Beneficial role of the GPR30 agonist G-1 in an animal model of multiple sclerosis

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Abstract

The beneficial effects of estrogens in multiple sclerosis are thought to be mediated exclusively by the classical nuclear estrogen receptors ERα and ERβ. However, recently many reports revealed that estrogens are able to mediate rapid signals through a G protein-coupled receptor (GPCR), known as GPR30. In the present study, we set out to explore whether effects mediated through this receptor were anti-inflammatory and could account for some of the beneficial effects of estrogen. We demonstrate that GPR30 is expressed in both human and mouse immune cells. Furthermore a GPR30-selective agonist, G-1, previously described by us, inhibits the production of lipopolysaccharide (LPS)-induced cytokines such as TNF-α and IL-6 in a dose-dependent manner in human primary macrophages and in a murine macrophage cell line. These effects are likely mediated solely through the estrogen-specific receptor GPR30 since the agonist G-1 displayed an IC50 far greater than 10 μM on the classical nuclear estrogen receptors as well as a panel of 25 other GPCRs. Finally, we show that the agonist G-1 is able to reduce the severity of disease in both active and passive EAE models of multiple sclerosis in SJL mice and that this effect is concomitant with a G-1-mediated decrease in proinflammatory cytokines, including IFN-γ and IL-17, in immune cells harvested from these mice. The effect of G-1 appears indirect, as the GPR30 agonist did not directly influence IFN-γ or IL-17 production by purified T cells. These data indicate that G-1 may represent a novel therapeutic agent for the treatment of chronic autoimmune, inflammatory diseases.

Introduction

There is an extensive literature centered on the beneficial effects of estrogens in multiple sclerosis. Overall, these studies support a role for estrogens as potent anti-inflammatory and neuroprotective agents. For example evidence from several studies in humans and animals (Abramsky, 1994, Damek and Shuster, 1997, Polanczyk et al., 2003, van Walderveen et al., 1994) has suggested that there is a reduction in the relapse rate of multiple sclerosis and in an experimental allergic encephalomyelitis (EAE) animal model during pregnancy. These initial observations were subsequently confirmed in the large Pregnancy In Multiple Sclerosis (PRIMS) study that demonstrated a 70% reduction in relapse rate during late pregnancy (Confavreux et al., 1998).

These and other studies performed by a number of different academic groups (Arnason and Richman, 1969, Jansson et al., 1994, Subramanian et al., 2003) have demonstrated independently and consistently that estrogen is a potent immunomodulator. There is some evidence that other steroids such as progesterone and testosterone also display immunomodulatory effects presumably through their cognate receptors (Shuster, 2008); however, the effects of estrogen have been far more extensively described. Nevertheless, both neurological and pathological findings have supported the notion that estrogens are potent anti-inflammatory as well as neuroprotective agents with a potential to serve as novel therapeutics for the treatment of autoimmune demyelination. In a disease such as multiple sclerosis, where the inflammatory process induces demyelination followed by secondary axonal damage and subsequent neurological disability, the potential neuroprotective therapeutic effect of estrogens can be considered an additional benefit to their anti-inflammatory properties.

Until now the beneficial effects of estrogens in multiple sclerosis were thought to be exclusively mediated by the classical nuclear estrogen receptors ERα (and possibly ERβ (Garidou et al., 2004, Polanczyk et al., 2003, Tiwari-Woodruff et al., 2007). However, in 2005 two reports demonstrated that estrogen is able to mediate rapid signals through the G protein-coupled receptor (GPCR) known as GPR30 (Revankar et al., 2005, Thomas et al., 2005). Together these studies revealed that GPR30 is selective for the physiologically active 17β isomer of estradiol as compared to the 17α isomer and furthermore does not bind other steroids such as progesterone, testosterone or cortisol. The discovery of an estrogen-responsive GPCR opened the possibility that such a receptor might play a beneficial role in multiple sclerosis and the present study set out to explore this notion. Here we show that GPR30 is expressed in both human and mouse immune cells. Furthermore a GPR30 specific agonist, previously described by us (Bologa et al., 2006), inhibits the production of LPS-induced cytokines such as TNF-α and IL-6 in a dose-dependent manner in human primary macrophages and in a murine macrophage cell line. These effects appear to be mediated through the estrogen-specific receptor GPR30 since the agonist G-1 displayed an IC50 far greater than 10 µM for the classical nuclear estrogen receptors as well as a panel of 25 other GPCRs. Finally, we show that the agonist G-1 is able to reduce the severity of disease in both active and passive EAE models of multiple sclerosis in SJL mice.

Section snippets

Materials

Estrogen and all laboratory chemicals were from Sigma unless otherwise noted. G-1 (1-[4-(6-bromobenzo[1,3]dioxol-5-yl)-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinolin-8-yl]-ethanone), the GPR30 specific agonist, was synthesized as previously described (Bologa et al., 2006).

Cell culture

RAW264.7 cells were cultured in DMEM supplemented with 10% FBS and 2 mM l-glutamine (no antibiotic) and plated in 24 well plates at a cell density of 2 × 105 cells/well. HL-60 cells were cultured in RPMI supplemented with 10%

Results

Although little is known regarding the expression of GPR30 within the immune system, preliminary studies in our laboratory to evaluate GPR30 expression by TaqMan demonstrated that the mRNA for GPR30 is expressed in human macrophages, although with some apparent donor-to-donor variability (data not shown). Based on these data, we studied the expression of the protein on a variety of cells using an anti-GPR30 antibody that we have previously described (Revankar et al., 2005). The antibody was

Discussion

The beneficial effects of estrogen in the autoimmune disease multiple sclerosis are well documented and have been thought to be a direct effect of the hormone acting through the classical nuclear receptors ERα and ERβ (Garidou et al., 2004, Polanczyk et al., 2003, Tiwari-Woodruff et al., 2007). However, not all of the activities of estrogen can be explained by activation of these receptors (Carmeci et al., 1997, Guo et al., 2002, Qiu et al., 2003). For example, estradiol can stimulate a rapid

Note added in proof

During the submission of our manuscript Wang et al. (2009) reported protective effects of GPR30 activation on EAE. Even though this work and the present study examined different aspects of immune regulation, they strongly reinforce each other and validate the idea that GPR30 can play an important protective role in the modulation of inflammation and neurodegeneration that occurs in multiple sclerosis. Furthermore, our manuscript shows the likely importance of GPR30 expressed on human

Acknowledgement

We wish to thank Dr. Hong-Tao Lu for providing human regulatory T cells.

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    Current address: Department of Applied Research, Bayer Healthcare, Richmond CA94806, United States.

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