Specific activation of the nuclear receptors PPARgamma and RORA by the antidiabetic thiazolidinedione BRL 49653 and the antiarthritic thiazolidinedione derivative CGP 52608.

The thiazolidinedione BRL 49653 and the thiazolidinedione derivative CGP 52608 are lead compounds of two pharmacologically different classes of compounds. BRL 49653 is a high affinity ligand of peroxisome proliferator-activated receptor gamma (PPARgamma) and a prototype of novel antidiabetic agents, whereas CGP 52608 activates retinoic acid receptor-related orphan receptor alpha (RORA) and exhibits potent antiarthritic activity. Both receptors belong to the superfamily of nuclear receptors and are structurally related transcription factors. We tested BRL 49653 and CGP 52608 for receptor specificity on PPARgamma, RORA, and retinoic acid receptor alpha, a closely related receptor to RORA, and compared their pharmacological properties in in vitro and in vivo models in which these compounds have shown typical effects. BRL 49653 specifically induced PPARgamma-mediated gene activation, whereas CGP 52608 specifically activated RORA in transiently transfected cells. Both compounds were active in nanomolar concentrations. Leptin production in differentiated adipocytes was inhibited by nanomolar concentrations of BRL 49653 but not by CGP 52608. BRL 49653 antagonized weight loss, elevated blood glucose levels, and elevated plasma triglyceride levels in an in vivo model of glucocorticoid-induced insulin resistance in rats, whereas CGP 52608 exhibited steroid-like effects on triglyceride levels and body weight in this model. In contrast, potent antiarthritic activity in rat adjuvant arthritis was shown for CGP 52608, whereas BRL 49653 was nearly inactive. Our results support the concept that transcriptional control mechanisms via the nuclear receptors PPARgamma and RORA are responsible at least in part for the different pharmacological properties of BRL 49653 and CGP 52608. Both compounds are prototypes of interesting novel therapeutic agents for the treatment of non-insulin-dependent diabetes mellitus and rheumatoid arthritis.

Platelet-derived growth factor and fibroblast growth factor differentially regulate interleukin 1beta- 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.

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

1. 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. 2. 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 1 beta (IL-1 beta) or tumour necrosis factor alpha and agents that elevate cellular levels of cyclic AMP. Treatment of mesangial cells with IL-1 beta for 24 h induced PLA2 activity secreted into cell culture supernatants by about 16 fold. Incubation of mesangial cells with cyclosporin A inhibited IL-1 beta-induced PLA2 section in a dose-dependent fashion, with an IC50 value of 4.3 microM. Cyclosporin A did not directly inhibit enzymatic activity of PLA2. 3. 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. 4. 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. 5. Previously we found that the nuclear transcription factor kappa B (NF kappa B) is an essential component of the IL-1 beta-dependent upregulation of PLA2 gene transcription. By electrophoretic mobility shift analysis, we demonstrated that cyclosporin A diminishes the formation of NF kappa B DNA-binding complexes, thus suggesting that this transcription factor is a target for cyclosporin A-mediated repression of PLA2 gene transcription. 6. The data presented in this study strongly suggest that the cellular mechanism involved in the IL1 beta-dependent transcriptional upregulation of the PLA2 gene in mesangial cells is a target for the action of cyclosporin A.

Tetranactin inhibits interleukin 1 beta 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.

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 of activating signals that may interact in a synergistic fashion. These two groups of activators comprise inflammatory cytokines such as interleukin-1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF alpha) 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 NF kappa B, we determined its role in cytokine--and cAMP--triggered group II PLA2 expression. Micromolar amounts of PDTC suppress the IL-1 beta- and TNF alpha-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-1 beta and TNF alpha, while only marginally affecting forskolin-induced PLA2 mRNA levels. Our data suggest that NF kappa B 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 NF kappa B. These observations may provide a basis to study the underlying mechanisms involved in the regulation of group II PLA2 gene expression.

The influence of anti-rheumatic drugs on hepatic mRNA levels of acute-phase proteins in rats with adjuvant arthritis.

