Selective induction of c-jun and jun-B but not c-fos or c-myc during mitogenesis in SV40-transformed cells at the predifferentiation growth arrest state.

Insulin has recently been reported to function as a complete mitogen for SV40 large T antigen-transformed 3T3 T-cells, designated CSV3-1, but not for nontransformed 3T3 T-cells (H. Wang and R. E. Scott, J. Cell. Physiol., 147: 102-110, 1991). It is now reported that sodium orthovanadate mimics this effect of insulin. For example, when exposed to 1-5 microM vanadate, most predifferentiation growth-arrested CSV3-1 cells undergo DNA synthesis within 24 h, but neither vanadate nor insulin induces mitogenesis in nontransformed 3T3 T-cells. To investigate the possible mechanisms by which mitogenesis is induced in CSV3-1 cells, the effects of insulin and vanadate on the expression of growth-related genes were examined. Whereas insulin and vanadate had no effect on the expression of c-fos, c-myc, c-jun, jun-B, or ornithine decarboxylase activity in nontransformed 3T3 T-cells, insulin and vanadate showed different effects on the expression of these genes in CSV3-1 cells. Insulin induced a rapid and transient accumulation of c-fos mRNA followed by induction of c-myc, c-jun, jun-B, and ornithine decarboxylase. In contrast, vanadate induced the expression of c-jun, jun-B, and ornithine decarboxylase without inducing c-fos and c-myc. These observations suggest that SV40 large T antigen may play an important role in insulin- and vanadate-induced mitogenesis and that insulin and vanadate may mediate their mitogenic effects by different signal transduction pathways.     

Distinct protein tyrosine phosphorylation during mitogenesis induced in quiescent sv40-transformed 3T3 T cells by insulin or vanadate

Insulin and vanadate selectively induce mitogenesis in quiescent SV40 large T antigen-transformed 3T3 T cells (CSV3–1) but not in quiescent nontransformed 3T3 T cells. Insulin and vanadate mediate this effect in CSV3–1 cells by distinct signal transduction mechanisms that involve protein tyrosine kinase activity. To further study these processes, changes in protein tyrosine phosphorylation induced by insulin and vanadate were investigated. Using immunoprecipitation and Western blotting techniques with antiphosphotyrosine antibodies, we report distinct protein phosphorylation characteristics in insulin- and vanadate-stimulated CSV3–1 cells. The insulin receptor β-subunit is phosphorylated within 2 min after insulin stimulation of transformed CSV3–1 cells. Insulin also stimulates a rapid increase in tyrosine phosphorylation of the 170 kDa insulin receptor substrate-1 and complex formation between the phosphorylated insulin receptor substrate-1 and the 85 kDa subunit of phosphatidylinositol 3'-kinase. In contrast, vanadate does not initially increase detectable phosphorylation of any proteins, including neither the insulin receptor nor the insulin receptor substrate-1. After 60 min, however, a marked increase in tyrosine phosphorylation of 55 and 64 kDa proteins is observed in vanadate-treated CSV3–1 cells. Furthermore, treatment of CSV3–1 cells with genistein abolishes the effects of vanadate on protein tyrosine phosphorylation but only minimally inhibits the effects of insulin. Finally, insulin stimulates the phosphorytion of a 33 kDa protein, whereas vanadate does not. By comparison, in nontransformed 3T3 T cells, insulin induces a delayed and weaker tyrosine phosphorylation of the insulin receptor β-subunit and vanadate does not enhance the tyrosine phosphorylation of the 55 and 64 kDa proteins. These data together indicate that the mitogenic effects of insulin and vanadate are associated with distinct protein phosphorylation patterns that appear to be differentially regulated in SV40-transformed and nontransformed 3T3 T cells. © 1994 Wiley-Liss, Inc.     

