©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Cell Type-specific Modulation of Cell Growth by Transforming Growth Factor 1 Does Not Correlate with Mitogen-activated Protein Kinase Activation (*)

(Received for publication, August 28, 1995; and in revised form, October 16, 1995)

Yuji Chatani (§) Susumu Tanimura Naomi Miyoshi Akira Hattori (§) Masahiro Sato Michiaki Kohno (¶)

From the Department of Biology, Gifu Pharmaceutical University, 5-6-1, Mitahora-higashi, Gifu 502, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Transforming growth factor beta1 (TGF-beta1) is a multifunctional cytokine that positively or negatively regulates the proliferation of various types of cells. In this study we have examined whether or not the activation of the mitogen-activated protein (MAP) kinases is involved in the transduction of cell growth modulation signals of TGF-beta1, as MAP kinase activity is known to be closely associated with cell cycle progression. Although TGF-beta1 stimulated the growth of quiescent Balb 3T3 and Swiss 3T3 cells, it failed to detectably stimulate the tyrosine phosphorylation and activation of the 41- and 43-kDa MAP kinases at any time point up to the reinitiation of DNA replication. TGF-beta1 also failed to stimulate the expression of the c-fos gene. Furthermore, TGF-beta1 synergistically enhanced the mitogenic action of epidermal growth factor (EGF) without affecting EGF-induced MAP kinase activation in these fibroblasts, and it inhibited the EGF-stimulated proliferation of mouse keratinocytes (PAM212) without inhibiting EGF-induced MAP kinase activation. Thus, the ability of TGF-beta1 to modulate cell proliferation is apparently not associated with the activation of MAP kinases. In this respect, TGF-beta1 is clearly distinct from the majority, if not all, of peptide growth factors, such as platelet-derived growth factor and EGF, whose ability to modulate cell proliferation is closely associated with the activation of MAP kinases. These results also suggest that the activation of MAP kinases is not an absolute requirement for growth factor-stimulated mitogenesis.


INTRODUCTION

Transforming growth factor-beta (TGF-beta) (^1)is the prototype of a large family of cytokines that regulate a wide variety of cellular processes including cell proliferation, cell differentiation, cell motility, cell organization, and extracellular matrix production (reviewed in (1, 2, 3, 4) ). The TGF-beta family includes three mammalian isoforms, TGF-beta1, TGF-beta2, and TGF-beta3, which have similar biochemical and biological characteristics. The effects of TGF-beta on cell growth control are complicated and vary dramatically depending on the target cell type, the cell density, and the presence of other growth factors in the culture medium. Although these biological functions of TGF-beta have been intensively studied over the past decade, the biochemical mechanisms that underlie these complex effects are largely unknown.

TGF-beta generates diverse cellular responses by interacting with specific membrane-bound proteins. Affinity labeling with radioiodinated TGF-beta has identified a number of different sizes of receptors and binding proteins; these include type I (M(r) = 53,000), type II (M(r) = 75,000), type III (M(r) = 280,000), type IV (M(r) = 60,000), type V (M(r) = 400,000), and type VI (M(r) = 180,000) receptors, as well as several other membrane binding proteins of M(r) = 40,000, 60,000, and 140,000 (reviewed in (5, 6, 7) ). Among these receptors and membrane binding proteins, the most widely distributed are the type I, II, and III receptors. The way these different receptors contribute to the multiple functions of TGF-beta is unclear. However, recent genetic and biochemical evidence suggests that the type I and type II receptors, both of which have transmembrane serine/threonine kinase structures, are essential for eliciting the many effects of TGF-beta(8, 9, 10, 11) , while the type III receptor (betaglycan) is involved in the presentation of the ligand to the signaling receptors(12, 13) .

