Agonist Propyl Pyrazole Triol Research Articles

Ethanol impairs estrogen receptor signaling resulting in accelerated activation of senescence pathways, whereas estradiol attenuates the effects of ethanol in osteoblasts.

Epidemiological and animal studies have suggested that chronic alcohol consumption is a major risk factor for osteoporosis. Using bone from cycling female rats infused chronically with ethanol (EtOH) in vivo and osteoblastic cells in vitro, we found that EtOH significantly increased estrogen receptor α (ERα) and β (ERβ) mRNA and ERα protein levels. Treatment with 17β-estradiol (E2) in vivo and in vitro interfered with these effects of EtOH on bone and osteoblastic cells. ERα agonist propylpyrazoletriol (PPT) and ERβ agonist diarylpropionitrile (DPN) attenuated EtOH-induced ERα and ERβ gene overexpression, respectively. Similar to the ER antagonist ICI 182780, EtOH blocked nuclear translocation of ERα-ECFP in the presence of E2 in UMR-106 osteoblastic cells. EtOH also downregulated ERE-luc reporter activity. On the other hand, EtOH by itself upregulated some common ERα- and ERβ-mediated genes apparently by an ER-independent pathway. EtOH also transactivated the luciferase activity of the p21 promoter region independent of additional exogenous ERα, activated p21 and p53, and stimulated senescence-associated β-galactosidase activity in rat stromal osteoblasts. E2 treatment attenuated these EtOH actions. We conclude that inhibitory cross-talk between EtOH and E2 in osteoblasts on ERs, p53/p21, and cell senescence provides a pathophysiologic mechanism underlying bone loss and the protective effects of estrogens in alcohol-exposed females.

Selective estrogen receptor-α and estrogen receptor-β agonists rapidly decrease pulmonary artery vasoconstriction by a nitric oxide-dependent mechanism

Both endogenous and exogenous estrogen decrease pulmonary artery (PA) vasoconstriction. Whether these effects are mediated via estrogen receptor (ER)-alpha or ER-beta, and whether the contribution of ERs is stimulus-dependent, remains unknown. We hypothesized that administration of the selective ER-alpha agonist propylpyrazole triol (PPT) and/or the selective ER-beta agonist diarylpropiolnitrile (DPN) rapidly decreases PA vasoconstriction induced by pharmacologic and hypoxic stimuli via a nitric oxide (NO)-dependent mechanism. PA rings (n = 3-10/group) from adult male Sprague-Dawley rats were suspended in physiologic organ baths. Force displacement was measured. Vasoconstrictor responses to phenylephrine (10(-8)M - 10(-5)M) and hypoxia (Po(2) 35-45 mmHg) were determined. Endothelium-dependent and -independent vasorelaxation were measured by generating dose-response curves to acetylcholine (10(-8)M - 10(-4)M) and sodium nitroprusside (10(-9)M - 10(-5)M). PPT or DPN (10(-9)M - 5 x 10(-5)M) were added to the organ bath in the presence and absence of the NO-synthase inhibitor N(omega)-nitro-l-arginine methyl ester (l-NAME) (10(-4)M). Selective ER-alpha activation (PPT, 5 x 10(-5)M) rapidly (<20 min) decreased phenylephrine-induced vasoconstriction. This effect, as well as PPT's effects on endothelium-dependent vasorelaxation, were neutralized by l-NAME. In contrast, selective ER-beta activation (DPN, 5 x 10(-5)M) rapidly decreased phase II of hypoxic pulmonary vasoconstriction (HPV). l-NAME eliminated this phenomenon. Lower PPT or DPN concentrations were less effective. We conclude that both ER-alpha and ER-beta decrease PA vasoconstriction. The immediate onset of effect suggests a nongenomic mechanism. The contribution of specific ERs appears to be stimulus specific, with ER-alpha primarily modulating phenylephrine-induced vasoconstriction, and ER-beta inhibiting HPV. NO inhibition eliminates these effects, suggesting a central role for NO in mediating the pulmonary vascular effects of both ER-alpha and ER-beta.

