Role of Ras and Mapks in TGFβ signaling

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Abstract

Normal signaling by TGFβ, in the absence of serum or exogenous factors, involves a rapid activation of Ras, Erks, and Sapks in proliferating cultures of TGFβ-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 TGFβ to induce AP-1 complex formation at the TGFβ1 promoter and to autoinduce its own production. The primary components present in this TGFβ-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 TGFβ1 promoter, supershift assays suggest that at least one of these does not bind Smad4, and the other is unable to bind factors activated by TGFβ. In contrast, TGFβ autoinduction is Smad3-dependent, as DN Smad3 inhibits the ability of TGFβ to stimulate TGFβ1 promoter activity. Our results indicate that TGFβ can activate both the MKK4/Sapk and MEK/Erk pathways, through Ras and TGFβ RI and RII, to induce TGFβ1 production; Smad4 does not appear to be involved, and Smad3 appears to function independently of this Smad4.

We also demonstrate that activation of the Ras/Mapk pathway by TGFβ positively modulates Smad1-signaling-pathway activation by TGFβ. In addition, Smad1 could enhance TGFβ activation of the SBE reporter SBE-luc and this effect could be blocked by co-expression of a DN TGFβ RI receptor or by the MEK1 inhibitor PD98059. This cross-talk between the MEK/Erk and Smad1 pathways was mediated through the four Erk consensus phosphorylation sites in the linker region of Smad1. Mutation of these sites resulted in a loss of the ligand-dependence of both Smad1–Smad4 interactions and nuclear accumulation of Smad1, as well as a loss of the ability of Smad1 to enhance TGFβ-mediated SBE activation. Our results provide evidence that Erk-mediated phosphorylation of Smad1 in response to TGFβ is critical for regulating Smad1 subcellular localization; this may be a key determinant in maintaining TGFβ-dependent transcriptional activation.

Introduction

The Ras GTPase functions as a transducer of cell signals from membrane receptors to intracellular pathways that control cell growth, survival, and differentiation. Activation of Ras is an essential step in the signaling of responses from a variety of extracellular signals. Some of the best characterized Ras signaling pathways are referred to as the mitogen-activated protein kinase (Mapk) cascade(s) [1], [2], [3]. In one of these cascades, Ras binds directly to Raf and recruits it to the membrane, where it undergoes activation. Activated Raf can then directly phosphorylate and activate MEK which, in turn, directly phosphorylates and activates Erk. Activation of Erk is essential for several Ras-induced cellular responses, including transcriptional activation of a number of genes.

There are actually three distinct groups of Mapks which have been identified in mammalian cells, referred to as extracellular signal-regulated kinases (Erks), c-JunN-terminal kinases/stress-activated protein kinases (JNKs/Sapks), and p38 Mapks. These Mapks are all proline-directed, serine-threonine kinases that are activated on threonine and tyrosine residues in response to a wide variety of extracellular stimuli. Mapks are mediators of signal transduction from the cytosol to the nucleus. A major nuclear target of these Mapk signaling pathways is the transcription factor, activator protein-1 (AP-1) [4]. Ras can also activate the Sapk/JNK and p38 types of Mapks, possibly involving some of the Rho proteins; a direct association of Ras with MEKK1 has also been demonstrated [5], [6].

According to several lines of evidence, the signaling properties of Ras appear to be dependent upon the cellular context within which Ras operates. For example, expression of an activated Ras in immortalized cell lines can lead to oncogenic transformation. In contrast, in primary cells, activated Ras can induce a permanent cell cycle arrest [7], [8]. In addition, Ras function is essential for proliferation of established fibroblast cells; in contrast, it causes differentiation of neuronal cells [9], [10]. The kinetics and levels of activation of different effector pathways also dictate the cellular outcome in response to Ras. That is, the ability of Ras to induce neuronal differentiation is dependent upon the duration of activation of the Mapk cascade [11], [12]. Moreover, while expression of activated Ras generally promotes mitogenesis in fibroblasts, activation of Ras in these cells induces apoptosis if protein kinase C activity is suppressed [13]. Finally, high levels of Ras activity can induce an apoptotic response, which requires the activation of both the JNK/Sapk and Erk/Mapk cascades [14].

