©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Transforming Growth Factor Activation of p44 in Proliferating Cultures of Epithelial Cells (*)

(Received for publication, November 21, 1994; and in revised form, January 20, 1995)

Melanie T. Hartsough (§) Kathleen M. Mulder (¶)

From the Department of Pharmacology, Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Transforming growth factor-beta (TGF-beta) is a potent growth inhibitor of a variety of epithelial cell types. The primary signaling mechanism involved in mediating this and other cellular effects of TGF-beta is still unknown. We report here that both TGF-beta(1) and TGF-beta(2) resulted in a rapid activation of mitogen-activated protein kinase (MAPK) p44, occurring within 5-10 min of growth factor addition. This effect occurred in exponentially proliferating cultures of intestinal epithelial (IEC) 4-1 cells under conditions in which DNA synthesis was inhibited by 95% to 98%. Furthermore, TGF-beta(2) induced a sustained activation of p44 under these conditions, lasting for at least 90 min after initial growth factor treatment. Another TGF-beta-sensitive epithelial cell line (CCL 64) displayed a similar rapid increase in p44 activity when treated with TGF-beta(1). In contrast, in IEC 4-6 cells that are resistant to TGF-beta effects on growth and DNA synthesis, TGF-beta(2) treatment did not result in an activation of p44. In contrast to the results in proliferating cultures, treatment of quiescent cultures of IEC 4-1 cells with TGF-beta(2) resulted in no significant change in either DNA synthesis or p44activity within 15 min of TGF-beta addition. In contrast, addition of the growth-stimulatory combination of factors (epidermal growth factor + insulin + transferrin = EIT) to quiescent and proliferating IEC 4-1 cells stimulated DNA synthesis and resulted in a sustained activation of p44. Together, our results suggest an association between activation of p44 and both TGF-beta-mediated growth inhibition and EIT-mediated growth stimulation. This suggests that the specificity for the cellular effects of growth factors may not occur at the level of MAPK activation per se, but rather at downstream events that include phosphorylation of distinct transcriptional complexes and activation of a select assortment of genes. With regard to TGF-beta specifically, we have proposed a model to explain how activation of p44 may be associated with a growth-inhibitory response.


INTRODUCTION

Transforming growth factor-beta (TGF-beta) (^1)regulates a multitude of biological functions in a variety of cell types and is a potent growth inhibitor of epithelial cells(1, 2, 3) . The TGF-beta family includes 3 mammalian isoforms (referred to as TGF-beta(1), TGF-beta(2), and TGF-beta(3)) that elicit cellular responses by interacting with specific membrane-bound proteins. Although several TGF-beta-binding proteins have currently been described, the type I and type II receptors are thought to be the primary signal transducing receptors in most cell types(4) . A number of species of the type I and II receptor classes have been cloned and have been shown to contain a serine/threonine kinase domain(5, 6, 7, 8, 9, 10, 11) . Recently, it was reported that in a yeast genetic screen immunophilin FKBP-12 specifically bound to the type I receptor(12) . In mammalian systems, however, no coupling components or endogenous substrates for the kinase activity have been reported. Moreover, the exact mechanism(s) for signaling from the receptors have not been elucidated.

Evidence has indicated that TGF-beta may signal exclusively through a receptor heterocomplex of type I and type II receptors, in which the cytoplasmic kinase domain of both receptors are essential for signal initiation(13, 14, 15, 16, 17, 18) . In contrast, additional findings suggest that TGF-beta may mediate some cellular responses by signaling through either the type I receptor or the type II receptor, indicating that two distinct receptor-associated signaling pathways may control a separate assortment of TGF-beta responses(19, 20) . Additional studies have reported the existence of several TGF-beta receptor complexes consisting of different receptor subtypes(8, 10, 11, 21, 22) . For example, TGF-beta receptor type II has been shown to complex with TGF-beta receptor type III and with different type I receptors, some of which also bind activin, a member of the TGF-beta superfamily(8, 10, 11, 21, 22) . Thus, TGF-beta may regulate its diverse actions by specifically engaging different receptor subtypes into functional multimeric complexes.

