Hydrogen sulfide inhibits nitric oxide production and nuclear factor-kappaB via heme oxygenase-1 expression in RAW264.7 macrophages stimulated with lipopolysaccharide

Hydrogen sulfide (H(2)S), a regulatory gaseous molecule that is endogenously synthesized by cystathionine gamma-lyase (CSE) and/or cystathionine beta-synthase (CBS) from L-cysteine (L-Cys) metabolism, is a putative vasodilator, and its role in nitric oxide (NO) production is unexplored. Here, we show that at noncytotoxic concentrations, H(2)S was able to inhibit NO production and inducible NO synthase (iNOS) expression via heme oxygenase (HO-1) expression in RAW264.7 macrophages stimulated with lipopolysaccharide (LPS). Both H(2)S solution prepared by bubbling pure H(2)S gas and NaSH, a H(2)S donor, dose dependently induced HO-1 expression through the activation of the extracellular signal-regulated kinase (ERK). Pretreatment with H(2)S or NaHS significantly inhibited LPS-induced iNOS expression and NO production. Moreover, NO production in LPS-stimulated macrophages that are expressing CSE mRNA was significantly reduced by the addition of L-Cys, a substrate for H(2)S, but enhanced by the selective CSE inhibitor beta-cyano-L-alanine but not by the CBS inhibitor aminooxyacetic acid. While either blockage of HO activity by the HO inhibitor, tin protoporphyrin IX, or down-regulation of HO-1 expression by HO-1 small interfering RNA (siRNA) reversed the inhibitory effects of H(2)S on iNOS expression and NO production, HO-1 overexpression produced the same inhibitory effects of H(2)S. In addition, LPS-induced nuclear factor (NF)-kappaB activation was diminished in RAW264.7 macrophages preincubated with H(2)S. Interestingly, the inhibitory effect of H(2)S on NF-kappaB activation was reversed by the transient transfection with HO-1 siRNA, but was mimicked by either HO-1 gene transfection or treatment with carbon monoxide (CO), an end product of HO-1. CO treatment also inhibited LPS-induced NO production and iNOS expression via its inactivation of NF-kappaB. Collectively, our results suggest that H(2)S can inhibit NO production and NF-kappaB activation in LPS-stimulated macrophages through a mechanism that involves the action of HO-1/CO.     

NF-kappa B regulates the LPS-induced expression of interleukin 12 p40 in murine peritoneal macrophages: roles of PKC, PKA, ERK, p38 MAPK, and proteasome

NF-kappa B plays a critical role in coordinating the control of gene expression during monocyte/macrophage activation. In this report we describe our investigation of the mechanisms of LPS-induced NF-kappa B activation and IL-12 expression in murine peritoneal suppressor macrophages. Treatment of these macrophages with LPS induced I kappa B alpha degradation and NF-kappa B activation. EMSAs demonstrated that NF-kappa B bound to a cis-acting element located in the murine IL-12 p40 promoter. LPS signal transduction has been shown to involve a variety of signal pathways. The results in this paper indicate that LPS-induced NF-kappa B binding activity was independent of PKC, PKA, ERK, and p38 MAPK, but was regulated by proteasome. Furthermore, Proteasome Inhibitor I abolished the LPS-induced mRNA expression of IL-12 p35 and p40, and SB203580 reduced these mRNA levels, whereas the blockade of PKC, PKA, and ERK had little effect. These data demonstrate that the LPS-induced activation of proteasome. I kappa B. NF-kappa B and p38 MAPK signal pathways regulate the IL-12 expression in murine peritoneal suppressor macrophages.     

Heme oxygenase-1 mediates the anti-inflammatory effect of interleukin-10 in mice

The mechanisms underlying the action of the potent anti-inflammatory interleukin-10 (IL-10) are poorly understood. Here we show that, in murine macrophages, IL-10 induces expression of heme oxygenase-1 (HO-1), a stress-inducible protein with potential anti-inflammatory effect, via a p38 mitogen-activated protein kinase-dependent pathway. Inhibition of HO-1 protein synthesis or activity significantly reversed the inhibitory effect of IL-10 on production of tumor necrosis factor-alpha induced by lipopolysaccharide (LPS). Additional experiments revealed the involvement of carbon monoxide, one of the products of HO-1-mediated heme degradation, in the anti-inflammatory effect of IL-10 in vitro. Induction of HO-1 by IL-10 was also evident in vivo. IL-10-mediated protection against LPS-induced septic shock in mice was significantly attenuated by cotreatment with the HO inhibitor, zinc protoporphyrin. The identification of HO-1 as a downstream effector of IL-10 provides new possibilities for improved therapeutic approaches for treating inflammatory diseases.     

Heat shock inhibits IL-12 p40 expression through NF-kappa B signalling pathway in murine macrophages.

