| 1. |
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| 2. |
- Kowanetz, Katarzyna, et al.
(författare)
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CIN85 associates with multiple effectors controlling intracellular trafficking of epidermal growth factor receptors.
- 2004
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Ingår i: Mol Biol Cell. - 1059-1524. ; 15:7, s. 3155-66
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Tidskriftsartikel (refereegranskat)abstract
- CIN85 is a multidomain adaptor protein involved in Cbl-mediated down-regulation of epidermal growth factor (EGF) receptors. CIN85 src homology 3 domains specifically bind to a proline-arginine (PxxxPR) motif in Cbl, and this association seems to be important for EGF receptor endocytosis. Here, we report identification of novel CIN85 effectors, all containing one or more PxxxPR motifs, that are indispensable for their mutual interactions. These effectors include phosphatidyl-inositol phosphatases SHIP-1 and synaptojanin 2B1, Arf GTPase-activating proteins ASAP1 and ARAP3, adaptor proteins Hip1R and STAP1, and a Rho exchange factor, p115Rho GEF. Acting as a molecular scaffold, CIN85 clusters its effectors and recruits them to high-molecular-weight complexes in cytosolic extracts of cells. Further characterization of CIN85 binding to ASAP1 revealed that formation of the complex is independent on cell stimulation. Overexpression of ASAP1 increased EGF receptor recycling, whereas ASAP1 containing mutated PxxxPR motif failed to promote this event. We propose that CIN85 functions as a scaffold molecule that binds to numerous endocytic accessory proteins, thus controlling distinct steps in trafficking of EGF receptors along the endocytic and recycling pathways.
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| 3. |
- Kowanetz, Katarzyna, et al.
(författare)
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Identification of a novel proline-arginine motif involved in CIN85-dependent clustering of Cbl and down-regulation of epidermal growth factor receptors
- 2003
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 278:41, s. 39735-39746
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Tidskriftsartikel (refereegranskat)abstract
- CIN85 is a multidomain adaptor protein implicated in Cbl-mediated down-regulation of receptor tyrosine kinases. CIN85 binding to Cbl is increased after growth factor stimulation and is critical for targeting receptor tyrosine kinases to clathrin-mediated endocytosis. Here we report the identification of a novel polyproline-arginine motif (PXXXPR), specifically recognized by the SH3 domains of CIN85 and its homologue CMS/CD2AP. This motif was indispensable for CIN85 binding to Cbl/Cbl-b, to other CIN85 SH3 domains' effectors, and for mediating an intramolecular interaction between the SH3-A domain and the proline-rich region of CIN85. Individual SH3 domains of CIN85 bound to PXXXPR peptides of Cbl/Cbl-b with micromolar affinities, whereas an extended structure of two or three SH3 domains bound with higher stoichiometry and increased affinity to the same peptides. This enabled full size CIN85 to simultaneously interact with multiple Cbl molecules, promoting their clustering in mammalian cells. The ability of CIN85 to cluster Cbl was important for ligand-induced stabilization of CIN85.Cbl.epidermal growth factor receptor complexes, as well as for epidermal growth factor receptor degradation in the lysosome. Thus, specific interactions of CIN85 SH3 domains with the PXXXPR motif in Cbl play multiple roles in down-regulation of receptor tyrosine kinases.
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| 4. |
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| 5. |
- Kowanetz, Marcin, et al.
(författare)
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TGFβ induces SIK to negatively regulate type I receptor kinase signaling
- 2008
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Ingår i: Journal of Cell Biology. - : The Rockefeller University Press. - 0021-9525 .- 1540-8140. ; 182:4, s. 655-662
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Tidskriftsartikel (refereegranskat)abstract
- Signal transduction by transforming growth factor beta (TGFbeta) coordinates physiological responses in diverse cell types. TGFbeta signals via type I and type II receptor serine/threonine kinases and intracellular Smad proteins that regulate transcription. Strength and duration of TGFbeta signaling is largely dependent on a negative-feedback program initiated during signal progression. We have identified an inducible gene target of TGFbeta/Smad signaling, the salt-inducible kinase (SIK), which negatively regulates signaling together with Smad7. SIK and Smad7 form a complex and cooperate to down-regulate the activated type I receptor ALK5. We further show that both the kinase and ubiquitin-associated domain of SIK are required for proper ALK5 degradation, with ubiquitin functioning to enhance SIK-mediated receptor degradation. Loss of endogenous SIK results in enhanced gene responses of the fibrotic and cytostatic programs of TGFbeta. We thus identify in SIK a negative regulator that controls TGFbeta receptor turnover and physiological signaling.