SELECTIVE ESTROGEN RECEPTOR AGONISTS ATTENUATE PULMONARY ARTERY VASOCONSTRICTION BY A NITRIC OXIDE-DEPENDENT MECHANISM

PURPOSE: Both endogenous and exogenous estrogen attenuates pulmonary artery (PA) vasoconstriction. Whether these effects are mediated via estrogen receptor (ER)-α or ER-β, remains unknown. A better understanding may result in novel, nonhormonal treatment options for pulmonary hypertension. We hypothesized that administration of the selective ER-α agonist propylpyrazole-triol (PPT) and/or the selective ER-β agonist diarylpropiolnitrile (DPN) attenuates PA vasoconstriction induced by pharmacologic and hypoxic stimuli.

Role of estrogen receptor subtypes in estrogen-induced organ-specific vasorelaxation after trauma-hemorrhage

Although endothelin-1 (ET-1)-induced organ hypoperfusion after trauma-hemorrhage is improved by estrogen administration, it remains unclear whether estrogen receptor (ER) subtypes play any role in the attenuation of ET-1-induced vasoconstriction in any specific organ bed. To investigate this, isolated perfusion experiments in the heart, liver, small intestine, kidney, and lung were carried out in sham, at the time of maximum bleedout (MBO; i.e., 5-cm midline incision, with removal of 60% of circulating blood volume over 45 min to maintain a mean blood pressure of 40 mmHg), and 2 h after trauma-hemorrhage and resuscitation (T-H/R). Organ-specific ET-1-induced vasoconstriction was evaluated, and the effects of 17beta-estradiol (E2) and ER-specific agonists propylpyrazole triol (PPT; ERalpha agonist) and diarylpropionitrile (DPN; ERbeta agonist) were determined. ET-1 induced the greatest vasoconstriction in sham animals, with the strongest response in the kidneys, followed by the small intestine and liver. ET-1-induced responses were weakest in the heart and lungs. ET-1-induced vasoconstriction was evident at the time of MBO but was significantly decreased at 2 h after T-H/R. ERbeta plays an important role in cardiac performance, as evidenced by improved heart performance (+dP/dt) in the presence of DPN. DPN also induced a greater effect than PPT in the reduction of ET-1-induced vasoconstriction in the kidneys and lungs. In contrast, PPT attenuated ET-1-induced vasoconstriction in the liver, whereas both DPN and PPT were equally effective in the small intestine. The increased +dP/dt values induced by E2, DPN, or PPT were evident at the time of MBO but were significantly decreased at 2 h after T-H/R. These data indicate that the effects of ET-1 on vasoconstriction and the role of ER subtypes in estrogen-induced vasorelaxation are organ specific and temporally specific after trauma-hemorrhage.

Selective estrogen receptor α activation disrupts sex organ differentiation and induces expression of vitellogenin II and very low-density apolipoprotein II in Japanese quail embryos

The Japanese quail (Coturnix japonica) is a widely used model species for studying the roles of steroid hormones in avian sex differentiation. The aim of the present study was to elucidate the significance of estrogen receptors alpha and beta (ERalpha and ERbeta) in normal sex differentiation of the reproductive organs in the Japanese quail and in xenoestrogen-induced disruption of reproductive organ differentiation. Real-time PCR indicated that ERalpha (ESR1) mRNA is expressed in both right and left gonads and Müllerian ducts (MDs) in both sexes during early morphological differentiation. ERbeta (ESR2) transcripts were also detected in gonads and MDs, but at very low levels. Both receptor subtypes were expressed in the liver and may therefore mediate the expression of estrogen-regulated egg-yolk proteins. Aromatase mRNA was expressed at much higher levels in female than male gonads as early as embryonic day 5, indicating early sex differences in estrogen synthesis. Treatment with the ERalpha-selective agonist propyl pyrazole triol showed that frequently reported xenoestrogen effects, such as ovotestis formation, abnormal MD development, and hepatic expression of egg-yolk proteins, were induced by selective activation of ERalpha. Taken together, our results suggest that activation of ERalpha is crucial for estrogen-dependent sex differentiation of the reproductive organs and that ERalpha mediates xenoestrogen-induced toxicity during reproductive development in birds.

