Steroid regulation of brain aromatase expression in glia: female preoptic and vocal motor nuclei

Expression of the enzyme aromatase, which converts androgens to estrogens, is known to be regulated by gonadal steroids in brain areas linked to reproduction and related behaviors in several groups of vertebrates. Previously, we demonstrated in a vocal fish, the plainfin midshipman, that both males and females undergo seasonal changes in brain aromatase mRNA expression in the preoptic area (POA) and the dimorphic sonic/vocal motor nucleus (SMN) that parallel seasonal variation in circulating steroid levels and reproductive behavior. We tested the hypothesis that steroids are directly responsible for seasonal modulation of aromatase in females because they show the most dramatic fluctuations of testosterone (T) and 17beta-estradiol (E2) throughout the year. Adult female midshipmen were ovariectomized and administered T, E2, or blank (control) implants. We then quantified aromatase mRNA expression within the POA and SMN by in situ hybridization. Both T- and E2-treated females had elevated mRNA expression levels in both brain areas compared to controls. T affected aromatase expression in a level-dependent manner, whereas E2 showed a decreased effect at higher circulating levels. This study demonstrates that seasonal differences in brain aromatase expression in female midshipman fish may be explained, in part, by changes in levels of circulating steroids.     

Gender differences in the inflammatory response and survival following haemorrhage and subsequent sepsis

Studies have shown gender dimorphism in cell-mediated immune responses following haemorrhage, with depressed responses in young males and maintained or enhanced responses in proestrus females. However, it remains unknown whether or not the sexually dimorphic immune response to haemorrhage provides any protection against a subsequent in vivo polymicrobial septic challenge. To study this, male and proestrus female C3H/HeN mice were subjected to haemorrhage (35+/-5 mmHg for 90 min followed by fluid resuscitation) or sham operation. Twenty-four hours thereafter, all mice were subjected to polymicrobial sepsis by cecal ligation and puncture (CLP) and survival was assessed over a 10 day period. Haemorrhage prior to CLP significantly increased mortality in males as compared to shams. In contrast, mortality in females following CLP was comparable between the sham and haemorrhage groups. Plasma levels of interleukin (IL-)6, tumour necrosis factor (TNF)-alpha and prostaglandin E(2)(PGE(2)) at 5 h after CLP were significantly increased in males subjected to prior haemorrhage. In contrast, plasma levels of IL-6 and TNF-alpha in females did not increase under such conditions. PGE(2)levels were comparable in males and females following CLP, however prior haemorrhage significantly reduced PGE(2)levels in females, whereas no change was observed in males. Liver and splenic expression of cyclooxygenase-2 protein paralleled the changes in plasma PGE(2). Female sex hormones, therefore, appear to play an important role not only in maintaining immune function following haemorrhage, but also provide a survival advantage against subsequent septic challenge.     

Bleomycin-induced E prostanoid receptor changes alter fibroblast responses to prostaglandin E2

Although PGE(2) is a potent inhibitor of fibroblast function, PGE(2) levels are paradoxically elevated in murine lungs undergoing fibrotic responses. Pulmonary fibroblasts from untreated mice expressed all four E prostanoid (EP) receptors for PGE(2). However, following challenge with the fibrogenic agent, bleomycin, fibroblasts showed loss of EP2 expression. Lack of EP2 expression correlated with an inability of fibroblasts from bleomycin-treated mice to be inhibited by PGE(2) in assays of proliferation or collagen synthesis and blunted cAMP elevations in response to PGE(2). PGE(2) was similarly unable to suppress proliferation or collagen synthesis in fibroblasts from EP2(-/-) mice despite expression of the other EP receptors. EP2(-/-), but not EP1(-/-) or EP3(-/-) mice, showed exaggerated fibrotic responses to bleomycin administration in vivo as compared with wild-type controls. EP2 loss on fibroblasts was verified in a second model of pulmonary fibrosis using FITC. Our results for the first time link EP2 receptor loss on fibroblasts following fibrotic lung injury to altered suppression by PGE(2) and thus identify a novel fibrogenic mechanism.     

