Inhibition of translation in cells infected with a poliovirus 2Apro mutant correlates with phosphorylation of the alpha subunit of eucaryotic initiation factor 2.

A poliovirus type 2 Lansing mutant was constructed by inserting 6 base pairs into the 2Apro region of an infectious cDNA clone, resulting in the addition of a leucine and threonine into the polypeptide sequence. The resulting small-plaque mutant, 2A-2, had a reduced viral yield in HeLa cells and synthesized viral proteins inefficiently. Infection with the mutant did not lead to specific inhibition of host cell protein synthesis early in infection, and this defect was attributed to a failure to induce cleavage of the cap-binding complex protein p220. At late times after infection with the mutant virus, both cellular and viral protein syntheses were severely inhibited. To explain this global inhibition of protein synthesis, the phosphorylation state of the alpha subunit of eucaryotic initiation factor 2 (eIF-2 alpha) was examined. eIF-2 alpha was phosphorylated in both R2-2A-2- and wild-type-virus-infected cells, indicating that poliovirus does not encode a function that blocks phosphorylation of eIF-2 alpha. The kinetics and extent of eIF-2 alpha phosphorylation correlated with the production of double-stranded RNA in infected cells, suggesting that eIF-2 alpha is phosphorylated by P1/eIF-2 alpha kinase. When HeLa cells were infected with R2-2A-2 in the presence of 2-aminopurine, a protein kinase inhibitor, much higher virus titers were produced, cleavage of p220 occurred, and host cell protein synthesis was specifically inhibited. Since phosphorylation of eIF-2 alpha was not inhibited by 2-aminopurine, we propose that 2-aminopurine rescues the ability of R2-2A-2 to induce cleavage of p220 by inhibition of a second as yet unidentified kinase.     

Mengovirus leader is involved in the inhibition of host cell protein synthesis.

The presence of a leader peptide in picornaviruses is restricted to the Cardiovirus and Aphthovirus genera. However, the leader peptides of these two genera are structurally and functionally unrelated. The aphthovirus leader is a protease involved in viral polyprotein processing and host cell translation shutoff. The function of the cardiovirus leader peptide is still unknown. To gain an insight into the function of the cardiovirus leader peptide, a mengovirus leader peptide deletion mutant was constructed. The deletion mutant was able to grow at a reduced rate in baby hamster kidney cells (BHK-21). Mutant virus production in mouse fibroblasts (L929 cells), however, could be demonstrated only after inoculation of BHK-21 cells with the transfected L929 cells. Analysis of cellular and viral protein synthesis in mutant virus-infected cells showed a delayed inhibition of host cell protein synthesis and a reduced production of viral proteins. In a single-cycle infection, mutant virus produced only 1% of wild-type virus yield at 8 h postinfection. Host cell translation shutoff in L929 cells infected with mutant virus was restored by the addition of the kinase inhibitor 2-aminopurine. Mutant virus production in 2-aminopurine-treated L929 cells was increased to 60% of wild-type virus yield at 8 h postinfection. Our results suggest that the cardiovirus leader peptide is involved in the inhibition of host cell protein synthesis.     

Regulation of proto-oncogene expression and deoxyribonucleic acid synthesis in granulosa cells of perifused immature rat ovaries.

