Here we show that the transcription-repressor DREAM binds to the A20 promoter to repress the expression of A20, the deubiquitinase suppressing inflammatory NF-κB signaling. DREAM-deficient (Dream−/−) 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, USF1 binding to the DRE-associated E-box domain activated A20 expression in response to inflammatory stimuli. These 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 in diseases such as acute lung injury associated with unconstrained NF-κB activity.

Background: STIM1 activates store-operated Ca 2ϩ entry (SOCE). The transcriptional regulation of STIM1 during sepsis is not known. Results: STIM1 expression occurs downstream of TLR4 via activation of NF-B and p38␣-mediated c-Fos expression in endothelial cells. Conclusion: Cooperative signaling of NF-B and p38 MAPK downstream of TLR4 is essential for STIM1 expression during sepsis. Significance: Selective p38␣ inhibitors may represent a potential therapeutic strategy to prevent sepsis-mediated lung vascular leaks.

Vascular endothelial protein tyrosine phosphatase (VE-PTP) stabilizes endothelial adherens junctions (AJs) through constitutive dephosphorylation of VE-cadherin. Here we investigated the role of stromal interaction molecule 1 (STIM1) activation of store-operated Ca entry (SOCE) in regulating AJ assembly. We observed that SOCE induced by STIM1 activated Pyk2 in human lung microvascular endothelial cells (ECs) and induced tyrosine phosphorylation of VE-PTP at Y1981. Pyk2-induced tyrosine phosphorylation of VE-PTP promoted binding to VE-PTP, activation, and subsequent VE-cadherin phosphorylation and thereby increased the endothelial permeability response. The increase in permeability was secondary to disassembly of AJs. Pyk2-mediated responses were blocked in EC-restricted knockout mice, indicating the requirement for STIM1 in initiating the signaling cascade. A peptide derived from the Pyk2 phosphorylation site on VE-PTP abolished the STIM1/SOCE-activated permeability response. Thus Pyk2 activation secondary to STIM1-induced SOCE causes tyrosine phosphorylation of VE-PTP, and VE-PTP, in turn, binds to and activates, thereby phosphorylating VE-cadherin to increase endothelial permeability through disassembly of AJs. Our results thus identify a novel signaling mechanism by which STIM1-induced Ca signaling activates Pyk2 to inhibit the interaction of VE-PTP and VE-cadherin and hence increase endothelial permeability. Therefore, targeting the Pyk2 activation pathway may be a potentially important anti-inflammatory strategy.

Prostate cancer (PCa) is the second most common solid tumor for cancer related deaths in American men. Genome wide association studies (GWAS) have identified single nucleotide polymorphisms (SNPs) associated with the increased risk of PCa. Because most of the susceptibility SNPs are located in noncoding regions, little is known about their functional mechanisms. We hypothesize that functional SNPs reside in cell type-specific regulatory elements that mediate the binding of critical transcription factors (TFs), which in turn result in changes in target gene expression. Using PCa-specific functional genomics data, here we identify 38 regulatory candidate SNPs and their target genes in PCa. Through risk analysis by incorporating gene expression and clinical data, we identify 6 target genes (ZG16B, ANKRD5, RERE, FAM96B, NAALADL2 and GTPBP10) as significant predictors of PCa biochemical recurrence. In addition, 5 SNPs (rs2659051, rs10936845, rs9925556, rs6057110 and rs2742624) are selected for experimental validation using Chromatin immunoprecipitation (ChIP), dual-luciferase reporter assay in LNCaP cells, showing allele-specific enhancer activity. Furthermore, we delete the rs2742624-containing region using CRISPR/Cas9 genome editing and observe the drastic downregulation of its target gene UPK3A. Taken together, our results illustrate that this new methodology can be applied to identify regulatory SNPs and their target genes that likely impact PCa risk. We suggest that similar studies can be performed to characterize regulatory variants in other diseases.

Objective: Recently, a new mutation in Caveolin‐1 (Cav‐1) was identified in a family with heritable pulmonary arterial hypertension (hPAH), but the underlying molecular mechanisms by which the Cav‐1 mutation leads to hPAH are poorly understood. Methods: Human dermal fibroblasts were obtained from 3 healthy control subjects and 3 hPAH patients with the Caveolin‐1 mutation. Results: Patient fibroblasts had a markedly higher proliferative rate than those of control subjects (34±5 vs. 19±4%, P<0.05). Since calcium is an important regulator of cell proliferation, we compared maximal cytoplasmic calcium influx in response to the SERCA inhibitor thapsigargin. We found that calcium influx was higher in fibroblasts with a Cav‐1 mutation (2.38‐fold increase, P<0.005). Also, fibroblasts with a Cav‐1 mutation exhibited higher oxidative stress (2.5‐fold increase in H2O2 P<0.01, 4‐fold increase in peroxynitrite P<0.001). Conclusions: The newly identified Caveolin‐1 in hPAH patients as associated higher fibroblast proliferation, aberrant calcium signaling, and high levels of oxidative stress. These signaling pathways may be promising targets for novel therapies in hPAH and validate the role of Cav‐1 in clinical PAH. Since Cav‐1 is a disease modifier in experimental PAH, our findings may also prove valuable for understanding the pathophysiology of non‐heritable PAH.