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Hildegonda P H Naber, Eliza Wiercinska,
Evangelia Pardali,
Theo van Laar,
Ella Nirmala,
Anders Sundqvist,
Hans van Dam,
Geertje van der Horst,
Gabri van der Pluijm,
Bertrand Heckmann,
Erik H J Danen,
Peter Ten Dijke
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ABSTRACT: The transforming growth factor (TGF)-β superfamily comprises cytokines such as TGF-β and Bone Morphogenetic Proteins (BMPs), which have a critical role in a multitude of biological processes. In breast cancer, high levels of TGF-β are associated with poor outcome, whereas inhibition of TGF-β-signaling reduces metastasis. In contrast, BMP-7 inhibits bone metastasis of breast cancer cells.
In this study, we investigated the effect of BMP-7 on TGF-β-induced invasion in a 3 dimensional invasion assay.
BMP-7 inhibited TGF-β-induced invasion of the metastatic breast cancer cell line MCF10CA1a, but not of its premalignant precursor MCF10AT in a spheroid invasion model. The inhibitory effect appears to be specific for BMP-7, as its closest homolog, BMP-6, did not alter the invasion of MCF10CA1a spheroids. To elucidate the mechanism by which BMP-7 inhibits TGF-β-induced invasion, we analyzed invasion-related genes. BMP-7 inhibited TGF-β-induced expression of integrin α(v)β(3) in the spheroids. Moreover, targeting of integrins by a chemical inhibitor or knockdown of integrin β(3) negatively affected TGF-β-induced invasion. On the other hand, overexpression of integrin β(3) counteracted the inhibitory effect of BMP7 on TGF-β-induced invasion.
Thus, BMP-7 may exert anti-invasive actions by inhibiting TGF-β-induced expression of integrin β(3).
Cellular oncology (Dordrecht). 09/2011; 35(1):19-28.
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ABSTRACT: Transforming growth factor-β (TGF-β) has opposing roles in breast cancer progression by acting as a tumor suppressor in the initial phase, but stimulating invasion and metastasis at later stages. In contrast to the mechanisms by which TGF-β induces growth arrest, the pathways that mediate tumor invasion are not well understood. Here, we describe a TGF-β-dependent invasion assay system consisting of spheroids of MCF10A1 normal breast epithelial cells (M1) and RAS-transformed (pre-)malignant derivatives (M2 and M4) embedded in collagen gels. Both basal and TGF-β-induced invasion of these cell lines was found to correlate with their tumorigenic potential; M4 showing the most aggressive behavior and M1 showing the least. Basal invasion was strongly inhibited by the TGF-β receptor kinase inhibitor SB-431542, indicating the involvement of autocrine TGF-β or TGF-β-like activity. TGF-β-induced invasion in premalignant M2 and highly malignant M4 cells was also inhibited upon specific knockdown of Smad3 or Smad4. Interestingly, both a broad spectrum matrix metalloproteinase (MMP) inhibitor and a selective MMP2 and MMP9 inhibitor mitigated TGF-β-induced invasion of M4 cells, while leaving basal invasion intact. In line with this, TGF-β was found to strongly induce MMP2 and MMP9 expression in a Smad3- and Smad4-dependent manner. This collagen-embedded spheroid system therefore offers a valuable screening model for TGF-β/Smad- and MMP2- and MMP9-dependent breast cancer invasion.