The present series of experiments examined the effects of follicle-stimulating hormone (FSH) and insulin (IN) on granulosa cell (GC) proto-oncogene expression and DNA synthesis. In the first study, GCs were harvested from immature rat ovaries after 15, 30, or 60 min of perifusion and DNA synthesis (3H-thymidine incorporation) and proto-oncogene mRNA levels were determined. The presence of c-myc and c-fos proteins was localized within GCs immunocytochemically. GCs of control ovaries exhibited modest levels of DNA synthesis and proto-oncogene expression. FSH/IN not only stimulated DNA synthesis but also increased c-myc, c-fos, and c-jun mRNA levels and the percentage of cells staining for c-fos and c-myc proteins. The protein kinase inhibitor, 2-aminopurine (2-AP), inhibited the FSH/IN-induced increases in c-myc and c-fos mRNA levels, the percentage of cells staining for Myc and Fos protein, and DNA and protein synthesis. The effects of 48 h of perifusion with FSH in the presence or absence of IN were also examined. These treatments were selected because after 48 h of continuous exposure to FSH alone, estradiol-17 beta (E2) secretion is enhanced and 3H-thymidine incorporation is inhibited. Conversely, FSH/IN maintains 3H-thymidine incorporation for up to 48 h of perifusion culture without stimulating E2 (Peluso et al., Endocrinology 1991; 128:191-196). After 48 h of perifusion, both FSH and FSH/IN stimulated c-fos mRNA and protein levels. However, high levels of c-jun mRNA and protein were detected only within GCs of FSH/IN-treated ovaries.(ABSTRACT TRUNCATED AT 250 WORDS)     

Role of [Ca2+]i in induction of c-fos, c-jun, and c-myc mRNA in rat PTE after oxidative stress.

Oxidative stress plays an important role in various types of cell injury and tumor promotion. Cells respond to oxidative stress in many ways including changes in membrane organization, ion movements, and altered gene expression, all of which contribute to the subsequent fate of affected cells. In this study, we investigated the expression of the proto-oncogenes c-fos, c-myc, and c-jun, which play a key role in proliferation and differentiation, using primary cultures of rat proximal tubular epithelium exposed to oxidative stress generated by the xanthine/xanthine oxidase system. This system generates superoxide and H2O2 in the extracellular space stimulating the release of active oxygen species from inflammatory cells. c-fos mRNA was expressed within 15 min, peaked at 30 min, and returned to constitutive levels by 3 h. c-jun mRNA began to rise after 30 min, peaked at 120 min, and remained above the constitutive levels up to 180 min. c-myc mRNA expression was less affected by the treatment, with levels increasing gradually over the 180 min period. The expression of c-fos was inhibited by superoxide dismutase but not by catalase and was super-induced by cycloheximide. H2O2 alone did not induce any c-fos mRNA in this system. Chelation of extracellular ionized calcium by EGTA or of intracellular ionized calcium by Quin 2/AM resulted in a marked decrease of c-fos expression. Two protein kinase C inhibitors, H-7 and staurosporine, partly diminished the expression of c-fos, whereas a third, 2-aminopurine, which has a broader spectrum of inhibiting protein kinases, almost completely abolished it. A poly ADP-ribosylation inhibitor, 3-aminobenzamide, had no effect on c-fos expression in this system. Our results show that oxidative stress provokes sequential expression of c-fos, c-jun, and c-myc, mRNA in this order. This c-fos expression appears to be largely controlled by calcium ion movement, which could include protein kinase C activation. Another protein kinase or kinases also appear to play an important role.     

c-fos基因在内皮素-1促肺泡Ⅱ型细胞表面活性物质合成中的作用

应用反义技术探讨ｃ－ｆｏｓ基因ＥＴ－１调控肺泡Ⅱ型细胞（ＡＴⅡ）表面活性物质（ＰＳ）合成的胞内信号转导中的作用,结果显示：（１）内皮素－１（ＥＴ－１）可提高ＡＴⅡ细胞的＾３Ｈ－胆碱掺入。（２）蛋白激酶Ｃ（ＰＫＣ）激活剂ＰＭＡ可使ＡＴⅡ细胞的＾３Ｈ－胆碱掺入量增加,ＰＫＣ抑制剂Ｈ７可抑制ＥＴ－１的促ＰＳ合成效应。（３）ＥＴ－１和ＰＭＡ可显著提高Ｆｏｓ蛋白表达量,Ｈ７和ｃ－ｆｏｓ反义寡核苷酸（ＯＤＮ）     

