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Daniel B. Rifkin - One of the best experts on this subject based on the ideXlab platform.
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An Osteoblast-Derived Proteinase Controls Tumor Cell Survival via TGF-Beta Activation in the Bone Microenvironment
PloS one, 2012Co-Authors: Sophie Thiolloy, Daniel B. Rifkin, James R. Edwards, Barbara Fingleton, Lynn M. Matrisian, Conor C. LynchAbstract:Background Breast to bone metastases frequently induce a “vicious cycle” in which osteoclast mediated bone resorption and proteolysis results in the release of bone matrix sequestered factors that drive tumor growth. While osteoclasts express numerous proteinases, analysis of human breast to bone metastases unexpectedly revealed that bone forming osteoblasts were consistently positive for the proteinase, MMP-2. Given the role of MMP-2 in extracellular matrix degradation and growth factor/cytokine processing, we tested whether osteoblast derived MMP-2 contributed to the vicious cycle of tumor progression in the bone microenvironment. Methodology/Principal Findings To test our hypothesis, we utilized murine models of the osteolytic tumor-bone microenvironment in immunocompetent wild type and MMP-2 null mice. In longitudinal studies, we found that host MMP-2 significantly contributed to tumor progression in bone by protecting against apoptosis and promoting cancer cell survival (caspase-3; immunohistochemistry). Our data also indicate that host MMP-2 contributes to tumor induced osteolysis (μCT, histomorphometry). Further ex vivo/in vitro experiments with wild type and MMP-2 null osteoclast and osteoblast cultures identified that 1) the absence of MMP-2 did not have a deleterious effect on osteoclast function (cd11B isolation, osteoclast differentiation, transwell migration and dentin resorption assay); and 2) that osteoblast derived MMP-2 promoted tumor survival by regulating the bioavailability of TGFβ, a factor critical for cell-cell communication in the bone (ELISA, immunoblot assay, clonal and soft agar assays). Conclusion/Significance Collectively, these studies identify a novel “mini-vicious cycle” between the osteoblast and metastatic cancer cells that is key for initial tumor survival in the bone microenvironment. In conclusion, the findings of our study suggest that the targeted inhibition of MMP-2 and/or TGFβ would be beneficial for the treatment of bone metastases.
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Tamoxifen and Estrogen Effects on TGF-β Formation: Role of Thrombospondin-1, αvβ3, and Integrin-Associated Protein
Biochemical and biophysical research communications, 2001Co-Authors: John G. Harpel, Stacey Schultz-cherry, Joanne E. Murphy-ullrich, Daniel B. RifkinAbstract:We have found that the enhanced Activation of latent TGF-Beta by human breast carcinoma cell lines either treated with tamoxifen or deprived of estrogen is dependent upon thrombospondin-1 (TSP-1) since Activation was blocked by anti-TSP-1 antibodies or by a TSP antagonist peptide. However, TGF-Beta formation upon tamoxifen exposure to estrogen withdrawal is associated with decreased levels of soluble TSP-1. A concomitant increase in the expression of the TSP-1 receptors alphavBeta3 and integrin-associated protein (IAP) occurs under these conditions, and antibodies to TSP-1 or to these receptors inhibit increased TGF-Beta formation. Therefore, increased cell surface associated TSP-1 enhances latent TGF-Beta Activation.
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Structure and Activation of the large latent transforming growth factor-Beta complex.
Journal of the American Optometric Association, 1998Co-Authors: Nunes I, Richard L. Shapiro, Pierre-emmanuel Gleizes, John S. Munger, Nagano Y, Daniel B. RifkinAbstract:BACKGROUND Many cytokines regulate processes involved in the pathogenesis of proliferative vitreoretinopathy. Transforming growth factor-Beta (TGF-Beta) is an example of a pluripotent growth factor that regulates cell proliferation, extracellular matrix (ECM) deposition, cell migration, and differentiation--all biological activities involved in the formation and progression of proliferative vitreoretinopathies. METHODS A review of experimental results that demonstrate how vascular cells generate biologically active TGF-Beta is presented. Most cell types--including endothelial cells and pericytes, which form the retinal microvasculature--express TGF-Beta as a large latent TGF-Beta complex. Mature TGF-Beta, the biologically active form, must be generated from the large latent complex before it can signal by binding to its high affinity cell surface receptors. RESULTS A critical step in regulating TGF-Beta effects may be the Activation of the large latent TGF-Beta complex. Activation of the complex can be achieved by chemical and enzymatic treatments, or by various cell systems. We have identified that co-culturing bovine smooth muscle cells or pericytes and endothelial cells generates active TGF-Beta. CONCLUSION The mechanism of latent TGF-Beta Activation self-regulates through effectors of plasmin generation. Studying TGF-Beta generation by co-cultures of pericytes and endothelial cells can provide us with insights into how disruption of latent TGF-Beta Activation may lead to unregulated endothelial proliferation, ECM deposition, and cellular infiltration, as observed clinically in neovascular- and fibrotic-related pathologies.
