The supernatant was transferred to a new Eppendorf tube (the cytoplasmic fraction). Nuclear pellets were then resuspended in 100 l of solution B (20 mM Hepes, pH 7.9, 0.4 NaCl, 1 mM EDTA, pH 8.0, 1 mM EGTA, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 5 g/ml aprotinin, and 5 g/ml leupeptin) and then incubated on ice for 20 min. The nuclei were pelleted by centrifugation at 14,000 rpm for 1 min. Supernatants containing the nuclear proteins were aliquoted in small fractions and stored at Ϫ70°C. Nuclear extract from mouse lung tissue was also prepared for electrophoretic mobility shift assay. Frozen lung tissue was thawed on ice and homogenized in Buffer A. Other steps used were similar to endothelial cells described above.
Electrophoretic Mobility Shift and Supershift Assays-NF-B oligonucleotide containing the NF-B consensus sequence 5Ј-AGTTGAGGGGACTTTCCCAGGC-3Ј was labeled with [␥-32 P]ATP using T4 polynucleotide kinase for 20 min at 37°C in the presence of 50 g of poly(dI-dC) and 10 mM Tris-HCl buffer, pH 7.5, containing 50 mM NaCl, 0.5 mM EDTA, 0.5 mM dithiothreitol, 4% (w/v) glycerol, and 1 mM MgCl 2 . The nuclear extracts (15 g of protein) were incubated with the radiolabeled NF-B oligonucleotide (80,000 cpm/reaction) and subjected to electrophoresis on a 6% native gel, dried onto Whatman paper, and then exposed to autoradiography. For supershift assays, nuclear extracts were incubated with indicated antibodies for 30 min subsequent to the addition of radiolabeled oligonucleotide probe (13).
Expression of Reporter Constructs-HPAECs grown to ϳ80% confluence in 6-well culture plates were used for reporter construct transfection. Plasmid DNA mixtures containing 1 g of NF-B-Luc in a minimal E1B vector and 0.035 g of pRL/TK were transfected using Lipofectamine (19). In some experiments, PKC␦-WT or PKC␦-T505A mutant plasmid DNA (1 g/well) co-transfected with NF-B-Luc and pRL/TK. The mixture containing Lipofectamine-DNA complexes was diluted with 0.8 ml of Opti-MEM I before being added to HPAECs and prewashed two times with Opti-MEM I for 2-4 h. To end transfection, 2 ml of endothelial growth medium supplemented with 10% FBS was added to each well.
Dual Luciferase Reporter Assay-At 24 h after transfection, the cells were incubated in 1% FBS medium containing for 2 h and then stimulated with thrombin. In some experiments, cells were pretreated with 40 M compound C for 30 min before thrombin treatment. After stimulation, cells were lysed, and 20 l of lysate was used to measure reporter gene expression. Firefly (Photinus pyralis) and sea pansy (R. reniformis) luciferase activity were assayed by the dual luciferase reagent assay system (19).
siRNA Transfection-HPAECs grown to ϳ70% confluence on gelatin-coated culture dishes were transfected with either 100 nM AMPK␣1 siRNA, TRPC1 siRNA, or Sc-siRNA using Santa Cruz Biotechnology transfection reagents. Seventy-two hours after transfection, cells were used for experiments.
Immunoblotting-Cells grown to confluence were incubated in Ca 2ϩ -containing Hanks' balanced salt solution for 2 h at 37°C. Following thrombin treatment in the presence or absence of Ca 2ϩ in Hanks' balanced salt solution, cells were washed, and then cytoplasmic and nuclear fractions were isolated separately as described above. Equal amounts of protein were resolved using SDS-PAGE on a 10% separating gel and subsequently transferred to polyvinylidene difluoride membrane. Membranes were incubated in blocking buffer (5% nonfat milk in 10 mM Tris-HCl, pH 7.4, 150 mM NaCl, and 0.05% Tween 20) for 60 min at room temperature. Membranes were then incubated with the indicated antibody in diluted blocking buffer overnight at 4°C. After two washes, the membranes were incubated with horseradish peroxidase-conjugated goat antimouse or -rabbit for 60 min at room temperature. Protein bands were detected by enhanced chemiluminescence.
Reverse Transcription (RT)-PCR-Confluent endothelial cell monolayers were washed with serum-free medium and then incubated with 1% FBS-containing medium for 2 h. After this period, the cells were treated with thrombin in 1% FBS-containing medium for various time intervals. Following treatment, total RNA was isolated using TRIzol reagent. RT was performed using oligo(dT) primers and superscript reverse transcript (Invitrogen), following the manufacturer's recommendation. ICAM-1 transcript expression in human and mouse endothelial cells was amplified using the following primer sets: human (forward, 5Ј-AGCAATGTGCAAGAAGATAGCCAA-3Ј; reverse, 5Ј-CGGCCTGCGTGTCCACC-3Ј) and mouse (forward, 5Ј-AGCATTTACCCTCAGCCACTTCCT-3Ј; reverse, 5Ј-TGAGGCTCGATTGTTCAGCTGCTA-3Ј). PCR product was 459 bp for human and 630 bp for mouse ICAM-1. The reaction conditions included an initial 95°C for 5 min and then 30 cycles of (94°C for 30 s, 59°C for 30 s, and 68°C for 1 min). Amplification of GAPDH used the following primers: forward, 5Ј-TATCGTGGAAGGACTCATGACCC-3Ј; reverse, 5Ј-TACATGGCAACTGTGAGGGG-3Ј. Reaction conditions were as follows: 30 cycles (94°C for 45 s, 60°C for 45 s and 72°C for 2 min). PCR product were resolved by using 1.5% agarose gel and identified by ethidium bromide staining. Normalization of ICAM-1 expression was achieved by comparing the expression of GAPDH for the matching sample (19).