The majority of reports pertaining to the growth-inhibitory effect of TGF-beta have focused on defining components that are regulated by this factor within the nucleus. For example, in epithelial cells, TGF-beta decreases c-myc, p34, cdk4, and B-myb expression(23, 24, 25, 26, 27, 28, 29, 30) , decreases the phosphorylation of the retinoblastoma protein and p34(31, 32, 33) , increases the expression of c-jun and c-fos(34, 35, 36) , stimulates the phosphorylation of cAMP-responsive element binding protein(37) , prevents cdk2 activation(38) , and regulates several G1 cyclins and cell cycle-associated cyclin-cdk inhibitors(39, 40, 41, 42) . In contrast to these nuclear effects of TGF-beta, we have reported the first direct evidence for a rapid activation of a cytoplasmic signaling component for TGF-beta in epithelial cells. That is, we have shown that TGF-beta(1) and TGF-beta(2) resulted in a rapid activation of p21 in TGF-beta-sensitive IEC 4-1 cells, but not in TGF-beta-resistant IEC 4-6 cells(43) . This finding was unexpected, since activation of Ras was thought to be associated with mitogenic responsiveness or stimulation of cellular growth(44) . However, multiple transducing signals are thought to converge downstream of Ras activity at the mitogen-activated protein kinases (MAPKs)(45) . Therefore, it is possible that other signaling molecules, functioning either in parallel with or downstream from Ras in the TGF-beta pathway, may alter or reverse the stimulatory signals transmitted by p21. Thus, it was of interest to examine the effect of TGF-beta on MAPK activity.

MAPKs, also known as extracellular signal regulated kinases (ERKs) constitute a family of serine/threonine kinases ranging in molecular mass from 42 kDa to 97 kDa. Two MAPK proteins, p42 and p44, require phosphorylation on both tyrosine and threonine residues for full enzymatic activation(46, 47) . Additionally, they are activated in association with stimulation of cell growth by a range of different agents, including insulin(48, 49) , thrombin(50) , epidermal growth factor(51) , and several hematopoietic growth factors(52, 53) . Moreover, nerve growth factor has been shown to activate these MAPK forms in association with the induction of differentiation in PC12 cells(49, 54) . Recently, several Ras-independent pathways have been shown to induce MAPK activity as well(55) . Once activated, p42 and p44 phosphorylate a variety of substrates found within the cytoplasm and the nucleus(45) . Recent studies have demonstrated that p42 and p44 can be translocated to the nucleus when they are persistently activated (56, 57) . With transient induction, however, MAPK activity is localized in the cytoplasm(56, 57) . Hence, p42 and p44are thought to serve as intermediaries connecting the two subcellular compartments.

In this report, we utilized the same two clonal intestinal epithelial (IEC) cell lines which were used to demonstrate an association between activation of Ras and growth inhibition by TGF-beta(43) . We have previously described the isolation of these TGF-beta-sensitive and -resistant cell clones(58) . Moreover, hypersensitivity of the IEC 4-1 cells to TGF-beta, relative to the parental cells from which they were derived, was explained by a 5- to 10-fold increase in both total receptor numbers per cell and TGF-beta binding to the type I and type II signaling receptors(58) . Here, we demonstrate that TGF-beta activates p44 in epithelial cells for which TGF-beta is growth-inhibitory. In contrast, TGF-beta did not activate this kinase in proliferating and quiescent cultures of epithelial cells that were resistant to the growth-inhibitory effects of this polypeptide.


EXPERIMENTAL PROCEDURES

Materials

Epidermal growth factor was purchased from UBI (Lake Placid, NY). Insulin, transferrin, and myelin basic protein (MBP) were obtained from Sigma. ERK1 antibody (SC-93), which has a higher affinity for p44 than p42, was purchased from Santa Cruz. [-P]ATP (3000 Ci/mmol, BLU002H) and [^3H]thymidine (20 Ci/mmol, NET-027X) were obtained from DuPont NEN. Nonimmune, whole molecule rabbit IgG was purchased from Jackson ImmunoResearch Laboratories, Inc. TGF-beta was a generous gift from P. R. Segarini (Celtrix Pharmaceuticals, Santa Clara, CA).

Cell Culture

The untransformed rat intestinal epithelial cell (IEC) clones, that are either sensitive(4-1) or resistant(4-6) to TGF-beta effects on growth inhibition, have been described previously (43, 58) . The clones were maintained in SMIGS medium consisting of McCoy's 5A (Life Technologies, Inc.) supplemented with amino acids, pyruvate, and antibiotics (streptomycin/penicillin) (SM), as well as insulin (4 µg/ml), glucose (4.5 mg/ml), and 5% fetal bovine serum. Mink lung epithelial CCL 64 cells were obtained from ATCC and were maintained in DMEM (Life Technologies, Inc.) containing 10% fetal bovine serum (DMEM-10).

