Mechanism of the nongenomic effects of estrogen on intestinal myeloperoxidase activity following trauma-hemorrhage: up-regulation of the PI-3K/Akt pathway

As studies indicate that genomic and nongenomic pathways are involved in mediating the salutary effects of 17beta-estradiol (E2) following trauma-hemorrhage, we examined if the nongenomic effects of E2 on attenuation of intestinal injury after trauma-hemorrhage involve the PI-3K/Akt pathway. Male Sprague-Dawley rats ( approximately 300 g body weight) underwent trauma-hemorrhage (mean blood pressure 40 mmHg for 90 min), followed by resuscitation. E2 conjugated to BSA (E2-BSA; 1 mg/Kg E2), with or without an estrogen receptor antagonist (ICI 182,780), a PI-3K inhibitor (Wortmannin), or vehicle, was injected i.v. during resuscitation. At 2 h after trauma-hemorrhage or sham operation, intestinal myeloperoxidase (MPO) activity, ICAM-1, cytokine-induced neutrophil chemoattractant (CINC)-1, CINC-3, and IL-6 levels were measured (n=6 rats/group). Intestinal PI-3K, phosphorylation of Akt (p-Akt), and Akt protein expressions were also determined. One-way ANOVA and Tukey's test were used for statistical analysis. The results indicated that trauma-hemorrhage increased intestinal MPO activity and ICAM-1, CINC-1, CINC-3, and IL-6 levels. These parameters were improved significantly in the E2- or E2-BSA-treated rats subjected to trauma-hemorrhage. Although trauma-hemorrhage decreased intestinal PI-3K and p-Akt protein expressions, E2 or E2-BSA treatment following trauma-hemorrhage prevented such decreases in intestinal PI-3K and p-Akt protein expressions. Coadministration of ICI 182,780 or Wortmannin abolished the beneficial effects of E2-BSA on attenuation of intestinal injury following trauma-hemorrhage. Thus, the PI-3K/Akt pathway plays a critical role in mediating the nongenomic, salutary effects of E2 on attenuation of shock-induced intestinal tissue damage.     

Mechanism of estrogen-mediated attenuation of hepatic injury following trauma-hemorrhage: Akt-dependent HO-1 up-regulation

Protein kinase B (Akt) is known to be involved in proinflammatory and chemotactic events in response to injury. Akt activation also leads to the induction of heme oxygenase (HO)-1. Up-regulation of HO-1 mediates potent, anti-inflammatory effects and attenuates organ injury. Although studies have shown that 17beta-estradiol (E2) prevents organ damage following trauma-hemorrhage, it remains unknown whether Akt/HO-1 plays any role in E2-mediated attenuation of hepatic injury following trauma-hemorrhage. To study this, male rats underwent trauma-hemorrhage (mean blood pressure, approximately 40 mmHg for 90 min), followed by fluid resuscitation. At the onset of resuscitation, rats were treated with vehicle, E2 (1 mg/kg body weight), E2 plus the PI-3K inhibitor (Wortmannin), or the estrogen receptor (ER) antagonist (ICI 182,780). At 2 h after sham operation or trauma-hemorrhage, plasma alpha-GST and hepatic tissue myeloperoxidase (MPO) activity, IL-6, TNF-alpha, ICAM-1, cytokine-induced neutrophil chemoattractant-1, and MIP-2 levels were measured. Hepatic Akt and HO-1 protein levels were also determined. Trauma-hemorrhage increased hepatic injury markers (alpha-GST and MPO activity), cytokines, ICAM-1, and chemokine levels. These parameters were markedly improved in the E2-treated rats following trauma-hemorrhage. E2 treatment also increased hepatic Akt activation and HO-1 expression compared with vehicle-treated, trauma-hemorrhage rats, which were abolished by coadministration of Wortmannin or ICI 182,780. These results suggest that the salutary effects of E2 on hepatic injury following trauma-hemorrhage are in part mediated via an ER-related, Akt-dependent up-regulation of HO-1.     

