OR08-6 RGS2 Expression Attenuates Contractile Signaling in Myometrial Smooth Muscle In Vitro

Abstract

Labour activation is regulated by a shift in the balance of pro-quiescent mechanisms that maintain pregnancy, and inflammatory and endocrine signals that stimulate uterine contractility. The prostaglandins (PG) and oxytocin act at Gq protein coupled receptors thought to drive labour contractions. However, emerging evidence suggests a twofold effect of PGE2 on the pregnant uterus, driving pro-labour or pro-quiescent signaling depending on the receptor subtype activated. It is possible that PGE2, abundant within the pregnant uterus, mediates pro-quiescent effects by activating Gs coupled receptors to upregulate regulator of G-protein signaling 2 (RGS2), a GTPase activating protein that can dampen smooth muscle contractility by turning off Gq signals. Approaching labour, inflammatory signals may downregulate RGS2 expression to derepress contractility. Understanding the regulatory role of RGS2 on uterine contractility may provide insights for improved clinical management of spontaneous preterm labour, to prevent premature birth. We hypothesise that RGS2, regulated by mediators that control quiescence and labour, reduces contractile functions of Gq coupled signals in the pregnant uterus. To test this hypothesis, we used primary myometrial smooth muscle (MSM) cells isolated from uterine biopsies collected at term (>37 weeks) non-labour caesarean deliveries. Treatment with 1μM PGE2 increased RGS2 mRNA and protein expression, peaking at 1 and 2 hours respectively. Pretreatment with 1ng/ml IL-1β or TNFα partially repressed the PGE2 effect, suggesting RGS2 may be downregulated in the inflammatory milieu that accompanies labour. To study the effect of RGS2 on MSM function, cells were loaded with Fluo-4 NW to detect intracellular calcium changes as a measure of contractility following challenge with 100nM oxytocin. Enhanced RGS2 expression, following PGE2 treatment or adenoviral overexpression, reduced oxytocin-mediated calcium flux, suggesting RGS2 attenuates this contractile signal. In addition to oxytocin, labour contractions likely integrate activation of many other GPCRs, signaling of which may also be regulated by RGS2. A PCR-based array analysis revealed expression of >300 GPCRs in MSM cells and tissue. The highly expressed histamine receptor 1 was targeted to begin exploring additional signals attenuated by RGS2 in MSM. Histamine (1μM) challenged cells generated a calcium response that was attenuated by PGE2-induced RGS2, and RGS2 adenoviral overexpression. Together our results show RGS2 is upregulated by pro-quiescent signals abundant in pregnancy, and downregulated by inflammatory mediators implicated in labour. Enhanced RGS2 expression reduces contractile responses to oxytocin and histamine, which we propose is consistent with a pro-quiescent role in pregnancy. Funding: CIHR, NSERC Project Grants; AIHS MD-PhD Studentship.