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.     

Growth factor-stimulated protein phosphorylation in 3T3-L1 cells. Evidence for protein kinase C-dependent and -independent pathways

Exposure of serum-deprived 3T3-L1 fibroblasts to phorbol 12-myristate 13-acetate (PMA), synthetic diacylglycerols, platelet-derived growth factor (PDGF), or pituitary fibroblast growth factor (FGF) resulted in stimulated phosphorylation of an acidic, multicomponent, soluble protein of Mr 80,000. Phosphorylation of this protein was promoted to a lesser extent by epidermal growth factor; however, neither insulin nor dibutyryl cAMP was effective. Phosphoamino acid analysis and peptide mapping of the Mr 80,000 32P-protein after exposure of fibroblasts to PDGF revealed identical patterns to those obtained with PMA or diacylglycerols. In contrast to the Mr 80,000 protein, proteins of Mr 22,000 (and pI 4.4) and Mr 31,000 were also phosphorylated in response to insulin as well as to PMA, diacylglycerols, epidermal growth factor, PDGF, and FGF in these cells. Similar findings were noted in fully differentiated 3T3-L1 adipocytes. Preincubation of the cells with high concentrations of active phorbol esters abolished specific [3H]phorbol 12,13-dibutyrate binding, protein kinase C activity, and immunoreactivity and also prevented stimulated phosphorylation of the Mr 80,000 protein by PMA, diacylglycerols, PDGF, or FGF, supporting the contention that this effect was mediated through protein kinase C. The stimulated phosphorylation of the Mr 22,000 and 31,000 proteins in response to PMA was also abolished by such pretreatment. In contrast, the ability of insulin, PDGF, and FGF to promote phosphorylation of the Mr 22,000 and 31,000 proteins was unaffected in the protein kinase C-deficient cells. We conclude that PDGF and FGF may exert some of their effects on these cells through at least two distinct pathways of protein phosphorylation, phorbol ester-like (P) activation of protein kinase C, and an insulin-like (I) pathway exemplified by phosphorylation of the Mr 22,000 and 31,000 proteins.     

Involvement of serum factor(s) adsorbed to the dish in the response of cycloheximide-pretreated BP-A31 cells to serum pulses

The mitogenic effect of serum pulses, observed previously in quiescent BP-A31 cells, is an artifact due to adsorption of (unknown) serum mitogens to the culture dish; the continuous presence of growth factors is necessary for these cells to traverse the G1 phase. When pretreated with cycloheximide (CH) during the last 8-24 h of quiescence, the BP-A31 cells are particularly sensitive to the adsorbed serum mitogens, as well as to low concentrations of fetal calf serum (less than 0.25%) and to basic fibroblast growth factor. In contrast, the mitogenic activity of insulin was not influenced by CH pretreatment. The expression of the "competence genes" c-myc and JE was only transiently elevated in quiescent BP-A31 cells during CH exposure and did not correlate with the enhancement of the cells' sensitivity to mitogens.     

Phorbol 12-myristate-13-acetate (PMA) stimulates a differential expression of cholecystokinin (CCK) and c-fos mRNA in a human neuroblastoma cell line.

Regulation of cholecystokinin (CCK) and the proto-oncogene c-fos mRNA expression was studied in the human neuroblastoma cell line SK-N-MC. Cells were treated either with the tumor promoting phorbol-ester phorbol-12-myristate-13-acetate (PMA), the phosphodiesterase inhibitor isobutyl-methylxanthine (IBMX), which results in an elevated intracellular cyclic AMP (cAMP) level, or with a combination of PMA and IBMX. The level of CCK and c-fos mRNA was determined by Northern-blot analysis with CCK and c-fos specific antisense RNA probes after 4-24 h of drug treatment. Treatment with PMA and IBMX for 4-24 hours transiently raised the CCK mRNA level approximately 1.5-3.5 times compared to the controls, and the combination PMA and IBMX had an additive effect and elevated CCK mRNA abundance 1.5-6.5 times. Under the same experimental conditions, both PMA and IBMX elevated the c-fos mRNA level approximately 3-5.5 times. The drug combination showed a pronounced synergistic effect and raised the c-fos mRNA level approximately 3-20 times as compared to controls. Apparently, CCK and c-fos mRNA expression appears to be regulated by similar protein kinase C (PKC) and cAMP-dependent mechanisms in SK-N-MC cells.     

