Convergence of Multiple Protein Kinase Pathways of Angiotensin II Type 1 Receptor on cAMP Response Element-Binding Protein (CREB) in Rat Vascular Smooth Muscle Cells
We previously reported a critical role of cyclic AMP response element (CRE) of interleukin-6 gene promoter in the induction of interleukin-6 gene expression by angiotensin II (AngII). We examined signaling pathways that are responsible for AngII-induced phosphorylation of CRE binding protein (CREB) at serine 133 that is a critical marker for the activation in rat vascular smooth muscle cells (VSMC). We performed Western blot analysis using an antibody against the phosphorylated form of CREB. AngII time-dependently induced phosphorylation of CREB with a peak at 5 minutes. The phosphorylation was dose-dependent. The AngII-induced phosphorylation of CREB was blocked by CV11974, an AngII type I receptor (AT1-R) antagonist but not by PD123319, an antagonist of Ang II type 2 receptor, suggesting that AT1-R mediates the phosphorylation of CREB. Inhibition of extracellular signal-regulated protein kinase (ERK) by PD98059 or inhibition of p38 mitogen activated protein kinase (MAPK) by SB203580 partially inhibited AngII-induced CREB phosphorylation. A protein kinase A (PKA) inhibitor, H89, also partially suppressed AngII-induced CREB phosphorylation. However, inhibition of calmodulin dependent kinase II or phosphatydilinositol 3 kinase did not affect Ang II-induced CREB phosphorylation. Transcriptional activation as measured by a CRE/luciferase reporter was increased by 1.7-fold by AngII stimulation. Infection of adenovirus vector expressing dominant negative form of CREB, which the serine residue at 133 is replaced with alanine to VSMC partially suppressed Ang II-induced thymidine incorporation. These findings suggest that AngII activates multiple protein kinase pathways involving two MAPK pathways and PKA, all of which contribute to the activation of CREB. And CREB-dependent gene transcription plays an important role for Ang II-induced mitogenesis. Activation of CREB-dependent gene transcription may play important roles in cardiovascular remodeling process.