Thiol-reactive metal compounds inhibit NF-kappa B activation by blocking I kappa B kinase.

Gold compounds are used in the treatment of rheumatoid arthritis. NF-kappa B is a transcription factor implicated in the expression of many inflammatory genes. NF-kappa B is activated by signal-induced phosphorylation and subsequent degradation of inhibitory I kappa B (inhibitory protein that dissociates from NF-kappa B) proteins, and a multisubunit I kappa B kinase (IKK) has been identified previously. We tested the effect of various gold compounds on the activation of NF-kappa B and IKK in LPS-stimulated RAW 264.7 mouse macrophages. A lipophilic gold compound, auranofin, suppressed the LPS-induced increase of nuclear kappa B-binding activity, degradation of I kappa B proteins, and IKK activation. Auranofin also blocked IKK activation induced by TNF and PMA/ionomycin, suggesting that the target of auranofin action is common among these diverse signal pathways. In vitro IKK activity was suppressed by addition of hydrophilic gold compounds, such as aurothiomalate, aurothioglucose, and AuCl3. Other thiol-reactive metal ions such as zinc and copper also inhibited IKK activity in vitro, and induction of IKK in LPS-stimulated macrophages. In vitro IKK activity required the presence of reducing agent and was blocked by addition of thiol group-reactive agents. Two catalytic subunits of IKK complex, IKK alpha and IKK beta, were both inhibited by these thiol-modifying agents, suggesting the presence of a cysteine sulfhydryl group in these subunits, which is critical for enzyme activity. The antiinflammatory activity of gold compounds in the treatment of rheumatoid arthritis may depend on modification of this thiol group by gold.

Nuclear factor kappaB (NF-kappaB) pathway as a therapeutic target in rheumatoid arthritis.

Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by persistent joint swelling and progressive destruction of cartilage and bone. Current RA treatments are largely empirical in origin and their precise mechanism of action is uncertain. Increasing evidence shows that chronic inflammatory diseases such as RA are caused by prolonged production of proinflammatory cytokines including tumor necrosis factor (TNF) and interleukin 1 (IL-1). The nuclear factor kappaB (NF-kappaB) plays an essential role in transcriptional activation of TNF and IL-1. NF-kappaB is induced by many stimuli including TNF and IL-1, forming a positive regulatory cycle that may amplify and maintain RA disease process. NF-kappaB and enzymes involved in its activation can be a target for anti-inflammatory treatment. Aspirin and sodium salicylate inhibit activation of NF-KB by blocking IkappaB kinase, a key enzyme in NF-kappaB activation. Glucocorticoids suppress expression of inflammatory genes by binding glucocorticoid receptor with NF-kappaB, and increasing expression of inhibitory protein of NF-kappaB, IkappaBalpha. Sulfasalazine and gold compounds also inhibit NF-kappaB activation. Continuing advances in our understanding of action mechanism of antirheumatic agents will benefit the future development of RA regimens with greater efficacy and less toxicity.

Cholesterol biosynthesis from lanosterol: regulation and purification of rat hepatic sterol 14-reductase.

We have previously characterized the membrane-bound sterol 14-reductase (14-reductase) that catalyzes anaerobically NADPH-dependent reduction of the 14-double bond of delta 8,14-diene or delta 7,14-diene sterols that are sterol intermediates in cholesterol biosynthesis in mammals (Paik et al. (1984) J. Biol. Chem. 259, 13413-13423). To elucidate the regulatory mechanism as well as molecular characteristics of the 14-reductase, we extended our investigation on the consequences of alteration of the enzymic activity under various physiological conditions. The enzymic activity of rat hepatic sterol 14-reductase was induced more than 11-fold by feeding 5% cholestyramine plus 0.1% lovastatin (the CL-diet) for 7 days but was severely suppressed by feeding 5% cholesterol or 0.01% AY-9944 (an inhibitor of 14-reductase) for the same period. The increase or decrease in the 14-reductase activity also parallels the same change in the cholesterol synthetic rate in hepatocytes from rats that had been fed either the CL-diet or 0.01% AY-9944. In vitro inhibition studies revealed that AY-9944 acts as a competitive inhibitor of the 14-reductase (Ki = 0.26 microM). A diurnal variation was observed for the 14-reductase with peak activity near the middle of the dark cycle (10 p.m.), which was abolished by administration of cycloheximide. With induced enzyme conditions 14-reductase has been further purified with chromatographic procedures to near homogeneity. Purified 14-reductase appears to be a M(r) = 70,000 protein that is composed of two equally-sized subunits having a M(r) = 38,000. All properties of the purified 14-reductase suggest that the solubilized enzyme is the principal 14-reductase of microsomes. Taken together, our results provide the first evidence in support of a previously unknown regulatory role for the 14-reductase in the overall cholesterol synthetic pathway.