Increased tyrosine phosphorylation of the insulin receptor,the insulin receptor substrate-1 and a 73 kDa protein associated with insulin-induced mitogenesis in SV40-transformed 3T3T cells

Insulin selectively induces mitogenesis in quiescent SV40 large T antigen-transformed murine 3T3T (CSV3-1) cells but not in quiescent nontransformed 3T3T cells. This mitogenic effect induced by insulin in CSV3-1 cells requires an induction of AP-1 activity associated with c-Jun and JunB. To further investigate the mechanisms that are involved in insulin-induced mitogenesis in CSV3-1 cells, the current experiments were performed. The results show that following insulin stimulation, the insulin receptor -subunit and the insulin receptor substrate-1 undergo a much more significant tyrosine phosphorylation in CSV3-1 cells than in 3T3T cells. Insulin also induces tyrosine phosphorylation of a 73 kDa protein that is coprecipitated with the tyrosine-phosphorylated insulin receptor in CSV3-1 cells but not in 3T3T cells. The increased tyrosine phosphorylation in response to insulin stimulation in CSV3-1 cells does not appear to be due to an increase in the level of expression of the insulin receptor and does not appear to result from a significant change in tyrosine phosphatase activity compared to nontransformed cells. The results also show that the insulin effect in CSV3-1 cells is not mediated by insulin-like growth factor 1 receptor because insulin at the concentrations that induce mitogenesis does not increase the tyrosine phosphorylation of the insulin-like growth factor 1 receptor and the expression level of the receptor is not significantly changed in CSV3-1 cells compared to nontransformed cells. These data together indicate that the selective mitogenic effect of insulin on CSV3-1 cells involves increased tyrosine phosphorylation of the insulin receptor, the insulin receptor substrate-1 and the 73 kDa protein, although the underlying mechanisms need to be further elucidated.     

Possible involvement of pertussis toxin-sensitive GTP-binding protein(s) in c-fos expression during differentiation of 3T3-L1 fibroblasts to adipocytes.

Following the differentiation of 3T3-L1 fibroblasts by insulin/dexamethasone/methylisobutylxanthine, marked increases in cAMP levels by isoproterenol but not forskolin and in 2-deoxyglucose uptake by insulin occurred. Pertussis toxin-pretreatment prior to addition of insulin/dexamethasone/methylisobutylxanthine and exposure of cells to pertussis toxin during differentiation attenuated glycerophosphate dehydrogenase activity as a differentiation marker enzyme and the responses to isoproterenol and insulin by approximately 50% of those in pertussis toxin-untreated cells. On the other hand, insulin/dexamethasone/methylisobutylxanthine caused induction of c-fos proto-oncogene in confluent 3T3-L1 fibroblasts. This induction was also reduced in pertussis toxin-pretreated cells. These results suggested that pertussis toxin-sensitive GTP-binding protein(s) is involved in expression of c-fos mRNA accompanied by differentiation. In addition, accumulation of c-fos mRNA by insulin/dexamethasone/methylisobutylxanthine was enhanced in protein kinase C-depleted cells pretreated with phorbol 12-myristate 13-acetate, indicating that protein kinase C may negatively regulate c-fos expression induced by insulin/dexamethasone/methylisobutylxanthine.     

Vasopressin rapidly stimulates protein kinase C in digitonin-permeabilized Swiss 3T3 cells: involvement of a pertussis toxin-insensitive guanine nucleotide binding protein

