Modulation of human mineralocorticoid receptor function by protein kinase A.

The mineralocorticoid receptor (MR) acts as a ligand-dependent transcription factor modulating specific gene expression in sodium-transporting epithelia. Physiological evidence suggest a cross-talk between the cAMP- and aldosterone-signaling pathways. We provide evidence that protein kinase A (PKA), a major mediator of signal transduction pathways, modulates transcriptional activity of the human MR (hMR). Using transient transfection assays in HepG2 cells, we show that 8-bromo-cAMP, a protein kinase A activator, stimulates glucocorticoid response element (GRE)-containing promoters in a ligand-independent manner. This effect was strictly MR dependent since no activation of the reporter gene was observed in the absence of cotransfected hMR expression plasmid. Furthermore, a synergistic activation was achieved when cells were treated with both aldosterone and cAMP. This synergistic effect was also observed in the CV1 and the stable hMR-expressing M cells but was dependent on the promoter used. In particular, synergism was less pronounced in promoters containing several GREs. We show that (protein kinase-inhibiting peptide (PKI), the peptide inhibitor of PKA, prevented both cAMP and aldosterone induction, which indicates that a functional cAMP pathway is required for stimulation of transcription by aldosterone. Using MR-enriched baculovirus extracts in gel shift assays, we have shown that the binding of the MR to a GRE-containing oligonucleotide was enhanced by PKA. Increased DNA binding of hMR is likely to reflect an increase in the number of active receptors, as measured by Scatchard analysis. Using a truncated MR, we show that the N-terminal domain is required for the effect. Finally, the N-terminal truncated MR was not directly phosphorylated by PKA in vitro. We conclude that PKA acts indirectly, probably by relieving the effect of an MR repressor.

Lithium chloride stimulates PLP and MBP expression in oligodendrocytes via Wnt/ß-catenin and Akt/CREB pathways.

Deciphering the molecular pathways involved in myelin gene expression is a major point of interest to better understand re/myelination processes. In this study, we investigated the role of Lithium Chloride (LiCl), a drug largely used for the treatment of neurological disorders, on the two major central myelin gene expression (PLP and MBP) in mouse oligodendrocytes. We show that LiCl enhances the expression of both PLP and MBP, by increasing mRNA amount and promoter activities. We investigated whether Wnt/ß-catenin and/or Akt/CREB pathways are modulated by LiCl to regulate myelin gene expression. We showed that ß-catenin is required both for PLP and MBP basal promoter activities and for LiCl-induced myelin gene stimulation. Furthermore, while CREB functionality does not influence PLP expression, MBP promoter activity depends on Akt/CREB activation. Finally, we show that LiCl can stimulate oligodendrocyte morphological maturation, and promote remyelination after lysolecithin-induced demyelination of organotypic cerebellar slice cultures. Our data provide mechanistic evidences that Akt/CREB together with ß-catenin participate in the transcriptional control of PLP and MBP exerted by LiCl. Therefore, the use of LiCl to balance between ß-catenin and CREB effectors could be considered as an efficient remyelinating strategy.

A single d(GpG) cisplatin adduct on the estrogen response element decreases the binding of the estrogen receptor.

Both cisplatin and the estrogen receptor (ER) are known to bend DNA. The influence of the bending of sequences by the d(GpG)cisPt adduct binding of ER to estrogen response element (ERE)-like sequences was examined. Three ERE-like oligonucleotides with different affinities for ER and which include a GG in the linker sequence were designed in order to form a single central d(GpG)cisPt adduct. Using electrophoretic mobility shift assay and Scatchard analysis, it was shown that the presence of a single d(GpG)cisPt adduct in the linker sequence decreases the ER affinity for DNA. These results do not support a critical role of a DNA bend in the initial recognition of ERE by ER. Then, the platination of DNA outside of the ERE half-sites decreases the interaction of ER with ERE.

The role of the sympathetic efferents in endotoxin-induced localized inflammatory hyperalgesia and cytokine upregulation.

The sympathetic system (SNS) is considered to be a major component of the neurogenic contribution to inflammation and hyperalgesia. We have investigated the role of the SNS in the local inflammatory pain induced by intraplantar (i.pl) injections of bacterial endotoxin (ET). Treatment of rats with an alpha-adrenoceptor antagonist (phentolamine, 0.25-1 mg/kg, i.p.), a beta-adrenoceptor antagonist (propranolol, 1-10 mg/kg, p.o.) or a sympathetic neuron-blocking agent (guanethedine, 30 mg/kg, s.c.) resulted in a dose-dependent reduction of the thermal hyperalgesia induced by ET. Mechanical hyperalgesia, however, was less sensitive to inhibition by propranolol and guanethedine but significantly inhibited by phentolamine. ET injection produced significant upregulation of tumor necrosis factor-alpha (TNF-alpha), interleukin-1 beta (IL-1 beta), IL-6, and nerve growth factor (NGF). Treatment with any one of the three sympatholytics abolished the upregulation of NGF and IL-6, while phentolamine and guanethedine also reversed the upregulation of TNF-alpha. IL-1 beta was resistant to all of the sympatholytic treatments. We conclude that the SNS can contribute to the local inflammation and hyperalgesia following injection of ET. The resistance to sympatholytics shown by IL-1 beta, known to play a key role in the inflammatory cascade, suggests that ET can initiate inflammation and hyperalgesia independently of peripheral and central sympathetic mechanisms.

