| 1. |
- Dahl, Markus, et al.
(author)
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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
- 2014
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In: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 9:8, s. e103651-
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Journal article (peer-reviewed)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.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.
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| 2. |
- Gélabert, Caroline, et al.
(author)
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The long non-coding RNA LINC00707 interacts with Smad proteins to regulate TGFβ signaling and cancer cell invasion
- 2023
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In: Cell Communication and Signaling. - : BioMed Central (BMC). - 1478-811X.
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Journal article (peer-reviewed)abstract
- Background: Long non-coding RNAs (lncRNAs) regulate cellular processes by interacting with RNAs or proteins. Transforming growth factor β (TGFβ) signaling via Smad proteins regulates gene networks that control diverse biological processes, including cancer cell migration. LncRNAs have emerged as TGFβ targets, yet, their mechanism of action and biological role in cancer remains poorly understood.Methods: Whole-genome transcriptomics identified lncRNA genes regulated by TGFβ. Protein kinase inhibitors and RNA-silencing, in combination with cDNA cloning, provided loss- and gain-of-function analyses. Cancer cell-based assays coupled to RNA-immunoprecipitation and protein screening sought mechanistic evidence. Functional validation of TGFβ-regulated lncRNAs was based on new transcriptomics and by combining RNAscope with immunohistochemical analysis in tumor tissue.Results: Transcriptomics of TGFβ signaling responses revealed down-regulation of the predominantly cytoplasmic long intergenic non-protein coding RNA 707 (LINC00707). Expression of LINC00707 required Smad and mitogen-activated protein kinase inputs. By limiting the binding of Krüppel-like factor 6 to the LINC00707 promoter, TGFβ led to LINC00707 repression. Functionally, LINC00707 suppressed cancer cell invasion, as well as key fibrogenic and pro-mesenchymal responses to TGFβ, as also attested by RNA-sequencing analysis. LINC00707 also suppressed Smad-dependent signaling. Mechanistically, LINC00707 interacted with and retained Smad proteins in the cytoplasm. Upon TGFβ stimulation, LINC00707 elimination allowed Smad accumulation in the nucleus. In vivo, LINC00707 expression was negatively correlated with Smad2 activation in tumor tissues.Conclusions: TGFβ signaling decreases LINC00707 expression, which facilitates Smad-dependent signaling, favoring cancer cell invasion.
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| 3. |
- Papoutsoglou, Panagiotis, et al.
(author)
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Long non-coding RNAs and TGF-beta signaling in cancer
- 2020
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In: Cancer Science. - : WILEY. - 1347-9032 .- 1349-7006. ; 111:8, s. 2672-2681
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Research review (peer-reviewed)abstract
- Cancer is driven by genetic mutations in oncogenes and tumor suppressor genes and by cellular events that develop a misregulated molecular microenvironment in the growing tumor tissue. The tumor microenvironment is guided by the excessive action of specific cytokines including transforming growth factor-beta (TGF-beta), which normally controls embryonic development and the homeostasis of young or adult tissues. As a consequence of the genetic alterations generating a given tumor, TGF-beta can preserve its homeostatic function and attempt to limit neoplastic expansion, whereas, once the tumor has progressed to an aggressive stage, TGF-beta can synergize with various oncogenic stimuli to facilitate tumor invasiveness and metastasis. TGF-beta signaling mechanisms via Smad proteins, various ubiquitin ligases, and protein kinases are relatively well understood. Such mechanisms regulate the expression of genes encoding proteins or non-coding RNAs. Among non-coding RNAs, much has been understood regarding the regulation and function of microRNAs, whereas the role of long non-coding RNAs is still emerging. This article emphasizes TGF-beta signaling mechanisms leading to the regulation of non-coding genes, the function of such non-coding RNAs as regulators of TGF-beta signaling, and the contribution of these mechanisms in specific hallmarks of cancer.
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| 4. |
- Papoutsoglou, Panagiotis, 1987-
(author)
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Regulation of TGFβ signaling by long non-coding RNAs and ADP-ribosylation
- 2018
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Doctoral thesis (other academic/artistic)abstract
- 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.
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| 5. |
- Papoutsoglou, Panagiotis, et al.
(author)
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TGFβ signaling down-regulates LINC00707 to inhibit inflammatory responses
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Other publication (other academic/artistic)abstract
- 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.
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| 6. |
- Papoutsoglou, Panagiotis, et al.
(author)
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The non-coding MIR100HG RNA mediates cytostatic responses of epithelial cells to transforming growth factor β
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Other publication (other academic/artistic)abstract
- 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.
