Translate this page to:
Arabic
In JoVE (1)
Other Publications (14)
- Gastroenterology
- Matrix Biology : Journal of the International Society for Matrix Biology
- Alcoholism, Clinical and Experimental Research
- Hepatology (Baltimore, Md.)
- Journal of Cellular and Molecular Medicine
- European Journal of Immunology
- Gastroenterology
- Growth Factors (Chur, Switzerland)
- Molecular Cell
- Cancer Research
- Cancer Letters
- Breast Cancer Research and Treatment
- Molecular Cancer
- Cellular Oncology (Dordrecht)
Automatic Translation
This translation into Arabic was automatically generated.
English Version | Other Languages
Articles by Eliza Wiercinska in JoVE
JoVE Clinical and Translational Medicine
كروي الفحص لقياس TGF - β بفعل الغزو
يوصف لفحص لقياس كميا تحويل عامل النمو (TGF) - β بفعل الغزو في المواد الهلامية الكولاجين 3 الأبعاد. هذا الاختبار يستفيد من سلسلة MCF10A من خطوط الخلايا التي تمثل مراحل مختلفة من تطور مرض السرطان الثدي. ويمكن اعتماد هذه الطريقة ليتم استخدامها مع خطوط الخلايا الأخرى ، ويمكن استخدامها لتفعيل التحقيق المحتملين الآخرين أو مثبطات الغزو.
Other articles by Eliza Wiercinska on PubMed
Smad7 Prevents Activation of Hepatic Stellate Cells and Liver Fibrosis in Rats
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).
Transdifferentiation-dependent Expression of Alpha-SMA in Hepatic Stellate Cells Does Not Involve TGF-beta Pathways Leading to Coinduction of Collagen Type I and Thrombospondin-2
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.
Anti-TGF-beta Strategies for the Treatment of Chronic Liver Disease
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.
Id1 is a Critical Mediator in TGF-beta-induced Transdifferentiation of Rat Hepatic Stellate Cells
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).
Liver Fibrogenesis Due to Cholestasis is Associated with Increased Smad7 Expression and Smad3 Signaling
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.
Transforming Growth Factor Beta1 Up-regulates Interferon Regulatory Factor 8 During Dendritic Cell Development
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.
The Bone Morphogenetic Protein Pathway is Inactivated in the Majority of Sporadic Colorectal Cancers
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.
Transforming Growth Factor-beta Induces Nerve Growth Factor Expression in Pancreatic Stellate Cells by Activation of the ALK-5 Pathway
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.
TMEPAI, a Transmembrane TGF-beta-inducible Protein, Sequesters Smad Proteins from Active Participation in TGF-beta Signaling
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.
Matrix Metalloproteinase-14 (MT1-MMP)-mediated Endoglin Shedding Inhibits Tumor Angiogenesis
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.
5-Aminosalicylic Acid Inhibits TGF-beta1 Signalling in Colorectal Cancer Cells
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.
The TGF-β/Smad Pathway Induces Breast Cancer Cell Invasion Through the Up-regulation of Matrix Metalloproteinase 2 and 9 in a Spheroid Invasion Model System
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.
Oncogenic Functions of HMDMX in in Vitro Transformation of Primary Human Fibroblasts and Embryonic Retinoblasts
In around 50% of all human cancers the tumor suppressor p53 is mutated. It is generally assumed that in the remaining tumors the wild-type p53 protein is functionally impaired. The two main inhibitors of p53, hMDM2 (MDM2) and hMDMX (MDMX/MDM4) are frequently overexpressed in wild-type p53 tumors. Whereas the main activity of hMDM2 is to degrade p53 protein, its close homolog hMDMX does not degrade p53, but it represses its transcriptional activity. Here we study the role of hMDMX in the neoplastic transformation of human fibroblasts and embryonic retinoblasts, since a high number of retinoblastomas contain elevated hMDMX levels.
BMP-7 Inhibits TGF-β-induced Invasion of Breast Cancer Cells Through Inhibition of Integrin β(3) Expression
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.
