Profibrotic TGFß responses require the cooperative action of PDGF and ErbB receptor tyrosine kinases.

Transforming growth factor ß (TGFß) has significant profibrotic activity both in vitro and in vivo. This reflects its capacity to stimulate fibrogenic mediators and induce the expression of other profibrotic cytokines such as platelet-derived growth factor (PDGF) and epidermal growth factor (EGF/ErbB) ligands. Here we address both the mechanisms by which TGFß induced ErbB ligands and the physiological significance of inhibiting multiple TGFß-regulated processes. The data document that ErbB ligand induction requires PDGF receptor (PDGFR) mediation and engages a positive autocrine/paracrine feedback loop via ErbB receptors. Whereas PDGFRs are essential for TGFß-stimulated ErbB ligand up-regulation, TGFß-specific signals are also required for ErbB receptor activation. Subsequent profibrotic responses are shown to involve the cooperative action of PDGF and ErbB signaling. Moreover, using a murine treatment model of bleomycin-induced pulmonary fibrosis we found that inhibition of TGFß/PDGF and ErbB pathways with imatinib plus lapatinib, respectively, not only prevented myofibroblast gene expression to a greater extent than either drug alone, but also essentially stabilized gas exchange (oxygen saturation) as an overall measure of lung function. These observations provide important mechanistic insights into profibrotic TGFß signaling and indicate that targeting multiple cytokines represents a possible strategy to ameliorate organ fibrosis dependent on TGFß.

Non-Smad transforming growth factor-ß signaling regulated by focal adhesion kinase binding the p85 subunit of phosphatidylinositol 3-kinase.

TGF-ß modulates numerous diverse cellular phenotypes including growth arrest in epithelial cells and proliferation in fibroblasts. Although the Smad pathway is fundamental for the majority of these responses, recent evidence indicates that non-Smad pathways may also have a critical role. Here we report a novel mechanism whereby the nonreceptor tyrosine focal adhesion kinase (FAK) functions as an adaptor necessary for cell type-specific responses to TGF-ß. We show that in contrast to Smad actions, non-Smad pathways, including c-Abl, PAK2, and Akt, display an obligate requirement for FAK. Interestingly, this occurs in Src null SYF cells and is independent of FAK tyrosine phosphorylation, kinase activity, and/or proline-rich sequences in the C-terminal FAT domain. FAK binds the phosphatidylinositol 3-kinase (PI3K) p85 regulatory subunit following TGF-ß treatment in a subset of fibroblasts but not epithelial cells and has an obligate role in TGF-ß-stimulated anchorage-independent growth and migration. Together, these results uncover a new scaffolding role for FAK as the most upstream component regulating the profibrogenic action of TGF-ß and suggest that inhibiting this interaction may be useful in treating a number of fibrotic diseases.

Transforming growth factor beta signaling via Ras in mesenchymal cells requires p21-activated kinase 2 for extracellular signal-regulated kinase-dependent transcriptional responses.

Transforming growth factor beta (TGF-beta) signaling via Smad proteins occurs in various cell types. However, whereas the biological response to TGF-beta can be as distinct as growth promoting (i.e., mesenchymal cells) versus growth inhibiting (i.e., epithelial cells), few discernible differences in TGF-beta signaling have been reported. In the current study, we examined the role of Ras in the proliferative response to TGF-beta and how it might interface with Smad-dependent and Smad-independent TGF-beta signaling targets. TGF-beta 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-beta 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-beta, 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-beta-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-beta proliferation and indicate that the mechanism(s) by which TGF-beta stimulates growth is not simply the opposite of its growth inhibitory actions.

Imatinib mesylate inhibits the profibrogenic activity of TGF-beta and prevents bleomycin-mediated lung fibrosis.

Idiopathic pulmonary fibrosis is a progressive and fatal fibrotic disease of the lungs with unclear etiology. Prior efforts to treat idiopathic pulmonary fibrosis that focused on anti-inflammatory therapy have not proven to be effective. Recent insight suggests that the pathogenesis is mediated through foci of dysregulated fibroblasts driven by profibrotic cytokine signaling. TGF-beta and PDGF are 2 of the most potent of these cytokines. In the current study, we investigated the role of TGF-beta-induced fibrosis mediated by activation of the Abelson (Abl) tyrosine kinase. Our data indicate that fibroblasts respond to TGF-beta by stimulating c-Abl kinase activity independently of Smad2/3 phosphorylation or PDGFR activation. Moreover, inhibition of c-Abl by imatinib prevented TGF-beta-induced ECM gene expression, morphologic transformation, and cell proliferation independently of any effect on Smad signaling. Further, using a mouse model of bleomycin-induced pulmonary fibrosis, we found a significant inhibition of lung fibrosis by imatinib. Thus, Abl family members represent common targets for the modulation of profibrotic cytokine signaling.

Internalization-dependent and -independent requirements for transforming growth factor beta receptor signaling via the Smad pathway.

