Kathleen M. Mulder

Role of Ras and Mapks in TGFβ signaling

Normal signaling by TGFb, in the absence of serum or exogenous factors, involves a rapid activation of Ras, Erks, and Sapks in proliferating cultures of TGFb-sensitive untransformed epithelial cells and human carcinoma cells. Expression of either RasN17 or dominant-negative (DN) MKK4, or addition of the MEK1 inhibitor PD98059, can block the ability of TGFb to induce AP-1 complex formation at the TGFb1 promoter and to autoinduce its own production. The primary components present in this TGFb-stimulated AP-1 complex are JunD and Fra-2, although c-Jun, and possibly Fos B, may also be present. While there are two potential Smad binding elements (SBE’s) in the TGFb1 promoter, supershift assays suggest that at least one of these does not bind Smad4, and the other is unable to bind factors activated by TGFb. In contrast, TGFb autoinduction is Smad3-dependent, as DN Smad3 inhibits the ability of TGFb to stimulate TGFb1 promoter activity. Our results indicate that TGFb can activate both the MKK4/Sapk and MEK/Erk pathways, through Ras and TGFb RI and RII, to induce TGFb1 production; Smad4 does not appear to be involved, and Smad3 appears to function independently of this Smad4.

Transforming growth factor-β signal transduction in epithelial cells

Transforming growth factor (TGF)-beta is a natural and potent growth inhibitor of a variety of cell types, including epithelial, endothelial, and hematopoietic cells. The ability of TGF-beta to potently inhibit the growth of many solid tumors of epithelial origin, including breast and colon carcinomas, is of particular interest. However, many solid tumor cells become refractory to the growth inhibitory effects of TGF-beta due to defects in TGF-beta signaling pathways. In addition, TGF-beta may stimulate the invasiveness of tumor cells via the paracrine effects of TGF-beta. Accordingly, in order to develop more effective anticancer therapeutics, it is necessary to determine the TGF-beta signal transduction pathways underlying the growth inhibitory effects and other cellular effects of TGF-beta in normal epithelial cells. Thus far, two primary signaling cascades downstream of the TGF-beta receptors have been elucidated, the Sma and mothers against decapentaplegic homologues and the Ras/mitogen-activated protein kinase pathways. The major objective of this review is to summarize TGF-beta signaling in epithelial cells, focusing on recent advances involving the Sma and mothers against decapentaplegic homologues and Ras/mitogen-activated protein kinase pathways. This review is particularly timely in that it provides a comprehensive summary of both signal transduction mechanisms and the cell cycle effects of TGF-beta.

Requirement of Ras/MAPK pathway activation by transforming growth factor β for transforming growth factor β1 production in a Smad-dependent pathway.

Our previous results have shown that transforming growth factor β (TGFβ) rapidly activates Ras, as well as both ERKs and SAPKs. In order to address the biological significance of the activation of these pathways by TGFβ, here we examined the role of the Ras/MAPK pathways and the Smads in TGFβ3 induction of TGFβ1 expression in untransformed lung and intestinal epithelial cells. Expression of either a dominant-negative mutant of Ras (RasN17) or a dominant-negative mutant of MKK4 (DN MKK4), or addition of the MEK1 inhibitor PD98059, inhibited the ability of TGFβ3 to induce AP-1 complex formation at the TGFβ1 promoter, and the subsequent induction of TGFβ1 mRNA. The primary components present in this TGFβ3-inducible AP-1 complex at the TGFβ1 promoter were JunD and Fra-2, although c-Jun and FosB were also involved. Furthermore, deletion of the AP-1 site in the TGFβ1 promoter or addition of PD98059 inhibited the ability of TGFβ3 to stimulate TGFβ1promoter activity. Collectively, our data demonstrate that TGFβ3 induction of TGFβ1 is mediated through a signaling cascade consisting of Ras, the MAPKKs MKK4 and MEK1, the MAPKs SAPKs and ERKs, and the specific AP-1 proteins Fra-2 and JunD. Although Smad3 and Smad4 were not detectable in TGFβ3-inducible AP-1 complexes at the TGFβ1promoter, stable expression of dominant-negative Smad3 could significantly inhibit the ability of TGFβ3 to stimulate TGFβ1 promoter activity. Transient expression of dominant-negative Smad4 also inhibited the ability of TGFβ3 to transactivate the TGFβ1 promoter. Thus, although the Ras/MAPK pathways are essential for TGFβ3 induction of TGFβ1, Smads may only contribute to this biological response in an indirect manner.

