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Liver ischemia-reperfusion (I/R) injury occurs through induction of oxidative stress and release of damage-associated molecular patterns (DAMPs), including cytosolic DNA released from dysfunctional mitochondria or from the nucleus. Cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) synthase (cGAS) is a cytosolic DNA sensor known to trigger stimulator of interferon genes (STING) and downstream type 1 interferon (IFN-I) pathways, which are pivotal innate immune system responses to pathogen. However, little is known about the role of cGAS/STING in liver I/R injury. We subjected C57BL/6 (WT), cGAS knockout (cGAS -/- ), and STING-deficient (STING gt/gt ) mice to warm liver I/R injury and that found cGAS -/- mice had significantly increased liver injury compared with WT or STING gt/gt mice, suggesting a protective effect of cGAS independent of STING. Liver I/R upregulated cGAS in vivo and also in vitro in hepatocytes subjected to anoxia/reoxygenation (A/R). We confirmed a previously published finding that hepatocytes do not express STING under normoxic conditions or after A/R. Hepatocytes and liver from cGAS -/- mice had increased cell death and reduced induction of autophagy under hypoxic conditions as well as increased apoptosis. Protection could be restored in cGAS -/- hepatocytes by overexpression of cGAS or by pretreatment of mice with autophagy inducer rapamycin. Our findings indicate a novel protective role for cGAS in the regulation of autophagy during liver I/R injury that occurs independently of STING. NEW & NOTEWORTHY Our studies are the first to document the important role of cGAS in the acute setting of sterile injury induced by I/R. Specifically, we provide evidence that cGAS protects liver from I/R injury in a STING-independent manner.

Whereas the ovary produces the majority of estradiol (E 2 ) in mature female primates, extraovarian sources contribute to E 2 synthesis and action, including the brain E 2 -regulating hypothalamic gonadotropin-releasing hormone. In ovary-intact female rodent models, aromatase inhibition (AI) induces a polycystic ovary syndrome-like hypergonadotropic hyperandrogenism due to absent E 2 -mediated negative feedback. To examine the role of extraovarian E 2 on nonhuman primate gonadotropin regulation, the present study uses letrozole to elicit AI in adult female marmoset monkeys. Sixteen female marmosets ( Callithrix jacchus ; >2 yr) were randomly assigned to ovary-intact or ovariectomy (OVX) conditions and subsequently placed on a daily oral regimen of either ~200 µl vehicle alone (ovary-intact Control, n = 3; OVX, n = 3) or 1 mg ⋅ kg -1 ⋅ day -1 letrozole in vehicle (ovary-intact AI, n = 4; OVX + AI, n = 6). Blood samples were collected every 10 days, and plasma chorionic gonadotropin (CG) and steroid hormone levels were determined by validated radioimmunoassay and liquid chromatography/tandem mass spectrometry, respectively. Ovary-intact, AI-treated and OVX females exhibited elevated CG ( P < 0.01, P = 0.004, respectively) compared with controls, and after 30 days, OVX + AI females exhibited a suprahypergonadotropic phenotype ( P = 0.004) compared with ovary-intact + AI and OVX females. Androstenedione ( P = 0.03) and testosterone ( P = 0.05) were also elevated in ovary-intact, AI-treated females above all other groups. The current study thus confirms in a nonhuman primate that E 2 depletion and diminished negative feedback in ovary-intact females engage hypergonadotropic hyperandrogenism. Additionally, we demonstrate that extraovarian estrogens, possibly neuroestrogens, contribute to female negative feedback regulation of gonadotropin release., (Copyright © 2017 the American Physiological Society.)