Breast Cancer Research and Treatment 08/2011; 128(3):657-66. · 4.43 Impact Factor
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ABSTRACT: TGF-β has opposing roles in breast cancer progression by acting as a tumor suppressor in the initial phase, but stimulating invasion and metastasis at later stage(1,2). Moreover, TGF-β is frequently overexpressed in breast cancer and its expression correlates with poor prognosis and metastasis (3,4). The mechanisms by which TGF-β induces invasion are not well understood. TGF-β elicits its cellular responses via TGF-β type II (TβRII) and type I (TβRI) receptors. Upon TGF-β-induced heteromeric complex formation, TβRII phosphorylates the TβRI. The activated TβRI initiates its intracellular canonical signaling pathway by phosphorylating receptor Smads (R-Smads), i.e. Smad2 and Smad3. These activated R-Smads form heteromeric complexes with Smad4, which accumulate in the nucleus and regulate the transcription of target genes(5). In addition to the previously described Smad pathway, receptor activation results in activation of several other non-Smad signaling pathways, for example Mitogen Activated Protein Kinase (MAPK) pathways(6). To study the role of TGF-β in different stages of breast cancer, we made use of the MCF10A cell system. This system consists of spontaneously immortalized MCF10A1 (M1) breast epithelial cells(7), the H-RAS transformed M1-derivative MCF10AneoT (M2), which produces premalignant lesions in mice(8), and the M2-derivative MCF10CA1a (M4), which was established from M2 xenografts and forms high grade carcinomas with the ability to metastasize to the lung(9). This MCF10A series offers the possibility to study the responses of cells with different grades of malignancy that are not biased by a different genetic background. For the analysis of TGF-β-induced invasion, we generated homotypic MCF10A spheroid cell cultures embedded in a 3D collagen matrix in vitro (Fig 1). Such models closely resemble human tumors in vivo by establishing a gradient of oxygen and nutrients, resulting in active and invasive cells on the outside and quiescent or even necrotic cells in the inside of the spheroid(10). Spheroid based assays have also been shown to better recapitulate drug resistance than monolayer cultures(11). This MCF10 3D model system allowed us to investigate the impact of TGF-β signaling on the invasive properties of breast cells in different stages of malignancy.
Journal of Visualized Experiments 01/2011;
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ABSTRACT: Endoglin is a transforming growth factor-beta coreceptor with a crucial role in angiogenesis. A soluble form of endoglin is present in the circulation, but the role of soluble endoglin (sEndoglin) is poorly understood. In addition, the endoglin shedding mechanism is not known. Therefore, we examined the role of sEndoglin in tumor angiogenesis and the mechanism by which the extracellular domain of endoglin is released from the membrane.In colorectal cancer specimens, we observed high endothelial endoglin protein expression, accompanied with slightly lower sEndoglin levels in the circulation, compared with healthy controls. In vitro analysis using endothelial sprouting assays revealed that sEndoglin reduced spontaneous and vascular endothelial growth factor-induced endothelial sprouting. Human umbilical vascular endothelial cells were found to secrete high levels of sEndoglin. Endoglin shedding was inhibited by matrix metalloproteinase (MMP) inhibitors and MMP-14 short hairpin RNA, indicating MMP-14 as the major endoglin shedding protease. Coexpression of endoglin and membrane-bound MMP-14 led to a strong increase in sEndoglin levels. Endoglin shedding required a direct interaction between endoglin and membrane-localized MMP-14. Using cleavage site mutants, we determined that MMP-14 cleaved endoglin at a site in close proximity to the transmembrane domain. Taken together, this study shows that MMP-14 mediates endoglin shedding, which may regulate the angiogenic potential of endothelial cells in the (colorectal) tumor microenvironment.
Cancer Research 05/2010; 70(10):4141-50. · 7.86 Impact Factor
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Yukihide Watanabe,
Susumu Itoh,
Toshiyasu Goto,
Eriko Ohnishi,
Masako Inamitsu,
Fumiko Itoh,
Kiyotoshi Satoh, Eliza Wiercinska,
Weiwen Yang,
Liang Shi,
Aya Tanaka,
Naoko Nakano,
A Mieke Mommaas,
Hiroshi Shibuya,
Peter Ten Dijke,
Mitsuyasu Kato
[show abstract]
[hide abstract]
ABSTRACT: Transforming growth factor-beta (TGF-beta) is a multifunctional cytokine of key importance for controlling embryogenesis and tissue homeostasis. How TGF-beta signals are attenuated and terminated is not well understood. Here, we show that TMEPAI, a direct target gene of TGF-beta signaling, antagonizes TGF-beta signaling by interfering with TGF-beta type I receptor (TbetaRI)-induced R-Smad phosphorylation. TMEPAI can directly interact with R-Smads via a Smad interaction motif. TMEPAI competes with Smad anchor for receptor activation for R-Smad binding, thereby sequestering R-Smads from TbetaRI kinase activation. In mammalian cells, ectopic expression of TMEPAI inhibited TGF-beta-dependent regulation of plasminogen activator inhibitor-1, JunB, cyclin-dependent kinase inhibitors, and c-myc expression, whereas specific knockdown of TMEPAI expression prolonged duration of TGF-beta-induced Smad2 and Smad3 phosphorylation and concomitantly potentiated cellular responsiveness to TGF-beta. Consistently, TMEPAI inhibits activin-mediated mesoderm formation in Xenopus embryos. Therefore, TMEPAI participates in a negative feedback loop to control the duration and intensity of TGF-beta/Smad signaling.