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| 6. |
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| 7. |
- Lönn, Peter, et al.
(författare)
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Transcriptional induction of salt-inducible kinase 1 by transforming growth factor β leads to negative regulation of type I receptor signaling in cooperation with the Smurf2 ubiquitin ligase
- 2012
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 287:16, s. 12867-12878
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Tidskriftsartikel (refereegranskat)abstract
- Transforming growth factor β (TGFβ)1 regulates many physiological processes and requires control mechanisms to safeguard proper and timely action. We have previously described how negative regulation of TGFβ signaling is controlled by the serine/threonine kinase salt-inducible kinase (SIK) 1. SIK1 forms complexes with the TGFβ type I receptor and with the inhibitory Smad7 and downregulates the type I receptor. We now demonstrate that TGFβ induces SIK1 levels via a direct transcriptional mechanism that implicates the Smad proteins and we have mapped a putative enhancer element on the SIK1 gene. We provide evidence that the ubiquitin ligase Smurf2 forms complexes and functionally cooperates with SIK1. Both the kinase activity of SIK1 and the ubiquitin ligase activity of Smurf2 are important for proper type I receptor turnover. We also show that knockdown of endogenous SIK1 and Smurf2 enhances physiological signaling by TGFβ that leads to epithelial growth arrest. In conclusion, TGFβ induces expression of Smad7, Smurf2 and SIK1, the products of which physically and functionally interlink to control the activity of this pathway.
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| 8. |
- Hägerstrand, Daniel, et al.
(författare)
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Characterization of an imatinib-sensitive subset of high-grade human glioma cultures
- 2006
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Ingår i: Oncogene. - : Springer Science and Business Media LLC. - 0950-9232 .- 1476-5594. ; 25:35, s. 4913-4922
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Tidskriftsartikel (refereegranskat)abstract
- High-grade gliomas, including glioblastomas, are malignant brain tumors for which improved treatment is urgently needed. Genetic studies have demonstrated the existence of biologically distinct subsets. Preliminary studies have indicated that platelet-derived growth factor (PDGF) receptor signaling contributes to the growth of some of these tumors. In this study, human high-grade glioma primary cultures were analysed for sensitivity to treatment with the PDGF receptor inhibitor imatinib/Glivec/Gleevec/STI571. Six out of 15 cultures displayed more than 40% growth inhibition after imatinib treatment, whereas seven cultures showed less than 20% growth inhibition. In the sensitive cultures, apoptosis contributed to growth inhibition. Platelet-derived growth factor receptor status correlated with imatinib sensitivity. Supervised analyses of gene expression profiles and real-time PCR analyses identified expression of the chemokine CXCL12/SDF-1 (stromal cell-derived factor 1) as a predictor of imatinib sensitivity. Exogenous addition of CXCL12 to imatinib-insensitive cultures conferred some imatinib sensitivity. Finally, coregulation of CXCL12 and PDGF alpha-receptor was observed in glioblastoma biopsies. We have thus defined the characteristics of a novel imatinib-sensitive subset of glioma cultures, and provided evidence for a functional relationship between imatinib sensitivity and chemokine signaling. These findings will assist in the design and evaluation of clinical trials exploring therapeutic effects of imatinib on malignant brain tumors.
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| 9. |
- Itoh, Susumu, et al.
(författare)
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Elucidation of Smad requirement in transforming growth factor-beta type I receptor-induced responses
- 2003
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 278:6, s. 3751-3761
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Tidskriftsartikel (refereegranskat)abstract
- Transforming growth factor-beta (TGF-beta) elicits cellular effects by activating specific Smad proteins that control the transcription of target genes. Whereas there is growing evidence that there are TGF-beta type I receptor-initiated intracellular pathways that are distinct from the pivotal Smad pathway, their physiological importance in TGF-beta signaling is not well understood. Therefore, we generated TGF-beta type I receptors (also termed ALK5s) with mutations in the L45 loop of the kinase domain, termed ALK5(D266A) and ALK5(3A). These mutants showed retained kinase activity but were unable to activate Smads. Characterization of their signaling properties revealed that the two L45 loop mutants did not mediate Smad-dependent transcriptional responses, TGF-beta-induced growth inhibition, and fibronectin and plasminogen activator-1 production in R4-2 mink lung epithelial cells lacking functional ALK5 protein. Mutation in the L45 loop region did not affect the binding of inhibitory Smads but did abrogate the weak binding of X-linked inhibitor of apoptosis protein and Disabled-2 to ALK5. This suggests that the L45 loop in the kinase domain is important for docking of other binding proteins. Interestingly, JNK MAP kinase activity was found to be activated by the ALK5(3A) mutant in various cell types. In addition, TGF-beta-induced inhibition of cyclin D1 expression and stimulation of PMEPA1 (androgen-regulated prostatic mRNA) expression were found to occur, albeit weakly, in an Smad-independent manner in normal murine mammary gland cells. However, the TGF-beta-induced epithelial to mesenchymal transdifferentiation was found to require an intact L45 loop and is likely to be dependent on the Smad pathways.
