Regulation of protooncogenes c-fos and c-myc expressions by protein tyrosine kinase, protein kinase C, and cyclic AMP mitogenic pathways in dog primary thyrocytes: a positive and negative control by cyclic AMP on c-myc expression

The proliferation of dog thyrocytes in primary culture is stimulated by three distinct intracellular signaling pathways: (1) the thyrotropin or forskolin-cyclic AMP-mediated cascade which is compatible with the differentiated state of the cell; (2) the protein kinase C pathway activated by diacylglycerol and phorbol esters; and (3) a protein tyrosine kinase system activated by epidermal growth factor. The two latter pathways also induce dedifferentiation. The activation of the three cascades induced the expression of the protooncogenes c-fos and c-myc with dose-response curves similar to those for DNA synthesis. After TPA and EGF, the time courses of stimulation of c-fos and c-myc were the same as those for mitogenically stimulated fibroblasts. However, after the cyclic AMP stimulation, c-myc expression was biphasic with an enhancement at 1 h followed by a down-regulation. A similar inhibition by cyclic AMP was also observed on the increased c-myc expression induced by EGF. This down-regulation is suppressed by cycloheximide, which suggests the involvement of a neosynthesized or a labile protein intermediate. The action of cyclic AMP on c-myc mRNA levels could be related to the opposite requirements of the stimulation of both proliferation and differentiation expression by the cyclic AMP pathway in the differentiated thyrocytes.     

Stimulation of protein kinase C or protein kinase A mediated signal transduction pathways shows three modes of response among serum inducible genes

Activation of the signal transduction pathways mediated by protein kinase A (PKA) or protein kinase C (PKC) led to different responses of several serum inducible genes including the jun gene family, c-fos, c-myc, krox 20 and krox 24. Whereas all of these genes were stimulated by the phorbol ester TPA, a chemical activator of protein kinase C, they were differently regulated upon cAMP stimulation of the PKA dependent pathway. The proto-oncogenes jun B, c-fos, and to a lesser extent jun D were stimulated by increasing the intracellular concentration of cAMP, whereas the TPA stimulation of c-jun and c-myc was inhibited under these conditions. Krox 20 and krox 24 were insensitive to this second messenger. This study allowed us to classify these growth stimulated genes into three distinct groups distinguished by their sensitivity to elevated concentrations of intracellular cAMP. The inhibition of c-jun and c-myc expression in the presence of increased cAMP levels may be at least partially responsible for the growth inhibitory effect of this agent in Balb/c-3T3 cells.     

Differential protein phosphorylation in induction of thyroid cell proliferation by thyrotropin, epidermal growth factor, or phorbol ester.

Protein phosphorylation was studied in primary cultures of thyroid epithelial cells after the addition of different mitogens: thyrotropin (TSH) acting through cyclic AMP, epidermal growth factor (EGF), or 12-O-tetradecanoylphorbol-13-acetate (TPA). EGF or TPA increased the phosphorylation of five common polypeptides. Among these, two 42-kilodalton proteins contained phosphotyrosine and phosphoserine with or without phosphothreonine. Their characteristics suggested that they are similar to the two 42-kilodalton target proteins for tyrosine protein phosphorylation demonstrated in fibroblasts in response to mitogens. No common phosphorylated proteins were detected in TSH-treated cells and in EGF- or TPA-treated cells. The differences in the protein phosphorylation patterns in response to TSH, EGF, and TPA suggested that the newly emerging cyclic AMP-mediated mitogenic pathway is distinct from the better known growth factor- and tumor promoter-induced pathways.     

Forskolin and 12-0-tetradecanoylphorbol-13-acetate mimic thyrotropin-stimulated protein iodination in mouse thyroid

In the present study, the capacity of the diterpene, forskolin, and the phorbol ester, 12-0-tetradecanoylphorbol-13-acetate (TPA), to stimulate protein iodination in freshly dispersed mouse thyroid open follicles was assessed. Although both agents stimulated 125I incorporation into TCA precipitable material, dose response curves (0.1 - 25 microM) showed that maximal concentrations of either agonist alone failed to reproduce the stimulatory effect of a maximal concentration of thyrotropin (TSH; 50 mU/ml). When a maximal concentration of forskolin (20 microM) and TPA (10 microM) were added in combination, the stimulatory effect was additive and mimicked the effect of TSH. TPA had no significant effect on either basal or forskolin-stimulated cyclic-AMP production. We conclude that the regulation of protein iodination by TSH may involve both the adenylate cyclase-cyclic-AMP-dependent protein kinase system and the diacylglycerol-activated calcium/phospholipid-dependent protein kinase C pathway.     