Kupffer cells and their mediators: the culprits in producing distant organ damage after trauma-hemorrhage

Posttraumatic activation of macrophages enhances development of systemic inflammation/immunosuppression and organ dysfunction. We hypothesized that Kupffer cells are the main source of monocyte chemoattractant protein-1 (MCP-1) production after trauma-hemorrhage, that administration of 17beta-estradiol (E2) after trauma-hemorrhage modulates MCP-1 release and reduces remote organ damage, and that salutary effects of E2 are mediated via estrogen receptor (ER)-alpha. To test these hypotheses, female B57BL/J6 mice received E2 (50 microg/25 g) or vehicle after trauma-hemorrhage and female 129 Sve ER-beta-/- transgenic mice and ovariectomized wild-type mice received E2 or ER-alpha agonist propyl pyrazole triol (50 microg/25 g) after trauma-hemorrhage. Systemic MCP-1 and interleukin-6 and their release by liver, spleen, and lung macrophages were determined by flow cytometry 4 hours after trauma-hemorrhage. Prior Kupffer cell depletion with gadolinium chloride significantly decreased systemic MCP-1 and interleukin-6 after trauma-hemorrhage and was associated with decreased edema/neutrophil infiltration in lung and liver. Kupffer cells were the only macrophages showing significant MCP-1 release, which was markedly reduced by E2 or propyl pyrazole triol in wild-type and in ER-beta-/- mice. Pretreatment of mice with anti-MCP-1 antiserum prevented an increase in myeloperoxidase and edema in lung and liver. These findings suggest that Kupffer cell-derived MCP-1 plays a major role in remote organ dysfunction after trauma-hemorrhage.     

17beta-Estradiol's salutary effects on splenic dendritic cell functions following trauma-hemorrhage are mediated via estrogen receptor-alpha

Although 17beta-estradiol administration following trauma-hemorrhage attenuates Kupffer cell, splenic and peritoneal macrophage functions, it remains unknown whether 17beta-estradiol has any salutary effects on splenic dendritic cell (DC) functions and if so, whether such effects are mediated via the estrogen receptors (ER). We hypothesized that 17beta-estradiol administration following trauma-hemorrhage has salutary effects on splenic DC functions. Male C3H/HeN (6-8 weeks) mice were randomly assigned to sham operation or trauma-hemorrhage. Trauma-hemorrhage was induced by midline laparotomy and approximately 90 min of hemorrhagic shock (blood pressure [BP] 35 mmHg), followed by fluid resuscitation (4x the shed blood volume in the form of Ringer's lactate). Estrogen receptor (ER)-alpha agonist propyl pyrazole triol (PPT; 5microg/kg), ER-beta agonist diarylpropionitrile (DPN; 5microg/kg), 17beta-estradiol (50microg/kg), or vehicle (10% DMSO) was injected subcutaneously during resuscitation. Two hours later, the mice were sacrificed, splenic DCs were isolated and the changes in their apoptosis, co-stimulating factors and MHC class II expression, ability to produce cytokines, and antigen presentation capacity were measured. Apoptosis of splenic DC increased following trauma-hemorrhage; however, 17beta-estradiol administration after trauma-hemorrhage normalized the rate of apoptosis. Moreover, splenic DC cytokines production, co-stimulating factors and MHC class II expression, and antigen presentation capacity were significantly decreased following trauma-hemorrhage; however, 17beta-estradiol as well as PPT also prevented these depressions. In contrast, DPN did not attenuate splenic DC functions following trauma-hemorrhage. Since PPT administration following trauma-hemorrhage was more effective in normalizing splenic DC functions than DPN, the salutary effects of 17beta-estradiol on splenic DC functions are mediated predominantly via ER-alpha.     