Thymidine Incorporation

For experiments using exponentially proliferating cultures, IEC 4-1 and IEC 4-6 cells were plated at densities of 1.3 times 10^4 cells/cm^2 and 2.4 times 10^4 cells/cm^2, respectively, in 12-well plates in SMIGS medium. CCL 64 cells were plated at a density of 2.5 times 10^4 cells/cm^2, in 12-well plates in DMEM-10 medium. One day later, the medium was changed to serum-free medium (IEC cells were placed in SM, while CCL 64 cells were placed in DMEM) for 12-15 h, in order to remove serum factors and binding proteins. This brief incubation in serum-free medium does not render the cells quiescent. TGF-beta (10 ng/ml) or EIT (E = EGF (10 ng/ml), I = insulin (20 µg/ml), and T = transferrin (4.0 µg/ml)) was then added to the appropriate wells for 24 h, after which time the cells were in late log phase (approximately 85-95% confluent). For experiments with quiescent cells, IEC 4-1 cells were plated at a density of 1.6 times 10^5 cells/cm^2 in SMIGS medium. Two days later, the medium was changed to McCoy's 5A (PM) medium for 9 days; this incubation period was shown previously to be required to produce a quiescent state in the IEC 4-1 cells, based upon the achievement of stable baseline levels of thymidine incorporation(43) . On the ninth day, TGF-beta (10 ng/ml) or EIT (concentrations mentioned above) was added to the wells for 24 or 21 h, respectively. [^3H]thymidine incorporation was then determined as described previously(24) . Briefly, cells were labeled for 1 h with [^3H]thymidine (25 µCi/well), washed once with SM, and treated for 10 min with 10% trichloroacetic acid. DNA was then extracted for 30 min with a solution of 40 µg/ml DNA, 0.2 M NaOH. Aliquots were analyzed for ^3H radioactivity in a Beckman scintillation counter.

In Vitro p44 Immunocomplex Assay

IEC 4-1, IEC 4-6, and CCL 64 cells were plated as for thymidine incorporation experiments, except that 75-cm^2 flasks were used. For experiments using exponentially proliferating cultures, 1 day after plating, cells were placed in serum-free medium (as indicated above) prior to TGF-beta (10 ng/ml) treatment. For experiments with quiescent cells, after the 9-day starve period, IEC 4-1 cells were treated with TGF-beta (10 ng/ml) or EIT (at concentrations described above) without a change of media, for the times indicated in the figure legend. In vitro p44 activity was determined by a modified version of the method described by Thomas and co-workers(59) . Briefly, cells were washed twice with phosphate-buffered saline and lysed in the nonionic detergent buffer described, modified by the addition of okadaic acid (100 nM) (Life Technologies, Inc.) and the reduction of the aprotinin concentration to 0.5%. After centrifugation and normalization of protein, lysates were incubated for 1 h at 4 °C with p44 antibody SC-93, which had been preadsorbed to protein A-agarose. Nonspecific phosphorylation of MBP was determined by incubating cell lysates with nonimmune rabbit IgG, followed by protein A-Sepharose. Immunoadsorbents were then washed once in lysis buffer and twice in Tris-buffered saline supplemented with sodium orthovanadate (1 mM) and benzamidine (5 mM). 30 µl of the reaction mixture (30 mM Hepes, 10 mM MgCl(2), 1 mM dithiothreitol, 20 µM ATP, 20 µCi of [-P]ATP, and 0.2 µg/ml MBP) was incubated with the immunoadsorbents for 30 min at 30 °C, and the reaction was terminated with SDS sample buffer. The eluted proteins were separated by SDS-polyacrylamide gel electrophoresis (15%). Gels were then Coomassie-stained, dried, and subjected to autoradiography. In some cases, the proteins were transferred to polyvinylidene difluoride and subjected to autoradiography. A Betagen betascope 603 blot analyzer (Betagen Corp., Waltham, MA) was used to quantitate the phosphorylation of MBP. Equal loading was determined by immunoblotting the polyvinylidene difluoride with p44 (SC-93) for 1 h at 25 °C followed by a second incubation with rabbit IgG-horseradish peroxidase conjugate for 1 h at 25 °C. MAPK (p44) protein was then visualized by enhanced chemiluminescence (ECL).


RESULTS

Inhibition of DNA Synthesis and Activation of p44 by TGF-beta(2) in Proliferating Cultures IEC 4-1 Cells

We have previously demonstrated the inhibitory effects of TGF-beta on cell number and growth rate of IEC 4-1 cells(43, 58) . Here, we examined the effect of TGF-beta(2) on the incorporation of [^3H]thymidine into DNA in exponentially proliferating IEC 4-1 cells. For these experiments, cells were incubated in serum-free medium prior to TGF-beta addition; this brief incubation does not render the cells quiescent(43) , but it does remove serum components that could interfere with TGF-beta responsiveness. Fig. 1A indicates that TGF-beta (10 ng/ml) addition to proliferating IEC 4-1 cells produced a 98% inhibition of DNA synthesis after 24 h.