The role of estrogen receptor subtypes on hepatic neutrophil accumulation following trauma-hemorrhage: Direct modulation of CINC-1 production by Kupffer cells

Although 17β-estradiol (E2) administration following trauma-hemorrhage (T-H) reduces liver injury by decreasing neutrophil accumulation via estrogen receptor (ER)-α, it remains unclear whether cytokine-induced neutrophil chemoattractant (CINC)-1 production by Kupffer cells (KC) is directly modulated by ER-α under such condition. Male rats underwent laparotomy and hemorrhagic shock (40 mmHg for 90 min), followed by resuscitation with four times the shed blood volume in the form of Ringer’s lactate. ER-α agonist propyl pyrazole triol (PPT; 5 μg/kg), ER-β agonist diarylpropionitrile (DPN; 5 μg/kg), E2 (50 μg/kg), or vehicle (10% DMSO) was administered subcutaneously during resuscitation; rats were sacrificed 24 h thereafter. KC were isolated and cultured with ER agonists to examine if they directly affect CINC-1 production. T-H increased plasma alanine aminotransferase (ALT; hepatic injury) and hepatic myeloperoxidase (MPO) activity. E2, PPT and DPN administration reduced increased ALT; however, PPT was more effective than DPN. PPT and E2, but not DPN significantly attenuated increased hepatic MPO activity and CINC-1 levels. PPT addition in vitro (10 −7 and 10 −6 M) significantly reduced KC CINC-1 production. In summary, the salutary effects of E2 against hepatic injury are mediated predominantly via ER-α which directly modulates KC CINC-1 production and hepatic neutrophil accumulation following T-H.

Estrogen Elicits Dorsal Root Ganglion Axon Sprouting via a Renin-Angiotensin System

Many painful conditions occur more frequently in women, and estrogen is a predisposing factor. Estrogen may contribute to some pain syndromes by enhancing axon outgrowth by sensory dorsal root ganglion (DRG) neurons. The objective of the present study was to define mechanisms by which estrogen elicits axon sprouting. The estrogen receptor-alpha agonist propyl pyrazole triol induced neurite outgrowth from cultured neonatal DRG neurons, whereas the estrogen receptor-beta agonist diarylpropionitrile was ineffective. 17beta-Estradiol (E2) elicited sprouting from peripherin-positive unmyelinated neurons, but not larger NF200-positive myelinated neurons. Microarray analysis showed that E2 up-regulates angiotensin II (ANGII) receptor type 2 (AT2) mRNA in vitro, and studies in adult rats confirmed increased DRG mRNA and protein in vivo. AT2 plays a central role in E2-induced axon sprouting because AT2 blockade by PD123,319 eliminated estrogen-mediated sprouting in vitro. We assessed whether AT2 may be responding to locally synthesized ANGII. DRG from adult rats expressed mRNA for renin, angiotensinogen, and angiotensin converting enzyme (ACE), and protein products were present and occasionally colocalized within neurons and other DRG cells. We determined if locally synthesized ANGII plays a role in estrogen-mediated sprouting by blocking its formation using the ACE inhibitor enalapril. ACE inhibition prevented estrogen-induced neuritogenesis. These findings support the hypothesis that estrogen promotes DRG nociceptor axon sprouting by up-regulating the AT2 receptor, and that locally synthesized ANGII can induce axon formation. Therefore, estrogen may contribute to some pain syndromes by enhancing the pro-neuritogenic effects of AT2 activation by ANGII.

