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1.
Oncotarget ; 8(48): 83354-83369, 2017 Oct 13.
Article in English | MEDLINE | ID: mdl-29137349

ABSTRACT

The genetic heterogeneity of acute myeloid leukemia (AML) and the variable responses of individual patients to therapy suggest that different AML genotypes may influence the bone marrow (BM) microenvironment in different ways. We performed gene expression profiling of bone marrow mesenchymal stromal cells (BM-MSC) isolated from normal C57BL/6 mice or mice inoculated with syngeneic murine leukemia cells carrying different human AML genotypes, developed in mice with Trp53 wild-type or nullgenetic backgrounds. We identified a set of genes whose expression in BM-MSC was modulated by all four AML genotypes tested. In addition, there were sets of differentially-expressed genes in AML-exposed BM-MSC that were unique to the particular AML genotype or Trp53 status. Our findings support the hypothesis that leukemia cells alter the transcriptome of surrounding BM stromal cells, in both common and genotype-specific ways. These changes are likely to be advantageous to AML cells, affecting disease progression and response to chemotherapy, and suggest opportunities for stroma-targeting therapy, including those based on AML genotype.

2.
JCI Insight ; 2(13)2017 Jul 06.
Article in English | MEDLINE | ID: mdl-28679949

ABSTRACT

Genotypic and phenotypic alterations in the bone marrow (BM) microenvironment, in particular in osteoprogenitor cells, have been shown to support leukemogenesis. However, it is unclear how leukemia cells alter the BM microenvironment to create a hospitable niche. Here, we report that acute myeloid leukemia (AML) cells, but not normal CD34+ or CD33+ cells, induce osteogenic differentiation in mesenchymal stromal cells (MSCs). In addition, AML cells inhibited adipogenic differentiation of MSCs. Mechanistic studies identified that AML-derived BMPs activate Smad1/5 signaling to induce osteogenic differentiation in MSCs. Gene expression array analysis revealed that AML cells induce connective tissue growth factor (CTGF) expression in BM-MSCs irrespective of AML type. Overexpression of CTGF in a transgenic mouse model greatly enhanced leukemia engraftment in vivo. Together, our data suggest that AML cells induce a preosteoblast-rich niche in the BM that in turn enhances AML expansion.

3.
J Cell Sci ; 126(Pt 10): 2164-75, 2013 May 15.
Article in English | MEDLINE | ID: mdl-23525012

ABSTRACT

Connective tissue growth factor (CTGF) plays an important role in the pathogenesis of chronic fibrotic diseases. However, the mechanism by which paracrine effects of CTGF control the cell fate of neighboring epithelial cells is not known. In this study, we investigated the paracrine effects of CTGF overexpressed in fibroblasts of Col1a2-CTGF transgenic mice on epithelial cells of skin and lung. The skin and lungs of Col1a2-CTGF transgenic mice were examined for phenotypic markers of epithelial activation and differentiation and stimulation of signal transduction pathways. In addition to an expansion of the dermal compartment in Col1a2-CTGF transgenic mice, the epidermis was characterized by focal hyperplasia, and basal cells stained positive for αSMA, Snail, S100A4 and Sox9, indicating that these cells had undergone a change in their genetic program. Activation of phosphorylated p38 and phosphorylated Erk1/2 was observed in the granular and cornified layers of the skin. Lung fibrosis was associated with a marked increase in cells co-expressing epithelial and mesenchymal markers in the lesional and unaffected lung tissue of Col1a2-CTGF mice. In epithelial cells treated with TGFß, CTGF-specific siRNA-mediated knockdown suppressed Snail, Sox9, S100A4 protein levels and restored E-cadherin levels. Both adenoviral expression of CTGF in epithelial cells and treatment with recombinant CTGF induced EMT-like morphological changes and expression of α-SMA. Our in vivo and in vitro data supports the notion that CTGF expression in mesenchymal cells in the skin and lungs can cause changes in the differentiation program of adjacent epithelial cells. We speculate that these changes might contribute to fibrogenesis.