TGF-beta has emerged as a positive or negative regulator of cell proliferation; it stimulates the growth of certain mesenchyme-derived cells but acts as a potent growth inhibitor of many other cell types such as epithelial and endothelial cells(1, 2, 3, 4) . All these effects of TGF-beta have been suggested to be mediated, as described above, by the activation of a heteromeric receptor complex that consists of the type I and type II receptors(8, 9, 10, 11) . Thus, in order to gain further insight into these dual effects of TGF-beta on cell growth regulation, it appears critical to determine the exact molecular signaling mechanism from the receptors to the nucleus. Up until now, the majority of reports pertaining to TGF-beta-regulated cell growth control have focused on the identification of the nuclear components that are regulated by this factor in inducing its growth-inhibitory effects. For example, in epithelial cells, TGF-beta decreases c-myc, p34, cdk4, and B-myb expression(14, 15, 16, 17, 18, 19) , decreases the phosphorylation of the retinoblastoma protein and p34(20, 21) , prevents cdk2 activation(22) , and regulates several G(1) cyclins and cell cycle-associated cyclin-cdk inhibitors(23, 24, 25, 26) . In contrast to these nuclear effects of TGF-beta, very little is known about how it regulates cytoplasmic signaling components in cells where TGF-beta is neither growth-stimulatory nor growth-inhibitory.

Mitogen-activated protein (MAP) kinases, also known as extracellular signal-regulated kinases (ERKs), are representative cytoplasmic signaling components. They are activated in many cell types by diverse extracellular stimuli that elicit a wide array of physiological responses such as cell division, differentiation, and secretion. MAP kinases are serine/threonine kinases that phosphorylate a variety of regulatory proteins, which include other protein kinases (p90, MAP kinase-activated protein kinase 2, etc.), cytoplasmic phospholipase A(2), cytoskeletal proteins (microtubule-associated proteins), and transcription factors (c-Jun, c-Myc, p62, etc.) (reviewed in (27, 28, 29, 30, 31, 32) ). Thus, MAP kinases are thought to function as key intermediaries in the intracellular signal transduction networks. The most widely studied members of the MAP kinase family are the 43- and 41-kDa MAP kinases (ERK1 and ERK2, respectively); these are activated by phosphorylation on both threonine and tyrosine residues (33, 34) by a dual specificity kinase, MAP kinase/ERK kinase (MEK)(35, 36) . MEK activity is in turn regulated by serine phosphorylation catalyzed by MEK activators; the major MEK activator is a serine/threonine kinase, Raf-1(37, 38, 39) . Furthermore, it has recently been reported that the activated Ras-mediated translocation of Raf-1 to the plasma membrane is one of the necessary events for Raf-1 to be activated following phosphorylation(40, 41, 42) . However, the identity of the kinase(s) that phosphorylate Raf-1 is poorly understood at present. Interesting in this respect, a rapid activation of Ras by TGF-beta has been reported in intestinal epithelial (IEC 4-1) cells, where TGF-beta was found to be growth-inhibitory(43) .

In this report, we have examined whether or not the effects of TGF-beta in modulating cell proliferation are associated with the activation of 41- and 43-kDa MAP kinases. Our results demonstrate that (i) TGF-beta1 alone can stimulate the growth of Swiss 3T3 and Balb 3T3 cells without MAP kinase activation, (ii) TGF-beta1 synergistically enhances the mitogenic action of EGF in these fibroblasts without affecting EGF-induced MAP kinase activation, and (iii) TGF-beta1 inhibits the EGF-stimulated proliferation of mouse keratinocytes without inhibiting EGF-induced MAP kinase activation.


EXPERIMENTAL PROCEDURES

Cell Culture

Swiss albino mouse fibroblasts (Swiss 3T3; American Type Culture Collection, CCL92) and Balb/c mouse fibroblasts (Balb 3T3, clone A31-1-1 (JCRB 0601)) were obtained through the Japanese Cancer Research Resources Bank. Mouse keratinocytes (PAM212) were kindly provided by Dr. T. Nakamura (Osaka University). Cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. For experimental use, confluent cell cultures in Dulbecco's modified Eagle's medium containing 10% fetal calf serum were rendered quiescent by incubation for 4048 h in serum-free medium (Dulbecco's modified Eagle's medium containing 2 mg/ml of bovine serum albumin (Boehringer Mannheim), 1 µg/ml of insulin, 2 µg/ml of transferrin, 20 nM Na(2)SeO(3), and 10 mM Hepes, pH 7.4), and then growth factors were added(44, 45) .