Perfused Mouse Lung Preparation-According to the approved protocol of University of the Illinois Animal Care Committee, male 129SvJ (wild type) and Trpc4 knock-out (Trpc4 Ϫ/Ϫ ) mice weighing 30 -35 g were used to make isolated perfused lung preparation as described (27). Lungs were perfused for 20 min to attain the isogravimetric condition, and then lungs were perfused for an additional 30 min with or without thrombin. At the end of this period, lungs were frozen in liquid nitrogen and then used for nuclear extract preparation, as described above.
Immunoprecipitation-HPAECs grown to confluence exposed to thrombin were washed three times with phosphatebuffered saline and lysed in lysis buffer as described (19). Insoluble material was removed by centrifugation (13,000 ϫ g for 15 min) prior to overnight immunoprecipitation with 1 g/ml antibody (as indicated) at 4°C. Protein AG-agarose beads were added to each sample and incubated for 1 h at 4°C. Immunoprecipitates were washed three times with wash buffer (Trisbuffered saline containing 0.05% Triton X-100, 1 mM Na 3 VO 4 , 1 mM NaF, 2 g/ml leupeptin, 2 g/ml pepstatin A, 2 g/ml aprotinin, and 44 g/ml phenylmethylsulfonyl fluoride). Immunoprecipitated proteins were resolved on SDS-PAGE and immunoblotted with the appropriate antibodies.
In Vitro Kinase Assay-In vitro kinase assays were performed using immunoprecipitated recombinant PKC␦ (26) and AMPK from endothelial cells. Briefly, HEK 293 cells in 100-mm dishes were transfected with the hemagglutinin-tagged expression constructs of PKC␦-WT or PKC␦-T505A (1 g/ml). At 24 h after transfection, cells were lysed and immunoprecipitated using an anti-hemagglutinin antibody. The precipitated proteins were used as substrate for an in vitro kinase assay (26). HPAECs treated with and without thrombin were washed and lysed, and the lysate (800 g protein) was incubated with anti-AMPK-␣ antibody for 60 min and then with 30 l of protein A/G-Sepharose for 3 h at 4°C. The immunoprecipitated proteins were washed twice with kinase buffer (50 mM Hepes, pH 7.5, 10 mM MgCl 2 , 1 mM dithiothreitol, 2.5 mM EGTA, 1 mM NaF, 0.1 mM Na 3 VO 4 , 10 mM ␤-glycerophosphate) and resuspended in 20 l of kinase buffer. The kinase assay was initiated by adding 20 l of the kinase buffer to a total volume of 40 l containing PKC␦ (PKC␦-WT or PKC␦-T505A), anti-AMPK-␣ antibody-precipitated proteins from endothelial cells, and 5 Ci of [␥-32 P]ATP. The reactions were continued at 30°C for 30 min and then terminated by adding Laemmli sample buffer. Samples were boiled for 5 min, the proteins were separated on 10% SDS-polyacrylamide gel and dried, and then an autoradiogram was performed. We also used recombinant constitutively active AMPK␣ (CA-AMPK␣) for an in vitro kinase assay. In this case, HEK293 cells were infected with Ad-CA-AMPK␣ (100 plaque-forming units/cell). At 36 h after the infection, the cells were lysed and immunoprecipitated using anti-Myc mAb to precipitate Myc-tagged-Ad-CA-AMPK␣, and the immunoprecipitate was used for an in vitro kinase assay as described above.
Statistical Analysis-Statistical comparisons were made using a 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.

Thrombin-induced Increase in [Ca 2ϩ ] i Mediates NF-B Activation in Endothelial
Cells-Inositol 1,4,5-trisphosphate receptor antagonist, 2-aminoethoxydiphenyl borate (2-APB) is known to inhibit stored Ca 2ϩ release as well as Ca 2ϩ entry (28). We first tested 2-APB for its ability to inhibit a thrombin-induced Ca 2ϩ rise in HPAECs. Cells were loaded with Fura-2/AM in the presence or absence of different concentrations of 2-APB and then stimulated with thrombin. In control cells, thrombin produced an increase in [Ca 2ϩ ] i , followed by a gradual decline to base line (see supplemental Store-operated Ca 2ϩ Entry (SOCE) Is Required for Thrombininduced NF-B Activation-We have shown previously that TRPC1 is the dominant TRPC isoform expressed and contributes to thrombin-induced Ca 2ϩ entry in human endothelial cells (13,18). In this study, we used a gene knockdown approach to address whether Ca 2ϩ entry is required for thrombin-induced NF-B activation. In TRPC1 siRNA-transfected cells, TRPC1 protein expression was suppressed by Ͼ80% compared with control or Sc-siRNA-transfected cells (Fig. 1A). Thrombin-induced Ca 2ϩ store depletion-mediated Ca 2ϩ influx was FIGURE 1. Store-operated Ca 2؉ influx in endothelial cells is required for thrombin-induced NF-B activation. A, HPAECs were transfected with either 100 nM Sc-siRNA or TRPC1 siRNA. At 72 h after transfection, cells were lysed and immunoblotted with anti-TRPC1 polyclonal Ab (top). The membrane was stripped and blotted with anti-␤-actin mAb (bottom). B, control HPAECs and HPAECs transfected with Sc-siRNA or TRPC1 siRNA were used to measure thrombin-induced store Ca 2ϩ release and Ca 2ϩ release-activated Ca 2ϩ influx (see details under "Experimental Procedures"). The results were compared with nontransfected control cells. C, control HPAECs and HPAECs transfected with Sc-siRNA or TRPC1 siRNA were exposed to thrombin (50 nM) or TNF-␣ (1000 units/ml) for the indicated time periods. EMSA was performed as described under "Experimental Procedures." Experiments were repeated 3-4 times with similar results. D, control HPAECs and HPAECs transfected with TRPC1 siRNA as described under "Experimental Procedures." At 72 h after transfection, cells were exposed to thrombin for different time intervals. After thrombin treatment, nuclear and cytoplasmic fractions prepared were used for immunoblotting. The cytosolic (Cy) fractions were immunoblotted with anti-IB␣ antibody (top panel). The membrane was stripped and probed for cytosolic marker with anti-␤ actin monoclonal antibody (second panel). The nuclear fractions (Nu) were immunoblotted with anti-p65 Ab (third panel). The membrane was stripped and probed with anti-MCM3 antibody (nuclear marker).
