Relaxin stimulates protein kinase C ζ translocation:: Requirement for cyclic adenosine 3′,5′-monophosphate production

Relaxin 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 zeta (PKC zeta) is a component of relaxin signaling in THP-1 cells. Sphingomyelinase increases cAMP production due to the release of ceramide, a direct activator of PKC zeta. Chelerythrine chloride (a general PKC inhibitor) inhibits relaxin induced cAMP production to the same degree (similar to 40%) as LY294002. Relaxin stimulates PKC zeta translocation to the plasma membrane in THP-1, MCF-7, pregnant human myometrial 1-31, and mouse mesangial cells, as shown by immunocytochemistry. PKC zeta translocation is phosphoinositide-3 kinase dependent and independent of cAMP production. Antisense PKC zeta oligodeoxynucleotides (PKC zeta-ODNs) deplete both PKC zeta transcript and protein levels in THP-1 cells. PKC zeta-ODNs abolish relaxin-mediated PKC zeta translocation and inhibit relaxin stimulation of cAMP by 40%, as compared with mock and random ODN controls. Treatment with LY294002 in the presence of PKC zeta-ODNs results in little further inhibition. In summary, we present a novel role for PKC zeta in relaxin-mediated stimulation of cAMP.