Measurement of Growth Stimulation

DNA synthesis was measured 12 h after exposure of the cells to mitogens by the addition of 5-bromo-2`-deoxyuridine (BrdU) (3 µg/ml) to the cultures (cells were grown on glass coverslips) followed by incubation for 16 h. After incorporation, the percentage of BrdU-labeled nuclei was determined by using a cell proliferation kit (Amersham International). The percentage of labeled nuclei was determined in a total of over 800 nuclei by using 2 coverslips/experimental condition(44, 45) .

Western Blot Analysis

Growth factor-stimulated cells were washed twice with ice-cold phosphate-buffered saline, scraped off plates into hypotonic cell lysis buffer (25 mM Tris-HCl, pH 7.4, 25 mM NaCl, 1 mM sodium orthovanadate, 10 mM NaF, 10 mM sodium pyrophosphate, 25 mM beta-glycerophosphate, 25 mMp-nitrophenylphosphate, 10 nM okadaic acid, 0.5 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, and 1% aprotinin), and then flash-frozen in liquid nitrogen. After three cycles of freeze-thaw, the cells were lysed by passage through a 25-gange needle followed by sonication for 60 s. Lysates were cleared by centrifugation at 12,000 times g for 30 min, and protein concentrations were determined using the BCA protein assay reagent (Pierce). Equal amounts of protein (usually 10 µg) were separated by SDS-PAGE, electrophoretically transferred to an Immobilon-P membrane (Millipore), and probed with either an anti-MAP kinase antibody (affinity-purified) or an anti-phosphotyrosine antibody (4G10) (Upstate Biotechnology Inc. The anti-MAP kinase antibody was raised against residues 299-321 (RIEVEQALAHPYLEQYYDPSDEP) of the 41-kDa MAP kinase (ERK2) and recognizes both the 43- and 41-kDa MAP kinases(34, 46) . Immunoreactive bands were visualized by enhanced chemiluminescence (ECL) (Amersham).

MAP Kinase Activity Assay

Cell lysates prepared as described above were pretreated with 0.1% SDS for 20 min at 4 °C (34, 46) , diluted 1:10 in cell lysis buffer, and then immunoprecipitated by incubating 3 h at 4 °C with the anti-MAP kinase antibody preadsorbed to protein-A Sepharose (Pharmacia Biotech Inc). The immunoprecipitates were washed 3 times with cell lysis buffer and twice with kinase buffer (50 mM Tris-HCl, pH 8.0, 25 mM MgCl(2), 1 mM dithiothreitol, 0.5 mM EGTA, 10% glycerol). Kinase assays were performed by incubating the immunoprecipitates in 30 µl of kinase buffer containing 20 µM ATP, 1 µCi [-P] ATP (3000 Ci/mmol) (Amersham), and 0.5 mg/ml myeline basic protein (MBP) (Sigma). After 30 min at 30 °C, the reaction was stopped by adding 10 µl of 0.6% HCl containing 1 mM ATP and 1% bovine serum albumin. Samples (30 µl) were then spotted onto 1.5 times 1.5-cm squares of phosphocellulose papers (P81, Whatman) and washed 5 times in 180 mM phosphoric acid, and the radioactivity incorporated into MBP was determined by liquid scintillation spectrometry. In some experiments, the phosphorylation reaction was terminated by the addition of 10 µl of 4 times SDS sample buffer, and the incorporation of P into MBP was examined by SDS-PAGE followed by autoradiography. Both assays always gave identical results (see Fig. 2and Fig. 3). Our anti-MAP kinase antibody recognized both the 41- and 43-kDa MAP kinases, and thus the MBP-phosphorylation activity of each of the immunoprecipitates is the sum of the activity of these two species of MAP kinases. Synchronous activation of both the 41- and 43-kDa MAP kinases in mitogen-stimulated cells was confirmed by using the kinase detection assay with MBP-containing polyacrylamide gels (data not shown, (34) ).