Dynamic interactions of c-fos protein in serum-stimulated 3T3 cells

The c-fos gene, the cellular homologue of the transforming gene of the FBJ osteosarcoma virus, v-fos, is strongly induced in quiescent BALB/c 3T3 cells by growth factors and in other cell types by a wide variety of transmembrane signalling agents. c-fos is a member of a family of structurally related proteins which includes the fos-related antigens (fra). We have studied the dynamic state of the c-fos protein with an antibody prepared by immunizing rabbits with a plasmid-encoded fos fusion protein. In serum-stimulated BALB/c 3T3 cells, the antibody recognizes a nuclear antigen which resolves on SDS-PAGE as a 60-68-kD group of bands corresponding to c-fos, a doublet at 44-45-kD corresponding to the noncovalently associated p39 protein, as well as an approximately 50-kD band corresponding to a fra. We show that although c-fos protein synthesis is only transiently induced by serum, the c-fos protein persists within the cell after its synthesis has ceased, and it decays with a half-life of 2 hours. Significantly, newly synthesized p39 continues to appear in the immune-precipitated complex even at times when c-fos is no longer synthesized. These kinetics indicate that even following shutoff of c-fos protein synthesis, p39 is newly synthesized and can complex with c-fos protein or a fos-related antigen. During this time, c-fos also undergoes an extensive posttranslational modification. The modification is partially reversed by phosphatase treatment, which implicates protein phosphorylation. Together these results suggest that both interaction with p39 and phosphorylation may progressively modify the properties of c-fos and/or the fos-related antigens over a period of 4-8 hours following the shutoff of fos synthesis. We discuss the implications of the dynamic state of c-fos and fra protein interactions for the function of these proteins.     

Role of Ca2+ influx in bombesin-induced mitogenesis in Swiss 3T3 fibroblasts

In Swiss 3T3 fibroblasts a peptide mitogen bombesin, which acts through the phospholipase C-protein kinase C signaling pathway, stimulates DNA synthesis in a manner strictly dependent on the medium calcium concentration: [3H]thymidine incorporation into DNA in the presence of a saturating concentration of bombesin (10(-8) M) is 4-fold greater at 3.0 mM extracellular calcium as compared with a value obtained at 0.03 mM calcium. In the present study we attempted to identify the site and the mechanism of action of Ca2+ influx along the bombesin-induced mitogenic signaling pathway, by comparing bombesin effects at 0.03 and 3.0 mM of medium calcium. Bombesin induces the same extent of increases in [3H]inositol phosphates after 1 min, and comparable sustained increases in the cellular content of 1,2-diacylglycerol for up to 4 h, at either 0.03 or 3.0 mM calcium. Bombesin induces the same extent of phosphorylation of MARCKS protein, the major cellular substrate for protein kinase C, irrespective of the medium calcium concentration for at least 4 h. Moreover, diverse cellular responses elicited by bombesin, including c-fos expression, activation of microtubule-associated protein 2 kinase and S6 kinase, glucose uptake, and protein synthesis but not the release of arachidonic acid and its metabolites, are induced similarly at either 0.03 or 3.0 mM calcium. Down-regulation of cellular protein kinase C nearly completely abolishes bombesin effects on c-fos expression, S6 kinase activation, glucose uptake, and DNA synthesis. These results suggest that the target of Ca2+ influx in bombesin-induced mitogenic signaling pathway is not located along the phospholipase C-protein kinase C signal transduction system including cellular events in early G1 phase that exist downstream to protein kinase C action.     

Role of protein phosphorylation in activation of interferon-stimulated gene factors.

