Mechanisms of Hormone Action

Multiple Signals Regulate PLC beta 3 in Human Myometrial Cells

Abstract
Phospholipase CB3 (PLCB3) Serine¹¹⁰⁵, 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-Serine¹¹⁰⁵ phosphorylation (P-S¹¹⁰⁵) was studied in immortalized and primary human myometrial cells. 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 PGF2alpha and ATP. Oxytocin-stimulated phosphorylation was blocked by the 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 shRNA constructs significantly attenuated oxytocin-stimulated increases in intracellular calcium. Oxytocin-induced phosphatidylinositol (PI) turnover was inhibited by CPT-cAMP and okadaic acid but 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.

Key words: Kinases • Phosphatases • myometrium • phospholipase C beta 3 • phosphorylation

This article has been cited by other articles:

				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]