Molecular cell 01/2010; 37(1):123-34. · 14.61 Impact Factor
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Stephan L Haas,
Brit Fitzner,
Robert Jaster, Eliza Wiercinska,
Haristi Gaitantzi,
Ralf Jesnowski,
Ralf Jesenowski,
J-Matthias Löhr,
Manfred V Singer,
Steven Dooley,
Katja Breitkopf
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ABSTRACT: Nerve growth factor (NGF), a survival factor for neurons enforces pain by sensitizing nociceptors. Also in the pancreas, NGF was associated with pain and it can stimulate the proliferation of pancreatic cancer cells. Hepatic stellate cells (HSC) respond to NGF with apoptosis. Transforming growth factor (TGF)-beta, one of the strongest pro-fibrogenic activators of pancreatic stellate cells (PSC) induced NGF and its two receptors in an immortalized human cell line (ihPSC) and primary rat PSC (prPSC) as determined by RT-PCR, western blot, and immunofluorescence. In contrast to HSC, PSC expressed both NGF receptors, although p75(NTR) expression was weak in prPSC. In contrast to ihPSC TGF-beta activated both Smad signaling cascades in prPSC. NGF secretion was diminished by the activin-like kinase (ALK)-5 inhibitor SB431542, indicating the predominant role of ALK5 in activating the NGF system in PSC. While NGF did not affect proliferation or survival of PSC it induced expression of Inhibitor of Differentiation-1. We conclude that under conditions of upregulated TGF-beta, like fibrosis, NGF levels will also increase in PSC which might contribute to pancreatic wound healing responses.
Growth factors (Chur, Switzerland) 08/2009; 27(5):289-99. · 2.47 Impact Factor
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ABSTRACT: The transforming growth factor-beta (TGF-beta) pathway is an important pathway in the initiation and progression of colorectal cancer. We aimed to determine the effects of 5-aminosalicylic acid (5-ASA) on TGF-beta signalling in colorectal cancer cells in vitro. 5-ASA inhibited TGF-beta1 signalling in HCT116 cells and colonic fibroblasts, as judged by a TGF-beta-specific reporter gene assay, plasminogen activator inhibitor-1 mRNA and protein levels, fibroblast trans-differentiation, Smad3 phosphorylation and nuclear translocation. We conclude that 5-ASA inhibits TGF-beta1 signalling in colorectal cancer cells, and might be a potent adjuvant therapeutic drug, interfering with aberrant TGF-beta signalling in colorectal cancer.
Cancer letters 07/2009; 287(1):82-90. · 4.86 Impact Factor
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Liudmila L Kodach, Eliza Wiercinska,
Noel F C C de Miranda,
Sylvia A Bleuming,
Alex R Musler,
Maikel P Peppelenbosch,
Evelien Dekker,
Gijs R van den Brink,
Carel J M van Noesel,
Hans Morreau,
Daniel W Hommes,
Peter Ten Dijke,
G Johan A Offerhaus,
James C H Hardwick
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ABSTRACT: The finding of bone morphogenetic protein (BMP) receptor 1a mutations in juvenile polyposis suggests that BMPs are important in colorectal cancer (CRC). We investigated the BMP pathway in sporadic CRC.
We investigated BMP receptor (BMPR) expression using immunoblotting and sequenced BMPR2 in CRC cell lines. We assessed the expression of BMPRs, SMAD4, and pSMAD1/5/8 in 72 sporadic CRCs using a tissue microarray and immunohistochemistry. We assessed the effect of reintroduction of wild-type BMPR2 on BMP pathway activity and the effect of wild-type or mutated BMPR2 3' untranslated region (UTR) sequences on protein expression by attachment to pCMV-Luc.
