Relaxin stimulates multiple signaling pathways: activation of cAMP, PI3K, and PKCzeta in THP-1 cells.

Relaxin has been shown previously to stimulate cyclic AMP production and the activation of MAPK. We reported that phosphoinositide-3 kinase (PI3K) activity is required for biphasic stimulation of cAMP by relaxin and that relaxin treatment increased PI3K activity in THP-1 cells. A downstream target of PI3K is protein kinase C zeta (PKCzeta). Relaxin stimulated translocation of PKCzeta to the plasma membrane in THP-1, MCF-7, pregnant human myometrial (PHM1-31), and mouse mesangial (MMC) cells. PKCzeta translocation is PI3K dependent and independent of cAMP production. Pharmacological and antisense approaches, utilized to inhibit or knock down PKCzeta, resulted in a 40% inhibition of relaxin-stimulated cAMP production. The stimulation of PKCzeta by relaxin therefore is downstream of PI3K leading to increased cAMP production. To determine the role of PI3K/PKCzeta stimulation by relaxin on downstream-mediated events, we examined the increase in vascular endothelial growth factor (VEGF) gene expression by relaxin. Treatment of THP-1 or MMC cells with the PI3K inhibitor, LY294002, abolished the relaxin-mediated stimulation of VEGF transcript levels. In summary, relaxin has pleiotropic signaling effects in THP-1 cells activating ERK1/2, cAMP, PI3K, and PKCzeta. We have described a novel bifurcated pathway by which relaxin stimulates Gs alpha and PI3K/PKCzeta leading to increased cAMP production and increased VEGF gene expression. Some, but not all, of these pathways are detected in other cell lines which may cause the unique diversity of downstream responses from this interesting hormone.