Phorbol ester mimics ACTH action in corticoadrenal cells stimulating steroidogenesis, blocking cell cycle, changing cell shape, and inducing c-fos proto-oncogene expression

Cells of the Y-1 corticoadrenal line are: (a) functional, (b) cell cycle-arrested by adrenocorticotropic hormone (ACTH), (c) tumorigenic, and (d) c-Ki-ras overexpressing. We here report that the phorbol ester phorbol 12-myristate 13-acetate (PMA) mimics all ACTH-specific effects in Y-1 cells, namely: (a) steroid-ogenesis stimulation, (b) cell cycle block, and (c) cell shape change. In addition, both ACTH and PMA caused a rapid and transient induction of the c-fos proto-oncogene while having no effect on c-Ki-ras mRNA steady state levels. Dibutyryl cAMP, known to elicit ACTH effects in Y-1 cells, was a poor inducer of the c-fos gene. PMA pretreatment rendered Y-1 cells unresponsive to ACTH. These results suggest that protein kinase C is likely to be involved in the mechanisms of action of ACTH.     

Phosphorylation of the MARCKS Protein (P87), a Major Protein Kinase C Substrate, Is Not an Obligatory Step in the Mitogenic Signaling Pathway of Basic Fibroblast Growth Factor in Rat Oligodendrocytes

Basic fibroblast growth factor (bFGF) is a well-characterized peptide hormone that has mitogenic activity for various cell types and elicits a characteristic set of responses on the cell types investigated. In this report we confirmed that bFGF is a potent mitogen for rat brain-derived oligodendrocyte (OL) precursor cells as well as for differentiated OL in secondary culture. bFGF was shown to induce expression of the protooncogene c-fos in OL. The role of protein kinase C (PKC) in mediating bFGF-stimulated proliferation as well as c-fos expression in OL was investigated. The PKC activator phorbol 12-myristate 13-acetate (PMA) stimulated c-fos expression but did not trigger cell proliferation. When PKC was down-regulated by pretreatment of OL with PMA for 20 h, the bFGF-mediated stimulations of OL proliferation and c-fos mRNA expression were still observed, whereas the induction of c-fos mRNA by PMA was totally inhibited. These data demonstrate that the bFGF mitogenic signaling pathway in OLs does not require PKC. On the other hand, bFGF was found to stimulate specifically the phosphorylation of a limited number of PKC substrates in oligodendroglial cells, including the MARCKS protein. The bFGF-dependent phosphorylation of MARCKS protein was totally inhibited when PKC was first down-regulated, indicating that the phosphorylation of this protein is PKC dependent. Tryptic digestion of the phosphorylated MARCKS protein revealed that bFGF stimulated specifically the phosphorylation of the MARCKS protein on a single phosphopeptide. We provide evidence that bFGF also stimulated fatty acylation of the MARCKS protein, which might explain the observed specific bFGF-dependent phosphorylation of this protein in OL. We propose that bFGF-dependent fatty acylation and phosphorylation of the MARCKS protein are not essential for the transduction of the bFGF mitogenic signal but are probably linked to differentiation processes elicited by bFGF on OL.     

c-myc gene expression is stimulated by agents that activate protein kinase C and does not account for the mitogenic effect of PDGF

