| 1. |
- Inamitsu, Masako, et al.
(författare)
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Methylation of Smad6 by protein arginine N-methyltransferase 1
- 2006
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Ingår i: FEBS Letters. - : Wiley. - 0014-5793 .- 1873-3468. ; 580:28-29, s. 6603-6611
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Tidskriftsartikel (refereegranskat)abstract
- Signal transduction pathways utilize posttranslational modifications to regulate the activity of their components in a temporal-spatial and efficient fashion. Arginine methylation is one of the posttranslational modifications that can result in monomethylated-, asymmetric dimethylated- and/or symmetric dimethylated-arginine residues in proteins. Here we demonstrate that inhibitory-Smads (Smad6 and Smad7), but not receptor-regulated- (R-)Smads and the common-partner Smad4, can be methylated by protein arginine N-methyltransferase (PRMT)1. Using mass-spectrometric analysis, we found that PRMT1 dimethylates arginine(74) (Arg(74)) in mouse Smad6. PRMT1 interacts with the N-terminal domain of Smad6 in which Arg(74) residue is located. Assays examined so far have shown no significant differences between the functions of Smad6 and those of methylation-defective Smad6 (Smad6R74A). Both wild-type and Smad6R74A were equally efficient in blocking BMP-induced growth arrest upon their ectopic expression in HS-72 mouse B-cell hybridoma cells.
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| 2. |
- Itoh, Fumiko, et al.
(författare)
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Poor vessel formation in embryos from knock-in mice expressing ALK5 with L45 loop mutation defective in Smad activation
- 2009
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Ingår i: Laboratory Investigation. - : Elsevier BV. - 1530-0307 .- 0023-6837. ; 89:7, s. 800-810
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Tidskriftsartikel (refereegranskat)abstract
- Transforming growth factor (TGF)-beta regulates vascular development through two type I receptors: activin receptor-like kinase (ALK) 1 and ALK5, each of which activates a different downstream Smad pathway. The endothelial cell (EC)-specific ALK1 increases EC proliferation and migration, whereas the ubiquitously expressed ALK5 inhibits both of these processes. As ALK1 requires the kinase activity of ALK5 for optimal activation, the lack of ALK5 in ECs results in defective phosphorylation of both Smad pathways on TGF-beta stimulation. To understand why TGF-beta signaling through ALK1 and ALK5 has opposing effects on ECs and whether this takes place in vivo, we carefully compared the phenotype of ALK5 knock-in (ALK5(KI/KI)) mice, in which the aspartic acid residue 266 in the L45 loop of ALK5 was replaced by an alanine residue, with the phenotypes of ALK5 knock-out (ALK5(-/-)) and wild-type mice. The ALK5(KI/KI) mice showed angiogenic defects with embryonic lethality at E10.5-11.5. Although the phenotype of the ALK5(KI/KI) mice was quite similar to that of the ALK5(-/-) mice, the hierarchical structure of blood vessels formed in the ALK5(KI/KI) embryos was more developed than that in the ALK5(-/-) mutants. Thus, the L45 loop mutation in ALK5 partially rescued the earliest vascular defects in the ALK5(-/-) embryos. This study supports our earlier observation that vascular maturation in vivo requires both TGF-beta/ALK1/BMP-Smad and TGF-beta/ALK5/activin-Smad pathways for normal vascular development. Laboratory Investigation (2009) 89, 800-810; doi:10.1038/labinvest.2009.37; published online 27 April 2009
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| 3. |
- Itoh, Fumiko, et al.
