Irmgard Wiesenberg

Pineal gland hormone melatonin binds and activates an orphan of the nuclear receptor superfamily.

Nuclear orpan receptors are members of the superfamily of structurally related, ligand-inducible transcription factors for which no ligand has yet been identified. Over the past few years many nuclear orphan receptors have been cloned, but only for the retinoid X receptor (RXR) has a natural ligand (9-cis-retinoic acid) been found. Here we report the identification of melatonin as a ligand for the recently cloned orphan receptor retinoid Z receptor beta (RZR beta). We found RZR beta expression in the rat brain nearly coincident with binding sites for the pineal gland hormone melatonin (5-methoxy-N-acetyltryptamine). We show here binding and activation of RZR beta by melatonin with Kd and EC50 values in the low nanomolar range. A nuclear signaling pathway for melatonin may contribute to some of the diverse and profound effects of this hormone, for example, in the context of circadian rhythmicity.

The orphan receptor family RZR/ROR, melatonin and 5‐lipoxygenase: An unexpected relationship

Abstract: The orphan receptors RZRoα, RZRβ, RORod, RZRα2, RORα3, and RORγform a subfamily within the superfamily of nuclear hormone receptors. Recently, experimental evidence that the pineal gland hormone melatonin is the natural ligand for these nuclear receptors has come to light. This discovery is rather surprising, given that most people in the field believed melatonin acts exclusively through membrane receptors. However, these new findings establish a nuclear signalling pathway for melatonin, i.e., direct ligand‐induced control of target gene transcription, which most probably mediates part of the physiological functions of the hormone. Interestingly, the very recently identified first RZR/melatonin responding gene, 5‐Iipoxygenase, is not expressed in the brain and is not involved in circadian rhythmicity, but rather acts in the periphery, mainly in myeloid cells, as one of the key enzymes of allergic and inflammatory reactions. Thus, nuclear melatonin signalling opens up a new perspective in understanding the actions of the pineal gland hormone.

Identification of Natural Monomeric Response Elements of the Nuclear Receptor RZR/ROR. THEY ALSO BIND COUP-TF HOMODIMERS

The receptor RZR/ROR is an important member of the nuclear receptor superfamily and has recently been shown to be the nuclear receptor for the pineal gland hormone melatonin. RZR/ROR binds as a monomer to DNA, and the human 5-lipoxygenase gene has been identified as the first RZR/ROR/melatonin-responding gene. Another prominent nuclear receptor is COUP-TF, which binds as a dimer to DNA. In this study, the sequences of known promoter regions of genes that may be involved in the physiological action of melatonin have been screened for putative monomeric RZR/ROR response elements. The binding of RZR/ROR and COUP-TF was compared and quantified on a set of 12 putative response elements. Interestingly, COUP-TF homodimers were found to bind with high affinity to some of the monomeric RZR/ROR response elements. Four RZR/ROR response elements, found in the genes of the mouse bifunctional enzyme, the rat bone sialoprotein, mouse Purkinje cell protein 2, and human p21WAF1/CIP1, were shown to be inducible by melatonin under conditions of low constitutive activity. Surprisingly, the constitutive activity of COUP-TF was also stimulated by an unknown serum compound. The novel Purkinje cell protein 2 and p21WAF1/CIP1 RZR/ROR/melatonin-responding genes may be the key for understanding the role of RZR/RORα in the mouse mutation staggerer and the antiproliferative action of melatonin, respectively.

Thiazolidine Diones, Specific Ligands of the Nuclear Receptor Retinoid Z Receptor/Retinoid Acid Receptor-related Orphan Receptor α with Potent Antiarthritic Activity

Rat adjuvant arthritis is a chronic T cell-dependent autoimmune disease with many similarities to rheumatoid arthritis. We have identified a class of thiazolidine diones with high potency in suppressing chronic inflammation and joint destruction in this experimental model. The lead compound CGP 52608 (1-(3-allyl-4-oxothiazolidine-2-ylidene)-4-methylthiosemicarbazone) exhibits antiarthritic activity at daily oral doses between 0.01 and 1 mg/kg and was shown to specifically activate the retinoid Z receptor/retinoid acid receptor-related orphan receptor α (RZR/RORα) in low nanomolar concentrations. This receptor is a novel member of the superfamily of ligand-inducible transcription factors, and we have recently identified the pineal gland hormone melatonin as a natural ligand. Structure-activity relationship studies with 13 closely related analogues of CGP 52608 revealed a striking correlation between RZR/RORα activation and antiarthritic activity. We therefore suggest that nuclear signaling via RZR/RORα is a key mechanism in mediating the antiarthritic effects of these thiazolidine diones and may open a novel therapeutic approach for the treatment of rheumatoid arthritis and other autoimmune diseases. The existence of a nuclear melatonin receptor may lead to a better understanding of the immunomodulatory actions of melatonin.