The role of the phosphoinositide turnover-protein kinase C pathway in mediating PDGF-stimulated c-myc expression and cell proliferation was studied. Both direct activators of kinase C (e.g. phorbol ester analogues) and hormones that activate kinase C via receptor-mediated phosphoinositide turnover (e.g. PDGF, bradykinin, or vasopressin) elicited a rapid increase in c-myc mRNA expression. Desensitization of the kinase C pathway by prolonged exposure to phorbol abolished the induction of c-myc by subsequent phorbol challenge and attenuated c-myc induction by PDGF and bradykinin, but did not affect PDGF-stimulated mitogenesis. Bradykinin and phorbol esters stimulated the same magnitude of c-myc expression as PDGF but elicited less than one-tenth the PDGF-induced mitogenic response. We conclude that stimulation of c-myc expression is a common response to a diverse group of agents that elicit phosphoinositide turnover and activate protein kinase C, and that neither activation of protein kinase C nor enhanced c-myc expression is sufficient for the mitogenic action of PDGF.     

Two signal transduction pathways mediate interleukin-1 receptor expression in Balb/c3T3 fibroblasts

Our previous studies showed that platelet-derived growth factor (PDGF) modulated interleukin-1 (IL-1) activity and IL-1 binding to Balb/c3T3 fibroblasts (Bonin, P. D., and Singh, J. P. (1988) J. Biol. Chem. 263, 11052-11055). Subsequent studies have demonstrated an action of PDGF at the level of IL-1 receptor (IL-1R) gene expression. PDGF treatment of Balb/c3T3 cells produces a 10-20-fold stimulation of mRNA for IL-1 receptor. Investigation of the signal transduction pathways shows that activation of either the protein kinase C pathway or the cAMP-mediated pathway leads to the stimulation of IL-1 receptor expression in Balb/c3T3 cells. Treatment of Balb/c3T3 cells with phorbol 12-myristate 13-acetate (PMA), a known activator of protein kinase C, produced an increased 125I-IL-1 binding to cells and stimulation of IL-1R mRNA. Staurosporine, an inhibitor of protein kinase C, blocked the induction of IL-1 binding by PDGF or PMA. Down-regulation of protein kinase C by pretreatment with PMA reduced the subsequent stimulation by PDGF. Chronic treatment with PMA, however, did not produce a complete inhibition of PDGF effect on IL-1R. Further studies showed that the agents that stimulate cAMP accumulation (isobutyl methylxanthine, dibutyryl), directly stimulate adenylate cyclase (forskolin), or activate G protein (choleragen) stimulated 125I-IL-1 binding and IL-1R mRNA accumulation in Balb/c3T3 cells. These studies suggest that potentially two signal transduction pathways mediate IL-1 receptor expression in Balb/c3T3 fibroblasts. Evidence is presented that suggests that stimulation of IL-1R through these two pathways (PMA/PDGF-stimulated and cAMP-stimulated) occurs independent of each other.     

The inhibition of phosphoenolpyruvate carboxykinase (guanosine triphosphate) gene expression by insulin is not mediated by protein kinase C

The role protein kinase C plays in the regulation of phosphoenolpyruvate carboxykinase (PEPCK) gene expression by insulin and phorbol esters was studied in H4IIE hepatoma cells (ATCC CRL 1548). The combined effects of phorbol 12-myristate 13-acetate (PMA) and insulin on the suppression of mRNA coding for PEPCK (mRNAPEPCK) synthesis were additive. A potent inhibitor of both cyclic nucleotide-dependent protein kinases and protein kinase C, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine, inhibited the cAMP and PMA-mediated regulation of mRNAPEPCK synthesis, but did not affect the action of insulin. Desensitization of the protein kinase C pathway by exposure to PMA for 16 h abolished the subsequent action of the phorbol ester, but did not affect insulin- or cAMP-mediated regulation of PEPCK gene expression. We conclude that insulin suppresses PEPCK gene expression independently from the protein kinase C-mediated pathway used by phorbol esters.     

