|Role of Rho family proteins in phospholipase D activation by growth factors.|
Hess, J A, et al.
J. Biol. Chem., 272: 1615-20 (1997)
Treatment of fibroblasts with growth factors results in activation of phospholipase D (PLD). In order to determine the role of the Rho family of small GTPases in growth factor-mediated PLD activation, we used cells transfected with wild type and mutant Rac1. In response to epidermal growth factor (EGF), PLD activity was greatly increased in Rat1 fibroblasts expressing wild type Rac1 (wtRac1), and completely abrogated in cells expressing dominant negative N17Rac1, consistent with Rac1 mediating the action of this growth factor. In contrast, in cells treated with platelet-derived growth factor (PDGF) or phorbol ester, the wtRac1 cells showed little or no enhancement of PLD activity, and the response was not affected in the N17Rac1 cells, implying that Rac1 played a minimal role in the activation of PLD by PDGF or protein kinase C. Both growth factors produced an attenuated PLD response in cells expressing constitutively active V12Rac1, but these cells showed other changes, including altered morphology, increased basal PLD, and decreased growth factor receptor autophosphorylation. The effects of EGF and PDGF on phosphoinositide phospholipase C activity were not enhanced in cells expressing wtRac1 or inhibited in those expressing N17Rac1. In cells expressing constitutively active V12Rac1, basal phosphoinositide phospholipase C was elevated, but there were no significant effects of EGF or PDGF. We used C3 transferase of Clostridium botulinum, which ADP-ribosylates and inactivates RhoA, to investigate the involvement of RhoA in the activation of PLD by PDGF. Cells expressing wtRac1 and N17Rac1 showed a decreased PLD in response to PDGF when treated with C3 transferase, indicating a role for RhoA. In summary, these data indicate a major role for Rac1 in the activation of PLD by EGF, but not PDGF or protein kinase C.
|Cell shape-dependent regulation of protein 4.1 alternative pre-mRNA splicing in mammary epithelial cells.|
Schischmanoff, P O, et al.
J. Biol. Chem., 272: 10254-9 (1997)
Expression of the complex gene encoding multiple isoforms of structural protein 4.1 is regulated by alternative pre-mRNA splicing. During erythropoiesis, developmental stage-specific inclusion of exon 16 generates protein 4.1 isoforms having a fully functional spectrin-actin binding domain. Here we show that human mammary epithelial cells (HMEC), coincident with the dramatic morphological changes induced by altered culture conditions, exhibit a novel pre-mRNA splicing switch involving a new exon (exon 17B, 450 nucleotides) in the COOH-terminal coding region. 4.1 RNA expressed in proliferating HMEC adherent to culture dishes mostly excluded exon 17B, whereas 4.1 transcripts processed in nondividing suspension cultures of HMEC strongly included this exon. This pre-mRNA splicing switch was reversible: cells transferred from poly(2-hydroxyethyl methacrylate) back to plastic resumed cell division and down-regulated exon 17B expression. More detailed studies revealed complex tissue-specific alternative splicing of exon 17B and another new exon 17A (51 nucleotides). These results predict the existence of multiple 4.1 protein isoforms with diverse COOH termini. Moreover, they strongly suggest that regulation of gene expression during differentiation of epithelial cells is mediated not only by transcriptional mechanisms, but also by post-transcriptional processes such as alternative pre-mRNA splicing.
|Sequence-independent inhibition of in vitro vascular smooth muscle cell proliferation, migration, and in vivo neointimal formation by phosphorothioate oligodeoxynucleotides.|
Wang, W, et al.
J. Clin. Invest., 98: 443-50 (1996)
Phosphorothioate oligodeoxynucleotides (PS oligos) are antisense (sequence-specific) inhibitors of vascular smooth muscle cell (SMC) proliferation when targeted against different genes. Recently an aptameric G-quartet inhibitory effect of PS oligos has been demonstrated. To determine whether PS oligos manifest non-G-quartet, non-sequence-specific effects on human aortic SMC, we examined the effects of S-dC28, a 28-mer phosphorothioate cytidine homopolymer, on SMC proliferation induced by several SMC mitogens. S-dC28 significantly inhibited SMC proliferation induced by 10% FBS as well as the mitogens PDGF, bFGF, and EGF without cytotoxicity. Moreover, S-dC28 abrogated PDGF-induced in vitro migration in a modified micro-Boyden chamber. Furthermore, S-dC28 manifested in vivo antiproliferative effects in the rat carotid balloon injury model. S-dC28 suppressed neointimal cross-sectional area by 73% and the intima/media area ratio by 59%. Therefore, PS oligos exert potent non-G-quartet, non-sequence-specific effects on in vitro SMC proliferation and migration as well as in vivo neointimal formation.