The CO-releasing molecule CORM-2 is a novel regulator of the inflammatory process in osteoarthritic chondrocytes.

OBJECTIVES: Previous work has shown that the CO-releasing molecule CORM-2 protects against cartilage degradation. The aim of this study was to examine whether CORM-2 can control the production of inflammatory mediators in osteoarthritic chondrocytes and determine the mechanisms involved. METHODS: Primary cultures of chondrocytes from OA patients were stimulated with IL-1beta. The production of reactive oxygen species, nitrite, PGE(2), TNF-alpha and IL-1 receptor antagonist (IL-1Ra) were measured in the presence or absence of CORM-2. The expression of nitric oxide synthase-2 (NOS-2), cyclo-oxygenase-2 (COX-2) and microsomal PG E synthase-1 (mPGES-1) was followed by western blot and real-time PCR. Activation of nuclear factor-kappaB (NF-kappaB) and hypoxia inducible factor-1alpha (HIF-1alpha), and phosphorylation of NF-kappaB inhibitory protein alpha (IkappaBalpha) were determined by ELISA. RESULTS: CORM-2 decreased the production of oxidative stress, nitrite and PGE(2). In addition, CORM-2 inhibited IL-1beta-induced TNF-alpha but enhanced IL-1Ra production. Treatment of chondrocytes with CORM-2 strongly down-regulated NOS-2 and mPGES-1 protein expression, whereas COX-2 was reduced to a lesser extent. These changes were accompanied by a significant decrease in mRNA expression for NOS-2 and mPGES-1. CORM-2 showed a concentration-dependent inhibition of DNA-binding activity for p65 NF-kappaB and HIF-1alpha. IkappaBalpha phosphorylation was also reduced by CORM-2 treatment. CONCLUSIONS: These data have opened new mechanisms of action for CORM-2, raising the prospect that CO-releasing molecules are an interesting strategy for the development of new treatments in articular conditions.

Anti-inflammatory actions of the heme oxygenase-1 pathway.

Heme oxygenase 1 (HO-1) is induced by oxidative or nitrosative stress, cytokines and other mediators produced during inflammatory processes, likely as part of a defence system in cells exposed to stress to provide a negative feedback for cell activation and the production of mediators, which could modulate the inflammatory response. HO-1 activity results in the inhibition of oxidative damage and apoptosis, with significant reductions in inflammatory events including edema, leukocyte adhesion and migration, and production of inflammatory cytokines. HO-1 is induced by nitric oxide (NO) in different biological systems and can control the increased production of this mediator observed in many inflammatory situations. Regulatory interactions between HO-1 and cyclooxygenase (COX) pathways have also been reported. Modulation of signal transduction pathways by HO-1 or products derived from its activity, such as carbon monoxide (CO), may mediate the anti-inflammatory effects of this protein. Regulation of HO-1 activity may be a therapeutical strategy for a number of inflammatory conditions.

Modulation of haem oxygenase-1 expression by nitric oxide and leukotrienes in zymosan-activated macrophages.

Phagocytosis of unopsonized zymosan by RAW 264.7 macrophages upregulated protein expression of haem oxygenase-1 (HO-1), inducible nitric oxide synthase (iNOS) and cyclo-oxygenase-2 (COX-2) in a time- and concentration-dependent manner. In the presence of zymosan, exogenous prostaglandin E(2) (PGE(2)) did not exert significant effects on the expression of these three enzymes. In contrast, exogenous leukotriene B(4) (LTB(4)) and LTC(4) in the nanomolar range inhibited HO-1 and iNOS expression, as well as nitrite accumulation. The COX inhibitors indomethacin and NS398 weakly inhibited HO-1 expression but had no effect on iNOS and COX-2 expression or nitrite. In contrast, the 5-lipoxygenase (5-LO) inhibitor ZM 230,487 significantly decreased HO-1, iNOS and nitrite, which were not affected by zileuton. Dexamethasone showed an inhibitory effect on HO-1 expression induced by zymosan. ZM 230,487 but not zileuton, inhibited the shift due to nuclear factor-kappaB (NF-kappaB), whereas they did not modify activator protein-1 (AP-1) binding. Our results suggest that inhibition of NF-kappaB binding could mediate the effects of ZM 230,487 on the modulation of HO-1 and iNOS protein expression. NOS inhibition by L-N(G)-nitroarginine methyl ester (L-NAME) or 1400 W abolished nitrite production and strongly reduced HO-1 expression. These results show an induction of HO-1 protein expression by zymosan phagocytosis in macrophages, with a positive modulatory role for endogenous NO and a negative regulation by exogenous LTs, likely dependent on the reduction of iNOS expression and NO production.

Oncostatin M down-regulates basal and induced cytochromes P450 in human hepatocytes.

