The relationship between hypoxia and regulation of nitric oxide synthase (NOS) in myocardial tissue is not well understood. We investigated the role of hypoxia inducible factor-1 (HIF-1) on expression of the inducible NOS (iNOS) in myocardial cells in vivo and in vitro. In situ hybridization in myocardial tissue from rats exposed to hypoxia for 3 weeks demonstrated increased iNOS mRNA expression. Northern analysis of RNA from hearts of those animals and from cells exposed to hypoxia for 12 hours in vitro demonstrated an increase of HIF-1 RNA expression. Electrophoretic mobility shift assays using oligonucleotides containing the iNOS HIF-1 DNA binding site and nuclear extracts from cardiac myocytes showed induction of specific DNA binding in cells subjected to hypoxia. Transient transfection of cardiac myocytes using the murine iNOS promoter resulted in a 3.43-fold increase in promoter activity under hypoxia compared with normoxia. Mutation or deletion of the HIF-1 site eliminated the hypoxic response. As cytokines have been shown to regulate iNOS expression in myocardial cells, cultured neonatal cardiac myocytes were stimulated with interleukin-1beta causing a dramatic induction of iNOS protein expression under normoxia, with further augmentation under hypoxia. Transient transfection of cells stimulated with interleukin-1beta showed an increased iNOS promoter activity under normoxic conditions compared with unstimulated cells, with a further increase in response to hypoxia, which was dependent on HIF-1. These results demonstrate that hypoxia causes an increase in iNOS expression in cardiac myocytes and that HIF-1 is essential for the hypoxic regulation of iNOS gene expression.

Pulmonary hypertension is characterized by structural and morphological changes to the lung vasculature. To determine the potential role of nitric oxide in the vascular remodeling induced by hypoxia, we exposed wild-type [WT(+/+)] and endothelial nitric oxide synthase (eNOS)-deficient [(-/-)] mice to normoxia or hypoxia (10% O(2)) for 2, 4, and 6 days or for 3 wk. Smooth muscle alpha-actin and von Willebrand factor immunohistochemistry revealed significantly less muscularization of small vessels in hypoxic eNOS(-/-) mouse lungs than in WT(+/+) mouse lungs at early time points, a finding that correlated with decreases in proliferating vascular cells (5-bromo-2'-deoxyuridine positive) at 4 and 6 days of hypoxia in the eNOS(-/-) mice. After 3 wk of hypoxia, both mouse types exhibited similar percentages of muscularized small vessels; however, only the WT(+/+) mice exhibited an increase in the percentage of fully muscularized vessels and increased vessel wall thickness. eNOS protein expression was increased in hypoxic WT(+/+) mouse lung homogenates at all time points examined, with significantly increased percentages of small vessels expressing eNOS protein after 3 wk. These results indicate that eNOS deficiency causes decreased muscularization of small pulmonary vessels in hypoxia, likely attributable to the decrease in vascular cell proliferation observed in these mice.

The nitric oxide (NO)-cGMP signal transduction pathway plays an important role in the regulation of pulmonary vascular tone and resistance in pulmonary hypertension. A number of studies have demonstrated that endothelial (e) and inducible nitric oxide synthases (NOS) are upregulated in hypoxia-exposed rat lung. These changes in NOS expression have been found to correlate with the process of pulmonary vascular remodeling in hypoxia-induced pulmonary hypertension, and remodeling is increased in the absence of eNOS. In this study, we examined the expression and localization of soluble guanylate cyclase (sGC), the primary receptor for NO, in hypoxia- and normoxia-treated rat lungs. Male Sprague-Dawley rats were exposed to hypoxia (10% O(2), normobaric) or normoxia for 1, 3, 5, and 21 days. The lungs were used for Western analysis of sGC protein, sGC enzyme activity, immunohistochemistry using antiserum against sGC alpha(1)- and beta(1)-subunits, and nonradioactive in situ hybridization (NRISH) using a digoxigenin-labeled sGC alpha(1)-subunit cRNA probe. Western blot analysis revealed a more than twofold increase of sGC protein alpha(1)-subunit in rat lungs exposed to 3, 5, and 21 days of hypoxia, correlating well with sGC enzyme activity. Immunohistochemistry and NRISH demonstrated increased expression of sGC in the smooth muscle cells of the pulmonary arteries and arterioles in the hypoxic rat lungs when compared with normoxic controls. Based on our results, the upregulation of sGC may play an important role in the regulation of smooth muscle tone and pressure in the pulmonary circulation during chronic hypoxia.