Figure 1: Effect of TGF-beta(2) on [^3H]thymidine incorporation and p44activity in proliferating cultures of IEC 4-1 cells. A, exponentially proliferating IEC 4-1 cells were treated with or without TGF-beta(2) (10 ng/ml) for 24 h in serum-free medium, after which time [^3H]thymidine incorporation was determined, as described under ``Experimental Procedures.'' Results are expressed as x ± S.D. (n = 3). B, near-confluent IEC 4-1 cells were treated for the indicated times with TGF-beta(2) (10 ng/ml). Cell lysates were incubated with either an antibody specific for p44 (SC-93) or nonimmune rabbit IgG (nonspecific control). In vitro phosphorylation of MBP by p44 was then analyzed as described under ``Experimental Procedures.'' Phosphorylated MBP was visualized by autoradiography. C, equal loading was determined by incubation of transferred proteins with p44 antibody (SC-93), followed by ECL detection. h.c. IgG = heavy chain IgG. D, plot of betagen scan results, x ± S.D. (n = 4 or 5); statistical significance was determined for all times except the 90-min point, which is represented as the mean of two experiments. *, p < 0.01.



In order to determine whether MAPK activity would be altered by TGF-beta, we examined the time-dependent effects of TGF-beta(2) on in vitro p44 activity. IEC 4-1 cells, plated as for Fig. 1A, but treated with TGF-beta(2) (10 ng/ml) for various times, were lysed and immunoprecipitated with either nonimmune rabbit IgG (nonspecific control) or with an antibody specific for p44. Immunoprecipitates were then incubated with MBP, and in vitro phosphorylation of MBP was examined by SDS-polyacrylamide gel electrophoresis and autoradiography (Fig. 1B). As Fig. 1B illustrates for the 0- and 5-min time points, MBP phosphorylation was not observed in the samples immunoprecipitated with nonimmune rabbit IgG. This was the case for all time points (data not shown). Equal loading was determined by immunoblotting transferred proteins with a p44-specific antibody (Fig. 1C). As Fig. 1C depicts, p44 was equally immunoprecipitated and equally loaded at all time points examined. Further, as expected, the nonimmune rabbit IgG immunoprecipitation control did not contain the p44 protein; however, the IgG heavy chain is visible in these lanes (Fig. 1C, Rab IgG lanes, 0 and 5 min). Relative levels of MBP phosphorylation were determined by betagen scanning. The mean MBP phosphorylation levels from 4 or 5 experiments are plotted in Fig. 1D, and the level of statistical significance at individual time points is provided. The results indicate that treatment of exponentially proliferating cultures of IEC 4-1 cells with TGF-beta(2) (10 ng/ml) resulted in a subtle, but sustained, activation of p44 (Fig. 1, B and D). As Fig. 1D depicts, p44 activity was increased by 1.6-fold within 5 min of TGF-beta(2) addition and then continued to increase as a function of incubation time. By 30 min, p44 activity had reached maximal levels of 1.9-fold above basal activity. This activity slightly decreased by 60 min, but was still sustained at levels 1.5-fold above basal activity for at least 90 min (Fig. 1, B and D). Thus, TGF-beta(2) produced a sustained activation of p44 under conditions that resulted in a 98% growth inhibition.

Inhibition of DNA Synthesis and Activation of p44 by TGF-beta(1) in Proliferating IEC 4-1 and CCL 64 Cells

In order to determine whether the effect of TGF-beta(2) on MAPK activity was TGF-beta-isoform-specific, we treated proliferating IEC 4-1 cells with the TGF-beta(1) isoform (10 ng/ml) for 10 min. As Fig. 2A indicates, TGF-beta caused a 95% inhibition in [H]thymidine incorporation in exponentially proliferating IEC 4-1 cells. In addition, a 1.8-fold induction of p44 activity was detected (Fig. 2, B and C). Thus, both TGF-beta and TGF-beta isoforms have similar effects on MAPK activity and DNA synthesis in IEC 4-1 cells.


Figure 2: Effect of TGF-beta(1) on [^3H]thymidine incorporation and p44activity in proliferating cultures of IEC 4-1 and CCL 64 cells. A, exponentially proliferating IEC 4-1 cells were incubated with or without TGF-beta(1) (10 ng/ml) for 24 h prior to determination of [^3H]thymidine incorporation, as in Fig. 1. Results are presented as x ± S.D. (n = 3). B, exponentially proliferating CCL 64 and IEC 4-1 cells were treated with TGF-beta(1) (10 ng/ml) for 10 min. In vitro phosphorylation of MBP by p44was determined as for Fig. 1. Equal loading was verified by Coomassie staining of the gels used for autoradiography. C, plot of betagen scan results are from two separate experiments, expressed as x ± range.