Role of prostaglandin E2 in the synthesis of the pro-inflammatory cytokine interleukin-6 in primary sensory neurons: an in vivo and in vitro study

Following various types of nerve injury, cyclooxygenase 2 and prostaglandin E2 (PGE2) are universally and chronically up-regulated in injured nerves and contribute to the genesis of neuropathic pain. Persistent high levels of PGE2 likely exert chronic effects on nociceptive dorsal root ganglion (DRG) neurons. In the present study, we tested the hypothesis that injured nerve-derived PGE2 contributes to the up-regulation of the pro-inflammatory cytokine interleukin-6 (IL-6) in DRG neurons following partial sciatic nerve ligation. In naive adult rats, IL-6 was expressed in only a few small size DRG neurons which all co-expressed EP4 receptors. Partial sciatic nerve ligation increased and shifted IL-6 expression from small to medium and large size damaged DRG neurons. Perineural injection of a selective cyclooxygenase 2 inhibitor or a selective EP4 receptor antagonist significantly suppressed the up-regulation of IL-6 in DRG, suggesting that injured nerve derived PGE2 contributes to the de novo synthesis of IL-6 in DRG neurons through EP4 receptors. In cultured sensory ganglion explants, a stabilized PGE2 analog increased IL-6 mRNA and protein levels through the activation of EP4, protein kinase A, protein kinase C, extracellular regulated protein kinase/MAPK, cAMP response element binding protein and NFκB signalling pathways. Taken together, these data indicate that facilitating the de novo synthesis of pain-related cytokines in injured medium and large size DRG neurons is a novel mechanism underlying the role of injured nerve derived PGE2 in the genesis of neuropathic pain.     

Obesity in transgenic female mice with constitutively elevated luteinizing hormone secretion

Transgenic (TG) female mice, expressing a chimeric bovine luteinizing hormone (LH) beta-subunit/human chorionic gonadotropin beta-subunit COOH-terminal extension (bLHbeta-CTP) gene, produce high levels of circulating LH and serve as a model for functional ovarian hyperandrogenism and follicular cysts. We report here that obesity is a typical feature of these female mice. The mean body weight of the bLHbeta-CTP females was significantly higher than in controls at, and beyond 5 wk of age, and at 5 mo, it was 32% increased. At this age, the amount of white adipose tissue in the bLHbeta-CTP females was significantly increased, as reflected by the weight difference of the retroperitoneal fat pad. In addition, the expression of leptin mRNA in white adipose tissue of the TG females was elevated about twofold. Serum leptin and insulin levels, and food intake, were also increased significantly in the TG females. Brown adipose tissue (BAT) thermogenic activity, as measured by GDP binding to BAT mitochondria, was reduced (P < 0.05). Ovariectomy at the age of 3 wk totally prevented the development of obesity. In summary, the present results show that intact female bLHbeta-CTP mice are obese, have increased food consumption, and reduced BAT thermogenic activity. The weight gain can be explained partly by elevated androgens but is probably also contributed to the increased adrenal steroidogenesis. Hence, the bLHbeta-CTP mice provide a useful model for studying obesity related to elevated LH secretion, with consequent alterations in ovarian and adrenal function.     

Periconceptional alcohol consumption-induced changes in embryonic prostaglandin E levels in mouse organogenesis: modulation by nitric oxide

The mechanisms of the teratogenic effects of maternal alcohol consumption remain unclear. The aim of the present work was to study the organogenic PGE(2) levels and the modulation of PGE(2) levels by NO after periconceptional alcohol ingestion. Female mice were intoxicated with a 10% ethanol in drinking water before pregnancy and up to day 10 of gestation. The PGE(2) released from organogenic embryos was measured by radio immunoassay following incubation with or without the addition of either a NO donor or a NO synthase (NOS) inhibitor. In the ethanol-treated females, we found increased percentages of retarded embryos, associated with a significantly elevated resorption rate (p<0.05), very high quantities of morphologically abnormal E.10 embryos (p<0.001) and significantly increased PGE(2) release, as compared to the embryo parameters of control females. While in the control-derived E.10 embryos the NO donor produced significantly increased PGE(2) release, in the ethanol-derived embryos decreased quantities of PGE(2) were observed. L-NMMA inhibited PGE(2) release in both control and ethanol-derived embryos at different concentrations, whereas it decreased PGE(2) content in controls but not in ethanol-derived embryos. The periconceptional alcohol ingestion produced excessive PGE(2) release, decreased PGE(2) content and disruption of the regulatory NO-PGE(2) pathways. These PGs alterations may be related to delayed organogenesis and abnormal neural tube development after chronic periconceptional consumption of alcohol.     