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TGF‐β Latency: Biological Significance and Mechanisms of Activation
Stem cells (Dayton Ohio), 1997Co-Authors: Pierre-emmanuel Gleizes, John S. Munger, Irene Nunes, John G. Harpel, Roberta Mazzieri, Irene Noguera, Daniel B. RifkinAbstract:Transforming growth factor (TGF-) Beta is secreted as a latent complex in which the mature growth factor remains associated with its propeptide. In order to elicit a biological response, the cytokine must be released from the latent complex, a process termed latent TGF-Beta Activation or TGF-Beta formation. Although latent TGF-Beta Activation is a critical step in the regulation of its activity, little is known about the molecular mechanisms that lead to the production of active TGF-Beta. In this article, we present an overview of the data available on this topic, and we propose a tentative model for the mechanism of TGF-Beta formation based upon the observations with different cell systems and on recent findings on the structure of the latent TGF-Beta complex.
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Characterization of latent TGF-Beta Activation by murine peritoneal macrophages.
Journal of immunology (Baltimore Md. : 1950), 1995Co-Authors: Nunes I, Richard L. Shapiro, Daniel B. RifkinAbstract:Transforming growth factor-Beta (TGF-Beta) is secreted by most cells as a biologically inactive complex, called the large latent TGF-Beta complex. The complex is comprised of latent TGF-Beta binding protein (LTBP) and latent TGF-Beta, which is mature TGF-Beta associated noncovalently with its amino-terminal propeptides. LTBP is disulfide-linked to the amino-terminal propeptide of latent TGF-Beta. Active TGF-Beta is generated by release of TGF-Beta from the complex. Generation of active TGF-Beta by macrophages has been reported, but the Activation mechanism has not been described. Latent TGF-Beta Activation by macrophages was characterized using serum-free cultures of resident and thioglycollate-elicited murine peritoneal macrophages that were either unstimulated or LPS-stimulated in vitro. Serum-free conditioned medium was assayed for TGF-Beta using a quantitative luciferase-based bioassay. LPS-stimulated thioglycollate-elicited macrophages activated endogenous latent TGF-Beta, whereas non-LPS-stimulated thioglycollate-elicited and resident macrophages generated undetectable levels of TGF-Beta. Latent TGF-Beta Activation required plasmin and urokinase (uPA), uPA binding to the uPA receptor, interaction with the cation-independent mannose 6-phosphate/insulin-like growth factor type II receptor, tissue type II transglutaminase, and LTBP. A time-course analysis of latent TGF-Beta Activation revealed that maximal TGF-Beta was generated after 24 h (25 +/- 5 pg/ml). TGF-Beta formed within the initial 24 h modulated the plasminogen activator system by down-regulating uPA, suggesting that TGF-Beta temporally modulated its own formation by regulating cell-associated uPA.
Joanne E. Murphy-ullrich - One of the best experts on this subject based on the ideXlab platform.
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Stainless steel ions stimulate increased thrombospondin-1-dependent TGF-Beta Activation by vascular smooth muscle cells: implications for in-stent restenosis.
Journal of vascular research, 2009Co-Authors: Manuel A. Pallero, Melissa Talbert Roden, Yiu-fai Chen, Peter G. Anderson, Jack E. Lemons, Brigitta C. Brott, Joanne E. Murphy-ullrichAbstract:Background/Aims: Despite advances in stent design, in-stent restenosis (ISR) remains a significant clinical problem. All implant metals exhibit corrosion, which results in release o
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Tamoxifen and Estrogen Effects on TGF-β Formation: Role of Thrombospondin-1, αvβ3, and Integrin-Associated Protein
Biochemical and biophysical research communications, 2001Co-Authors: John G. Harpel, Stacey Schultz-cherry, Joanne E. Murphy-ullrich, Daniel B. RifkinAbstract:We have found that the enhanced Activation of latent TGF-Beta by human breast carcinoma cell lines either treated with tamoxifen or deprived of estrogen is dependent upon thrombospondin-1 (TSP-1) since Activation was blocked by anti-TSP-1 antibodies or by a TSP antagonist peptide. However, TGF-Beta formation upon tamoxifen exposure to estrogen withdrawal is associated with decreased levels of soluble TSP-1. A concomitant increase in the expression of the TSP-1 receptors alphavBeta3 and integrin-associated protein (IAP) occurs under these conditions, and antibodies to TSP-1 or to these receptors inhibit increased TGF-Beta formation. Therefore, increased cell surface associated TSP-1 enhances latent TGF-Beta Activation.