Differential sensitivity of subclasses of human colon carcinoma cell lines to the growth inhibitory effects of transforming growth factor-β1

In this study we have employed a model system comprising three groups of colon carcinoma cell lines to examine the growth-inhibitory effects of two molecular forms of transforming growth factor-beta (TGF-beta), TGF-beta 1 and TGF-beta 2. Aggressive, poorly differentiated colon carcinoma cells of group I did not respond to growth inhibitory effects of TGF-beta 1 or TGF-beta 2, while less aggressive, well-differentiated cells of group III displayed marked sensitivity to both TGF-beta 1 and TGF-beta 2 in monolayer culture as well as in soft agarose. One moderately well-differentiated cell line from group II which has intermediate growth characteristics failed to respond to TGF-beta 1 or TGF-beta 2, but the growth of two other cell lines in this group was inhibited. TGF-beta 1 and TGF-beta 2 were equally potent, 50% growth inhibition for responsive cell lines being observed at a concentration of 1 ng/ml (40 pM). Antiproliferative effects of TGF-beta 1 and TGF-beta 2 in responsive cell lines of groups II and III were associated with morphological alterations and enhanced, concentration-dependent secretion of carcinoembryonic antigen. Radiolabeled TGF-beta 1 bound to all three groups of colon carcinoma cells with high affinity (Kd between 42 and 64 pM). These data indicate for the first time a strong correlation between the degree of differentiation of colon carcinoma cell lines and sensitivity to the antiproliferative and differentiation-promoting effects of TGF-beta 1 and TGF-beta 2.

Transforming Growth Factor-β Signaling in Epithelial Cells

Transforming growth factor (TGF)-beta is a potent growth suppressor of epithelial cells. Resistance to TGF-beta, however, occurs frequently in solid tumors of epithelial origin and contributes to the uncontrolled growth of these tumors. Although mutant receptor proteins contribute to TGF-beta insensitivity, deregulation of TGF-beta signaling cascades represents an equally important mechanism underlying TGF-beta resistance. Identification of abnormal regulation of signaling components in tumor epithelial cells will lead to the development of selective therapeutic approaches to repair the relevant signaling cascade(s) and reverse the growth anomaly. Within the past few years, great strides have been made in defining signaling pathways for TGF-beta. For example, our laboratory has demonstrated a direct correlation between TGF-beta-mediated growth inhibition of epithelial cells and activation of Ras and three members of the mitogen-activated protein kinase (MAPK) superfamily. The TGF-beta signaling events were sustained, dose-dependent, and absent in TGF-beta-resistant cells. Further, up-regulation of both p27Kip1 and p21Cip1, nuclear events important for the growth inhibitory effect of TGF-beta, are completely dependent upon the activation of Ras. However, Ras-independent pathways are also activated simultaneously with the Ras/MAPK pathways to mediate the final TGF-beta growth inhibitory outcome. One such pathway includes the SMAD signaling components that control TGF-beta-mediated gene transcription, currently under active study by a number of laboratories, including our own. Future efforts in this field will focus on defining the significance of these signaling proteins and pathways in mediating specific TGF-beta responses. Moreover, additional novel signaling proteins are sure to be identified.