In light of the diversity of biological responses that can be mediated by the Ras/Mapk cascades, it is not surprising that a growth inhibitor such as TGFβ might also be able to activate these pathways (see Fig. 1). However, there has been some controversy in the TGFβ field with regard to the role of the Ras/Mapk pathways in mediating TGFβ responses. These experimental differences have occurred for a number of reasons. First, many of the earlier studies had focused on the ability of TGFβ to block the regulation of the Ras/Mapk cascades which had been induced by tyrosine kinase receptor signaling [15], [16]. For these types of studies, the cells were first made quiescent by removal of serum or growth factors, followed by stimulation of re-entry into the cell cycle by the addition of serum or growth stimulatory factors, in the absence or presence of TGFβ. Clearly, this type of experimental protocol would be expected to yield different results from studies examining the direct activation of these cascades by TGFβ, under conditions where the cells are in exponential phase growth. The differences in the experimental protocols utilized for these studies demonstrates the importance of cellular context when examining growth factor activation of signaling cascades.

Another factor that has contributed to discrepancies among different laboratories examining TGFβ regulation of these cascades is the presence of serum or other growth factors in the medium, at the time the TGFβ is added. This type of experimental approach not only alters the cellular context of TGFβ during activation of these pathways, but also may result in an inability to detect the direct effects of TGFβ on these cascades. That is, it is well known that many external signals can regulate these cascades, as discussed above. The levels of activation of these signaling components by growth stimulatory factors can often greatly exceed the level of activation observed after TGFβ addition to cells [17]. Thus, the presence of serum, exogenous growth factors, or autocrine factors can all serve to mask detection of the activation of these cascades by TGFβ itself.

The cell type under investigation can also drastically influence the regulation of these cascades by TGFβ. It would be expected that different cell types would regulate different signaling components to achieve specific biologic responses. For example, under conditions in which TGFβ stimulated the DNA synthesis of quiescent Balb 3T3 and swiss 3T3 fibroblast cells, TGFβ did not detectably increase Mapk activation or Fos expression [18]. For this reason, this review will focus on TGFβ regulation of these pathways in epithelial cells, specifically.

Another factor that contributes to the differences among studies of the role of the Ras/Mapk cascade in TGFβ signaling relates to the transformation state of the cells. Many transformed cells have lost growth inhibitory responsiveness to TGFβ and, therefore, normal signaling pathways activated by TGFβ would be expected to be altered [19]. In addition, many investigators have performed studies in cells in which mutant Ras genes have been overexpressed. An over-activity of Ras signaling cascades often times leads to cellular transformation, as well as to resistance to the growth inhibitory effects of TGFβ [20], [21], [22], [23], [24], [25]. Moreover, multiple effectors, which are certainly not specific to TGFβ signaling, will have been activated upon over-expression of activated Ras; it is well known that many external stimuli activate Ras to mediate distinct biological outcomes. Overall then, expression of activated Ras genes will not only stimulate non-TGFβ signaling events, but may lead to transformation of the cells, with associated TGFβ resistance, thereby precluding studies of TGFβ-dependent signaling events.

Similar concerns exist for studies in which constitutively active forms of various members of these cascades are expressed in different cell types [26]. As indicated [26], a potential limitation of the use of constitutively activated forms of signaling kinases relates to the production of nonspecific or promiscuous effects. The distinction between activation of normal Ras and subsequent signaling, vs experiments in which oncogenic, activated Ras has been expressed, was also made with regard to experimental discrepancies among non-TGFβ signaling events [27]. In order to clarify the role of the Ras/Mapk cascades in mediating normal signaling through TGFβ, this review will focus on studies in epithelial cells in which the cells are cultured in the absence of serum or other exogenous factors, yet are still in exponential phase growth (for additional background information, see Ref. [19]). Further, the majority of the studies described herein involve the expression of dominant-negative mutants of signaling component, rather than the expression of constitutively active forms. The dominant-negative approach would appear to be superior to expressing constitutively active mutants for studies in which ligand-dependent events are to be investigated.

