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 [(35)S]GTPgammaS binding, was different from that of already known CysLT and P2Y receptors, with EC(50) 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.

Deciphering the mechanisms regulating the generation of new neurons and new oligodendrocytes, the myelinating cells of the central nervous system, is of paramount importance to address new strategies to replace endogenous damaged cells in the adult brain and foster repair in neurodegenerative diseases. Upon brain injury, the extracellular concentrations of nucleotides and cysteinyl-leukotrienes (cysLTs), two families of endogenous signaling molecules, are markedly increased at the site of damage, suggesting that they may act as “danger signals” to alert responses to tissue damage and start repair. Here we show that, in brain telencephalon, GPR17, a recently deorphanized receptor for both uracil nucleotides and cysLTs (e.g., UDP-glucose and LTD4), is normally present on neurons and on a subset of parenchymal quiescent oligodendrocyte precursor cells. We also show that induction of brain injury using an established focal ischemia model in the rodent induces profound spatiotemporal-dependent changes of GPR17. In the lesioned area, we observed an early and transient up-regulation of GPR17 in neurons expressing the cellular stress marker heat shock protein 70. Magnetic Resonance Imaging in living mice showed that the in vivo pharmacological or biotechnological knock down of GPR17 markedly prevents brain infarct evolution, suggesting GPR17 as a mediator of neuronal death at this early ischemic stage. At later times after ischemia, GPR17 immuno-labeling appeared on microglia/macrophages infiltrating the lesioned area to indicate that GPR17 may also acts as a player in the remodeling of brain circuitries by microglia. At this later stage, parenchymal GPR17+ oligodendrocyte progenitors started proliferating in the peri-injured area, suggesting initiation of remyelination. To confirm a specific role for GPR17 in oligodendrocyte differentiation, the in vitro exposure of cortical pre-oligodendrocytes to the GPR17 endogenous ligands UDP-glucose and LTD4 promoted the expression of myelin basic protein, confirming progression toward mature oligodendrocytes. Thus, GPR17 may act as a “sensor” that is activated upon brain injury on several embryonically distinct cell types, and may play a key role in both inducing neuronal death inside the ischemic core and in orchestrating the local remodeling/repair response. Specifically, we suggest GPR17 as a novel target for therapeutic manipulation to foster repair of demyelinating wounds, the types of lesions that also occur in patients with multiple sclerosis.

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-E 2 (PGE 2 ), and metalloproteinase-9 (MMP-9). These effects are dose-dependent, maximal at 1 nM 17␤-estradiol, and can be blocked by the estrogen receptor (ER) antagonist ICI 182,780. The demonstration of ER␣ and ER␤ 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.

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 17␤-estradiol (E 2 ) on neurons. Here we demonstrate that brain macrophages are also involved in E2 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 ER␣ but not of ER␤. The specific role of ER␣ 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 ER␣ is associated with a spontaneous reactive phenotype of microglia in specific brain regions of adult ER␣-null mice. Altogether, these results reveal a previously undescribed function for E2 in brain and provide a mechanism for its beneficial activity on neuroinflammatory pathologies. They also underline the key role of ER␣ in brain macrophage reactivity and hint toward the usefulness of ER␣-specific drugs in hormone replacement therapy of inflammatory diseases.

Through intracellular receptors, estrogens control growth, differentiation and function of not only reproductive tissues, but also other systems. Estrogen receptors are ligand-dependent transcription factors whose activity is modulated either by estrogens, or by alternative intracellular signaling pathways downstream of growth factors and neurotransmitters. To determine the dynamics of estrogen receptor activity and the dependence of estrogen receptor on 17beta-estradiol in vivo, we generated a transgenic mouse that expresses a luciferase reporter gene under the control of activated estrogen receptors. As expected, luciferase activity, monitored with a cooled charged coupled device camera, paralleled circulating estrogen levels in reproductive tissues and in liver, indicating that the peak transcriptional activity of the estrogen receptor occurred at proestrus. In contrast, in tissues such as bone and brain, the peak activity of estrogen receptors was observed at diestrus. These tissue-specific responses are masked when mice undergo conventional hormone treatment. We also demonstrate that estrogen receptors are active in immature mice before gonadal production of sex hormones as well as in ovariectomized adult mice. These findings emphasize the importance of hormone-independent activation of the estrogen receptor, and have implications for the therapeutic use of estrogens, such as hormone replacement therapy.