17Beta-estradiol downregulates Kupffer cell TLR4-dependent p38 MAPK pathway and normalizes inflammatory cytokine production following trauma-hemorrhage

Although studies have shown that 17beta-estradiol (estradiol) normalized Kupffer cell function following trauma-hemorrhage, the mechanism by which E2 maintains immune function remains unclear. Activation of Toll-like receptor 4 (TLR4) initiates an inflammatory cascade, involving activation of p38 mitogen-activated protein kinase (MAPK), phosphatidylinositol 3-kinase (PI3K), and nuclear factor-kappaB (NF-kappaB). This leads to the release of proinflammatory cytokines. Thus, we hypothesized that the salutary effects of estradiol on Kupffer cell function following trauma-hemorrhage are mediated via negative regulation of TLR4-dependent p38 MAPK and NF-kappaB. TLR4 mutant (C3H/HeJ) and wild type (C3H/HeOuJ) mice were subjected to trauma-hemorrhage (mean BP 35+/-5 mmHg approximately 90 min, then resuscitation) or sham operation. Administration of estradiol following trauma-hemorrhage in wild type mice decreased Kupffer cell TLR4 expression as well as prevented the phosphorylation of p38 MAPK and NF-kappaB. This was accompanied by normalization of Kupffer cell production capacities of IL-6, TNF-alpha, macrophage inflammatory protein (MIP)-1alpha, and MIP-2 and the decrease in plasma cytokine levels. In contrast, TLR4 mutant mice did not exhibit the increase in Kupffer cell p38 MAPK and NF-kappaB activation, cytokine production, or the increase in circulating cytokine levels following trauma-hemorrhage. No difference was observed in activation of PI3K among groups. These results suggest that the protective effect of estradiol on Kupffer cell function is mediated via downregulation of TLR4-dependent p38 MAPK and NF-kappaB signaling following trauma-hemorrhage, which prevents the systemic release of cytokines.     

Mechanism of the salutary effects of flutamide on intestinal myeloperoxidase activity following trauma-hemorrhage: up-regulation of estrogen receptor-{beta}-dependent HO-1

Hemeoxygenase (HO)-1 induction following adverse circulatory conditions is known to be protective, and precastrated males have less intestinal damage than sham-operated males following trauma-hemorrhage (T-H). Previous studies have also shown that administration of flutamide up-regulated estrogen receptor (ER) expression in males following T-H. We hypothesized that flutamide administration in males following T-H up-regulates HO-1 via an ER-dependent pathway and protects against intestinal injury. Male Sprague-Dawley rats underwent T-H [mean blood pressure (MBP) 40 mmHg for 90 min and then resuscitation]. A single dose of flutamide (25 mg/kg body weight), with or without an ER antagonist (ICI 182,780), a HO enzyme inhibitor [chromium-mesoporphyrin (CrMP)], or vehicle, was administered subcutaneously during resuscitation. At 2 h after T-H or sham operation, intestinal myeloperoxidase (MPO) activity, intercellular adhesion molecule (ICAM)-1, cytokine-induced neutrophil chemoattractant (CINC)-1, and CINC-3 levels were measured. Intestinal ER-alpha, ER-beta, androgen receptor, and HO-1 mRNA/protein levels were also determined. Results showed that T-H increased intestinal MPO activity, ICAM-1, CINC-1, and CINC-3 levels. These parameters were improved significantly in the flutamide-treated rats subjected to T-H. Flutamide treatment increased intestinal HO-1 and ER-beta mRNA/protein levels as compared with vehicle-treated T-H rats. Administration of the ER antagonist ICI 182,780 or the HO inhibitor CrMP prevented the flutamide-induced attenuation of shock-induced intestinal damage. Thus, the salutary effects of flutamide administration on attenuation of intestinal injury following T-H are mediated via up-regulation of ER-beta-dependent HO-1 expression.     