Subject(s)
Connective Tissue Growth Factor/metabolism , Epithelial-Mesenchymal Transition , Fibroblasts/physiology , Focal Epithelial Hyperplasia/physiopathology , Pulmonary Fibrosis/physiopathology , Animals , Biomarkers/metabolism , Cell Differentiation/genetics , Cells, Cultured , Collagen Type I/genetics , Connective Tissue Growth Factor/genetics , Epithelial-Mesenchymal Transition/genetics , Lung/pathology , MAP Kinase Signaling System/genetics , Mice , Mice, Transgenic , Paracrine Communication , RNA, Small Interfering/genetics , Signal Transduction/genetics , Skin/pathology , Transforming Growth Factor beta/immunology , Transgenes/genetics
4.
Ann Rheum Dis ; 71(5): 761-7, 2012 May.
Article in English | MEDLINE | ID: mdl-22328737

ABSTRACT

OBJECTIVES: Pathologic fibroblast activation drives fibrosis of the skin and internal organs in patients with systemic sclerosis (SSc). ß-catenin is an integral part of adherens junctions and a central component of canonical Wnt signaling. Here, the authors addressed the role of ß-catenin in fibroblasts for the development of SSc dermal fibrosis. METHODS: Nuclear accumulation of ß-catenin in fibroblasts was assessed by triple staining for ß-catenin, prolyl-4-hydroxylase-ß and 4',6-diamidino-2-phenylindole (DAPI). The expression of Wnt proteins in the skin was analysed by real-time PCR and immunohistochemistry. Mice with fibroblast-specific stabilisation or fibroblast-specific depletion were used to evaluate the role of ß-catenin in fibrosis. RESULTS: The auhors found significantly increased nuclear levels of ß-catenin in fibroblasts in SSc skin compared to fibroblasts in the skin of healthy individuals. The accumulation of ß-catenin resulted from increased expression of Wnt-1 and Wnt-10b in SSc. The authors further showed that the nuclear accumulation of ß-catenin has direct implications for the development of fibrosis: Mice with fibroblast-specific stabilisation of ß-catenin rapidly developed fibrosis within 2 weeks with dermal thickening, accumulation of collagen and differentiation of resting fibroblasts into myofibroblasts. By contrast, fibroblast-specific deletion of ß-catenin significantly reduced bleomycin-induced dermal fibrosis. CONCLUSIONS: The present study findings identify ß-catenin as a key player of fibroblast activation and tissue fibrosis in SSc. Although further translational studies are necessary to test the efficacy and tolerability of ß-catenin/Wnt inhibition in SSc, the present findings may have clinical implications, because selective inhibitors of ß-catenin/Wnt signaling have recently entered clinical trials.


Subject(s)
Fibroblasts/metabolism , Fibrosis/metabolism , Scleroderma, Systemic/metabolism , Wnt Proteins/metabolism , Wnt Signaling Pathway , beta Catenin/metabolism , Adult , Aged , Cell Nucleus/metabolism , Cell Nucleus/pathology , Female , Fibroblasts/pathology , Fibrosis/pathology , Humans , Male , Middle Aged , Scleroderma, Systemic/pathology , Skin/metabolism , Skin/pathology , Wnt Proteins/genetics , Young Adult
5.
Matrix Biol ; 30(7-8): 396-403, 2011 Sep.
Article in English | MEDLINE | ID: mdl-21864682