Additionally, we wished to determine whether p44 activation by TGF-beta occurred in epithelial cell types other than IEC 4-1 cells. Thus, we chose to examine the effects of TGF-beta(1) on the mink lung epithelial cell line CCL 64. TGF-beta(1) has previously been shown to reversibly inhibit the DNA synthesis of CCL 64 cells by more than 90% at a concentration of 2.5 ng/ml(60) . Under the conditions we utilized, a 97% inhibition in DNA synthesis was observed with TGF-beta(1) (10 ng/ml) treatment (data not shown). Moreover, a 10-min treatment with TGF-beta(1) (10 ng/ml) induced p44 activity by approximately 2.0-fold in the CCL 64 cells (Fig. 2, B and C). Thus, the stimulation of MAPK by TGF-beta is not limited to intestinal epithelial cells and can be detected in lung epithelial cells as well.

Effects of TGF-beta(2) on DNA Synthesis and p44 Activity in TGF-beta-resistant IEC 4-6 Cells

In order to determine whether the increase in p44 activity by TGF-beta was associated with the growth-inhibitory responsiveness of the cells, we utilized a TGF-beta-resistant intestinal epithelial clone (IEC 4-6), previously derived from the IEC-18 cell line(58) . Although we have demonstrated that the IEC 4-6 clone displayed altered ratios of receptor binding to the TGF-beta signaling receptors (type I and type II) by TGF-beta(1) and TGF-beta(2), overall levels of binding to the type I and II receptors were similar to those observed in the parental IEC-18 cells. Thus, post-receptor mechanisms were proposed to explain the TGF-beta resistance observed in the IEC 4-6 cell line(58) . In contrast, the IEC 4-1 cells, for which IC values were a least 1/9 those of the parental cells, displayed 5-10 times as many signaling receptors as did the parental cells(58) . The results in Fig. 3A demonstrate that TGF-beta (10 ng/ml) treatment of cycling IEC 4-6 cells had no significant effect on DNA synthesis after 24 h. Additionally, TGF-beta did not activate p44 in the TGF-beta-resistant IEC 4-6 cells, yet a 1.7-fold activation of p44 was observed in the IEC 4-1 cells after 10 min (Fig. 3, B and C). Taken together, these results indicate that p44 is activated by TGF-beta in IEC cells that are sensitive to the effects of TGF-beta on cellular growth, yet not in cells that are resistant to these TGF-beta-mediated effects.


Figure 3: Effect of TGF-beta(2) on [^3H]thymidine incorporation and p44activity in proliferating cultures of IEC 4-6 cells. A, exponentially proliferating IEC 4-6 cells were incubated for 24 h with or without TGF-beta(2) (10 ng/ml). Thymidine incorporation was determined as for Fig. 1. Results are expressed as x ± S.D. (n = 3). B, near-confluent IEC cells were treated for the indicated times with TGF-beta(2) (10 ng/ml). In vitro phosphorylation of MBP by p44 was then determined as for Fig. 1. Equal loading was determined as for Fig. 2. C, plot of betagen scan results for IEC 4-1 cells is expressed as x ± S.D. (n = 5); IEC 4-6 results plotted are from two separate experiments, expressed as x ± range.



Effects of TGF-beta(2) on DNA Synthesis and p44 Activity in Quiescent IEC 4-1 Cells

In order to determine whether the activity of p44 would be altered by TGF-beta in the absence of an effect of TGF-beta on DNA synthesis, quiescent IEC 4-1 cells were treated with TGF-beta for various times. For these experiments, quiescence was established by placing confluent cultures of IEC 4-1 cells on PM medium for 9 days. TGF-beta(2) (10 ng/ml) was then added to the cells without replenishment of medium, and thymidine incorporation was measured 24 h later. Cells made quiescent by nutrient and growth factor deprivation in this fashion are arrested in the cell cycle in early G(0)/G(1). Upon treatment with TGF-beta, the cells would be expected to remain in the G(1) phase of the cell cycle, since TGF-beta is known to arrest cell growth at a restriction point in late G(1)(1, 2, 3) . Accordingly, no change in DNA synthesis (S phase) would be expected after treatment of quiescent cells with TGF-beta; TGF-beta addition, under these conditions, could then serve as a negative control. As expected, Fig. 4A indicates that, under these conditions, TGF-beta had no significant effect on DNA synthesis. Further, Fig. 4, B and C, depicts the kinetics for TGF-beta effects on p44 activity in quiescent IEC 4-1 cells. The results indicate that, when quiescent cells were treated with TGF-beta (10 ng/ml) for time periods that resulted in activation of MAPK in proliferating cells (0-15 min), no significant change in p44 activity was observed.