BMPR2 and SMAD4 protein expression is abrogated in microsatellite unstable (MSI) and microsatellite stable (MSS) cell lines, respectively. BMPR2 3'UTR is mutated in all MSI and in none of the MSS cell lines. Mutant BMPR2 3'UTR sequences reduced luciferase expression 10-fold compared with wild-type BMPR2 3'UTR. BMPR2 expression is impaired more frequently in MSI CRCs than MSS (85% vs 29%; P < .0001) and shows a mutually exclusive pattern of impaired expression compared with SMAD4. Nine of 11 MSI cancers with impaired expression of BMPR2 have microsatellite mutations. The BMP pathway is inactivated, as judged by nuclear pSMAD1/5/8 expression, in 70% of CRCs, and this correlates with BMPR and SMAD4 loss.
Our data suggest that the BMP pathway is inactivated in the majority of sporadic CRCs. In MSI CRC this is associated predominantly with impaired BMPR2 expression and in MSS CRC with impaired SMAD4 expression.
Gastroenterology 06/2008; 134(5):1332-41. · 11.68 Impact Factor
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ABSTRACT: Langerhans cells (LC) represent the cutaneous contingent of dendritic cells (DC). Their development critically depends on transforming growth factor beta1 (TGF-beta1) as demonstrated by analysis of TGF-beta1(-/-) mice, which lack LC. Here we used a two-step culture system and transcriptional profiling by DNA microarrays to search for TGF-beta1 target genes in DC. The study identified interferon regulatory factor 8 (IRF-8) as a novel target gene of TGF-beta1 signaling in DC. TGF-beta1 effectively induced Smad2/3 phosphorylation and IRF-8 RNA and protein expression. Blocking the TGF-beta1/Smad pathway by ectopic expression of inhibitory Smad7 and by SB431542 inhibitor abolished TGF-beta1 induced up-regulation of IRF-8. Furthermore, TGF-beta1-dependent induction of IRF-8 occurred in the absence of protein biosynthesis, suggesting a direct action of TGF-beta1/Smad signaling on IRF-8 gene activity. TGF-beta1 also induced expression of the chemokine receptor CCR7 and enhanced DC migration towards CCR7 ligand ELC. DC of IRF-8(-/-) mice show reduced CCR7 expression and migratory activity, thereby implicating the TGF-beta1/Smad/IRF-8 signaling pathway in CCR7 regulation. Thus, this study identified a novel TGF-beta1/Smad/IRF-8 signaling pathway with an impact on DC phenotype and function.
European Journal of Immunology 06/2007; 37(5):1174-83. · 5.10 Impact Factor
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Eliza Wiercinska,
Lucia Wickert,
Bernd Denecke,
Harun M Said,
Jafar Hamzavi,
A M Gressner,
Midori Thorikay,
Peter ten Dijke,
Peter R Mertens,
Katja Breitkopf,
Steven Dooley
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ABSTRACT: Transforming growth factor (TGF)-beta is critically involved in the activation of hepatic stellate cells (HSCs) that occurs during the process of liver damage, for example, by alcohol, hepatotoxic viruses, or aflatoxins. Overexpression of the TGF-beta antagonist Smad7 inhibits transdifferentiation and arrests HSCs in a quiescent stage. Additionally, bile duct ligation (BDL)-induced fibrosis is ameliorated by introducing adenoviruses expressing Smad7 with down-regulated collagen and alpha-smooth muscle actin (alpha-SMA) expression. The aim of this study was to further characterize the molecular details of TGF-beta pathways that control the transdifferentiation process. In an attempt to elucidate TGF-beta target genes responsible for fibrogenesis, an analysis of Smad7-dependent mRNA expression profiles in HSCs was performed, resulting in the identification of the inhibitor of differentiation 1 (Id1) gene. Ectopic Smad7 expression in HSCs strongly reduced Id1 mRNA and protein expression. Conversely, Id1 overexpression in HSCs enhanced cell activation and circumvented Smad7-dependent inhibition of transdifferentiation. Moreover, knock-down of Id1 in HSCs interfered with alpha-SMA fiber formation, indicating a pivotal role of Id1 for fibrogenesis. Treatment of HSCs with TGF-beta1 led to increased Id1 protein expression, which was not directly mediated by the ALK5/Smad2/3, but the ALK1/Smad1 pathway. In vivo, Id1 expression and Smad1 phosphorylation were co-induced during fibrogenesis. In conclusion, Id1 is identified as TGF-beta/ALK1/Smad1 target gene in HSCs and represents a critical mediator of transdifferentiation that might be involved in hepatic fibrogenesis. Supplementary material for this article can be found on the HEPATOLOGY website (http://interscience.wiley.com/jpages/0270-9139/suppmat/index.html).