An Estrogen Receptor-α Knock-In Mutation Provides Evidence of Ligand-Independent Signaling and Allows Modulation of Ligand-Induced Pathways in Vivo

Estrogen-nonresponsive estrogen receptor-alpha (ERalpha) knock-in (ENERKI) mice were generated to distinguish between ligand-induced and ligand-independent ER-alpha actions in vivo. These mice have a mutation [glycine 525 to leucine (G525L)] in the ligand-binding domain of ERalpha, which significantly reduces ERalpha interaction with and response to endogenous estrogens, whereas not affecting growth factor activation of ligand-independent pathways. ENERKI mice had hypoplastic uterine tissues and rudimentary mammary gland ductal trees. Females were infertile due to anovulation, and their ovaries contained hemorrhagic cystic follicles because of chronically elevated levels of LH. The ENERKI phenotype confirmed that ligand-induced activation of ERalpha is crucial in the female reproductive tract and mammary gland development. Growth factor treatments induced uterine epithelial proliferation in ovariectomized ENERKI females, directly demonstrating that ERalpha ligand-independent pathways were active. In addition, the synthetic ERalpha selective agonist propyl pyrazole triol (PPT) and ER agonist diethylstilbestrol (DES) were still able to activate ligand-induced G525L ERalpha pathways in vitro. PPT treatments initiated at puberty stimulated ENERKI uterine development, whereas neonatal treatments were needed to restore mammary gland ductal elongation, indicating that neonatal ligand-induced ERalpha activation may prime mammary ducts to become more responsive to estrogens in adult tissues. This is a useful model for in vivo evaluation of ligand-induced ERalpha pathways and temporal patterns of response. DES did not stimulate an ENERKI uterotrophic response. Because ERbeta may modulate ERalpha activation and have an antiproliferative function in the uterus, we hypothesize that ENERKI animals were particularly sensitive to DES-induced inhibition of ERalpha due to up-regulated uterine ERbeta levels.

Genome-Wide Identification of Estrogen Receptor α-Binding Sites in Mouse Liver

We report the genome-wide identification of estrogen receptor alpha (ERalpha)-binding regions in mouse liver using a combination of chromatin immunoprecipitation and tiled microarrays that cover all nonrepetitive sequences in the mouse genome. This analysis identified 5568 ERalpha-binding regions. In agreement with what has previously been reported for human cell lines, many ERalpha-binding regions are located far away from transcription start sites; approximately 40% of ERalpha-binding regions are located within 10 kb of annotated transcription start sites. Almost 50% of ERalpha-binding regions overlap genes. The majority of ERalpha-binding regions lie in regions that are evolutionarily conserved between human and mouse. Motif-finding algorithms identified the estrogen response element, and variants thereof, together with binding sites for activator protein 1, basic-helix-loop-helix proteins, ETS proteins, and Forkhead proteins as the most common motifs present in identified ERalpha-binding regions. To correlate ERalpha binding to the promoter of specific genes, with changes in expression levels of the corresponding mRNAs, expression levels of selected mRNAs were assayed in livers 2, 4, and 6 h after treatment with ERalpha-selective agonist propyl pyrazole triol. Five of these eight selected genes, Shp, Stat3, Pdgds, Pck1, and Pdk4, all responded to propyl pyrazole triol after 4 h treatment. These results extend our previous studies using gene expression profiling to characterize estrogen signaling in mouse liver, by characterizing the first step in this signaling cascade, the binding of ERalpha to DNA in intact chromatin.

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

Although 17β-estradiol administration following trauma–hemorrhage attenuates Kupffer cell, splenic and peritoneal macrophage functions, it remains unknown whether 17β-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 17β-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 ∼90min of hemorrhagic shock (blood pressure [BP] 35mmHg), followed by fluid resuscitation (4× the shed blood volume in the form of Ringer's lactate). Estrogen receptor (ER)-α agonist propyl pyrazole triol (PPT; 5μg/kg), ER-β agonist diarylpropionitrile (DPN; 5μg/kg), 17β-estradiol (50μg/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, 17β-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, 17β-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 17β-estradiol on splenic DC functions are mediated predominantly via ER-α.