Members of the transforming growth factor beta (TGF-beta) family of proteins signal through cell surface transmembrane serine/threonine protein kinases known as type I and type II receptors. The TGF-beta signal is extended through phosphorylation of receptor-associated Smad proteins by the type I receptor. Although numerous investigations have established the sequence of events in TGF-beta receptor (TGF-beta R) activation, none have examined the role of the endocytic pathway in initiation and/or maintenance of the signaling response. In this study we investigated whether TGF-beta R internalization modulates type I receptor activation, the formation of a functional receptor/Smad/SARA complex, Smad2/3 phosphorylation or nuclear translocation, and TGF-beta-dependent reporter gene activity. Our data provide evidence that, whereas type I receptor phosphorylation and association of SARA and Smad2 with the TGF-beta R complex take place independently of clathrin lattice formation, Smad2 or Smad3 activation and downstream signaling only occur after endocytic vesicle formation. Thus, TGF-beta R endocytosis is not simply a way to dampen the signaling response but instead is required to propagate signaling via the Smad pathway.

Transforming growth factor-beta activation of phosphatidylinositol 3-kinase is independent of Smad2 and Smad3 and regulates fibroblast responses via p21-activated kinase-2.

Transforming growth factor-beta (TGF-beta) 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-beta in essentially all cell types, it is unlikely to be the sole mediator of cell type-specific outcomes to TGF-beta stimulation. In the current study, we determined that TGF-beta 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-beta in vitro.

ERBB receptor activation is required for profibrotic responses to transforming growth factor beta.

Engagement of the transforming growth factor-ß (TGF-ß) receptor complex activates multiple signaling pathways that play crucial roles in both health and disease. TGF-ß is a key regulator of fibrogenesis and cancer-associated desmoplasia; however, its exact mode of action in these pathologic processes has remained poorly defined. Here, we report a novel mechanism whereby signaling via members of the ERBB or epidermal growth factor family of receptors serves as a central requirement for the biological responses of fibroblasts to TGF-ß. We show that TGF-ß triggers upregulation of ERBB ligands and activation of cognate receptors via the canonical SMAD pathway in fibroblasts. Interestingly, activation of ERBB is commonly observed in a subset of fibroblast but not epithelial cells from different species, indicating cell type specificity. Moreover, using genetic and pharmacologic approaches, we show that ERBB activation by TGF-ß is essential for the induction of fibroblast cell morphologic transformation and anchorage-independent growth. Together, these results uncover important aspects of TGF-ß signaling that highlight the role of ERBB ligands/receptors as critical mediators in fibroblast responses to this pleiotropic cytokine.

Type II transforming growth factor-beta receptor recycling is dependent upon the clathrin adaptor protein Dab2.

Transforming growth factor (TGF)-ß family proteins form heteromeric complexes with transmembrane serine/threonine kinases referred to as type I and type II receptors. Ligand binding initiates a signaling cascade that generates a variety of cell type-specific phenotypes. Whereas numerous studies have investigated the regulatory activities controlling TGF-ß signaling, there is relatively little information addressing the endocytic and trafficking itinerary of TGF-ß receptor subunits. In the current study we have investigated the role of the clathrin-associated sorting protein Disabled-2 (Dab2) in TGF-ß receptor endocytosis. Although small interfering RNA-mediated Dab2 knockdown had no affect on the internalization of various clathrin-dependent (i.e., TGF-ß, low-density lipoprotein, or transferrin) or -independent (i.e., LacCer) cargo, TGF-ß receptor recycling was abrogated. Loss of Dab2 resulted in enlarged early endosomal antigen 1-positive endosomes, reflecting the inability of cargo to traffic from the early endosome to the endosomal recycling compartment and, as documented previously, diminished Smad2 phosphorylation. The results support a model whereby Dab2 acts as a multifunctional adaptor in mesenchymal cells required for TGF-ß receptor recycling as well as Smad2 phosphorylation.

Distinct roles for mammalian target of rapamycin complexes in the fibroblast response to transforming growth factor-beta.

Transforming growth factor-beta (TGF-beta) promotes a multitude of diverse biological processes, including growth arrest of epithelial cells and proliferation of fibroblasts. Although the TGF-beta signaling pathways that promote inhibition of epithelial cell growth are well characterized, less is known about the mechanisms mediating the positive response to this growth factor. Given that TGF-beta has been shown to promote fibrotic diseases and desmoplasia, identifying the fibroblast-specific TGF-beta signaling pathways is critical. Here, we investigate the role of mammalian target of rapamycin (mTOR), a known effector of phosphatidylinositol 3-kinase (PI3K) and promoter of cell growth, in the fibroblast response to TGF-beta. We show that TGF-beta activates mTOR complex 1 (mTORC1) in fibroblasts but not epithelial cells via a PI3K-Akt-TSC2-dependent pathway. Rapamycin, the pharmacologic inhibitor of mTOR, prevents TGF-beta-mediated anchorage-independent growth without affecting TGF-beta transcriptional responses or extracellular matrix protein induction. In addition to mTORC1, we also examined the role of mTORC2 in TGF-beta action. mTORC2 promotes TGF-beta-induced morphologic transformation and is required for TGF-beta-induced Akt S473 phosphorylation but not mTORC1 activation. Interestingly, both mTOR complexes are necessary for TGF-beta-mediated growth in soft agar. These results define distinct and overlapping roles for mTORC1 and mTORC2 in the fibroblast response to TGF-beta and suggest that inhibitors of mTOR signaling may be useful in treating fibrotic processes, such as desmoplasia.