Rapid communicationEvidence of a novel intracrine mechanism in angiotensin II-induced cardiac hypertrophy
Introduction
Classically, peptide growth factors and hormones act in the extracellular space by binding to specific receptors on the cell surface and initiating intracellular signaling events, which lead to the biological effect. However, there is evidence for another mechanism in which some of these peptides act intracellularly, without binding to receptors on the extracellular surface of the cell [1], [2]. The latter represents an intracrine mechanism, a term more commonly used in conjunction with steroid hormone actions [3]. The significance of an intracrine action is that the intracellular presence of a peptide hormone can either modulate traditional signaling events, originating from the plasma membrane receptor, by potentiation or antagonism or have an altogether different effect [4], [5].
The renin–angiotensin system (RAS) has a significant role in cardiac hypertrophy. Angiotensin II (Ang II), the bioactive component of the RAS, binds to two types of specific cell surface receptors, AT1 and AT2, which are seven-transmembrane G-protein coupled receptors (GPCRs). AT1, the predominant receptor in adult tissue, links to growth stimulatory signaling; while AT2 is more prominent in fetal tissue and is generally growth inhibitory [6]. In addition to plasma membrane, AT1-like Ang II receptors have been detected in the sarcolemma, T-tubules and nuclei of rat cardiac myocytes using electron microscopic and immunofluorescence–cytochemistry techniques [7]. Several studies have shown that intracellular Ang II receptors are functionally active. In rat hepatocytes, the AT1-like nuclear receptor couples to gene transcription and is blocked by the specific nonpeptide receptor antagonist, losartan [8], [9]. In vascular smooth muscle cells, intracellular application of Ang II leads to a rapid increase in intracellular calcium concentration and induces cell growth [10], [11]. In cardiomyopathic hamsters, at an advanced stage of the disease, microinjection of Ang II in cardiac myocytes abolishes cell coupling [12]. These effects of iAng II could be suppressed by intracellular, but not extracellular losartan or candesartan, suggesting a role for intracellular AT1. Ang II may also have effects independent of the AT1 receptor. Utilizing immunohistochemical techniques, Ang II was localized to nuclei in cerebellar neurons and to the transcriptionally active euchromatin in endothelial and granule cells, suggesting that Ang II directly regulates gene expression [13]. Ang II binding to solubilized rat liver chromatin fragments, the existence of a discrete Ang II-binding nucleoprotein particle and direct effects of nuclear localized Ang II on transcription have also been demonstrated [14], [15]. In hepatoma cells, intracellularly generated Ang II is mitogenic, a response that involves Ang II interaction with an intracellular AT1-like receptor [16], [17].
In cardiac tissue, Ang II has been depicted as an endocrine, as well as an autocrine and a paracrine factor. The possibility of an intracrine mode of action has not been studied with respect to cardiac hypertrophy. Since RAS components in the heart are increased in cardiac hypertrophy [18], [19], it is likely that Ang II levels are also increased inside the cell which might induce intracrine effects. In this study, we tested the hypothesis that intracellular angiotensin II (iAng II) participates in the cardiac hypertrophic process via an intracrine mechanism. We produced Ang II peptide intracellularly using recombinant adenoviral and plasmid expression vectors and studied the hypertrophic growth in vitro and in vivo.
Section snippets
Adenoviral expression vectors
The adenoviral expression vectors, for in vitro studies, were constructed using the Adeno-X Tet-Off gene expression system (Clontech, Palo Alto, CA). Two complimentary oligonucleotides containing coding sequence for the eight amino acids of Ang II, start and stop codons and flanked by BamHI and XbaI sites (sense strand: 5′-GATCCATGGACCGCGTATACATCCACCCCTTTTAAT-3′), were annealed and cloned into the pTRE-Shuttle plasmid. The expression cassette from the recombinant shuttle plasmid was ligated to
Intracellular expression of Ang II
To express Ang II intracellularly, we used vectors that coded only for the Ang II peptide, instead of the complete angiotensinogen (Ao) gene, to make certain that Ang II generation did not depend upon the availability of other RAS components. The omission of the signal sequence ensured that the recombinant Ang II was retained intracellularly. Adenoviral vectors, controlled by a tetracycline regulated minimal CMV promoter (Fig. 1), were used for in vitro experiments to overcome the inefficient
Discussion
In the present investigation, we identify a novel mechanism of Ang II induced cardiac hypertrophy that does not require Ang II-AT1 receptor interaction at the plasma membrane. Ang II was expressed intracellularly by removing the signal peptide. The possibility that iAng II ‘leaked’ into the extracellular space and had a growth effect was excluded by the fact that no significant increase in extracellular Ang II was observed in culture medium of Ad-AngII infected myocytes. Similarly, in vivo
Acknowledgements
This work was supported by NIH grant HL44883.
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