Using specific cDNA probes, we have investigated drug-induced changes in hepatic mRNA levels of the major acute-phase proteins (APP) fibrinogen, alpha 2-macroglobulin (alpha 2-MG), albumin and alpha 1-acid glycoprotein (alpha 1-AGP) in male Lewis rats with adjuvant arthritis. Test compounds were given orally from day 0 to 20 and hepatic mRNA analysis was performed at day 21. Prednisolone (1, 3, 10 mg/kg), Cyclosporine A (1, 3, 10 mg/kg) and cyclophosphamide (3 mg/kg) dose-dependently normalized hepatic mRNA levels of all four APP. Equipotent anti-inflammatory doses of indomethacin (0.3, 1 mg/kg) significantly downregulated alpha 2-MG mRNA levels but were much less effective in influencing albumin and alpha 1-AGP mRNA levels and even slightly increased hepatic fibrinogen mRNA levels. These results suggest that cytokine over-production, which is thought to be responsible for the acute-phase response in rats with adjuvant arthritis, can be effectively downregulated by immunosuppressive drugs, but is distinctly less affected by the cyclooxygenase inhibitor indomethacin.

Cytokine regulation of group II phospholipase A2 expression in glomerular mesangial cells.

Phospholipase A2 (PLA2) is believed to play an essential role in inflammatory processes by releasing arachidonic acid from membrane phospholipids for synthesis of important lipid mediators, such as prostaglandins, leukotrienes and platelet activating factor. We have used glomerular mesangial cells as a model system to study the regulation of PLA2 under inflammatory conditions. Potent pro-inflammatory cytokines, such as interleukin 1 (IL-1) and tumour necrosis factor alpha (TNF alpha), as well as agents that increase cellular cAMP levels have been found to increase Group II PLA2 gene expression in a time- and dose-dependent manner. In all cases cytokine-induced synthesis of PLA2 occurred in parallel with cytokine-stimulated prostaglandin (PG) E2 synthesis. Three important classes of compounds that potently antagonise the stimulatory effect of IL-1, TNF alpha and cAMP on Group II PLA2 expression in mesangial cells have been identified, namely, glucocorticoids, transforming growth factors (TGF) type-beta and platelet-derived growth factor (PDGF). Those agents may act sequentially to protect the kidney from damage resulting from cytokine-stimulated mediator release and the subsequent inflammatory reactions.

Changes in hepatic mRNA levels of acute phase proteins during rat adjuvant arthritis.

Using specific cDNA probes, we have investigated changes in hepatic mRNA concentrations of the major acute phase proteins fibrinogen, alpha 2-macroglobulin (alpha 2-MG), albumin and alpha 1-acid glycoprotein (alpha 1-AGP) during developing adjuvant arthritis in Lewis rats. Continuously increasing levels in the mRNA of the positive reactants beta-fibrinogen, alpha 2-MG and alpha 1-AGP were found during developing disease with peak levels from day 15 to 21, whereas mRNA concentrations of the negative reactant albumin decreased, reaching their lowest levels on day 11 to 15. As early as 4 days after arthritis induction, the hepatic mRNA levels of beta-fibrinogen, alpha 1-AGP and albumin were distinctly different from control values. The most dramatic changes in the hepatic mRNA levels and plasma concentrations of acute phase reactants were seen between days 11 and 21. These results indicate that overproduction of the major inflammatory cytokines IL-1, TNF-alpha and IL-6, which are now felt to be largely responsible for the acute phase response in the rat, is an early event during adjuvant arthritis and that the highest amounts are produced during the inflammatory phase of the disease. mRNA changes in the acute phase proteins alpha 1-AGP and albumin, which are mainly regulated by IL-1/TNF alpha, were more pronounced than those of alpha 2-MG and beta-fibrinogen, which are predominantly controlled by IL-6.(ABSTRACT TRUNCATED AT 250 WORDS)

Transforming growth factors type-beta and dexamethasone attenuate group II phospholipase A2 gene expression by interleukin-1 and forskolin in rat mesangial cells.

Treatment of rat mesangial cells with interleukin-1 beta (IL-1 beta) and forskolin induced, in a synergistic fashion, the expression of group II phospholipase A2 (PLA2) mRNA, with subsequent increased synthesis and secretion of PLA2. In contrast, interleukin-6 did not increase PLA2 mRNA levels of PLA2 activity. Transforming growth factor (TGF) beta 1, TGF beta 2 and TGF beta 3 equipotently attenuated the IL-1 beta- and forskolin-induced elevation of PLA2 mRNA, as well as PLA2 synthesis and secretion. The glucocorticoid dexamethasone only partially suppressed the IL-1 beta- and forskolin-induced elevation of PLA2 mRNA, but totally inhibited PLA2 synthesis and secretion.

Dermatopharmacologic investigations of halobetasol propionate in comparison with clobetasol 17-propionate.

