Edens, Maryanne

Abstract Transforming growth factor β (TGF-β) signaling via Smad proteins occurs in various cell types. However, whereas the biological response to TGF-β can be as distinct as growth promoting (i.e., mesenchymal cells) versus growth inhibiting (i.e., epithelial cells), few discernible differences in TGF-β signaling have been reported. In the current study, we examined the role of Ras in the proliferative response to TGF-β and how it might interface with Smad-dependent and Smad-independent TGF-β signaling targets. TGF-β stimulated Ras activity in a subset of mesenchymal, but not epithelial, cultures and was required for extracellular signal-regulated kinase (ERK)–dependent transcriptional responses. Although dominant negative Ras had no effect on TGF-β internalization or Smad-dependent signaling (i.e., phosphorylation, nuclear translocation, or SBE-luciferase activity), it did prevent the hyperphosphorylation of the Smad transcriptional corepressor TG-interacting factor (TGIF). This was not sufficient, however, to overcome the mitogenic response stimulated by TGF-β, which was dependent on signals downstream of p21-activated kinase 2 (PAK2). Moreover, although the initial activation of Ras and PAK2 are distinctly regulated, TGF-β–stimulated PAK2 activity is required for Ras-dependent ERK phosphorylation and Elk-1 transcription. These findings show the requirement for crosstalk between two Smad-independent pathways in regulating TGF-β proliferation and indicate that the mechanism(s) by which TGF-β stimulates growth is not simply the opposite of its growth inhibitory actions. [Cancer Res 2007;67(8):3673–82]

Abstract Transforming growth factor-β (TGF-β) stimulates cellular proliferation and transformation to a myofibroblast phenotype in vivo and in a subset of fibroblast cell lines. As the Smad pathway is activated by TGF-β in essentially all cell types, it is unlikely to be the sole mediator of cell type–specific outcomes to TGF-β stimulation. In the current study, we determined that TGF-β receptor signaling activates phosphatidylinositol 3-kinase (PI3K) in several fibroblast but not epithelial cultures independently of Smad2 and Smad3. PI3K activation occurs in the presence of dominant-negative dynamin and is required for p21-activated kinase-2 kinase activity and the increased proliferation and morphologic change induced by TGF-β in vitro.

Epithelial cells in vivo form tight cell-cell associations that spatially separate distinct apical and basolateral domains. These domains provide discrete cellular processes essential for proper tissue and organ development. Using confocal imaging and selective plasma membrane domain activation, the type I and type II transforming growth factor-β (TGFβ) receptors were found to be localized specifically at the basolateral surfaces of polarized Madin-Darby canine kidney (MDCK) cells. Receptors concentrated predominantly at the lateral sites of cell-cell contact, adjacent to the gap junctional complex. Cytoplasmic domain truncations for each receptor resulted in the loss of specific lateral domain targeting and dispersion to both the apical and basal domains. Whereas receptors concentrate basolaterally in regions of direct cell-cell contact in nonpolarized MDCK cell monolayers, receptor staining was absent from areas of noncell contact. In contrast to the defined basolateral polarity observed for the TGFβ receptor complex, TGFβ ligand secretion was found to be from the apical surfaces. Confocal imaging of MDCK cells with an antibody to TGFβ1 confirmed a predominant apical localization, with a stark absence at the basal membrane. These findings indicate that cell adhesion regulates the localization of TGFβ receptors in polarized epithelial cultures and that the response to TGFβ is dependent upon the spatial distribution and secretion of TGFβ receptors and ligand, respectively.