Inhibition of cyclooxygenase-2 enhances myocardial damage in a mouse model of viral myocarditis

To determine critical role of cyclooxygenase-2 (COX-2) for development of viral myocarditis, a mouse model of encephalomyocarditis virus-induced myocarditis was used. The virus was intraperitoneally given to COX-2 gene-deficient heterozygote mice (COX-2+/-) and wild-type mice (WT). We examined differences in heart weights, cardiac histological scores, numbers of infiltrating or apoptotic cells in myocardium, cardiac expression levels of COX-2, tumor necrosis factor-alpha (TNF-alpha), and adiponectin mRNA, immunoreactivity of COX-2, TNF-alpha, and adiponectin in myocytes, cardiac concentrations of TNF-alpha and adiponectin, prostaglandin E2 (PGE2) levels in hearts, and viral titers in tissues between COX-2+/- and WT. We observed significantly decreased expression of COX-2 mRNA and reactivity in hearts from COX-2+/- on day 8 after viral inoculation as compared with that from WT, together with elevated cardiac weights and severe inflammatory myocardial damage in COX-2+/-. Cardiac expression of TNF-alpha mRNA, reactivity, and protein on day 8 was significantly higher in COX-2+/- than in WT, together with reciprocal expression of adiponectin mRNA, reactivity, and protein in hearts. Significantly reduced cardiac PGE2 levels on day 8 were found in COX-2+/- compared with those in WT. There was no difference in local viral titers between both groups on day 4. Infected WT treated with a selective COX-2 inhibitor, NS-398, also showed the augmented myocardial damage on day 8. These results suggest that inhibition of COX-2 may enhance myocardial damage through reciprocal cardiac expression of TNF-alpha and adiponectin in a mouse model of viral myocarditis.     

Protective role of adiponectin against ethanol-induced gastric injury in mice

Adiponectin is an anti-inflammatory molecule released from adipocytes, and serum adiponectin concentrations are reduced in obesity. We previously reported that gastric erosion occurs in association with obesity and low serum adiponectin levels. In the present study, we examined adiponectin-knockout (APN-KO) mice to elucidate the role of adiponectin in gastric mucosal injury. Gastric injury was induced by oral administration of ethanol in wild-type (WT) and APN-KO mice. Ethanol treatment induced severe gastric injury in APN-KO mice compared with WT mice. In APN-KO mice, increased apoptotic cells and decreased expression of prostaglandin E(2) (PGE(2)) were detected in the injured stomach. We next assessed the effect of adiponectin on the cellular response to ethanol treatment and wound repair in rat gastric mucosal cells (RGM1). Adiponectin induced the expression of PGE(2) and cyclooxygenase 2 (COX-2) in ethanol-treated RGM1 cells. RGM1 cells exhibited efficient wound repair accompanied by increased PGE(2) expression in the presence of adiponectin. Coadministration of adiponectin with celecoxib, a COX-2 inhibitor, inhibited efficient wound repair. These findings indicate that adiponectin has a protective role against ethanol-induced gastric mucosal injury in mice. This effect may be partially mediated by the efficient wound repair of epithelial cells via increased PGE(2) expression.     

Sexual dimorphism and growth hormone regulation of a hybrid gene in transgenic mice.

The sexually dimorphic expression of the urinary protein genes of mice (Mup genes) in the liver is mediated by the different male and female temporal patterns of circulating GH. Normal females were induced to male levels when GH was administered by injection to mimic the male GH pattern, showing that expression at the male level does not require a male sex steroid status in addition to intermittent GH. Two Mup-alpha 2u-globulin hybrid transgenes with different Mup gene promoters showed sexually dimorphic expression, and their expression in females increased to male levels upon testosterone treatment. GH-deficient (lit/lit) mice did not express these transgenes, and GH-deficient females did not respond to testosterone treatment, showing that GH was required for induction. Both normal and GH-deficient females were induced to male levels when GH was administered by injection. This is the first report of a transgene responsive to GH. A transgene consisting of a Mup promoter fused to a Herpes simplex virus thymidine kinase reporter sequence also showed sexual dimorphism, although to a lesser degree. It was expressed at the same level in normal females and GH-deficient mice of both sexes and was induced when GH-deficient mice were treated with GH. We propose that this transgene has a basal constitutive expression, possibly due to the absence of any rodent DNA downstream of the promoter. Since expression of the transgene was significantly induced by GH, the GH response is due at least in part to sequences in the promoter region.     

