Original article
Nuclear accumulation of the AT1 receptor in a rat vascular smooth muscle cell line: effects upon signal transduction and cellular proliferation

https://doi.org/10.1016/j.yjmcc.2005.11.014Get rights and content

Abstract

The objective of the study was to identify the functional outcome of intracellular versus extracellular angiotensin II–AT1 receptor interactions in vascular cells. Rat vascular smooth muscle cell line A10 was transfected, independently and concurrently, with plasmids encoding fluorescent fusion proteins of rat angiotensin II (pECFP/AII, encodes AII fused downstream of enhanced cyan fluorescent protein) and the rat AT1a receptor (pAT1R/EYFP, encodes the rat AT1a receptor fused upstream of enhanced yellow fluorescent protein). The AII fluorescent fusion protein possesses no secretory signal peptide and deconvolution microscopy established that is maintained within these cells predominantly in the nucleus. AT1R/EYFP was absent from the nucleus when expressed exclusively or in untreated cells but accumulated in the nucleus following exogenous AII treatment or when co-expressed with ECFP/AII. Furthermore, expression of ECFP/AII stimulated proliferation of A10 vascular smooth muscle cells (VSMCs) 1.6-fold (P < 0.05). Transfection of a control, pECFP/AIIC (which encodes a scrambled AII peptide fused to ECFP) had no growth effect. In light of the intracellular growth effects of ECFP/AII, we sought to elucidate the underlying signaling pathways. We found that extracellular AII treatment of A10 cells activated cAMP response element-binding protein (CREB) as determined by one-hybrid assays and immunoblots. Expression of intracellular ECFP/AII similarly activated CREB. However, intracellular and extracellular AII activated CREB through different phosphorylation pathways. Exogenous AII treatment of A10 cells activated p38MAPK and ERK1/2 phosphorylation as determined by Western blot analyses and one-hybrid assays. The p38MAPK inhibitor, SB203580, and the ERK kinase inhibitor, PD98059 each partially inhibited exogenous AII-conferred CREB activation confirming that p38MAPK and ERK1/2 mediate CREB phosphorylation in this system. In contrast, expression of ECFP/AII (intracellular AII) in A10 VSMCs activated p38MAPK but not ERK1/2; inhibition of p38MAPK by SB203580 inhibited intracellular AII-induced CREB phosphorylation. In summary, extracellular AII stimulates at least one pathway common to intracellular AII. This common pathway, in the case of exogenous AII, likely reflects intracellular signaling following internalization of receptor–ligand complex. Extracellular AII also stimulates a unique pathway, apparently reflecting interaction with plasma membrane-associated AT1R.

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.

References (29)

  • T. Kambe et al.

    Basal transcriptional regulation of rat AT1 angiotensin II receptor gene expression

    Clin. Exp. Pharmacol. Physiol.

    (2004)
  • C. Savoia et al.

    Negative regulation of RhoA/Rho kinase by angiotensin II type 2 receptor in vascular smooth muscle cells: role in angiotensin II-induced vasodilation in stroke-prone spontaneously hypertensive rats

    J. Hypertens.

    (2005)
  • M. Cammarota et al.

    Angiotensin II promotes the phosphorylation of cyclic AMP-responsive element binding protein (CREB) at Ser133 through an ERK1/2-dependent mechanism

    J. Neurochem.

    (2001)
  • A.I. Gotlieb et al.

    Mechanochemical proteins, cell motility and cell–cell contacts: the localization of mechanochemical proteins inside cultured cells at the edge of an in vitro “wound”

    J. Cell. Physiol.

    (1979)
  • Cited by (0)

    View full text