Professional Education


  • Bachelor of Science, James Cook University (2001)
  • Doctor of Philosophy, James Cook University (2016)

Stanford Advisors


Patents


  • Edward Leof, Mark Wilkes, Claire Repellin, Jeong-Han Kang, Xueqian Yin, Mahefian Andrianifahanana. "United States Patent 62/297,277 Polypeptide Inhibitors of Smad3 Polypeptide Activities", Mayo Clinic and Foundation, Feb 19, 2016
  • Edward Leof, Mark Wilkes, Claire Repellin, Jeong-Han Kang, Xueqian Yin, Mahefian Andrianifahanana. "United States Patent 62/295,843 Polypeptide Inhibitors of Smad3 Polypeptide Activities", Mayo Clinic and Foundation, Feb 16, 2016

All Publications


  • Sorting nexin 9 differentiates ligand-activated Smad3 from Smad2 for nuclear import and transforming growth factor beta signaling MOLECULAR BIOLOGY OF THE CELL Wilkes, M. C., Repellin, C. E., Kang, J., Andrianifahanana, M., Yin, X., Leof, E. B. 2015; 26 (21): 3879-3891

    Abstract

    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.

    View details for DOI 10.1091/mbc.E15-07-0545

    View details for Web of Science ID 000366322200023

    View details for PubMedID 26337383

  • Profibrotic TGF beta responses require the cooperative action of PDGF and ErbB receptor tyrosine kinases FASEB JOURNAL Andrianifahanana, M., Wilkes, M. C., Gupta, S. K., Rahimi, R. A., Repellin, C. E., Edens, M., Wittenberger, J., Yin, X., Maidl, E., Becker, J., Leof, E. B. 2013; 27 (11): 4444-4454

    Abstract

    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.

    View details for DOI 10.1096/fj.12-224907

    View details for Web of Science ID 000329937500012

    View details for PubMedID 23913859

  • Retromer maintains basolateral distribution of the type II TGF-beta receptor via the recycling endosome MOLECULAR BIOLOGY OF THE CELL Yin, X., Murphy, S. J., Wilkes, M. C., Ji, Y., Leof, E. B. 2013; 24 (14): 2285-2298

    Abstract

    Transforming growth factor β (TGF-β) is critical for the development and maintenance of epithelial structures. Because receptor localization and trafficking affect the cellular and organismal response to TGF-β, the present study was designed to address how such homeostatic control is regulated. To that end, we identify a new role for the mammalian retromer complex in maintaining basolateral plasma membrane expression of the type II TGF-β receptor (TβRII). Retromer and TβRII associate in the presence or absence of TGF-β ligand. After retromer knockdown, although TβRII internalization and trafficking to a Rab5-positive compartment occur as in wild-type cells, receptor recycling is inhibited. This results in TβRII mislocalization from the basolateral to both the basolateral and apical plasma membranes independent of Golgi transit and the Rab11-positive apical recycling endosome. The data support a model in which, after initial basolateral TβRII delivery, steady-state polarized TβRII expression is maintained by retromer/TβRII binding and delivery to the common recycling endosome.

    View details for DOI 10.1091/mbc.E13-02-0093

    View details for Web of Science ID 000322159400012

    View details for PubMedID 23720763

  • Imatinib mesylate causes genome-wide transcriptional changes in systemic sclerosis fibroblasts in vitro CLINICAL AND EXPERIMENTAL RHEUMATOLOGY Hinchcliff, M., Huang, C. C., Ishida, W., Fang, F., Lee, J., Jafari, N., Wilkes, M., Bhattacharyya, S., LEOF, E., Varga, J. 2012; 30 (2): S86-S96

    Abstract

    Systemic sclerosis (SSc) is a heterogeneous multifactorial disease dominated by progressive skin and internal organ fibrosis that is driven in part by transforming growth factor-beta (TGF-β). An important downstream target of TGF-β is the Abelson (c-Abl) tyrosine kinase, and its inhibition by imatinib mesylate (Gleevec) attenuates fibrosis in mice. Here we examined the effect of c-Abl activation and blockade in explanted healthy control and SSc fibroblasts.Skin biopsies and explanted fibroblasts from healthy subjects and patients with SSc were studied. Changes in genome-wide expression patterns in imatinib-treated control and SSc fibroblasts were analysed by DNA microarray.Treatment of control fibroblasts with TGF-β resulted in activation of c-Abl and stimulation of fibrotic gene expression that was prevented by imatinib. Moreover, imatinib reduced basal collagen gene expression in SSc but not control fibroblasts. No significant differences in tissue levels of c-Abl and phospho-c-Abl were detected between SSc and control skin biopsies. In vitro, imatinib induced dramatic changes in the expression of genes involved in fibrosis, cardiovascular disease, inflammation, and lipid and cholesterol metabolism. Remarkably, of the 587-imatinib-responsive genes, 91% showed significant change in SSc fibroblasts, but only 12% in control fibroblasts.c-Abl plays a key role in fibrotic responses. Imatinib treatment results in dramatic changes in gene expression in SSc fibroblasts but has only modest effects in control fibroblasts. These data provide novel insights into the mechanisms underlying the antifibrotic effect of imatinib in SSc.