Section snippets

Activation of Ras by TGFβ

My laboratory was the first to provide direct evidence of the rapid activation of a cytoplasmic signaling component by TGFβ in 1992 [28]. In this report we demonstrated that both TGFβ1 and TGFβ2 could result in a rapid (within 3–6 min) stimulation of GTP bound to Ras in TGFβ-sensitive intestinal and lung epithelial cells (see Fig. 1). These effects were not observed in TGFβ-resistant cells. The activation of Ras by TGFβ was concentration-dependent and occurred in proliferating cultures in the

TGFβ activation of Erk Mapks

My laboratory was also the first to demonstrate a rapid activation of p44Mapk, occurring within 5–10 min of TGFβ1 or TGFβ2 addition [17] (see Fig. 1). This effect occurred in exponentially proliferating cultures of untransformed epithelial cells under conditions in which DNA synthesis was inhibited by 95–98% [31]. The effect was also sustained, indicative of nuclear translocation of the Mapk. These findings were similar to those observed for nerve growth factor stimulation of sustained Mapk

Ras requirement for TGFβ regulation of downstream events

In order to demonstrate that Ras was required for TGFβ activation of both Erk and downstream components associated with TGFβ-mediated growth inhibition, untransformed intestinal epithelial cells (IECs) were stably transfected with a dominant-negative mutant of the Ras protein (RasN17) under the control of an inducible metallothionein promoter. Several clones were selected and characterized as described previously [43]. In this report we demonstrated that induction of Ras expression by at least

TGFβ activation of the stress-activated protein kinase/Jun N-terminal kinase (Sapk/JNK) type of Mapks

My laboratory was also the first to demonstrate a rapid activation of the Sapk/JNK type of Mapks [36], [37]. Collectively, our results demonstrated that TGFβ could affect a sustained activation of Sapk/JNK in untransformed epithelial cells, as well as in TGFβ-responsive human breast and colon cancer cells [36], [59], [60]. These effects occurred within 5–10 min of TGFβ addition to proliferating cell cultures (see Fig. 1). Additional details regarding the methodology used for these studies have

Role of Erk and Sapk

As previously described, we have provided definitive evidence that both the Erk and Sapk types of Mapks are rapidly activated after TGFβ addition to proliferating cultures of epithelial cells. In addition, we have demonstrated that Ras is essential for the activation of Erks by TGFβ, and that MEK1 is the upstream activator of Erk in our systems [43], [62], [63]. More recently, we have utilized our untransformed epithelial cells, stably expressing the dominant-negative mutant of Ras under the

TGFβ stimulation of Smad1 activation events

We have cloned a rat homolog of the Drosophila Mad gene (rSmad1) by screening an intestinal epithelial cell cDNA library [62]. This rat Smad1 sequence differed from the previously published rat sequence [75] by four amino acids. Our rSmad1 sequence had four potential Erk consensus phosphorylation sites, similar to the human Smad1. This represents one additional site not previously described for rat Smad1 [75]. Using an in vitro kinase assay with rSmad1 as the substrate, we demonstrated that the

Concluding remarks

It is clear from the above discussion that TGFβ rapidly activates Ras, as well as both the Erk and Sapk types of Mapks. These effects occur in proliferating cultures of TGFβ-sensitive untransformed epithelial cells in the absence of serum or other exogenous factors [19]. Functional TGFβ RI and RII receptors are required for these activation events [60]. In addition, the biological significance of the activation of these pathways by TGFβ is becoming increasingly clear. First, we have

Acknowledgements

This work was supported by NIH grants, R01 CA51452, R01 CA68444, and R01 CA54816.

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