The glucocorticoid receptor (NR3C1, also known as GR) binds to specific DNA sequences and directly induces transcription of anti-inflammatory genes that contribute to cytokine repression, frequently in cooperation with NF-kB. Whether inflammatory repression also occurs through local interactions between GR and inflammatory gene regulatory elements has been controversial. Here, using global run-on sequencing (GRO-seq) in human airway epithelial cells, we show that glucocorticoid signaling represses transcription within 10 min. Many repressed regulatory regions reside within “hyper-ChIPable” genomic regions that are subject to dynamic, yet nonspecific, interactions with some antibodies. When this artifact was accounted for, we determined that transcriptional repression does not require local GR occupancy. Instead, widespread transcriptional induction through canonical GR binding sites is associated with reciprocal repression of distal TNF-regulated enhancers through a chromatin-dependent process, as evidenced by chromatin accessibility and motif displacement analysis. Simultaneously, transcriptional induction of key anti-inflammatory effectors is decoupled from primary repression through cooperation between GR and NF-kB at a subset of regulatory regions. Thus, glucocorticoids exert bimodal restraints on inflammation characterized by rapid primary transcriptional repression without local GR occupancy and secondary anti-inflammatory effects resulting from transcriptional cooperation between GR and NF-kB.
Ca 2+ signaling in pulmonary arterial smooth muscle cells (PASMCs) plays an important role in pulmonary hypertension (PH). However, the underlying specific ion channel mechanisms remain largely unknown. Here, we report ryanodine receptor (RyR) channel activity and Ca 2+ release both are increased, and association of RyR2 by FK506 binding protein 12.6 (FKBP12.6) is decreased in PASMCs from mice with chronic hypoxia (CH)-induced PH. Smooth muscle cell (SMC)-specific RyR2 knockout (KO) or Rieske iron-sulfur protein (RISP) knockdown inhibits the altered Ca 2+ signaling, increased nuclear factor (NF)-κB/cyclin D1 activation and cell proliferation, and CH-induced PH in mice. FKBP12.6 KO or FK506 treatment enhances CH-induced PH, while S107 (a specific stabilizer of RyR2/FKBP12.6 complex) produces an opposite effect. In conclusion, CH causes RISP-dependent ROS generation and FKBP12.6/ RyR2 dissociation, leading to PH. RISP inhibition, RyR2/FKBP12.6 complex stabilization and Ca 2+ release blockade may be potentially beneficial for the treatment of PH.
The glucocorticoid receptor (GR) binds to specific DNA sequences and directly induces transcription of anti-inflammatory genes that contribute to cytokine repression, frequently in cooperation with NF-kB. Whether inflammatory repression also occurs through local interactions between GR and inflammatory gene regulatory elements remains controversial.Here, using Global Run-on Sequencing (GRO-seq) in human airway epithelial cells, we show that glucocorticoid signaling represses transcription within 10 minutes. Many repressed regulatory regions reside within 'hyper-ChIPable' genomic regions that are subject to nonspecific interactions with some antibodies. When this was accounted for, we determined that transcriptional repression occurs without local GR occupancy. Instead, widespread transcriptional induction through canonical GR binding sites is associated with reciprocal repression of distal TNF-regulated enhancers through a chromatin-dependent process, as evidenced by chromatin accessibility and enhancer-reporter assays. Simultaneously, transcriptional induction of key anti-inflammatory effectors is decoupled from primary repression through cooperation between GR and NF-kB at a subset of regulatory regions.Thus, glucocorticoids exert bimodal restraints on inflammation characterized by rapid primary transcriptional repression without local GR occupancy and secondary anti-inflammatory effects resulting from transcriptional cooperation between GR and NF-kB.3