Guanine nucleotides and pertussis toxin were used to test for the involvement of a guanine nucleotide binding protein in the vasopressin V1 receptor-mediated stimulation of protein kinase C activity in Swiss 3T3 cells. Addition of vasopressin in the presence of [gamma-32P]ATP and digitonin caused a marked and rapid increase (8 +/- 1-fold after 1 min) in the phosphorylation of an Mr = 80,000 cellular protein (80K), a specific marker for protein kinase C activation. This phosphorylation was selectively blocked by the V1 receptor antagonist Pmp1-0-Me-Tyr2 [Arg8] vasopressin, indicating that the effect was mediated through the vasopressin V1 receptor. Down regulation of protein kinase C by prior prolonged pretreatment of intact cells with phorbol 12,13-dibutyrate (PBt2) blocked the ability of vasopressin to stimulate the phosphorylation of 80K in digitonin-permeabilized cells. Addition of a submaximal concentration of vasopressin together with the GTP analogue GTP-gamma-S caused a synergistic stimulation of 80K phosphorylation. The GDP analogue GDP-beta-S caused a 50% inhibition of the phosphorylation of 80K induced by a saturating concentration of vasopressin and shifted the vasopressin dose-response curve to the right. GDP-beta-S had no effect on the dose-response for the stimulation of 80K phosphorylation induced by PBt2. Prior incubation of intact quiescent cultures of Swiss 3T3 cells with pertussis toxin did not impair either vasopressin-induced increase in cytosolic [Ca2+] or activation of protein kinase C. These findings provide functional evidence for the involvement of a pertussis toxin-insesitive G protein in the vasopressin V1 receptor-mediated stimulation of protein kinase C in Swiss 3T3 cells.     

Regulation of glucose transport by insulin, bombesin, and bradykinin in Swiss 3T3 fibroblasts: involvement of protein kinase C-dependent and -independent mechanisms

Glucose transport stimulation by insulin, bombesin, and bradykinin in Swiss 3T3 fibroblasts was compared with the phosphoinositide hydrolysis effects of the same stimulants in a variety of experimental paradigms known to affect generation and/or functioning of intracellular second messengers: short- and long-term treatments with phorbol dibutyrate, that cause activation and down-regulation of protein kinase C, respectively; cell loading with high [quin2], that causes clamping of [Ca2+]i near the resting level; poisoning with pertussis toxin, that affects the GTP binding proteins of the Go/Gi class; treatment with Ca2+ ionophores. Glucose transport stimulation by maximal [insulin] was affected by neither pertussis toxin nor protein kinase C down-regulation. The latter, however, partially blocked the action of suboptimal [insulin]; moreover, acute phorbol dibutyrate treatment caused responses more than additive at all [insulin]. Thus, the insulin action on glucose transport in 3T3 cells appears to be synergistically potentiated by a protein kinase C-dependent mechanism, and not directly mediated by the enzyme. This result correlates with the lack of effect of insulin on phosphoinositide hydrolysis. In contrast, part of the glucose transport responses induced by bombesin and bradykinin appeared to be mediated by protein kinase C in proportion with the stimulation induced by these peptides on the phosphoinositide hydrolysis. The protein kinase C-independent portion of the response to bradykinin was found to be inhibitable by pertussis toxin. This latter result might suggest an interaction between the bradykinin receptor and a glucose transporter, mediated by a protein of the Go/Gi class.     

Unique and selective mitogenic effects of vanadate on SV40-transformed cells

Vanadate and insulin both function as unique complete mitogens for SV40-transformed 3T3T cells, designated CSV3-1, but not for nontransformed 3T3T cells. The mitogenic effects induced by vanadate and insulin in CSV3-1 cells are mediated by different signaling mechanisms. For example, vanadate does not stimulate the tyrosine phosphorylation of the insulin receptor -subunit nor the 170 kDa insulin receptor substrate-1. Instead, vanadate induces a marked increase in tyrosine phosphorylation of 55 and 64 kDa proteins that is not observed in insulin-stimulated CSV3-1 cells. Perhaps most interestingly, vanadate-induced mitogenesis is associated with the selective induction ofc-jun andjunB expression without significantly inducingc-fos orc-myc. Furthermore, treatment of CSV3-1 cells with genistein abolishes the effects of vanadate on protein tyrosine phosphorylation andc-jun induction. These and related data suggest that modulation of protein tyrosine phosphorylation andc-jun andjunB expression may serve the critical roles in mediating vanadate-induced mitogenesis in SV40-transformed cells.     