Figure 4: Effect of TGF-beta(2) on [^3H]thymidine incorporation and p44activity in quiescent cultures of IEC 4-1 cells. A, quiescent IEC 4-1 cells were incubated for 24 h with or without TGF-beta(2) (10 ng/ml). Thymidine incorporation was determined as for Fig. 1. Results are expressed as x ± S.D. (n = 3). B, quiescent cells were treated for the indicated times with TGF-beta(2) (10 ng/ml). In vitro phosphorylation of MBP by p44 was determined as for Fig. 1. Equal loading was determined as for Fig. 2. C, plot of betagen scan results shown in B. Results are representative of two experiments.



EIT Activation of DNA Synthesis and 44 in Quiescent and Proliferating IEC 4-1 Cells

Since TGF-beta had no effect on p44 in quiescent cells, we wished to verify whether growth-stimulatory factors would alter p44 activity at quiescence in our system, as has been previously reported for other cell types(50, 51, 56) . Thus, a combination of growth-stimulatory factors (epidermal growth factor + insulin + transferrin = EIT) was added to quiescent IEC 4-1 cells. As Fig. 5A indicates, incubation of quiescent IEC 4-1 cells with EIT resulted in a 72-fold increase in DNA synthesis. Fig. 5, B and C, depicts the time-dependent effects of EIT on p44 activity in quiescent 4-1 cells. Within 2 min of EIT addition, a rapid increase in p44 activity, to levels 2.4-fold above baseline values, was observed. MAPK activity peaked at levels 3.8-fold above baseline values after 5 min and then slightly declined to levels 2.5-fold above basal levels thereafter. The elevation of p44 activity by EIT in IEC 4-1 cells was sustained, in that the phosphorylation of MBP by p44 remained at levels 2.5-fold above baseline levels for at least 60 min.


Figure 5: The effect of the growth- stimulatory combination of factors (E + I + T = EIT) on [^3H]thymidine incorporation and p44activity in quiescent and proliferating cultures of IEC 4-1 cells. A, quiescent IEC 4-1 cells were incubated for 21 h with or without EIT, after which time [^3H]thymidine incorporation levels were determined as for Fig. 1. Results are presented as x ± S.D. (n = 3). B, quiescent IEC 4-1 cells were treated with EIT for the indicated times, and p44 activity was determined as for Fig. 1. C, plot of betagen scan results shown in B. D, proliferating cultures of IEC 4-1 cells were incubated with the growth-stimulatory combination EIT for 24 h. Thymidine incorporation was determined as for Fig. 1. Results are expressed as x ± S.D. (n = 3). E, near-confluent IEC 4-1 cells were treated with EIT for the indicated times. In vitro phosphorylation of MBP by p44was then determined as for Fig. 1. F, plot of betagen scan results shown in E. Equal loading was determined by immunoblotting, as in Fig. 1. Results are representative of two experiments.



Additionally, we wished to investigate whether these growth factors would modulate p44 in proliferating cultures of IEC 4-1 cells, as we have observed for TGF-beta. Fig. 5D indicates that treatment with EIT for 24 h stimulated a 3.0-fold increase in DNA synthesis in proliferating cultures of IEC 4-1 cells. Furthermore, a 3.1-fold induction of p44 was detected within 5 min of growth factor addition, which then decreased slightly to levels 2.8-fold above basal activity for at least 20 min (Fig. 5, E and F). All lanes were equally loaded, as determined by incubating transferred proteins with p44 antibody (SC-93), followed by ECL detection (data not shown). Thus, this growth-stimulatory combination of factors (EIT) stimulated DNA synthesis and activated p44 in both proliferating and quiescent IEC 4-1 cells.


DISCUSSION

In this report, we have demonstrated that both TGF-beta(1) and TGF-beta(2) result in a rapid activation of p44, beginning as early as 5 min after addition of the growth factor to proliferating cultures of epithelial cells. In untransformed intestinal epithelial cells, maximal activation of p44 by TGF-beta occurred at 30 min, at which time kinase activity was stimulated by 1.9-fold. Moreover, this effect occurred under conditions in which the growth of the cells was inhibited by at least 95-98%. This report is the first description of an activation of this form of MAPK by a growth inhibitor. Although a previous report described a slight activation of a 57-kDa Erk-like protein by TGF-beta(1) in colon carcinoma cells(61) , only a transient activation was observed. In the untransformed epithelial cells that we have examined, TGF-beta(2) affected a sustained activation of p44, lasting for at least 90 min.

Recent evidence has indicated that the level and duration of activation of MAPK are important factors in determining which specific cellular effects will be elicited(56, 57) . For example, a persistent activation of MAPKs was coupled to the mitogenic potential of Chinese hamster lung (CCL 39) fibroblast cells, whereas non-mitogens, such as phorbol esters and alpha-thrombin-receptor synthetic peptides, only induced a transient activation of MAPK(56) . Conversely, EGF, a growth stimulator of PC12 pheochromocytoma cells, resulted in a transient activation of MAPK in these cells, whereas the induction of differentiation by nerve growth factor in PC-12 cells was associated with a sustained activation of MAPK(57) . Hence, the duration of MAPK activation is not definitively correlated with a specific cellular function, but it does appear to be regulated in a cell-type and stimulus-specific manner. In our studies, the kinetics for TGF-beta activation of p44 closely resembled the sustained activation displayed by the differentiation factor nerve growth factor in PC-12 cells(57) .

