Endothelial TrkB receptor activation controls vascular tone of rat middle cerebral artery.

Little is known on the cerebrovascular BDNF (brain-derived neurotrophic factor)/TrkB (tropomyosin related kinase B) pathway. This study investigated the contribution of endogenous endothelial BDNF to the control of vascular tone of rat middle cerebral artery (MCA) and the capacity of exogenous agonist of TrkB receptors to induce their relaxation. Endothelial cells constitutively expressed both BDNF and activated TrkB receptors. Supporting endothelial BDNF as an autocrine regulator of basal myogenic tone, incubation of MCA with the TrkB antagonist cyclotraxin B induced contraction as observed with incubation in the presence of inhibitors of nitric oxide (NO) and endothelium-derived hyperpolarizing factor (EDHF) production. Exposure of MCA with the TrkB agonist LM22A-4 that increased expression of TrkB receptors phosphorylated at tyrosine 816 induced relaxation of preconstricted MCA (EC50 6.7 × 10-8 mol/L) as efficiently than acetylcholine (EC50 5.3 × 10-8 mol/L). Finally, endothelium removal, exposure to a TrkB antagonist or to inhibitors of NO and EDHF production prevented the relaxant effect of LM22A-4. In conclusion, our study identified endothelial BDNF as a new autocrine regulator of vascular tone of MCA, thus making the endothelial BDNF/TrkB pathway an attractive target for strategies aiming to improve blood supply to the brain.

Glucocorticoids improve endothelial function in rheumatoid arthritis: a study in rats with adjuvant-induced arthritis.

To determine the effect of glucocorticoids (GCs) on endothelial dysfunction (ED) and on traditional cardiovascular (CV) risk factors in the adjuvant-induced arthritis (AIA) rat model. At the first signs of AIA, a high dose (HD) [10 mg/kg/day, intraperitoneally (i.p.), GC-HD] or low dose (LD) (1 mg/kg/day, i.p., GC-LD) of prednisolone was administered for 3 weeks. Endothelial function was studied in aortic rings relaxed with acetylcholine (Ach) with or without inhibitors of nitric oxide synthase (NOS), cyclooxygenase 2 (COX-2), arginase, endothelium derived hyperpolarizing factor (EDHF) and superoxide anions ( O2-°) production. Aortic expression of endothelial NOS (eNOS), Ser1177-phospho-eNOS, COX-2, arginase-2, p22phox and p47phox was evaluated by Western blotting analysis. Arthritis scores, blood pressure, heart rate and blood levels of cytokines, triglycerides, cholesterol and glucose were measured. GC-HD but not GC-LD reduced arthritis score significantly and improved Ach-induced relaxation (P < 0·05). The positive effect of GC-HD resulted from increased NOS activity and EDHF production and decreased COX-2/arginase activities and O2-° production. These functional effects relied upon increased phospho-eNOS expression and decreased COX-2, arginase-2 and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase expression. Despite the lack of effect of GC-LD on ED, it increased NOS and EDHF and down-regulated O2-° pathways but did not change arginase and COX-2 pathways. GC-HD increased triglycerides levels and blood pressure significantly (P < 0·05). Both doses of GCs decreased to the same extent as plasma interleukin (IL)-1ß and tumour necrosis factor (TNF)-α levels (P < 0·05). Our data demonstrated that subchronic treatment with prednisolone improved endothelial function in AIA via pleiotropic effects on endothelial pathways. These effects occurred independently of the deleterious cardiometabolic effects and the impact of prednisolone on systemic inflammation.

Brain-derived neurotrophic factor of the cerebral microvasculature: a forgotten and nitric oxide-dependent contributor of brain-derived neurotrophic factor in the brain.

