Gaby Walker

THERAPEUTIC STRATEGIES FOR THE INHIBITION OF INDUCIBLE NITRIC OXIDE SYNTHASE—POTENTIAL FOR A NOVEL CLASS OF ANTI-INFLAMMATORY AGENTS

In recent years, NO, a gas previously considered a potentially toxic chemical, has become established as a diffusible universal messenger mediating cell—cell communication throughout the body. In mammals, NO is a recognized mediator of blood vessel relaxation that helps to maintain blood pressure. In the central nervous system NO acts as a non‐conventional neurotransmitter and participates in the establishment of long‐term plasticity required for memory formation. In addition, NO is responsible for some parts of the host response to sepsis and inflammation and contributes to certain disease states. A number of strategies have emerged with regard to a pharmacological control of pathological NO overproductions. This review will discuss these novel therapeutic approaches that may provide new means for clinical medicine.

Proteolytic cleavage of inducible nitric oxide synthase (iNOS) by calpain I

Proteolytic degradation of inducible nitric oxide synthase (iNOS or NOS2; EC 1.14.13.39) is one of the key steps by which the synthetic glucocorticoid dexamethasone controls the amount of iNOS protein and thus the production of nitric oxide (NO) in interferon-gamma-stimulated RAW 264.7 cells. In the present study we examined the role of the calmodulin (CaM)-binding site present within iNOS protein for the proteolytic degradation by the calcium-dependent neutral cysteine protease calpain I (EC 3.4.22.17). Using pulse chase experiments as well as cell-free degradation assays we show that the iNOS monomer is a direct substrate for cleavage by calpain I. Two structural determinants are involved in proteolytic cleavage, the canonical CaM-binding domain present at amino acids 501-532 and a conformational determinant located within iNOS. The access of the CaM-binding region appears to be critical for substrate cleavage as incubation of in vitro synthesized iNOS with purified CaM inhibits iNOS degradation by calpain I. Moreover, cytosolic CaM levels are decreased upon treatment of RAW 264.7 cells with dexamethasone as assessed by immunoprecipitation. The data shown herein provide novel insights into the underlying mechanisms involved in the anti-inflammatory actions of glucocorticoids.

Pyrrolidine dithiocarbamate differentially affects cytokine- and cAMP-induced expression of group II phospholipase A2 in rat renal mesangial cells

Renal mesangial cells express group II phospholipase A2 in response to two principal classes activating signals that may interact in a synergistic fashion. These two groups of activators comprise inflammatory cytokines such as interleukin‐1β (IL‐1β) and tumor necrosis factor‐α (TNFα) and agents that elevate cellular levels of cAMP such as forskolin, an activator of adenylate cyclase. Using pyrrolidine dithiocarbamate (PDTC), a potent inhibitor of nuclear factor NFκB, we determined its role in cytokine — and cAMP — triggered group II PLA2 expression. Micromolar amounts of PDTC suppress the IL‐1β‐ and TNF α‐dependent, but not the forskolin‐stimulated group II PLA2 activity in mesangial cells. Furthermore, PDTC inhibited the increase of group II PLA2 mRNA steady state levels in response to IL‐1β and TNFα, while only marginally affecting forskolin‐induced PLA2 mRNA level. Our data suggest that NFκB activation is an essential component of the cytokine signalling pathway responsible for group II PLA2 gene regulation and that cAMP triggers a separate signalling cascade not involving NFκB. These observations may provide a basis to study the underlying mechanisms involved in the regulation of group II PLA2 gene expression.

Suppression by cyclosporin A of interleukin 1β-induced expression of group II phospholipase A2 in rat renal mesangial cells

We investigated whether cyclosporin A, a potent immunosuppressive drug, affects group II phospholipase A2 (PLA2; EC 3.1.1.4) induction in rat renal mesangial cells. Previously we showed that the expression of group II PLA2 in rat renal mesangial cells is triggered by exposure of the cells to inflammatory cytokines such as interleukin 1β (IL‐1β) or tumour necrosis factor α and agents that elevate cellular levels of cyclic AMP. Treatment of mesangial cells with IL‐1β for 24 h induced PLA2 activity secreted into cell culture supernatants by about 16 fold. Incubation of mesangial cells with cyclosporin A inhibited IL‐1β‐induced PLA2 section in a dose‐dependent fashion, with an IC50 value of 4.3 μM. Cyclosporin A did not directly inhibit enzymatic activity of PLA2. Immunoprecipitation of radioactively labelled PLA2 protein from mesangial cell supernatants revealed that the inhibition of PLA2 activity is due to a suppression of PLA2 protein levels. This effect was preceded by a reduction of PLA2 mRNA steady state levels, as demonstrated by Northern blot analyses of total cellular RNA isolated from stimulated mesangial cells. In order to evaluate whether cyclosporin A would affect the transcriptional activity of the PLA2 gene, we performed nuclear run on transcription experiments and provided evidence that the transcription rate of the PLA2 gene is reduced by cyclosporin A. Previously we found that the nuclear transcription factor κB (NFκB) is an essential component of the IL‐1β‐dependent upregulation of PLA2 gene transcription. By electrophoretic mobility shift analysis, we demonstrated that cyclosporin A diminishes the formation of NFκB DNA‐binding complexes, thus suggesting that this transcription factor is a target for cyclosporin A‐mediated repression of PLA2 gene transcription. The data presented in this study strongly suggest that the cellular mechanism involved in the IL1β ‐ dependent transcriptional upregulation of the PLA2 gene in mesangial cells is a target for the action of cyclosporin A.