Potently immunosuppressive 5-fluorouracil-resistant mesenchymal stromal cells completely remit an experimental autoimmune disease

We treated mice with 5-fluorouracil (5-FU) to isolate a quiescent and undifferentiated mesenchymal stromal cell (MSC) population from the bone marrow. We examined these 5-FU-resistant MSCs (5-FU-MSCs) free from hematopoietic components for CFU fibroblasts (CFU-Fs) and assessed their immunosuppressive potential in vitro and in vivo. We differentiated fibroblastic CFU-Fs (Fibro-CFU-Fs) from mixed CFU-Fs, based on the absence of in situ expression of CD11b and CD45 hematopoietic markers, as well as on their differentiation capacity. Fibro-CFU-Fs were associated with increased numbers of large-sized Fibro-CFU-Fs (≥9 mm(2)) that displayed enhanced capacity for differentiation into adipogenic and osteogenic mesenchymal lineages. Administration of these 5-FU-resistant CD11b(-)CD45(-) MSCs 6 d after myelin oligodendrocyte glycoprotein (MOG) immunization completely remitted MOG-induced experimental autoimmune encephalomyelitis after initial development of mild disease. The remission was accompanied by reduced CNS cellular infiltration and demyelination, as well as a significant reduction in anti-MOG Ab and splenocyte proliferation to MOG. MOG-stimulated splenocytes from these mice showed elevated levels of Th2 cytokines (IL-4, IL-5, and IL-6) and decreased IL-17. Compared with untreated MSCs, 5-FU-MSCs demonstrated potent immunosuppression of Con A-stimulated splenocytes in vitro, even at a 1:320 MSC/splenocyte ratio. Immunosuppression was accompanied by elevated IL-1ra, IL-10, and PGE(2). Blocking IL-1ra, IL-10, and PGE(2), but not IL-6, heme oxygenase-1, and NO, attenuated 5-FU-MSC-induced immunosuppression. Together, our findings suggested that immunosuppression by 5-FU-MSC is mediated by a combination of elevated IL-1ra, IL-10, and PGE(2), anti-inflammatory Th2 cytokines, and decreased IL-17. Our findings suggested that 5-FU treatment identifies a population of potently immunosuppressive 5-FU-MSCs that have the potential to be exploited to remit autoimmune diseases.     

Prostaglandin E2 protects lower airways against bronchoconstriction

Prostaglandin E2 (PGE2), similar to beta-adrenergic receptor agonists, can protect airways from bronchoconstriction and resulting increase in airway resistance induced by a number of agents, including cholinergic receptor agonists and antigen. We examined the impact of sustained alterations in PGE2 pathways on changes in airway resistance. Genetic methods were utilized to alter PGE2 metabolism and signal transduction in the murine lung. PGE2 levels were elevated by generating mice lacking 15-hydroxyprostaglandin (Hpgd-/-), the major catabolic enzyme of PGE2, and by generating a transgenic line in which mouse PGE2 synthase (Ptges) expression is driven by a human lung-specific promoter, hSP-C. Conversely, to determine the impact of loss of PGE2 on airway reactivity, we examined mice lacking this synthase (Ptges-/-) and receptors that mediate the actions of PGE2, particularly the PGE2 EP2 receptor (Ptger2). Diminished capacity to produce and respond to PGE2 did not alter the response of mice to cholinergic stimuli. In contrast, the responsiveness to cholinergic stimulation was dramatically altered in animals with elevated PGE2 levels. The Hpgd-/- and hSP-C-Ptges transgenic lines both showed attenuated airway responsiveness to methacholine as measured by lung resistance. Thus, whereas compromise of the Ptges/PGE2/Ptger2 pathway does not alter airway responsiveness, genetic modulation that elevates PGE2 levels in the lung attenuates airway responsiveness.     