Hepatology 06/2006; 43(5):1032-41. · 11.66 Impact Factor
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ABSTRACT: Permanent alcohol abuse may lead to chronic liver injury with deleterious sequelae such as liver cirrhosis and hepatocellular carcinoma. Mechanisms of fibrogenesis encompass recruitment of inflammatory cells at the site of injury and cytokine mediated activation of hepatic stellate cells (HSC) with accumulation of interstitial collagens. HSC transdifferentiation and accompanying apoptosis result in destruction of liver architecture and are therefore key steps of disease progression. TGF-beta represents the main profibrogenic cytokine in liver fibrosis and other fibroproliferative disorders by inducing extracellular matrix deposition as part of the wound healing response. In parallel, TGF-beta triggers hepatocytes that are strongly responsive for this cytokine, to undergo apoptosis, thereby providing space for HSC proliferation and generation of a collagenous matrix. Anti TGF-beta approaches were established and successfully utilized for the treatment of experimental fibrogenesis. Dominant negative TGF-beta receptors (TbetaR), generated by fusing the Fc domain of human IgG and the N-terminal (extracellular) fragment of TbetaRII (Fc:TbetaRII) were applied to suppress fibrosis. Similarly TGF-beta binding proteins like decorin, antagonistic cytokines such as bone morphogenetic protein-7, hepatocyte growth factor, IL-10, or IFN-gamma were as efficient as camostat mesilate, a protease inhibitor that possibly abrogated proteolytic activation of TGF-beta. Further, our group recently overexpressed Smad7 in bile duct ligation induced liver fibrosis and achieved efficient inhibition of intracellular TGF-beta signaling, thereby counteracting profibrogenic effects in cultured HSC and in vivo. A direct link between the effect of alcohol and TGF-beta exists through reactive oxygen species that are generated in liver cells by alcohol metabolism and represent activators of TGF-beta signaling. Thus, soluble TbetaRII expression reduced experimental fibrogenesis in vitro and in vivo partially by decreasing intracellular ROS and inhibiting NADH oxidase. Approaches that specifically target profibrogenic TGF-beta signaling are promising to treat alcoholic liver disease in the future. However, to ensure safety for the patients to be treated, approaches with strong specificity need to be established. Therefore, it is essential to delineate the profibrogenic actions of TGF-beta and the influence of alcohol abuse in molecular detail.
Alcoholism Clinical and Experimental Research 12/2005; 29(11 Suppl):121S-131S. · 3.34 Impact Factor
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ABSTRACT: Hepatic stellate cells (HSC) cultured on plastic spontaneously transdifferentiate to a myofibroblast-like cell type (MFB). This model system of hepatic fibrogenesis is characterized by phenotypic changes of the cells and increased matrix synthesis. Here, we analyzed if transdifferentiation-dependent induction of ECM components, e.g., collagen type I and thrombospondin-2 (TSP-2), and phenotypic changes are coregulated events and if both processes are mediated via TGF-beta pathway(s). Blocking the TGF-beta-dependent p38 MAPK pathway in HSC with the specific inhibitor SB203580 strongly reduces collagen I and TSP-2 mRNA expression without inhibiting upregulation of the typical MFB-marker, alpha-smooth-muscle actin (alpha-SMA). Similarly, interference with the Smad2/3/4 pathway using dexamethasone also heavily decreased expression of collagen type I and TSP-2 whereas transdifferentiation of HSC to the typical morphology of MFB with loss of fat droplets and increasing alpha-SMA was unchanged. Further, p38 MAPK mediated induction of collagen I and TSP-2 expression by TGF-beta1 was still achieved in the presence of dexamethasone, showing that dexamethasone does not block p38 while it delays Smad2 phosphorylation and antagonizes stimulation of a Smad3/Smad4 dependent TGF-beta reporter construct. Interestingly, in contrast to SB203580 and dexamethasone, overexpression of the TGF-beta antagonist Smad7 reduced ECM expression and simultaneously inhibited morphologic transdifferentiation, indicating that Smad7 fulfills additional features in HSC. In conclusion, our data show that phenotypic changes of transdifferentiating HSC and induction of matrix synthesis are independent processes, the latter being stimulated by both, Smad dependent and MAPK dependent TGF-beta signaling.