Estrogen receptor-α predominantly mediates the salutary effects of 17β-estradiol on splenic macrophages following trauma-hemorrhage

Although 17beta-estradiol administration following trauma-hemorrhage prevents the suppression in splenic macrophage cytokine production, it remains unknown whether the salutary effects are mediated via estrogen receptor (ER)-alpha or ER-beta and which signaling pathways are involved in such 17beta-estradiol effects. Utilizing ER-alpha- or ER-beta-specific agonists, this study examined the role of ER-alpha and ER-beta in 17beta-estradiol-mediated restoration of macrophage cytokine production following trauma-hemorrhage. In addition, since MAPK and NF-kappaB are known to regulate macrophage cytokine production, we also examined the activation of those signaling molecules. Male rats underwent trauma-hemorrhage (mean arterial pressure of 40 mmHg for 90 min) and fluid resuscitation. The ER-alpha agonist propyl pyrazole triol (PPT; 5 microg/kg), the ER-beta agonist diarylpropionitrile (DPN; 5 microg/kg), 17beta-estradiol (50 microg/kg), or vehicle (10% DMSO) was injected subcutaneously during resuscitation. Twenty-four hours thereafter, splenic macrophages were isolated, and their IL-6 and TNF-alpha production and activation of MAPK and NF-kappaB were measured. Macrophage IL-6 and TNF-alpha production and MAPK activation were decreased, whereas NF-kappaB activity was increased, following trauma-hemorrhage. PPT or 17beta-estradiol administration after trauma-hemorrhage normalized those parameters. DPN administration, on the other hand, did not normalize the above parameters. Since PPT but not DPN administration following trauma-hemorrhage was as effective as 17beta-estradiol in preventing the suppression in macrophage cytokine production, it appears that ER-alpha plays the predominant role in mediating the salutary effects of 17beta-estradiol on macrophage cytokine production following trauma-hemorrhage and that such effects are likely mediated via normalization of MAPK but not NF-kappaB signaling pathways.

Sex and drugs: Comment on “Evidence for involvement of erβ and rgs9-2 in 17-β estradiol enhancement of amphetamine-induced place preference behavior” by Silverman and Koenig

Sex and drugs: Comment on “Evidence for involvement of erβ and rgs9-2 in 17-β estradiol enhancement of amphetamine-induced place preference behavior” by Silverman and Koenig

The integrated action of oestrogen receptor isoforms and sites with progesterone receptor in the gonadotrope modulates LH secretion: evidence from tamoxifen-treated ovariectomized rats

The specific role of each oestrogen receptor (ER) isoform (alpha and beta ) and site (nucleus and plasma membrane) in LH release was determined in ovariectomized (OVX) rats injected over 6 days (days 15-20 after OVX) with a saturating dose (3 mg/day) of tamoxifen (TX), a selective ER modulator with nuclear ERalpha agonist actions in the absence of oestrogen. This pharmacological effect of TX was demonstrated by the fact that it was blocked by the selective ERalpha antagonist methyl-piperidinopyrazole. Over the past 3 days of the 6-day TX treatment, rats received either 25 microg/day oestradiol benzoate (EB), 1.5 mg/day selective ERalpha agonist propylpyrazole triol (PPT) and the selective ERbeta agonist diarylpropionitrile (DPN), or a single 3 mg injection of the antiprogestin onapristone (ZK299) administered on day 20. Blood samples were taken to determine basal and progesterone receptor (PR)-dependent LH-releasing hormone (LHRH)-stimulated LH secretion and to evaluate LHRH self-priming, the property of LHRH that increases gonadotrope responsiveness to itself. Blood LH concentration was determined by RIA and gonadotrope PR expression by immunohistochemistry. Results showed that i) EB and DPN potentiated the negative feedback of TX on basal LH release; ii) DPN reduced TX-induced PR expression; iii) EB and PPT blocked TX-elicited LHRH self-priming and iv) ZK299 reduced LHRH-stimulated LH secretion and blocked LHRH self-priming. These observations suggest that oestrogen action on LH secretion in the rat is exerted at the classic ERalpha pool and that this action might be modulated by both ERbeta and membrane ERalpha through their effects on PR expression and action respectively.

