Relaxin Stimulates Protein Kinase C ζ Translocation: Requirement for Cyclic Adenosine 3′,5′-Monophosphate Production

Abstract

AbstractRelaxin is a polypeptide hormone that activates the leucine-rich repeat containing G protein-coupled receptors, LGR7 and LGR8. In an earlier study, we reported that relaxin produces a biphasic time course and the second wave of cAMP is highly sensitive to phosphoinositide-3 kinase inhibitors (LY294002 and wortmannin). LY294002 inhibits relaxin-mediated increases in cAMP production by 40–50% across a large range of relaxin concentrations. Here we show that protein kinase C ζ (PKCζ) is a component of relaxin signaling in THP-1 cells. Sphingomyelinase increases cAMP production due to the release of ceramide, a direct activator of PKCζ. Chelerythrine chloride (a general PKC inhibitor) inhibits relaxin induced cAMP production to the same degree (∼40%) as LY294002. Relaxin stimulates PKCζ translocation to the plasma membrane in THP-1, MCF-7, pregnant human myometrial 1–31, and mouse mesangial cells, as shown by immunocytochemistry. PKCζ translocation is phosphoinositide-3 kinase dependent and independent of cAMP production. Antisense PKCζ oligodeoxynucleotides (PKCζ-ODNs) deplete both PKCζ transcript and protein levels in THP-1 cells. PKCζ-ODNs abolish relaxin-mediated PKCζ translocation and inhibit relaxin stimulation of cAMP by 40%, as compared with mock and random ODN controls. Treatment with LY294002 in the presence of PKCζ-ODNs results in little further inhibition. In summary, we present a novel role for PKCζ in relaxin-mediated stimulation of cAMP.