Transforming growth factor- (TGF-) ()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- family includes 3 mammalian isoforms (referred to as TGF-, TGF-, and TGF-) that elicit cellular responses by interacting with specific membrane-bound proteins. Although several TGF--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- 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- 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- responses(19, 20) . Additional studies have reported the existence of several TGF- receptor complexes consisting of different receptor subtypes(8, 10, 11, 21, 22) . For example, TGF- receptor type II has been shown to complex with TGF- receptor type III and with different type I receptors, some of which also bind activin, a member of the TGF- superfamily(8, 10, 11, 21, 22) . Thus, TGF- 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- have focused on defining components that are regulated by this factor within the nucleus. For example, in epithelial cells, TGF- 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-, we have reported the first direct evidence for a rapid activation of a cytoplasmic signaling component for TGF- in epithelial cells. That is, we have shown that TGF- and TGF- resulted in a rapid activation of p21 in TGF--sensitive IEC 4-1 cells, but not in TGF--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- pathway, may alter or reverse the stimulatory signals transmitted by p21. Thus, it was of interest to examine the effect of TGF- 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-(43) . We have previously described the isolation of these TGF--sensitive and -resistant cell clones(58) . Moreover, hypersensitivity of the IEC 4-1 cells to TGF-, 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- binding to the type I and type II signaling receptors(58) . Here, we demonstrate that TGF- activates p44 in epithelial cells for which TGF- is growth-inhibitory. In contrast, TGF- 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

Cell Culture

Thymidine Incorporation

In Vitro p44 Immunocomplex Assay

RESULTS

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

Figure 1: Effect of TGF- on [H]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- (10 ng/ml) for 24 h in serum-free medium, after which time [H]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- (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-, we examined the time-dependent effects of TGF- on in vitro p44 activity. IEC 4-1 cells, plated as for Fig. 1A, but treated with TGF- (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- (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- 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- 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- in Proliferating IEC 4-1 and CCL 64 Cells

Figure 2: Effect of TGF- on [H]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- (10 ng/ml) for 24 h prior to determination of [H]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- (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- occurred in epithelial cell types other than IEC 4-1 cells. Thus, we chose to examine the effects of TGF- on the mink lung epithelial cell line CCL 64. TGF- 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- (10 ng/ml) treatment (data not shown). Moreover, a 10-min treatment with TGF- (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- is not limited to intestinal epithelial cells and can be detected in lung epithelial cells as well.

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

Figure 3: Effect of TGF- on [H]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- (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- (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- on DNA Synthesis and p44 Activity in Quiescent IEC 4-1 Cells

Figure 4: Effect of TGF- on [H]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- (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- (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

Figure 5: The effect of the growth- stimulatory combination of factors (E + I + T = EIT) on [H]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 [H]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-. 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- and TGF- 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- 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- in colon carcinoma cells(61) , only a transient activation was observed. In the untransformed epithelial cells that we have examined, TGF- 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 -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- 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- (sustained for at least 90 min) suggests that TGF- may result in translocation of this form of MAPK to the nucleus. Although we have not yet examined TGF- effects on subcellular localization of p44, the kinetic profile for the activation of this kinase suggests that this TGF--mediated cellular effect may provide an important link between the cytoplasmic and nuclear events associated with TGF- responses. Furthermore, our results indicate that the activation of p44 by TGF- may be associated with the growth-inhibitory responsiveness of the IEC 4-1 cells to TGF-. That is, while this effect was observed in IEC 4-1 cells that are growth-inhibited by TGF-, the activation of p44 by TGF- was not observed in IEC 4-6 cells that are insensitive to the growth-inhibitory effects of TGF-.

Our experiments involving TGF- 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- and activation of p44. That is, addition of TGF- 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- addition, respectively. It was not surprising that [H]thymidine incorporation into DNA did not change under the conditions of these experiments (see Fig. 4A), since treatment with TGF- 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- addition to quiescent IEC 4-1 cells, as was observed after TGF- addition to proliferating cultures. A previous report (62) indicated that TGF- inhibited the activation of p44 (for periods up to 30 min) in quiescent CCL 64 mink lung epithelial cells, released from TGF--induced G growth arrest by addition of serum and EGF. In these experiments, EGF and serum were added in the absence and presence of TGF- to cells that had been arrested in late G by growth of the cells on TGF- for 24 h prior to subsequent additions. In contrast, our results demonstrate direct effects of TGF- alone (i.e. in the absence of serum or other growth factors) on p44, a potential signaling component for mediating growth inhibition by TGF-.

As discussed earlier, the kinetics for TGF- activation of p44 closely resemble those obtained in PC-12 cells, following addition of the differentiation factor nerve growth factor (57) . Further, TGF- 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- may be involved in regulating cellular differentiation, rather than cell growth. Evidence against this possibility includes the fact that TGF- activated p44 in CCL 64 cells, which do not appear to undergo morphological differentiation in response to TGF-. 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- responsiveness in IEC-6 cells(63, 66) .

Although the data in Fig. 1Fig. 2Fig. 3Fig. 4indicate an association between TGF- 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-. 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- addition. Moreover, EIT stimulated p44 activity to levels 3-4-fold above baseline values, whereas TGF- 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--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- specifically, one possible explanation for the observed activation of p44 by TGF-, in association with an inhibition of cell growth, involves an associated phosphorylation and activation of Elk-1/p62, ultimately leading to autoinduction of TGF- (see model in Fig. 6). This hypothesis is based upon several previous reports. That is, it has been demonstrated that autoinduction of TGF- is mediated by the AP-1 (Jun-Fos) complex in the TGF- promoter, and that both Jun and Fos components are required for transcriptional activation(34) . TGF- has also been shown to autoinduce TGF- 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- 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- promoter, and increased production of TGF- (see Fig. 6). In cell types that have the potential to activate the latent, secreted TGF-(1, 2, 3) , the growth-inhibitory effects of TGF- would be amplified. In this way, the activation of p44 by TGF- may be indirectly associated with the growth-inhibitory effects of TGF-. However, additional experiments are required to test this model.

Figure 6: Model for association between activation of p44 by TGF- and growth inhibition. TGF- 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- promoter AP-1 activity, thereby inducing TGF- production. This autoinduction of TGF- results in amplification of the growth-inhibitory effect of this growth factor in cell types that can activate the secreted TGF-. R and R = TGF- 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-) can activate a kinase known for its involvement in growth-stimulatory pathways (p44). The activation began within 5 min of TGF- 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- 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-. 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- autoinduction via the AP-1 sites in the TGF- promoter. In keeping with this model, the TGF--sensitive IEC 4-1 cells produce and activate TGF-, whereas the TGF--resistant IEC 4-6 cells do not produce any detectable TGF-(58) . Thus, the IEC 4-6 cells may be resistant to the growth-inhibitory effects of TGF- due to their inability to activate p44 and to amplify production of TGF-.

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.

()

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

ACKNOWLEDGEMENTS

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