Phosphorylation of the Cyclic AMP Response Element Binding Protein Mediates Transforming Growth Factor β-Induced Downregulation of Cyclin A in Vascular Smooth Muscle Cells

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Abstract
Transforming growth factor β (TGFβ), a multifunctional cytokine associated with vascular injury, is a potent inhibitor of cell proliferation. The current results demonstrate that the TGFβ-induced growth arrest of vascular smooth muscle cells (VSMCs) is associated with cyclin A downregulation. TGFβ represses the cyclin A gene through a cyclic AMP (cAMP) response element, which complexes with the cAMP response element binding protein (CREB). The CREB-cyclin A promoter interaction is hindered by TGFβ, preceded by a TGFβ receptor-dependent CREB phosphorylation. Induction of CREB phosphorylation with forskolin or 6bnz-cAMP mimics TGFβ's inhibitory effect on cyclin A expression. Conversely, inhibition of CREB phosphorylation with a CREB mutant in which the phosphorylation site at serine 133 was changed to alanine (CREB-S133A) upregulated cyclin A gene expression. Furthermore, the CREB-S133A mutant abolished TGFβ-induced CREB phosphorylation, cyclin A downregulation, and growth inhibition. Since we have previously shown that the novel PKC isoform protein kinase C delta (PKCδ) is activated by TGFβ in VSMCs, we tested the role of this kinase in CREB phosphorylation and cyclin A downregulation. Inhibition of PKCδ by a dominant-negative mutant or by targeted gene deletion blocked TGFβ-induced CREB phosphorylation and cyclin A downregulation. Taken together, our data indicate that phosphorylation of CREB stimulated by TGFβ is a critical step leading to the inhibition of cyclin A expression and, thus, VSMC proliferation.