ABSTRACT

Accumulation of type I collagen is a key event in renal interstitial fibrosis. As there is no effective treatment, understanding the site where collagen is transcribed and the factors driving it in response to disease in vivo is critical for designing future therapies. The present research investigated the transcriptional activity of the COL1A2 gene in a mouse model of progressive fibrosis induced by aristolochic acid (aristolochic acid nephropathy, AAN). To achieve this we genetically modified mice to express a reporter gene (LacZ) and CCN2 (connective tissue growth factor) under the transcriptional control of the COL1A2 promoter /enhancer sequences. Using these mice we asked where is collagen actively transcribed and secondly, what is the role of CCN2 in AAN. Here, we report that de-novo transcription of the COL1A2 gene occurred predominantly in damaged tubular epithelial cells during progressive interstitial fibrosis in vivo. The activation of COL1A2 was studied by detection of the reporter gene LacZ and COL1A2 mRNA in interstitial, glomerular, vascular, and tubular epithelial tissue from laser capture microscopy. We also demonstrated that LacZ-positive cells co-express E-Cadherin a marker of epithelial origin which is consistent with an epithelial phenotype which is capable of collagen expression during injury. There was no evidence of detachment of these cells from tubules to become myofibroblasts. Moreover, we showed that the transgenic mice show a modest enhancement of CCN2 expression; however fibrosis induced by AA is the same in transgenics and controls suggesting that CCN2, at this level of expression, is not sufficient to enhance fibrogenesis. Overall our study provides a better understanding into the expression patterns and roles of two major extracellular matrix proteins: type I collagen and CCN2.


Subject(s)
Aristolochic Acids/adverse effects , Collagen Type I/metabolism , Epithelial Cells/metabolism , Fibrosis/pathology , Kidney/pathology , Animals , Aristolochic Acids/administration & dosage , Aristolochic Acids/pharmacology , Biomarkers/analysis , Blood Urea Nitrogen , Cadherins/metabolism , Collagen Type I/genetics , Connective Tissue Growth Factor/genetics , Connective Tissue Growth Factor/metabolism , Disease Models, Animal , Disease Progression , Epithelial Cells/drug effects , Epithelial Cells/pathology , Female , Fibrosis/etiology , Fibrosis/metabolism , Kidney Tubules/cytology , Laser Capture Microdissection , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Promoter Regions, Genetic , Transcriptional Activation , Transgenes
6.
Arthritis Res Ther ; 12(2): R60, 2010.
Article in English | MEDLINE | ID: mdl-20359365

ABSTRACT

INTRODUCTION: SPARC is a matricellular protein, which, along with other extracellular matrix components including collagens, is commonly over-expressed in fibrotic diseases. The purpose of this study was to examine whether inhibition of SPARC can regulate collagen expression in vitro and in vivo, and subsequently attenuate fibrotic stimulation by bleomycin in mouse skin and lungs. METHODS: In in vitro studies, skin fibroblasts obtained from a Tgfbr1 knock-in mouse (TBR1CA; Cre-ER) were transfected with SPARC siRNA. Gene and protein expressions of the Col1a2 and the Ctgf were examined by real-time RT-PCR and Western blotting, respectively. In in vivo studies, C57BL/6 mice were induced for skin and lung fibrosis by bleomycin and followed by SPARC siRNA treatment through subcutaneous injection and intratracheal instillation, respectively. The pathological changes of skin and lungs were assessed by hematoxylin and eosin and Masson's trichrome stains. The expression changes of collagen in the tissues were assessed by real-time RT-PCR and non-crosslinked fibrillar collagen content assays. RESULTS: SPARC siRNA significantly reduced gene and protein expression of collagen type 1 in fibroblasts obtained from the TBR1CA; Cre-ER mouse that was induced for constitutively active TGF-beta receptor I. Skin and lung fibrosis induced by bleomycin was markedly reduced by treatment with SPARC siRNA. The anti-fibrotic effect of SPARC siRNA in vivo was accompanied by an inhibition of Ctgf expression in these same tissues. CONCLUSIONS: Specific inhibition of SPARC effectively reduced fibrotic changes in vitro and in vivo. SPARC inhibition may represent a potential therapeutic approach to fibrotic diseases.