Mastoparan, a novel mitogen for Swiss 3T3 cells, stimulates pertussis toxin-sensitive arachidonic acid release without inositol phosphate accumulation

Mastoparan, a basic tetradecapeptide isolated from wasp venom, is a novel mitogen for Swiss 3T3 cells. This peptide induced DNA synthesis in synergy with insulin in a concentration-dependent manner; half-maximum and maximum responses were achieved at 14 and 17 microM, respectively. Mastoparan also stimulated DNA synthesis in the presence of other growth promoting factors including bombesin, insulin-like growth factor-1, and platelet-derived growth factor. The synergistic mitogenic stimulation by mastoparan can be dissociated from activation of phospholipase C. Mastoparan did not stimulate phosphoinositide breakdown, Ca2+ mobilization or protein kinase C-mediated phosphorylation of a major cellular substrate or transmodulation of the epidermal growth factor receptor. In contrast, mastoparan stimulated arachidonic acid release, prostaglandin E2 production, and enhanced cAMP accumulation in the presence of forskolin. These responses were inhibited by prior treatment with pertussis toxin. Hence, mastoparan stimulates arachidonic acid release via a pertussis toxin-sensitive G protein in Swiss 3T3 cells. Arachidonic acid, like mastoparan, stimulated DNA synthesis in the presence of insulin. The ability of mastoparan to stimulate mitogenesis was reduced by pertussis toxin treatment. These results demonstrate, for the first time, that mastoparan stimulates reinitiation of DNA synthesis in Swiss 3T3 cells and indicate that this peptide may be a useful probe to elucidate signal transduction mechanisms in mitogenesis.     

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.     

Human IL-3 induction of c-jun in normal monocytes is independent of tyrosine kinase and involves protein kinase C.

We have used normal human monocytes as a model system to begin elucidating the signal transduction mechanism associated with the IL-3R. Normal human monocytes deprived of human serum and CSF become quiescent in vitro. Stimulation of these cells with rIL-3 induces expression of the c-jun protooncogene, as detected by Northern blotting of total monocyte RNA. This protooncogene is also induced in these cells by phorbol ester through direct stimulation of protein kinase C. Concentrations of the protein kinase C inhibitor I-(5-isoquindinyl-sulfonyl)-2 methylpiperazine (H-7) between 30 and 100 microM (5-20 x Ki) inhibit this induction by phorbol ester. The same concentration-range of H-7 completely inhibited the induction of c-jun by human IL-3. A structural analog of H-7 designated HA-1004 preferentially inhibits cyclic nucleotide-dependent protein kinase rather that protein kinase C. HA-1004 at 5 to 20 x Ki did not inhibit IL-3-induced c-jun mRNA accumulation. Further 30 microM genistein that is an effective inhibitor of cellular tyrosine kinases did not inhibit IL-3-induced c-jun expression. Immunoprecipitation of lysates from [32P]orthophosphate labeled cells with antiphosphotyrosine polyclonal antibody showed that IL-3-stimulated phosphorylation of a 70-kDa protein and a 110-kDa protein on tyrosine, and that these protein phosphorylations were completely inhibited by 30 microM genistein. As further confirmation that IL-3 is stimulating protein kinase C in human monocytes we have found that IL-3 stimulates phosphorylation of the unique protein kinase C substrate myristoylated alanine-rich C kinase substrate in these cells. It is therefore likely that the interaction of IL-3 with its receptor generates diacylglycerol and stimulates the Ca2+/phospholipid-dependent protein kinase C.     

Effects of insulin,pertussis toxin and cholera toxin on protein synthesis and diacylglycerol production in 3T3 fibroblasts: Evidence for a G-protein mediated activation of phospholipase C in the insulin signal mechanism

The rapid increase in protein synthesis that occurs on addition of insulin (1 mU/ml) to stepped-down 3T3 cells was blocked by pre-incubation of the cells with pertussis toxin. Cholera toxin on the other hand stimulated protein synthesis and this effect was insensitive to actinomycin D and inhibited by pro-treatment of the cells with phorbol dibutyrate to deplete cell protein kinase C. Insulin was found to cause a rapid and transient increase in diacylglycerol (DAG) synthesis. The insulin-induced increase in diacylglycerol was blocked by pertussis toxin. Exogenous DAG (10 M) stimulated protein synthesis within 1 hour. The results suggest that insuIin stimulates ribosomal activity through a signal mechanism that involves a G-protein mediated activation of phospholipase C to increase DAG levels.     