prevented in TRPC1 siRNA-transfected cells compared with control cells (Fig. 1B). Recent studies have implicated that TRPC1 associates with STIM1 to form a functional SOCE channel complex (24). To address the STIM1 involvement in the mechanism of thrombin-induced Ca 2ϩ entry, we suppressed STIM1 expression by transfecting STIM1-specific siRNA and measured thrombin-induced Ca 2ϩ entry in HPAECs. Like TRPC1 knockdown, STIM1 knockdown also prevented thrombin-induced Ca 2ϩ entry in HPAECs (data not shown). Since the knockdown of either TRPC1 or STIM1 prevented thrombininduced Ca 2ϩ entry in HPAECs, we used TRPC1 knockdown cells for further experiments to measure thrombin-induced NF-B activation. In TRPC1 knockdown cells, thrombin-induced p65/RelA binding to DNA was prevented (Fig. 1C), but TNF-␣-induced NF-B activation was not significantly altered (Fig. 1C). We also determined thrombin-induced IB␣ degradation and nuclear localization of p65 in control and TRPC1 siRNA-transfected HPAECs. In control cells, thrombin stimulation caused a time-dependent degradation of IB␣ and p65 nuclear translocation (Fig. 1D). In contrast to control cells, thrombin-induced IB␣ degradation and p65 nuclear translocation were prevented in TRPC1 knockdown cells (Fig. 1D), suggesting that Ca 2ϩ influx signaling is required for thrombininduced p65/RelA activation in HPAECs.
Impairment of p65/RelA Nuclear Translocation in Trpc4 Ϫ/Ϫ Mouse Lung Endothelial Cells-We next investigated the importance of store-operated Ca 2ϩ entry signal in the mechanism of NF-B activation using Trpc4 Ϫ/Ϫ mouse lungs and endothelial cells. First, we measured thrombin-induced NF-B-DNA binding activities in the lung preparations (see details under "Experimental Procedures"). In this experiment, lungs were perfused with or without thrombin for 30 min. After perfusion, nuclear extracts were prepared and were used for EMSA. EMSA results showed the absence of NF-B-DNA complex formation when lungs were perfused only with buffer in both Trpc4 Ϫ/Ϫ and WT mice ( Fig. 2A). A strong NF-B binding signal to DNA was observed in WT lungs perfused with thrombin ( Fig. 2A). In contrast, Trpc4 Ϫ/Ϫ lungs exhibited a marked reduction in NF-B binding to DNA ( Fig. 2A). Next, we measured thrombin-induced NF-B activation in lung endothelial cells (LECs) from WT and Trpc4 Ϫ/Ϫ mice. We observed a marked reduction in thrombin-induced NF-B-DNA complex formation in Trpc4 Ϫ/Ϫ LECs compared with WT LECs (Fig. 2B). Interestingly, TNF-␣-induced NF-B-DNA complex formation was not altered in Trpc4 Ϫ/Ϫ LECs (Fig. 2B). A supershift assay was carried out utilizing NF-B-specific antibodies to identify NF-B proteins translocated to the nucleus. These data showed a shift in the presence of the anti-p65 antibody in thrombin-stimulated LECs, whereas the shift was observed with both anti-p65 antibody and anti-p50 antibody in TNF-␣stimulated cells. These results indicate that thrombin induces nuclear localization of p65 homodimer, but TNF-␣ induces p65/p50 heterodimer (Fig. 2B). We also determined the NF-B target gene ICAM-1 expression in mouse LECs in response to thrombin. Total RNA was isolated from mouse LECs, and RT-PCR was performed. In WT LECs, thrombin produced an ϳ10fold increase in ICAM-1 mRNA over basal after 4 h of stimulation (Fig. 2C), and the ICAM-1 mRNA level returned to basal at 6 h after thrombin challenge (Fig. 2C). In Trpc4 Ϫ/Ϫ LECs, no significant increase was seen in response to thrombin (Fig. 2C). We exposed LECs from WT and Trpc4 Ϫ/Ϫ mice with the Ca 2ϩ ionophore ionomycin to increase intracellular Ca 2ϩ and measured ICAM-1 transcript expression. We observed an equal increase in ICAM-1 transcript expression in LECs from WT and Trpc4 Ϫ/Ϫ mice (Fig. 2D). These results show an obligatory role for store-operated Ca 2ϩ entry in signaling thrombin-induced NF-B activation and ICAM-1 expression in endothelial cells.
Requirement for Ca 2ϩ /Calmodulin Signaling for Nuclear Localization of NF-B-We next investigated the role of CaM as a downstream Ca 2ϩ target mediating NF-B activation. HPAECs were treated with CaM-specific antagonist, W-7, and thrombin-induced NF-B binding to DNA was measured by EMSA. W-7 inhibited thrombin-induced NF-B translocation to the nucleus as well as IB␣ degradation (see supplemental Fig. 2, A and B). The Ca 2ϩ /CaM complex activates CaM-dependent protein kinases, which include CaMKI, CaMKII, CaMKIV, and CaM-kinase kinase (29,30). Based on studies showing that CaMKII and CaMKIV are involved in signaling NF-B-dependent gene expression (31,32), we investigated whether CaMKII or CaMKIV signaling is required for thrombin-induced NF-B activation. Inhibition of CaMKII and CaMKIV by incubating HPAECs with KN93 before stimulating with thrombin produced no significant effect on thrombin-induced NF-B nuclear translocation (see supplemental Fig. 2C). We also studied the effects of the CaMKK␤-specific inhibitor STO-609 on thrombin-induced NF-B nuclear translocation. STO-609 pretreatment markedly reduced thrombin-induced nuclear localization of NF-B (Fig. 3A).
