TGF-beta 1 induces human alveolar epithelial to mesenchymal cell transition (EMT)

Background: Fibroblastic foci are characteristic features in lung parenchyma of patients with idiopathic pulmonary fibrosis (IPF). They comprise aggregates of mesenchymal cells which underlie sites of unresolved epithelial injury and are associated with progression of fibrosis. However, the cellular origins of these mesenchymal phenotypes remain unclear. We examined whether the potent fibrogenic cytokine TGF-β1 could induce epithelial mesenchymal transition (EMT) in the human alveolar epithelial cell line, A549, and investigated the signaling pathway of TGF-β1-mediated EMT. Methods: A549 cells were examined for evidence of EMT after treatment with TGF-β1. EMT was assessed by: morphology under phase-contrast microscopy; Western analysis of cell lysates for expression of mesenchymal phenotypic markers including fibronectin EDA (Fn-EDA), and expression of epithelial phenotypic markers including E-cadherin (E-cad). Markers of fibrogenesis, including collagens and connective tissue growth factor (CTGF) were also evaluated by measuring mRNA level using RT-PCR, and protein by immunofluorescence or Western blotting. Signaling pathways for EMT were characterized by Western analysis of cell lysates using monoclonal antibodies to detect phosphorylated Erk1/2 and Smad2 after TGF-β1 treatment in the presence or absence of MEK inhibitors. The role of Smad2 in TGF-β1-mediated EMT was investigated using siRNA. Results: The data showed that TGF-β1, but not TNF-α or IL-1β, induced A549 cells with an alveolar epithelial type II cell phenotype to undergo EMT in a time-and concentration-dependent manner. The process of EMT was accompanied by morphological alteration and expression of the fibroblast phenotypic markers Fn-EDA and vimentin, concomitant with a downregulation of the epithelial phenotype marker E-cad. Furthermore, cells that had undergone EMT showed enhanced expression of markers of fibrogenesis including collagens type I and III and CTGF. MMP-2 expression was also evidenced. TGF-β1-induced EMT occurred through phosphorylation of Smad2 and was inhibited by Smad2 gene silencing; MEK inhibitors failed to attenuate either EMT-associated Smad2 phosphorylation or the observed phenotypic changes. Conclusion: Our study shows that TGF-β1 induces A549 alveolar epithelial cells to undergo EMT via Smad2 activation. Our data support the concept of EMT in lung epithelial cells, and suggest the need for further studies to investigate the phenomenon

Testing the role of predicted gene knockouts in human anthropometric trait variation

National Heart, Lung, and Blood Institute (NHLBI) S.L. is funded by a Canadian Institutes of Health Research Banting doctoral scholarship. G.L. is funded by Genome Canada and Génome Québec; the Canada Research Chairs program; and the Montreal Heart Institute Foundation. C.M.L. is supported by Wellcome Trust (grant numbers 086596/Z/08/Z, 086596/Z/08/A); and the Li Ka Shing Foundation. N.S. is funded by National Institutes of Health (grant numbers HL088456, HL111089, HL116747). The Mount Sinai BioMe Biobank Program is supported by the Andrea and Charles Bronfman Philanthropies. GO ESP is supported by NHLBI (RC2 HL-103010 to HeartGO, RC2 HL-102923 to LungGO, RC2 HL-102924 to WHISP). The ESP exome sequencing was performed through NHLBI (RC2 HL-102925 to BroadGO, RC2 HL- 102926 to SeattleGO). EGCUT work was supported through the Estonian Genome Center of University of Tartu by the Targeted Financing from the Estonian Ministry of Science and Education (grant number SF0180142s08); the Development Fund of the University of Tartu (grant number SP1GVARENG); the European Regional Development Fund to the Centre of Excellence in Genomics (EXCEGEN) [grant number 3.2.0304.11-0312]; and through FP7 (grant number 313010). EGCUT were further supported by the US National Institute of Health (grant number R01DK075787). A.K.M. was supported by an American Diabetes Association Mentor-Based Postdoctoral Fellowship (#7-12-MN- 02). The BioVU dataset used in the analyses described were obtained from Vanderbilt University Medical Centers BioVU which is supported by institutional funding and by the Vanderbilt CTSA grant ULTR000445 from NCATS/NIH. Genome-wide genotyping was funded by NIH grants RC2GM092618 from NIGMS/OD and U01HG004603 from NHGRI/NIGMS. Funding to pay the Open Access publication charges for this article was provided by a block grant from Research Councils UK to the University of Cambridge

Testing the role of predicted gene knockouts in human anthropometric trait variation