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| 10. |
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| 11. |
- Kowanetz, Marcin, et al.
(författare)
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Id2 and Id3 define the potency of cell proliferation and differentiation responses to transforming growth factor β and bone morphogenetic protein
- 2004
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Ingår i: Molecular and Cellular Biology. - : American Society for Microbiology. - 0270-7306 .- 1098-5549. ; 24:10, s. 4241-4254
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Tidskriftsartikel (refereegranskat)abstract
- Transforming growth factors beta (TGF-betas) inhibit growth of epithelial cells and induce differentiation changes, such as epithelial-mesenchymal transition (EMT). On the other hand, bone morphogenetic proteins (BMPs) weakly affect epithelial cell growth and do not induce EMT. Smad4 transmits signals from both TGF-beta and BMP pathways. Stimulation of Smad4-deficient epithelial cells with TGF-beta 1 or BMP-7 in the absence or presence of exogenous Smad4, followed by cDNA microarray analysis, revealed 173 mostly Smad4-dependent, TGF-beta-, or BMP-responsive genes. Among 25 genes coregulated by both factors, inhibitors of differentiation Id2 and Id3 showed long-term repression by TGF-beta and sustained induction by BMP. The opposing regulation of Id genes is critical for proliferative and differentiation responses. Hence, ectopic Id2 or Id3 expression renders epithelial cells refractory to growth inhibition and EMT induced by TGF-beta, phenocopying the BMP response. Knockdown of endogenous Id2 or Id3 sensitizes epithelial cells to BMP, leading to robust growth inhibition and induction of transdifferentiation. Thus, Id genes sense Smad signals and create a permissive or refractory nuclear environment that defines decisions of cell fate and proliferation.
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| 12. |
- Kowanetz, Marcin, 1977-
(författare)
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Novel Regulators of the TGF-β Signaling Pathway
- 2005
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The transforming growth factor-β (TGF-β) superfamily consists of related multifunctional cytokines, which include TGF-βs, activins, and bone morphogenetic proteins (BMPs) and coordinate several biological responses in diverse cell types. The biological activity of TGF-β members is executed by transmembrane serine/threonine kinase receptors and intracellular Smad proteins. The effects of TGF-β on the epithelium are of high interest. Carcinomas (tumors of epithelial origin) are the most common type of human cancer and frequently exhibit aberrant responses to TGF-β. Therefore, TGF-β can be defined as tumor suppressor as it inhibits growth of normal epithelial cells. However, TGF-β also induces an epithelial-mesenchymal transition (EMT), a key component of metastasis, and thus promotes cancer spread.The scope of this thesis is the mechanism of TGF-β signaling in epithelial cells. We established that only TGF-β, but not BMP pathways can elicit EMT. Moreover, we found that Smad signaling is critical for regulation of EMT. In a transcriptomic analysis, we identified a large group of novel genes, whose regulation is pivotal for TGF-β-induced EMT and metastasis. We focused on two of such genes, Id2 and Id3. Interestingly, we found that TGF-β-induced repression of Ids is necessary for inducing EMT and potent cell cycle arrest. BMP increases expression of Ids and therefore it cannot induce the same biological responses as TGF-β. Hence, knock-down of endogenous Id2 and Id3 proteins sensitized epithelial cell to BMP-7. We proposed a model, in which Id2 and Id3 are important components controlling concerted regulation of cell proliferation and EMT downstream of TGF-β pathways.Furthermore, we identified a serine/threonine kinase, SNF1LK, whose mRNA is rapidly induced by TGF-β in epithelial cells. We found that SNF1LK is a negative regulator of the TGF-β pathway and it promotes TGF-β receptor turnover. Subsequently, we demonstrated that SNF1LK together with Smad7 and Smurf2 targets TGF-β receptor for ubiquitin-dependent degradation. Furthermore, SNF1LK interacts with proteasomes, suggesting that SNF1LK serves as bridge between ubiquitinated receptors and proteasomes, helping proteasomes to recognize the ubiquitinated cargo destined for degradation. We therefore established a novel negative feedback regulatory mechanism of TGF-β signaling.