Subject(s)
Fibroblasts/pathology , Lung/pathology , Osteonectin/genetics , Pulmonary Fibrosis/pathology , RNA, Small Interfering/genetics , Skin/pathology , Animals , Cells, Cultured , Collagen Type I/genetics , Collagen Type I/metabolism , Connective Tissue Growth Factor/genetics , Connective Tissue Growth Factor/metabolism , Disease Models, Animal , Female , Fibroblasts/drug effects , Fibroblasts/metabolism , Fibrosis , Gene Expression , Gene Knock-In Techniques , Gene Silencing , Lung/drug effects , Lung/metabolism , Mice , Mice, Inbred C57BL , Mice, Transgenic , Osteonectin/antagonists & inhibitors , Protein Serine-Threonine Kinases/genetics , Pulmonary Fibrosis/genetics , Pulmonary Fibrosis/metabolism , RNA, Small Interfering/pharmacology , Receptor, Transforming Growth Factor-beta Type I , Receptors, Transforming Growth Factor beta/genetics , Skin/drug effects , Skin/metabolism , Transfection
7.
Arthritis Rheum ; 62(5): 1523-32, 2010 May.
Article in English | MEDLINE | ID: mdl-20213804

ABSTRACT

OBJECTIVE: Connective tissue growth factor (CTGF) is a cysteine-rich secreted matricellular protein involved in wound healing and tissue repair. Enhanced and prolonged expression of CTGF has been associated with tissue fibrosis in humans. However, questions remain as to whether CTGF expression alone is sufficient to drive fibrosis. This study was undertaken to investigate whether CTGF alone is sufficient to cause fibrosis in intact animals and whether its effects are mediated through activation of transforming growth factor beta (TGFbeta) signaling or through distinct signal transduction pathways. METHODS: We generated mice overexpressing CTGF in fibroblasts under the control of the fibroblast-specific collagen alpha2(I) promoter enhancer. Tissues such as skin, lung, and kidney were harvested for histologic analysis. Mouse embryonic fibroblasts were prepared from embryos (14.5 days postcoitum) for biochemical analysis. RESULTS: Mice overexpressing CTGF in fibroblasts were susceptible to accelerated tissue fibrosis affecting the skin, lung, kidney, and vasculature, most notably the small arteries. We identified a marked expansion of the myofibroblast cell population in the dermis. RNA analysis of transgenic dermal fibroblasts revealed elevated expression of key matrix genes, consistent with a fibrogenic response. CTGF induced phosphorylation of p38, ERK-1/2, JNK, and Akt, but not Smad3, in transgenic mouse fibroblasts compared with wild-type mouse fibroblasts. Transfection experiments showed significantly increased basal activity of the CTGF and serum response element promoters, and enhanced induction of the CTGF promoter in the presence of TGFbeta. CONCLUSION: These results demonstrate that selective expression of CTGF in fibroblasts alone causes tissue fibrosis in vivo through specific signaling pathways, integrating cues from the extracellular matrix into signal transduction pathways to orchestrate pivotal biologic responses relevant to tissue repair and fibrosis.


Subject(s)
Connective Tissue Growth Factor/genetics , Connective Tissue Growth Factor/metabolism , Fibroblasts/pathology , Liver Cirrhosis , Pulmonary Fibrosis , Animals , Basement Membrane/pathology , Blood Vessels/pathology , Cell Differentiation/physiology , Collagen/genetics , Collagen Type I , Fibroblasts/metabolism , Gene Expression/physiology , Glomerulosclerosis, Focal Segmental/metabolism , Glomerulosclerosis, Focal Segmental/pathology , Glomerulosclerosis, Focal Segmental/physiopathology , Kidney/pathology , Lac Operon , Liver Cirrhosis/metabolism , Liver Cirrhosis/pathology , Liver Cirrhosis/physiopathology , Lymphokines , Mice , Mice, Inbred C57BL , Mice, Inbred CBA , Mice, Transgenic , Promoter Regions, Genetic/physiology , Pulmonary Fibrosis/metabolism , Pulmonary Fibrosis/pathology , Pulmonary Fibrosis/physiopathology , Signal Transduction/physiology , Skin/pathology , Transforming Growth Factor beta/metabolism
8.
PLoS One ; 4(10): e7438, 2009 Oct 13.
Article in English | MEDLINE | ID: mdl-19823586