Involvement of substance P, CGRP and histamine in the hyperalgesia and cytokine upregulation induced by intraplantar injection of capsaicin in rats.

Intraplantar (i.pl.) injection of small doses of capsaicin has been shown to produce hyperalgesia and upregulation of the levels of proinflammatory cytokines. The present work aimed at investigating the possible mediation of these effects by sensory neuropeptides and mast cells. Various groups of rats received i.pl. injection of capsaicin alone or preceded by the injection of antagonists to substance P (SP), calcitonin gene-related protein (CGRP) and histamine (H1, H2) or the mast cell blocker ketotifen. All pretreatments prevented, in a dose-related manner, the capsaicin-induced hyperalgesia. The SP, H2 antagonists and ketotifen prevented the upregulation of all cytokines and nerve growth factor (NGF) levels, while the CGRP and H1 antagonists showed only attenuation of the NGF level.

Thymulin reverses inflammatory hyperalgesia and modulates the increased concentration of proinflammatory cytokines induced by i.c.v. endotoxin injection.

The immunomodulatory thymic hormone thymulin has been shown previously to possess anti-inflammatory actions in the periphery. In this study, we have examined the effect of i.c.v. injections of either endotoxin (ET) or thymulin, in separate groups of conscious rats, on pain-related behavior and cytokine levels in different areas of the brain. Furthermore, we investigated the effect of pretreatment with either i.c.v. or i.p. injections of thymulin on endotoxin-induced hyperalgesia and the effect of pretreatment with i.c.v. thymulin on endotoxin-induced up-regulation of cytokine levels. Our results demonstrate that i.c.v. injection of endotoxin (1 microg in 5 microl saline) resulted in a significant decrease in the nociceptive thresholds as assessed by different pain tests, with peak hyperalgesia at 3 h. However, thymulin at different doses, when injected (i.c.v.), had no significant effect on pain related behavior. Pretreatment (i.c.v.) with thymulin (0.1, 0.5 and 1 microg in 5 microl saline) 20 min before endotoxin (i.c.v.) injection (1 microg in 5 microl saline) reduced, in a dose dependent manner, the endotoxin-induced hyperalgesia and exerted differential effects on the up-regulated levels of cytokines in different areas of the brain. The results provide behavioral and immunochemical characterization of a rat model for intracerebral inflammation and indicates a neuroprotective role for thymulin in the CNS.

Modulation of ultraviolet-induced hyperalgesia and cytokine upregulation by interleukins 10 and 13.

1. Exposure to midrange ultraviolet radiation (UVB) is known to produce skin inflammation similar to sunburn. The aim of this study was to characterize the hyperalgesia and cytokine upregulation induced by UVB and their modulation by antiinflammatory cytokines. 2. Acute exposure of the dorsal skin of mice to UVB (200, 250 and 300 mJ cm(2)) resulted in a dose-dependent decrease in the latencies of the hot plate and tail flick tests, without evident signs of skin lesions. 3. The observed hyperalgesia displayed a biphasic temporal evolution with an acute phase (3 - 6 h) and a late (48 - 96 h) phase. 4. Exposure to UVB (300 mJ cm(2)) elicited significant upregulation of interleukin (IL)-1 beta, tumour necrosis factor (TNF)-alpha and nerve growth factor (NGF), determined by ELISA in the exposed skin. This upregulation was more important during the acute phase of hyperalgesia. 5. Daily treatment of mice, with i.p. injections of either IL-10 or IL-13 (1.5, 7.5 and 15 ng in 100 microl saline) produced a dose-dependent attenuation of the UVB-induced hyperalgesia. 6. Treatment with the highest doses of either IL-10 or IL-13, produced significant attenuation of the levels of the cytokines and NGF by UVB, with relatively more pronounced effects by IL-13. 7. Acute exposure to moderate amounts of UVB results in a systemic hyperalgesia related to the upregulation of cytokine and NGF levels, since both were prevented by treatment with antiinflammatory cytokines.

An assay for the detection of xenoestrogens based on a promoter containing overlapping EREs.

