The transcription factor DREAM represses the deubiquitinase A20 and mediates inflammation.

Here we found that the transcription repressor DREAM bound to the promoter of the gene encoding A20 to repress expression of this deubiquitinase that suppresses inflammatory NF-κB signaling. DREAM-deficient mice displayed persistent and unchecked A20 expression in response to endotoxin. DREAM functioned by transcriptionally repressing A20 through binding to downstream regulatory elements (DREs). In contrast, binding of the transcription factor USF1 to the DRE-associated E-box domain in the gene encoding A20 activated its expression in response to inflammatory stimuli. Our studies define the critical opposing functions of DREAM and USF1 in inhibiting and inducing A20 expression, respectively, and thereby the strength of NF-κB signaling. Targeting of DREAM to induce USF1-mediated A20 expression is therefore a potential anti-inflammatory strategy for the treatment of diseases associated with unconstrained NF-κB activity, such as acute lung injury.

Ca2+ influx via TRPC channels induces NF-kappaB-dependent A20 expression to prevent thrombin-induced apoptosis in endothelial cells.

NF-kappaB signaling is known to induce the expression of antiapoptotic and proinflammatory genes in endothelial cells (ECs). We have shown recently that Ca(2+) influx through canonical transient receptor potential (TRPC) channels activates NF-kappaB in ECs. Here we show that Ca(2+) influx signal prevents thrombin-induced apoptosis by inducing NF-kappaB-dependent A20 expression in ECs. Knockdown of TRPC1 expressed in human umbilical vein ECs with small interfering RNA (siRNA) suppressed thrombin-induced Ca(2+) influx and NF-kappaB activation in ECs. Interestingly, we observed that thrombin induced >25% of cell death (apoptosis) in TRPC1-knockdown ECs whereas thrombin had no effect on control or control siRNA-transfected ECs. To understand the basis of EC survival, we performed gene microarray analysis using ECs. Thrombin stimulation increased only a set of NF-kappaB-regulated genes 3- to 14-fold over basal levels in ECs. Expression of the antiapoptotic gene A20 was the highest among these upregulated genes. Like TRPC1 knockdown, thrombin induced apoptosis in A20-knockdown ECs. To address the importance of Ca(2+) influx signal, we measured thrombin-induced A20 expression in control and TRPC1-knockdown ECs. Thrombin-induced p65/RelA binding to A20 promoter-specific NF-kappaB sequence and A20 protein expression were suppressed in TRPC1-knockdown ECs compared with control ECs. Furthermore, in TRPC1-knockdown ECs, thrombin induced the expression of proapoptotic proteins caspase-3 and BAX. Importantly, thrombin-induced apoptosis in TRPC1-knockdown ECs was prevented by adenovirus-mediated expression of A20. These results suggest that Ca(2+) influx via TRPC channels plays a critical role in the mechanism of cell survival signaling through A20 expression in ECs.

NF-kappaB regulates thrombin-induced ICAM-1 gene expression in cooperation with NFAT by binding to the intronic NF-kappaB site in the ICAM-1 gene.

Activation of NF-kappaB is essential for protease-activated receptor-1 (PAR-1)-mediated ICAM-1 expression in endothelial cells. Here we show that PAR-1 activation induces binding of both p65/RelA and NFATc1 to the NF-kappaB binding site localized in intron-1 of the ICAM-1 gene to initiate transcription in endothelial cells. We discovered the presence of two NF-kappaB binding sites in intron-1 (+70, NF-kappaB site 1; +611, NF-kappaB site 2) of the human ICAM-1 gene. Chromatin immunoprecipitation results showed that thrombin induced binding of p65/RelA and of NFATc1 specifically to intronic NF-kappaB site 1 of the ICAM-1 gene. Electrophoretic mobility shift and supershift assays confirmed the binding of p65/RelA and NFATc1 to the intronic NF-kappaB site 1 in thrombin-stimulated cells. Thrombin increased the expression of ICAM-1-promoter-intron 1-reporter (-1,385 to +234) construct approximately 25-fold and mutation of intronic NF-kappaB site 1 markedly reduced thrombin-induced reporter expression. Moreover, inhibition of calcineurin, knockdown of either NFATc1 or p65/RelA with siRNA significantly reduced thrombin-induced ICAM-1 expression and polymorphonuclear leukocyte adhesion to endothelial cells. In contrast, NFATc1 knockdown had no effect on TNF-alpha-induced ICAM-1 expression. Thus these results suggest that p65/RelA and NFATc1 bind to the intronic NF-kappaB site 1 sequence to induce optimal transcription of the ICAM-1 gene in response to thrombin in endothelial cells.

Ca2+ entry via TRPC channels is necessary for thrombin-induced NF-kappaB activation in endothelial cells through AMP-activated protein kinase and protein kinase Cdelta.

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-kappaB 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 Cdelta (PKCdelta) to mediate NF-kappaB activation in endothelial cells. We observed that thrombin-induced p65/RelA, AMPK, and PKCdelta 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 beta downstream of the Ca2+ influx or knockdown of the downstream Ca2+/calmodulin-dependent protein kinase kinase beta target kinase, AMPK, also prevented NF-kappaB activation. Further, we observed that AMPK interacted with PKCdelta and phosphorylated Thr505 in the activation loop of PKCdelta in thrombin-stimulated endothelial cells. Expression of a PKCdelta-T505A mutant suppressed the thrombin-induced but not the TNF-alpha-induced NF-kappaB activation. These findings demonstrate a novel mechanism for TRPC channels to mediate NF-kappaB activation in endothelial cells that involves the convergence of the TRPC-regulated signaling at AMPK and PKCdelta and that may be a target of interference of the inappropriate activation of NF-kappaB associated with thrombosis.