Tissue-specific expression of estrogen receptors and their role in the regulation of neutrophil infiltration in various organs following trauma-hemorrhage

Although 17beta-estradiol (E2) administration after trauma-hemorrhage (T-H) reduces tissue neutrophil sequestration in male rodents, it remains unknown which of the estrogen receptor (ER) subtypes mediates this effect and whether the same ER subtype is involved in all the tissues. We hypothesized that the salutary effects of E2 on attenuation of neutrophil accumulation following T-H are tissue and receptor subtype-specific. Male Sprague-Dawley rats underwent sham operation or T-H (mean blood pressure, 40 mmHg for 90 min and then resuscitation). E2 (50 microg/kg), ER-alpha agonist propyl pyrazole triol (PPT; 5 microg/kg), ER-beta agonist diarylpropiolnitrile (DPN; 5 microg/kg), or vehicle (10% dimethyl sulfoxide) was administered subcutaneously during resuscitation. Twenty-four hours thereafter, tissue myeloperoxidase (MPO) activity (a marker of neutrophil sequestration), cytokine-induced neutrophil chemoattractant (CINC)-1, CINC-3, and intercellular adhesion molecule (ICAM)-1 levels in the liver, intestine, and lung were measured (n = 6 rats/group). ER-alpha and ER-beta mRNA levels in sham-operated rats were also determined. T-H increased MPO activity, CINC-1, CINC-3, and ICAM-1 levels in the liver, intestine, and lung. These parameters were improved significantly in rats receiving E2 after T-H. Administration of the ER-alpha agonist PPT but not the ER-beta agonist DPN improved the measured parameters in the liver. In contrast, DPN but not PPT significantly improved these parameters in the lung. In the intestine, ER subtype specificity was not observed. ER-alpha mRNA expression was highest in the liver, whereas ER-beta mRNA expression was greatest in the lung. Thus, the salutary effects of E2 administration on tissue neutrophil sequestration following T-H are receptor subtype and tissue-specific.     

A role for multiple estrogen receptors in immune regulation of common carp

Estrogens are important for bi-directional neuroendocrine-immune interaction. They act via nuclear estrogen receptors (ERα and ERβ) and/or G-protein coupled receptor - GPR30.We found expression of ERα, ERβ and GPR30 in carp lymphoid tissues and head kidney monocytes/macrophages, neutrophils and lymphocytes. Interestingly, ERβ is also expressed in some head kidney lymphocytes but not in naive PBLs. Immune stimulation altered the cell type specific profile of expression of these receptors, which depends on both activation and maturation stage.This implies direct leukocyte responsiveness to estrogen stimulation and therefore in vitro effects of 17β-estradiol (E2) on reactive oxygen species (ROS) production in monocytes/macrophages were determined. Short-time incubation with E2 increased ROS production in PMA-stimulated cells. Results comply with mediation by GPR30, partially functioning via phosphoinositide 3-kinase activation.These results furthermore demonstrate that neuroendocrine-immune communication via estrogen receptors is evolutionary conserved.     

Are the protective effects of 17beta-estradiol on splenic macrophages and splenocytes after trauma-hemorrhage mediated via estrogen-receptor (ER)-alpha or ER-beta?

The depression in cell-mediated immune function following trauma-hemorrhage is shown to be restored by 17beta-estradiol (E2) administration. However, it remains unknown which of the two estrogen-receptors, (ER)-alpha or ER-beta, plays the predominant role in mediating the beneficial effects of E2. Female B57BL/J6 ER-beta(-/-) transgenic mice [knockout (KO)] and corresponding ovariectomized wild-type (WT) mice were subjected to laparotomy and hemorrhagic shock (35.0+/-5.0 mmHg for 90 min) and treated with E2 (50 microg/25 g) or ER-alpha agonist propyl pyrazole triol (PPT; 50 microg/25 g) following trauma-hemorrhage. Four hours after resuscitation, systemic cytokine concentrations and cytokine release by splenocytes and splenic macrophages were determined by cytometric bead array. Trauma-hemorrhage resulted in a significant increase in plasma tumor necrosis factor alpha (TNF-alpha), interleukin (IL)-6, and IL-10. In contrast, the release of these cytokines by splenic macrophages was decreased significantly in WT and KO animals. Administration of E2 or PPT following trauma-hemorrhage produced a significant reduction in systemic TNF-alpha and IL-6 concentrations in WT and KO mice. Although the suppression in the productive capacity of these cytokines following trauma-hemorrhage by macrophages and splenocyte was also prevented in E2- and PPT-treated WT mice, the release of cytokines by macrophages and splenocytes in E2- and PPT-treated KO mice was not restored to the levels observed in sham animals. These findings collectively suggest that both receptors appear to play a significant role in mediating the immunoprotective effects of E2 in different tissue compartments following trauma-hemorrhage.     

