Repellin, Claire E.

The primary mediator of transforming growth factor β (TGFβ) responses are the Smad proteins. Here, we identify sorting nexin 9 (SNX9) as being required for Smad3 (but not Smad2) nuclear translocation and cell stimulation. SNX9 principally interacts with phosphorylated (p) Smad3 and promotes more rapid nuclear delivery from that observed independent of ligand. Although SNX9 does not bind Imp7, Impb, Nup153 or Nup214, it mediates the association of pSmad3 with Imp8 and the nuclear membrane. This facilitates recruitment of pSmad3/SNX9 to the nuclear pore, heterodimerization of Imp8 with Impb, and nuclear translocation of pSmad3, but not SNX9. The demonstration that Smad3 is regulated in a distinct manner from Smad2 provides the opportunity to develop intervention strategies to enhance or dampen specific aspects of the cellular response to TGFβ. Support or Funding Information This work was supported by Public Health Service grants GM‐055816 and GM‐054200 from National Institutes of General Medical Sciences.

Transforming growth factor β (TGFβ) is a pleiotropic protein secreted from essentially all cell types and primary tissues. While TGFβ’s actions reflect the activity of a number of signaling networks, the primary mediator of TGFβ responses are the Smad proteins. Following receptor activation, these cytoplasmic proteins form hetero-oligomeric complexes that translocate to the nucleus and affect gene transcription. Here, through biological, biochemical, and immunofluorescence approaches, sorting nexin 9 (SNX9) is identified as being required for Smad3-dependent responses. SNX9 interacts with phosphorylated (p) Smad3 independent of Smad2 or Smad4 and promotes more rapid nuclear delivery than that observed independent of ligand. Although SNX9 does not bind nucleoporins Nup153 or Nup214 or some β importins (Imp7 or Impβ), it mediates the association of pSmad3 with Imp8 and the nuclear membrane. This facilitates nuclear translocation of pSmad3 but not SNX9.

Transforming growth factor β (TGFβ) 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 TGFβ induced ErbB ligands and the physiological significance of inhibiting multiple TGFβ‐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 TGFβ‐stimulated ErbB ligand up‐regulation, TGFβ‐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 TGFβ/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 TGFβ signaling and indicate that targeting multiple cytokines represents a possible strategy to ameliorate organ fibrosis dependent on TGFβ.—Andrianifahanana, M., Wilkes, M. C., Gupta, S. K., Rahimi, R. R., Repellin, C. E., Edens, M., Wittenberger, J., Yin, X., Maidl, E., Becker, J., Leof, E. B. Profibrotic TGFβ responses require the cooperative action of PDGF and ErbB receptor tyrosine kinases. FASEB J . 27, 4444‐4454 (2013). www.fasebj.org

Transforming growth factor (TGF)-β 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-β signaling, there is relatively little information addressing the endocytic and trafficking itinerary of TGF-β receptor subunits. In the current study we have investigated the role of the clathrin-associated sorting protein Disabled-2 (Dab2) in TGF-β receptor endocytosis. Although small interfering RNA-mediated Dab2 knockdown had no affect on the internalization of various clathrin-dependent (i.e., TGF-β, low-density lipoprotein, or transferrin) or -independent (i.e., LacCer) cargo, TGF-β 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-β receptor recycling as well as Smad2 phosphorylation.

Abstract Engagement of the transforming growth factor-β (TGF-β) receptor complex activates multiple signaling pathways that play crucial roles in both health and disease. TGF-β 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-β. We show that TGF-β 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-β is essential for the induction of fibroblast cell morphologic transformation and anchorage-independent growth. Together, these results uncover important aspects of TGF-β signaling that highlight the role of ERBB ligands/receptors as critical mediators in fibroblast responses to this pleiotropic cytokine. Cancer Res; 70(19); 7421–30. ©2010 AACR.