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 ER␣ is required for uterine responses to IGF-1 in vivo, both wild-type (WT) and ER␣ knockout (␣ERKO) mice were administered IGF-1, and various uterine responses to IGF-1 were compared. In both WT and ␣ERKO 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 ␣ERKO mice. However, IGF-1 treatment stimulated BrdUrd incorporation and proliferating cell nuclear antigen expression in WT uteri only. To determine whether ER␣ 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 ␣ERKO mouse uterus, 2) ER␣ 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.Epithelial cells of the mammalian uterus undergo a wave of DNA synthesis followed by mitosis in response to 17␤-estradiol (E 2 ), 1 which regulates the transcription of numerous target genes by binding to and activating the nuclear estrogen receptor (ER). Among the genes identified as targets for regulation by the E 2 /ER complex in the uterus is that encoding insulinlike growth factor-1 (IGF-1). Studies have demonstrated that rodent uterine IGF-1 mRNA levels increase after exposure to E 2 (1, 2). Furthermore, presumably through increasing local production of IGF-1, E 2 has been shown to stimulate uterine IGF-1 receptor (IGF-1R) signaling as measured by tyrosine phosphorylation of IGF-1R and the formation of a signaling complex composed of IGF-1R, insulin receptor substrate-1 (IRS-1), and p85, the regulatory subunit of phosphatidylinositol 3-kinase (PI 3-kinase) (3, 4). These studies suggested that IGF-1 signaling is involved in E 2 -induced uterine growth, and in support of this mechanism, other studies have shown that, like E 2 , IGF-1 can induce DNA synthesis in cells of the rodent uterus (5). A more recent study further demonstrated a role for IGF-1 in E 2 -induced uterine proliferation by demonstrating that IGF-1 is required for E 2 -induced uterine epithelial cell mitosis (6). In that study, DNA synthesis occurred in IGF-1 knockout (IGF-1KO) m...

Estrogens have well-documented effects on lung development and physiology. However, the classical estrogen receptor ␣ (ER␣) is undetectable in the lung, and this has left many unanswered questions about the mechanism of estrogen action in this organ. Here we show, both in vivo and in vitro, that ER␤ is abundantly expressed and biologically active in the lung. Comparisons of lungs from wild-type mice and mice with an inactivated ER␤ gene (ER␤ ؊/؊ ) revealed decreased numbers of alveoli in adult female ER␤ ؊/؊ mice and findings suggesting deficient alveolar formation as well as evidence of surfactant accumulation. Plateletderived growth factor A (PDGF-A) and granulocyte-macrophage colony-stimulating factor (GM-CSF), key regulators of alveolar formation and surfactant homeostasis, respectively, were decreased in lungs of adult female ER␤ ؊/؊ mice, and direct transcriptional regulation of these genes by ER␤ was demonstrated. This suggests that estrogens act via ER␤ in the lung to modify PDGF-A and GM-CSF expression. These results provide a potential molecular mechanism for the gender differences in alveolar structure observed in the adult lung and establish ER␤ as a previously unknown regulator of postnatal lung development and homeostasis.The vital function of the lung is to provide a gas-exchange surface to meet the organism's needs for oxygen uptake and carbon dioxide elimination. Several parameters in lung biology and pathology, both during development and in the adult, are sexually dimorphic. A role for estrogen in these dimorphisms was suggested in 1980 when Mendelson et al. (21) showed an estrogen-binding component in human fetal lung tissue. Lung maturation during fetal development is more rapid in female fetuses than in male fetuses, and the onset of surfactant synthesis occurs later in the male fetus. This difference appears to be mediated mainly by inhibitory effects of androgens, but stimulatory effects of estrogens have also been demonstrated (2). Postnatal sex differences in the rodent lung have been described by Massaro et al. (20). Adult females have a larger number of alveoli, smaller in size, than males, probably to allow for elevated oxygen consumption during pregnancy and lactation. This difference develops as animals reach sexual maturity and seems to be mediated mainly by estrogens (19). In the human population, women are more prone than men to developing chronic obstructive pulmonary disease (29) and incur a higher risk of developing lung cancer (13, 41), indicating that women are more susceptible to the deleterious effects of tobacco smoking. The reasons for these sex differences are unknown, but estrogens are likely to play a major role, since in animal models, there are estrogen-dependent gender differences in susceptibility towards tobacco-associated lung carcinogens (23), and furthermore, epidemiological studies suggest that hormone replacement therapy with estrogen is associated with a higher risk of lung cancer in postmenopausal women (1,39).Although previous data suggest that estrogen...

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.