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Fine-Tuning of Smad Protein Function by Poly(ADP-Ribose) Polymerases and Poly(ADP-Ribose) Glycohydrolase during Transforming Growth Factor β Signaling
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research. Uppsala University, Science for Life Laboratory, SciLifeLab.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research. Uppsala University, Science for Life Laboratory, SciLifeLab.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research. Uppsala University, Science for Life Laboratory, SciLifeLab.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research. Uppsala University, Science for Life Laboratory, SciLifeLab.
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2014 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 9, no 8, p. e103651-Article in journal (Refereed) Published
Abstract [en]

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.

CONCLUSION:

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.

Place, publisher, year, edition, pages
2014. Vol. 9, no 8, p. e103651-
National Category
Clinical Medicine
Identifiers
URN: urn:nbn:se:uu:diva-231920DOI: 10.1371/journal.pone.0103651ISI: 000341302700014PubMedID: 25133494OAI: oai:DiVA.org:uu-231920DiVA, id: diva2:745828
Available from: 2014-09-11 Created: 2014-09-11 Last updated: 2017-12-05Bibliographically approved
In thesis
1. Gene regulation by different proteins of TGFβ superfamily
Open this publication in new window or tab >>Gene regulation by different proteins of TGFβ superfamily
2018 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The present thesis discusses how gene regulation by transforming growth factor β (TGFβ) family cytokines is affected by post-translational modifications of different transcription factors. The thesis also focuses on gene regulation by transcription factors involved in TGFβ signaling.

The importance of the poly ADP-ribose polymerase (PARP) family in controlling gene expression in response to TGFβ and bone morphogenetic protein (BMP) is analyzed first. PARP2, along with PARP1, ADP-ribosylates Smad2 and Smad3, the signaling mediators of TGFβ. On the other hand, poly ADP-ribose glycohydrolase (PARG) removes the ADP-ribose from Smad2/3 and antagonizes PARP1 and PARP2. ADP-ribosylation of Smads in turn affects their DNA binding capacity. We then illustrate how PARP1 and PARG can regulate gene expression in response to BMP that signals via Smad1, 5. Over-expression of PARP1 suppressed the transcriptional activity of Smad1/5. Knockdown of PARP1 or over-expression of PARG enhanced the transcriptional activity of BMP-Smads on target genes. Hence our data suggest that ADP-ribosylation of Smad proteins controls both TGFβ and BMP signaling. 

I then focus on elucidating novel genes that are regulated by ZEB1 and Snail1, two key transcriptional factors in TGFβ signaling, known for their ability to induce EMT and cancer metastasis. Chromatin immunoprecipitation-sequencing (ChIP-seq) and targeted whole genome transcriptomics in triple negative breast cancer cells were used, to find binding regions and the functional impact of ZEB1 and Snail1 throughout the genome. ZEB1 binds to the regulatory sequences of a wide range of genes, not only related to cell invasion, pointing to new functions of ZEB1. On the other hand, Snail1 regulated only a few genes, especially related to signal transduction and cellular movement. Further functional analysis revealed that ZEB1 could regulate the anchorage-independent growth of the triple negative breast cancer cells, whereas Snail1 could regulate the expression of BMP6 in these cells. We have therefore elucidated novel functional roles of the two transcription factors, Snail1 and ZEB1 in triple negative breast cancer cells.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2018. p. 50
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1404
Keyword
EMT, Snail1, ZEB1, TGFβ, BMP, Gene regulation
National Category
Medical and Health Sciences Biochemistry and Molecular Biology Cell Biology
Identifiers
urn:nbn:se:uu:diva-334411 (URN)978-91-513-0172-3 (ISBN)
Public defence
2018-01-29, Room B42, BMC, Husargatan 3, Uppsala, 09:15 (English)
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Supervisors
Available from: 2017-12-29 Created: 2017-11-23 Last updated: 2018-03-08

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Maturi, VarunLönn, PeterPapoutsoglou, PanagiotisZieba, AgataVanlandewijck, MichaelSöderberg, OlaHeldin, Carl-HenrikMoustakas, Aristidis

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Maturi, VarunLönn, PeterPapoutsoglou, PanagiotisZieba, AgataVanlandewijck, MichaelSöderberg, OlaHeldin, Carl-HenrikMoustakas, Aristidis
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