Obligatory Role of AMPK in Mediating NF-B Nuclear Translocation and NF-B-dependent Gene Expression-Since thrombin may activate AMPK signaling through Ca 2ϩ /CaMdependent CaMKK␤ (33), we determined the effects of the AMPK inhibitor compound C on NF-B activation. Compound C prevented thrombin-induced NF-B binding to DNA in a dose-dependent manner (Fig. 3B). Further, we observed the inhibition of thrombin-induced IB␣ phosphorylation by exposing cells to Compound C (Fig. 3C). Next we determined the effect of Compound C on NF-B-driven reporter expression in endothelial cells (see details under "Experimental Procedures"). Thrombin-induced NF-B-driven reporter expression was markedly suppressed in cells exposed to Compound C (Fig. 3D). In addition, the AMPK inhibitor prevented thrombin-induced ICAM-1 mRNA and protein expression (Fig. 3E).
To further address the role of AMPK in signaling NF-B activation, we used siRNA to knockdown the endogenous AMPK␣ subunit in HPAECs. Transfection of HPAECs with siRNA specific to AMPK␣ inhibited Ͼ85% of AMPK␣ expression compared with control, whereas scrambled siRNA (control siRNA) had no effect (Fig. 4C). Thrombin-induced nuclear localization of NF-B was prevented in AMPK␣-knockdown cells compared with control cells (Fig. 4, D and E). Thus, AMPK signaling is critical in the regulation of thrombin-induced IB␣ degradation and NF-B binding to nuclear DNA.
Inhibition of AMPK Prevents Thrombin-induced p38 MAP Kinase Activation-Studies have shown that p38 MAP kinase activation can increase the binding of TATA-binding protein to DNA as well as increase the phosphorylation of p65/RelA to induce NF-B-dependent gene expression (34,35). To study whether AMPK is upstream of p38 MAP kinase, we exposed HPAECs with AMPK inhibitor compound C and measured thrombin-induced p38 MAP kinase phosphorylation. Thrombin increased phosphorylation of p38 MAP kinase in a time-dependent manner, whereas AMPK inhibitor treatment prevented the response (Fig. 5A). We also showed that knockdown of AMPK␣ using siRNA markedly reduced thrombin-induced p38 MAP kinase phosphorylation, whereas the control siRNA had no effect (Fig. 5B). In addition, we measured thrombininduced phosphorylation of p65/RelA in HPAECs transfected with AMPK␣-specific siRNA. We observed that thrombin-induced phosphorylation of p65 at Ser 536 was prevented in cells transfected with AMPK␣-specific siRNA compared with scrambled siRNA (Fig. 5C), indicating that an AMPK-dependent p38 MAP kinase activation signal mediates p65/Rel phosphorylation. FIGURE 2. Impairment of thrombin-induced p65/RelA nuclear localization in Trpc4 knock-out (Trpc4 ؊/؊ ) mouse lung endothelial cells. A, lungs from WT or Trpc4 Ϫ/Ϫ were perfused with thrombin (50 nM) for 30 min, and nuclear extracts prepared were used to measure NF-B activation utilizing EMSA (see details under "Experimental Procedures"). The autoradiograph was quantified using a densitometer, and the results were expressed in arbitrary units (left). For WT, n ϭ 6; Trpc4 Ϫ/Ϫ , n ϭ 6. *, p Ͻ 0.05, different from WT. B, LECs from WT and Trpc4 Ϫ / Ϫ mice were challenged with thrombin (50 nM) or TNF-␣ (1000 units/ml) for the indicated periods and then used for EMSA and supershift assays. C, LECs from WT and Trpc4 Ϫ / Ϫ mice type were challenged with thrombin (50 nM) for the indicated times. Total RNA was isolated, and RT-PCR was performed to determine ICAM-1 and GAPDH transcript expression. ICAM-1 mRNA induction -fold in response to thrombin was calculated by measuring the ratio of ICAM-1 to GAPDH. The results are the mean Ϯ S.E. from four separate experiments shown. *, p Ͻ 0.05, significantly different from the control. D, effects of TNF-␣ and ionomycin on ICAM-1 mRNA expression. LECs from WT and Trpc4 Ϫ/Ϫ mice were treated with TNF-␣ (1000 units/ml) or ionomyocin (5 M) for 4 h. Total RNA was isolated, and RT-PCR was performed to determine ICAM-1 and GAPDH expression levels. The experiment was repeated three times with similar results.

Ca 2ϩ Influx-dependent AMPK Activation Leads to Thrombin-induced PKC␦ Activation-Since we observed that thrombin-induced p65/RelA activation was prevented in both TRPC1
and AMPK␣ knockdown HPAECs, we investigated the possibility that thrombin-induced Ca 2ϩ influx signal is a prerequisite for PKC␦ activation in endothelial cells. PKC␦ activation is shown by agonist-induced phosphorylation of Thr 505 in its activation loop (26,36). In control cells, thrombin induced PKC␦ phosphorylation at Thr 505 in a time-dependent manner (Fig.  5D). In contrast, thrombin-induced PKC␦ phosphorylation was abolished in TRPC1 siRNA-transfected endothelial cells (Fig.   5D), which lacked Ca 2ϩ influx following thrombin stimulation (Fig.  1B). A similar observation was made in thrombin-induced PKC␦ phosphorylation in LECs obtained from WT and Trpc4 Ϫ/Ϫ mice. Thrombin-induced PKC␦ phosphorylation was markedly reduced in Trpc4 Ϫ/Ϫ LECs compared with WT LECs (Fig.  5E). To determine whether Ca 2ϩdependent AMPK activation is upstream of PKC␦ activation, we measured thrombin-induced PKC␦ phosphorylation in AMPK␣ siRNAtransfected cells. As expected, thrombin-induced PKC␦ phosphorylation was prevented in these cells compared with control cells (Fig.  5F). Moreover, inhibition of AMPK with Compound C also prevented thrombin-induced PKC␦ phosphorylation (data not shown).