Effects of mitogens on ornithine decarboxylase activity and messenger RNA levels in normal and protein kinase C-deficient NIH-3T3 fibroblasts

Ornithine decarboxylase activity was assessed in serum-deprived quiescent NIH-3T3 murine fibroblasts after exposure to a variety of growth-promoting factors. Ornithine decarboxylase activity increased after treatment with phorbol 12-myristate 13-acetate (PMA), fetal calf serum, bovine pituitary fibroblast growth factor (FGF), platelet-derived growth factor (PDGF), and the synthetic diacyglycerol sn-1,2-dioctanolyglycerol but not after treatment with epidermal growth factor, insulin, 4 alpha-phorbol 12,13-didecanoate, sn-1,2-dibutyrylglycerol, or the calcium ionophore A23187. Activity peaked at 3-4 h and returned to basal levels after 8 h. To determine the importance of protein kinase C in this increase, cells were pretreated with PMA for 16 h to make the cells effectively deficient in protein kinase C; this deficiency was documented by direct measurement of enzyme activity and immunoreactivity. The ornithine decarboxylase response to each mitogen was then compared in cells pretreated with PMA or control conditions. PMA pretreatment abolished the increase in ornithine decarboxylase activity due to additional PMA and decreased but did not eliminate the ability of serum, FGF, and PDGF to cause increases in ornithine decarboxylase activity. Similarly, pretreatment with PMA abolished the ability of additional PMA to increase ornithine decarboxylase mRNA levels but did not prevent the increases in these mRNA levels caused by FGF or serum. These data suggest that the increases in ornithine decarboxylase activity and mRNA levels that occur in quiescent fibroblasts in response to serum, FGF, or PDGF are due to activation of at least two separate pathways, one involving protein kinase C and the other independent of protein kinase C.     

Regulation of platelet-derived growth factor A and B chain gene expression in bone marrow stromal cells

MBA-2, bone marrow-derived endothelial stromal cells, express platelet-derived growth factor (PDGF) A and B chain mRNAs and secrete PDGF activity that is induced by TGF-beta. Either chain of the PDGF molecule could modulate hematopoiesis and stromal cell growth. Intracellular pathways that regulate PDGF expression in the marrow microenvironment are unknown. In the present study, we examined the mechanisms that mediate PDGF A and B chain mRNA induction by TGF-beta and the role of protein kinase C (PKC) and cyclic AMP in PDGF regulation. TGF-beta was tested in parallel with PMA, an activator of phorbol ester-dependent PKC isoforms. Both PMA (10^?7M) and TGF-beta (2.5 ng/ml) increased PDGF A and B chain mRNA levels. The serine/threonine protein kinase inhibitor, H7, blocked PDGF A and B chain mRNA induction in response to TGF-beta. However, down-regulation of PKC by prolonged incubation with PMA failed to abolish TGF-beta induction of PDGF A and B chain mRNAs. These findings indicate that induction of PDGF A and B chain mRNAs can be mediated via phorbol ester-dependent PKC pathway. In contrast, H7-sensitive protein kinase(s) other than phorbol ester-sensitive protein kinase C mediate the effect of TGF-beta. Agents that increase cAMP were also tested for their effect on PDGF gene expression. TGF-beta-mediated induction of PDGF A and B chain mRNAs was markedly inhibited by cAMP. cAMP also blocked stimulation of PDGF A chain mRNA by PMA. The positive and negative signaling mechanisms involved in modulating PDGF in the microenvironment may be important for determining hematopoietic and stromal cell responses in vivo. © 1995 Wiley-Liss, Inc.     

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.     

Phorbol ester downregulates PDGFbeta receptor via PKCbeta1 in vascular smooth muscle cells

The role of protein kinase C (PKC) and their isoforms in cell growth regulation remains elusive. Here we showed that in cultured human vascular smooth muscle cells (SMC), the PKC stimulator phorbol 12-myristate 13-acetate (PMA) inhibited [(3)H]thymidine incorporation in response to the growth factor PDGF associated with downregulation of PDGFbeta (but not alpha) receptors, which was recovered to normal level after PKC was depleted. The changes in PDGFbeta receptor were inversely correlated with PKCbeta1 protein levels regulated by PMA. The downregulation of PDGFbeta receptor by PMA was fully prevented by the PKCbeta inhibitor LY379196, however, without recovery of [(3)H]thymidine incorporation to PDGF. In contrast, [(3)H]thymidine incorporation was fully recovered after depletion of PKCs. These results indicate that in human SMC PKCbeta1 mediates PDGFbeta receptor downregulation. Other PKC isoforms activated by phorbol ester also contribute to the inhibitory effects on cell growth.     