(författare)
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Smad2/Smad3 in endothelium is indispensable for vascular stability via S1PR1 and N-cadherin expressions
- 2012
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Ingår i: Blood. - : American Society of Hematology. - 1528-0020 .- 0006-4971. ; 119:22, s. 5320-5328
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Tidskriftsartikel (refereegranskat)abstract
- Transforming growth factor-beta (TGF-beta) is involved in vascular formation through activin receptor-like kinase (ALK)1 and ALK5. ALK5, which is expressed ubiquitously, phosphorylates Smad2 and Smad3, whereas endothelial cell (EC)-specific ALK1 activates Smad1 and Smad5. Because ALK5 kinase activity is required for ALK1 to transduce TGF-beta signaling via Smad1/5 in ECs, ALK5 knockout (KO) mice were not able to give us the precise mechanisms by which TGF-beta/ALK5/Smad2/3 signaling is implicated in angiogenesis. To delineate the role of Smad2/3 signaling in endothelium, the Smad2 gene in Smad3 KO mice was selectively deleted in ECs using Tie2-Cre transgenic mice, termed EC-specific Smad2/3 double KO (EC-Smad2/3KO) mice. EC-Smad2/3KO embryos revealed hemorrhage leading to embryonic lethality around E12.5. EC-Smad2/3KO embryos exhibited no abnormality of vasculogenesis and angiogenesis in both the yolk sac and the whole embryo, whereas vascular maturation was incomplete because of inadequate assembly of mural cells in the vasculature. Wide gaps between ECs and mural cells could be observed in the vasculature of EC-Smad2/3KO mice because of reduced expression of N-cadherin and sphingosine-1-phosphate receptor-1 (S1PR1) in ECs from those mice. These results indicated that Smad2/3 signaling in ECs is indispensable for maintenance of vascular integrity via the fine-tuning of N-cadherin, VE-cadherin, and S1PR1 expressions in the vasculature. (Blood. 2012;119(22):5320-5328)
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| 4. |
- Okita, Yukari, et al.
(författare)
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The transcription factor MAFK induces EMT and malignant progression of triple-negative breast cancer cells through its target GPNMB
- 2017
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Ingår i: Science Signaling. - : AMER ASSOC ADVANCEMENT SCIENCE. - 1945-0877 .- 1937-9145. ; 10:474
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Tidskriftsartikel (refereegranskat)abstract
- Triple-negative breast cancer (TNBC) is particularly aggressive and difficult to treat. For example, the transforming growth factor-beta (TGF-beta) pathway is implicated in TNBC progression and metastasis, but its opposing role in tumor suppression in healthy tissues and early-stage lesions makes it a challenging target. Therefore, additional molecular characterization of TNBC may lead to improved patient prognosis by informing the development and optimum use of targeted therapies. We found that musculoaponeurotic fibrosarcoma (MAF) oncogene family protein K (MAFK), a member of the small MAF family of transcription factors that are induced by the TGF-beta pathway, was abundant in human TNBC and aggressive mouse mammary tumor cell lines. MAFK promoted tumorigenic growth and metastasis by 4T1 cells when implanted subcutaneously in mice. Overexpression of MAFK in mouse breast epithelial NMuMG cells induced epithelial-mesenchymal transition (EMT) phenotypes and promoted tumor formation and invasion in mice. MAFK induced the expression of the gene encoding the transmembrane glycoprotein nmb(GPNMB). Similar to MAFK, GPNMB overexpression in NMuMG cells induced EMT, tumor formation, and invasion, in mice, whereas knockdown of MAFK in tumor cells before implantation suppressed tumor growth and progression. MAFK and GPNMB expression correlated with poor prognosis in TNBC patients. These findings suggest that MAFK and its target gene GPNMB play important roles in the malignant progression of TNBC cells, offering potentially new therapeutic targets for TNBC patients.
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| 5. |
- Sonkoly, Enikö, et al.