Myocytes are nonhemopoietic in origin and functionally essential in generating gastrointestinal motility. In endotoxemia, a rapid-onset nonhemopoietic mechanism potently triggers early ileus in a Toll-like receptor 4 (TLR4)/myeloid differentiation primary response gene 88 (MyD88)-dependent manner. Moreover, synergistically with hemopoietic cells, nonhemopoietic cells escalate late ileus via an IL-6 receptor-dependent inflammation-driven pathway. We therefore specifically investigated the role of myocytes in TLR4-triggered inflammation and ileus. TLR4(+/+), TLR4(-/-), bmTLR4(+/+)/TLR4(-/-) chimera, SM22-Cre(-/-)TLR4(flox/flox), and selective myocyte TLR4-deficient (SM22-Cre(+/-)TLR4(flox/flox)) mice were injected intraperitoneally with purified lipopolysaccharide. SM22-driven Cre recombinase activity was selectively detected in cardiac, gastrointestinal, skeletal, and vascular myocytes, of small-sized vessels in a two-color fluorescent Cre reporter mouse. In contrast to nonhemopoietic TLR4 deficiency, deletion of myocyte TLR4 signaling prevented neither endotoxin-induced suppression of spontaneous jejunal contractility in vitro nor early ileus in vivo at 6 h. Circulating plasma colony-stimulating factor 3 was greatly elevated during endotoxemia, independent of myocyte TLR4 signaling or time. TLR4 activation of myocytes contributed significantly to an early enteric IL-6 mRNA induction and systemic IL-6 release, as well as to a late increase in circulating chemokine (C-X-C motif) ligand 1 (CXCL1) and IL-17. Consequently, inhibition of myocyte TLR4 signaling allowed functional recovery of motility by preventing inflammation-driven late ileus at 24 h. Direct TLR4 activation of myocytes is not responsible for nonhemopoietic-mediated early ileus. However, myocytes are proinflammatory cells that potently drive enteric and systemic inflammation, subsequently fueling late mediator-triggered ileus. Specifically, the myocyte TLR4-dependent inflammatory signature of elevated plasma IL-6, CXCL1, and IL-17 is strongly associated with late rodent ileus., (Copyright © 2015 the American Physiological Society.)

Nitric oxide (NO) acts as a vasoregulatory molecule that inhibits vascular smooth muscle cell (SMC) proliferation. Studies have illustrated that NO inhibits SMC proliferation via the extracellular signal-regulated kinase (ERK) pathway, leading to increased protein levels of the cyclin-dependent kinase inhibitor p21(Waf1/Cip1). The ERK pathway can be pro- or antiproliferative, and it has been demonstrated that the activation status of the small GTPase RhoA determines the proliferative fate of ERK signaling, whereby inactivation of RhoA influences ERK signaling to increase p21(Waf1/Cip1) and inhibit proliferation. The purpose of these investigations was to examine the effect of NO on RhoA activation/S-nitrosation and to test the hypothesis that inhibition of SMC proliferation by NO is dependent on inactivation of RhoA. NO decreases activation of RhoA, as demonstrated by RhoA GTP-binding assays, affinity precipitation, and phalloidin staining of the actin cytoskeleton. Additionally, these effects are independent of cGMP. NO decreases SMC proliferation, and gene transfer of constitutively active RhoA (RhoA(63L)) diminished the antiproliferative effects of NO, as determined by thymidine incorporation. Western blots of p21(Waf1/Cip1) correlated with changes in proliferation. S-nitrosation of recombinant RhoA protein and immunoprecipitated RhoA was demonstrated by Western blotting for nitrosocysteine and by measurement of NO release. Furthermore, NO decreases GTP loading of recombinant RhoA protein. These findings indicate that inactivation of RhoA plays a role in NO-mediated SMC antiproliferation and that S-nitrosation is associated with decreased GTP binding of RhoA. Nitrosation of RhoA and other proteins likely contributes to cGMP-independent effects of NO.

Cytokine-stimulated inducible nitric oxide synthase (iNOS) gene expression is dependent on nuclear factor-kappa B (NF-kappa B) activation and is suppressed by glucocorticoids (GC). In this study we examined the molecular mechanisms of GC inhibition of iNOS expression in rat hepatocytes. Combinations of tumor necrosis factor-alpha, interleukin-1 beta, and interferon-gamma (cytokine mixture CM) induced high levels of iNOS mRNA and NO synthesis. The synthetic GC dexamethasone markedly repressed iNOS mRNA and protein expression, and nuclear run-on assays showed that this inhibition was occurring at the level of transcription. In addition, transfection studies showed that CM-stimulated activity of a 1.6-kb murine iNOS promoter fragment linked upstream of luciferase was suppressed by dexamethasone. Electromobility shift assays demonstrated that CM induced the appearance of an NF-kappa B complex composed of p50 and p65 subunits; the addition of dexamethasone markedly decreased this band shift. I-kappa B alpha expression was decreased by CM and upregulated in the presence of dexamethasone. Subsequently, nuclear p65 levels were decreased by dexamethasone compared with CM-treated cells. Thus GC repress NF-kappa B DNA-binding activity in rat hepatocytes in part through the upregulation of its inhibitor I-kappa B alpha. These data indicate that one mechanism by which GC block iNOS expression is through the inhibition of NF-kappa B activation resulting in decreased iNOS transcription.