ABSTRACT

BACKGROUND: Fibrosis, the excessive deposition of scar tissue by fibroblasts, is one of the largest groups of diseases for which there is no therapy. Fibroblasts from lesional areas of scleroderma patients possess elevated abilities to contract matrix and produce alpha-smooth muscle actin (alpha-SMA), type I collagen and CCN2 (connective tissue growth factor, CTGF). The basis for this phenomenon is poorly understood, and is a necessary prerequisite for developing novel, rational anti-fibrotic strategies. METHODS AND FINDINGS: Compared to healthy skin fibroblasts, dermal fibroblasts cultured from lesional areas of scleroderma (SSc) patients possess elevated Rac activity. NSC23766, a Rac inhibitor, suppressed the persistent fibrotic phenotype of lesional SSc fibroblasts. NSC23766 caused a decrease in migration on and contraction of matrix, and alpha-SMA, type I collagen and CCN2 mRNA and protein expression. SSc fibroblasts possessed elevated Akt phosphorylation, which was also blocked by NSC23766. Overexpression of rac1 in normal fibroblasts induced matrix contraction and alpha-SMA, type I collagen and CCN2 mRNA and protein expression. Rac1 activity was blocked by PI3kinase/Akt inhibition. Basal fibroblast activity was not affected by NSC23766. CONCLUSION: Rac inhibition may be considered as a novel treatment for the fibrosis observed in SSc.


Subject(s)
Actins/metabolism , Fibroblasts/metabolism , Fibroblasts/pathology , Fibrosis , Gene Expression Regulation , Muscle, Smooth/metabolism , rac GTP-Binding Proteins/metabolism , rac1 GTP-Binding Protein/antagonists & inhibitors , rac1 GTP-Binding Protein/metabolism , Cell Movement , Collagen/metabolism , Connective Tissue Growth Factor/metabolism , Fibrosis/metabolism , Humans , Phenotype , Phosphorylation , Proto-Oncogene Proteins c-akt/metabolism , Scleroderma, Systemic/metabolism , Scleroderma, Systemic/pathology , rac GTP-Binding Proteins/antagonists & inhibitors
9.
Curr Opin Rheumatol ; 21(6): 630-5, 2009 Nov.
Article in English | MEDLINE | ID: mdl-19730378

ABSTRACT

PURPOSE OF REVIEW: The underlying pathogenesis of systemic sclerosis (SSc; scleroderma) involves a complex interplay of inflammation, fibrosis and vasculopathy that is incompletely understood. In this article, we highlight the important contributions that recent preclinical research has made to the knowledge base of pathogenesis and therapeutics in SSc, describe some of the newly developed models available for further investigation and discuss future research opportunities in this fascinating area. RECENT FINDINGS: Several well characterized SSc models are available for the study of fibrosis. However, recent study on transgenic and knockout models has advanced knowledge both in fibrosis research and in vascular disease in SSc. In the present review, we focus on models in which altered signalling, particularly transforming growth factor-beta (TGF-beta), is limited to fibroblasts. We discuss contemporary models of SSc vascular disease, transgenesis in fibrocyte research, the contribution to neurological signalling research and provide examples of how preclinical models have contributed to novel therapeutics development in SSc. We also look at how research from related disciplines impacts on the SSc knowledge base. SUMMARY: These new models represent exciting advances. However, none completely recapitulates the vasculopathic and inflammatory components of this disease. These advances help to delineate the relative contributions of specific ligands, receptors, their signalling pathways and feedback mechanisms, in fibrotic and inflammatory processes and this will provide new targets for potential therapies in SSc.