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| 7. |
- Papoutsoglou, Panagiotis, et al.
(author)
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The noncoding MIR100HG RNA enhances the autocrine function of transforming growth factor beta signaling
- 2021
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In: Oncogene. - : Springer Nature. - 0950-9232 .- 1476-5594. ; 40:21, s. 3748-3765
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Journal article (peer-reviewed)abstract
- Activation of the transforming growth factor beta (TGF beta) pathway modulates the expression of genes involved in cell growth arrest, motility, and embryogenesis. An expression screen for long noncoding RNAs indicated that TGF beta induced mir-100-let-7a-2-mir-125b-1 cluster host gene (MIR100HG) expression in diverse cancer types, thus confirming an earlier demonstration of TGF beta-mediated transcriptional induction of MIR100HG in pancreatic adenocarcinoma. MIR100HG depletion attenuated TGF beta signaling, expression of TGF beta-target genes, and TGF beta-mediated cell cycle arrest. Moreover, MIR100HG silencing inhibited both normal and cancer cell motility and enhanced the cytotoxicity of cytostatic drugs. MIR100HG overexpression had an inverse impact on TGF beta signaling responses. Screening for downstream effectors of MIR100HG identified the ligand TGF beta 1. MIR100HG and TGFB1 mRNA formed ribonucleoprotein complexes with the RNA-binding protein HuR, promoting TGF beta 1 cytokine secretion. In addition, TGF beta regulated let-7a-2-3p, miR-125b-5p, and miR-125b-1-3p expression, all encoded by MIR100HG intron-3. Certain intron-3 miRNAs may be involved in TGF beta/SMAD-mediated responses (let-7a-2-3p) and others (miR-100, miR-125b) in resistance to cytotoxic drugs mediated by MIR100HG. In support of a model whereby TGF beta induces MIR100HG, which then enhances TGF beta 1 secretion, analysis of human carcinomas showed that MIR100HG expression correlated with expression of TGFB1 and its downstream extracellular target TGFBI. Thus, MIR100HG controls the magnitude of TGF beta signaling via TGF beta 1 autoinduction and secretion in carcinomas.
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| 8. |
- Papoutsoglou, Panagiotis, et al.
(author)
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The TGFB2-AS1 lncRNA Regulates TGF-beta Signaling by Modulating Corepressor Activity
- 2019
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In: Cell Reports. - : CELL PRESS. - 2211-1247. ; 28:12, s. 3182-3198.E11
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Journal article (peer-reviewed)abstract
- Molecular processes involving lncRNAs regulate cell function. By applying transcriptomics, we identify lncRNAs whose expression is regulated by transforming growth factor beta (TGF-beta). Upon silencing individual lncRNAs, we identify several that regulate TGF-beta signaling. Among these lncRNAs, TGFB2-antisense RNA1 (TGFB2-AS1) is induced by TGF-beta through Smad and protein kinase pathways and resides in the nucleus. Depleting TGFB2-AS1 enhances TGF-beta/Smad-mediated transcription and expression of hallmark TGF-beta-target genes. Increased dose of TGFB2-AS1 reduces expression of these genes, attenuates TGF-beta-induced cell growth arrest, and alters BMP and Wnt pathway gene profiles. Mechanistically, TGFB2-AS1, mainly via its 3' terminal region, binds to the EED adaptor of the Polycomb repressor complex 2 (PRC2), promoting repressive histone H3K27me(3) modifications at TGF-beta-target gene promoters. Silencing EED or inhibiting PRC2 methylation activity partially rescues TGFB2-AS1-mediated gene repression. Thus, the TGF-beta-induced TGFB2-AS1 lncRNA exerts inhibitory functions on TGF-beta/BMP signaling output, supporting auto-regulatory negative feedback that balances TGF-beta/BMP-mediated responses.
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| 9. |
- Papoutsoglou, Panagiotis, et al.
(author)
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The TGFB2-AS1 lncRNA regulates TGFβ signaling by modulating corepressor activity
- 2019
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In: Cell Reports. - : Elsevier. - 2211-1247. ; 28:12, s. 3182-3198.e11
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Journal article (peer-reviewed)abstract
- 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.
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| 10. |
- Watanabe, Yukihide, et al.
(author)
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Regulation of Bone Morphogenetic Protein Signaling by ADP-ribosylation
- 2016
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In: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 291:24, s. 12706-12723
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Journal article (peer-reviewed)abstract
- 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.
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