Next we determined the effect of the PKC␦-specific inhibitor rottlerin on thrombin-induced AMPK␣ phosphorylation (see above). Rottlerin had no effect on thrombin-induced AMPK␣ phosphorylation (data not shown), but rottlerin prevented thrombin-induced p65/RelA binding to DNA (nuclear translocation) in endothelial cells (data not shown). These results place PKC␦ downstream of AMPK and upstream of the NF-B activation pathway, including IKK␤ and p38 MAP kinase. Therefore, Ca 2ϩ influx-dependent AMPK activation mediates PKC␦ activation and subsequent NF-B activation in endothelial cells. Furthermore, AMPK-mediated PKC␦ activation also can induce p65/ RelA transactivation via p38 MAP kinase activation.
Thrombin-induced PKC␦ Phosphorylation at Thr 505 Is Mediated by AMPK and Not by PDK1-To confirm the relationship between AMPK and PKC␦ in thrombin-induced NF-B activation in endothelial cells, first, we determined thrombin-induced association of AMPK with PKC␦ by immunoprecipitation. We observed the co-immunoprecipitation of PKC␦ with AMPK␣ in thrombin-stimulated cells (Fig. 6A). Since PKC␦ phosphorylation in its activation loop where Thr 505 is located is a mechanism of regulation of the kinase activity (26,36), we performed an in vitro kinase assay to determine thrombin-induced and AMPK-mediated PKC␦ phosphorylation (see details under "Experimental Procedures"). Thrombin activation of The experiment was repeated four times. NF-B-DNA binding activity was quantified by scanning the autoradiograph using densitometer and expressed in arbitrary units (bottom). *, p Ͻ 0.05, significantly different from thrombin-treated control cells. B, HPAECs were pretreated with varying concentrations of AMPK inhibitor compound C (Comp C) for 30 min, and then cells were challenged with thrombin (50 nM). After thrombin treatment, nuclear extracts prepared were used for EMSA. The experiment was repeated three times. C, HPAECs preincubated with compound C (40 M) for 30 min were stimulated with thrombin (50 nM) for 0, 15, 30, and 60 min. After thrombin treatment, cells were lysed and immunoblotted with anti-phospho-IB␣ antibody. The membrane was stripped and probed with anti-␤ actin mAb for loading control. D, HPAECs were transfected with NF-B-luciferase (NF-B pro-luc) expression constructs (see details under "Experimental Procedures"). At 24 h after transfection, cells were pretreated with 40 M compound C for 30 min and then stimulated with thrombin (50 nM) for 6 h. Cells were lysed and reporter activity was measured. Data are mean Ϯ S.E.; n ϭ 4 for each condition. *, p Ͼ 0.05 significantly different from control cells (not treated with compound C). E, HPAECs exposed to compound C for 30 min challenged with thrombin for 4 and 16 h were used for RT-PCR and immunoblot (IB), respectively, to determine ICAM-1 expression.
HPAECs induced phosphorylation of PKC␦-WT protein (Fig.  6B); however, phosphorylation was not observed in PKC␦-T505A mutant protein (Fig. 6B). We also used recombinant constitutively active AMPK␣ for an in vitro kinase assay, and we observed phosphorylation in PKC␦-WT protein but not in PKC␦-T505A mutant protein (Fig. 6C). These results collectively suggest that AMPK activation is required for PKC␦ phosphorylation at Thr 505 in the activation loop in endothelial cells.
PDK1 (phosphoinositide-dependent protein kinase-1) is known to phosphorylate PKC␦ at Thr 505 in the activation loop (36). Phosphoinositide 3-kinase is an upstream kinase that phosphorylates PDK1 and also activates AKT either directly or indirectly via activating PDK1 (37,38). Thrombin is known to activate phosphoinositide 3-kinase and thus presumably can mediate phosphorylation of PDK1 (39,40). To address whether PDK1 is involved in thrombin-induced phosphorylation of PKC␦ at Thr 505 , first, we measured thrombin-induced PDK1 phosphorylation at Ser 241 (which is required for its activity (41)) in HPAECs. Surprisingly, we did not observe inducible phosphorylation of PDK1 in response to thrombin; however, we observed the expression of constitutively phosphorylated PDK1 in HPAECs (supplemental Fig. 3A). As a positive control, we measured thrombin-induced AKT phosphorylation in HPAECs. We observed phosphorylation of AKT in a time-dependent manner in thrombin-stimulated cells, and this effect was blocked by the phosphoinositide 3-kinase inhibitor LY294002 (supplemental Fig. 3A). The phosphoinositide 3-kinase inhibitor had no effect on the constitutive phosphorylation of PDK1 in endothelial cells (supplemental Fig. 3A). In addition, we observed that thrombin-induced PKC␦ phosphorylation was not altered by preexposure of endothelial cells to phosphoinositide 3-kinase inhibitor LY294002. Next, we determined the association of PDK1 with PKC␦ and AMPK␣ in thrombin-stimulated HPAECs by immunoprecipitation. We observed that PDK1 was not immunoprecipitated with either PKC␦ or AMPK (supplemental Fig. 3B). Thus, these results suggest that PDK1 is unlikely to mediate PKC␦ phosphorylation at Thr 505 in thrombin-stimulated endothelial cells.