Possible involvement of protein phosphorylation in interferon (IFN)-mediated activation of IFN-stimulated gene factor 3 (ISGF3) was investigated. For this purpose, in vivo experiments with specific inhibitors of protein kinases and in vitro experiments with protein phosphatases were carried out. In HeLaM cells, 2-aminopurine, an inhibitor of double-stranded RNA-dependent protein kinase, blocked the induction of ISGF3 gamma subunit but not the activation of ISGF3 alpha subunit. A series of experiments using combinations of protein and RNA synthesis inhibitors and 2-aminopurine indicated that the block elicited by 2-aminopurine was at the level of ISGF3 gamma mRNA synthesis. Activation of ISGF3 alpha, although insensitive to 2-aminopurine, was completely blocked by 10 nM staurosporine, an inhibitor of protein kinase C. On the other hand, even 500 nM staurosporine did not block the induction of ISGF3 gamma. Incubation of cytoplasmic or nuclear extracts of IFN-treated HeLaM cells in vitro with alkaline phosphatase completely eliminated their ability to form the ISGF3 complex but not the ISGF1 complex. Treatment with acid phosphatase, on the other hand, changed the electrophoretic mobility of the ISGF3 complex but did not obliterate it. Complementation experiments revealed that ISGF3 alpha was the alkaline phosphatase-sensitive component of the complex. These results suggest that a protein kinase C-mediated phosphorylation step is involved in ISGF3 alpha activation and a 2-aminopurine-sensitive component is involved in ISGF3 gamma mRNA induction.     

Competition between c-fos and 1,25(OH)2 vitamin D3 in the transcriptional control of type I collagen synthesis in MC3T3-E1 osteoblastic cells

Interaction between c-fos and 1,25(OH)₂ vitamin D₃ (VD) on the type I collagen synthesis was studied. VD inhibited collagen synthesis and type I collagen mRNA expression in MC3T3-E1 osteoblastic cells. In contrast, VD reversed the inhibition of collagen synthesis and mRNA expression of the c-fos transfectants that overexpressed c-fos gene to a comparable level as those of the control transfectants. The gel shift assay showed that vitamin D receptor (VDR) complex binding to vitamin D responsive element (VDRE) was inhibited under constitutively expressed c-fos gene, suggesting that c-fos gene product, c-Fos, may inhibit the binding of VDR complex to VDRE by making a c-Fos-VDR complex. The result suggests the existence of a fine tuning between c-fos and VD in the bone metabolism which may be relevant to the pathogenesis of rheumatoid bone lesion. © 1995 Wiley-Liss, Inc.     

Heparin suppresses specific second messenger pathways for protooncogene expression in rat vascular smooth muscle cells.

We investigated the molecular mechanisms underlying the ability of heparin to inhibit vascular smooth muscle cell (VSMC) growth. Previous experiments have shown that heparin inhibits induction of c-fos and c-myc protooncogene mRNA in rat VSMC stimulated by phorbol 12-myristate 13-acetate (PMA) but not when stimulated by epidermal growth factor (EGF) (Pukac, L. A., Castellot, J. J., Wright, T. C., Caleb, B. L., and Karnovsky, M. J. (1990) Cell Regul. 1, 435-443). The present experiments show that these mitogens activate distinct second messenger pathways in VSMC, because PMA but not EGF induction of c-fos and c-myc mRNA was suppressed in protein kinase C (PKC) down-regulated VSMC; this suggests that EGF does not act through a PKC-dependent pathway for induction of these genes. Heparin inhibited serum stimulation of c-fos mRNA in control VSMC, but heparin did not inhibit the smaller but significant serum stimulation of c-fos mRNA in PKC down-regulated VSMC, indicating that heparin may selectively inhibit PKC-dependent, but not PKC-independent, stimulation of gene expression. To further determine if heparin inhibits non-PKC pathways, VSMC were treated with dibutyryl cAMP, 3-isobutyl-1-methyl-xanthine, and Ca2+ ionophore A23187; stimulation of c-fos mRNA by this treatment was not inhibited by heparin. DNA synthesis and cell proliferation were inhibited in rat VSMC exposed briefly to heparin during the G0/G1 phase of the cell cycle. These experiments indicate heparin can act early in the cell cycle and suggest PKC-dependent but not PKC-independent signaling pathways for gene expression are selectively sensitive to heparin inhibition.     