Matrix Biology 06/2005; 24(3):198-207. · 3.30 Impact Factor
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ABSTRACT: Numerous studies implicate transforming growth factor (TGF)-beta signaling in liver fibrogenesis. To perturb the TGF-beta pathway during this process, we overexpressed Smad7, an intracellular antagonist of TGF-beta signaling, in vivo and in primary-cultured hepatic stellate cells (HSCs).
Ligation of the common bile duct (BDL) was used to induce liver fibrosis in rats. Animals received injections of an adenovirus carrying Smad7 cDNA into the portal vein during surgery and via the tail vein at later stages. The effect of Smad7 on TGF-beta signaling and activation of HSC was further analyzed in primary-cultured cells.
Smad7-overexpressing BDL rats displayed reduced collagen and alpha-SMA expression and reduced hydroxyproline content in the liver, when compared with animals administered AdLacZ. Such a beneficial effect was also observed when Smad7 was expressed in animals with established fibrosis. Accordingly, Smad7 arrested transdifferentiation of primary-cultured HSCs. AdSmad7 infected cells remained in a quiescent stage and retained storage of vitamin A droplets. Smad7 expression totally blocked TGF-beta signal transduction, shown by inhibiting Smad2/3 phosphorylation, nuclear translocation of activated Smad complexes, and activation of (CAGA)(9)-MLP-Luc, resulting in decreased collagen I expression. Smad7 also abrogated TGF-beta-dependent proliferation inhibition of HSC. Smad7 did not decrease expression of alpha-SMA, but immunofluorescent staining with anti alpha-SMA antibodies displayed destruction of the fibrillar organization of the actin cytoskeleton.
In summary, gene transfer of Smad7 inhibits experimental fibrogenesis in vivo. Studies with isolated HSC suggest that the underlying mechanisms involve inhibition of TGF-beta signaling and HSC transdifferentiation.
Gastroenterology 08/2003; 125(1):178-91. · 11.68 Impact Factor
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ABSTRACT: Profibrogenic TGF-beta signaling in hepatic stellate cells is modulated during transdifferentiation. Strategies to abrogate TGF-beta effects provide promising antifibrotic results, however, in vivo data regarding Smad activation during fibrogenesis are scarce.
Here, liver fibrosis was assessed subsequent to bile duct ligation by determining liver enzymes in serum and collagen deposition in liver tissue. Activated hepatic stellate cells were identified by immunohistochemistry and immunoblots for alpha smooth muscle actin. Cellular localization of Smad3 and Smad7 proteins was demonstrated by immunohistochemistry. RTPCR for Smad4 and Smad7 was conducted with total RNA and Northern blot analysis for Smad7 with mRNA. Whole liver lysates were prepared to detect Smad2/3/4 and phospho- Smad2/3 by Western blotting.
Cholestasis induces TGF-beta signaling via Smad3 in vivo, whereas Smad2 phosphorylation was only marginally increased. Smad4 expression levels were unchanged. Smad7 expression was continuously increasing with duration of cholestasis. Hepatocytes of fibrotic lesions exhibited nuclear staining Smad3. In contrast to this, Smad7 expression was localized to activated hepatic stellate cells.
Hepatocytes of damaged liver tissue display increased TGF-beta signaling via Smad3. Further, negative feedback regulation of TGF-beta signaling by increased Smad7 expression in activated hepatic stellate cells occurs, however does not interfere with fibrogenesis.
