Original articleNuclear accumulation of the AT1 receptor in a rat vascular smooth muscle cell line: effects upon signal transduction and cellular proliferation
Introduction
We have reported that co-expression of a fluorescent fusion protein of AII (ECFP/AII) with a fluorescent fusion protein of the AT1 receptor (AT1R/EYFP) alters the receptor distribution and increases proliferation in COS-7 and CHO-K1 cells [1]. The present studies were designed to confirm these physiological effects in vascular smooth muscle cells (VSMCs) and to identify downstream signaling pathways affected by expression of intracellular AII (ECFP/AII). Since exogenous AII has been reported to stimulate CREB phosphorylation in several systems, we investigated the potential for intracellular AII–AT1 receptor interactions to stimulate CREB activation in A10 VSMCs (which express the AT1R [2], [3], [4], [5]). We further investigated the kinase pathways involved in CREB phosphorylation activation by intracellular AII (IC AII). Cammarota et al. [6] report that extracellular AII stimulates CREB phosphorylation in bovine adrenal chromaffin cells through an ERK1/2-dependent mechanism. In VSMCs, however, exogenous AII induces activation of CREB and transcription from the fibronectin promoter via p38MAPK activation [7]. Furthermore, studies by Ichiki and associates (Funakoshi et al. [8]) suggest that p38MAPK, ERK1/2 and PKA may all be involved in AII-mediated CREB phosphorylation and downstream c-fos expression, protein synthesis and VSMC hypertrophy. Based on these principles, we sought to determine whether intracellular AII similarly activates multiple signaling pathways in VSMCs and to what extent these overlap with pathways activated by extracellular AII.
Section snippets
Plasmids
pECFP-C1 and pEYFP-N1 (otherwise referred to as pECFP and pEYFP in this paper) are control vehicles into which desired fusion protein-encoding DNA sequences can be cloned (Clontech, Palo Alto, CA, USA). In the C1 vector, encoded fused proteins are present at the C-terminus of the fluorescent protein moiety; in the N1 vector, encoded fused proteins are present at the N-terminus. pAT1R/EYFP (encodes a fusion protein of the rat AT1R), pECFP/AII (encodes a fusion protein of AII) and pECFP/AIIC
Microscopy
In order to determine the effects of IC AII on A10 cells we used a fusion construct of the AII peptide-encoding sequence ligated downstream from enhanced cyan fluorescent protein (blue fluorescent protein) [1]. As a control, we used a similar plasmid encoding a peptide of scrambled AII sequence fused to ECFP. In each case, the encoded peptide is separated from ECFP by a 10-amino-acid spacer arm. In previous studies, ECFP/AII proved to be reactive to both anti-AII and anti-EGFP antibodies as
Discussion
In adrenal medulla cells, AII regulates catecholamine biosynthesis in a CREB-dependent manner. Cammarota et al. [6] have determined that AII-mediated CREB phosphorylation in bovine adrenal chromaffin cells is blocked by an ERK1/2 inhibitor but not by inhibitors of p38MAPK. Furthermore, prior studies have suggested that the AT1R induces Src-dependent increases in Ras activation and formation of Ras-RAF1 complexes [17]. CREB has been found to be critical for AII-induced hypertrophy of VSMCs [8].
Acknowledgments
This work was supported by Ochsner Clinic Foundation and NIH/NHLBI HL072795.
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