Subject(s)
Scleroderma, Systemic/etiology , Animals , Collagen/genetics , Disease Models, Animal , Fibroblasts/metabolism , Fibroblasts/pathology , Fibrosis , Hematopoietic Stem Cells/metabolism , Hematopoietic Stem Cells/pathology , Mice , Mice, Knockout , Mice, Transgenic , Models, Biological , Neuropeptides/metabolism , Platelet-Derived Growth Factor/metabolism , Scleroderma, Systemic/metabolism , Scleroderma, Systemic/pathology , Scleroderma, Systemic/therapy , Signal Transduction , Transforming Growth Factor beta/metabolism , Vascular Diseases/etiology , Vascular Diseases/pathology
10.
Arthritis Rheum ; 56(1): 334-44, 2007 Jan.
Article in English | MEDLINE | ID: mdl-17195237

ABSTRACT

OBJECTIVE: Increased signaling by transforming growth factor beta (TGFbeta) has been implicated in systemic sclerosis (SSc; scleroderma), a complex disorder of connective tissues characterized by excessive accumulation of collagen and other extracellular matrix components in systemic organs. To directly assess the effect of sustained TGFbeta signaling in SSc, we established a novel mouse model in which the TGFbeta signaling pathway is activated in fibroblasts postnatally. METHODS: The mice we used (termed TBR1(CA); Cre-ER mice) harbor both the DNA for an inducible constitutively active TGFbeta receptor I (TGFbetaRI) mutation, which has been targeted to the ROSA locus, and a Cre-ER transgene that is driven by a fibroblast-specific promoter. Administration of 4-hydroxytamoxifen 2 weeks after birth activates the expression of constitutively active TGFbetaRI. RESULTS: These mice recapitulated clinical, histologic, and biochemical features of human SSc, showing pronounced and generalized fibrosis of the dermis, thinner epidermis, loss of hair follicles, and fibrotic thickening of small blood vessel walls in the lung and kidney. Primary skin fibroblasts from these mice showed elevated expression of downstream TGFbeta targets, reproducing the hallmark biochemical phenotype of explanted SSc dermal fibroblasts. The mouse fibroblasts also showed elevated basal expression of the TGFbeta-regulated promoters plasminogen activator inhibitor 1 and 3TP, increased Smad2/3 phosphorylation, and enhanced myofibroblast differentiation. CONCLUSION: Constitutive activation of TGFbeta signaling in fibroblastic cells of mice after birth caused a marked fibrotic phenotype characteristic of SSc. These mice should be excellent models with which to test therapies aimed at correcting excessive TGFbeta signaling in human scleroderma.


Subject(s)
Disease Models, Animal , Fibroblasts/metabolism , Scleroderma, Systemic/metabolism , Transforming Growth Factor beta/metabolism , Activin Receptors, Type I/genetics , Activin Receptors, Type I/metabolism , Animals , Cell Differentiation/drug effects , Cells, Cultured , Female , Fibroblasts/drug effects , Fibroblasts/pathology , Gene Targeting/methods , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Phosphorylation , Plasminogen Activator Inhibitor 1/metabolism , Protein C Inhibitor/metabolism , Protein Serine-Threonine Kinases , Receptor, Transforming Growth Factor-beta Type I , Receptors, Transforming Growth Factor beta/genetics , Receptors, Transforming Growth Factor beta/metabolism , Scleroderma, Systemic/genetics , Scleroderma, Systemic/pathology , Signal Transduction , Skin/drug effects , Skin/metabolism , Skin/pathology , Smad2 Protein/metabolism , Smad3 Protein/metabolism , Tamoxifen/analogs & derivatives , Tamoxifen/pharmacology , Transforming Growth Factor beta/genetics
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