An increase in intracellular calcium concentration ([Ca 2ϩ ]i) in pulmonary arterial smooth muscle cells (PASMCs) induces hypoxic cellular responses in the lungs; however, the underlying molecular mechanisms remain incompletely understood. We report, for the first time, that acute hypoxia significantly enhances phospholipase C (PLC) activity in mouse resistance pulmonary arteries (PAs), but not in mesenteric arteries. Western blot analysis and immunofluorescence staining reveal the expression of PLC-␥1 protein in PAs and PASMCs, respectively. The activity of PLC-␥1 is also augmented in PASMCs following hypoxia. Lentiviral shRNA-mediated gene knockdown of mitochondrial complex III Rieske iron-sulfur protein (RISP) to inhibit reactive oxygen species (ROS) production prevents hypoxia from increasing PLC-␥1 activity in PASMCs. Myxothiazol, a mitochondrial complex III inhibitor, reduces the hypoxic response as well. The PLC inhibitor U73122, but not its inactive analog U73433, attenuates the hypoxic vasoconstriction in PAs and hypoxic increase in [Ca 2ϩ ]i in PASMCs. PLC-␥1 knockdown suppresses its protein expression and the hypoxic increase in [Ca 2ϩ ]i. Hypoxia remarkably increases inositol 1,4,5-trisphosphate (IP3) production, which is blocked by U73122. The IP3 receptor (IP3R) antagonist 2-aminoethoxydiphenyl borate (2-APB) or xestospongin-C inhibits the hypoxic increase in [Ca 2ϩ ]i. PLC-␥1 knockdown or U73122 reduces H2O2-induced increase in [Ca 2ϩ ]i in PASMCs and contraction in PAs. 2-APB and xestospongin-C produce similar inhibitory effects. In conclusion, our findings provide novel evidence that hypoxia activates PLC-␥1 by increasing RISP-dependent mitochondrial ROS production in the complex III, which causes IP3 production, IP3R opening, and Ca 2ϩ release, playing an important role in hypoxic Ca 2ϩ and contractile responses in PASMCs.hypoxia; phospholipase c-␥1; mitochondria; Rieske iron-sulfur protein; reactive oxygen species IN RESPONSE TO LOW ALVEOLAR oxygen content, termed hypoxia, pulmonary arteries (PAs) constrict to increase vascular resistance and help direct blood to well-ventilated lung alveoli. This phenomenon is known as hypoxic pulmonary vasoconstriction (HPV), an important physiological response. Persistent HPV can be a critical pathological factor in the development of pulmonary hypertension and right ventricular failure. An increase in intracellular Ca 2ϩ concentration ([Ca 2ϩ ] i ) plays an essential role in producing HPV; however, the underlying signaling mechanisms are not fully understood, and identification of the important molecular players involved in the hypoxic increase in [Ca 2ϩ ] i in pulmonary arterial smooth muscle cells (PASMCs) and attendant HPV as well as their mechanisms is imperative (6,22,30). Growing evidence suggests that the hypoxic increase in [Ca 2ϩ ] i in PASMCs is attributed to an increase in intracellular reactive oxygen species (ROS), which are mainly produced by mitochondria and NADPH oxidase (20,30). The hypoxic generation of mitochondrial...
Hypoxia-induced pulmonary vasoconstriction (HPV) is attributed to an increase in intracellular Ca concentration ([Ca]) in pulmonary artery smooth muscle cells (PASMCs). We have reported that phospholipase C-γ1 (PLCγ1) plays a significant role in the hypoxia-induced increase in [Ca] in PASMCs and attendant HPV. In this study, we intended to determine molecular mechanisms for hypoxic Ca and contractile responses in PASMCs. Our data reveal that hypoxic vasoconstriction occurs in pulmonary arteries, but not in mesenteric arteries. Hypoxia caused a large increase in [Ca] in PASMCs, which is diminished by the PLC inhibitor U73122 and not by its inactive analog U73433 . Hypoxia augments PLCγ1-dependent inositol 1,4,5-trisphosphate (IP) generation. Exogenous ROS, hydrogen peroxide (HO), increases PLCγ1 phosphorylation at tyrosine-783 and IP production. IP receptor-1 (IPR1) knock-down remarkably diminishes hypoxia- or HO-induced increase in [Ca]. Hypoxia or HO increases the activity of IPRs, which is significantly reduced in protein kinase C-ε (PKCε) knockout PASMCs. A higher PLCγ1 expression, activity, and basal [Ca] are found in PASMCs, but not in mesenteric artery smooth muscle cells from mice exposed to chronic hypoxia (CH) for 21 days. CH enhances HO- and ATP-induced increase in [Ca] in PASMCs and PLC-dependent, norepinephrine-evoked pulmonary vasoconstriction. In conclusion, acute hypoxia uniquely causes ROS-dependent PLCγ1 activation, IP production, PKCε activation, IPR1 opening, Ca release, and contraction in mouse PASMCs; CH enhances PASM PLCγ1 expression, activity, and function, playing an essential role in pulmonary hypertension in mice.
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