We report the effect of heat shock on lipopolysaccharide (LPS)-induced interleukin 12 (IL-12) expression. The augmentation of LPS-induced IL-12 p40 mRNA and p70 protein was significantly suppressed in both peritoneal macrophages and RAW264.7 cells after heat shock at 43 degrees C. The binding activity of nuclear factor kappa B (NF-kappa B) was reduced by prior heat shock. LPS did not induce degradation of the inhibitory protein I-kappa B alpha in the shocked cells, which might be a potential mechanism to block NF-kappa B activation. Furthermore, transient transfection assay in RAW264.7 cells demonstrated that LPS-induced activation of DM703 and DM138 (contains NF-kappa B motif) was highly sensitive to heat shock. These data suggest that heat shock influences expression of IL-12 through the I-kappa B/NF-kappa B pathway.     

15-deoxy-delta12,14-prostaglandin J2-mediated ERK signaling inhibits gram-negative bacteria-induced RelA phosphorylation and interleukin-6 gene expression in intestinal epithelial cells through modulation of protein phosphatase 2A activity

We have previously shown that non-pathogenic Gram-negative Bacteroides vulgatus induces transient RelA phosphorylation (Ser-536), NF-kappaB activity, and pro-inflammatory gene expression in native and intestinal epithelial cell (IEC) lines. We now demonstrate that 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)) but not prostaglandin E(2) inhibits lipopolysaccharide (LPS) (B. vulgatus)/LPS (Escherichia coli)-induced RelA phosphorylation and interleukin-6 gene expression in the colonic epithelial cell line CMT-93. This inhibitory effect of 15d-PGJ(2) was mediated independently of LPS-induced IkappaBalpha phosphorylation/degradation and RelA nuclear translocation as well as RelA DNA binding activity. Interestingly, although B. vulgatus induced nuclear expression of peroxisome proliferator-activated receptor gamma (PPARgamma) in native epithelium of monoassociated Fisher rats, PPARgamma-specific knock-down in CMT-93 cells using small interference RNA failed to reverse the inhibitory effects of PPARgamma agonist 15d-PGJ(2), suggesting PPARgamma-independent mechanisms. In addition, 15d-PGJ(2) but not the synthetic high affinity PPARgamma ligand rosiglitazone triggered ERK1/2 phosphorylation in IEC, and most importantly, MEK1 inhibitor PD98059 reversed the inhibitory effect of 15dPGJ(2) on LPS-induced RelA phosphorylation and interleukin-6 gene expression. Calyculin A, a specific phosphoserine/phospho-threonine phosphatase inhibitor increased the basal phosphorylation of RelA and reversed the inhibitory effect of 15d-PGJ(2) on LPS-induced RelA phosphorylation. We further demonstrated in co-immunoprecipitation experiments that 15d-PGJ(2) triggered protein phosphatase 2A activity, which directly dephosphorylated RelA in LPS-stimulated CMT-93 cells. We concluded that 15d-PGJ(2) may help to control NF-kappaB signaling and normal intestinal homeostasis to the enteric microflora by modulating RelA phosphorylation in IEC through altered protein phosphatase 2A activity.     

RA-XII exerts anti-oxidant and anti-inflammatory activities on lipopolysaccharide-induced acute renal injury by suppressing NF-κB and MAPKs regulated by HO-1/Nrf2 pathway

Acute kidney injury (AKI) is an abrupt loss of kidney function and severe AKI needs renal replacement therapeutic strategy and has high mortality. RA-XII is a natural cyclopeptide, isolated from the traditional Chinese medicine Rubia yunnanensis, exerting anti-inflammatory and anti-tumor activities. The present study aimed to explore the effects of RA-XII on LPS-induced ACI and the underlying molecular mechanism in TCMK-1?cells in vitro. The results indicated that RA-XII delayed the animal death caused by LPS in mice. The kidney histological changes were markedly attenuated by RA-XII. RA-XII also reduced the serum uric acid, creatinine, BUN and renal 8-OHdG. In addition, RA-XII suppressed LPS-induced oxidative stress in kidney, as evidenced by the up-regulation of superoxide dismutase (SOD), catalase (CAT) and glutathione (GSH) levels, and the down-regulation of malondialdehyde (MDA) levels. Additionally, RA-XII enhanced heme oxygenase (HO)-1 and nuclear factor erythroid 2-related factor 2 (Nrf2) expressions in renal tissue sections. Further, RA-XII reduced the release of pro-inflammatory cytokines, including tumor necrosis factor-alpha (TNF-α), interleukin-1β (IL-1β), IL-6 and IL-18, in renal, which was linked to the inhibition of inhibitor of alpha/nuclear factor kappa B (IκBα/NF-κB) and mitogen-activated protein kinases (MAPKs) pathways. The in vitro study illustrated that the anti-inflammatory effects of RA-XII were partially reversed following Nrf2 and HO-1 inhibition. Together, these findings strongly suggested that RA-XII is a potential agent against acute kidney injury.     