    View details for Web of Science ID 000304976600015

    View details for PubMedID 22691216

  • Non-Smad Transforming Growth Factor-beta Signaling Regulated by Focal Adhesion Kinase Binding the p85 Subunit of Phosphatidylinositol 3-Kinase JOURNAL OF BIOLOGICAL CHEMISTRY Hong, M., Wilkes, M. C., Penheiter, S. G., Gupta, S. K., Edens, M., Leof, E. B. 2011; 286 (20): 17841-17850

    Abstract

    TGF-β 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-β. 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-β treatment in a subset of fibroblasts but not epithelial cells and has an obligate role in TGF-β-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-β and suggest that inhibiting this interaction may be useful in treating a number of fibrotic diseases.

    View details for DOI 10.1074/jbc.M111.233676

    View details for Web of Science ID 000290585200047

    View details for PubMedID 21454615

  • Type II Transforming Growth Factor-beta Receptor Recycling Is Dependent upon the Clathrin Adaptor Protein Dab2 MOLECULAR BIOLOGY OF THE CELL Penheiter, S. G., Singh, R. D., Repellin, C. E., Wilkes, M. C., Edens, M., Howe, P. H., Pagano, R. E., Leof, E. B. 2010; 21 (22): 4009-4019

    Abstract

    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.

    View details for DOI 10.1091/mbc.E09-12-1019

    View details for Web of Science ID 000284216800051

    View details for PubMedID 20881059

  • ERBB Receptor Activation Is Required for Profibrotic Responses to Transforming Growth Factor beta CANCER RESEARCH Andrianifahanana, M., Wilkes, M. C., Repellin, C. E., Edens, M., Kottom, T. J., Rahimi, R. A., Leof, E. B. 2010; 70 (19): 7421-7430

    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.

    View details for DOI 10.1158/0008-5472.CAN-10-0232

    View details for Web of Science ID 000282647700007

    View details for PubMedID 20841477

  • Noncanonical TGF-beta pathways, mTORC1 and Ab1, in renal interstitial fibrogenesis AMERICAN JOURNAL OF PHYSIOLOGY-RENAL PHYSIOLOGY Wang, S., Wilkes, M. C., Leof, E. B., Hirschberg, R. 2010; 298 (1): F142-F149

    Abstract

    Renal interstitial fibrosis is a major determinant of renal failure in the majority of chronic renal diseases. Transforming growth factor-beta (TGF-beta) is the single most important cytokine promoting renal fibrogenesis. Recent in vitro studies identified novel non-smad TGF-beta targets including p21-activated kinase-2 (PAK2), the abelson nonreceptor tyrosine kinase (c-Abl), and the mammalian target of rapamycin (mTOR) that are activated by TGF-beta in mesenchymal cells, specifically in fibroblasts but less in epithelial cells. In the present studies, we show that non-smad effectors of TGF-beta including PAK2, c-Abl, Akt, tuberin (TSC2), and mTOR are activated in experimental unilateral obstructive nephropathy in rats. Treatment with c-Abl or mTOR inhibitors, imatinib mesylate and rapamycin, respectively, each blocks noncanonical (non-smad) TGF-beta pathways in the kidney in vivo and diminishes the number of interstitial fibroblasts and myofibroblasts as well as the interstitial accumulation of extracellular matrix proteins. These findings indicate that noncanonical TGF-beta pathways are activated during the early and rapid renal fibrogenesis of obstructive nephropathy. Moreover, the current findings suggest that combined inhibition of key regulators of these non-smad TGF-beta pathways even in dose-sparing protocols are effective treatments in renal fibrogenesis.

    View details for DOI 10.1152/ajprenal.00320.2009

    View details for Web of Science ID 000272924500018

    View details for PubMedID 19846571

  • Erbin and the NF2 Tumor Suppressor Merlin Cooperatively Regulate Cell-Type-Specific Activation of PAK2 by TGF-beta DEVELOPMENTAL CELL Wilkes, M. C., Repellin, C. E., Hong, M., Bracamonte, M., Penheiter, S. G., Borg, J., Leof, E. B. 2009; 16 (3): 433-444

    Abstract

    Transforming growth factor beta (TGF-beta) family ligands are pleotropic proteins with diverse cell-type-specific effects on growth and differentiation. For example, PAK2 activation is critical for the proliferative/profibrotic action of TGF-beta on mesenchymal cells, and yet it is not responsive to TGF-beta in epithelial cells. We therefore investigated the regulatory constraints that prevent inappropriate PAK2 activation in epithelial cultures. The results show that the epithelial-enriched protein Erbin controls the function of the NF2 tumor suppressor Merlin by determining the output of Merlin's physical interactions with active PAK2. Whereas mesenchymal TGF-beta signaling induces PAK2-mediated inhibition of Merlin function in the absence of Erbin, Erbin/Merlin complexes bind and inactivate GTPase-bound PAK2 in epithelia. These results not only identify Erbin as a key determinant of epithelial resistance to TGF-beta signaling, they also show that Erbin controls Merlin tumor suppressor function by switching the functional valence of PAK2 binding.

