title: Fine-tuning of Smad protein function by poly(ADP-ribose) polymerases and poly(ADP-ribose) glycohydrolase during transforming growth factor β signaling.
abstract: BACKGROUND
  Initiation, amplitude, duration and termination of transforming growth factor β (TGFβ) signaling via Smad proteins is regulated by post-translational modifications, including phosphorylation, ubiquitination and acetylation. We previously reported that ADP-ribosylation of Smads by poly(ADP-ribose) polymerase 1 (PARP-1) negatively influences Smad-mediated transcription. PARP-1 is known to functionally interact with PARP-2 in the nucleus and the enzyme poly(ADP-ribose) glycohydrolase (PARG) can remove poly(ADP-ribose) chains from target proteins. Here we aimed at analyzing possible cooperation between PARP-1, PARP-2 and PARG in regulation of TGFβ signaling.
  
  
  METHODS
  A robust cell model of TGFβ signaling, i.e. human HaCaT keratinocytes, was used. Endogenous Smad3 ADP-ribosylation and protein complexes between Smads and PARPs were studied using proximity ligation assays and co-immunoprecipitation assays, which were complemented by in vitro ADP-ribosylation assays using recombinant proteins. Real-time RT-PCR analysis of mRNA levels and promoter-reporter assays provided quantitative analysis of gene expression in response to TGFβ stimulation and after genetic perturbations of PARP-1/-2 and PARG based on RNA interference.
  
  
  RESULTS
  TGFβ signaling rapidly induces nuclear ADP-ribosylation of Smad3 that coincides with a relative enhancement of nuclear complexes of Smads with PARP-1 and PARP-2. Inversely, PARG interacts with Smads and can de-ADP-ribosylate Smad3 in vitro. PARP-1 and PARP-2 also form complexes with each other, and Smads interact and activate auto-ADP-ribosylation of both PARP-1 and PARP-2. PARP-2, similar to PARP-1, negatively regulates specific TGFβ target genes (fibronectin, Smad7) and Smad transcriptional responses, and PARG positively regulates these genes. Accordingly, inhibition of TGFβ-mediated transcription caused by silencing endogenous PARG expression could be relieved after simultaneous depletion of PARP-1.
  
  
  CONCLUSIONS
  Nuclear Smad function is negatively regulated by PARP-1 that is assisted by PARP-2 and positively regulated by PARG during the course of TGFβ signaling.
authors.affiliation: Ludwig Institute for Cancer Research, Science for Life Laboratory, Uppsala University, Uppsala, Sweden.
authors.forename: Markus
authors.initials: M
authors.lastname: Dahl
authors.affiliation: Ludwig Institute for Cancer Research, Science for Life Laboratory, Uppsala University, Uppsala, Sweden.
authors.forename: Varun
authors.initials: V
authors.lastname: Maturi
authors.affiliation: Ludwig Institute for Cancer Research, Science for Life Laboratory, Uppsala University, Uppsala, Sweden.
authors.forename: Peter
authors.initials: P
authors.lastname: Lönn
authors.affiliation: Ludwig Institute for Cancer Research, Science for Life Laboratory, Uppsala University, Uppsala, Sweden.
authors.forename: Panagiotis
authors.initials: P
authors.lastname: Papoutsoglou
authors.affiliation: Department of Immunology, Genetics and Pathology, Science for Life Laboratory, Uppsala University, Uppsala, Sweden.
authors.forename: Agata
authors.initials: A
authors.lastname: Zieba
authors.affiliation: Ludwig Institute for Cancer Research, Science for Life Laboratory, Uppsala University, Uppsala, Sweden.
authors.forename: Michael
authors.initials: M
authors.lastname: Vanlandewijck
authors.affiliation: Ludwig Institute for Cancer Research, Science for Life Laboratory, Uppsala University, Uppsala, Sweden.
authors.forename: Lars P
authors.initials: LP
authors.lastname: van der Heide
authors.affiliation: Ludwig Institute for Cancer Research, Science for Life Laboratory, Uppsala University, Uppsala, Sweden.
authors.forename: Yukihide
authors.initials: Y
authors.lastname: Watanabe
authors.affiliation: Department of Immunology, Genetics and Pathology, Science for Life Laboratory, Uppsala University, Uppsala, Sweden.
authors.forename: Ola
authors.initials: O
authors.lastname: Söderberg
authors.affiliation: Institute of Veterinary Biochemistry and Molecular Biology, University of Zurich, Zurich, Switzerland.
authors.forename: Michael O
authors.initials: MO
authors.lastname: Hottiger
authors.affiliation: Ludwig Institute for Cancer Research, Science for Life Laboratory, Uppsala University, Uppsala, Sweden.
authors.forename: Carl-Henrik
authors.initials: CH
authors.lastname: Heldin
authors.affiliation: Ludwig Institute for Cancer Research, Science for Life Laboratory, Uppsala University, Uppsala, Sweden; Department of Medical Biochemistry and Microbiology, Science for Life Laboratory, Uppsala University, Uppsala, Sweden.
authors.forename: Aristidis
authors.initials: A
authors.lastname: Moustakas
mesh_terms: D005353:Fibronectins / Q000235:genetics / Q000378:metabolism; D005786:Gene Expression Regulation; D006026:Glycoside Hydrolases / Q000502:physiology*; D057809:HEK293 Cells; D006801:Humans; D000071137:Poly (ADP-Ribose) Polymerase-1; D011065:Poly(ADP-ribose) Polymerases / Q000502:physiology*; D011499:Protein Processing, Post-Translational; D015398:Signal Transduction; D051899:Smad2 Protein / Q000378:metabolism; D051900:Smad3 Protein / Q000378:metabolism*; D051906:Smad7 Protein / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic; D016212:Transforming Growth Factor beta / Q000502:physiology*
chemical_list: D005353:Fibronectins; C497782:SMAD2 protein, human; C497790:SMAD3 protein, human; C497806:SMAD7 protein, human; D051899:Smad2 Protein; D051900:Smad3 Protein; D051906:Smad7 Protein; D016212:Transforming Growth Factor beta; C499320:PARP1 protein, human; C483041:PARP2 protein, human; D000071137:Poly (ADP-Ribose) Polymerase-1; D011065:Poly(ADP-ribose) Polymerases; D006026:Glycoside Hydrolases; C022407:poly ADP-ribose glycohydrolase