Highlights d CNTF induces neuronal production of IL-6 upon peripheral nerve injury d CNTF-STAT3-IL-6 mediates neuroinflammation from peripheral to central nervous systems d Schwann cells-neurons-microglia transmit neuroinflammation to the central nervous system d CNTF acts as a danger signal for nerve injury response and pain enhancement

Microglial activation occurs in divergent neuropathological conditions. Such microglial event has the key involvement in the progression of CNS diseases. However, the transcriptional mechanism governing microglial activation remains poorly understood. Here, we investigate the microglial response to traumatic injury-induced neurodegeneration by the 3D fluorescence imaging technique. We show that transcription factors IRF8 and PU.1 are both indispensible for microglial activation, as their specific post-developmental deletion in microglia abolishes the process. Mechanistically, we reveal that IRF8 and PU.1 directly target the gene transcription of each other in a positive feedback to sustain their highly enhanced expression during microglial activation. Moreover, IRF8 and PU.1 dictate the microglial response by cooperatively acting through the composite IRF-ETS motifs that are specifically enriched on microglial activation-related genes. This action of cooperative transcription can be further verified biochemically by the synergetic binding of IRF8 and PU.1 proteins to the composite-motif DNA. Our study has therefore elucidated the central transcriptional mechanism of microglial activation in response to neurodegenerative condition.Electronic supplementary materialThe online version of this article (10.1007/s13238-018-0599-3) contains supplementary material, which is available to authorized users.

Background:Corticotrophin-releasing hormone (CRH) is considered to be the central driving force of the hypothalamic-pituitary-adrenal axis, which plays a key role in the stress response and depression. Clinical reports have suggested that excess retinoic acid (RA) is associated with depression. Abscisic acid (ABA) and RA are direct derivatives of carotenoids and share a similar molecular structure. Here, we proposed that ABA also plays a role in the regulation of CRH activity sharing with the RA signaling pathway.Methods:[3H]-ABA radioimmunoassay demonstrated that the hypothalamus of rats shows the highest concentration of ABA compared with the cortex and the hippocampus under basal conditions.Results:Under acute stress, ABA concentrations increased in the serum, but decreased in the hypothalamus and were accompanied by increased corticosterone in the serum and c-fos expression in the hypothalamus. Moreover, chronic ABA administration increased sucrose intake and decreased the mRNA expression of CRH and retinoic acid receptor alpha (RARα) in the hypothalamus of rats. Furthermore, ABA improved the symptom of chronic unpredictable mild stress in model rats, as indicated by increased sucrose intake, increased swimming in the forced swim test, and reduced mRNA expression of CRH and RARα in the rat hypothalamus. In vitro, CRH expression decreased after ABA treatment across different neural cells. In BE(2)-C cells, ABA inhibited a series of retinoid receptor expression, including RARα, a receptor that could facilitate CRH expression directly.Conclusions:These results suggest that ABA may play a role in the pathogenesis of depression by downregulating CRH mRNA expression shared with the RA signaling pathway.

Oxaliplatin (OXL) is a third-generation chemotherapeutic agent commonly used to treat metastatic digestive tumors; however, one of the main limiting complications of OXL is neuropathic pain. In this study, the underlying mechanisms responsible for OXL evoked-neuropathic pain were examined. Using a rat model, the results demonstrated that intraperitoneal (i.p.) injection of OXL significantly increased mechanical pain and cold sensitivity as compared with control animals (P < 0.05 vs. control rats). Blocking proteinase-activated receptor 2 (PAR2) significantly attenuated mechanical pain and cold sensitivity observed in control rats and OXL rats (P < 0.05 vs. vehicle control). The attenuating effect of PAR2 on mechanical pain and cold sensitivity were significantly smaller in OXL-rats than in control rats. The role played by PAR2 downstream signaling pathways [namely, transient receptor potential ankyrin 1 (TRPA1)] in regulating OXL evoked-neuropathic pain was also examined. The data shows that TRPA1 expression was upregulated in the lumbar dorsal root ganglion (DRG) of OXL rats and blocking TRPA1 inhibited mechanical pain and heightened cold sensitivity (P < 0.05 vs. control rats). Blocking PAR2 also significantly decreased TRPA1 expression in the DRG. Findings in this study show that OXL intervention amplifies mechanical hyperalgesia and cold hypersensitivity and PAR2 plays an important role in regulating OXL-induced neuropathic pain via TRPA1 pathways.

Retinoid acid (RA) plays critical roles in regulating differentiation and apoptosis in a variety of cancer cells. Abscisic acid (ABA) and RA are direct derivatives of carotenoids and share structural similarities. Here we proposed that ABA may also play a role in cellular differentiation and apoptosis by sharing a similar signaling pathway with RA that may be involved in glioma pathogenesis. We reported for the first time that the ABA levels were twofold higher in low‐grade gliomas compared with high‐grade gliomas. In glioma tissues, there was a positive correlation between the ABA levels and the transcription of cellular retinoic acid‐binding protein 2 (CRABP2) and a negative correlation between the ABA levels and transcription of fatty acid‐binding protein 5 (FABP5). ABA treatment induced a significant increase in the expression of CRABP2 and a decrease in the expression of peroxisome proliferator‐activated receptor (PPAR) in glioblastoma cells. Remarkably, both cellular apoptosis and differentiation were increased in the glioblastoma cells after ABA treatment. ABA‐induced cellular apoptosis and differentiation were significantly reduced by selectively silencing RAR‐α, while RAR‐α overexpression exaggerated the ABA‐induced effects. These results suggest that ABA may play a role in the pathogenesis of glioma by promoting cellular apoptosis and differentiation through the RA signaling pathway.