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| 13. |
- Kurisaki, Akira, et al.
(författare)
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The mechanism of nuclear export of smad3 involves exportin 4 and Ran
- 2006
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Ingår i: Molecular and Cellular Biology. - 0270-7306 .- 1098-5549. ; 26:4, s. 1318-1332
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Tidskriftsartikel (refereegranskat)abstract
- Transforming growth factor beta (TGF-beta) receptors phosphorylate Smad3 and induce its nuclear import so it can regulate gene transcription. Smad3 can return to the cytoplasm to propagate further cycles of signal transduction or to be degraded. We demonstrate that Smad3 is exported by a constitutive mechanism that is insensitive to leptomycin B. The Mad homology 2 (MH2) domain is responsible for Smad3 export, which requires the GTPase Ran. Inactive, GDP-locked RanT24N or nuclear microinjection of Ran GTPase activating protein 1 blocked Smad3 export. Inactivation of the Ran guanine nucleotide exchange factor RCC1 inhibited Smad3 export and led to nuclear accumulation of phosphorylated Smad3. A screen for importin/exportin family members that associate with Smad3 identified exportin 4, which binds a conserved peptide sequence in the MH2 domain of Smad3 in a Ran-dependent manner. Exportin 4 is sufficient for carrying the in vitro nuclear export of Smad3 in cooperation with Ran. Knockdown of endogenous exportin 4 completely abrogates the export of endogenous Smad3. A short peptide representing the minimal interaction domain in Smad3 effectively competes with Smad3 association to exportin 4 and blocks nuclear export of Smad3 in vivo. We thus delineate a novel nuclear export pathway for Smad3.
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| 14. |
- Moustakas, Aristidis, et al.
(författare)
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TGFß/Smad signaling in epithelial to mesenchymal transition
- 2006
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Ingår i: Smad Signal Transduction. - Dordrecht : Springer. - 9781402047091 ; , s. 131-150
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Bokkapitel (refereegranskat)abstract
- Epithelial-mesenchymal transition (EMT) allows polarized epithelial layers to locally dissolve and create fibroblast-like or myofibroblastic cell derivatives. EMT resembles other processes of transdifferentiation from one differentiated cell type to another and is critical for proper embryonic development. Furthermore, EMT describes similar processes between endothelial and myoepithelial cells occurring during blood vessel remodeling. All such processes are triggered and controlled by the concerted action of multiple extracellular growth/morphogenetic factors, TGF-β being one of them. The mechanism by which TGF-β elicits EMT has been a major topic of current research. This is due to the relevance EMT has to the processes of tumor cell invasiveness and metastasis on the one hand, and to fibrotic conditions on the other. Here we emphasize on mechanisms of signal transduction and specific gene targets of the TGF-β pathway that are critical effectors of EMT.
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| 15. |
- Pardali, Katerina, et al.
(författare)
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Smad pathway-specific transcriptional regulation of the cell cycle inhibitor p21Waf1/Cip1
- 2005
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Ingår i: Journal of Cellular Physiology. - : WILEY-LISS, INC.. - 0021-9541 .- 1097-4652. ; 204:1, s. 260-272
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Tidskriftsartikel (refereegranskat)abstract
- Transforming growth factor-beta (TGF-beta) inhibits epithelial cell growth, in part via transcriptional induction of the cell cycle inhibitor p21(WAF1/Cip1) (p21). We show that bone morphogenetic protein (BMP)-7 induces higher p21 expression than TGF-beta1 in various epithelial cells. Despite this, BMP-7 only weakly suppresses epithelial cell proliferation, as Id2, a cell cycle-promoting factor, becomes concomitantly induced by BMP-7. Signaling studies with all type I receptors of the TGF-beta superfamily show that BMP receptors induce higher p21 expression than TGF-beta/activin receptors. Smad4 is essential for p21 regulation by all receptor pathways. Based on the previously known ability of c-Myc to block p21 expression and epithelial growth arrest in response to TGF-beta1, we demonstrate that ectopic c-Myc expression can abrogate Smad-mediated p21 induction by all TGF-beta and BMP receptors. Furthermore, p21 induction by all receptor pathways can be blocked by the natural inhibitors of the TGF-beta superfamily. Smad7 inhibits all pathways whereas Smad6 selectively inhibits the BMP pathways. The observed pathway specificity reflects the efficiency by which BMP Smads, compared to TGF-beta Smads, transactivate the p21 promoter. In addition, BMP-specific Smads, Smad1, Smad5, and especially Smad8, induce endogenous p21 mRNA and protein levels, while they fail to induce epithelial growth inhibition when compared to TGF-beta receptor-phosphorylated Smads (R-Smads), Smad2 and Smad3. Thus, p21 is a common target of all TGF-beta superfamily pathways. However, the ability of TGF-beta superfamily members to induce cell growth arrest depends on the regulation of additional gene targets.