Figure 2: Mitogen-induced activation of 41- and 43-kDa MAP kinases in Balb 3T3 and Swiss 3T3 cells. Growth-arrested Balb 3T3 or Swiss 3T3 cells were treated with 20 ng/ml of PDGF-BB, 20 ng/ml of PDGF-AA, or 2.5 ng/ml of TGF-beta1 for the indicated periods of time. Cells were then lysed, and cell lysates (20 µg protein) were resolved by SDS-PAGE, blotted, and probed with anti-MAP kinase antibody or anti-phosphotyrosine antibody, followed by ECL detection. Closed arrowheads indicate positions of the phosphorylated (activated) forms of 41- and 43-kDa MAP kinases (pp41, pp43), while open arrowheads indicate positions of the unphosphorylated form of these MAP kinases (p41, p43). MAP kinase assay was performed by incubating cell lysates (10 µg of protein) with anti-MAP kinase antibody, followed by the kinase reaction and resolution on SDS-PAGE; phosphorylated MBP was detected by autoradiography (P-MBP). Data shown are representative of three to five separate experiments that gave essentially the same results.




Figure 3: Kinetics of MAP kinase activation by PDGF-BB and TGF-beta1 in Balb 3T3 cells. Growth-arrested Balb 3T3 cells were treated with 20 ng/ml of PDGF-BB or 2.5 ng/ml of TGF-beta1 for the indicated periods of time. MAP kinase assay was performed by incubating cell lysates (10 µg of protein) with anti-MAP kinase antibody followed by the kinase reaction; radioactivity incorporated into MBP was determined as described under ``Experimental Procedures.'' Inset shows the prolonged kinetics of MAP kinase activation. Each value represents the mean ± S.E. of triplicate determinations of a representative experiment. Similar results were obtained in five independent experiments.



RNA Isolation and Northern Blot Analysis

Total RNA was isolated from growth factor-stimulated fibroblasts according to the method of Chomczynski and Sacchi(47) . Twenty µg of total RNA was fractionated on 1.2% agarose gels containing 2.2 M formaldehyde and then transferred to a Zeta-probe membrane (Bio-Rad). The filter-bound RNA was hybridized with randomly primed, P-labeled v-fos cDNA probe (pfos-1 obtained through Japanese Cancer Research Resources Bank) (48) for 16 h at 42 °C. The filters were then washed under stringent conditions according to the instruction manual (Bio-Rad).

Materials

Recombinant human TGF-beta1 was purchased from Wako Chemical Co. (Osaka, Japan); recombinant human PDGF-AA, recombinant human PDGF-BB, and EGF purified from mouse submaxillary glands were from Toyobo Co. (Osaka, Japan). Other chemicals and reagents were of the purest grade available.


RESULTS

TGF-beta1 Stimulates the Proliferation of Fibroblasts without Activating the 41- and 43-kDa MAP Kinases

The addition of recombinant human TGF-beta1 to growth-arrested cultures of mouse fibroblasts such as Balb 3T3 and Swiss 3T3 cells stimulated the reinitiation of DNA synthesis. The mitogenic activity of TGF-beta1 was dose-dependent; as little as 0.025 ng/ml of TGF-beta1 stimulated the growth of these cells, and growth stimulation was maximal at a concentration of 1 ng/ml (Fig. 1). Although TGF-beta1 was a good mitogen for both fibroblast cell lines and stimulated DNA synthesis to approximately the same level as did PDGF-AA, its effect was weaker than that of PDGF-BB or EGF at maximal effective concentrations.


Figure 1: Mitogen-induced DNA synthesis reinitiation in Balb 3T3 and Swiss 3T3 cells. Growth-arrested Balb 3T3 or Swiss 3T3 cells were exposed to varying concentrations or 2.5 ng/ml of TGF-beta1, 20 ng/ml of PDGF-AA, 20 ng/ml of PDGF-BB, or 20 ng/ml of EGF. The rate of DNA synthesis was measured 12 h after exposure of the cells to mitogens by adding BrdU to the cultures followed by incubation for 16 h. BrdU-labeled nuclei were determined in a total of more than 800 nuclei using 2 coverslips/experimental condition. Data shown are representative of four separate experiments that gave essentially the same results.