(författare)
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Protein kinase C-dependent upregulation of miR-203 induces the differentiation of human keratinocytes
- 2010
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Ingår i: Journal of Investigative Dermatology. - : Elsevier BV. - 0022-202X .- 1523-1747. ; 130:1, s. 124-134
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Tidskriftsartikel (refereegranskat)abstract
- Terminal differentiation of keratinocytes is a multistep process that requires a coordinated program of gene expression. We aimed to explore the possible involvement of a previously unreported class of non-coding RNA genes, microRNAs (miRNAs) in keratinocyte differentiation by using miRNA expression profiling. Out of 365 miRNAs tested, 7 showed significant change between keratinocytes cultured in low or high calcium concentration. The highest-ranked upregulated gene was miR-203, whose expression was significantly upregulated in response to calcium and other inducers of keratinocyte differentiation such as 12-O-tetradecanoylphorbol-13-acetate (TPA) and vitamin D(3). Differentiation-induced upregulation of miR-203 expression was blocked by treatment with specific inhibitors of protein kinase C (PKC), GF109203X, and Ro31-8220. Moreover, our results showed that the activator protein-1 (AP-1) proteins c-Jun and JunB regulate miR-203 expression in keratinocytes. In contrast to inducers of keratinocyte differentiation, epidermal growth factor and keratinocyte growth factor suppressed miR-203 expression in keratinocytes below the basal level. Overexpression of miR-203 in keratinocytes resulted in enhanced differentiation, whereas inhibition of miR-203 suppressed calcium-induced terminal differentiation as judged by involucrin expression. These results suggest that upregulation of miR-203 in human keratinocytes is required for their differentiation and is dependent on the activation of the PKC/AP-1 pathway.
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| 6. |
- Tsubakihara, Yutaro, et al.
(författare)
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TGF beta selects for pro-stemness over pro-invasive phenotypes during cancer cell epithelial-mesenchymal transition
- 2022
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Ingår i: Molecular Oncology. - : John Wiley & Sons. - 1574-7891 .- 1878-0261. ; 16:12, s. 2330-2354
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Tidskriftsartikel (refereegranskat)abstract
- Transforming growth factor beta (TGF(beta) induces epithelial-mesenchymal transition (EMT), which correlates with sternness and invasiveness. Mesenchymal-epithelial transition (MET) is induced by TGF beta withdrawal and correlates with metastatic colonization. Whether TGF beta promotes sternness and invasiveness simultaneously via EMT remains unclear. We established a breast cancer cell model expressing red fluorescent protein (RFP) under the E-cadherin promoter. In 2D cultures, TGF beta induced EMT, generating RFPlow cells with a mesenchymal transcriptome, and regained RFP, with an epithelial transcriptome, after MET induced by TGF beta withdrawal. RFPlow cells generated robust mammospheres, with epithelio-mesenchymal cell surface features. Mammospheres that were forced to adhere generated migratory cells, devoid of RFP, a phenotype which was inhibited by a TGF beta receptor kinase inhibitor. Further stimulation of RFPlow mammospheres with TGF beta suppressed the generation of motile cells, but enhanced mammosphere growth. Accordingly, mammary fat-pad-transplanted mammospheres, in the absence of exogenous TGF beta treatment, established lung metastases with evident MET (RFPhigh cells). In contrast, TGF beta-treated mammospheres revealed high tumour-initiating capacity, but limited metastatic potential. Thus, the biological context of partial EMT and MET allows TGF beta to differentiate between pro-sternness and pro-invasive phenotypes.
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| 7. |
- Tzavlaki, Kalliopi, et al.
(författare)
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The liver kinase B1 inhibits transforming growth factor β signaling during mammary epithelial morphogenesis
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- The liver kinase B1 (LKB1) controls cellular metabolism, including autophagy, and epithelialcell polarity across species. We previously established a mechanism of negative regulation oftransforming growth factor β (TGFβ) signaling by LKB1. In an effort to achieve a deeperunderstanding of LKB1 as a regulator of TGFβ signaling during the establishment of epithelial polarity, we employed the immortalized mammary epithelial cell model MCF10A, which hasbeen widely used for such studies based on its ability to form 3D acinar organoids. CRISPR/Cas9-mediated loss-of-function mutations to the endogenous STK11 (LKB1) gene inMCF10A cells, led to profound loss of the ability to form 3D organoids, resulting in amorphousoutgrowth and loss of rotation of young spheroids embedded in matrigel. This defect wasassociated with a relative enhancement of signaling by TGFβ, resulting in enhanced generesponses, including TGFβ auto-induction and induction of transcription factors that mediateepithelial-mesenchymal transition (EMT). Protein marker analysis confirmed a more efficient EMT response to TGFβ signaling in LKB1 knockout cells. Accordingly, a chemical inhibitor ofthe TGFβ type I receptor kinase restored to a great extent the polarity defect of the LKB1 knockout cells. In contrast, a chemical inhibitor of the tank-binding kinase 1, another EMTregulator could only partially restore the polarity defect of the LKB1 knockout cells. Weconclude that the contribution of LKB1 to mammary epithelial polarity is dependent on theregulation of autogenous TGFβ signaling, whose hyperactivity is physiologically limited by LKB1 during mammary epithelial morphogenesis.
