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Multiple Signals Regulate Phospholipase CBeta3 in Human Myometrial Cells¹

Department of Biomedical Sciences, Colorado State University, Fort Collins, Colorado 80523

ABSTRACT

Phospholipase CB3 (PLCB3) serine¹¹⁰⁵ (S¹¹⁰⁵), a substrate for multiple protein kinases, represents a potential point of convergence of several signaling pathways in the myometrium. To explore this hypothesis, the regulation of PLCB3-S¹¹⁰⁵ phosphorylation (P-S¹¹⁰⁵) was studied in immortalized and primary human myometrial cells. 8-[4-chlorophenylthio] (CPT)-cAMP and calcitonin gene-related peptide (CALCA) transiently increased P-S¹¹⁰⁵. Relaxin also stimulated P-S¹¹⁰⁵; this effect was partially blocked by the protein kinase A (PRKA) inhibitor, Rp-8-CPT-cAMPS. Oxytocin, which stimulates Galphaq-mediated pathways, also rapidly increased P-S¹¹⁰⁵, as did prostaglandin F2alpha and ATP. Oxytocin-stimulated phosphorylation was blocked by protein kinase C (PRKC) inhibitor Go6976 and by pretreatment overnight with a phorbol ester. Cypermethrin, a PP2B phosphatase inhibitor, but not okadaic acid, a PP1/PP2A inhibitor, prolonged the effect of CALCA on P-S¹¹⁰⁵, whereas the reverse was the case for the oxytocin-stimulated increase in P-S¹¹⁰⁵. PLCB3 was the predominant PLC isoform expressed in the myometrial cells and PLCB3 short hairpin RNA constructs significantly attenuated oxytocin-stimulated increases in intracellular calcium. oxytocin-induced phosphatidylinositol (PI) turnover was inhibited by CPT-cAMP and okadaic acid, but was enhanced by pretreatment with Go6976. CPT-cAMP inhibited oxytocin-stimulated PI turnover in the presence of overexpressed PLCB3, but not overexpressed PLCB3-S¹¹⁰⁵A. These data demonstrate that both negative crosstalk from the cAMP/PRKA pathway and a negative feedback loop in the oxytocin/G protein/PLCB pathway involving PRKC operate in myometrial cells and suggest that different protein phosphatases predominate in mediating P-S¹¹⁰⁵ dephosphorylation in these pathways. The integration of multiple signal components at the level of PLCB3 may be important to its function in the myometrium.

kinases, myometrium, phosphatases, phospholipase CB3, phosphorylation, protein kinase A, protein kinase C, protein phosphatase

¹Supported in part by National Institutes of Health grant NIH-HD09618.

Correspondence: ²Barbara M. Sanborn, Department of Biomedical Sciences, 102 Physiology Campus Delivery 1680, Colorado State University, Fort Collins, CO 80523. FAX: 970 491 7569; e-mail: Barbara.Sanborn{at}colostate.edu

³These authors contributed equally to this work.

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				K. Heng, R. Ivell, P. Wagaarachchi, and R. Anand-Ivell Relaxin signalling in primary cultures of human myometrial cells Mol. Hum. Reprod., October 1, 2008; 14(10): 603 - 611. [Abstract] [Full Text] [PDF]