Both halobetasol propionate and clobetasol 17-propionate exerted very marked antiinflammatory, antiproliferative, and vasoconstrictive effects during evaluation in a range of dermatopharmacologic models. Halobetasol propionate was distinctly more potent than clobetasol 17-propionate in the ultraviolet-induced dermatitis inhibition assay in guinea pigs and in the rat model of oxazolone-induced late inflammatory reaction. Halobetasol propionate was slightly more potent than clobetasol 17-propionate in inhibiting croton oil-induced ear edema in rats and mice and in the mouse model of oxazolone-induced early inflammatory reaction. In the cotton-pellet granuloma assay in rats and the epidermal hyperplasia inhibition assay in guinea pigs, halobetasol propionate was distinctly superior to clobetasol 17-propionate. There was a trend in favor of halobetasol propionate in the cutaneous vasoconstriction assay performed in volunteers with ethanol solutions of halobetasol propionate and clobetasol 17-propionate. In a further vasoconstriction assay, performed with a 0.05% concentration of both halobetasol propionate and clobetasol 17-propionate in cream and ointment formulations, halobetasol propionate ointment yielded the highest blanching score. In a hypothalamic-pituitary-adrenal axis study in volunteers, effects of 0.05% halobetasol propionate ointment and 0.05% clobetasol 17-propionate ointment on serum cortisol levels were similar. The overall efficacy trends demonstrated in these dermatopharmacologic studies are in agreement with predictions made from corticosteroid structure and activity relationships and the results of two clinical trials comparing halobetasol propionate and clobetasol 17-propionate ointments in the treatment of plaque psoriasis.

PDGF suppresses the activation of group II phospholipase A2 gene expression by interleukin 1 and forskolin in mesangial cells.

Treatment of rat mesangial cells with interleukin 1 beta (IL-1 beta) and forskolin greatly enhanced the expression of group II phospholipase A2 (PLA2) mRNA, with subsequent increased synthesis and secretion of PLA2, as detected by PLA2 activity measurements and immunoprecipitation of culture media of [35S]methionine-labelled mesangial cells. PDGF-BB dose-dependently suppressed the IL-1 beta- and forskolin-induced elevation of PLA2 mRNA, as well as PLA2 synthesis and secretion. In contrast, PDGF-AA had no inhibitory effect. The tyrosine kinase inhibitor genistein dose-dependently antagonized the inhibitory effect of PDGF-BB on IL-1 beta-stimulated PLA2 secretion, thus suggesting that tyrosine phosphorylation may be required for PDGF-BB inhibition of PLA2 gene expression in mesangial cells.

Cyclic AMP mimics, but does not mediate, interleukin-1- and tumour-necrosis-factor-stimulated phospholipase A2 secretion from rat renal mesangial cells.

We have previously shown that recombinant interleukin 1 (IL-1) and recombinant tumour necrosis factor (TNF) synergistically stimulate phospholipase A2 release from mesangial cells. We now report that treatment of mesangial cells with the beta-agonist salbutamol, prostaglandin E2 (PGE2), cholera toxin or forskolin, which all activate adenylate cyclase, increased release of phospholipase A2 activity. Likewise, addition of a membrane-permeant cyclic AMP (cAMP) analogue or the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine enhanced release of phospholipase A2 activity from mesangial cells. There was a lag period of about 8 h before a significantly enhanced secretion could be detected. Furthermore, actinomycin D or cycloheximide completely suppressed cAMP-stimulated secretion of phospholipase A2. Angiotensin II, the phorbol ester phorbol 12-myristate 13-acetate, the Ca2+ ionophore A23187 and a membrane-permeant cGMP analogue did not stimulate phospholipase A2 release from the cells. Treatment with indomethacin completely inhibited IL-1 beta- and TNF-stimulated PGE2 synthesis, without having any effect on phospholipase A2 secretion, thus excluding cytokine-induced PGE2 synthesis as the mediator of phospholipase A2 release. Neither IL-1 beta nor TNF induced any increase in intracellular cAMP in mesangial cells. Furthermore, incubation of the cells with 2',5'-dideoxyadenosine, an inhibitor of adenylate cyclase, did not block cytokine-stimulated phospholipase A2 secretion. In addition, IL-1 beta and TNF synergistically interacted with forskolin to stimulate phospholipase A2 release from the cells. The protein kinase inhibitors H-8, staurosporine, K252a and amiloride inhibited IL-1 beta- and TNF-stimulated phospholipase A2 secretion. However, high concentrations that inhibit other protein kinases were needed. These observations suggest that IL-1 beta and TNF cause secretion of phospholipase A2 by a mechanism independent of cAMP. The signalling pathways used by IL-1 beta and TNF may involve a protein kinase that is probably different from protein kinase A or protein kinase C.

