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Regulation of TGFβ signaling by long non-coding RNAs and ADP-ribosylation
Uppsala University, Science for Life Laboratory, SciLifeLab.
2018 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Transforming growth factor β (TGFβ) signaling pathways participate in embryonic development and tissue homeostasis and have a dual role in cancer. TGFβ acts as a tumor suppressor that promotes cell cycle arrest and apoptosis at initial stages of tumorigenesis. In contrast, TGFβ, induces epithelial to mesenchymal transition (EMT), a normal embryonic process which is employed by advanced cancers, in order to acquire mesenchymal traits and metastasize.

Bone morphogenetic protein (BMP) family members belong to the TGFβ superfamily and are involved in cell differentiation, development and bone formation.

Non-coding RNAs (ncRNAs) are not translated into proteins, are important regulators of gene expression and physiological processes and are often de-regulated in cancer. They control gene expression through physical association with chromatin, DNA, RNA molecules or proteins.

Poly(ADP-ribose) polymerases (PARPs) catalyze the poly (ADP)-ribosylation of proteins, whereas the enzyme poly(ADP-ribose) glycohydrolase (PARG) removes ADP-ribose units. Members of the PARP family function in the DNA damage response, regulation of transcription and cell death.

In this thesis, we investigated the importance of the TGFβ signaling pathway in regulating the expression of long non-coding RNAs (lncRNAs). We identified TGFβ-regulated lncRNAs and observed that a substantial number of them act in a feedback loop to modulate the magnitude of TGFβ signaling. Interestingly, the nuclear lncRNA TGFB2-antisense RNA 1 (TGFB2-AS1) is induced by TGFβ and negatively regulates expression of members of the TGFβ and BMP pathways, through interaction with EED, a protein of the polycomb repressor complex 2 (PRC2). Also, TGFβ signaling promoted the expression of mir-100-let-7a-2-mir-125b-1 cluster host gene (MIR100HG), which enhanced TGFβ signaling and affected TGFβ-mediated cell cycle arrest. The MIR100HG-derived miRNAs let-7a-2-3p, miR125b-5p and miR-125b-1-3p, were also induced by TGFβ. In contrast, the long intergenic non-protein coding RNA 707 (LINC00707), was reduced in response to TGFβ and affected the expression of a group of genes related to inflammatory responses and interferon-γ (IFN-γ) signaling.

We also report that TGFβ and BMP pathways are regulated by ADP-ribosylation of Smad proteins, the signaling mediators of these pathways. We observed that PARP1 and PARP2 attenuated, while PARG favored TGFβ signaling. Furthermore, PARP1 negatively regulated BMP signaling, by ADP-ribosylating Smad1 and Smad5, whereas PARG enhanced BMP signaling by de-ADP-ribosylating Smads.

Collectively, we provide evidence that lncRNAs and ADP-ribosylating enzymes modulate TGFβ and BMP signaling pathways and propose models for their molecular mechanisms and functional roles.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2018. , p. 65
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1513
Keywords [en]
TGFβ, signal transduction, long non-coding RNAs, ADP-ribosylation, transcriptional regulation, chromatin remodeling
National Category
Cell and Molecular Biology
Research subject
Medical Science
Identifiers
URN: urn:nbn:se:uu:diva-364107ISBN: 978-91-513-0497-7 (print)OAI: oai:DiVA.org:uu-364107DiVA, id: diva2:1258105
Public defence
2018-12-17, B42, Biomedical Center, Husargatan 3, Uppsala, 13:00 (English)
Opponent
Supervisors
Available from: 2018-11-22 Created: 2018-10-23 Last updated: 2018-11-30
List of papers
1. The TGFB2-AS1 lncRNA regulates TGFβ signaling by modulating corepressor activity
Open this publication in new window or tab >>The TGFB2-AS1 lncRNA regulates TGFβ signaling by modulating corepressor activity
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2018 (English)Article in journal (Refereed) Submitted
Abstract [en]

LncRNAs regulate cell function through many physiological processes. We have identified lncRNAs whose expression is regulated by transforming growth factor β (TGFβ), by a transcriptomic screen. We focused on TGFB2-antisense RNA1 (TGFB2-AS1), which was induced by TGFβ through Smad and protein kinase pathways, and exhibited predominantly nuclear localization. Depleting TGFB2-AS1 enhanced TGFβ/Smad-mediated transcription and expression of the TGFβ-target genes FN1 and SERPINE1. Overexpression of TGFB2-AS1 reduced expression of these genes, attenuated TGFβ-induced cell growth arrest, and altered BMP and Wnt pathway gene profiles. Mechanistically, TGFB2-AS1 mainly via its 3’ terminal region, bound to EED, an adaptor of the Polycomb repressor complex 2 (PRC2), promoting repressive histone H3K27me3 modifications at TGFβ-target gene promoters. Silencing EED or inhibiting PRC2 methylation activity, partially rescued TGFB2-AS1 mediated gene repression. Our observations support the notion that TGFB2-AS1 is a TGFβ-induced lncRNA with inhibitory functions on TGFβ and BMP pathways output, constituting an auto-regulatory negative feedback mechanism that balances TGFβ- and BMP-mediated responses.