Salutary effects of 17β-estradiol on T-cell signaling and cytokine production after trauma-hemorrhage are mediated primarily via estrogen receptor-α

Although 17beta-estradiol (E2) administration following trauma-hemorrhage prevents the suppression in splenocyte cytokine production, it remains unknown whether the salutary effects of 17beta-estradiol are mediated via estrogen receptor (ER)-alpha or ER-beta. Moreover, it is unknown which signaling pathways are involved in 17beta-estradiol's salutary effects. Utilizing an ER-alpha- or ER-beta-specific agonist, we examined the role of ER-alpha and ER-beta in E2-mediated restoration of T-cell cytokine production following trauma-hemorrhage. Moreover, since MAPK, NF-kappaB, and activator protein (AP)-1 are known to regulate T-cell cytokine production, we also examined the activation of MAPK, NF-kappaB, and AP-1. Male rats underwent trauma-hemorrhage (mean arterial pressure 40 mmHg for 90 min) and fluid resuscitation. ER-alpha agonist propyl pyrazole triol (PPT; 5 microg/kg), ER-beta agonist diarylpropionitrile (DPN; 5 microg/kg), 17beta-estradiol (50 microg/kg), or vehicle (10% DMSO) was injected subcutaneously during resuscitation. Twenty-four hours thereafter, splenic T cells were isolated, and their IL-2 and IFN-gamma production and MAPK, NF-kappaB, and AP-1 activation were measured. T-cell IL-2 and IFN-gamma production was decreased following trauma-hemorrhage, and this was accompanied with a decrease in T-cell MAPK, NF-kappaB, and AP-1 activation. PPT or 17beta-estradiol administration following trauma-hemorrhage normalized those parameters, while DPN administration had no effect. Since PPT, but not DPN, administration following trauma-hemorrhage was as effective as 17beta-estradiol in preventing the T-cell suppression, it appears that ER-alpha plays a predominant role in mediating the salutary effects of 17beta-estradiol on T cells following trauma-hemorrhage, and that such effects are likely mediated via normalization of MAPK, NF-kappaB, and AP-1 signaling pathways.

Estrogen modulates TNF-α-induced inflammatory responses in rat aortic smooth muscle cells through estrogen receptor-β activation

We have previously shown that 17beta-estradiol (E2) attenuates responses to endoluminal injury of the rat carotid artery, at least in part, by decreasing inflammatory mediator expression and neutrophil infiltration into the injured vessel, with a major effect on the neutrophil-specific chemokine cytokine-induced neutrophil chemoattractant (CINC)-2 beta. Current studies tested the hypothesis that activated rat aortic smooth muscle cells (RASMCs) express these same inflammatory mediators and induce neutrophil migration in vitro and that E2 inhibits these processes by an estrogen receptor (ER)-dependent mechanism. Quiescent RASMCs treated with E2, the ER alpha-selective agonist propyl pyrazole triol (PPT), the ER beta-selective agonist diarylpropiolnitrile (DPN), or vehicle for 24 h were stimulated with tumor necrosis factor (TNF)-alpha and processed for real-time RT-PCR, ELISA, or chemotaxis assays 6 h later. TNF-alpha stimulated and E2 attenuated mRNA expression of inflammatory mediators, including P-selectin, intercellular adhesion molecule (ICAM)-1, vascular cell adhesion molecule (VCAM)-1, monocyte chemoattractant protein (MCP)-1, and CINC-2 beta. DPN dose dependently attenuated TNF-alpha-induced mRNA expression of CINC-2 beta, whereas PPT had no effect. The anti-inflammatory effects of DPN and E2 were blocked by the nonselective ER-inhibitor ICI-182,780. ELISA confirmed the TNF-alpha-induced increase and E2-induced inhibition of CINC-2 beta protein secretion. TNF-alpha treatment of RASMCs produced a twofold increase in neutrophil chemotactic activity of conditioned media; E2 and DPN treatment markedly inhibited this effect. E2 inhibits activated RASMC proinflammatory mediator expression and neutrophil chemotactic activity through an ER beta-dependent mechanism.