Estrogen receptors in human bladder cells regulate innate cytokine responses to differentially modulate uropathogenic E. coli colonization

The bladder epithelial cells elicit robust innate immune responses against urinary tract infections (UTIs) for preventing the bacterial colonization. Physiological fluctuations in circulating estrogen levels in women increase the susceptibility to UTI pathogenesis, often resulting in adverse health outcomes. Dr adhesin bearing Escherichia coli (Dr E. coli) cause recurrent UTIs in menopausal women and acute pyelonephritis in pregnant women. Dr E. coli bind to epithelial cells via host innate immune receptor CD55, under hormonal influence. The role of estrogens or estrogen receptors (ERs) in regulating the innate immune responses in the bladder are poorly understood. In the current study, we investigated the role of ERα, ERβ and GPR30 in modulating the innate immune responses against Dr E. coli induced UTI using human bladder epithelial carcinoma 5637 cells (HBEC). Both ERα and ERβ agonist treatment in bladder cells induced a protection against Dr E. coli invasion via upregulation of TNFα and downregulation of CD55 and IL10, and these effects were reversed by action of ERα and ERβ antagoinsts. In contrast, the agonist-mediated activation of GPR30 led to an increased bacterial colonization due to suppression of innate immune factors in the bladder cells, and these effects were reversed by the antagonist-mediated suppression of GPR30. Further, siRNA-mediated ERα knockdown in the bladder cells reversed the protection against bacterial invasion observed in the ERα positive bladder cells, by modulating the gene expression of TNFα, CD55 and IL10, thus confirming the protective role of ERα. We demonstrate for the first time a protective role of nuclear ERs, ERα and ERβ but not of membrane ER, GPR30 against Dr E. coli invasion in HBEC 5637 cells. These findings have many clinical implications and suggest that ERs may serve as potential drug targets towards developing novel therapeutics for regulating local innate immunity and treating UTIs.     

Estrogen receptor alpha and G-protein coupled receptor 30 mediate the neuroprotective effects of 17β-estradiol in novel murine hippocampal cell models

The hippocampus is a multifaceted, complex brain structure considered to be the learning center. The use of primary hippocampal cell cultures has uncovered important cellular mechanisms involved in overall physiological function. Yet, the use of primary culture is inherently difficult, and the lack of immortalized cell lines from the murine hippocampus for mechanistic studies at the molecular level is evident. We have immortalized cell lines from embryonic (E18) and adult-derived hippocampal primary cell culture using retroviral infection of SV40 T-antigen. Four clonal embryonic lines, mHippoE-2, mHippoE-5, mHippoE-14, mHippoE-18, and one mixed adult line, mHippoA-mix, exhibited neuronal morphologies with neurite extensions and expression of neuronal markers, with unique gene expression profiles. We used these cell models to study the neuroprotective effects of 17β-estradiol (E2) on glutamate-induced neurotoxicity. The cell lines express a relevant array of genes and receptors suggested to play a role in neuroprotection, including estrogen receptors ERα, ERβ, and GPR30. We find that pretreatment with E2 (10 or 100 nM) for 24 h significantly reduced cell death induced by glutamate mHippoE-14 and mHippoE-18 cells, but not the mHippoA-mix. Using 24 h pretreatment with the specific estrogen receptor (ER) agonists, 4,4′,4′′-(4-propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol (PPT) and diarylpropionitrile, 2,3-bis(4-Hydroxyphenyl)-propionitrile (DPN), we linked the E2-mediated neuroprotection to ERα, but only in the mHippoE-18 cells. Since E2 activated both PI3K/Akt and STAT3 signaling pathways, we also tested whether the membrane-bound E2 receptor GPR30 was involved in its neuroprotective action. Pretreatment with the GPR30 agonist G-1 (10 and 100 nM) for 1 h, but not 24 h, significantly attenuated cell death in both mHippoE-14 and mHippoE-18 cells. The use of specific ER antagonist ICI 182780 and GPR30 antagonist G-15 linked these effects to both ER and GPR30 receptors. This is the first evidence that GPR30 may play a role in the protective effects of estrogen in hippocampal neurons.     