Xenoestrogens could be implicated in the decrease of male fertility and in the increased incidence of testicular and breast cancers in humans. To predict their deleterious effects, various in vivo or in vitro tests have been proposed to assay the xenoestrogenic activity. We have designed an assay for the detection of xenoestrogens based on a novel estrogen responsive unit formed by two overlapping estrogen response elements (overEREs). This construct is able to mediate a synergistic activation of transcription by 17ss-estradiol. We have used the overERE unit to assay the estrogenic activity of synthetic compounds, mostly organochlorine compounds. By using the overERE construct, we were able to detect the estrogenic activity of compounds at concentrations 10- to 100-fold lower than a single ERE (i.e., we detected the estrogenic effect of endosulfan at a concentration of 10(-5) M with ERE, whereas the overERE unit allowed us to detect a significant estrogenic activity of endosulfan at a lower concentration (10(-6) M). Some compounds did not exhibit any estrogenic activity when tested with a classical ERE, whereas they were potent xenoestrogens when the overERE was used (i.e., Betanal). The assays we have developed are very sensitive and can be performed quickly. Moreover, because the promoter that we used contains only an overlapping ERE as a regulatory unit, the interference of the tested molecules with other regulatory pathways can be avoided.

Testicular and epididymal dual origin of hCAP-18/SOB3, a human sperm protein.

The expression and localization of the human sperm protein hCAP-18/SOB3 were evaluated in human testis and epididymis through in situ hybridization and immunohistochemistry with the use of an anti-recombinant hCAP-18/SOB3 polyclonal antibody. Both hCAP-18/SOB3 messenger RNA and hCAP-18/SOB3 protein were detected in testis germinal cells (from late spermatogonia to spermatozoa) and in the epididymal epithelium. This localization is in agreement with the antimicrobial properties previously described and supports its involvement in zona pellucida binding, as we had previously suggested.

Cell-specific, promoter-dependent mineralocorticoid agonist activity of spironolactone.

The agonist activity of the antimineralocorticoid spironolactone was evaluated in various cell lines through the use of transfection experiments. The target promoters were derived from the deltaMTV promoter in which one or several glucocorticoid-responsive elements (GRE) were inserted in tandem. Spironolactone at 100 nM activated by 6-fold the GRE/deltaMTV promoter in the human hepatoma HepG2 cell line and only partially prevented the 10-fold activation of this promoter by 0.1 nM aldosterone. Both effects were completely dependent on the cotransfection of an expression vector for the mineralocorticoid receptor. The half-maximal agonist effect of spironolactone was similar to its half-maximal antagonist effect (approximately 10 nM). For the GRE-2/deltaMTV, GRE-4/deltaMTV, and wild-type MMTV promoters, the activation by aldosterone was much more potent (70-, 100-, and 110-fold, respectively), whereas spironolactone elicited a 10-, 24-, and 25-fold activation, respectively. Thus, the effect of both compounds and the relative efficiency of spironolactone, compared with that of aldosterone, were dependent on the number of GREs present in the regulatory region of the promoter. The agonist effect of spironolactone was cell specific. Indeed, although spironolactone agonist activity was observed in H5 kidney tubule cells, none could be detected at concentrations of < or = 1 microM in the CV1 monkey fibroblast cells. In contrast, the antagonist effect was observed in all cells. Furthermore, other antimineralocorticoids, such as RU 26752 and progesterone, also displayed mineralocorticoid receptor-dependent agonist activity in the HepG2 cells. The antiprogesterone RU 486 and the antiandrogen cyproterone acetate were ineffective at < or = 1 microM. In conclusion, we show that under certain experimental conditions, several antimineralocorticoids display significant agonist activity in a cell-specific and promoter-dependent manner.

Properties of overlapping EREs: synergistic activation of transcription and cooperative binding of ER.

We have designed a novel estrogen-responsive unit, overERE, which consists of two overlapping ERE separated by 5 bp (center-to-center). In gel retardation assays, this sequence forms a low-mobility complex that migrates like an estrogen receptor tetramer. The receptor-overERE complex was specific and was supershifted by anti-ER H222 antibodies. Dose response studies showed that the formation of the receptor tetramer-overERE complex was cooperative. Truncated receptors were used to assess the contribution of the receptor domains. Deletion of the E domain of the ER prevented the formation of an ER-tetramer complex, which reflects a novel function of this receptor domain. In transfection experiments, 17-beta-estradiol activated transcription from an overERE-containing promoter 4-6 times better than from an ERE-containing promoter. This synergistic effect was observed using either the natural hormone (17-beta-estradiol) or xenoestrogens (phenol red, chlordane). We conclude that two overlapping estrogen-responsive elements can elicit synergistic induction of transcription.

A natural sequence consisting of overlapping glucocorticoid-responsive elements mediates glucocorticoid, but not androgen, regulation of gene expression.