Interrelationships of platelet-derived growth factor isoform-induced changes in c-fos expression, intracellular free calcium, and mitogenesis

Both increases in c-fos proto-oncogene expression and intracellular free calcium ([Ca2+]i) have been implicated as necessary components of the signal transduction pathway by which platelet-derived growth factor (PDGF) stimulates DNA synthesis in cultured BALB/c3T3 fibroblasts. To determine the interrelationship between PDGF-induced increases in c-fos proto-oncogene expression and [Ca2+]i, purified, recombinant BB and AA homodimeric isoforms of PDGF were used to evaluate the dose-response relationships and mechanisms of growth factor-induced changes in these two parameters as well as DNA synthesis. Concentration-dependent increases in [Ca2+]i, c-fos expression, and [3H]thymidine incorporation were observed with both BB and AA PDGF isoforms. BB PDGF was consistently more potent and efficacious than the AA isoform in eliciting a given response. The [Ca2+]i dependency of PDGF-induced increases in c-fos expression and DNA synthesis was determined by pretreatment of cells with agents that inhibit increases in [Ca2+]i: BAPTA, Quin-2, and TMB-8. Under these conditions, PDGF-induced DNA synthesis was blocked, whereas c-fos expression was enhanced. Conversely, in cells made deficient in protein kinase C (PKC) activity by prolonged treatment with phorbol ester, BB and AA PDGF-induced c-fos expression was inhibited by 75-80%, while PDGF-induced increases in [Ca2+]i and DNA synthesis were unaffected or enhanced. Additionally, the PKC-independent component of PDGF-stimulated c-fos expression was found to be independent of increases in [Ca2+]i. These data suggest that 1) both BB and AA PDGF isoforms elicit alterations in [Ca2+]i and c-fos proto-oncogene expression through the same or similar mechanisms in BALB/c3T3 fibroblasts, 2) PDGF-stimulated increases in [Ca2+]i are not required for c-fos expression, and 3) distinct pathways regulate PDGF-induced c-fos expression and mitogenesis, with c-fos expression being substantially PKC-dependent yet [Ca2+]i-independent, while mitogenesis is [Ca2+]i-dependent yet PKC-independent.     

Bombesin induction of c-fos and c-myc proto-oncogenes in Swiss 3T3 cells: significance for the mitogenic response

Bombesin is a potent mitogen for Swiss 3T3 cells and acts synergistically with insulin and other growth factors. We show here that addition of bombesin to quiescent Swiss 3T3 cells causes a striking increase in the levels of c-fos and c-myc mRNAs. Enhanced expression of c-fos (122 +/- 14-fold) occurred within minutes of peptide addition followed by increased expression of c-myc (82 +/- 16-fold). The concentrations of peptide required for half-maximal increase in the levels of c-fos and c-myc mRNAs were 1.0 and 0.9 nM, respectively. The peptide [D-Arg1, D-Pro2, D-Trp7,9, Leu11] substance P which inhibits the binding of bombesin to its receptor and bombesin-stimulated DNA synthesis in Swiss 3T3 cells blocked the increase in c-fos and c-myc mRNA levels promoted by bombesin. Down-regulation of protein kinase C by long-term exposure to phorbol esters prevented c-fos and c-myc induction by bombesin. This and other results indicate that the induction of these proto-oncogenes by bombesin could be mediated by the coordinated effects of protein kinase C activation and Ca2+ mobilization. The marked synergistic effect between bombesin and insulin was used to assess whether the increase in the induction of c-fos and c-myc is an obligatory event in cell activation. In the presence of insulin, bombesin stimulated DNA synthesis at subnanomolar concentrations but had only a small effect on c-fos and c-myc mRNA levels. This apparent dissociation of mitogenesis from proto-oncogene induction was even more dramatic in 3T3 cells with down-regulated protein kinase C. In these cells bombesin stimulated DNA synthesis in the presence of insulin but failed to enhance c-fos and c-myc mRNA levels at comparable concentrations. Thus, the induction of c-fos and c-myc may be a necessary step in the mitogenic response initiated by ligands that act through activation of protein kinase C but the expression of these proto-oncogenes may not be an obligatory event in the stimulation of mitogenesis in 3T3 cells by mitogens that utilise other signalling pathways.