PDGF-AA, PDGF-BB, and EGF rapidly activated both of the 41- and 43-kDa MAP kinases in growth-arrested Balb 3T3 and Swiss 3T3 cells. Activation of these MAP kinases was detected by the appearance of their active forms, which show reduced mobility in SDS-PAGE due to phosphorylation of specific threonine and tyrosine residues(33, 34) , by analyzing their tyrosine phosphorylation, and by a direct in vitro kinase assay of MAP kinase immunoprecipitates using MBP as the substrate. These analyses always gave essentially the same time course profile of MAP kinase activation. As shown in Fig. 2and Fig. 3, the activation of the MAP kinases in PDGF-AA/-BB-stimulated Balb 3T3 cells reached a maximal level within 10 min of the addition of PDGF-AA/-BB and then declined gradually. However, considerable activation (20% of maximum) could still be detected 3 h after stimulation. In contrast, TGF-beta1 stimulation of quiescent Balb 3T3 and Swiss 3T3 cells did not significantly induce the tyrosine phosphorylation or the activation of 41- and 43-kDa MAP kinases at any time point up to 16 h, although the addition of TGF-beta1 to these cells led to the reinitiation of DNA synthesis after a lag time of 12 h (data not shown).

TGF-beta1 Does Not Induce the Expression of Protooncogene c-fos

It has recently been reported that the ternary complex factor (p62/Elk-1) is phosphorylated by MAP kinases. This phosphorylation results in enhanced ternary complex formation, which then induces c-fos expression(49, 50) . We therefore wished to see if TGF-beta1 could stimulate the expression of c-fos in Balb 3T3 and Swiss 3T3 cells. Treatment of these cells with PDGF-AA/-BB or EGF apparently induced the rapid and transient expression of c-fos gene (Fig. 4) In contrast, TGF-beta1 did not stimulate c-fos gene expression significantly in these cells at any time point up to 6 h after treatment, although TGF-beta1 stimulated the reinitiation of DNA synthesis in Balb 3T3 cells to about the same or even higher levels than did PDGF-AA or EGF under these experimental conditions.


Figure 4: Mitogen-induced expression of c-fos gene in Balb 3T3 and Swiss 3T3 cells. Growth-arrested Balb 3T3 cells were treated with of 2.5 ng/ml of TGF-beta1, 20 ng/ml of PDGF-AA, or 1 ng/ml of EGF for the indicated periods of time, while growth-arrested Swiss 3T3 cells were treated with 2.5 ng/ml of TGF-beta1 or 20 ng/ml of PDGF-BB. Twenty µg of total RNA was separated by formaldehyde/agarose gel electrophoresis, blotted, and hybridized with P-labeled v-fos cDNA probe. The amounts of 28 and 18 S ribosomal RNA are shown as internal standards. The values for the percentage of labeled nuclei in each mitogen-stimulated Balb 3T3 cells were 0.3% (unstimulated), 15.5% (TGF-beta1), 17.6% (PDGF-AA), or 8.8% (EGF), which were determined by using sister cultures corresponding to those shown in the figure. Similar results were obtained in three independent experiments.



TGF-beta1 Synergistically Enhances the Mitogenic Action of EGF in Fibroblasts without Affecting EGF-induced MAP Kinase Activation

The possible enhancing effect of combinations of growth factors on the reinitiation of DNA synthesis and MAP kinase activation was examined in Balb 3T3 cells and Swiss 3T3 cells. For this analysis, cells were treated with a suboptimal concentration of each of the growth factors, as the enhancing effect of each mitogen on DNA synthesis was not clearly observed when combinations of optimal concentrations were employed (data not shown). As shown in Fig. 5, the simultaneous addition of EGF and TGF-beta1 stimulated the reinitiation of DNA synthesis in Balb 3T3 cells to a much greater extent than did each mitogen alone. This effect was apparently synergistic since the sum of the number of cells that reinitiated DNA synthesis when treated simultaneously with EGF and TGF-beta1 exceeded by approximately 2-fold the sum of the number of cells that reinitiated DNA synthesis when the cells were stimulated with EGF and TGF-beta1 separately. These synergistic effects were also observed between EGF and PDGF-BB and between EGF and PDGF-AA (data not shown), although to a lesser extent compared with those of EGF and TGF-beta1. In contrast, treatment of Swiss 3T3 and Balb 3T3 cells with combinations of PDGF-BB/-AA and TGF-beta1 were at best additive, and no synergistic effect on the reinitiation of DNA synthesis was observed.