Sex differences in the response to influenza virus infection: modulation by stress

Influenza virus infection is a significant public health problem; however factors affecting the incidence and severity of disease have not been fully elucidated. The present study sought to examine the role of sex and stress in mediating susceptibility to an influenza viral infection in mice. Male and female mice underwent repeated cycles of restraint (RST) stress, followed by an influenza A/PR8 virus infection. Following these manipulations, levels of circulating corticosterone, lung proinflammatory cytokine gene expression and sickness behavior were examined. The data indicate sex differences in several aspects of the response to the A/PR8 virus infection. The kinetics of lung interleukin-1β mRNA expression were faster in infected males compared to females, while circulating corticosterone levels were elevated in infected females, but not in males. Anorexia and reduced saccharin consumption began earlier and symptoms were more pronounced in infected males than in females. In addition, RST modulated the response to the A/PR8 virus infection. Proinflammatory cytokine gene expression in response to infection was enhanced and sickness behavior was modulated by RST in both males and females. These data suggest that males mount more vigorous immune and behavioral responses to influenza viral infection compared to females, and stress exacerbates the response in both males and females. In conclusion, complex interactions between biological and behavioral factors are involved in mediating individual differences in health and disease. Additional studies may help uncover some of the factors contributing to the individual differences in susceptibility to influenza infection.     

Burn trauma alters calcium transporter protein expression in the heart.

We have shown previously that burn trauma produces significant cardiac dysfunction, which is first evident 8 h postburn and is maximal 24 h postburn. Because calcium handling by the cardiomyocyte is essential for cardiac function, one mechanism by which burn injury may cause cardiac abnormalities is via calcium dyshomeostasis. We hypothesized that major burn injury alters cardiomyocyte calcium handling through changes in calcium transporter expression. Sprague-Dawley rats were given either burn injury or no burn injury (controls). Cardiomyocyte intracellular calcium and sodium were quantified at various times postburn by fura 2-AM or sodium-binding benzofuran isophthalate fluorescent indicators, respectively. In addition, hearts freeze-clamped at various times postburn (2, 4, 8, and 24 h) were used for Western blot analysis using antibodies against the sarcoplasmic reticulum calcium-ATPase (SERCA), the L-type calcium-channel, the ryanodine receptor, the sodium/calcium exchanger, or the sodium-potassium-ATPase. Intracellular calcium levels were elevated significantly 8-24 h postburn, and intracellular sodium was increased significantly 4 through 24 h postburn. Expression of SERCA was significantly reduced 1-8 h postburn, whereas L-type calcium-channel expression was diminished 1 and 2 h postburn (P < 0.05) but returned toward control levels 4 h postburn. Ryanodine receptor protein was significantly reduced at 1 and 2 h postburn, returning to baseline by 4 h postburn. Sodium/calcium exchanger expression was significantly elevated 2 h postburn but was significantly reduced 24 h postburn. An increase in sodium-potassium-ATPase expression occurred 2-24 h postburn. These data confirm that burn trauma alters calcium transporter expression, likely contributing to cardiomyocyte calcium loading and cardiac contractile dysfunction.     

Autism‐related behaviors in the cyclooxygenase‐2‐deficient mouse model

Prostaglandin E2 (PGE2) is an endogenous lipid molecule involved in normal brain development. Cyclooxygenase‐2 (COX2) is the main regulator of PGE2 synthesis. Emerging clinical and molecular research provides compelling evidence that abnormal COX2/PGE2 signaling is associated with autism spectrum disorder (ASD). We previously found that COX2 knockout mice had dysregulated expression of many ASD genes belonging to important biological pathways for neurodevelopment. The present study is the first to show the connection between irregular COX2/PGE2 signaling and autism‐related behaviors in male and female COX2‐deficient knockin, (COX)‐2^?, mice at young (4‐6 weeks) or adult (8‐11 weeks) ages. Autism‐related behaviors were prominent in male (COX)‐2^? mice for most behavioral tests. In the open field test, (COX)‐2^? mice traveled more than controls and adult male (COX)‐2^? mice spent less time in the center indicating elevated hyperactive and anxiety‐linked behaviors. (COX)‐2^? mice also buried more marbles, with males burying more than females, suggesting increased anxiety and repetitive behaviors. Young male (COX)‐2^? mice fell more frequently in the inverted screen test revealing motor deficits. The three‐chamber sociability test found that adult female (COX)‐2^? mice spent less time in the novel mouse chamber indicative of social abnormalities. In addition, male (COX)‐2^? mice showed altered expression of several autism‐linked genes: Wnt2, Glo1, Grm5 and Mmp9. Overall, our findings offer new insight into the involvement of disrupted COX2/PGE2 signaling in ASD pathology with age‐related differences and greater impact on males. We propose that (COX)‐2^? mice might serve as a novel model system to study specific types of autism.     

