Intracellular angiotensin II increases the long isoform of PDGF mRNA in rat hepatoma cells

被引:33
作者
Cook, JL [1 ]
Giardina, JF [1 ]
Zhang, Z [1 ]
Re, RN [1 ]
机构
[1] Alton Ochsner Med Fdn & Ochsner Clin, Div Res, New Orleans, LA 70121 USA
关键词
angiotensin II; intracrine; enhanced green fluorescent protein; PDGF; amidation;
D O I
10.1006/jmcc.2002.2106
中图分类号
R5 [内科学];
学科分类号
1002 ; 100201 ;
摘要
J. L. COOK, J. F. GIARDINA, Z. ZHANG AND R. N. RE. Intracellular Angiotensin II Increases the Long Isoform of PDGF mRNA in Rat Hepatoma Cells. Journal of Molecular and Cellular Cardiology (2002) 34, 1525-1537. Our recent published studies suggest that angiotensin II (All), generated and retained intracellularly, enhances growth of H4-II-E-C3 rat hepatoma cells, an average of 33%. Proliferation conferred by introduction of a plasmid [Ang(-S)Exp/pSVL] encoding a signal sequence-depleted angiotensinogen [Ang(-S)Exp] into these cells (which we have shown possess ACE and renin mRNAs) is mediated, at least in part, by enhanced PDGF-A chain mRNA production and protein secretion. The mitogenic effect is inhibited by losartan suggesting that it involves All interaction with an AT(1)-like receptor. Introduction of anti-All antibodies into the medium of these transfected cells has no effect upon growth of the cells, suggesting that All is retained by the cells and that intracellular All is growth stimulatory. In the present study, we sought to further characterize the intracellular localization and mode of action of Ang(-S)Exp. Consistent with our expectations, we now show that a fusion product of Ang(-S)Exp with green fluorescent protein [Ang(-S)Exp/EGFP], generated from an expression plasmid, is abundant and primarily cytoplasmic. Wild-type angiotensinogen/EGFP, in contrast. is only detectable following a cold-block (which acts to enhance folding-kinetics and slow secretion) and is largely restricted to the secretory pathway. We further show, using semi-quantitative RT/PCR that the long isoform of PDGF mRNA is elevated in Ang(-S)Exp transfected cells and in All-treated naive cells but not in losartan-treated Ang(-S)Exp transfected cells. We identify C-terminal amidation recognition sites within the long-form protein (that are not present in the short-form) and show that these cells possess PAM (amidating enzyme precursor) and carboxypeptidase E mRNAs (the corresponding proteins of which are sufficient for amidation). Inhibitors of amidation inhibit growth of naive and Ang(-S)Cntr/pSVL-transfected cells (2.6-fold for phenylbutenoic acid and 3.5-fold for disulfiram treatment) but more profoundly inhibit growth of Ang(-S)Exp/pSVL-transfected cells (6.7-fold for phenylbutenoic acid and 13-fold for disulfiram). In conclusion, these data confirm that signal sequence-depleted Ang(-S)Exp is retained within cells and is largely cytoplasmic. Because C-terminal amidation is absolutely required for full biological potency of a number of peptide hormones (including oxytocin, gastrin and calcitonin), we postulate that growth effects of both intracellular All and exogenous All can be conferred by PDGF long-form, possibly through an amidation-dependent mechanism. (C) 2002 Published by Elsevier Science Ltd.
引用
收藏
页码:1525 / 1537
页数:13
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