Increase in number of functional release sites by cyclic AMP-dependent protein kinase in cultured neurons isolated from hippocampal dentate gyrus

The enhancement of synaptic exocytosis is one form of long-term potentiation (LTP) of synaptic transmission. As possible mechanisms underlying this enhancement, increases in the release probability and/or the number of release sites are suggested. To obtain direct evidence for the increase in the number of functional release sites induced by protein kinase A (PKA) cascade, we attempted to visualize functional release sites using styryl dyes, FM4-64 and FM1-43, and investigated the effects of PKA on the release sites. A PKA activator FSK increased the number of active release sites by approximately 20-30%. A direct PKA activator, Sp-cAMPS, showed the same effect, which was blocked by a PKA inhibitor, KT5720, suggesting that this effect was mediated by PKA. This PKA-dependent increase in the number of release sites requires Ca(2+) in the bath solution, and Sr(2+) can not be a substitute for Ca(2+). Since the number of functional release sites is approximately half the total number of synaptophysin-immunoreactive sites, the PKA dependent activation of silent release sites of DG neuron terminals is suggested.     

Proliferative activity, apoptosis and expression of oestrogen receptor and Bcl-2 oncoprotein in canine mammary gland tumours

Samples of 39 canine mammary gland tumours (MGTs) were examined immunohistochemically for oestrogen receptor (ER-alpha), Bcl-2 protein and Ki67 antigen, and by TUNEL assay for apoptosis. ER-alpha was expressed by 80% (31/39) of the tumours, including all of the 15 benign tumours and 67% (16/42) of the malignant tumours. ER-alpha expression was greater in the benign than in the malignant tumours (P<0.01). Bcl-2 protein was detected in 62% (24/39) of the MGTs, of which 67% (10/15) were benign and 58% (14/24) malignant. No significant difference in Bcl-2 expression between benign and malignant tumours was detected. The Ki67 and TUNEL indices were greater in malignant than in benign tumours (P<0.01). Correlation analysis suggested that ER-alpha and Bcl-2 expression were related, but this observation lacked statistical significance. The levels of cell proliferation and apoptosis did not appear to be significantly correlated with the expression of Bcl-2. A positive relationship was apparent between cell proliferation and apoptosis, whilst a negative correlation between ER-alpha and cell proliferation was demonstrated. In conclusion, the suggestion of a positive correlation between ER-alpha and Bcl-2 in canine MGTs indicates that ER may be the regulator of Bcl-2 protein, as in human breast cancer. In contrast to cell proliferation and apoptosis, ER-alpha and Bcl-2 expression were greater in benign MGTs than in their malignant counterparts.     

Effects of 17beta-estradiol and flutamide on inflammatory response and distant organ damage following trauma-hemorrhage in metestrus females