Different sensitivity to phorbol esters and pertussis toxin of bombesin- and platelet-derived growth factor-induced, phospholipase C-mediated hydrolysis of phosphoinositides in NIH/3T3 cells

Incubation of the serum-deprived cultures of NIH/3T3 cells with bombesin or platelet-derived growth factor (PDGF) induced the phospholipase C-mediated hydrolysis of phosphoinositides. Protein kinase C-activating 12-O-tetradecanoylphorbol 13-acetate (TPA) and pertussis toxin inhibited the bombesin-induced phospholipase C reactions. AlF4-, a direct activator of GTP-binding proteins (G proteins), also induced the phospholipase C reactions and TPA inhibited the AlF4- -induced reactions. These results suggest that a pertussis toxin-sensitive G protein is involved in the coupling of the bombesin receptor to the phospholipase C and that the coupling of the G protein to the phospholipase C is inhibited by protein kinase C. In contrast, neither TPA nor pertussis toxin inhibited the PDGF-induced phospholipase C reactions, indicating that a pertussis toxin-sensitive G protein is not involved in the coupling of the PDGF receptor to the phospholipase C and that this coupling is insensitive to protein kinase C. These results suggest that the regulatory mechanism of the PDGF receptor for the phospholipase C activation is different from that of the bombesin receptor.     

Insulin-induced mitogenesis associated with transformation by the SV40 large T antigen.

Simian virus 40 (SV40) large T antigen-transformed cells typically show a markedly reduced serum requirement for growth and the inability to growth arrest and differentiate. An SV40 large T antigen-transformed 3T3 T cell line, CSV3-1, that can growth arrest and differentiate into adipocytes with high efficiency has, however, recently been described (Scott et al: Proc. Natl. Acad. Sci. U.S.A. 86:1652-1656, 1989; Estervig et al: J. Virol. 63:2718-2725, 1989; J. Cell. Physiol. 142:552-558, 1990). The results of the current studies using these cells show that whereas quiescent 3T3 T cells show no mitogenic response to insulin, quiescent CSV3-1 cells show a highly significant insulin-induced mitogenic responsiveness in the absence of other added growth factors. Maximum mitogenesis was observed at an insulin concentration of 1 microgram/ml, which induced 40-70% of the cells to undergo DNA synthesis within 48 hours. The half maximum response was achieved with 1-10 ng/ml of insulin. Insulin's mitogenic effect on CSV3-1 cells was evident under several different culture conditions that induce quiescence and was not mediated by any detectable autocrine growth factors that might make CSV3-1 cells competent to respond to insulin. In CSV3-1 cells insulin appears to act on its own receptor rather than on the IGF-1 receptor, because at comparable dosages IGF-1 is 10- to 100-fold less effective than insulin. Insulin also is shown to be a mitogen for another SV40-transformed cell line, CSV3-35, which can be growth arrested; in contrast insulin has no mitogenic effect on two control cell lines that are stably transfected with pSV2neo, a plasmid containing SV40 early promoter/enhancer but lacking large T antigen gene: These results suggest a significant relationship between SV40 T antigen-associated transformation and the expression of mitogenic responsiveness to insulin.     

Inhibition of bombesin-induced mitogenesis by pertussis toxin: dissociation from phospholipase C pathway

Prior incubation of quiescent cultures of Swiss 3T3 cells with pertussis toxin selectively inhibited the stimulation of DNA synthesis induced by peptides of the bombesin family. While pertussis toxin blocked mitogenesis at an early stage in the action of the peptide, the toxin did not impair the rapid stimulation of polyphosphoinositide breakdown, Ca2+ mobilization or activation of protein kinase C promoted by bombesin. Thus, inhibition of bombesin-induced mitogenesis by pertussis toxin can be dissociated from inactivation of the phospholipase C signalling pathway.     