Deletion of microsomal prostaglandin E2 (PGE2) synthase-1 reduces inducible and basal PGE2 production and alters the gastric prostanoid profile

Microsomal prostaglandin E synthase-1 (mPGES-1) is an inducible protein recently shown to be an important source of inflammatory PGE2. Here we have used mPGES-1 wild type, heterozygote, and null mice to assess the impact of reduction or absence mPGES-1 protein on the production of PGE2 and other prostaglandins in lipopolysaccharide (LPS)-treated macrophages and mice. Thioglycollate-elicited peritoneal macrophages with mPGES-1 deficiency were found to lose their ability to produce PGE2 upon LPS stimulation. Resident mPGES-1(-/-) peritoneal macrophages exhibited severely impaired PGE2-releasing activity but retained some LPS-inducible PGE2 production capacity. Both macrophage types showed a 50% decrease in PGE2 production with removal of one copy of the mPGES-1 gene. In vivo, mPGES-1 deletion abolished the LPS-stimulated production of PGE2 in spleen, kidney, and brain. Surprisingly, lack of mPGES-1 activity resulted in an 80-90% decrease in basal, cyclooxygenase-1 (COX-1)-dependent PGE2 production in stomach and spleen, and a 50% reduction in brain and kidney. Other prostaglandins (thromboxane B2, PGD2, PGF(2alpha), and 6-keto-PGF(1alpha)) were significantly elevated in stomachs of mPGES-1-null mice but not in other tissues. Examination of mRNA for several terminal prostaglandin synthases did not reveal changes in expression levels associated with mPGES-1 deficiency, indicating that gastric prostaglandin changes may be due to shunting of cyclooxygenase products to other terminal synthases. These data demonstrate for the first time a dual role for mPGES-1 in both inflammatory and COX-1-mediated PGE2 production and suggest an interdependence of prostanoid production with tissue-specific alterations of prostaglandin levels in the absence of mPGES-1.     

Coupling of COX-1 to mPGES1 for prostaglandin E2 biosynthesis in the murine mammary gland

The mammary gland, like most tissues, produces measurable amounts of prostaglandin E2 (PGE2), a metabolite of arachidonic acid produced by sequential actions of two cyclooxygenases (COX-1 and COX-2) and three terminal PGE synthases: microsomal prostaglandin E2 synthase-1 (mPGES1), mPGES2, and cytosolic prostaglandin E2 synthase (cPGES). High PGE2 levels and COX-2 overexpression are frequently detected in mammary tumors and cell lines. However, less is known about PGE2 metabolic enzymes in the context of normal mammary development. Additionally, the primary COX partnerships of terminal PGE synthases and their contribution to normal mammary PGE2 biosynthesis are poorly understood. We demonstrate that expression of COX-1, generally considered constitutive, increases dramatically with lactogenic differentiation of the murine mammary gland. Concordantly, total PGE2 levels increase throughout mammary development, with highest levels measured in lactating tissue and breast milk. In contrast, COX-2 expression is extremely low, with only a modest increase detected during mammary involution. Expression of the G(s)-coupled PGE2 receptors, EP2 and EP4, is also temporally regulated, with highest levels detected at stages of maximal proliferation. PGE2 production is dependent on COX-1, as PGE2 levels are nearly undetectable in COX-1-deficient mammary glands. Interestingly, PGE2 levels are similarly reduced in lactating glands of mPGES1-deficient mice, indicating that PGE2 biosynthesis results from the coordinated activity of COX-1 and mPGES1. We thus provide evidence for the first time of functional coupling between COX-1 and mPGES1 in the murine mammary gland in vivo.     