Butyrate blocks the accumulation of CDC2 mRNA in late G1 phase but inhibits both the early and late G1 progression in chemically transformed mouse fibroblasts BP-A31

Sodium butyrate (6 mM) blocks the resumption of the cell division cycle in serum-deprived chemically transformed Balb/c-3T3 mouse fibroblasts (BP-A31). The inhibition of G1 progression by sodium butyrate is not restricted to a specific mitogenic signaling pathway and is equally effective when tetradecanoyl phorbol acetate (TPA), insulin, or fetal calf serum (FCS) is used as inducer. The inhibitor acts in early as well as late G1 phase as indicated by experiments in which inhibitor was added and withdrawn at different times after restimulation of quiescent cells by FCS. At the gene expression level, sodium butyrate does not affect the inducibility of early cell cycle-related genes (c-myc, c-jun) while blocking the induction of cdc 2 mRNA, a late G1 marker. We conclude that sodium butyrate does not interfere with the growth factor signaling pathways regulating the (early) cell cycle-related gene expression. However, the presence of sodium butyrate early in G1 phase inhibits the cascade of events leading eventually to the expression of late G1-characteristic genes such as cdc2. The antimitogenic activity of sodium butyrate may be related to its interference with an (unknown) process involved in the "mitogenic" cascade.     

Salicylic acid reverses phorbol 12-myristate-13-acetate (PMA)- and tumor necrosis factor alpha (TNFalpha)-induced insulin receptor substrate 1 (IRS1) serine 307 phosphorylation and insulin resistance in human embryonic kidney 293 (HEK293) cells

Salicylates, including aspirin, have been shown to improve insulin sensitivity both in human and animal models. Although it has been suggested that salicylates sensitize insulin action by inhibiting IkappaB kinase beta (IKKbeta), the detailed mechanisms remain unclear. Protein kinase C isoforms and tumor necrosis factor alpha (TNFalpha) signaling pathways are well described mediators of insulin resistance; they are implicated in the activation of IKKbeta and the subsequent inhibition of proximal insulin signaling via insulin receptor substrate 1 (IRS1) and Akt. This study investigated the effect of salicylic acid on phorbol 12-myristate 13-acetate (PMA)- and TNFalpha-induced insulin resistance in a human embryonic kidney 293 (HEK293) cell line stably expressing recombinant human IRS1. The results showed that both PMA and TNFalpha inhibited insulin-induced Akt phosphorylation and promoted IRS1 phosphorylation on Ser-307. Salicylic acid pretreatment completely reversed the effects of PMA and TNFalpha on both Akt and IRS1. Whereas PMA activated protein kinase C isoforms and IKKbeta, TNFalpha activated neither. On the other hand, both PMA and TNFalpha activated the c-Jun N-terminal kinase (JNK), which has been reported to directly phosphorylate IRS1 Ser-307. SP600125, a JNK inhibitor, prevented PMA and TNFalpha-induced IRS1 Ser-307 phosphorylation. Finally, salicylic acid inhibited JNK activation induced by both PMA and TNFalpha. Taken together, these observations suggest that salicylic acid can reverse the inhibitory effects of TNFalpha on insulin signaling via an IKKbeta-independent mechanism(s), potentially involving the inhibition of JNK activation. The role of JNK in salicylic acid-mediated insulin sensitization, however, requires further validation because the JNK inhibitor SP600125 appears to have other nonspecific activity in addition to inhibiting JNK activity.