To address whether AMPK-induced Thr 505 phosphorylation in the activation loop of PKC␦ is critical for NF-B activation, we measured thrombin-induced NF-B-driven reporter (luciferase) expression in PKC␦-WTor PKC␦-T505A mutanttransfected endothelial cells. Thrombin stimulation induced FIGURE 4. Ca 2؉ -dependent AMPK activation signals NF-B activation in endothelial cells. A, HPAECs were exposed to PAR-1 agonist peptide (40 M) for different time intervals. After PAR-1 agonist peptide treatment, cells were lysed, and the lysates were immunoblotted with anti-phosphospecific Thr 172 AMPK␣ antibody. The membrane was stripped and blotted with AMPK␣-specific antibody. The phosphorylation of AMPK␣ was quantified densitometrically, and the results were normalized to AMPK␣ level (bottom). Representative immunoblot and the densitometry mean data of four independent experiments are shown. B, HPAECs transfected with TRPC1 siRNA (see details under "Experimental Procedures") were challenged with PAR-1 agonist peptide, and phosphorylation of AMPK␣ was determined as described above. C, HPAECs were transfected with the Sc-siRNA (100 nM) or AMPK␣ siRNA (100 nM). At 72 h after transfection, cells were lysed and immunoblotted with anti-AMPK␣ antibody. The membrane was stripped and probed with anti-actin mAb for to monitor loading. D, HPAECs transfected with the Sc-siRNA (100 nM) or AMPK␣ siRNA (100 nM) were challenged with thrombin (50 nM) for 60 min. After thrombin treatment, nuclear extracts prepared were used for EMSA. The experiment was repeated four times. The autoradiograph from four independent experiments were quantified by densitometry and expressed in arbitrary units (E). *, p Ͻ 0.05, significantly different from control or Sc-siRNA-transfected cells.
NF-B-driven reporter expression Ͼ6-fold in control cells compared with cells not stimulated with thrombin (Fig. 6D). In PKC␦-WT transfected cells, thrombin-induced NF-B-driven reporter expression was increased compared with control cells (Fig. 6D). Interestingly, thrombin-induced NF-B-driven reporter expression was markedly reduced in PKC␦-T505A mutant-transfected endothelial cells (Fig. 6D), indicating that the thrombin-induced Thr 505 phosphorylation in the activation loop of PKC␦ is essential for thrombin-induced NF-B activation in endothelial cells. This activation mechanism appears to be specific for the thrombin-induced NF-B activation, since expression of PKC␦-T505A mutant in endothelial cells failed to prevent TNF-␣-induced NF-B activation (Fig.  6E), suggesting that thrombin-induced Ca 2ϩ signaling specifically targets the PKC␦ signaling pathway to activate NF-B in endothelial cells.

DISCUSSION
PAR-1 activation by thrombin induces Ca 2ϩ mobilization in endothelial cells that occurs in two stages, initially ER-Ca 2ϩ store depletion followed by Ca 2ϩ entry (also called SOCE) in endothelial cells (13,20). The second phase of Ca 2ϩ mobilization accounts for the sustained increase in intracellular Ca 2ϩ required for signaling the increase in endothelial permeability and other responses generally classified as "endothelial activation." We have shown that the canonical TRPC isoforms are essential for thrombin-induced Ca 2ϩ entry in endothelial cells (13,18,20). Here we describe a fundamental role of Ca 2ϩ entry in signaling NF-B activation in endothelial cells secondary to CaMKK␤-mediated activation of the AMPK/PKC␦ pathway.
Thrombin elicits multiple NF-B-mediated responses, including the expression of ICAM-1, VCAM-1, and TRPC1 in endothelial cells (3,4,12,13). The role of increased intracellular Ca 2ϩ in the mechanism of NF-B activation and gene expression is not well understood. In the present study, we showed this using pharmacological agent 2-APB, which prevented both stages of Ca 2ϩ mobilization in HPAECs. Thrombin-induced NF-B activation as well as ICAM-1 transcript expression were also markedly reduced by 2-APB. However, HPAECs treated with 2-APB showed no effect on TNF-␣-induced NF-B activation and ICAM-1 transcript expression, indicating that the effects of PAR-1 activation cannot be generalized to prototypic inflammatory cytokines, such as TNF-␣. We have shown previously that TNF-␣ failed to induce a rise in [Ca 2ϩ ] i in endothelial cells (42); thus, TNF-␣ activates NF-B through a Ca 2ϩindependent pathway.
All of the TRPC members contain six transmembrane domains, and the Ca 2ϩ -permeable pore region is located between transmembrane helices 5 and 6 (43). TRPC members assemble to homo-and heterotetrameric structure to form a putative SOCE channel complex (44). Further, recent findings suggest that TRPC channels also interact with STIM1 and ORAI1 to form SOCE channel or CRAC channel (24,25). Since the TRPC channels are essential components of the SOCE channel complex, we used TRPC channel knockdown and knock-out strategies to address the role of TRPC channels in the mechanism of thrombin-induced NF-B activation. Thrombin-induced Ca 2ϩ entry was prevented by knockdown of either TRPC1 or STIM1 in HPAECs. Interestingly, we observed that thrombin-induced p65/RelA nuclear translocation and IB␣ degradation were both markedly reduced in TRPC1 knockdown HPAECs. Next, we utilized Trpc4 knockout mice (20,21) to investigate the role of TRPC-mediated Ca 2ϩ influx in the mechanism of thrombin-induced NF-B After thrombin treatment, cells were lysed and immunoblotted with anti-phospho-p38 MAP kinase mAb. The membrane was stripped and blotted with anti-p38 MAP kinase Ab for total p38 MAP kinase. B, HPAECs transfected with the Sc-siRNA or AMPK␣ siRNA were challenged with thrombin (50 nM) for different time intervals. After thrombin treatment, cells were lysed and immunoblotted with anti-phospho-p38 MAP kinase mAb and p38 MAP kinase Ab. The experiment was repeated three times. The results from representative experiments are shown. C, HPAECs transfected with the Sc-siRNA or AMPK␣ siRNA were challenged with thrombin (50 nM) for 0, 10, and 30 min. After thrombin treatment, cells were lysed and immunoblotted with anti-phospho-Ser 536 -p65 mAb and anti-p65 Ab. D, control HPAECs and HPAECs transfected with TRPC1 siRNA were challenged with thrombin for different time intervals. After thrombin challenge, cells were lysed and immunoblotted with anti-phospho-PKC␦ (Thr 505 ) Ab. The blots were stripped and probed with anti-PKC␦ Ab. The experiment was repeated three times. The results from representative experiments are shown. E, lung endothelial cells from WT and Trpc4 Ϫ / Ϫ mice type were challenged with thrombin (50 nM) for the indicated times. After thrombin challenge, cells were lysed and immunoblotted with anti-phospho-PKC␦ (Thr 505 ) Ab. The experiment was repeated three times. F, control HPAECs and HPAECs transfected with AMPK␣ siRNA were challenged with thrombin for different time intervals. After thrombin challenge, cells were lysed and immunoblotted with anti-phospho-PKC␦ (Thr 505 ) or anti-PKC␦ antibodies. The experiment was repeated three times. The results from representative experiments are shown.