Further evidence for a phospholipase C-coupled G protein in hamster fibroblasts. Induction of inositol phosphate formation by fluoroaluminate and vanadate and inhibition by pertussis toxin

We have previously reported that alpha-thrombin induces in resting hamster fibroblasts (CCL39) the formation of inositol phosphates (IP) by activating a GTP-binding protein (G protein) sensitive to pertussis toxin (Paris, S., and Pouysségur, J. (1986) EMBO J. 5, 55-60). Here we show that IP formation in CCL39 cells can also be induced by NaF with AlCl3 and by vanadate. In the presence of Li+, IP accumulation is linear over 30 min with no detectable lag and is concentration-dependent. NaF alone is slightly stimulatory, but a marked potentiation is observed in the presence of AlCl3, by itself without effect. Maximal stimulation is obtained with 10 mM NaF and 3 microM AlCl3, and with vanadate half-maximal effect is achieved at 0.3 mM. Both stimulations are markedly inhibited (up to 80%) by pertussis toxin (half-maximal inhibition at 1-2 ng/ml). We therefore conclude that phospholipase C is stimulated by NaF plus AlCl3 (presumably acting as AlF-4) and by vanadate by direct activation of the regulatory G protein. In addition, NaF inhibits the inositol-1-phosphatase, but this effect is not potentiated by AlCl3. Similarly, vanadate inhibits inositol trisphosphate degradation. Maximal stimulations of phospholipase C by AlF-4 and vanadate are not additive, whereas they are both additive with thrombin effects. Pretreatment of cells for 15 min with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate nearly completely abolishes induction of IP formation by AlF-4 and vanadate, suggesting that protein kinase C exerts a feedback negative control either on the G protein or on phospholipase C itself. An increase in cellular cyclic AMP similarly results in a marked attenuation of AlF-4-induced IP formation, indicating that activation of phospholipase C can be controlled also by cyclic AMP. However, the stimulatory effect of AlF-4 on phospholipase C is clearly dissociated from its effect on the adenylate cyclase system.     

Inhibition of protein kinase C-dependent cellular proliferation by interaction of endogenous ganglioside GM1 with the B subunit of cholera toxin

In quiescent Swiss 3T3 fibroblasts, the B subunit of cholera toxin, a protein which binds specifically to ganglioside GM1 on the cell surface, stimulates DNA synthesis and potentiates the effects of several other growth factors such as insulin, epidermal growth factor, bombesin, and even unfractionated serum. In contrast to its synergistic effect with other known growth factors, the B subunit markedly inhibited DNA synthesis induced by the phorbol ester, 12-O-tetradecanoyl-phorbol 13-acetate (TPA). The inhibitory effect of the B subunit was observed even in the presence of insulin, which greatly potentiates the mitogenic response to TPA or the B subunit. In contrast to the effect of the B subunit, calcium ionophores and cholera toxin stimulated DNA synthesis induced by TPA. The antagonism between the B subunit and TPA is not simply due to their abilities to modify their mutual binding sites or known effector systems. TPA did not block the early rise in cytosolic free calcium in response to the B subunit, and conversely, the B subunit did not modify the ability of TPA to activate protein kinase C. However, in protein kinase C-deficient cells, the antagonistic effect between TPA and the B subunit was abolished. In addition, there was no indication for the involvement of a pertussis toxin-sensitive G protein in the antagonism. Maximum inhibition was found when the B subunit was added 2 h after the addition of TPA. Significant inhibition was still evident when the time of addition of the B subunit was delayed until 6 h after the addition of TPA. This suggests that the cross-talk between signal transduction induced through endogenous gangliosides and protein kinase C is a late step in mitogenesis.     

Effect of a dominant inhibitory Ha-ras mutation on mitogenic signal transduction in NIH 3T3 cells.