Phorbol ester prevents the thyroid-stimulating-hormone-induced but not the forskolin-induced decrease of cAMP-dependent protein kinase activity in thyroid cell cultures

The potent tumor promoter 12-O-tetradecanoyl-phorbol 13-acetate (TPA) affects several thyroid cell functions and interacts with thyroid-stimulating hormone (TSH) either by inhibiting or potentiating its action on different cellular parameters. Since phorbol ester acts mainly through the activation of protein kinase C, which is its receptor, we studied this activation and its interaction with TSH and forskolin in suspension cultures of porcine thyroid cells. In thyroid cell cultures, TPA has a dual effect on protein kinase C activity: immediately (2-5 min) after exposure of cells to TPA, it began to be translocated from the cytosol to the particulate fraction. The transfer of the cytosolic enzyme was total and could occur with or without a loss of activity. The translocated enzyme still needed Ca2+ and phospholipids for its activation. The basal activity increased transiently (2-4 h) in both the cytosol and particulate fractions during translocation. The peak activity in the particulate fraction was reached 10-30 min after exposure of cells to TPA, and was followed by down-regulation of protein kinase C and almost complete disappearance of its activity. The residual activity was about 13% of control after a 2-day exposure to TPA. It was unequally distributed between cytosol (4%) and particulate fraction (9%). Prolonged exposure of cells to TPA did not affect either the activity or the subcellular distribution of the cAMP-dependent protein kinase activity. TPA interacted with TSH and prevented the decrease of this activity induced by prolonged exposure of cells to the hormone not only when it was introduced simultaneously with TSH, but also when it was added 24 h after TSH. However, the forskolin-induced decrease in cAMP-dependent protein kinase activity was not prevented by the presence of TPA. TPA also affected the increases in cAMP accumulation mediated by TSH and forskolin. The TSH-induced increase was significantly stimulated by TPA after short contacts (5-15 min), while longer preincubations of cells with TPA provoked a very strong inhibition of the TSH action. However, the forskolin-induced stimulation of the cAMP accumulation was maintained and even further increased in the presence of TPA. Consequently, the actions of TSH and TPA are apparently interdependent, while those of forskolin and TPA seem to be parallel and independent. Neither TSH nor forskolin prevented the TPA-induced down regulation of protein kinase C. The biologically inactive phorbol ester analogue 4 alpha-phorbol 12,13-didecanoate had no effect on protein kinase C activity, and did not interact with either TSH or forskolin.(ABSTRACT TRUNCATED AT 400 WORDS)     

Mitogenic signaling pathways of growth factors can be distinguished by the involvement of pertussis toxin-sensitive guanosine triphosphate-binding protein and of protein kinase C.

We have examined the possible involvements of pertussis toxin (PT)-sensitive guanosine triphosphate (GTP)-binding protein (Gp) and protein kinase C (PKC) in the mitogenic signaling pathways of various growth factors by the use of PT-pretreated and/or 12-O-tetradecanoyl phorbol-13-acetate (TPA)-pretreated mouse fibroblasts. Effects of PT pretreatment (inactivation of PT-sensitive Gp) and TPA pretreatment (depletion of PKC) on mitogen-induced DNA synthesis varied significantly and systematically in response to growth factors: mitogenic responses of cells to thrombin, bombesin, and bradykinin were almost completely abolished both in PT- and TPA-pretreated cells; responses to epidermal growth factor (EGF), platelet-derived growth factor (PDGF), and vanadate were reduced to approximately 50% both in PT- and TPA-pretreated cells compared with native cells; response to basic fibroblast growth factor (bFGF) was not affected in PT-pretreated cells but was inhibited to some extent in TPA-pretreated cells. Thus, growth factors examined have been classified into three groups with regard to the involvements of PT-sensitive Gp and PKC in their signal transduction pathways. Binding of each growth factor to its receptor was not affected significantly by pretreatment of cells with PT or TPA. Inhibitory effects of PT and TPA pretreatment on each mitogen-induced DNA synthesis were not additive, suggesting that the functions of PT-sensitive Gp and PKC lie on an identical signal transduction pathway. Although all three groups of mitogens activated PKC, signaling of each growth factor depends to a varying extent on the function of PKC. Our results indicate that a single peptide growth factor such as EGF, PDGF, or bFGF acts through multiple signaling pathways to induce cell proliferation.     