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| 8. |
- Tzavlaki, Kalliopi, et al.
(författare)
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The liver kinase B1 supports mammary epithelial morphogenesis by inhibiting critical factors that mediate epithelial-mesenchymal transition
- 2023
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Ingår i: Journal of Cellular Physiology. - : John Wiley & Sons. - 0021-9541 .- 1097-4652. ; 238:4, s. 790-812
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Tidskriftsartikel (refereegranskat)abstract
- The liver kinase B1 (LKB1) controls cellular metabolism and cell polarity across species. We previously established a mechanism for negative regulation of transforming growth factor β (TGFβ) signaling by LKB1. The impact of this mechanism in the context of epithelial polarity and morphogenesis remains unknown. After demonstrating that human mammary tissue expresses robust LKB1 protein levels, whereas invasive breast cancer exhibits significantly reduced LKB1 levels, we focused on mammary morphogenesis studies in three dimensional (3D) acinar organoids. CRISPR/Cas9-introduced loss-of-function mutations of STK11 (LKB1) led to profound defects in the formation of 3D organoids, resulting in amorphous outgrowth and loss of rotation of young organoids embedded in matrigel. This defect was associated with an enhanced signaling by TGFβ, including TGFβ auto-induction and induction of transcription factors that mediate epithelial-mesenchymal transition (EMT). Protein marker analysis confirmed a more efficient EMT response to TGFβ signaling in LKB1 knockout cells. Accordingly, chemical inhibition of the TGFβ type I receptor kinase largely restored the morphogenetic defect of LKB1 knockout cells. Similarly, chemical inhibition of the bone morphogenetic protein pathway or the TANK-binding kinase 1, or genetic silencing of the EMT factor SNAI1, partially restored the LKB1 knockout defect. Thus, LKB1 sustains mammary epithelial morphogenesis by limiting pathways that promote EMT. The observed downregulation of LKB1 expression in breast cancer is therefore predicted to associate with enhanced EMT induced by SNAI1 and TGFβ family members.
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| 9. |
- Valcourt, Ulrich, et al.
(författare)
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Analysis of Epithelial-Mesenchymal Transition Induced by Transforming Growth Factor β
- 2016
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Ingår i: TGF-Beta Signaling. - New York, NY : Springer. ; , s. 147-181
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Bokkapitel (refereegranskat)abstract
- In recent years, the importance of the cell biological process of epithelial-mesenchymal transition (EMT) has been established via an exponentially growing number of reports. EMT has been documented during embryonic development, tissue fibrosis, and cancer progression in vitro, in animal models in vivo and in human specimens. EMT relates to many molecular and cellular alterations that occur when epithelial cells undergo a switch in differentiation that generates mesenchymal-like cells with newly acquired migratory and invasive properties. In addition, EMT relates to a nuclear reprogramming similar to the one occurring in the generation of induced pluripotent stem cells. Via such a process, EMT is gradually established to promote the generation and maintenance of adult tissue stem cells which under disease states such as cancer, are known as cancer stem cells. EMT is induced by developmental growth factors, oncogenes, radiation, and hypoxia. A prominent growth factor that causes EMT is transforming growth factor β (TGF-β).A series of molecular and cellular techniques can be applied to define and characterize the state of EMT in diverse biological samples. These methods range from DNA and RNA-based techniques that measure the expression of key EMT regulators and markers of epithelial or mesenchymal differentiation to functional assays of cell mobility, invasiveness and in vitro stemness. This chapter focuses on EMT induced by TGF-β and provides authoritative protocols and relevant reagents and citations of key publications aiming at assisting newcomers that enter this prolific area of biomedical sciences, and offering a useful reference tool to pioneers and aficionados of the field.
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