Figure 5: Effect of combinations of growth factors on DNA synthesis reinitiation and MAP kinase activation in Balb 3T3 cells. Growth-arrested Balb 3T3 cells were treated with 0.25 ng/ml of TGF-beta1, 1 ng/ml of EGF, or 2.5 ng/ml of PDGF-BB, separately or in combination (TGF-beta1/EGF (A); TGF-beta1/PDGF-BB (B); EGF/PDGF-BB (C)). The rate of DNA synthesis was measured as described in the Fig. 1legend. Dotted lines/arrowheads indicate values for sum of the percentage of BrdU-labeled nuclei in the cells stimulated with these growth factors separately (right panel). MAP kinase activity was determined after stimulating cells with growth factors for the indicated periods of time as described in the Fig. 3legend, using sister cultures corresponding to those used for the measurement of DNA synthesis. Each value represents the mean ± S.E. of duplicate determinations of a representative experiment (left panel). Similar results were obtained in three independent experiments.



Thus, although TGF-beta1 influenced the mitogenic potential of EGF and PDGF-AA/-BB in Balb 3T3 and Swiss 3T3 cells quite differently, it did not affect significantly the degree of MAP kinase activation induced by all of these growth factors at any time point analyzed (Fig. 5, A and B). In sharp contrast, the degree of MAP kinase activation, especially at the sustained phase (3060 min following stimulation), was markedly enhanced in those fibroblasts treated simultaneously with EGF and PDGF-AA/-BB compared with fibroblasts treated separately with these mitogens. Fig. 5C shows the typical results of such an analysis for Balb 3T3 cells stimulated with a combination of EGF and PDGF-BB.

TGF-beta1 Inhibits the EGF-stimulated Proliferation of Mouse Keratinocytes without Inhibiting EGF-induced MAP Kinase Activation

Addition of EGF to growth-arrested mouse keratinocytes (PAM212) markedly stimulated the reinitiation of DNA synthesis. On the other hand, TGF-beta1 alone did not affect significantly the proliferation of serum-starved PAM 212 cells. However, TGF-beta1 decreased the mitogenic potential of EGF by more than 60% when the cells were treated with these growth factors together (Fig. 6A).


Figure 6: Effect of combination of TGF-beta1 and EGF on DNA synthesis reinitiation and MAP kinase activation in PAM212 cells. Growth-arrested PAM212 cells were exposed to 2.5 ng/ml of TGF-beta1 (T) or 20 ng/ml of EGF (E), separately or in combination (E/T). A, the rate of DNA synthesis was measured 12 h after exposure of the cells to growth factors by adding BrdU to the cultures followed by incubation for 10 h. The percentage of labeled nuclei were determined in 400 nuclei/coverslip, and each value represents the mean ± S.E. of determinations on three coverslips/experimental condition of a representative experiment. B, cell lysates (10 µg protein) of PAM212 cells treated with growth factors for the indicated periods of time were resolved by SDS-PAGE, blotted, and probed with anti-MAP kinase antibody (Anti-MAPK) or anti-phosphotyrosine antibody (Anti-pTyr), followed by ECL detection. Closed arrowheads indicate positions of the phosphorylated (activated) forms of 41- and 43-kDa MAP kinases (pp41, pp43), while open arrowheads indicate positions of the unphosphorylated form of these MAP kinases (p41, p43). C, MAP kinase assay was performed by incubating cell lysates (10 µg of protein) with anti-MAP kinase antibody followed by the kinase reaction; radioactivity incorporated into MBP was determined as described under ``Experimental Procedures.'' Each value represents the mean ± S.E. of duplicate determinations of a representative experiment, using sister cultures corresponding to those used to measure DNA synthesis (A). Similar results were obtained in two independent experiments.