AIM: Evidence that brain-derived neurotrophic factor (BDNF), a neurotrophin largely involved in cognition, is expressed by cerebral endothelial cells led us to explore in rats the contribution of the cerebral microvasculature to BDNF found in brain tissue and the link between cerebrovascular nitric oxide (NO) and BDNF production. METHODS: Brain BDNF protein levels were measured before and after in situ removal of the cerebral endothelium that was achieved by brain perfusion with a 0.2% CHAPS (3-[(3-cholamidopropyl) dimethylammonio]-1-propane sulphonate) solution. BDNF protein and mRNA levels as well as levels of endothelial NO synthase phosphorylated at serine 1177 (P-eNOSser1177 ) were measured in cerebral microvessel-enriched fractions. These fractions were also exposed to glycerol trinitrate. Hypertension (spontaneously hypertensive rats) and physical exercise training were used as experimental approaches to modulate cerebrovascular endothelial NO production. RESULTS: CHAPS perfusion resulted in a marked decrease in brain BDNF levels. Hypertension decreased and exercise increased P-eNOSser1177 and BDNF protein levels. However, BDNF mRNA levels that were increased by exercise did not change after hypertension. Finally, in vitro exposure of cerebral microvessel-enriched fractions to glycerol trinitrate enhanced BDNF production. CONCLUSION: These data reveal that BDNF levels measured in brain homogenates correspond for a large part to BDNF present in cerebral endothelial cells and that cerebrovascular BDNF production is dependent on cerebrovascular endothelial eNOS activity. They provide a paradigm shift in the cellular source of brain BDNF and suggest a new approach to improve our understanding of the link between endothelial function and cognition.

Deferoxamine blocks death induced by glutathione depletion in PC 12 cells.

The purpose of the present work was to investigate the mechanisms by which glutathione depletion induced by treatment with buthionine sulfoximine (BSO) led within 24-30 h to PC 12 cells apoptosis. Our results showed that treatment by relatively low concentrations (10-30 µM) of deferoxamine (DFx), a natural iron-specific chelator, almost completely shielded the cells from BSO-induced toxicity and that DFx still remained protective when added up to 9-12h after BSO treatment. On the other hand, phosphopeptides derived from milk casein and known to carry iron across cell membranes, markedly potentiated the toxic action of BSO when loaded with iron but were ineffective in sodium form. Kept for 24 h in serum-free medium, the cells underwent a decrease in glutathione content after BSO treatment, but remained viable. However, these BSO-pre-treated cells showed a rapid (90-120 min) decrease in cell viability when incubated with low doses of iron, whereas a great proportion of them remained viable in the presence of higher concentrations of copper and zinc. We also observed in PC 12 cells an early (4-8 h) and transient increase in the expression of ferritin subunits following BSO addition. Taken together these results suggest that BSO-induced glutathione depletion leads to an alteration of cellular iron homeostasis, which may contribute to its toxicity.

Ipsilateral versus contralateral spontaneous post-stroke neuroplastic changes: involvement of BDNF?

Stroke is a leading cause of death and disability in industrialized countries. Although surviving patients exhibit a certain degree of restoration of function attributable to brain plasticity, the majority of stroke survivors has to struggle with persisting deficits. In order to potentiate post-stroke recovery, several rehabilitation therapies have been undertaken and many experimental studies have reported that brain-derived neurotrophic factor (BDNF) is central to many facets of neuroplastic processes. However, although BDNF role in brain plasticity is well characterized through strategies that manipulate its content, the involvement of this neurotrophin in spontaneous post-stroke recovery remains to be clarified. Besides, while the neuroplastic role of BDNF is restricted to its mature form, most studies investigating the proper effect of ischemia on post-stroke BDNF metabolism focused on mRNA or total protein expressions. In addition, these studies are mainly performed in brain regions collected either at or around the lesion site. Therefore, the objective of the present study was to investigate in both hemispheres, the long-term expression (up to one month) of both pro- and mature BDNF forms in rats subjected to photothrombotic ischemia. These assessments were performed in the cortex and in the hippocampus, two regions known to subserve functional recovery after stroke and were coupled to the study of synaptophysin expression, a marker of synaptogenesis. Our study reports that stroke induces an early and transient increase (4h) in mature BDNF expression in the cortex of both hemispheres that was associated with a delayed rise (30d) in synaptophysin levels ipsilateraly. In both hippocampal territories, the pattern of mature BDNF expression shows a more delayed increase (from 8 to 30d), which coincides with the evolution of synaptophysin expression. Interestingly, in these hippocampal territories, pro-BDNF levels evolve differently suggesting a differential gene regulation between the two hemispheres. While highlighting the complexity of changes in BDNF metabolism after stroke, our data suggest that BDNF involvement in spontaneous post-stroke plasticity is region-dependent.