‘Antiflammins’: Two nonapeptide fragments of uteroglobin and lipocortin I have no phospholipase A2 ‐inhibitory and anti‐inflammatory activity

The ‘antiflammin’ nonapeptides P1 and P2 [(1988) Nature 335, 726‐730] were synthesized and tested for inhibition of phospholipase A2 and release of prostaglandin E2, and leukotriene C4 in stimulated cells in vitro, and in vivo for anti‐inflammatory activity in rats with carrageenan‐induced paw oedema. Porcine pancreatic phospholipase A2, was not inhibited at concentrations of 0.5–50 μM. Prostaglandin E2, and leukotriene C4 release by mouse macrophages stimulated with zymosan or ATP was not affected up to a concentration of 10 μm, nor was prostaglandin release by interleukin 1β‐stimulated mesangial cells and angiotensin II‐stimulated smooth muscle cells. Both peptides exhibited no anti‐inflammatory activity in carrageenan‐induced rat paw oedema after topical (250 μg/paw) or systemic administration (1 or 4 s.c.). These results do not support the claim of potent phospholipase A2‐inhibitory and anti‐imflammatory activity of the ‘antiflammins’ P1 and P2 [1].

The analgesic drug buprenorphine inhibits osteoclastic bone resorption in vitro, but is proinflammatory in rat adjuvant arthritis.

We have examined the effect of the μ-opioid analgesic buprenorphine on osteoclastic bone resorption in vitro and in the rat adjuvant arthritis model. In the bone slice assay buprenorphine inhibited osteoclastic bone resorption with an IC50 of 1 μM. This effect was not mimicked by the μ-opioid agonist ([D-Ala,N-Me-Phe, Gly-ol]-enkephalin and was not prevented by the μ-opioid antagonist naloxone. Since other agents that inhibit osteoclastic bone resorption, such as bisphosphonates and calcitonin prevent bone erosion in the rat adjuvant arthritis model, we also examined the effect of buprenorphine in this model. Surprisingly, buprenorphine exacerbated inflammation measured by paw volume and increased joint destruction assessed by X-ray scores, in the injected paws and particularly in the non-injected paws. These studies also show that attempts to ameliorate animal suffering in this chronic model by using centrally acting analgesics such as buprenorphine may lead to complications in interpreting screening results obtained with novel, potential anti-arthritic compounds.

Time-dependent cytokine production in the croton oil-induced mouse ear oedema and inhibition by prednisolone

The croton oil-induced mouse ear oedema is a well established model for investigating anti-inflammatory effects of nonsteroidal and steroidal compounds [1]. Corticosteroids are highly effective inhibitors of this irritant-induced local inflammation [2]. They are still the most potent inhibitors of cytokine biosynthesis and are widely used as reference compounds for discovering and profiling novel inhibitors. This study was undertaken to investigate whether measurable amounts of IL-113 are produced during croton oil-induced skin inflammation and whether prednisolone is able to inhibit IL-1/3 production in this model. The kinetics of IL-I~ production, leukocyte infiltration (myeloperoxidase activity, MPO) and oedema development were compared.

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.

CGP 47969A: A novel inhibitor of the synthesis of inflammatory cytokines

CGP 47969A is a novel piperazine derivative that inhibits the synthesis of inflammatory cytokines, such as interleukin-1α (IL-1), IL-1β and tumor necrosis factor α (TNF), in human monocytes stimulated with lipopolysaccharide (LPS), zymosan or IL-1 itself. IC50 values are in the range of 0.3–5 μmol/l. CGP 47969A does not inhibit total protein or RNA synthesis indicating selectivity for cytokine inhibition. CGP 47969A exerts its inhibitory effect at a post-transcriptional level, most probably by reducing translational efficiency of IL-β mRNA, as steady-state levels of IL-1β mRNA are not inhibited while the primary translation product, the 31 kD IL-1β precursor molecule, is dose-dependently inhibited by CGP 47969A. The compound is devoid of cyclooxygenase and phospholipase A2 inhibitory activity but efficiently inhibits the generation of PGE2 and LTC4 in zymosanstimulated mouse macrophages with an IC50 of 1.2 and 0.6 μmol/l, respectively. Antagonism of IL-1 and/or TNF is thought to have a beneficial effect on the course of inflammatory diseases. CGP 47969A may therefore represent a mechanistically new approach to the treatment of such diseases.

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.