activation. In this study, we observed that thrombin-induced NF-B binding to DNA as well as ICAM-1 transcript expression were significantly reduced in Trpc4 knock-out mouse endothelial cells compared with endothelial cells from wild type mice.
CaM, the Ca 2ϩ -binding protein expressed in mammalian cells, senses changes in intracellular Ca 2ϩ concentration and thereby participates in Ca 2ϩ signaling (29 -32). Ca 2ϩ binding to EF-hand motifs on CaM is responsible for activating downstream target molecules (29,30). To study the role of CaM in NF-B activation, we determined the effects of CaM antagonist W-7. W-7 inhibited thrombin-induced NF-B binding to DNA in a dose-dependent manner and concomitantly prevented IB␣ degradation, suggesting a key role of Ca 2ϩ /CaM complex formation in signaling NF-B activation. The CaMKK␤-specific inhibitor STO-609 also prevented thrombin-induced NF-B activation in endothelial cells, suggesting the role of CaMKK␤ downstream of CaM in signaling NF-B activation.
AMPK is a serine/threonine kinase composed of the catalytic ␣ subunit and the regulatory ␤ and ␥ subunits (45)(46)(47). Phosphorylation of the ␣ subunit at threonine 172 is required for the catalytic activity of AMPK (45)(46)(47). Ca 2ϩ /CaM-dependent CaMKK␤ was shown to activate AMPK in multiple cell types (46,47). Since PAR-1 has been shown to activate AMPK signaling via Ca 2ϩ /CaM-dependent CaMKK␤ in endothelial cells (33), we surmised that Ca 2ϩ /CaM signaling and resultant AMPK activation may regulate NF-B activation. We observed that an AMPK-specific inhibitor compound C inhibited thrombin-induced NF-B binding to DNA in a dose-dependent manner. AMPK␣ subunit was also phosphorylated at Thr 172 in response to PAR-1 activation induced by PAR-1 agonist peptide (TFLLRNPNDK) or thrombin. In TRPC1 knockdown endothelial cells, PAR-1 agonist peptide-induced AMPK␣ phosphorylation was prevented. Further, chelation of intracellular Ca 2ϩ with 1,2-bis(2-aminophenoxy)ethane-N,N,NЈ,NЈ-FIGURE 6. AMPK-mediated Thr 505 phosphorylation in the activation loop of PKC␦ signals NF-B activation in endothelial cells. A, HPAECs exposed to thrombin (50 nM) for the indicated time intervals were lysed and immunoprecipitated (IP) with control IgG or anti-PKC␦ Ab. The precipitated proteins were immunoblotted (IB) with anti-AMPK␣ Ab (top). The membrane was stripped and probed with anti-PKC␦ Ab (bottom). B, AMPK-mediated PKC␦ phosphorylation at Thr 505 was determined by in vitro kinase assay (see details under "Experimental Procedures"). Hemagglutinin-tagged PKC␦ proteins (PKC␦-WT and PKC␦-T505A) expressed in HEK293 cells were immunoprecipitated and used as substrates. HPAECs were stimulated with thrombin, lysed, and immunoprecipitated using anti-AMPK␣ Ab or control IgG. The anti-AMPK␣ Ab or control IgG precipitated proteins served as kinase for in vitro phosphorylation of PKC␦. The autoradiograph (top) shows 32 P incorporation in PKC␦-WT but not in PKC␦-T505A mutant. An equal volume of the assay mixture was immunoblotted with anti-AMPK␣ (middle) and anti-PKC␦ (bottom) antibodies. The experiment was repeated three times. The results from representative experiments are shown. C, recombinant constitutively active AMPK␣-mediated PKC␦ phosphorylation at Thr 505 was determined by in vitro kinase assay. HEK293 cells infected with adenovirus expressing Myc epitopetagged CA-AMPK␣ was immunoprecipitated with anti-Myc mAb as described under "Experimental Procedures." The anti-Myc mAb precipitated protein was used for in vitro phosphorylation of PKC␦. The autoradiograph (top) shows 32 P incorporation in PKC␦-WT but not in the PKC␦-T505A mutant. Equal volume of the assay mixture was immunoblotted with anti-PKC␦ (middle) and anti-Myc (bottom) antibodies. The results from representative experiments are shown. D and E, HPAECs grown to 80% confluence were transfected with PKC␦-WT or PKC␦-T505A mutant and NF-B pro-luc constructs (see details under "Experimental Procedures"). At 24 h after transfection, cells were stimulated with thrombin (50 nM) or TNF-␣ (1000 units/ml) for 6 h. After this treatment, cells were lysed, and reporter activity was measured. Also, the cell lysates were immunoblotted with anti-PKC␦ Ab to determine PKC␦ expression. The mean Ϯ S.E. from four experiments repeated in triplicate is shown in D and E. The asterisk indicates statistically significant different from PKC␦-WT-expressing cells stimulated with thrombin (*, p Ͻ 0.001). tetraacetic acid acetoxymethyl ester or inhibition of CaM function with W-7 blocked thrombin-induced AMPK activation. These results collectively show that CaMKK␤-dependent AMPK activation is an important pathway mediating thrombin-induced NF-B activation in endothelial cells. This observation was confirmed by depleting endogenous AMPK␣ expression using AMPK␣-specific siRNA, which reduced AMPK␣ expression (by 85%) and prevented thrombin-induced NF-B binding to DNA. Previous studies have shown that AMPK activation with pharmacological agents such as metformin and 5-amino-4-imidazole carboxamide riboside prevented TNF-␣-induced NF-B activation in endothelial cells (48,49). Our results showed that Ca 2ϩ -dependent AMPK activation is required for thrombin-induced p65/RelA binding to nuclear DNA. These findings support the notion that distinct signaling pathways are activated in endothelial cells to induce NF-B signaling by thrombin and TNF-␣.