Tissue compartment-specific role of estrogen receptor subtypes in immune cell cytokine production following trauma-hemorrhage

Although 17beta-estradiol administration following trauma-hemorrhage attenuates plasma cytokines and alteration in immune cell cytokine production, it is not known whether the salutary effects are mediated via estrogen receptor (ER)-alpha or ER-beta. Accordingly, we examined which ER subtype predominantly mediates the salutary effects of 17beta-estradiol on systemic inflammatory response/immune cell cytokine production in various tissues following trauma-hemorrhage. Male rats underwent trauma-hemorrhage (mean blood pressure: 40 mmHg for 90 min) and fluid resuscitation. The ER-alpha agonist propyl pyrazole triol (PPT; 5 microg/kg), the ER-beta agonist diarylpropionitrile (DPN; 5 microg/kg), 17beta-estradiol (50 microg/kg), or vehicle (10% DMSO) was injected subcutaneously during resuscitation, and various measurements were made 24 h thereafter. 17beta-Estradiol or PPT administration following trauma-hemorrhage prevented the increase in plasma IL-6 and IL-10 levels that were observed in vehicle-treated animals. IL-6 and TNF-alpha production by Kupffer cells increased; however, splenic macrophages (SMPhi), alveolar macrophages (AMPhi), and peripheral blood mononuclear cells (PBMC) had decreased release of these cytokines after trauma-hemorrhage. IL-10 production, however, increased in all macrophage populations. Administration of 17beta-estradiol following trauma-hemorrhage prevented all of these alterations. PPT had the same effects as 17beta-estradiol on IL-6 and TNF-alpha production by Kupffer cells and SMPhi, and DPN had the same effects on AMPhi and PBMC. The same effects as 17beta-estradiol on IL-10 production were observed by PPT on Kupffer cells and DPN on PBMC. Both agonists were equally effective on SMPhi and AMPhi. Thus ER subtypes have tissue compartment-specific roles in mediating the effects of 17beta-estradiol on immune cell functions following trauma-hemorrhage.

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.

17β-estradiol administration following trauma-hemorrhage prevents the increase in Kupffer cell cytokine production and MAPK activation predominately via estrogen receptor-α

17 beta-estradiol (E2) administration following trauma-hemorrhage (T-H) attenuates the elevation in plasma cytokines and Kupffer cell (KC) cytokine production; however, it remains unknown whether the salutary effects are mediated via estrogen receptor (ER)-alpha or ER-beta. We hypothesized that E2 mediates its salutary effects via ER-alpha and normalization of MAPK under those conditions. Male rats underwent T-H (mean blood pressure [BP] 40 mmHg for 90 min) and fluid resuscitation. ER-alpha agonist propyl pyrazole triol (PPT; 5 microg/kg), ER-beta agonist diarylpropionitrile (DPN; 5 microg/kg), E2 (50 microg/kg), or vehicle (10% DMSO) was injected subcutaneously during resuscitation. Twenty-four hours thereafter, KCs were isolated and their cytokine production (IL-6, TNF-alpha, IL-10) and MAPK activation were measured. Cytokine production increased after T-H, however, PPT or E2 administration after T-H normalized KC cytokine production. Although DPN attenuated increased production of these cytokines, KC capacity to produce the cytokines remained significantly higher than sham. PPT or E2 also prevented T-H-mediated activation of MAPK in KC. However, DPN did not prevent MAPK activation. Since PPT administration after T-H was more effective in decreasing KC cytokine production and MAPK activation than DPN, the salutary effects of E2 on KC functions are mediated predominantly via ER-alpha and normalization of MAPK following T-H.