The effects of oncostatin M on the expression of different cytochrome P450 (CYP) isozymes has been investigated in human hepatocytes. The dose-response and time-course analyses of effects on CYP1A2 and CYP3A4 isozymes revealed that maximal inhibition was reached after 48 hr of exposure of human hepatocytes to 25 units/ml oncostatin M. Reductions in CYP1A2 and CYP3A4 activity produced by oncostatin M correlated with decreases in protein content, de novo protein synthesis and specific mRNA levels, thus suggesting that oncostatin M could down-regulate CYP expression at the transcriptional level. The inhibitory potency of oncostatin M on CYP expression was compared with that of other cytokines belonging to the interleukin-6 receptor family (interleukin-6, interleukin-11 and leukemia inhibitory factor), and interferon-gamma, which is recognized to inhibit human CYP expression, and granulocyte colony-stimulating factor, a cytokine that shares structural homology with the interleukin-6 family but has a different transduction signal. Maximal reductions in CYP1A2 activity were reached after 48 hr of treatment with cytokines. At that time, oncostatin M showed the highest inhibitory effects on CYP1A2 activity (38% of control), followed by interferon (49% of control) and interleukin-6 (60% of control), whereas minor effects were produced by the other cytokines (74-80%). Comparable decreases were observed for CYP2A6, CYP2B6 and CYP3A4 activities. Enzymatic activity and de novo protein synthesis of 3-methylcholanthrene-induced CYP1A2 and dexamethasone-induced CYP3A4 were also reduced to a much greater extent by oncostatin M than by other cytokines. The results show that oncostatin M is the most effective cytokine in down-regulating CYP isozymes in human hepatocytes, and its effects were evident even after removal of the cytokine from the culture medium.

Nitric oxide-mediated inhibition of cytochrome P450 by interferon-gamma in human hepatocytes.

The role of nitric oxide in the inhibition of the cytochrome P450 system produced by interferon-gamma in human hepatocytes has been examined. Nitric oxide exogenously released from S-nitroso-N-acetylpenicillamine produced a dose-dependent decrease in cytochrome P4501A2 activity, assessed as 7-ethoxy resorufin O-deethylation. After 24 hr of treatment with 300 U/ml interferon-gamma, a rise in nitric oxide release (200% over control cells) and a parallel inhibition in 7-ethoxyresorufin O-deethylase activity (50% of control) were observed in human hepatocytes. This inhibition was concentration-dependently prevented by N(G)-monomethyl-L-arginine, a competitive inhibitor of nitric oxide biosynthesis. Comparable results were observed for cytochrome P4502A6 (7-coumarin hydroxylation), 2B6 (7-benzoxyresorufin O-dealkylation) and 3A4 (testosterone 6beta-hydroxylation) activities. Decreases in CYP1A2 activity found after exposure of 3-methylcholanthrene-treated hepatocytes to interferon-gamma were also reversed in the presence of N(G)-monomethyl-L-arginine. Down-regulation of cytochrome P4501A2 and 3A4 expression by interferon-gamma was observed in parallel. This study suggests that at least some of the interferon-gamma effects on human hepatocyte cytochrome P450 isoenzymes are mediated by nitric oxide biosynthesis.

The hepatocyte growth factor regulates the synthesis of acute-phase proteins in human hepatocytes: divergent effect on interleukin-6-stimulated genes.

Our study addressed the role of the human hepatocyte growth factor (HGF), a potent mitogen for mature rat and human hepatocytes, in the regulation of specific hepatic genes. The experimental evidence obtained in primary cultured human hepatocytes indicates that HGF regulates the synthesis of plasma proteins in a dose-response fashion. It stimulates the synthesis of the negative acute-phase proteins albumin, transferrin, and fibronectin, decreases that of alpha1-antichymotrypsin (ACT) and haptoglobin, and stimulates that of alpha2-macroglobulin (AMG), which in man is insensitive to inflammatory mediators. HGF had no effect on C-reactive protein (CRP) synthesis. These effects differ from those elicited by interleukin-6 (IL-6). The effects of HGF on fibrinogen and alpha1-antitrypsin were, however, similar to those induced by IL-6. The effects of HGF were also observed at the messenger RNA (mRNA) level. Time-course induction experiments showed that the effects of HGF on protein synthesis were delayed by about 48 to 72 hours, in contrast with the 12-hour lag found after IL-6 stimulation. Although the presence of glucocorticoids was not absolutely necessary for HGF to affect plasma protein synthesis, it moderately extended the effects. In pulse-chase experiments, it was found that the action of HGF was not due to an alteration of the rate of secretion of the proteins. The effects of HGF on the synthesis of albumin, transferrin, fibronectin, alpha1-antichymotrypsin, and haptoglobin could be counteracted by the simultaneous presence of IL-6 in the incubation media. A clear additive effect was observed only in the case of fibrinogen. No interaction was observed in the cases of CRP and AMG. The results of this study indicate that the effects of HGF on human hepatocytes may not simply be limited to its mitogenic activity, but that it also regulates hepatic-specific genes and antagonizes, in part, the action of IL-6.