Tetranactin inhibits interleukin 1β and cAMP induction of group II phospholipase A2 in rat renal mesangial cells

Renal mesangial cells express secretory phospholipase A2 in response to two principal classes of activating signals that may interact in a synergistic fashion. These two groups of activators comprise inflammatory cytokines, such as interleukin 1 beta, and agents that elevate cellular levels of cAMP. Treatment of mesangial cells with tetranactin, a cyclic antibiotic produced by Streptomyces aureus with a molecular structure similar to cyclosporin A inhibits interleukin 1 beta- and cAMP-dependent group II phospholipase A2 secretion in a dose-dependent manner with IC50 values of 43 and 33 nM, respectively. However, tetranactin does not directly inhibit group II phospholipase A2 activity. Western blot analyses of mesangial cell supernatants reveal that the inhibition of phospholipase A2 activity is due to suppression of phospholipase A2 protein synthesis. This effect is preceded by the reduction of phospholipase A2 mRNA steady-state levels as shown by Northern blot analyses of total cellular RNA isolated from stimulated mesangial cells. Thus, tetranactin is a potent inhibitor of group II phospholipase A2 expression in cytokine- and cAMP-stimulated mesangial cells and represents a new class of group II phospholipase A2 inhibitors with IC50 values in the low nanomolar range. This compound may be useful in the therapy of diseases associated with increased group II phospholipase A2 secretion.

Platelet-derived growth factor and fibroblast growth factor differentially regulate interleukin 1β- and cAMP-induced group II phospholipase A2 expression in rat renal mesangial cells

Expression of group II phospholipase A2 (PLA2; EC 3.1.1.4) in rat renal mesangial cells is triggered in response to two principal classes of activating signals. These two groups of activators comprise inflammatory cytokines such as interleukin 1beta (IL-1beta) or tumor necrosis factor alpha and agents that elevate cellular levels of cyclic AMP (cAMP) such as forskolin, an activator of adenylate cyclase. Treatment of mesangial cells with IL-1beta or forskolin for 24 h induces group II PLA2 activity secreted into cell culture supernatants by about 15-fold and 11-fold, respectively. Platelet-derived growth factor (PDGF)-BB potently inhibits secretion of IL-1beta- and forskolin-induced group II PLA2 activity. By Western and Northern blot analyses, we demonstrate that this is due to a reduction of PLA2 protein levels and the corresponding PLA2 mRNA steady-state levels. Basic fibroblast growth factor (bFGF) virtually does not inhibit IL-1beta-stimulated group II PLA2 activity, but markedly inhibits forskolin-induced expression of group II PLA2 activity. These effects are caused by changes in the corresponding PLA2 protein and PLA2 mRNA steady-state levels. Inhibition of protein kinase C (PKC) by the potent and selective PKC inhibitor calphostin C converted the inhibitory action of PDGF into a bFGF-type of response thus suggesting that PKC is a major effector in PDGF-induced inhibition of IL-1beta-stimulated group II sPLA2 expression. In summary, our data suggest that PDGF and bFGF differentially modulate in a stimulus-specific manner the expression of group II PLA2 in mesangial cells.

Transforming growth factor-β2 inhibits interleukin 1β-induced expression of inducible nitric oxide synthase in rat renal mesangial cells

Abstract.Objective and Design: We have investigated whether transforming growth factor β2 (TGF-β2) influences the expression of inducible nitric oxide synthase (iNOS).¶Material: Rat renal mesangial cells exposed to the inflammatory cytokine interleukin 1β (IL-1β).¶Results: Addition of TGF-β2 dose-dependently suppresses IL-1β-induced nitrite formation. Western and Northern blot analyses of mesangial cell extracts reveal that the inhibition of IL-1β-induced nitrite formation by TGF-β2 is due to decreased iNOS protein and iNOS mRNA steady state levels. Reduction of iNOS mRNA levels is due to decreased transcriptional activity of the iNOS gene under the action of TGF-β2 as shown by nuclear run on experiments.¶Conclusions: TGF-β2 is a potent inhibitor of iNOS expression acting by reducing the transcriptional activity of the iNOS gene.

Inhibition by tetranactin of interleukin 1β- and cyclic AMP-induced nitric oxide synthase expression in rat renal mesangial cells

1 . We have investigated whether tetranactin, a cyclic antibiotic produced by Streptomyces aureus with a molecular structure related to cyclosporin A, influences inducible nitric oxide synthase (iNOS; EC 1.14.13.39) induction in rat glomerular mesangial cells. 2 . Previously we have shown that iNOS is expressed in renal mesangial cells in response to two principal classes of activating signals comprising inflammatory cytokines such as interleukin 1 (IL‐1) or tumour necrosis factor α and agents that elevate cellular levels of cyclic AMP. Treatment of mesangial cells with IL‐1β or the membrane‐permeable cyclic AMP analogue, N6, 0–2′‐dibutyryladenosine 3′,5′‐phosphate (Bt2 cyclic AMP) for 24 h induces iNOS activity measured as nitrite levels in cell culture supernatants by 44 fold or 33 fold, respectively. Incubation of mesangial cells with tetranactin inhibits IL‐1β‐ and cyclic AMP‐dependent production of nitrite in a dose‐dependent fashion with IC50 values of 50 nM and 10 nM, respectively. 3 . Western‐blot analyses of mesangial cell extracts reveal that the inhibition of nitrite synthesis by tetranactin is due to a suppression of iNOS protein levels. This effect is preceded by a reduction of iNOS mRNA steady state levels as demonstrated by Northern blot analyses of total cellular RNA isolated from stimulated mesangial cells. 4 . Thus, tetranactin is a potent inhibitor of iNOS expression in cytokine‐ and cyclic AMP‐stimulated mesangial cells and represents a new class of iNOS inhibitors with IC50s in the low nanomolar range. This compound may be useful in the therapy of diseases associated with pathological NO overproduction due to iNOS expression.