National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-363700 (URN)
Available from: 2018-10-20 Created: 2018-10-20 Last updated: 2018-12-03
2. TGFβ signaling down-regulates LINC00707 to inhibit inflammatory responses
Open this publication in new window or tab >>TGFβ signaling down-regulates LINC00707 to inhibit inflammatory responses
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

The class of long non-coding RNAs (lncRNAs) consists of RNA molecules, which lack protein coding potential and regulate a wide variety of cellular processes. At the molecular level, lncRNAs act as regulators of gene expression by interacting with chromatin, other types of RNA or proteins. Transforming growth factor β (TGFβ) plays pivotal roles in diverse biological processes, such as cell growth arrest, embryonic development and regulation of the immune system. In this study, we describe the long intergenic non-protein coding RNA 707 (LINC00707) as a TGFβ responsive gene. By combining transcriptomic data from human keratinocytes and glioblastoma cancer stem cells, we observed that TGFβ signaling down-regulates the expression of LINC00707. RNA sequencing revealed that in keratinocytes knockdown of LINC00707 or stimulation by TGFβ, affected expression of genes involved in inflammatory responses and interferon-γ-mediated signaling. In summary, we suggest that the immune suppressive actions of TGFβ involve suppression of the pro-inflammatory LINC00707.

National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-363898 (URN)
Available from: 2018-10-19 Created: 2018-10-19 Last updated: 2018-12-03
3. The non-coding MIR100HG RNA mediates cytostatic responses of epithelial cells to transforming growth factor β
Open this publication in new window or tab >>The non-coding MIR100HG RNA mediates cytostatic responses of epithelial cells to transforming growth factor β
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Transforming growth factor β (TGFβ) stimulation modulates the expression of many epithelial genes involved in cell growth arrest, epithelial-to-mesenchymal transition and development. Many recent reports provide evidence that TGFβ signaling regulates the expression of long non-coding RNAs (lncRNAs), i.e. RNAs lacking protein coding potential. After screening for lncRNAs whose expression is regulated by TGFβ signaling, we observed that TGFβ induced the expression of the mir-100-let-7a-2-mir-125b-1 cluster host gene (MIR100HG), a genetic locus which gives rise to multiple lncRNAs (MIR100HG splice variants), as well as the micro-RNA clusters miR-100, let-7a-2 and miR-125b-1. In addition, TGFβ stimulation led to increased levels of mature let-7a-2-3p, miR-125b-5p and miR-125b-1-3p miRNAs. MIR100HG depletion attenuated the TGFβ/Smad-mediated transcriptional responses, the expression of the TGFβ-target genes SERPINE1 (PAI-1) and fibronectin 1 (FN1), and TGFβ-mediated cell growth arrest. Moreover, overexpressing let-7a-2-3p, but not miR-125b-5p or miR-125b-1-3p miRNAs, mimicked enhanced TGFβ/Smad-mediated transcription and inhibited cell proliferation, while inhibition of let-7a-2-3p slightly reduced PAI-1 and fibronectin expression. Thus, we identified MIR100HG and the miRNA clusters generated by its locus as TGFβ-target non-coding RNAs, and ascribed to them a potential role in mediating cytostatic responses by modulating the magnitude of TGFβ signaling.

National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-363899 (URN)
Available from: 2018-10-19 Created: 2018-10-19 Last updated: 2018-10-23
4. Fine-Tuning of Smad Protein Function by Poly(ADP-Ribose) Polymerases and Poly(ADP-Ribose) Glycohydrolase during Transforming Growth Factor β Signaling
Open this publication in new window or tab >>Fine-Tuning of Smad Protein Function by Poly(ADP-Ribose) Polymerases and Poly(ADP-Ribose) Glycohydrolase during Transforming Growth Factor β Signaling
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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.

National Category
Clinical Medicine
Identifiers
urn:nbn:se:uu:diva-231920 (URN)10.1371/journal.pone.0103651 (DOI)000341302700014 ()25133494 (PubMedID)
Available from: 2014-09-11 Created: 2014-09-11 Last updated: 2018-10-23Bibliographically approved
5. Regulation of Bone Morphogenetic Protein Signaling by ADP-ribosylation
Open this publication in new window or tab >>Regulation of Bone Morphogenetic Protein Signaling by ADP-ribosylation
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2016 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 291, no 24, p. 12706-12723Article in journal (Refereed) Published
Abstract [en]

We previously established a mechanism of negative regulation of transforming growth factor beta signaling mediated by the nuclear ADP-ribosylating enzyme poly-(ADP-ribose) polymerase 1 (PARP1) and the deribosylating enzyme poly-(ADP-ribose) glycohydrolase (PARG), which dynamically regulate ADP-ribosylation of Smad3 and Smad4, two central signaling proteins of the pathway. Here we demonstrate that the bone morphogenetic protein (BMP) pathway can also be regulated by the opposing actions of PARP1 and PARG. PARG positively contributes to BMP signaling and forms physical complexes with Smad5 and Smad4. The positive role PARG plays during BMP signaling can be neutralized by PARP1, as demonstrated by experiments where PARG and PARP1 are simultaneously silenced. In contrast to PARG, ectopic expression of PARP1 suppresses BMP signaling, whereas silencing of endogenous PARP1 enhances signaling and BMP-induced differentiation. The two major Smad proteins of the BMP pathway, Smad1 and Smad5, interact with PARP1 and can be ADP-ribosylated in vitro, whereas PARG causes deribosylation. The overall outcome of this mode of regulation of BMP signal transduction provides a fine-tuning mechanism based on the two major enzymes that control cellular ADP-ribosylation.

National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-299724 (URN)10.1074/jbc.M116.729699 (DOI)000378119900024 ()27129221 (PubMedID)
Funder
Swedish Research Council, K2010-67X-14936-07-3 K2013-66X-14936-10-5
Available from: 2016-07-26 Created: 2016-07-26 Last updated: 2018-10-23Bibliographically approved

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