Effect of early decrease in the lesion size on late brain tissue loss, synaptophysin expression and functionality after a focal brain lesion in rats.

The purpose of the present study is to determine the effects of early decrease in the lesion size on late brain tissue loss, synaptogenesis and functionality after a focal brain lesion in rats. The lesion was induced either to the cortex using the photothrombotic ischemic stroke or to the striatum using the malonate poisoning model. The cortical and striatal lesions amounted to 66-80 mm(3) at day 1 post-lesion and were reduced by 50% after the acute administration of dipyridyl (a liposoluble iron chelator) and aminoguanidine (an inhibitor of the inducible nitric oxide synthase), respectively. Loss of histologically intact tissue and synaptophysin expression as an indicator of synaptogenesis were examined at day 35 post-lesion. Both types of lesion resulted in synaptophysin upregulation in contralateral and ipsilateral cortical areas. On the contrary, brain tissue loss was greater after the striatal (-17%) than the cortical lesion (-5%). Synaptophysin expression and tissue loss were not different between drug- and vehicle-treated rats. Moreover, a set of standard neurological tests revealed a difference in deficit between the both types of lesion, yet only in the acute post-lesion stage. However, it did not distinguish between vehicle- and drug-treated rats whatever the lesion location. Our results indicate that late histological endpoints measurements are not recommended to probe the potential neuroprotective properties of a drug administered within the acute post-lesion stage. They also suggest that inhibition of cytotoxic mechanisms involved in lesion growth is of no clinical interest when it cannot lead to a long-term histological protection and/or increased synaptogenesis.

Cytoprotective efficacy and mechanisms of the liposoluble iron chelator 2,2'-dipyridyl in the rat photothrombotic ischemic stroke model.

We examined the efficacy of the liposoluble iron chelator 2,2'-dipyridyl (DP) in reducing histological damage in rats submitted to cerebral ischemia and the mechanisms involved in the potential cytoprotection. For this purpose, DP (20 mg/kg, i.p.) was administered 15 min before and 1 h after induction of cortical photothrombotic vascular occlusion in rat. Histological studies were performed to assess infarct volume (at days 1 and 3 postischemia) and astromicroglial activation (at day 3 postischemia). Damage to endothelial and neuronal cells was evaluated at day 1 postischemia by quantitative measurements of Evans Blue extravasation and N-acetylaspartate levels, respectively. Cerebral blood flow was recorded in the ischemic core by laser-Doppler flowmetry within the 15 min to 2 h period after photothrombosis. At 4-h postischemia, radical oxygen species (ROS) production was evaluated by measuring brain glutathione concentrations. The cortical expression of the proteins heme oxygenase-1 (HO-1) and hypoxia-inducible factor-1alpha (HIF-1alpha) was analyzed by Western blotting at day 1 postischemia. Infarct volume and ischemic damage to endothelial and neuronal cells were significantly reduced by DP treatment. This cytoprotection was associated with a reduction in ROS production, perfusion deficits, and astrocytic activation. DP treatment also resulted in significant changes in HO-1 (+100%) and HIF-1alpha (-50%) protein expression at the level of the ischemic core. These results report the efficacy of the liposoluble iron chelator DP in reducing histological damage induced by permanent focal ischemia.

Differential MnSOD and HO-1 expression in cerebral endothelial cells in response to sublethal oxidative stress.

The two inducible enzymes, manganese superoxide dismutase (MnSOD) and heme-oxygenase-1 (HO-1) may participate in the cellular defense of brain endothelium against oxidative stress. The time-dependent expression of MnSOD and HO-1 mRNAs and proteins was investigated in vitro in rat cerebral endothelial cells (CEC) subjected to sublethal mild or moderate hydroxyl radical-induced oxidative stress. Mild oxidative stress induced increases in both MnSOD and HO-1 mRNA and protein expression. Moderate oxidative stress resulted in a significant reduction in HO-1 mRNA and protein expression, whereas MnSOD expression pattern was similar to that observed after mild oxidative stress. A profound protein loss of both MnSOD and HO-1 was detected 24 h after exposure of CEC to a moderate oxidative stress. The data indicate that cerebral endothelial cells respond by increasing the expression of antioxidant defense enzymes in a manner dependent on the oxidative stress intensity.