National Heart, Lung, and Blood Institute (NHLBI) S.L. is funded by a Canadian Institutes of Health Research Banting doctoral scholarship. G.L. is funded by Genome Canada and Génome Québec; the Canada Research Chairs program; and the Montreal Heart Institute Foundation. C.M.L. is supported by Wellcome Trust (grant numbers 086596/Z/08/Z, 086596/Z/08/A); and the Li Ka Shing Foundation. N.S. is funded by National Institutes of Health (grant numbers HL088456, HL111089, HL116747). The Mount Sinai BioMe Biobank Program is supported by the Andrea and Charles Bronfman Philanthropies. GO ESP is supported by NHLBI (RC2 HL-103010 to HeartGO, RC2 HL-102923 to LungGO, RC2 HL-102924 to WHISP). The ESP exome sequencing was performed through NHLBI (RC2 HL-102925 to BroadGO, RC2 HL- 102926 to SeattleGO). EGCUT work was supported through the Estonian Genome Center of University of Tartu by the Targeted Financing from the Estonian Ministry of Science and Education (grant number SF0180142s08); the Development Fund of the University of Tartu (grant number SP1GVARENG); the European Regional Development Fund to the Centre of Excellence in Genomics (EXCEGEN) [grant number 3.2.0304.11-0312]; and through FP7 (grant number 313010). EGCUT were further supported by the US National Institute of Health (grant number R01DK075787). A.K.M. was supported by an American Diabetes Association Mentor-Based Postdoctoral Fellowship (#7-12-MN- 02). The BioVU dataset used in the analyses described were obtained from Vanderbilt University Medical Centers BioVU which is supported by institutional funding and by the Vanderbilt CTSA grant ULTR000445 from NCATS/NIH. Genome-wide genotyping was funded by NIH grants RC2GM092618 from NIGMS/OD and U01HG004603 from NHGRI/NIGMS. Funding to pay the Open Access publication charges for this article was provided by a block grant from Research Councils UK to the University of Cambridge

Transforming growth factor-β and breast cancer: Lessons learned from genetically altered mouse models

Transforming growth factor (TGF)-βs are plausible candidate tumor suppressors in the breast. They also have oncogenic activities under certain circumstances, however. Genetically altered mouse models provide powerful tools to analyze the complexities of TGF-βaction in the context of the whole animal. Overexpression of TGF-β can suppress tumorigenesis in the mammary gland, raising the possibility that use of pharmacologic agents to enhance TGF-β function locally might be an effective method for the chemoprevention of breast cancer. Conversely, loss of TGF-β response increases spontaneous and induced tumorigenesis in the mammary gland. This confirms that endogenous TGF-βs have tumor suppressor activity in the mammary gland, and suggests that the loss of TGF-β receptors seen in some human breast hyperplasias may play a causal role in tumor development

Testing the role of predicted gene knockouts in human anthropometric trait variation

National Heart, Lung, and Blood Institute (NHLBI) S.L. is funded by a Canadian Institutes of Health Research Banting doctoral scholarship. G.L. is funded by Genome Canada and Génome Québec; the Canada Research Chairs program; and the Montreal Heart Institute Foundation. C.M.L. is supported by Wellcome Trust (grant numbers 086596/Z/08/Z, 086596/Z/08/A); and the Li Ka Shing Foundation. N.S. is funded by National Institutes of Health (grant numbers HL088456, HL111089, HL116747). The Mount Sinai BioMe Biobank Program is supported by the Andrea and Charles Bronfman Philanthropies. GO ESP is supported by NHLBI (RC2 HL-103010 to HeartGO, RC2 HL-102923 to LungGO, RC2 HL-102924 to WHISP). The ESP exome sequencing was performed through NHLBI (RC2 HL-102925 to BroadGO, RC2 HL- 102926 to SeattleGO). EGCUT work was supported through the Estonian Genome Center of University of Tartu by the Targeted Financing from the Estonian Ministry of Science and Education (grant number SF0180142s08); the Development Fund of the University of Tartu (grant number SP1GVARENG); the European Regional Development Fund to the Centre of Excellence in Genomics (EXCEGEN) [grant number 3.2.0304.11-0312]; and through FP7 (grant number 313010). EGCUT were further supported by the US National Institute of Health (grant number R01DK075787). A.K.M. was supported by an American Diabetes Association Mentor-Based Postdoctoral Fellowship (#7-12-MN- 02). The BioVU dataset used in the analyses described were obtained from Vanderbilt University Medical Centers BioVU which is supported by institutional funding and by the Vanderbilt CTSA grant ULTR000445 from NCATS/NIH. Genome-wide genotyping was funded by NIH grants RC2GM092618 from NIGMS/OD and U01HG004603 from NHGRI/NIGMS. Funding to pay the Open Access publication charges for this article was provided by a block grant from Research Councils UK to the University of Cambridge