Journal of Cellular and Molecular Medicine 10(4):922-32. · 4.13 Impact Factor
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Yukihide Watanabe,
Susumu Itoh,
Toshiyasu Goto,
Eriko Ohnishi,
Masako Inamitsu,
Fumiko Itoh,
Kiyotoshi Satoh, Eliza Wiercinska,
Weiwen Yang,
Liang Shi,
Aya Tanaka,
Naoko Nakano,
A. Mieke Mommaas,
Hiroshi Shibuya,
Peter ten Dijke,
Mitsuyasu Kato,
幸秀 渡邊,
進 伊東,
光保 加藤
[show abstract]
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ABSTRACT: Transforming growth factor-β (TGF-β) is a multifunctional cytokine of key importance for controlling embryogenesis and tissue homeostasis. How TGF-β signals are attenuated and terminated is not well understood. Here, we show that TMEPAI, a direct target gene of TGF-β signaling, antagonizes TGF-β signaling by interfering with TGF-β type I receptor (TβRI)-induced R-Smad phosphorylation. TMEPAI can directly interact with R-Smads via a Smad interaction motif. TMEPAI competes with Smad anchor for receptor activation for R-Smad binding, thereby sequestering R-Smads from TβRI kinase activation. In mammalian cells, ectopic expression of TMEPAI inhibited TGF-β-dependent regulation of plasminogen activator inhibitor-1, JunB, cyclin-dependent kinase inhibitors, and c-myc expression, whereas specific knockdown of TMEPAI expression prolonged duration of TGF-β-induced Smad2 and Smad3 phosphorylation and concomitantly potentiated cellular responsiveness to TGF-β. Consistently, TMEPAI inhibits activin-mediated mesoderm formation in Xenopus embryos. Therefore, TMEPAI participates in a negative feedback loop to control the duration and intensity of TGF-β/Smad signaling.
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Eliza Wiercinska
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ABSTRACT: Der transformierende Wachstumsfaktor beta (Transforming growth factor beta; TGF-beta) spielt eine wichtige Role in der Aktivierung von hepatischen Sternzellen (hepatic stellate cells, HSCs) und der Freisetzung von extrazellularer Matrix während der Leberfibrose. Ziel der vorliegenden Studie war durch Expression des intrazellulären Antagonisten, Smad7, den TGF-beta Signalweg zu blockieren. Darüber hinaus wurde ein neues TGF-beta Zielgen identifiziert, das an der Transdifferenzierung von HSCs beteiligt ist. Die in vivo Fibrogenese wurde in einem Galengangsligatur-Model in der Ratte untersucht. Die Tiere wurden mit Smad7 codierenden Adenoviren infiziert. Die Kollagen-Expression und der Hydroxyprolin-Gehalt war in den Lebern von Smad7 behandelten Ratten verglichen mit Kontrollratten stark reduziert. Smad7 zeigte auch starke hemmende Wirkung auf die Transdifferenzierung von HSCs in vitro und blockiert den TGF-beta Signalweg in den Zellen. Die Phosphorylierung von Smad2/3 ist runterreguliert und Smad7-exprimierende Zellen verloren die Vitamin A Tröpfchen nicht. Die Rolle von TGF-beta bei der Transdifferenzierung von HSCs wurde in einer Smad7 abhängigen Microarray-Analyse untersucht, was zur Identifizierung des Differenzierungsinhibitors 1 (inhibitor of differentiation; Id) führte. Smad7 führte zu starker Reduktion der Id1 Expression, wohingegen Id1 Überexpression Aktivierung von HSCs verstärkt und hinreichend ist, um den Smad7-Effekt zu überkommen. Si-RNA abhängiger Id1 knock down interferiert mit HSC-Aktivierung, was eine zentrale Rolle von Id1 während der Fibrogenese unterstreicht. Ich klärte weiter auf, dass die TGF-beta-abhängige Expression von Id1 in HSCs über einen neuen ALK1/Smad1/5/8 Signalweg reguliert wird. Ein ALK1 si-RNA knock down blockierte die TGF-beta induzierte Smad1-Phosphorylierung und Id1-Expression, hatte jedoch keinen Einfluss auf den ALK5/Smad2/3 Signalweg. Zusammenfassend hemmt adenoviraler Transfer von Smad7 die Leberfibrose in vivo. Für den Effekt ist wahrscheinlich die Blockade TGF-beta induzierter HSC-Aktivierung verantwortlich. Zusätzlich wurde ein neuer TGF-beta Signalweg über ALK1 und Smad1 untersucht, der zu Id1 Expression. Diese neuen Erkenntnisse sollen dazu beitragen in Zukunft Leberfibrose besser