Effects of iNOS-related NO on hearts exposed to liposoluble iron.

Inducible nitric oxide synthase (iNOS) protects heart against ischemia/reperfusion injury. However, it is unknown whether the beneficial effects of iNOS are mediated by the interaction of NO with radical oxygen species (ROS). To address this issue, we examined the effects of liposoluble iron-induced ROS generation in isolated perfused hearts from rats treated with lipopolysaccharide (LPS). LPS administration (10 mg/kg, i.p., 6 h before heart removal) induced iNOS expression and increased NO production as indicated by a 3-fold elevation of nitrite level in coronary effluents relative to control hearts. An enhanced expression of hemeoxygenase 1 protein was also observed in septic hearts compared to control. Iron-induced perfusion and contractile deficits were ameliorated by LPS with more important coronary than myocardial benefits. In iron-loaded hearts, oxidative stress as measured by the 2,3 dihydroxybenzoic acid/salicylic acid concentration ratio in cardiac tissue was 23% lower in septic than in control heart although the difference did not reach significance. In addition, the presence of the NO synthase inhibitor N-nitro-L-arginine in the perfusion medium totally blocked NO production but did not reverse the protective effects of LPS. The results indicate that LPS protects from iron-induced cardiac dysfunction by mechanisms independent on ex vivo NO production and suggest that NO acts as a trigger rather than a direct mediator of the cardioprotective effects of LPS in heart exposed to iron.

Effects of a direct injection of liposoluble iron into rat striatum. Importance of the rate of iron delivery to cells.

For a better understanding of the role of iron imbalance in neuropathology, a liposoluble iron complex (ferric hydroxyquinoline, FHQ) was injected into striatum of rats. The effects of two modalities of iron injections on brain damage, hydroxyl radical (*OH) production (assessed by the salicylate method coupled to microdialysis) and tissue reactive iron level (evaluated ex vivo by the propensity of the injected structure for lipid peroxidation) were examined. Rapid injection of FHQ (10 nmoles of 5 mM FHQ pH 3 solution over 1-min period) but not that of corresponding vehicle led to extensive damage associated with increased tissue free iron level in the injected region. Conversely, neither lesion nor free iron accumulation was observed after slow FHQ injection (10 nmoles of a 100 microM FHQ pH 7 solution over 1-h period) as compared to corresponding vehicle injection. Production of *OH was induced by slow FHQ injection but not by rapid FHQ injection, probably as a result of in vivo abolition of iron-induced *OH formation by acid pH. Indeed, rapid injection of FAC pH 7 (ferric ammonium citrate, 5 mM in saline) was associated with *OH formation whereas rapid injection of FAC pH 3 did not. Our results identify the rate of iron delivery to cells as an important determinant of iron toxicity and do not support a major role for extracellular *OH in damage associated with intracerebral iron injection.

Chemical preconditioning with 3-nitropropionic acid: lack of induction of neuronal tolerance in gerbil hippocampus subjected to transient forebrain ischemia.

Chemical preconditioning using the mitochondrial toxin, 3-nitropropionic acid (3-NP) has been reported to induce neuroprotection against subsequent global ischemia. To investigate the underlying mechanisms, Mongolian gerbils were pretreated with either vehicle or 3-NP at the dose of 3 or 10 mg/kg, intraperitoneal, 3 days prior to a 5-min bilateral carotid artery occlusion followed by either 48 h or 7 days of blood recirculation. Neuronal damage was assessed by a cresyl violet/fuchsin acid staining. Induction of heat shock protein 72 (HSP72) and manganese superoxide dismutase (MnSOD) expression was evaluated by Western blotting. Astroglial and microglial activation was detected by immunohistochemistry (glial fibrillary acid protein) and by histochemistry (isolectin B4 staining), respectively. Present data show that the hippocampal neuronal damage induced by ischemia were of similar extent between the vehicle- and 3-NP-treated gerbils, whatever the dose tested, indicating that 3-NP did not induce tolerance to transient forebrain ischemia under our experimental conditions. The lack of difference in the post-ischemic level of HSP72 and MnSOD protein expression and in the intensity of astroglial and microglial activation represents further indirect indications of the absence of 3-NP preconditioning effect. In conclusion, although chemical preconditioning with 3-NP is a well-established phenomenon at least in vitro and in models of focal ischemia, the relevance of 3-NP as a preconditioning molecule towards global brain ischemia remains an open question.