A comprehensive 1000 Genomes-based genome-wide association meta-analysis of coronary artery disease

Existing knowledge of genetic variants affecting risk of coronary artery disease (CAD) is largely based on genome-wide association studies (GWAS) analysis of common SNPs. Leveraging phased haplotypes from the 1000 Genomes Project, we report a GWAS meta-analysis of 185 thousand CAD cases and controls, interrogating 6.7 million common (MAF>0.05) as well as 2.7 million low frequency (0.005<MAF<0.05) variants. In addition to confirmation of most known CAD loci, we identified 10 novel loci, eight additive and two recessive, that contain candidate genes that newly implicate biological processes in vessel walls. We observed intra-locus allelic heterogeneity but little evidence of low frequency variants with larger effects and no evidence of synthetic association. Our analysis provides a comprehensive survey of the fine genetic architecture of CAD showing that genetic susceptibility to this common disease is largely determined by common SNPs of small effect siz

Investigation of three new mouse mammary tumor cell lines as models for transforming growth factor (TGF)-β and Neu pathway signaling studies: identification of a novel model for TGF-β-induced epithelial-to-mesenchymal transition

INTRODUCTION: This report describes the isolation and characterization of three new murine mammary epithelial cell lines derived from mammary tumors from MMTV (mouse mammary tumor virus)/activated Neu + TβRII-AS (transforming growth factor [TGF]-β type II receptor antisense RNA) bigenic mice (BRI-JM01 and BRI-JM05 cell lines) and MMTV/activated Neu transgenic mice (BRI-JM04 cell line). METHODS: The BRI-JM01, BRI-JM04, and BRI-JM05 cell lines were analyzed for transgene expression, their general growth characteristics, and their sensitivities to several growth factors from the epidermal growth factor (EGF) and TGF-β families (recombinant human EGF, heregulin-β(1 )and TGF-β(1)). The BRI-JM01 cells were observed to undergo a striking morphologic change in response to TGF-β(1), and they were therefore further investigated for their ability to undergo a TGF-β-induced epithelial-to-mesenchymal transition (EMT) using motility assays and immunofluorescence microscopy. RESULTS: We found that two of the three cell lines (BRI-JM04 and BRI-JM05) express the Neu transgene, whereas, unexpectedly, both of the cell lines that were established from MMTV/activated Neu + TβRII-AS bigenic tumors (BRI-JM01 and BRI-JM05) do not express the TβRII-AS transgene. The cuboidal BRI-JM01 cells exhibit a short doubling time and are able to form confluent monolayers. The BRI-JM04 and BRI-JM05 cell lines are morphologically much less uniform, grow at a much slower rate, and do not form confluent monolayers. Only the BRI-JM05 cells can form colonies in soft agar. In contrast, all three cell lines form colonies in Matrigel, although the BRI-JM04 and BRI-JM05 cell lines do so more efficiently than the BRI-JM01 cell line. All three cell lines express the cell surface marker E-cadherin, confirming their epithelial character. Proliferation assays showed that the three cell lines respond differently to recombinant human EGF and heregulin-β(1), and that all are growth inhibited by TGF-β(1), but that only the BRI-JM01 cell line undergoes an EMT and exhibits increased motility upon TGF-β(1 )treatment. CONCLUSION: We suggest that the BRI-JM04 and BRI-JM05 cell lines can be used to investigate Neu oncogene driven mammary tumorigenesis, whereas the BRI-JM01 cell line will be useful for studying TGF-β(1)-induced EMT

PINCH1 Is Transcriptional Regulator in Podocytes That Interacts with WT1 and Represses Podocalyxin Expression

Background: PINCH1, an adaptor protein containing five LIM domains, plays an important role in regulating the integrin-mediated cell adhesion, migration and epithelial-mesenchymal transition. PINCH1 is induced in the fibrotic kidney after injury, and it primarily localizes at the sites of focal adhesion. Whether it can translocate to the nucleus and directly participate in gene regulation is completely unknown. Methodology/Principal Findings: Using cultured glomerular podocytes as a model system, we show that PINCH1 expression was induced by TGF-β1, a fibrogenic cytokine that promotes podocyte dysfunction. Interestingly, increased PINCH1 not only localized at the sites of focal adhesions, but also underwent nuclear translocation after TGF-β1 stimulation. This nuclear translocation of PINCH1 was apparently dependent on the putative nuclear export/localization signals (NES/NLS) at its C-terminus, as deletion or site-directed mutations abolished its nuclear shuttling. Co-immunoprecipitation and pull-down experiments revealed that PINCH1 interacted with Wilms tumor 1 protein (WT1), a nuclear transcription factor that is essential for regulating podocyte-specific gene expression in adult kidney. Interaction of PINCH1 and WT1 was mediated by the LIM1 domain of PINCH1 and C-terminal zinc-finger domain of WT1, which led to the suppression of the WT1-mediated podocalyxin expression in podocytes. PINCH1 also repressed podocalyxin gene transcription in a promoter-luciferase reporter assay. Conclusion/Significance: These results indicate that PINCH1 can shuttle into the nucleus from cytoplasm in podocytes, wherein it interacts with WT1 and suppresses podocyte-specific gene expression. Our studies reveal a previously unrecognized, novel function of PINCH1, in which it acts as a transcriptional regulator through controlling specific gene expression. © 2011 Wang et al