Regulation of the prostaglandin pathway during development of invasive bladder cancer in mice

Prostaglandin E(2) (PGE(2)) is reported to play an important role in tumor development. We explored the differential expression of genes governing production of, and response to, PGE(2) during development of invasive bladder cancer. N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN) or vehicle-treated mice (n=4-5) were euthanized after 4-8 weeks (period 1, P1), 12-16 weeks (P2), and 20-23 weeks (P3). Half of each bladder was analyzed histologically and the other half extracted for mRNA analysis by quantitative real-time PCR. Bladders from BBN-treated mice showed progression from submucosal inflammation (P1) to squamous metaplasia/focal CIS (P2) to poorly differentiated, invasive cancer (P3). mRNA levels for the inducible cyclooxygenase, COX-2, were elevated three to fourfold at all time points in BBN-treated mice compared to controls. In contrast, mRNA levels for constitutive COX-1 and cytosolic phospholipase A(2) (cPLA(2)), which releases substrate for COX, were either unchanged or decreased in BBN-treated mice relative to controls. Downstream of COX, mRNA levels of membrane-bound PGE(2) synthase (mPGES-1) were increased 1.7-fold at P1 in BBN bladders but returned to control levels at P2 and P3. mRNA levels for 15-prostaglandin dehydrogenase (PGDH), which inactivates PGE(2), were reduced 50-80% in BBN-treated bladders at all time points. mRNA levels for EP2R and EP4R, receptors for PGE(2), were two to threefold increased at P1, but returned to control levels or below at P3. Hence, increased COX-2 and decreased PDGH expression occurred throughout tumor development, while mPGES-1, EP2R and EP4R were elevated only before development of invasive cancer. We compared expression of these genes in the malignant human urothelial cell lines, HTB-5 and HT-1376, with expression in a benign urothelial cell line, UROtsa. Neither malignant cell line reproduced the complete in vivo pattern, relative to benign cells, but each showed abnormal basal expression of several of the genes downstream of COX-2, but not COX-2 itself. We conclude that components involved in PGE(2) synthesis and activity are differentially regulated during bladder tumor development and the therapeutic efficacy of targeting the various components may vary with stage of tumor development.     

The role of progesterone in pregnancy-induced aggression in mice

A series of six experiments was performed in order to explore the potential involvement of progesterone (P) in pregnancy-induced aggression (PIA) displayed by Rockland-Swiss mice toward adult male intruders. In Experiment 1, circulating levels of P and aggression were low on gestation Days 6 and 10 while both the behavior and the steroid reached peak levels by gestation Day 14. By gestation Day 18 (the day prior to parturition), serum P was at its lowest level yet aggressive behavior was still intense. Also, individual differences in the display of fighting behavior by pregnant females were not related to circulating P. Experiments 2 and 3 showed that supplemental P treatment to early pregnant female mice did not advance the onset of aggression. Experiment 4 showed that P treatment promoted the onset and elevated the incidence of aggression in virgin mice, but only in those females with intact ovaries. Experiment 5 showed that the aggressive behavior of P-stimulated virgin females was qualitatively and quantitatively different from that exhibited by pregnant mice in that the former exhibited fewer attacks and lunges than the latter. Finally, Experiment 6 showed that the removal of P from aggressive, P-stimulated virgins dramatically attenuated levels of the behavior. This contrasts sharply with the continued fighting behavior observed in late pregnant P-deficient mice. Thus, although P augments aggression in female mice it apparently is not a sufficient stimulus for producing pregnancy-like aggressive behavior.     

Enhanced expression of cyclooxygenase-2 and prostaglandin E2 in response to endotoxin after trauma is dependent on MAPK and NF-kappaB mechanisms

Macrophage prostaglandin E2 (PGE2) production is important in cellular immune suppression and in affecting the potential development of sepsis after trauma. We hypothesized that macrophage PGE2 production after trauma is regulated by mitogen-activated protein kinase (MAPK) and nuclear factor kappa B (NF-kappaB). Mice were subjected to trauma and splenic macrophages isolated 7 days later. Macrophages from traumatized mice showed increased cyclooxygenase-2 (COX-2) mRNA, protein expression, and PGE2 production compared with controls. Increased phosphorylation of extracellular signal-regulated kinase (ERK), c-jun N-terminal kinase (JNK), and p38 kinase was observed in macrophages from traumatized mice. Pharmacologic inhibition of MAPK blocked trauma-induced COX-2 expression, and PGE2 production. Trauma macrophages showed increased IkappaBalpha phosphorylation and NF-kappaB binding to DNA. Inhibiting IkappaBalpha blocked trauma-induced NF-kappaB activity, COX-2 expression and PGE2 production. This suggests that trauma-induced PGE2 production is mediated through MAPK and NF-kappaB activation and offers potential for modifying the macrophages' responses following injury.