Na+/K+/Cl- cotransporter activates MAP-kinase cascade downstream to protein kinase C, and upstream to MEK

In this study, we demonstrated that the specific inhibitors of the Na+/K+/Cl- cotransporter (NKCC1), bumetanide and furosemide, inhibited extracellular regulated kinase (ERK) phosphorylation in Balb/c 3T3 fibroblasts, stimulated with a variety of mitogens. In addition to fibroblast growth factor (FGF) shown before, the various mitogens tested in the present study (endothelial growth factor (EGF), platelet-derived growth factor (PDGF), insulin, thrombin, and the phorbol ester, 12-O-tetradecanoyl-phorbol-13-acetate (TPA)). Enter, the Ras/Raf/MEK/ERK cascade via different growth factors receptors and through one of the two main routes. The results of the present study provide evidence that have led us to conclude that the target protein which is controlled by the Na+/K+/Cl- cotransporter, is downstream of tyrosine kinase receptors, as well as of the G-protein-coupled receptor (GPCR). Several additional lines of evidence supported the above conclusion: (i) furosemide inhibits phosphorylation of MAPK kinase (MEK) induced by receptor tyrosine kinase (RTK) ligands, such as PDGF, FGF, and EGF. (ii) Furosemide also inhibited ERK phosphorylation, induced by thrombin, a GPCR. (iii) Furosemide inhibited MEK and ERK phosphorylation even when ERK phosphorylation was induced by direct activation of protein kinase C (PKC) by TPA, which bypasses early steps of the mitogenic cascade. In addition, we found that furosemide did not affect PKC phosphorylation induced directly by TPA. Taken together, the results of the present study indicate that the signal transduction protein, controlled by the Na+/K+/Cl- cotransporter, must be downstream of the PKC, and at/or upstream to MEK in the Ras/Raf/MEK/ERK cascade.     

Estrogen-induced genes,WISP-2 and pS2, respond divergently to protein kinase pathway

Recently, we identified WISP-2 (Wnt-1 inducible signaling pathway protein 2) as a novel estrogen-inducible gene in the MCF-7 human breast cancer cell line. In this study, we examined whether WISP-2 expression is modulated by PK activators. Treatment with protein kinase A (PKA) activators [cholera toxin plus 3-isobutyl-1-methylxanthine (CT/IBMX)] induced WISP-2 expression. CT/IBMX induced expression of the other estrogen-responsive gene, pS2, more dramatically than maximum stimulation by 17beta-estradiol (E2). Treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA), which directly stimulates protein kinase C (PKC) activity, completely prevented WISP-2 mRNA induction by E2, whereas it increased pS2 mRNA expression more dramatically than maximum stimulation by E2. Results of treatments with the protein synthesis inhibitor cycloheximide and the pure antiestrogen ICI182,780 suggest that these PK pathways modulate WISP-2 gene expression via different molecular mechanisms than those for pS2. Because TPA inhibits cell proliferation, we investigated whether WISP-2 induction was dependent on cell growth. Cells were treated with insulin-like growth factor-1 (IGF-1) or interleukin-1alpha (IL-1alpha) to stimulate or inhibit cell growth, respectively. These treatments had no effect on WISP-2 mRNA expression either alone or in combination with E2, suggesting that WISP-2 induction is independent of cell growth.     

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.     