In both of the reports discussed above, the prolonged phase of MAPK activation was linked to translocation of the enzyme into the nucleus (56, 57) . Thus, the prolonged activation of p44 by TGF-beta (sustained for at least 90 min) suggests that TGF-beta may result in translocation of this form of MAPK to the nucleus. Although we have not yet examined TGF-beta effects on subcellular localization of p44, the kinetic profile for the activation of this kinase suggests that this TGF-beta-mediated cellular effect may provide an important link between the cytoplasmic and nuclear events associated with TGF-beta responses. Furthermore, our results indicate that the activation of p44 by TGF-beta may be associated with the growth-inhibitory responsiveness of the IEC 4-1 cells to TGF-beta. That is, while this effect was observed in IEC 4-1 cells that are growth-inhibited by TGF-beta, the activation of p44 by TGF-beta was not observed in IEC 4-6 cells that are insensitive to the growth-inhibitory effects of TGF-beta.

Our experiments involving TGF-beta addition to quiescent cells, in the absence of other exogenous growth factors or serum, also support an association between inhibition of DNA synthesis by TGF-beta and activation of p44. That is, addition of TGF-beta to IEC 4-1 cells, made quiescent by growing the cells on basal medium alone for 9 days (nutrient and growth factor deprivation), resulted in no change in DNA synthesis and no activation of p44 within 24 h and 15 min after TGF-beta addition, respectively. It was not surprising that [^3H]thymidine incorporation into DNA did not change under the conditions of these experiments (see Fig. 4A), since treatment with TGF-beta would not result in entry of the cells into the S phase of the cell cycle. However, it was of interest that p44 was not activated within 15 min of TGF-beta addition to quiescent IEC 4-1 cells, as was observed after TGF-beta addition to proliferating cultures. A previous report (62) indicated that TGF-beta inhibited the activation of p44 (for periods up to 30 min) in quiescent CCL 64 mink lung epithelial cells, released from TGF-beta-induced G(1) growth arrest by addition of serum and EGF. In these experiments, EGF and serum were added in the absence and presence of TGF-beta to cells that had been arrested in late G(1) by growth of the cells on TGF-beta for 24 h prior to subsequent additions. In contrast, our results demonstrate direct effects of TGF-beta alone (i.e. in the absence of serum or other growth factors) on p44, a potential signaling component for mediating growth inhibition by TGF-beta.

As discussed earlier, the kinetics for TGF-beta activation of p44 closely resemble those obtained in PC-12 cells, following addition of the differentiation factor nerve growth factor (57) . Further, TGF-beta has been shown to induce a more differentiated phenotype in some cell types, including untransformed IEC and human colon carcinoma cells, under certain circumstances(63, 64, 65) . Thus, it is possible that the activation of p44 by TGF-beta may be involved in regulating cellular differentiation, rather than cell growth. Evidence against this possibility includes the fact that TGF-beta activated p44 in CCL 64 cells, which do not appear to undergo morphological differentiation in response to TGF-beta. Further, although we have not examined markers of differentiation in the IEC 4-1 cells, the induction of intestinal-specific enzymes is not always associated with TGF-beta responsiveness in IEC-6 cells(63, 66) .

Although the data in Fig. 1Fig. 2Fig. 3Fig. 4indicate an association between TGF-beta activation of p44 and growth inhibition, other results in Fig. 5, A-F, indicate that the growth factor combination EIT can also activate p44 in IEC 4-1 cells. However, the kinetics for MAPK activation by EIT in both proliferating and quiescent IEC 4-1 cells were different from those for TGF-beta. That is, activation of p44 was maximal by 5 min after EIT addition, whereas maximal levels of activation were not attained until 30 min after TGF-beta addition. Moreover, EIT stimulated p44 activity to levels 3-4-fold above baseline values, whereas TGF-beta induced kinase activity by only 2-fold (approximately) in IEC 4-1 cells. Thus, the temporal regulation and threshold levels of MAPK activity may specify some growth factor responses. However, MAPK activation was sustained in association with both TGF-beta-mediated growth inhibition and EIT-mediated growth stimulation. Thus, it appears that the specificity for the cellular effects of growth factors may largely be determined, not at the level of MAPK activation per se, but by subsequent events (i.e. the formation of various complexes between MAPKs and transcription factors, the phosphorylation and activation of distinct transcription factors, and the temporal activation of select classes of genes).

