Transforming growth factor-beta (TGF-beta) regulates a wide variety of cellular processes including cell growth, apoptosis, differentiation, migration, and extracellular matrix production among others. The canonical signaling pathway induced by the TGF-beta receptor complex involves the phosphorylation of Smad proteins which upon activation accumulate in the nucleus and regulate transcription. Interestingly, the cellular response to TGF-beta can be extremely variable depending on the cell type and stimulation context. TGF-beta causes epithelial cells to undergo growth arrest and apoptosis, responses which are critical to suppressing carcinogenesis, whereas it can also induce epithelial-mesenchymal transition and mediate fibroblast activation, responses implicated in promoting carcinogenesis and fibrotic diseases. However, TGF-beta induces all these responses via the same receptor complex and Smad proteins. To address this apparent paradox, during the last few years a number of additional signaling pathways have been identified which potentially regulate the different cellular responses to TGF-beta. The identification of these signaling pathways has shed light onto the mechanisms whereby Smad and non-Smad pathways collaborate to induce a particular cellular phenotype. In this article, we review TGF-beta signaling in epithelial cells and fibroblasts with a focus on understanding the mechanisms of TGF-beta versatility.

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-β and PDGF are 2 of the most potent of these cytokines. In the current study, we investigated the role of TGF-β-induced fibrosis mediated by activation of the Abelson (Abl) tyrosine kinase. Our data indicate that fibroblasts respond to TGF-β by stimulating c-Abl kinase activity independently of Smad2/3 phosphorylation or PDGFR activation. Moreover, inhibition of c-Abl by imatinib prevented TGF-β-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.

Proteins in the transforming growth factor-␤ (TGF-␤) family recognize transmembrane serine/threonine kinases known as type I and type II receptors. Binding of TGF-␤ to receptors results in receptor down-regulation and signaling. Whereas previous work has focused on activities controlling TGF-␤ signaling, more recent studies have begun to address the trafficking properties of TGF-␤ receptors. In this report, it is shown that receptors undergo recycling both in the presence and absence of ligand activation, with the rates of internalization and recycling being unaffected by ligand binding. Recycling occurs as receptors are most likely internalized through clathrin-coated pits, and then returned to the plasma membrane via a rab11-dependent, rab4-independent mechanism. Together, the results suggest a mechanism wherein activated TGF-␤ receptors are directed to a distinct endocytic pathway for down-regulation and clathrin-dependent degradation after one or more rounds of recycling. INTRODUCTIONTGF-␤ is a ubiquitous 25-kDa polypeptide that regulates a variety of cellular processes, including matrix deposition, mitosis, development, differentiation, and apoptosis (Roberts, 1992;ten Dijke et al., 1996). The response to TGF-␤ treatment usually depends on the cell type involved, with effects as diverse as growth and growth inhibition (Massagué, 1996;Moses and Serra, 1996). TGF-␤ binds to singlepass transmembrane serine/threonine kinases referred to as type I and II TGF-␤ receptors (Bassing et al., 1994;ten Dijke et al., 1994). On binding of TGF-␤ to the constitutively active type II receptor (T2R), the type I receptor (T1R) is recruited and phosphorylated by T2R. The activated T1R then phosphorylates downstream signaling intermediates such as the Smad proteins, which translocate to the nucleus and function as transcriptional comodulators (Franzén et al., 1993;Macias-Silva et al., 1996;Yingling et al., 1996).Study of the endocytic response of TGF-␤ receptors to ligand has been difficult due to nonspecific TGF-␤ binding and the fact that different receptor complexes form on the cell surface (heteromers vs. homomers) and undergo distinct endocytic fates (Anders et al., 1997). To overcome these problems, our laboratory created a chimeric receptor system where the ligand binding extracellular domains of the granulocyte/macrophage-colony stimulating factor (GM-CSF) receptors were fused to the transmembrane and cytoplasmic domains of the type I and type II TGF-␤ receptors (Anders and Leof, 1996). Using a number of well-established assays for TGF-␤ action, it could be shown that TGF-␤ signaling is fully recapitulated by the chimeric system (Anders and Leof, 1996;Anders et al., 1997Anders et al., , 1998. In addition, use of the chimeric system (Anders et al., 1997) facilitated TGF-␤ endocytic assays, which showed, similar to other studies (Ehrlich et al., 2001;Yao et al., 2002), that ligand-induced internalization of TGF-␤ receptor complexes occurs through a clathrindependent process. Other reports, however, have proposed roles for...

Transforming growth factor-beta (TGF-beta) is the single most important cytokine promoting renal fibrogenesis. p21-activated kinase-2 (PAK2) and activation of abelson nonreceptor tyrosine kinase (c-abl) have been shown recently to be smad-independent, fibroblast-specific targets downstream of the activated TGF-beta receptor. In the current study we show that in cultured NRK49F-renal fibroblasts (but not in tubular or mesangial cells) TGF-beta similarly activates PAK2 as well as c-abl and induces cell proliferation. Inhibition of the c-abl kinase with imatinib mesylate prevents increased proliferation after TGF-beta addition without affecting PAK2. These in vitro findings were extended to rats with unilateral obstructive nephropathy, a disease model of TGF-beta-driven renal fibrogenesis. In obstructed kidneys, PAK2 and c-abl activity were increased but only c-abl activation was blocked by imatinib. Treatment with imatinib did not prevent renal interstitial infiltration of macrophages or phosphorylation and nuclear translocation of smad2/3 in obstructed kidneys. In contrast, imatinib substantially inhibited an increase in the number of interstitial fibroblasts and myofibroblasts and reduced the expression and interstitial accumulation of collagen type III, collagen type IV and fibronectin. These findings indicate that TGF-beta-induced activation of the nonreceptor c-abl tyrosine kinase regulates fibroblast proliferation and, by this means, is a costimulatory signal in TGF-beta-dependent renal fibrogenesis. Inhibition of c-abl activity with imatinib mesylate ameliorates experimental renal fibrosis in rats.

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-β and PDGF are 2 of the most potent of these cytokines. In the current study, we investigated the role of TGF-β-induced fibrosis mediated by activation of the Abelson (Abl) tyrosine kinase. Our data indicate that fibroblasts respond to TGF-β by stimulating c-Abl kinase activity independently of Smad2/3 phosphorylation or PDGFR activation. Moreover, inhibition of c-Abl by imatinib prevented TGF-β-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.