We hypothesized that administration of androgen receptors antagonist flutamide following trauma-hemorrhage (T-H) in metestrus females will maintain immune function and reduce remote organ damage under those conditions. Female B57BL/J6 mice (metestrus state, 8-12 weeks old) underwent laparotomy and hemorrhagic shock (35.0+/-5.0 mmHg for 90 min) and then received 17beta-estradiol (E2; 50 microg/25 g), flutamide (625 microg/25 g), or E2 + flutamide. Four hours after resuscitation, plasma cytokine and chemokine (TNF-alpha, IL-6, IL-10, IFN-gamma, and MCP-1) concentrations and their release in vitro by hepatic and pulmonary tissue macrophages (M Phi) were determined by flow cytometry. Organ damage was assessed by edema formation (wet-to-dry weight ratio) and neutrophil infiltration [myeloperoxidase (MPO) activity]. Administration of E2, flutamide, or E2 + flutamide following T-H resulted in a significant decrease in systemic TNF-alpha, IL-6, and MCP-1 concentrations under those conditions. This was accompanied by significantly decreased in vitro TNF-alpha release by Kupffer cells after administration of E2, flutamide, or E2 + flutamide. The in vitro release of proinflammatory cytokines by alveolar M Phi, however, was reduced significantly only by the addition of E2 or E2 + flutamide but not by the addition of flutamide. A significant decrease in pulmonary and hepatic edema formation as well as neutrophil infiltration in the lung was observed after E2, flutamide and E2 + flutamide administration. In contrast, hepatic neutrophil infiltration was only significantly reduced following E2 and E2 + flutamide administration. Thus, although flutamide does not produce synergistic, salutary effects with E2, its administration in females following T-H also produces salutary effects on the immune and organ function, similar to E2 administration under those conditions.     

A G protein-associated ERK pathway is involved in LPS-induced proliferation and a PTK-associated p38 MAPK pathway is involved in LPS-induced differentiation in resting B cells

We, previously, showed that PKC-dependent ERK/p38 MAPK activation was inhibited by treating the resting B cell line 38B9 with an anti-MHC class II antibody. Further studies in this work demonstrated that PKA was involved in lipopolysaccharide (LPS)-induced proliferation of the cells, such that the PKC inhibitor activated PKA with concomitant LPS-induced proliferation but not IgG secretion. Consequently, the PKA inhibitor down-regulated ERK and p38 MAPK, and decreased cell proliferation. In addition, the treatment of LPS-stimulated 38B9 cells with PTK inhibitor reduced PKC- and PKA-dependent p38 MAPK activation and reduced the level of IgG secretion rather than the level of proliferation. However, the treatment of LPS-stimulated 38B9 cells with pertussis toxin (PTX), an inhibitor for the G protein-coupled receptor, inhibited the activation of both PKC- and PKA-dependent ERK and significantly reduced LPS-induced proliferation but not IgG secretion. Furthermore, ERK and p38 MAPK inhibitors reduced LPS-induced proliferation and differentiation, respectively, in 38B9 cells in a dose-dependent manner. These results suggest that LPS-induced proliferation of resting B cells is mainly mediated through a G protein-associated PKA/PKC-dependent ERK pathway and that a PTK-associated PKC/PKA-dependent p38 MAPK pathway is mostly involved in LPS-induced differentiation of the resting B cells.     

Beta2‐adrenergic receptor signaling in CD4+ Foxp3+ regulatory T cells enhances their suppressive function in a PKA‐dependent manner

Beta2‐adrenergic receptor (B2AR) signaling is known to impair Th1‐cell differentiation and function in a cAMP‐dependent way, leading to inhibition of cell proliferation and decreased production of IL‐2 and IFN‐γ. CD4⁺ Foxp3⁺ Treg cells play a key role in the regulation of immune responses and are essential for maintenance of self‐tolerance. Nevertheless, very little is known about adrenergic receptor expression in Treg cells or the influence of noradrenaline on their function. Here we show that Foxp3⁺ Treg cells express functional B2AR. B2AR activation in Treg cells leads to increased intracellular cAMP levels and to protein kinase A (PKA)‐dependent CREB phosphorylation. We also found that signaling via B2AR enhances the in vitro suppressive activity of Treg cells. B2AR‐mediated increase in Treg‐cell suppressive function was associated with decreased IL‐2 mRNA levels in responder CD4⁺ T cells and improved Treg‐cell‐induced conversion of CD4⁺ Foxp3^? cells into Foxp3⁺ induced Treg cells. Moreover, B2AR signaling increased CTLA‐4 expression in Treg cells in a PKA‐dependent way. Finally, we found that PKA inhibition totally prevented the B2AR‐mediated increase in Treg‐cell suppressive function. Our data suggest that sympathetic fibers are able to regulate Treg‐cell suppressive activity in a positive manner through B2AR signaling.     