Cross-talk between the Smad1 and Ras/MEK signaling pathways for TGFβ

Our previous data demonstrated that Ras activation was necessary and sufficient for transforming growth factor-β (TGFβ)-mediated Erk1 activation, and was required for TGFβ up-regulation of the Cdk inhibitors (CKIs) p27Kip1 and p21Cip1 (KM Mulder and SL Morris, J. Biol. Chem., 267, 5029 – 5031, 1992; MT Hartsough and KM Mulder, J. Biol. Chem., 270, 7117 – 7124, 1995; MT Hartsough et al., J. Biol. Chem., 271, 22368 – 22375, 1996 and J Yue et al., Oncogene, 17, 47 – 55, 1998). Here we examined the role of Ras in TGFβ-mediated effects on a rat homolog of Smad1 (termed RSmad1). We demonstrate that both TGFβ and bone morphogenetic protein (BMP) can induce endogenous Smad1 phosphorylation in intestinal epithelial cells (IECs). The combination of transient expression of RSmad1 and TGFβ treatment had an additive effect on induction of the TGFβ-responsive reporter 3TP-lux. Either inactivation of Ras by stable, inducible expression of a dominant-negative mutant of Ras (RasN17) or addition of MAP and ERK kinase (MEK) inhibitor PD98059 to cells significantly decreased the ability of both TGFβ and BMP to induce phosphorylation of endogenous Smad1 in IECs. Moreover, either inactivation of Ras or addition of PD98059 to IEC 4-1 cells inhibited the ability of RSmad1 to regulate 3TP luciferase activity in both the presence and absence of TGFβ. Collectively, our data indicate that TGFβ can regulate RSmad1 function in epithelial cells, and that the Ras/MEK pathway is partially required for TGFβ-mediated regulation of RSmad1.

TGFβ regulation of mitogen-activated protein kinases in human breast cancer cells

We demonstrate herein the ability of transforming growth factor-beta-2 (TGFbeta2) to potently activate extracellular signal-regulated kinase 2 (ERK2) in the highly TGFbeta-sensitive breast cancer cell (BCC) line Hs578T. The ERK2 isoform was activated by 3-fold within 5 min of TGFbeta2 addition to Hs578T cells. However, TGFbeta2 only slightly activated ERK2 (1.5-fold) in the partially TGFbeta-responsive BCC line MDA-MB-23 1. The magnitude of the difference in activation of ERK2 by TGFbeta2 in the two cell lines paralleled the difference in the IC50 values for TGFbeta inhibition of DNA synthesis; the IC50 value in the MDA-MB-231 cells was 32-fold greater than that in the Hs578T cells. Further, our data demonstrate that TGFbeta2 activated the stress-activated protein kinase/Jun N-terminal kinase (SAPK/JNK) type of mitogen-activated protein kinases (MAPKs); maximal induction levels were 2.5-fold above basal values and were attained at 30 min after TGFbeta2 treatment. Transient co-transfection of a luciferase reporter construct (3TP-Lux) containing three AP-1 sites and the plasminogen activator inhibitor-1 (PAI-1) promoter, in conjunction with a construct that directs expression of a dominant-negative mutant ERK2 (TAYF) protein, did not block the ability of TGFbeta to induce AP-1 or PAI-1 activity. In contrast, TAYF ERK2 was able to block EGF and insulin-induced 3TP-Lux-reporter activity. These results indicate that in these BCCs, the activation of ERK2 by TGFbeta is more tightly linked to the ability of TGFbeta to inhibit DNA synthesis than to the ability to stimulate promoter regions important for TGFbeta production and control of the extracellular matrix. In addition, this is the first demonstration that TGFbeta can activate the SAPK/JNK type of MAPK in TGFbeta-sensitive human BCCs.