Regulation of VL30 gene expression by activators of protein kinase C

The mouse genome contains a retrovirus-like sequence, designated VL30, which is expressed at high levels in transformed cells and which can be induced by exogenously supplied epidermal growth factor (EGF). Binding of EGF to the EGF receptor produces changes in intracellular calcium levels and phospholipase activity which indirectly lead to activation of protein kinase C. We treated AKR-2B cells, Swiss 3T3 cells, and the 3T3 variants NR6 (EGF receptorless) and TNR9 (phorbol ester nonresponsive) with various phorbol ester tumor promoters and with the synthetic diacylglycerol sn-1,2-dioctanoylglycerol. Tumor-promoting phorbol esters (e.g. 12-O-tetradecanoyl phorbol acetate (TPA] increased the level of VL30 expression. Stimulation with either TPA or EGF produced a similar time course of VL30 expression. TPA induced VL30 expression in the EGF-receptorless NR6 cell line, indicating that neither EGF ligand-receptor binding nor phosphorylation of the EGF receptor was required for induction of VL30 expression. Protein synthesis was not required for the TPA-mediated increase in VL30 expression, as pretreatment with cycloheximide did not block or reduce the TPA effect. VL30 expression was also stimulated by treatment with sn-1,2-dioctanoylglycerol, an analog of a probable endogenous activator of protein kinase C. These results suggest that activation of protein kinase C plays a direct role in regulating VL30 expression.     

Differential activation of the Ras/extracellular-signal-regulated protein kinase pathway is responsible for the biological consequences induced by the Axl receptor tyrosine kinase.

To understand the mechanism of Axl signaling, we have initiated studies to delineate downstream components in interleukin-3-dependent 32D cells by using a chimeric receptor containing the recombinant epidermal growth factor (EGF) receptor extracellular and transmembrane domains and the Axl kinase domain (EAK [for EGF receptor-Axl kinase]). We have previously shown that upon exogenous EGF stimulation, 32D-EAK cells are capable of proliferation in the absence of interleukin-3. With this system, we determined that EAK-induced cell survival and mitogenesis are dependent upon the Ras/extracellular-signal-regulated protein kinase (ERK) cascade. Although the phosphatidylinositol-3 kinase pathway is activated upon EAK signaling, it appears to be dispensable for the biological actions of the Axl kinase. Furthermore, we demonstrated that different threshold levels of Ras/ERK activation are needed to induce a block to apoptosis or proliferation in 32D cells. Recently, we have identified an Axl ligand, GAS6. Surprisingly, GAS6-stimulated 32D-Axl cells exhibited no blockage to apoptosis or mitogenic response which is correlated with the absence of Ras/ERK activation. Taken together, these data suggest that different extracellular domains dramatically alter the intracellular response of the Axl kinase. Furthermore, our data suggest that the GAS6-Axl interaction does not induce mitogenesis and that its exact role remains to be determined.     

Multiple intracellular pathways induce expression of a zinc-finger encoding gene (EGR1): relationship to activation of the Na/H exchanger

Intracellular pathways that rapidly stimulate the expression of a mitogen-inducible, zinc-finger encoding gene, EGR1 (Sukhatme et al., Cell 53:37-43), have been characterized in two human fibroblasts strains (WI-38 and HSWP). Serum and epidermal growth factor (EGF) were each found to strongly stimulate EGR1 expression in both cell types. Comparably high levels of expression could also be induced by treatment with the phorbol ester TPA. In cells rendered deficient in PK-C, serum and EGF were each still capable of inducing high levels of EGR1 mRNA, demonstrating that additional non-protein kinase C pathways are capable of stimulating EGR1 expression. In both fibroblasts strains, stimulation of EGR1 expression by all these agents exhibited rapid, transient kinetics and could be superinduced if protein synthesis was inhibited through the addition of cycloheximide. Finally, various agents, known to stimulate/inhibit the activation of another early mitogenic response, the activation of Na/H exchange, were analyzed for their effect on EGR1 expression. Interestingly bradykinin, vasopressin, and Ca ionophores, which dramatically stimulate Na/H exchange, were only weak stimulants of EGR1 expression. Conversely, EGF, which stimulates Na/H exchange poorly, strongly activated EGR1 expression. Hence while EGR1 expression could be triggered by multiple intracellular pathways, its expression does not appear to require the prior activation of Na/H exchange.     