PRL antiapoptotic effect in the rat decidua involves the PI3K/protein kinase B-mediated inhibition of caspase-3 activity.

During gestation, the uterus undergoes severe changes to accommodate and protect the developing conceptus. In particular, stromal endometrial cells proliferate and differentiate to form the decidual tissue, which produces PRL. Once the conceptus begins to grow, extensive regression by apoptosis take place in the decidua coincident with the loss of the PRL receptor in this tissue. In this report we have established for the first time that PRL, acting through the long form of the PRL receptor and the PI3K pathway, exerts an antiapoptotic effect in rat decidua. We have also shown that protein kinase B phosphorylation on serine 473 as well as its nuclear translocation are stimulated by PRL in decidual cells. Moreover, we have found that caspase-3, a well known effector of apoptosis, becomes expressed and active in the rat decidua just at a time when this tissue undergoes extensive apoptosis. PRL was able to down-regulate both caspase-3 mRNA levels as well as activity. Furthermore, using a protein kinase B dominant-negative expression vector, we provide evidence that PRL inhibition of caspase-3 requires an intact protein kinase B pathway. Finally, we have also found that rat placental lactogen I and II dose-dependently inhibit caspase-3 mRNA, suggesting multiple sources of PRL in the hormonal control of rat decidual regression. In summary, the results of this study have defined an important role for decidual PRL in the normal progress of pregnancy, specifically in the regression and reorganization of the decidua.

Stress response to hypoxia in gerbil brain: HO-1 and Mn SOD expression and glial activation.

Hypoxic preconditioning has been shown to induce neuroprotection against a subsequent damaging insult. In order to study the underlying molecular and cellular mechanisms of hypoxic preconditioning, we investigated, in gerbil hippocampus, the effects in vivo of transient exposure to hypoxia (4% O(2) for 6 min followed by either 48 h or 7 days of reoxygenation) (i) on the induction of 72 kDa heat shock protein (HSP72), heme oxygenase-1 (HO-1) and manganese superoxide dismutase (Mn SOD) as assessed by Western immunoblotting and (ii) on the astroglial and microglial activation as detected by both immunohistochemistry and Western immunoblotting for GFAP, and histochemistry for isolectin B4, respectively. Our data show that, although hypoxia and subsequent reoxygenation led to neither neuronal damage nor HSP72 induction in gerbil hippocampus, it induced a progressive and sustained expression of HO-1 and Mn SOD. As expected from the absence of neuronal death, hypoxia was not associated with microglial activation but led to a significant astrocytic activation. These findings demonstrate that transient hypoxia enhances the antioxidative enzymatic defenses of the brain, which are susceptible to increased tolerance against a subsequent damaging insult.

Characterization of a rat uterine cell line, U(III) cells: prolactin (PRL) expression and endogenous regulation of PRL-dependent genes; estrogen receptor beta, alpha(2)-macroglobulin, and decidual PRL involving the Jak2 and Stat5 pathway.

