Transforming growth factor-β1 stimulates protein kinase A in mesangial cells

We recently demonstrated that transforming growth factor-β (TGF-β) stimulates phosphorylation of the type I inositol 1,4,5-trisphosphate receptor (Sharma, K., Wang, K., Zhu, Y., Bokkala, S., and Joseph, S. (1997) J. Biol. Chem. 272, 14617-14623), possibly via protein kinase A (PKA) activation in murine mesangial cells. In the present study, we evaluated whether TGF-β stimulates PKA activation. Utilizing a specific PKA kinase assay, we found that TGF-β increases PKA activity by 3-fold within 15 min of TGF-β1 treatment, and the enhanced kinase activity was completely reversed by the inhibitory peptide for PKA (PKI; 1 μM). In mesangial cells transfected with a PKI expression vector, enhanced PKA activity could not be demonstrated with TGF-β1 treatment. TGF-β1 was also found to stimulate translocation of the α-catalytic subunit of PKA to the nucleus by Western analysis of nuclear protein as well as by confocal microscopy. TGF-β1- mediated phosphorylation of cAMP response element-binding protein was completely reversed by H-89 (3 μM), a specific inhibitor of PKA. Stimulation of fibronectin mRNA by TGF-β1 was also attenuated in cells overexpressing PKI. We thus conclude that TGF-β stimulates the PKA signaling pathway in mesangial cells and that PKA activation contributes to TGF-β stimulation of cAMP response element-binding protein phosphorylation and fibronectin expression.