Another component of the thrombin-induced NF-B activation signaling pathway delineated in these studies is the role of p38 MAP kinase. Li et al., (50) showed that activated AMPK interacts with the scaffold protein TAB1, and the resulting AMPK-TAB1 complex associates with p38 MAP kinase and thus promotes p38 MAP kinase autophosphorylation in ischemic hearts. We observed in the present study that inhibition of AMPK in endothelial cells prevented thrombin-induced p38 MAP kinase activation, demonstrating that AMPK lies upstream of p38 MAP kinase. Further, knockdown of AMPK in endothelial cells prevented the thrombin-induced phosphorylation of p65/RelA mediated by p38 MAP kinase. Thus, from these observations, we can conclude that AMPK-dependent p38 MAP kinase activation plays a key role in thrombin-induced p65/RelA transactivation.
The novel PKC isoform PKC␦ has been shown to play an important role in the mechanism of NF-B activation. Minami et al. (15) showed that thrombin-induced VCAM-1 expression was dependent on PKC␦-mediated NF-B activation in endothelial cells. We previously demonstrated that the thrombininduced PKC␦ activation signal induced NF-B activation and ICAM-1 expression in endothelial cells (4). In another study, thrombin-induced PKC␦ activation induced p65/RelA transactivation signaling via p38 MAP kinase to up-regulate ICAM-1 (14). PKC␦ activation is Ca 2ϩ -indedependent but dependent on diacylglycerol (4,36). Several studies have shown that besides diacylglycerol binding to the regulatory domain, phosphorylation at the catalytic domain of PKC␦ is essential for its activity (14,26,36,51). Cheng et al. (26) have recently shown that phosphorylation at Thr 505 in the catalytic domain of the activation loop is critical for the catalytic function of PKC␦. Thus, we addressed the possibility that PKC␦ activation downstream of the Ca 2ϩ influx signaling pathway elaborated above was involved in regulating thrombin-induced NF-B activation. We observed that Trpc4 knock-out, TRPC1 knockdown, or AMPK␣ knockdown in endothelial cells prevented thrombininduced phosphorylation of PKC␦ at the catalytic domain Thr 505 , indicating that Ca 2ϩ influx-dependent AMPK activation is required for PKC␦ activation. We also showed co-immunoprecipitation of PKC␦ with AMPK in thrombin-stimulated endothelial cells. Further, we showed that AMPK specifically phosphorylated PKC␦ at Thr 505 by in vitro kinase assay. In addition, we provide evidence in this study that PDK1 signaling is not involved in thrombin-induced PKC␦ phosphorylation at Thr 505 in endothelial cells. Moreover, in this study, we addressed the relationship between AMPK-mediated PKC␦ phosphorylation and NF-B activation in endothelial cells. We showed that PKC␦-T505A mutant expression suppressed thrombin-induced but not TNF-␣-induced NF-B activation in endothelial cells. These results demonstrate that the Ca 2ϩ influx-dependent AMPK activation and resultant PKC␦ activation is an important pathway mediating NF-B activity in thrombin-stimulated endothelial cells. This novel mechanism of activation complements another pathway involving thrombin-induced Ca 2ϩ -dependent PKC␣ activation that could induce NF-B signaling by activating PKC␦ via Rho signaling (11,52,53).
In summary, we have shown that PAR-1-mediated Ca 2ϩ entry via TRPC channels results in AMPK activation, which is required for activation of the downstream target PKC␦ and induction of p65/RelA signaling in endothelial cells (Fig. 7). NF-B activation by the TRPC channel signaling pathway FIGURE 7. Signaling pathway mediating thrombin-induced NF-B activation in endothelial cells. PAR-1 activation-induced ER store Ca 2ϩ depletion via phospholipase C␤ (PLC␤) activates Ca 2ϩ entry through TRPC channels to cause sustained increase in intracellular Ca 2ϩ in endothelial cells. Ca 2ϩ binding to CaM activates CaMKK␤, which in turn phosphorylates AMPK␣ (which is required for catalytic function of AMPK). Activated AMPK phosphorylates PKC␦ at Thr 505 to activate PKC␦, which in turn targets IKK␤ to mediate NF-B binding to nuclear DNA by inducing IB␣ degradation. Both the activated AMPK and PKC␦ can induce p65/RelA transactivation signaling via p38 MAP kinase.
therefore provides an important link between activation of the coagulation cascade and NF-B-regulated innate immunity response and inflammatory mechanisms.