Tumor necrosis factor-alpha induces nuclear factor-kappa B-dependent TRPC1 expression in endothelial cells
ABSTRACT We investigated the role of tumor necrosis factor-alpha (TNF-alpha) in activating the store-operated Ca2+ channels in endothelial cells via the expression of transient receptor potential channel (TRPC) isoforms. We observed that TNF-alpha exposure of human umbilical vein endothelial cells resulted in TRPC1 mRNA and protein expression, whereas it had no effect on TRPC3, TRPC4, or TRPC5 expression. The TRPC1 expression was associated with increased Ca2+ influx after intracellular Ca2+ store depletion with either thrombin or thapsigargin. We cloned the 5'-regulatory region of the human TRPC1 (hTRPC1) gene which contained a TATA box and CCAAT sequence close to the transcription initiation site. We also identified four nuclear factor-kappaB (NF-kappaB)-binding sites in the 5'-regulatory region. To address the contribution of NF-kappaB in the mechanism of TRPC1 expression, we determined the effects of TNF-alpha on expression of the reporter luciferase after transfection of hTRPC1 promoter-luciferase (hTRPC1-Pro-Luc) construct in the human dermal microvascular endothelial cell line. Reporter activity increased >4-fold at 4 h after TNF-alpha challenge. TNF-alpha-induced increase in reporter activity was markedly reduced by co-expression of either kinase-defective IKKbeta kinase mutant or non-phosphorylatable IkappaB mutant. Treatment with NEMO-binding domain peptide, which prevents NF-kappaB activation by selectively inhibiting IKKgamma interaction with IKK complex, also blocked the TNF-alpha-induced TRPC1 expression. Thus, TNF-alpha induces TRPC1 expression through an NF-kappaB-dependent pathway in endothelial cells, which can trigger augmented Ca2+ entry following Ca2+ store depletion. The augmented Ca2+ entry secondary to TRPC1 expression may be an important mechanism of endothelial injury induced by TNF-alpha.
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ABSTRACT: Introduction: Acute pancreatitis (AP) is known to induce injuries to extra-pancreatic organs. Because respiratory dysfunction is the main cause of death in patients with severe AP, acute pancreatitis-associated lung injury (APALI) is a great challenge for clinicians. This study aimed to investigate the potential role of hydrogen sulfide (H2S) in the pathogenesis of APALI. Material and methods: Fifty-four SD rats were randomly divided into three groups: the AP group of rats that received injection of sodium deoxycholate into the common bile duct, the control group that underwent a sham operation, and the treatment group made by intraperitoneal injection of propargylglycine (PAG), an inhibitor of cystathionine-gamma-lyase (CSE), into rats with AP. Histopathology of the lung was examined and the expression of CSE and TNF-alpha mRNA in lung tissue was detected by real-time polymerase chain reaction. The H2S level in the serum was detected spectrophotometrically. Results: The serum concentration of H2S and CSE and TNF-alpha expression in the lung were increased in AP rats modeled after 3 h and 6 h than in control rats (p < 0.05). Intraperitoneal injection of PAG could reduce the serum concentration of H2S, reduce CSE and TNF-alpha expression, and alleviate the lung pathology (p < 0.05). Conclusions: Taken together, our findings suggest that the H2S/CSE system is crucially involved in the pathological process of APALI and represents a novel target for the therapy of APALI.Archives of Medical Science 08/2014; 10(4):825-829. DOI:10.5114/aoms.2014.44873 · 1.89 Impact Factor
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ABSTRACT: In contrast to other Classical Transient Receptor Potential TRPC channels the function of TRPC1 as an ion channel is a matter of debate, because it is often difficult to obtain substantial functional signals over background in response to over-expression of TRPC1 alone. Along these lines, heterologously expressed TRPC1 is poorly translocated to the plasma membrane as a homotetramer and may not function on its own physiologically, but may rather be an important linker and regulator protein in heteromeric TRPC channel tetramers. However, due to the lack of specific TRPC1 antibodies able to detect native TRPC1 channels in primary cells, identification of functional TRPC1 containing heteromeric TRPC channel complexes in the plasma membrane is still challenging. Moreover, an extended TRPC1 cDNA, which was recently discovered, may seriously question results obtained in heterologous expression systems transfected with shortened cDNA versions. Therefore, this review will focus on the current status of research on TRPC1 function obtained in primary cells and a TRPC1-deficient mouse model.12/2014; 3(4):939-962. DOI:10.3390/cells3040939
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ABSTRACT: Stromal interacting molecule 1 (STIM1) regulates store-operated Ca2+ entry (SOCE). Here, we show that STIM1 expression in endothelial cells (ECs) is increased during sepsis and thus contributes to hyper-permeability. LPS induced STIM1 mRNA and protein expression in human and mouse lung ECs. The induced STIM1 expression was associated with augmented SOCE as well as permeability increase in both in vitro and in vivo models. Since activation of both NF-κB and p38 MAPK signaling pathways downstream of TLR4 amplify vascular inflammation, we studied the influence of these two pathways on LPS-induced STIM1 expression. Inhibition of either NF-κB or p38 MAPK activation by pharmacological agents prevented LPS-induced STIM1 expression. Silencing of the NF-κB proteins (p65/RelA or p50/NF-κB1) or the p38 MAPK isoform p38α prevented LPS-induced STIM1 expression and increased SOCE in ECs. In support of these findings, we found NF-κB and AP1 binding sites in the 5-regulatory region of human and mouse STIM1 genes. Further, we demonstrated that LPS induced time-dependent binding of the transcription factors NF-κB (p65/RelA) and AP1 (c-Fos/c-Jun) to the STIM1 promoter. Interestingly, silencing of c-Fos, but not c-Jun markedly reduced LPS-induced STIM1 expression in ECs. Also, we observed that silencing of p38α prevented c-Fos expression in response to LPS in ECs, suggesting that p38α signaling mediates expression of c-Fos. These results support the proposal that cooperative signaling of both NF-κB and AP1 (via p38α) amplifies STIM1 expression in ECs and thereby contributes to the lung vascular hyper-permeability response during sepsis.