The combination of ischemic preconditioning and liver Bcl-2 overexpression is a suitable strategy to prevent liver and lung damage after hepatic ischemia-reperfusion

The present study evaluates the effectiveness of ischemic preconditioning and Bcl-2 overexpression against the liver and lung damage that follow hepatic ischemia-reperfusion and investigates the underlying protective mechanisms. Preconditioning and Bcl-2, respectively, reduced the increased tumor necrosis factor (TNF) and macrophage inflammatory protein-2 (MIP)-2 levels observed after hepatic reperfusion. Bcl-2 overexpression or anti-MIP-2 pretreatment seems to be more effective than preconditioning or anti-TNF pretreatment against inflammatory response, microcirculatory disorders, and subsequent hepatic ischemia-reperfusion injury. Furthermore, each one of these strategies individually was unable to completely inhibit hepatic injury. The combination of preconditioning and Bcl-2 overexpression as well as the combined anti-TNF and anti-MIP-2 pretreatment totally prevented hepatic injury, whereas the benefits of preconditioning and Bcl-2 were abolished by TNF and MIP-2. In contrast to preconditioning, Bcl-2 did not modify lung damage induced by hepatic reperfusion. This could be explained by the differential effect of both treatments on TNF release. Anti-TNF therapy or preconditioning, by reducing TNF release, reduced pulmonary inflammatory response, whereas the benefits of preconditioning on lung damage were abolished by TNF. Thus, the induction of both Bcl-2 overexpression in liver and preconditioning, as well as pharmacological strategies that simulated their benefits, such as anti-TNF and anti-MIP-2 therapies, could be new strategies aimed to reduce lung damage and inhibit the hepatic injury associated with hepatic ischemia-reperfusion.     

17Beta-estradiol induced Ca2+ influx via L-type calcium channels activates the Src/ERK/cyclic-AMP response element binding protein signal pathway and BCL-2 expression in rat hippocampal neurons: a potential initiation mechanism for estrogen-induced neuroprotection

Our group and others have demonstrated that 17beta-estradiol (E2) induces neurotrophic and neuroprotective responses in hippocampal and cortical neurons which are dependent upon the Src/extracellular signal-regulated kinase (ERK) signaling pathways. The purpose of this study was to determine the upstream mechanism(s) that initiates the signaling cascade leading to E2-inducible neuroprotection. We tested the hypothesis that E2 activates rapid Ca(2+) influx in hippocampal neurons, which would lead to activation of the Src/ERK signaling cascade and up-regulation of Bcl-2 protein expression. Using fura-2 ratiometric Ca(2+) imaging, we demonstrated that E2 induced a rapid rise of intracellular Ca(2+) concentration ([Ca(2+)](i)) within minutes of exposure which was blocked by an L-type Ca(2+) channel antagonist. Inhibition of L-type Ca(2+) channels resulted in a loss of E2 activation of the Src/ERK cascade, activation of cyclic-AMP response element binding protein (CREB) and subsequent increase in Bcl-2. Real-time intracellular Ca(2+) imaging combined with pERK immunofluorescence, demonstrated that E2 induced [Ca(2+)](i) was coincident with ERK activation in the same neuron. Small interfering RNA knockdown of CREB resulted in a loss of E2 activation of CREB and subsequent E2-induced increase of Bcl-2 expression. We further demonstrated the presence of specific membrane E2 binding sites in hippocampal neurons. Together, these data indicate that E2-induced Ca(2+) influx via the L-type Ca(2+) channel is required for E2 activation of the Src/ERK/CREB/Bcl-2 signaling. Implications of these data for understanding estrogen action in brain and use of estrogen therapy for prevention of neurodegenerative disease are discussed.