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| 16. |
- Rodriguez, Alejandro, et al.
(författare)
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Phenotypical differences in connective tissue cells emerging from microvascular pericytes in response to over-expression of PDGF-B and TGF-beta in normal skin in vivo
- 2013
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Ingår i: American Journal of Pathology. - : Elsevier BV. - 0002-9440 .- 1525-2191. ; 182:6, s. 2132-2146
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Tidskriftsartikel (refereegranskat)abstract
- Fibrosis is a deleterious consequence of chronic inflammation in a number of human pathological states ultimately leading to, if not perturbed to organ dysfunction and failure. Two key processes in fibrosis are activation of blood vessels and connective tissue cells leading to excess deposition of ECM components; collagen type I being the most abundant. In the present study the effects of two growth factors known to be important in the fibrotic process, namely PDGF-B and TGF-β1, were studied. Adenoviral vectors engineered to express PDGF-B or TGF-β1 were introduced into mouse skin thereby inducing a transient over-expression of either growth factor. After 3, 7 and 14 days post injection, tissues were harvested and morphology and protein expression were examined on plastic embedded 1 µm thick sections and by immunohistochemistry on frozen sections, respectively. Over-expression of both TGF-β1 and PDGF-B lead to a fibrotic response consisting of a marked macrophage influx as well as an expansion of the connective tissue cell population. In both conditions the latter originated from microvascular pericytes. The resulting phenotype of the emerging connective tissue cell population differed between PDGF-B and TGF-β1 treated skin. In tissues over expressing PDGF-B but not TGF-β1 the fibrotic process was partially reversible. Both growth factors were able to initiate, but neither were able to sustain the process of angiogenesis, which lead to vascular regression. PDGF-B but not TGF-β1 was able to stimulate but not maintain a distinct form of angiogenesis i.e. glomeruloid body formation. In conclusion, over-expression of PDGF-B and TGF-β1 in normal skin resulted in the emergence of connective tissue cells differing in their phenotype, originating in both cases from a common cell namely the microvascular pericyte. Therefore the vasculature and the fibrotic process are linked in a previously unrecognized way.
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| 17. |
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| 18. |
- Valcourt, Ulrich, et al.
(författare)
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TGF-β and the Smad signaling pathway support transcriptomic reprogramming during epithelial-mesenchymal cell transition
- 2005
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Ingår i: Molecular Biology of the Cell. - : The American Society for Cell Biology. - 1059-1524 .- 1939-4586. ; 16:4, s. 1987-2002
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Tidskriftsartikel (refereegranskat)abstract
- Epithelial-mesenchymal transition (EMT) contributes to normal tissue patterning and carcinoma invasiveness. We show that transforming growth factor (TGF)-beta/activin members, but not bone morphogenetic protein (BMP) members, can induce EMT in normal human and mouse epithelial cells. EMT correlates with the ability of these ligands to induce growth arrest. Ectopic expression of all type I receptors of the TGF-beta superfamily establishes that TGF-beta but not BMP pathways can elicit EMT. Ectopic Smad2 or Smad3 together with Smad4 enhanced, whereas dominant-negative forms of Smad2, Smad3, or Smad4, and wild-type inhibitory Smad7, blocked TGF-beta-induced EMT. Transcriptomic analysis of EMT kinetics identified novel TGF-beta target genes with ligand-specific responses. Using a TGF-beta type I receptor that cannot activate Smads nor induce EMT, we found that Smad signaling is critical for regulation of all tested gene targets during EMT. One such gene, Id2, whose expression is repressed by TGF-beta1 but induced by BMP-7 is critical for regulation of at least one important myoepithelial marker, alpha-smooth muscle actin, during EMT. Thus, based on ligand-specific responsiveness and evolutionary conservation of the gene expression patterns, we begin deciphering a genetic network downstream of TGF-beta and predict functional links to the control of cell proliferation and EMT.
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