EGF rapidly and markedly induced tyrosine phosphorylation and activation of the 41- and 43-kDa MAP kinases in PAM 212 cells as it did in fibroblasts (Fig. 6, B and C). TGF-beta1 alone did not induce MAP kinase activation in PAM 212 cells at any time point analyzed. Also, TGF-beta1 did not affect EGF-induced MAP kinase activation in these epithelial cells when the two growth factors were added simultaneously, although under these conditions EGF's capacity to stimulate cell proliferation was markedly reduced.


DISCUSSION

We have examined whether or not the MAP kinase cascade is involved in the signaling pathway of TGF-beta in eliciting its effects to modulate cell proliferation. The MAP kinase cascade is the major cytoplasmic kinase pathway activated commonly by numerous mitogenic stimuli that interact with a diversity of structurally distinct receptors(27, 28, 29, 30, 31, 32) , and it has recently been shown that the activation of the cascade is necessary for the proliferation of fibroblasts(51, 52) . For the analysis, we focused on the activation of the 41- and 43-kDa MAP kinases, as these kinases stand at a key position in the cascade and play a role in integrating multiple mitogenic signaling pathways that involve Ras, Raf-1, Mos, MEK kinase-1, protein kinase C, and even certain heterotrimeric G proteins(27, 28, 29, 30, 31, 32) .

We demonstrate in this report that TGF-beta1 fails to detectably activate the 41- and 43-kDa MAP kinases in Swiss 3T3 and Balb 3T3 cells where TGF-beta1 is clearly mitogenic. TGF-beta1 also failed to induce c-fos gene expression in these cells (Fig. 4). This response is mediated by the serum-response element, which is bound in a ternary complex containing the transcription factors p67 and p62(53) , and phosphorylation of the p62 by MAP kinases results in enhanced ternary complex formation with consequent induction of c-fos expression(49, 50) . In some of our experiments, induction of a minimum level of the c-fos gene expression was observed in Swiss/Balb 3T3 cells treated with TGF-beta1 for 30 min but only on overexposed autoradiograms (more than a 10 times longer exposure than that shown in Fig. 4). More significantly, under our experimental conditions, TGF-beta1 stimulated DNA synthesis reinitiation in Balb 3T3 cells to an approximately equal or even higher level than did PDGF-AA or EGF. Thus, it seems unlikely that such an extremely low level of c-fos gene expression could have any consequences for the cell or represent a significant response to the extracellular stimuli. Furthermore, TGF-beta1 had neither a synergistic nor an antagonistic effect on the activation of the 41- and 43-kDa MAP kinases when used in combination with other growth factors, although TGF-beta1 apparently synergized (in fibroblasts) or antagonized (in epithelial cells) the mitogenic potential of several growth factors such as EGF (Fig. 5A and Fig. 6).

All these findings show no apparent association between MAP kinase activation and the ability of TGF-beta1 to modulate cell proliferation. The cytoplasmic signaling pathway of TGF-beta1 appears to be totally independent of the MAP kinase cascade. Thus, TGF-beta1 is clearly distinct from the majority, if not all, of other peptide growth factors such as EGF, PDGF, and fibroblast growth factor whose ability to modulate cell proliferation is closely associated with the activation of MAP kinases(54) . In this respect, interleukin-4(55) , activin A (another member of the TGF-beta superfamily)(56) , and thyrotropin (57, 58) have been reported to induce cellular proliferation without activation of MAP kinases, although thyrotropin has also been shown to induce the activation of MAP kinases in human thyroid cells(59) .

