Ca²⁺/calmodulin-dependent protein kinase IV activates cysteine-rich protein 1 through adjacent CRE and CArG elements

Abstract

Smooth muscle-specific transcription is controlled by a multitude of transcriptional regulators that cooperate to drive expression in a temporospatial manner. Previous analysis of the cysteine-rich protein 1 (CRP1/Csrp) gene revealed an intronic enhancer that is sufficient for expression in arterial smooth muscle cells and requires a serum response factor-binding CArG element for activity. The presence of a CArG box in smooth muscle regulatory regions is practically invariant; however, it stands to reason that additional elements contribute to the modulation of transcription in concert with the CArG. Because of the potential importance of other regulatory elements for expression of the CRP1 gene, we sought to identify additional motifs within the enhancer that are necessary for expression. In this effort, we identified a conserved cAMP response element (CRE) that, when mutated, diminishes the expression of the enhancer in cultured vascular smooth muscle cells. Using transfection and electrophoretic mobility shift assays, we have shown that the CRE binds the cAMP response element-binding protein (CREB) and is activated by Ca2+/calmodulin-dependent protein kinase IV (CaMKIV), but not by CaMKII. Furthermore, our data demonstrate that CaMKIV stimulates CRP1 expression not only through the CRE but also through the CArG box. These findings represent evidence of a functional CRE within a smooth muscle-specific gene and provide support for a mechanism in which CREB functions as a smooth muscle determinant through CaMKIV activation.

Loss of the Serum Response Factor Cofactor, Cysteine-Rich Protein 1, Attenuates Neointima Formation in the Mouse

“…The ability of the wildtype and Csrp1 −/− cells to activate the SMA-reporter was SRF-dependent, as a SMA-reporter with a mutated CArG was not induced in either cell type by TGF-beta. Consistent with our studies with the SMA reporter, a luciferase reporter containing 3 copies of the intronic CArG element from the Csrp1 gene 12, 26 was also upregulated by TGF-beta in the WT and Csrp1 −/− cells, while the Csrp2 −/− cells were unresponsive (supplementary data, Figure S1, panel A). …”

Protein Kinase A-regulated Assembly of a MEF2·HDAC4 Repressor Complex Controls c-Jun Expression in Vascular Smooth Muscle Cells

“…This observation likely explains the absence of widespread MEF2 activation in vivo following vascular injury. Of the various CaMKs, CaMKII␦ appears to be the most likely kinase involved in c-Jun induction, given that recent evidence has demonstrated a critical role of this isoform during neointima formation, whereas CaMKIV has been implicated in VSMC differentiation (55,56). In addition, we demonstrate that PKA can enhance the repression of c-jun by increasing the nuclear localization of HDAC4 through inhibition of SIK1.…”

CRP1, a Protein Localized in Filopodia of Growth Cones, Is Involved in Dendritic Growth

“…Although several transcriptional mechanisms have been identified for the Ca 2ϩ -dependent dendritic arborization, few transcriptionally regulated effectors have been reported. In smooth muscle cells, CRP1 expression is increased after depolarization induced by high K ϩ (Najwer and Lilly, 2005), suggesting that CRP1 may be involved in postdepolarization modifications. Because our data showed that CRP1 is required for dendritic growth in neurons, we hypothesized that CRP1 may act as a mediator for dendritic growth induced by Ca 2ϩ influx.…”