Transforming growth factor beta 2 differentially modulates interleukin-1 beta- and tumour-necrosis-factor-alpha-stimulated phospholipase A2 and prostaglandin E2 synthesis in rat renal mesangial cells.

Treatment of rat glomerular mesangial cells with transforming growth factor beta 2 (TGF beta 2) stimulates prostaglandin E2 (PGE2) synthesis. Actinomycin D, cycloheximide and diclofenac attenuate the TGF beta 2-induced PGE2 formation. As shown previously, two proinflammatory cytokines, interleukin 1 beta (IL-1 beta) and tumour necrosis factor alpha (TNF alpha), are potent stimuli for PGE2 and phospholipase A2 secretion from mesangial cells. We report here that, whereas TGF beta 2 potentiates the IL-1 beta- and TNF alpha-evoked PGE2 production, it strongly inhibits the phospholipase A2 secretion induced by both cytokines. In addition, the inhibitory effect of TGF beta 2 on phospholipase A2 secretion is not due to the augmented PGE2 formation.

Determination of human serum phospholipase A2. Comparison of two methods.

A photometric assay for serum phospholipase A is compared with a radiometric assay specific for phospholipase A2. The methods show a high correlation. The increased phospholipase A activity in sera of patients with inflammatory and non-pancreatic necrotizing diseases is attributable to a neutral-active, calcium-sensitive phospholipase A2 but not to pancreatic phospholipase A2. Phospholipase A1 did not seem to contribute significantly to the overall activity of phospholipase A.

Release of phospholipase A2 activity from rat vascular smooth muscle cells mediated by cAMP.

Primary cultures of smooth muscle cells (SMC) derived from rat aorta release a phospholipase A2 activity into the culture medium. Phospholipase A2 activity was determined with [1-14C]oleate-labelled Escherichia coli as substrate. The enzyme has a neutral pH optimum and the activity is critically dependent on the free calcium concentration, with significant activity in the micromolar range of free calcium. Treatment of SMC with the beta agonist salbutamol, forskolin or cholera toxin, which all activate adenylate cyclase and increase intracellular cAMP concentration, increase the release of phospholipase A2 activity in a dose-dependent manner. Likewise, the addition of the membrane-permeable cAMP analogues, (Sp)-adenosine 3',5'-[thio]phosphate and N6,O-2'-dibutyryladenosine 3',5'-phosphate, enhance the release of phospholipase A2 activity from SMC in a dose-dependent manner. There is a lag period of about 4 h before a significant secretion of phospholipase A2 can be detected under basal, as well as under stimulated conditions. The forskolin analogue 1,9-dideoxyforskolin, which is inactive as a stimulator of adenylate cyclase, has no effect on phospholipase A2 secretion. Likewise, the potent vasoconstrictive peptide angiotensin II activates inositol phospholipid turnover in SMC, but has no effect on phospholipase A2 release. Pretreatment of SMC with actinomycin D or cycloheximide completely suppresses basal and cAMP-stimulated secretion of phospholipase A2 activity, thus demonstrating that transcription and protein synthesis are necessary for enzyme release.

Interleukin 1 and tumor necrosis factor synergistically stimulate prostaglandin synthesis and phospholipase A2 release from rat renal mesangial cells.

Treatment of rat glomerular mesangial cells with recombinant human interleukin 1 alpha (rIL-1 alpha), recombinant human interleukin 1 beta (rIL-1 beta) or recombinant human tumor necrosis factor (rTNF) induces prostaglandin E2 (PGE2) synthesis and the release of a phospholipase A2 (PLA2) activity. rIL-1 beta is significantly more potent than rIL-1 alpha or rTNF in stimulating PGE2 as well as PLA2 release from mesangial cells. When given together, rTNF interacts in a synergistic fashion with rIL-1 alpha and rIL-1 beta to enhance both, PGE2 synthesis and PLA2 release. The released PLA2 has a neutral pH optimum and is calcium-dependent. Pretreatment of cells with actinomycin D or cycloheximide inhibits basal and cytokine-stimulated PGE2 and PLA2 release.