In Vivo Delivery of Gremlin siRNA Plasmid Reveals Therapeutic Potential against Diabetic Nephropathy by Recovering Bone Morphogenetic Protein-7

Diabetic nephropathy is a complex and poorly understood disease process, and our current treatment options are limited. It remains critical, then, to identify novel therapeutic targets. Recently, a developmental protein and one of the bone morphogenetic protein antagonists, Gremlin, has emerged as a novel modulator of diabetic nephropathy. The high expression and strong co-localization with transforming growth factor- β1 in diabetic kidneys suggests a role for Gremlin in the pathogenesis of diabetic nephropathy. We have constructed a gremlin siRNA plasmid and have examined the effect of Gremlin inhibition on the progression of diabetic nephropathy in a mouse model. CD-1 mice underwent uninephrectomy and STZ treatment prior to receiving weekly injections of the plasmid. Inhibition of Gremlin alleviated proteinuria and renal collagen IV accumulation 12 weeks after the STZ injection and inhibited renal cell proliferation and apoptosis. In vitro experiments, using mouse mesangial cells, revealed that the transfect ion of gremlin siRNA plasmid reversed high glucose induced abnormalities, such as increased cell proliferation and apoptosis and increased collagen IV production. The decreased matrix metalloprotease level was partially normalized by transfection with gremlin siRNA plasmid. Additionally, we observed recovery of bone morphogenetic protein-7 signaling activity, evidenced by increases in phosphorylated Smad 5 protein levels. We conclude that inhibition of Gremlin exerts beneficial effects on the diabetic kidney mainly through maintenance of BMP-7 activity and that Gremlin may serve as a novel therapeutic target in the management of diabetic nephropathy

Rosiglitazone Inhibits Transforming Growth Factor-β1 Mediated Fibrogenesis in ADPKD Cyst-Lining Epithelial Cells

BACKGROUND: Interstitial fibrosis plays an important role in progressive renal dysfunction in autosomal dominant polycystic kidney disease (ADPKD). In our previous studies, we confirmed that PPAR-γ agonist, rosiglitazone could protect renal function and prolong the survival of a slowly progressive ADPKD animal model by reducing renal fibrosis. However, the mechanism remains unknown. METHODS: Primary culture epithelial cells pretreated with TGF-β1 were incubated with rosiglitazone. Extracellular matrix proteins were detected using real-time PCR and Western blotting. MAPK and Smad2 phosphorylation were measured with western blot. ERK1/2 pathway and P38 pathway were inhibited with the specific inhibitors PD98059 and SB203580. The Smad2 pathway was blocked with the siRNA. To address whether PPAR-γ agonist-mediated inhibition of TGF-β1-induced collagen type I expression was mediated through a PPAR-γ dependent mechanism, genetic and pharmaceutical approaches were used to block the activity of endogenous PPARγ. RESULTS: TGF-β1-stimulated collagen type I and fibronectin expression of ADPKD cyst-lining epithelia were inhibited by rosiglitazone in a dosage-dependent manner. Smad2, ERK1/2 and P38 pathways were activated in response to TGF-β1; however, TGF-β1 had little effect on JNK pathway. Rosiglitazone suppressed TGF-β1 induced Smad2 activation, while ERK1/2 and P38MAPK signals remained unaffected. Rosiglitazone could also attenuate TGF-β1-stimulated collagen type I and fibronectin expression in primary renal tubular epithelial cells, but had no effect on TGF-β1-induced activation of Smad2, ERK1/2 and P38 pathways. There was no crosstalk between the Smad2 and MAPK pathways in ADPKD cyst-lining epithelial cells. These inhibitory effects of rosiglitazone were reversed by the PPARγ specific antagonist GW9662 and PPARγ siRNA. CONCLUSION: ADPKD cyst-lining epithelial cells participate in TGF-β1 mediated fibrogenesis. Rosiglitazone could suppress TGF-β1-induced collagen type I and fibronectin expression in ADPKD cyst-lining epithelia through modulation of the Smad2 pathway. Our study may provide therapeutic basis for clinical applications of rosiglitazone in retarding the progression of ADPKD