Intracellular @entity1808 increases the long isoform of PDGF mRNA in @entity35 XXXX cells.

@entity9113 @entity1808 @entity70979 @entity3449 @entity1129 @entity35 @entity157 @entity10340
Our recent published studies suggest that @entity1808 ( @entity1808 ), generated and retained intracellularly, enhances growth of H4-II-E-C3 @entity35 @entity157 cells, an average of 33%. Proliferation conferred by introduction of a plasmid [ @entity10340 (-S)Exp/pSVL ] encoding a signal sequence-depleted angiotensinogen [ @entity10340 (-S)Exp] into these cells (which we have shown possess ACE and @entity3449 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 @entity1808 interaction with an @entity1129 (1)-like receptor. Introduction of anti- @entity1808 antibodies into the medium of these transfected cells has no effect upon growth of the cells, suggesting that @entity1808 is retained by the cells and that intracellular @entity1808 is growth stimulatory. In the present study, we sought to further characterize the intracellular localization and mode of action of @entity10340 (-S)Exp. Consistent with our expectations, we now show that a fusion product of @entity10340 (-S)Exp with green fluorescent protein [ @entity10340 (-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 @entity10340 (-S)Exp transfected cells and in @entity1808 -treated naive cells but not in losartan-treated @entity10340 (-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 @entity70979 mRNAs (the corresponding proteins of which are sufficient for amidation). Inhibitors of amidation inhibit growth of naive and @entity10340 (-S)Cntr/ pSVL -transfected cells (2.6-fold for phenylbutenoic acid and 3.5-fold for disulfiram treatment) but more profoundly inhibit growth of @entity10340 (-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 @entity10340 (-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, @entity9113 and calcitonin), we postulate that growth effects of both intracellular @entity1808 and exogenous @entity1808 can be conferred by PDGF long-form, possibly through an amidation-dependent mechanism.