Cytosolic aspartate aminotransferase (cAspAT) is regulated by glucocorticoids in rat liver and kidney. Part of this regulation is mediated by an unusual glucocorticoid-responsive element (GRE)-like sequence called GRE A. GRE A is composed of two overlapping imperfect GREs, each comprising a conserved half-site (half-sites 1 and 4 respectively) and a poorly conserved half-site (half-sites 2 and 3 respectively). The sequence binds co-operatively two dimers of the glucocorticoid receptor (GR) and mediates efficient glucocorticoid regulation of gene expression. Analysis of deletions of the cAspAT gene promoter and subcloning of GRE A upstream of the thymidine kinase promoter indicate that this sequence is responsive to glucocorticoids, but not to androgens. Electrophoretic mobility shift assays indicate that the GRE A unit does not bind the androgen receptor (AR). The modification of three nucleotides in the poorly conserved half-sites 2 and 3, converting GRE A into two overlapping high-affinity GREs (ov-cGRE), resulted in co-operative binding of the AR. Furthermore, ov-cGRE efficiently mediated androgen regulation of the thymidine kinase promoter. A single base modification in half-site 2 or 3 in GRE A allowed the binding of the AR as one or two dimers respectively, and restored transcriptional activation by androgens only in the latter case. Thus the poor affinity of the AR for half-sites 2 and 3 prevented its binding to GRE A, indicating that the overlapping GRE A sequence of the cAspAT gene promoter discriminates a glucocorticoid-mediated from an androgen-mediated response.

Induction of secreted type IIA phospholipase A2 gene transcription by interleukin-1beta. Role of C/EBP factors.

Secreted type IIA phospholipase A(2), which is involved in arachidonic acid release, is abundantly produced by chondrocytes and secreted in the synovial fluids of patients affected by rheumatoid arthritis. Transfection experiments showed that interleukin-1beta stimulates the phospholipase A(2) [-1614; +20] promoter activity by 6-7-fold and that the [-210; -176] fragment is critical for this stimulation. CAAT enhancer-binding protein (C/EBP) beta and C/EBPdelta transcription factors bind to this element as shown by bandshift experiments. Interleukin-1beta increased the levels of C/EBPdelta mRNA as soon as 2 h and up to 24 h without affecting those of C/EBPbeta. Higher amounts of C/EBPdelta proteins correlate with the stimulation of C/EBPdelta mRNA. Mutations or 5' deletions in the upstream [-247; -210] region reduced by 2-fold the basal and interleukin-1beta-stimulated transcription activities. Two types of factors bind to overlapping sequences on this fragment: NF1-like proteins and the glucocorticoid receptor. The glucocorticoid receptor is responsible for a moderate stimulation of the promoter activity by dexamethasone and may interact with C/EBP factors to achieve a full transcription activity in basal conditions and in the presence of interleukin-1beta. A [-114; -85] proximal regulatory element forms three complexes in bandshift experiments, the slowest mobility one involving the Sp1 zinc finger factor. Mutation of this sequence reduced to 2-fold the stimulation of the promoter activity by interleukin-1beta or the C/EBP factors. Induction of the transcription of secreted type IIA phospholipase A(2) gene by interleukin-1beta in chondrocytes absolutely requires C/EBPbeta and C/EBPdelta factors but does not involve NF-kappaB.

Upregulation of proinflammatory cytokines and nerve growth factor by intraplantar injection of capsaicin in rats.

Capsaicin-sensitive primary afferents (CSPA) are known to be involved in nociception and neurogenic inflammation. Extensive research has been devoted to the sensory role of these fibres but less attention has been paid to their local effector function. This study aimed at gaining more insight into the molecular mechanisms underlying the neurogenic inflammation induced by this special group of afferent fibres. Different groups of rats (n = 5 in each group), either naive or subjected to selective ablation of their CSPA, received individual intraplantar injections of saline, capsaicin, its vehicle or capsaicin preceded by its antagonist, capsazepine. Acute tests for nociception were used to assess the variations of the nociceptive thresholds. Variations of the levels of proinflammatory cytokines and nerve growth factor (NGF) were measured by enzyme-linked immunosorbent assay (ELISA). Intraplantar injection of capsaicin (10 microg in 50 microl) produced a sustained thermal and mechanical hyperalgesia that peaked at 3-6 h and disappeared 24 h following the injection. Similar capsaicin injection in further groups of rats produced an early upregulation of the proinflammatory cytokines and NGF, which peaked at 30-60 min and returned to control levels within 2-5 h. Similar effects were observed following the application of either capsaicin or intense electrical stimulation on the cut end of the distal portion of the sciatic nerve. The effects of capsaicin were abolished in rats subjected to selective ablation of their CSPA. These results demonstrate that CSPA can simultaneously challenge the immune system through the release of proinflammatory mediators and the central nervous system through nociceptive signalling and can therefore serve as a common afferent pathway to both immune and nervous systems.