Decidualization of endometrial stroma in the rat induces the expression and secretion of rat decidual PRL (rdPRL). Recently, we have generated a nontransformed rat uterine stromal cell line (U(III)) that decidualizes spontaneously in culture. In this report, we have established by immunocytochemistry, RT-PCR, Western blot analysis, labeled amino acid incorporation and RIA that these cells express the rat PRL messenger RNA as well as synthesize and secrete PRL. We have also cloned by RT-PCR a 403-bp complementary DNA fragment whose sequence is identical with that of rat pituitary PRL. In addition, U(III) cells express the PRL receptor (PRL-R) long form, all the components involved in the PRL signal transduction pathway, estrogen receptor beta (ER beta) and alpha(2)-macroglobulin (alpha(2)-MG), which are known to be PRL-regulated genes. However, when U(III) cells were treated with PRL, no regulation of these genes was observed. Moreover, in these cells, the PRL signaling components: the tyrosine kinase Jak2 and the transcription factor Stat5 were endogenously phosphorylated and their phosphorylation states were not enhanced in the presence of exogenous PRL. To examine whether the endogenously secreted PRL affects the expression of PRL-regulated genes, U(III) cells were treated with either an anti-PRL receptor antibody or a Jak2 inhibitor, AG490. The anti-PRL receptor antibody decreased alpha(2)-MG expression. AG490 inhibited Jak2 and Stat5 phosphorylation, prevented Stat5 binding to its DNA consensus sequence, and also caused a dose-dependent down-regulation of alpha(2)-MG and ER beta expression. In contrast, AG490 enhanced PRL mRNA levels. In summary, we have established that the U(III) stromal cells of uterine origin produce PRL. Furthermore, we have shown for the first time that decidual PRL may act locally to activate the Jak2/Stat5 pathway and up-regulate important genes involved in decidual growth and placentation.

Estrogen receptors alpha and beta in rat decidua cells: cell-specific expression and differential regulation by steroid hormones and prolactin.

Estradiol is known to play an important role in the growth and differentiation of rat uterine stromal cells into decidual cells. In particular, this hormone with progesterone is necessary for blastocyst implantation and subsequent decidualization in the rat. Although binding experiments have demonstrated the presence of estrogen-binding sites, no evidence exists as to whether the rat decidua expresses both isoforms of the estrogen receptor (ER), alpha and beta. In this investigation, we analyzed the expression of decidual ERalpha and ERbeta, studied their regulation by PRL and steroid hormones and examined the ability of decidual ERp to transduce the estradiol signal to the progesterone receptor. Immunocytochemistry, RT-PCR, and Northern blot analysis showed that both ER species are coexpressed in the decidua during pseudopregnancy. Interestingly, these genes were preferentially found in a cell population localized in the antimesometrial site of the uterus where blastocyst implantation takes place. Using decidual cells in primary culture obtained from pseudopregnant rats and a decidua-derived cell line (GG-AD), we show a differential regulation of ERalpha and ERbeta by PRL and ovarian steroid hormones. Whereas PRL, estradiol, and progesterone all increased ERbeta messenger RNA (mRNA) expression in a dose-dependent manner, only PRL up-regulated the mRNA levels of ERa. Estradiol had no effect on ERalpha expression, whereas progesterone markedly decreased its mRNA levels. Interestingly, progesterone, which up-regulates the ability of PRL to signal to a PRL-regulated gene in mammary-gland derived cells, prevented PRL stimulation of decidual ERalpha and had no synergistic effect on ERbeta expression. The use of GG-AD cells, which express only ERbeta, allowed us to demonstrate that this receptor subtype is functional and transduces estradiol signal to the progesterone receptor. In summary, the results of this investigation have revealed that ERbeta is expressed in addition to ERalpha in the rat decidua, and that the expression of both ERs are cell specific and differentially regulated by PRL and steroids. One salient finding of this investigation is that progesterone down-regulates ERalpha, but concomitantly increases the expression of a functional ERbeta that mediates estradiol up-regulation of the decidual progesterone receptor.

Involvement of SOCS-1, the suppressor of cytokine signaling, in the prevention of prolactin-responsive gene expression in decidual cells.