A Transforming Growth Factor-β Receptor–Interacting Protein Frequently Mutated in Human Ovarian Cancer

Ovarian carcinomas, particularly recurrent forms, are frequently resistant to transforming growth factor-beta (TGF-beta)-mediated growth inhibition. However, mutations in the TGF-beta receptor I and receptor II (TbetaR-I and TbetaR-II) genes have only been reported in a minority of ovarian carcinomas, suggesting that alterations in TGF-beta-signaling components may play an important role in the loss of TGF-beta responsiveness. Using laser-capture microdissection and nested reverse-transcription-PCR, we found that km23, which interacts with the TGF-beta receptor complex, is altered at a high frequency in human ovarian cancer patients. A novel form of km23, missing exon 3 (Deltaexon3-km23), was found in 2 of 19 tumor tissues from patients with ovarian cancer. In addition to this alteration, a stop codon mutation (TAA --> CAC) was detected in two patients. This alteration results in an elongated protein, encoding 107-amino-acid residues (Delta107km23), instead of the wild-type 96-amino-acid form of km23. Furthermore, five missense mutations (T38I, S55G, T56S, I89V, and V90A) were detected in four patients, providing a total alteration rate of 42.1% (8 of 19 cases) in ovarian cancer. No km23 alterations were detected in 15 normal tissues. Such a high alteration rate in ovarian cancer suggests that km23 may play an important role in either TGF-beta resistance or tumor progression in this disease. In keeping with these findings, the functional studies described herein indicate that both the Deltaexon3-km23 and S55G/I89V-km23 mutants displayed a disruption in binding to the dynein intermediate chain in vivo, suggesting a defect in cargo recruitment to the dynein motor complex. In addition, the Deltaexon3-km23 resulted in an inhibition of TGF-beta-dependent transcriptional activation of both the p3TP-lux and activin responsive element reporters. Collectively, our results suggest that km23 alterations found in ovarian cancer patients result in altered dynein motor complex formation and/or aberrant transcriptional regulation by TGF-beta.

Modulation of C-myc by transforming growth factor-β in human colon carcinoma cells☆

Previous work indicated that transforming growth factor-beta elicits proliferation-inhibitory and differentiation-like effects in the human colon carcinoma cell line MOSER. We report for the first time that the proto-oncogene c-myc is repressed in response to transforming growth factor-beta in a human colon carcinoma cell line. We also describe a subline of these cells which are relatively resistant to the transforming growth factor-beta-induced effects on proliferation in monolayer and in soft agarose, but which retain the ability to specifically bind transforming growth factor-beta. Analysis of molecular and cellular alterations in this subline may aid in elucidating the mechanism of action of transforming growth factor-beta.

Requirement of Smad3 and CREB-1 in Mediating Transforming Growth Factor-β (TGFβ) Induction of TGFβ3 Secretion

Because increased transforming growth factor-β (TGFβ) production by tumor cells contributes to cancer progression through paracrine mechanisms, identification of critical points that can be targeted to block TGFβ production is important. Previous studies have identified the precise signaling components and promoter elements required for TGFβ induction of TGFβ1 expression in epithelial cells (Yue, J., and Mulder, K. M. (2000) J. Biol. Chem. 275, 30765–30773). To determine how regulation of TGFβ3 expression differs from that of TGFβ1, we identified the precise signaling pathways and transcription factor-binding sites that are required for TGFβ3 gene expression. By using mutational analysis in electrophoresis mobility shift assays (EMSAs), we demonstrated that the c-AMP-responsive element (CRE) site in the TGFβ3 promoter was required for TGFβ-inducible TGFβ3 expression. Electrophoresis mobility supershift assays indicated that CRE-binding protein 1 (CREB1) and Smad3 were the major components present in this TGFβ-inducible complex. Furthermore, by using chromatin immunoprecipitation assays, we demonstrated that CREB-1, ATF-2, and c-Jun bound constitutively at the TGFβ3 promoter (–100 to +1), whereas Smad3 bound at this site only after TGFβ stimulation. In addition, inhibition of JNK and p38 suppressed TGFβ induction of TGFβ3 transactivation, whereas inhibition of ERK and protein kinase A had no effect. Small interfering RNA-CREB1 and small interfering RNA-Smad3 significantly inhibited TGFβ stimulation of TGFβ3 promoter reporter activity and TGFβ3 production. Our results indicate that TGFβ activation of the TGFβ3 promoter CRE site, which leads to TGFβ3 production, is required for TGFβRII, JNK, p38, and Smad3 but was independent of protein kinase A, ERK, and Smad4.