We used a dominant inhibitory mutation of c-Ha-ras which changes Ser-17 to Asn-17 in the gene product p21 [p21(Asn-17)Ha-ras] to investigate ras function in mitogenic signal transduction. An NIH 3T3 cell line [NIH(M17)] was isolated that displayed inducible expression of the mutant Ha-ras gene (Ha-ras Asn-17) via the mouse mammary tumor virus long terminal repeat and was growth inhibited by dexamethasone. The effect of dexamethasone induction on response of quiescent NIH(M17) cells to mitogens was then analyzed. Stimulation of DNA synthesis by epidermal growth factor (EGF) and 12-O-tetradecanoylphorbol-13-acetate (TPA) was completely blocked by p21(Asn-17) expression, and stimulation by serum, fibroblast growth factor, and platelet-derived growth factor was partially inhibited. However, the induction of fos, jun, and myc by EGF and TPA was not significantly inhibited in this cell line. An effect of p21(Asn-17) on fos induction was, however, demonstrated in transient expression assays in which quiescent NIH 3T3 cells were cotransfected with a fos-cat receptor plasmid plus a Ha-ras Asn-17 expression vector. In this assay, p21(Asn-17) inhibited chloramphenicol acetyltransferase expression induced by EGF and other growth factors. In contrast to its effect on DNA synthesis, however, Ha-ras Asn-17 expression did not inhibit fos-cat expression induced by TPA. Conversely, downregulation of protein kinase C did not inhibit fos-cat induction by activated ras or other oncogenes. These results suggest that ras proteins are involved in at least two parallel mitogenic signal transduction pathways, one of which is independent of protein kinase C. Although either pathway alone appears to be sufficient to induce fos, both appear to be necessary to induce the full mitogenic response.     

Studies on the mechanisms of signalling and inhibition by pertussis toxin of fibroblast growth factor-stimulated mitogenesis in Balb/c 3T3 cells.

Basic fibroblast growth factor (bFGF) stimulates mitogenesis of Balb/c 3T3 fibroblast cells. This stimulation may be mediated by multiple signal pathways as it is accompanied by the formation of inositol phosphates, activation of PKC (protein kinase C) and a decrease in intracellular cAMP levels. The multiple positive and negative pathways implicated for FGF-induced mitogenesis may interact and each may contribute in varying degrees to the final cellular response. At least two types of G-proteins may be involved in the intracellular signalling pathways of FGF. Pertussis toxin blocks FGF and TPA (12-O-tetradecanoylphorbol-13-acetate) induced. PKC-mediated mitogenesis and also the associated fall in intracellular cAMP levels. However, pertussis toxin has no effect upon FGF-induced inositol phosphates formation. Thus, inhibition of mitogenesis by pertussis toxin may involve pertussis toxin sensitive G-proteins which may affect at least two separate putative signal pathways involving adenylate cyclase and protein kinase C. Pertussis toxin insensitive G-proteins may also be involved in coupling the FGF receptor to phosphoinositidase C.     

Fibroblast growth factor-dependent mitogenic signal transduction pathway in chemically transformed mouse fibroblasts is similar to but distinct from that initiated by phorbol esters.

Treatment of the quiescent, chemically transformed Balb/c mouse 3T3 cells (BP-A31) with fibroblast growth factor (FGF) leads to reinitiation of the cell division cycle in a large proportion of the cells. The characteristics of the mitogenic action of FGF closely resemble those of phorbol esters (activators of protein kinases type C) and differ from those of insulin (mediated by insulin-like growth factor 1 receptors). In particular, the effects of FGF as well as of phorbol-2-myristate-13-acetate (PMA), unlike the effects of insulin, are prevented by a low concentration (7.5 nM) of staurosporin (an efficient inhibitor of protein kinase C) as well as by 3-isobutyl-1-methyl xanthin (IBMX). Both FGF and PMA are good inducers of the accumulation of c-fos and c-jun mRNAs, whereas insulin has little effect. However, FGF was fully active (both as a mitogen and as inducer of c-fos mRNA accumulation) also in cells where the protein kinase C-mediated pathway had been downregulated by a long exposure to phorbol dibutyrate. We propose that the mitogenic effect of FGF does not require activation of protein kinase C, but that the subsequent events in the transduction pathways initiated by FGF and PMA, respectively, are (in part) coincident.