The cells forming the rat decidua produce PRL and PRL-related proteins and express both the long and short forms of the PRL receptor. Yet, only a defined subpopulation, the mesometrial cells, express the PRL-dependent alpha2-macroglobulin gene. This gene is silenced in vivo in the antimesometrial cells and in the GG-AD cell line, derived from antimesometrial cells. To examine whether the lack of alpha2-macroglobulin expression is due to defective components in the PRL signaling pathway, we compared the relative expression of Janus kinase 2 (Jak2), signal transducer and activator of transcription 5 a and b (Stat5 a and b), suppressor of cytokine signaling-1 (SOCS-1), and the tyrosine phosphatase SHP-2 mRNA in mesometrial and antimesometrial decidua on days 12 and 13 of pseudopregnancy, the time of maximal alpha2-macroglobulin expression. We found no significant differences in the relative expression of either Jak2, Stat5 (a and b), or SHP-2 in the two cell populations. However, we discovered a profound difference in the expression of SOCS-1, an inhibitor of the Jak/Stat pathway. This gene was highly expressed in the antimesometrial cells and in the GG-AD cells, which do not produce alpha2-macroglobulin. Immunoprecipitation experiments with GG-AD cells revealed that although Jak2 and Stat5 coprecipitate in response to PRL stimulation, no phosphorylation of Jak2 and Stat5 could be observed. To examine whether SOCS-1 plays a role in silencing the alpha2-macroglobulin gene, we cultured GG-AD cells in the presence of either a SOCS-1 antisense oligonucleotide or an irrelevant oligonucleotide for 4, 12, and 28 h. Cells were also treated with PRL. Within 4 h of SOCS-1 antisense treatment, alpha2-macroglobulin mRNA expression was initiated. After 28 h, only cells treated with PRL and SOCS-1 antisense oligonucleotide retained the ability to express the alpha2-macroglobulin gene. In summary, results of this study reveal that constitutive expression of SOCS-1 can prevent PRL signaling and that the lack of PRL-induced expression of alpha2-macroglobulin in a defined decidual cell population is largely due to SOCS-1 expression in these cells.

Rat decidual prolactin. Identification, molecular cloning, and characterization.

Establishment and maintenance of pregnancy require the activity of a highly specialized maternal tissue, the decidua. It is well established that the human decidua synthesizes and releases prolactin. However, in the rat, no study has been able to demonstrate the production of prolactin by the decidua. In this report, we established for the first time using Northern blot analysis and reverse transcription-polymerase chain reaction, Western blot analysis, immunocytochemistry, and enzyme-linked immunosorbent assay, that a defined cell population located in the rat antimesometrial decidua expresses prolactin mRNA, as well as synthesizes and secretes this hormone. By reverse transcription-polymerase chain reaction and rapid amplification of cDNA ends, we cloned a full-length cDNA for rat decidua prolactin, whose sequence was identical to that of pituitary prolactin. Our results also showed that pituitary prolactin appeared to down-regulate decidual prolactin levels. Under these circumstances, inhibition of pituitary prolactin secretion led to a rise in both decidual prolactin mRNA and protein expression. Moreover, addition of exogenous prolactin to primary decidual cells in culture also caused a marked decrease in decidual prolactin mRNA expression. Finally, treatment of primary decidual cells with steroid hormones or 8-bromo-cAMP revealed a differential regulation of decidual prolactin expression from that of pituitary suggesting a tissue-specific regulation of prolactin gene expression, possibly through the use of an alternative promoter in rat decidua.

The expression of interleukin-6 (IL-6), IL-6 receptor, and gp130-kilodalton glycoprotein in the rat decidua and a decidual cell line: regulation by 17beta-estradiol and prolactin.

The cytokine interleukin 6 (IL-6), a major mediator of immune and acute phase responses of the liver, has been implicated in the termination of pregnancy once expressed in the uterus. This study was undertaken to investigate the expression and regulation of genes encoding IL-6 and IL-6 receptor (IL-6R) in rat decidual tissue. Total RNA obtained from rat decidual tissue on different days of pseudopregnancy was analyzed by RT-PCR using specific primers for IL-6, IL-6R, and 130-kDa glycoprotein (gp130). Ribosomal L19 primers served as an internal control. IL-6R and gp130 were found to be expressed in the decidua throughout development, while no messenger RNA (mRNA) for IL-6 was detected. Interestingly, within several hours of culture, decidual explants acquired the ability to express IL-6. The apparent ability of decidual cells to express IL-6 and its lack of expression in vivo led us to examine whether the IL-6 gene is actively inhibited. Primary decidual cells were cultured in the presence of estradiol, progesterone, or PRL. Progesterone showed no effect, whereas estradiol and PRL reduced the level of IL-6 mRNA expression. To examine the mechanism by which these hormones inhibit IL-6 expression, we used a simian virus 40-transformed decidual cell line (GG-AD), which expresses only estrogen receptor-beta (ERbeta). Like primary decidual cells in culture, GG-AD cells express IL-6, IL-6R, and gp130 mRNA. When cultured in the presence of estradiol (0-100 ng/ml), mRNA for IL-6 and its receptor components were down-regulated in a dose-dependent manner. Estradiol also caused a dose-dependent decrease in IL-6 protein secretion into the culture medium. The inhibitory effect of estradiol on IL-6 mRNA expression was reversed by the antiestrogen ICI-164,384. Similar inhibition of IL-6 and gp130 mRNA expression was observed with PRL treatment. However, PRL had no effect on IL-6R mRNA levels. PRL inhibition of IL-6 expression was totally reversed by tyrphostin AG490, a JAK2 inhibitor. In summary, the results of this investigation indicate that IL-6 expression, which is detrimental to the maintenance of pregnancy, is inhibited in the rat decidual tissue. This inhibition is induced by PRL and estradiol, which down-regulate not only IL-6 expression, but also the expression of IL-6 receptor and signaling proteins. The results also suggest that PRL signaling to the IL-6 gene is mediated through the long form of PRL receptor and involves JAK2 activation, whereas that of estradiol can be transduced by estrogen receptor-beta.

