title: Distinct role of endocytosis for Smad and non-Smad TGF-β signaling regulation in hepatocytes.
abstract: OBJECTIVE
  In injured liver, TGF-β affects all hepatic cell types and participates in wound healing and fibrogenesis. TGF-β downstream signaling is highly complex and cell type dependent, involving Smad and non-Smad signaling cascades thus requiring tight regulation. Endocytosis has gained relevance as important mechanism to control signaling initiation and termination. In this study, we investigated endocytic mechanisms for TGF-β mediated Smad and non-Smad signaling in hepatocytes.
  
  
  METHODS
  Endocytosis in hepatocytes was elucidated using chemical inhibitors, RNAi, viral gene transfer and caveolin-1-/- mice. TGF-β signaling was monitored by Western blot, reporter assays and gene expression analysis.
  
  
  RESULTS
  In hepatocytes, Smad activation is to a large degree accomplished AP-2 complex dependent on the hepatocyte surface without the necessity of clathrin coated pit formation or an endocytic step. In contrast, non-Smad/AKT pathway activation required functional dynamin mediated endocytosis and the presence of caveolin-1, an essential protein for caveolae formation. Furthermore, these two TGF-β signaling initiation platforms discriminate distinct signaling routes that integrate at the transcriptional level as shown for TGF-β target genes, Id1, Smad7, and CTGF. Endocytosis inhibition increased canonical Smad signaling and culminated in a superinduction of Id1 and Smad7 expression, whereas caveolin-1 mediated AKT pathway activation was required for maximal CTGF induction.
  
  
  CONCLUSIONS
  Endocytosis is critical for TGF-β signaling regulation in hepatocytes and determines gene expression signature and (patho)physiological outcome.
authors.affiliation: Molecular Hepatology - Alcohol Associated Diseases, II. Medical Clinic, Medical Faculty Mannheim, Heidelberg University, Theodor-Kutzer-Ufer 1-3, 68167 Mannheim, Germany. christoph.meyer@medma.uni-heidelberg.de
authors.forename: Christoph
authors.initials: C
authors.lastname: Meyer
authors.forename: Patricio
authors.initials: P
authors.lastname: Godoy
authors.forename: Anastasia
authors.initials: A
authors.lastname: Bachmann
authors.forename: Yan
authors.initials: Y
authors.lastname: Liu
authors.forename: David
authors.initials: D
authors.lastname: Barzan
authors.forename: Iryna
authors.initials: I
authors.lastname: Ilkavets
authors.forename: Patrick
authors.initials: P
authors.lastname: Maier
authors.forename: Carsten
authors.initials: C
authors.lastname: Herskind
authors.forename: Jan G
authors.initials: JG
authors.lastname: Hengstler
authors.forename: Steven
authors.initials: S
authors.lastname: Dooley
mesh_terms: D021581:Active Transport, Cell Nucleus; D000818:Animals; D001483:Base Sequence; D051242:Caveolin 1 / Q000172:deficiency / Q000235:genetics / Q000378:metabolism; D003034:Coated Pits, Cell-Membrane / Q000378:metabolism; D055513:Connective Tissue Growth Factor / Q000235:genetics / Q000378:metabolism; D004705:Endocytosis / Q000502:physiology*; D022781:Hepatocytes / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D008954:Models, Biological; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010766:Phosphorylation; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics; D015398:Signal Transduction; D051785:Smad Proteins / Q000378:metabolism*; D016212:Transforming Growth Factor beta / Q000378:metabolism*
chemical_list: D051242:Caveolin 1; D055513:Connective Tissue Growth Factor; D019869:Phosphatidylinositol 3-Kinases; D051057:Proto-Oncogene Proteins c-akt; D034741:RNA, Small Interfering; D051785:Smad Proteins; D016212:Transforming Growth Factor beta; C526318:CCN2 protein, mouse