After neuronal injury and in several neurodegenerative diseases, activated microglia secrete proinflammatory molecules that can contribute to the progressive neural damage. The recent demonstration of a protective role of estrogen in neurodegenerative disorders in humans and experimental animal models led us to investigate whether this hormone regulates the inflammatory response in the CNS. We here show that estrogen exerts an anti-inflammatory activity on primary cultures of rat microglia, as suggested by the blockage of the phenotypic conversion associated with activation and by the prevention of lipopolysaccharide-induced production of inflammatory mediators: inducible form of NO synthase (iNOS), prostaglandin-E₂ (PGE₂), and metalloproteinase-9 (MMP-9). These effects are dose-dependent, maximal at 1 nm 17β-estradiol, and can be blocked by the estrogen receptor (ER) antagonist ICI 182,780. The demonstration of ERα and ERβ expression in microglia and macrophages and the observation of estrogen blockade of MMP-9 mRNA accumulation and MMP-9 promoter induction further support the hypothesis of a genomic activity of estrogen via intracellular receptors. This is the first report showing an anti-inflammatory activity of estrogen in microglia. Our study proposes a novel explanation for the protective effects of estrogen in neurodegenerative and inflammatory diseases and provides new molecular and cellular targets for the screening of ER ligands acting in the CNS.

Nucleotides and cysteinyl‐leukotrienes (CysLTs) are unrelated signaling molecules inducing multiple effects through separate G‐protein‐coupled receptors: the P2Y and the CysLT receptors. Here we show that GPR17, a Gi‐coupled orphan receptor at intermediate phylogenetic position between P2Y and CysLT receptors, is specifically activated by both families of endogenous ligands, leading to both adenylyl cyclase inhibition and intracellular calcium increases. Agonist‐response profile, as determined by [35S]GTPγS binding, was different from that of already known CysLT and P2Y receptors, with EC50 values in the nanomolar and micromolar range, for CysLTs and uracil nucleotides, respectively. Both rat and human receptors are highly expressed in the organs typically undergoing ischemic damage, that is, brain, heart and kidney. In vivo inhibition of GPR17 by either CysLT/P2Y receptor antagonists or antisense technology dramatically reduced ischemic damage in a rat focal ischemia model, suggesting GPR17 as the common molecular target mediating brain damage by nucleotides and CysLTs. In conclusion, the deorphanization of GPR17 revealed a dualistic receptor for two endogenous unrelated ligand families. These findings may lead to dualistic drugs of previously unexplored therapeutic potential.

Beyond the key role in reproductive and cognitive functions, estrogens have been shown to protect against neurodegeneration associated with acute and chronic injuries of the adult brain. Current hypotheses reconcile this activity with a direct effect of 17β-estradiol (E ₂ ) on neurons. Here we demonstrate that brain macrophages are also involved in E ₂ action on the brain. Systemic administration of hormone prevents, in a time- and dose-dependent manner, the activation of microglia and the recruitment of peripheral monocytes induced by intraventricular injection of lipopolysaccharide. This effect occurs by limiting the expression of neuroinflammatory mediators, such as the matrix metalloproteinase 9 and lysosomal enzymes and complement C3 receptor, as well as by preventing morphological changes occurring in microglia during the inflammatory response. By injecting lipopolysaccharide in estrogen receptor (ER)-null mouse brains, we demonstrate that hormone action is mediated by activation of ERα but not of ERβ. The specific role of ERα is further confirmed by comparing the effects of ERs on the matrix metalloproteinase 9 promoter activity in transient transfection assays. Finally, we report that genetic ablation of ERα is associated with a spontaneous reactive phenotype of microglia in specific brain regions of adult ERα-null mice. Altogether, these results reveal a previously undescribed function for E ₂ in brain and provide a mechanism for its beneficial activity on neuroinflammatory pathologies. They also underline the key role of ERα in brain macrophage reactivity and hint toward the usefulness of ERα-specific drugs in hormone replacement therapy of inflammatory diseases.

Although 17β-estradiol (E₂) anti-inflammatory activity has been well described, very little is known about the effects of this hormone on the resolution phase of the inflammatory process. Here, we identified a previously unreported ERα-mediated effect of E₂ on the inflammatory machinery. The study showed that the activation of the intracellular estrogen receptor shortens the LPS-induced pro-inflammatory phase and, by influencing the intrinsic and extrinsic programs, triggers the resolution of inflammation in RAW 264.7 cells. Through the regulation of the SOCS3 and STAT3 signaling pathways, E₂ facilitates the progression of the inflammatory process toward the IL10-dependent “acquired deactivation” phenotype, which is responsible for tissue remodeling and the restoration of homeostatic conditions. The present study may provide an explanation for increased susceptibility to chronic inflammatory diseases in women after menopause and it suggests novel anti-inflammatory treatments for such disorders.

In the uterus insulin-like growth factor-1 (IGF-1) signaling can be initiated by estradiol acting through its nuclear receptor (estrogen receptor (ER)) to stimulate the local synthesis of IGF-1. Conversely, in vitro studies have demonstrated that estradiol-independent ER transcriptional activity can be induced by IGF-1 signaling, providing evidence for a cross-talk mechanism between IGF-1 and ER. To investigate whether ERα is required for uterine responses to IGF-1 in vivo, both wild-type (WT) and ERα knockout (αERKO) mice were administered IGF-1, and various uterine responses to IGF-1 were compared. In both WT and αERKO mice, IGF-1 treatment resulted in phosphorylation of uterine IGF-1 receptor (IGF-1R) and formation of an IGF-1R/insulin receptor substrate-1/ phosphatidylinositol 3-kinase signaling complex. In addition, IGF-1 stimulated phosphorylation of uterine Akt and MAPK in both WT and αERKO mice. However, IGF-1 treatment stimulated BrdUrd incorporation and proliferating cell nuclear antigen expression in WT uteri only. To determine whether ERα can be activated in vivo by IGF-1 signaling, transgenic mice carrying a luciferase gene driven by two estrogen response elements (ERE-luciferase mice) were utilized. Treatment of ovariectomized ERE-luciferase mice with IGF-1 resulted in an increase in uterine luciferase activity that was attenuated in the presence of the ER antagonist ICI 182,780. Together these data demonstrate that 1) functional signaling proximal to IGF-1R is maintained in the αERKO mouse uterus, 2) ERα is necessary for IGF-1 induction of uterine nuclear proliferative responses, and 3) cross-talk between IGF-1R and ER signaling pathways exists in vivo.