Contribution of protein kinase A and protein kinase C signalling pathways to the regulation of HSD11B2 expression and proliferation of MCF-7 cells

Contribution of the protein kinase A (PKA) and protein kinase C (PKC) signalling pathways to the regulation of 11beta-hydroxysteroid dehydrogenase type II (HSD11B2) gene expression was investigated in human breast cancer cell line MCF-7. Treatment of the cells with an adenylyl cyclase activator, forskolin, known to stimulate the PKA pathway, resulted in an increase in HSD11B2 mRNA content. Semi-quantitative RT-PCR revealed attenuation of the effect of forskolin by phorbol ester, tetradecanoyl phorbol acetate (TPA), an activator of the PKC pathway. It was also demonstrated that specific inhibitors significantly reduced the effect of activators of the two pathways. Stimulation of the PKA pathway did not affect, whereas stimulation of the PKC pathway significantly reduced MCF-7 cell proliferation in a time-dependent manner. A cell growth inhibitor, dexamethasone, at high concentrations, caused a 40% decrease in proliferation of MCF-7 cells and this effect was abolished under conditions of increased HSD11B2 expression. It was concluded that in MCF-7 cells, stimulation of the PKA signal transduction pathway results in the induction of HSD11B2 expression and that this effect is markedly reduced by activation of the PKC pathway. Activation of the PKC pathway also resulted in inhibition of cell proliferation, while activation of the PKA pathway abolished the antiproliferative effect of dexamethasone. These effects might be due to oxidation of dexamethasone by the PKA-inducible HSD11B2.     

Mitogenic growth factors regulate differentially early gene mRNA expression: a study on two clones of 3T3 fibroblasts.

The relationship between cell proliferation and mRNA levels of the immediate early genes c-fos, c-jun, and jun B has been investigated in two clones of 3T3 fibroblasts (D1-3T3 and N2-3T3) upon treatment with basic fibroblast growth factor (bFGF), thrombin, phorbol 12-myristate 13-acetate (PMA) and dibutyryl cyclic AMP (Bt2cAMP). The 3T3-derived clone D1-3T3 almost stops dividing upon serum deprivation, while the N2-3T3 clone does not. The proliferation of the two clones was stimulated by thrombin and PMA and inhibited by Bt2cAMP. Basic FGF stimulated the growth of D1-3T3 but partly inhibited that of N2-3T3 cells. In spite of variable mitogenic response, immediate early genes, c-fos, c-jun, jun B, and c-myc, were induced by the growth factors and by PMA in both cell clones. In our experimental conditions the early gene mRNAs were expressed independently; i.e., the expression of one protooncogene had no bearing on the expression of the other. The cell growth was not directly related to the expression of a particular protooncogene mRNA. Data are presented showing that early gene mRNA expression induced by bFGF or thrombin was not mediated by protein kinase C activation while thrombin-induced mitosis was. Basic FGF induced a part of c-jun mRNA expression, but not mitosis, through a pertussis toxin-sensitive mechanism.     

Induction of tumor promotor-inducible genes in murine 3T3 cell lines and tetradecanoyl phorbol acetate-nonproliferative 3T3 variants can occur through protein kinase C-dependent and -independent pathways.

We isolated a group of genes that are rapidly and transiently induced in 3T3 cells by tetradecanoyl phorbol acetate (TPA). These genes are called TIS genes (for TPA-inducible sequences). Epidermal growth factor (EGF), fibroblast growth factor (FGF), and TPA activated TIS gene expression with similar induction kinetics. TPA pretreatment to deplete protein kinase C activity did not abolish the subsequent induction of TIS gene expression by epidermal growth factor or fibroblast growth factor; both peptide mitogens can activate TIS genes through a protein kinase C-independent pathway(s). We also analyzed TIS gene expression in three TPA-nonproliferative variants (3T3-TNR2, 3T3-TNR9, and A31T6E12A). The results indicate that (i) modulation of a TPA-responsive sodium-potassium-chloride transport system is not necessary for TIS gene induction either by TPA or by other mitogens and (ii) TIS gene induction is not sufficient to guarantee a proliferative response to mitogenic stimulation.