Upregulation of MIP-2 (CXCL2) expression by 15-deoxy-Delta(12,14)-prostaglandin J(2) in mouse peritoneal macrophages

A peroxisome proliferator-activated receptor gamma (PPARgamma) ligand, 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)), has been reported to possess anti-inflammatory activity in activated monocytes/macrophages. In this study, we investigated the effect of 15d-PGJ(2) on the lipopolysaccharide (LPS)-induced expression of chemokine mRNAs, especially macrophage inhibitory protein (MIP)-2 (CXCL2), in mouse peritoneal macrophages. The inhibitory actions of the natural PPARgamma ligands, 15d-PGJ(2) and prostaglandin A1 (PGA1), on the expression of RANTES (regulated upon activation, normal T expressed and secreted; CCL5), MIP-1beta (CCL4), MIP-1alpha (CCL3), IFN-gamma-inducible protein 10 kilodaltons (IP-10; CXCL10) and monocyte chemoattractant protein-1 (MCP-1; CCL2) mRNA in LPS-treated cells were stronger than those of the synthetic PPARgamma ligands troglitazone and ciglitazone. However, 15d-PGJ(2) enhanced the expression of LPS-induced MIP-2 (CXCL2) mRNA. A specific PPARgamma antagonist (GW9662) had no effect on the inhibitory action of 15d-PGJ(2) and PGA1 in LPS-induced chemokine mRNA expression and on the synergistic action of 15d-PGJ(2) in LPS-induced MIP-2 (CXCL2) expression. Moreover, LPS itself reduced the expression of PPARgamma. Although the synergistic effect of 15d-PGJ(2) on LPS-induced MIP-2 (CXCL2) mRNA expression was remarkable, the production of MIP-2 (CXCL2) in cells treated with 15d-PGJ(2) and LPS did not increase compared to the production in cells treated with LPS alone. The synergistic action of 15d-PGJ(2) on LPS-induced MIP-2 (CXCL2) mRNA expression was dependent on the activation of nuclear factor-kappaB (NF-kappaB), and 15d-PGJ(2) increased the phosphorylation of p38 and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) in cells stimulated with LPS. These results suggest that the synergistic effect of 15d-PGJ(2) on LPS-induced MIP-2 (CXCL2) expression is PPARgamma-independent, and is mediated by the p38 and SAPK/JNK pathway in mitogen-activated protein kinase signaling pathways, which activates NF-kappaB. Our data may give more insights into the different mechanisms contrary to the anti-inflammatory effect of 15d-PGJ(2) on the expression of chemokine genes.     

Heme oxygenase modulates selectin expression in different regional vascular beds

Heme oxygenase (HO) catalyzes the degradation of heme to biliverdin, iron, and CO. The inducible isoform (HO-1) has been implicated as a modulator of the inflammatory response. HO-1 activity can be induced by hemin and inhibited with zinc protoporphyrin IX (ZnPP). Using these reagents, we assessed the possibility that HO-1 modulates the inflammatory response by altering the expression of endothelial cell adhesion molecules. Endotoxin (lipopolysaccharide, LPS)-induced expression of P- and E-selectin expression was quantified in different vascular beds of the rat using the dual radiolabeled monoclonal antibody technique. Pretreatment with hemin attenuated, whereas ZnPP treatment exacerbated, the increased selectin expression normally elicited by LPS. Biliverdin, at an equimolar dosage, was as effective as hemin in attenuating LPS-induced selectin expression in the lung, kidneys, liver, and intestines. These findings indicate that the anti-inflammatory properties of HO-1 may be related to an inhibitory action of P- and E-selectin expression in the vasculature. Biliverdin (or its metabolite, bilirubin), rather than CO, may account for this action of HO-1 on endothelial cell adhesion molecule expression.     

Heme oxygenase/carbon monoxide signaling pathways: regulation and functional significance

Carbon monoxide (CO), a gaseous second messenger, arises in biological systems during the oxidative catabolism of heme by the heme oxygenase (HO) enzymes. HO exists as constitutive (HO-2, HO-3) and inducible isoforms (HO-1), the latter which responds to regulation by multiple stress-stimuli. HO-1 confers protection in vitro and in vivo against oxidative cellular stress. Although the redox active compounds that are generated from HO activity (i.e. iron, biliverdin-IXalpha, and bilirubin-IXa) potentially modulate oxidative stress resistance, increasing evidence points to cytoprotective roles for CO. Though not reactive, CO regulates vascular processes such as vessel tone, smooth muscle proliferation, and platelet aggregation, and possibly functions as a neurotransmitter. The latter effects of CO depend on the activation of guanylate cyclase activity by direct binding to the heme moiety of the enzyme, stimulating the production of cyclic 3':5'-guanosine monophosphate. CO potentially interacts with other intracellular hemoprotein targets, though little is known about the functional significance of such interactions. Recent progress indicates that CO exerts novel anti-inflammatory and anti-apoptotic effects dependent on the modulation of the p38 mitogen activated protein kinase (MAPK)-signaling pathway. By virtue of these effects, CO confers protection in oxidative lung injury models, and likely plays a role in HO-1 mediated tissue protection.