Arachidonic acid up-regulates and prostaglandin E2 down-regulates the expression of pancreatic-type phospholipase A2 and prostaglandin-endoperoxide synthase 2 in uterine stromal cells.

It is well known that arachidonic acid, as a substrate of prostaglandin G/H synthase (PGHS), is converted into prostaglandins of the two-series. In this work, we attempted to determine whether arachidonic acid and prostaglandin E2 might regulate the expression of PGHS and the pancreatic-type phospholipase A2 (PLA2I), which may be involved in the liberation of arachidonic acid from membrane phospholipids. For this purpose, we used the uterine stromal cell line UIII, which produces prostaglandin E2 and expresses both the constitutive and inducible PGHS enzymes (PGHS1 and PGHS2) and PLA2 I. The results show that PGHS1, which is expressed at a high level in UIII cells, was not modified by arachidonic acid. The expression of PGHS2 and PLA2 I was up-regulated by increasing arachidonate concentrations (1-10 microM). The maximal response was obtained at 24 h, reaching a 2.3-fold and 2.6-fold increase for PGHS2 and PLA2 I expression, respectively, compared to the control level. To discriminate between the effect of arachidonic acid and that of prostaglandins, which are highly increased in the presence of exogenous arachidonic acid, we treated the cells with two inhibitors of PGHS activity, aspirin and meclofenamic acid. Both inhibitors failed to suppress the arachidonate-induced increase of PLA2 I and PGHS2 expression and even enhanced it either in the presence or absence of arachidonic acid. In contrast, the addition of prostaglandin E2 to the culture medium decreased the expression of both enzymes in a dose-dependent manner, the maximal response being reached at 1 microM. We conclude that arachidonic acid up-regulates the expression of PLA2 I and PGHS2 in the uterine stromal cells, independently of prostanoids, and that prostaglandin E2 is capable of down-regulating enzyme expression.

Prolactin up-regulates prostaglandin E2 production through increased expression of pancreatic-type phospholipase A2 (type I) and prostaglandin G/H synthase 2 in uterine cells.

Uterine stromal cells produce and release PGE2, both processes being regulated by hormonal factors. In this study, we examined the effect of PRL on the PGE2 production and release measured by radioimmunoassay. For this purpose, we used a rat uterine stromal cell line, UIII cells, which produce PGE2 and contain PRL receptors. The expression of sPLA2I and PGHS (PGHS1 and PGHS2), enzymes required for PGE2 production, was also estimated by immunocytochemistry and 'Western blotting' in response to PRL. PRL (10 to 60 ng/ml) significantly increased the PGE2 release (up to 6-fold) and production, in a dose-dependent manner. Results show that PGHS1 and PGHS2 are both expressed constitutively in the uterine UIII cells, although PGHS2 is expressed at a low level. PRL did not increase PGHS1 expression, but stimulated the expression of sPLA2I and PGHS2 by 3.5- and 2.5-fold, respectively. These data show for the first time a regulation of sPLA2I and PGHS2 expression by PRL and may indicate that, in uterine cells, PRL enhances the PGE2 release and production by increasing the expression of both sPLA2I and PGHS2.