Although it has previously been proposed that TGF-beta promotes the growth of cells via the induced expression of PDGF-AA(60, 61, 62) , recent studies have demonstrated that TGF-beta can stimulate cell proliferation by a PDGF-independent mechanism(63) . Our results showing that TGF-beta1-treatment of cells did not induce MAP kinase activation and c-fos gene expression at any time point analyzed (Fig. 2Fig. 3Fig. 4) suggest that the action of TGF-beta to stimulate proliferation of Swiss 3T3 and Balb 3T3 cells is unrelated to that of PDGF-AA/-BB, because these mitogens clearly induced the activation of MAP kinases and c-fos gene expression soon after binding to their receptors. The mitogenic potential of TGF-beta1 was not inhibited by the depletion of protein kinase C in Balb 3T3 and Swiss 3T3 cells, which were pretreated with 200 ng/ml of phorbol 12-myristate 13-acetate for 24 h (data not shown), would also support the above conclusion; the capacity of PDGF-AA/-BB to reinitiate DNA synthesis was reduced to 50% in such phorbol 12-myristate 13-acetate-pretreated cells compared with the untreated native cells(45) . However, we cannot exclude the possibility that TGF-beta1 induces the production of a novel growth factor, which acts as a direct mitogen in fibroblasts through a MAP kinase-/protein kinase C-independent mechanism.

As far as we know, TGF-beta1 does not induce the activation of other recently identified cytoplasmic signaling pathways such as the JAK/STAT pathway or the JNK(SAPK) pathway in Swiss 3T3 cells. (^2)Activation of the former has been commonly observed in cells stimulated with a variety of cytokines (interferons, interleukins, erythropoitin, etc.) and also with EGF and PDGF (reviewed in (64) ), while the later pathway has been reported to be activated by treatment of cells with UV radiation, proinflammatory cytokines, and environmental stress (reviewed in Refs. 31, 32, and 65). Thus, the signaling pathway of TGF-beta1, beginning with the cell surface receptors that have intrinsic serine/threonine kinase activity, seems quite unique and apparently independent of those well-characterized cytoplasmic kinase cascades. The precise cytoplasmic signaling pathway for TGF-beta1 through which it elicits its complex effects on the regulation of cellular growth remains to be elucidated.

Recently, TGF-beta has been shown to induce the rapid activation of p21(43) , and of a MAP kinase (p44) (66) in proliferating cultures but not in quiescent cultures of intestinal epithelial (IEC 4-1) cells for which TGF-beta is growth inhibitory. However, activation of the p44 was 2-fold at most, and appearance of the active form of p44, which has reduced mobility in SDS-PAGE due to the phosphorylation of specific threonine and tyrosine residues was not clearly observed in the TGF-beta-treated IEC 4-1 cells. It seems likely that only a marginal part of the cellular pp44 was activated. Such a small level of MAP kinase activation could play a role only if it exceeds the threshold level in each cell.

In conclusion, we have demonstrated in this report that TGF-beta1 is able to modulate cellular proliferation by a signaling pathway that is totally independent of the MAP kinase cascade. Our results also suggest that the activation of MAP kinases is not an absolute requirement for the transition of cells from the arrested state (G(0)) through G(1) into S phase.


FOOTNOTES

*
This work was supported in part by grants-in-aid from the Ministry of Education, Science and Culture of Japan and a cancer research grant from the Research Foundation for Pharmaceutical Sciences of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Recipient of Fellowships from the Japan Society for the Promotion of Science for Japanese Junior Scientists.

To whom all correspondence should be addressed. Tel.: 81-58-237-3931 (ext. 207); Fax: 81-58-237-5979.

(^1)
The abbreviations used are: TGF-beta, transforming growth factor-beta; MAP kinase, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; MEK, MAP kinase/ERK kinase; EGF, epidermal growth factor; PDGF, platelet-derived growth factor; PAGE, polyacrylamide gel electrophoresis; ECL, enhanced chemiluminescence; MBP, myeline basic protein; BrdU, 5-bromo-2`-deoxyuridine.

(^2)
S. Iwasaki, N. Miyoshi, A. Hattori, M. Tsujimoto, and M. Kohno, manuscript in preparation.


ACKNOWLEDGEMENTS

We thank Dr. T. Nakamura for supplying us with PAM212 cells and Drs. P. Hughes and S. Toyama for critical reading of the manuscript.


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