behandeln zu können. Transforming growth factor (TGF)-beta is a key mediator of hepatic stellate cell (HSC) activation and extracellular matrix accumulation leading to fibrosis. Aim of the present study was to perturb the TGF-beta pathway in this process by overexpression of Smad7, an intracellular antagonist of TGF-beta signaling. Moreover, a new TGF-beta response gene was identified, which participates in the control of profibrogenic transdifferentiation to myofibroblast-like cells. Ligation of the common bile duct was used to induce liver fibrosis in rats. Animals received injections of an adenovirus overexpressing Smad7. The effect of Smad7 on TGF-beta signaling and activation of HSC was further analyzed in primary cultured cells. AdSmad7 infected rats displayed reduced collagen deposition and hydroxyproline content in the liver, when compared with animals administered AdLacZ. Such a beneficial effect was also observed, when Smad7 was expressed in animals with established fibrosis. Accordingly, Smad7 arrested transdifferentiation of primary cultured HSC. AdSmad7 infected cells remained in a quiescent stage and retained storage of vitamin A droplets. Smad7 expression totally blocked TGF-beta signal transduction, shown by inhibiting Smad2/3 phosphorylation. In an attempt to elucidate TGF-beta target genes responsible for fibrogenesis, an analysis of Smad7-dependent mRNA expression profiles in HSC was performed, resulting in identification of the inhibitor of differentiation 1 (Id1) gene. Ectopic Smad7 expression reduced Id1 expression. Conversely, Id1 overexpression in HSC enhanced cell activation and circumvented Smad7-dependent inhibition of transdifferentiation. Moreover, knock-down of Id1 in HSC interfered with the transdifferentiation process, indicating a pivotal role of Id1 for fibrogenesis. Additionally, treatment of HSC with TGF-beta led to increased Id1 protein expression, which was dependent on activation of the novel ALK1/Smad1/5/8 pathway. Knocking-down expression of ALK1 in HSC interfered with TGF-beta dependent Smad1 phosphorylation and Id1 upregulation, but did not block the activation of the Smad2/3 pathway. In summary, gene transfer of Smad7 inhibits experimental fibrogenesis in vivo. Studies with isolated HSC suggest that the underlying mechanisms involve inhibition of TGF-beta signaling and HSC transdifferentiation. Moreover, a novel TGF-beta pathway involving ALK1 and Smad1 was shown to mediate HSC activation via Id1 upregulation. These new findings represent valuable information for the development of more efficient antifibrotic strategies to treat liver damage in the future.
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Susumu Itoh,
Yukihide Watanabe,
Kiyotoshi Satoh,
Masako Inamitsu,
Liang Shi,
Naoko Nakano,
Aya Tanaka, Eliza Wiercinska,
Hiroshi Shibuya,
Peter ten Dijke,
Mitsuyasu Kato
[show abstract]
[hide abstract]
ABSTRACT: Transforming growth factor-[beta] (TGF-[beta]) is a multifunctional cytokine of key importance for controlling embryogenesis and tissue homeostasis. How TGF-[beta] signals are attenuated and terminated is not well understood. Here, we show that TMEPAI, a direct target gene of TGF-[beta] signaling, antagonizes TGF-[beta] signaling by interfering with TGF-[beta] type I receptor (T[beta]RI)-induced R-Smad phosphorylation. TMEPAI can directly interact with R-Smads via a Smad interaction motif (SIM). TMEPAI competes with Smad anchor for receptor activation (SARA) for R-Smad binding, thereby sequestering R-Smads from T[beta]RI kinase activation. In mammalian cells, ectopic expression of TMEPAI inhibited TGF-[beta]-induced PAI-1 production, whereas specific siRNA-mediated knockdown of TMEPAI expression potentiated TGF-[beta]-induced Smad2 phosphorylation and cellular responsiveness by TGF-[beta]. Consistently, TMEPAI inhibits activin-mediated mesoderm formation in _Xenopus_ embryos. Taken together, TMEPAI participates in a negative feedback loop to control the duration and intensity of TGF-[beta] signaling.
Nature Precedings.