    View details for DOI 10.1016/j.devcel.2009.01.009

    View details for Web of Science ID 000264368400013

    View details for PubMedID 19289088

  • A non-Smad mechanism of fibroblast activation by transforming growth factor-beta via c-Abl and Egr-1: selective modulation by imatinib mesylate ONCOGENE Bhattacharyya, S., Ishida, W., Wu, M., Wilkes, M., Mori, Y., Hinchcliff, M., LEOF, E., Varga, J. 2009; 28 (10): 1285-1297

    Abstract

    The nonreceptor protein tyrosine kinase c-Abl regulates cell proliferation and survival. Recent studies provide evidence that implicate c-Abl as a mediator for fibrotic responses induced by transforming growth factor-beta (TGF-beta), but the precise mechanisms underlying this novel oncogene function are unknown. Here, we report that when expressed in normal fibroblasts, a constitutively active mutant of Abl that causes chronic myelogenous leukemia (CML) stimulated the expression and transcriptional activity of the early growth response factor 1 (Egr-1). Mouse embryonic fibroblasts (MEFs), lacking c-Abl, were resistant to TGF-beta stimulation. Responsiveness of these MEFs to TGF-beta could be rescued by wild-type c-Abl, but not by a kinase-deficient mutant form of c-Abl. Furthermore, Abl kinase activity was necessary for the induction of Egr-1 by TGF-beta in normal fibroblasts, and Egr-1 was required for stimulation of collagen by Bcr-Abl. Lesional skin fibroblasts in mice with bleomycin-induced fibrosis of skin displayed evidence of c-Abl activation in situ, and elevated phospho-c-Abl correlated with increased local expression of Egr-1. Collectively, these results position Egr-1 downstream of c-Abl in the fibrotic response, delineate a novel Egr-1-dependent intracellular signaling mechanism that underlies the involvement of c-Abl in certain TGF-beta responses, and identify Egr-1 as a target of inhibition by imatinib. Furthermore, the findings show in situ activation of c-Abl paralleling the upregulated tissue expression of Egr-1 that accompanies fibrosis. Pharmacological targeting of c-Abl and its downstream effector pathways may, therefore, represent a novel therapeutic approach to blocking TGF-beta-dependent fibrotic processes.

    View details for DOI 10.1038/onc.2008.479

    View details for Web of Science ID 000264116000001

    View details for PubMedID 19151753

  • Distinct Roles for Mammalian Target of Rapamycin Complexes in the Fibroblast Response to Transforming Growth Factor-beta CANCER RESEARCH Rahimi, R. A., Andrianifahanana, M., Wilkes, M. C., Edens, M., Kottom, T. J., Blenis, J., Leof, E. B. 2009; 69 (1): 84-93

    Abstract

    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.

    View details for DOI 10.1158/0008-5472.CAN-08-2146

    View details for Web of Science ID 000262273100013

    View details for PubMedID 19117990

  • Transforming growth factor beta signaling via ras in mesenchymal cells requires p21-activated kinase 2 for extracellular signal-regulated kinase- dependent transcriptional responses CANCER RESEARCH Suzuki, K., Wilkes, M. C., Garamszegi, N., Edens, M., Leof, E. B. 2007; 67 (8): 3673-3682

    Abstract

    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.

    View details for DOI 10.1158/0008-5472.CAN-06-3211

    View details for Web of Science ID 000245779600028

    View details for PubMedID 17440079

  • Transforming growth factor beta activation of c-Abl is independent of receptor internalization and regulated by phosphatidylinositol 3-kinase and PAK2 in mesenchymal cultures JOURNAL OF BIOLOGICAL CHEMISTRY Wilkes, M. C., Leof, E. B. 2006; 281 (38): 27846-27854

    Abstract

    Transforming growth factor beta (TGF-beta) modulates a number of cellular phenotypes as divergent as growth stimulation and growth inhibition. Although the Smad pathway is critical for many of these responses, recent evidence indicates that Smad-independent pathways may also have a critical role. One such protein previously shown to regulate TGF-beta action independent of the Smad proteins is the c-Abl nonreceptor tyrosine kinase. In the current study we determined that TGF-beta receptor signaling activates c-Abl kinase activity in a subset of fibroblast but not epithelial cultures. This cell type-specific response occurs in a membrane-proximal locale independent of receptor internalization and upstream of dynamin action. Although c-Abl activation by TGF-beta is independent of Smad2 or Smad3, it is prevented by inhibitors of phosphatidylinositol 3-kinase or PAK2. Thus, c-Abl represents a target downstream of phosphatidylinositol 3-kinase-activated PAK2, which differentiates TGF-beta signaling in fibroblasts and epithelial cell lines and integrates serine/threonine receptor kinases with tyrosine kinase pathways.