With regard to TGF-beta specifically, one possible explanation for the observed activation of p44 by TGF-beta, in association with an inhibition of cell growth, involves an associated phosphorylation and activation of Elk-1/p62, ultimately leading to autoinduction of TGF-beta (see model in Fig. 6). This hypothesis is based upon several previous reports. That is, it has been demonstrated that autoinduction of TGF-beta(1) is mediated by the AP-1 (Jun-Fos) complex in the TGF-beta(1) promoter, and that both Jun and Fos components are required for transcriptional activation(34) . TGF-beta(1) has also been shown to autoinduce TGF-beta(1) expression in IEC-6 cells (66) and to induce both c-jun and c-fos expression in other cell types (34, 35, 36) . In addition, it has been shown that p44/p42 can efficiently phosphorylate and activate the transactivation potential of Elk-1/p62in vitro and in vivo(67, 68, 69) . Phosphorylation of specific sites within the C-terminal domain of Elk-1/p62 enhances the formation of a ternary complex composed of Elk-1/p62, p67, and the serum response element in the c-fos promoter, thereby increasing c-fos transcription, and subsequently AP-1-dependent gene transcription(67, 68, 69) . Thus, TGF-beta addition to untransformed epithelial cells may result in activation and nuclear translocation of p44, followed by phosphorylation and activation of Elk-1/p62, increased transcription of c-fos, elevated AP-1 activity of the TGF-beta(1) promoter, and increased production of TGF-beta(1) (see Fig. 6). In cell types that have the potential to activate the latent, secreted TGF-beta(1, 2, 3) , the growth-inhibitory effects of TGF-beta would be amplified. In this way, the activation of p44 by TGF-beta may be indirectly associated with the growth-inhibitory effects of TGF-beta. However, additional experiments are required to test this model.


Figure 6: Model for association between activation of p44 by TGF-beta and growth inhibition. TGF-beta activates p44 within minutes of its addition to proliferating cultures. This activation results in phosphorylation of transcription factors such as Elk-1/p62 and in transcriptional activation of genes such as c-fos. Elevated expression of c-fos induces an increase in the TGF-beta(1) promoter AP-1 activity, thereby inducing TGF-beta(1) production. This autoinduction of TGF-beta(1) results in amplification of the growth-inhibitory effect of this growth factor in cell types that can activate the secreted TGF-beta. R and R = TGF-beta receptor type I and receptor type II, Ras = p21, MAPKK = mitogen-activated protein kinase kinase, SRF = p67, Elk-1 = Elk-1/p62, SRE = serum response element, and Fos/Jun = heterocomplex of c-Fos and c-Jun.



In summary, we have shown that a growth inhibitor (TGF-beta) can activate a kinase known for its involvement in growth-stimulatory pathways (p44). The activation began within 5 min of TGF-beta addition to proliferating cultures of epithelial cells, in the absence of serum or other growth stimulators, and was sustained for at least 90 min. Moreover, the activation occurred in cells that responded to TGF-beta with an inhibition of DNA synthesis and cell growth, but not in cells that did not display these effects. We have proposed a model to account for the association between activation of this kinase and the growth-inhibitory responsiveness of the cells to TGF-beta. The model involves phosphorylation and activation of transcription factors (such as Elk-1/p62) that can ultimately increase AP-1-dependent gene transcription, thereby leading to TGF-beta(1) autoinduction via the AP-1 sites in the TGF-beta(1) promoter. In keeping with this model, the TGF-beta-sensitive IEC 4-1 cells produce and activate TGF-beta(1), whereas the TGF-beta-resistant IEC 4-6 cells do not produce any detectable TGF-beta(1)(58) . Thus, the IEC 4-6 cells may be resistant to the growth-inhibitory effects of TGF-beta due to their inability to activate p44 and to amplify production of TGF-beta(1).


FOOTNOTES

*
This work was supported by National Institutes of Health Grants CA51452 and CA54816 (to K. M. M.). 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 a Merck graduate fellowship.

Recipient of National Institutes of Health Research Career Development Award K04 CA59552. To whom correspondence and reprint requests should be addressed: Dept. of Pharmacology, Pennsylvania State University College of Medicine, 500 University Dr., Hershey, PA 17033. Tel.: 717-531-6789; Fax: 717-531-5013.

(^1)
The abbreviations used are: TGF-beta, transforming growth factor-beta; MAPK, mitogen-activated protein kinase; MBP, myelin basic protein; DMEM, Dulbecco's modified Eagle's medium; EGF, epidermal growth factor.


ACKNOWLEDGEMENTS

We thank P. R. Segarini (Celtrix Pharmaceuticals) for generously supplying the TGF-beta and Eric Dauter for his assistance in the preparation of this manuscript.


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