Upregulation of mitochondrial respiratory complex IV by estrogen receptor-β is critical for inhibiting mitochondrial apoptotic signaling and restoring cardiac functions following trauma–hemorrhage

Our recent study showed that estrogen receptor (ER) β plays a major role in mediating the salutary effects of 17β-estradiol (E2) on cardiac function following trauma–hemorrhage (T–H). E2 is known to regulate mitochondrial DNA (mtDNA)-encoded genes including the mitochondrial respiratory complex (MRC) proteins. Depressed MRC activity has been reported to promote the release of cytochrome c from mitochondria and induce apoptosis. We hypothesized that E2 and ERβ-mediated cardioprotection following T–H is dependent on mtDNA transcription encoding for MRC activity. To test this, male rats underwent T–H (mean BP 40 mm Hg ∼ 90 min, then resuscitation). During resuscitation, rats received either ERα agonist propylpyrazole triol (PPT; 5 μg/kg), ERβ agonist diarylpropionitrile (DPN; 5 μg/kg), E2 (50 μg/kg), or vehicle (10% DMSO). Another group of rats received mitochondrial respiratory complex-IV (MRC-IV) inhibitor sodium cyanide (SCN; 6 mg/kg) with or without DPN. The results indicated that 24 h after T–H, cardiac functions were depressed in the vehicle-treated but were normal in the DPN-treated rats. Moreover, E2 or DPN treatment after T–H normalized cardiac mitochondrial ERβ expression and increased mitochondrial ERβ DNA-binding activity. This was accompanied by an increase in MRC-IV gene expressions and activity, while MRC-I gene expression remained unchanged. Inhibition of MRC-IV in DPN-treated T–H rats by SCN abolished the DPN-mediated cardioprotection, ATP production, mitochondrial cytochrome c release, caspase-3 cleavage, and apoptosis. Thus, E2 and ERβ-mediated cardioprotection following T–H appears to be mediated via mitochondrial ERβ-dependent MRC-IV activity and inhibition of mitochondrial apoptotic signaling pathways.

Inhibition of cardiac PGC‐1α expression abolishes ERβ agonist‐mediated cardioprotection following trauma‐hemorrhage

PGC-1alpha (peroxisome proliferator-activated receptor [PPARgamma] coactivator-1alpha) activates PPARalpha and mitochondrial transcription factor A (Tfam), which regulate proteins, fatty acid and ATP metabolism (i.e., FAT/CD36, MCAD, and COX I). Recently we found that the salutary effects of estradiol (E2) on cardiac function following trauma-hemorrhage (T-H) are mediated via estrogen receptor (ER)beta. In this study we tested the hypothesis that ERbeta-mediated cardioprotection is induced via up-regulation of PGC-1alpha through PPARalpha or Tfam-dependent pathway. Male rats underwent T-H and received ERalpha agonist propylpyrazole-triol (PPT), ERbeta agonist diarylpropionitrile (DPN), E2, or vehicle. Another group was treated with antisense PGC-1alpha oligonucleotides prior to administration of DPN. E2 and DPN treatments attenuated the decrease in cardiac mitochondrial ATP, abrogated the T-H-induced lipid accumulation, and normalized PGC-1alpha, PPARalpha, FAT/CD36, MCAD, Tfam, and COX I after T-H. In contrast, PPT administration did not abrogate lipid accumulation. Moreover, in PPT-treated animals mitochondrial ATP remained significantly lower than those observed in DPN- or E2-treated animals. Prior administration of antisense PGC-1alpha prevented DPN-mediated cardioprotection and increase in ATP levels and Tfam but not in PPARalpha following T-H. These findings suggest that the salutary effects of E2 on cardiac function following T-H are mediated via ERbeta up-regulation of PGC-1alpha through Tfam-dependent pathway.