    View details for DOI 10.1074/jbc.M603721200

    View details for Web of Science ID 000240534400020

    View details for PubMedID 16867995

  • Transforming growth factor-beta activation of phosphatidylinositol 3-kinase is independent of Smad2 and Smad3 and regulates fibroblast responses via p21-activated kinase-2 CANCER RESEARCH Wilkes, M. C., Mitchell, H., Penheiter, S. G., Dore, J. J., Suzuki, K., Edens, M., Sharma, D. K., Pagano, R. E., Leof, E. B. 2005; 65 (22): 10431-10440

    Abstract

    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.

    View details for DOI 10.1158/0008-5472.CAN-05-1522

    View details for Web of Science ID 000233418900042

    View details for PubMedID 16288034

  • Imatinib mesylate blocks a non-smad TGF-beta pathway and reduces renal fibrogenesis in vivo FASEB JOURNAL Wang, S. N., Wilkes, M. C., LEOF, E. B., Hirschberg, R. 2005; 19 (1): 1-11

    Abstract

    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.

    View details for DOI 10.1096/fj.04-2370com

    View details for Web of Science ID 000226576600036

    View details for PubMedID 15629889

  • Imatinib mesylate inhibits the profibrogenic activity of TGF-beta and prevents bleomycin-mediated lung fibrosis JOURNAL OF CLINICAL INVESTIGATION Daniels, C. E., Wilkes, M. C., Edens, M., Kottom, T. J., Murphy, S. J., Limper, A. H., Leof, E. B. 2004; 114 (9): 1308-1316

    Abstract

    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.

    View details for DOI 10.1175/JCI200419603

    View details for Web of Science ID 000224872400020

    View details for PubMedID 15520863

  • Differential trafficking of transforming growth factor-beta receptors and ligand in polarized epithelial cells MOLECULAR BIOLOGY OF THE CELL Murphy, S. J., Dore, J. J., Edens, M., Coffey, R. J., BARNARD, J. A., Mitchell, H., Wilkes, M., Leof, E. B. 2004; 15 (6): 2853-2862

    Abstract

    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-beta (TGFbeta) 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 TGFbeta receptor complex, TGFbeta ligand secretion was found to be from the apical surfaces. Confocal imaging of MDCK cells with an antibody to TGFbeta1 confirmed a predominant apical localization, with a stark absence at the basal membrane. These findings indicate that cell adhesion regulates the localization of TGFbeta receptors in polarized epithelial cultures and that the response to TGFbeta is dependent upon the spatial distribution and secretion of TGFbeta receptors and ligand, respectively.

    View details for DOI 10.1091/mbc.E04-02-0097

    View details for Web of Science ID 000221778300030

    View details for PubMedID 15075369

  • Cell-type-specific activation of PAK2 by transforming growth factor beta independent of Smad2 and Smad3 MOLECULAR AND CELLULAR BIOLOGY Wilkes, M. C., Murphy, S. J., Garamszegi, N., LEOF, E. B. 2003; 23 (23): 8878-8889

    Abstract

    Transforming growth factor beta (TGF-beta) causes growth arrest in epithelial cells and proliferation and morphological transformation in fibroblasts. Despite the ability of TGF-beta to induce various cellular phenotypes, few discernible differences in TGF-beta signaling between cell types have been reported, with the only well-characterized pathway (the Smad cascade) seemingly under identical control. We determined that TGF-beta receptor signaling activates the STE20 homolog PAK2 in mammalian cells. PAK2 activation occurs in fibroblast but not epithelial cell cultures and is independent of Smad2 and/or Smad3. Furthermore, we show that TGF-beta-stimulated PAK2 activity is regulated by Rac1 and Cdc42 and dominant negative PAK2 or morpholino antisense oligonucleotides to PAK2 prevent the morphological alteration observed following TGF-beta addition. Thus, PAK2 represents a novel Smad-independent pathway that differentiates TGF-beta signaling in fibroblast (growth-stimulated) and epithelial cell (growth-inhibited) cultures.

    View details for DOI 10.1128/MCB.23.23.8878-8889.2003

    View details for Web of Science ID 000186618300039

    View details for PubMedID 14612425