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Thrombospondin causes Activation of latent transforming growth factor-Beta secreted by endothelial cells by a novel mechanism.
The Journal of cell biology, 1993Co-Authors: Stacey Schultz-cherry, Joanne E. Murphy-ullrichAbstract:Thrombospondin (TSP) forms specific complexes with transforming growth factor-Beta (TGF-Beta) in the alpha granule releasate of platelets and these TSP-TGF-Beta complexes inhibit the growth of bovine aortic endothelial cells (BAE). In these studies, we report that TSP stripped of associated TGF-Beta (sTSP) retained growth inhibitory activity which was partially reversed by a neutralizing antibody specific for TGF-Beta. Since BAE cells secrete latent TGF-Beta, we determined whether sTSP activates the latent TGF-Beta secreted by BAE cells. Cells were cultured with or without sTSP and then the conditioned medium was tested for the ability to support TGF-Beta-dependent normal rat kidney (NRK) colony formation in soft agar. Medium conditioned with sTSP showed a dose- and time-dependent ability to stimulate BAE-secreted TGF-Beta activity, reaching maximal Activation by 1-2 h with 0.4 micrograms/ml (0.9 nM) sTSP. The sTSP-mediated stimulation of TGF-Beta activity is not dependent on serum factors and is not a general property of extracellular matrix molecules. The sTSP-mediated stimulation of TGF-Beta activity was blocked by a mAb specific for sTSP and by neutralizing antibodies to TGF-Beta. Activation of BAE cell secreted latent TGF-Beta by sTSP can occur in the absence of cells and apparently does not require interactions with cell surface molecules, since in conditioned medium removed from cells and then incubated with sTSP, Activation occurs with kinetics and at levels similar to what is seen when sTSP is incubated in the presence of cells. Serine proteases such as plasmin are not involved in sTSP-mediated Activation of TGF-Beta. Factors that regulate the conversion of latent to active TGF-Beta are keys to controlling TGF-Beta activity. These data suggest that TSP is a potent physiologic regulator of TGF-Beta Activation.
E J Ehrhart - One of the best experts on this subject based on the ideXlab platform.
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latent transforming growth factor Beta1 Activation in situ quantitative and functional evidence after low dose gamma irradiation
The FASEB Journal, 1997Co-Authors: E J Ehrhart, Monica L.-s. Tsang, Patricia Segarini, Alan G Carroll, Mary Helen BarcelloshoffAbstract:The biological activity of transforming growth factor Beta1 (TGF-Beta) is controlled by its secretion as a latent complex in which it is noncovalently associated with latency-associated peptide (LAP). Activation is the extracellular process in which TGF-Beta is released from LAP, and is considered to be a primary regulatory control. We recently reported rapid and persistent changes in TGF-Beta immunoreactivity in conjunction with extracellular matrix remodeling in gamma-irradiated mouse mammary gland. Our hypothesis is that these specific changes in immunoreactivity are indicative of latent TGF-Beta Activation. In the present study, we determined the radiation dose response and tested whether a functional relationship exists between radiation-induced TGF-Beta and collagen type III remodeling. After radiation exposures as low as 0.1 Gy, we detected increased TGF-Beta immunoreactivity in the mammary epithelium concomitant with decreased LAP immunostaining, which are events consistent with Activation. Quanti...
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Latent transforming growth factor Beta1 Activation in situ: quantitative and functional evidence after low-dose gamma-irradiation.
FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 1997Co-Authors: E J Ehrhart, Monica L.-s. Tsang, Patricia Segarini, Alan G Carroll, Mary Helen Barcellos-hoffAbstract:The biological activity of transforming growth factor Beta1 (TGF-Beta) is controlled by its secretion as a latent complex in which it is noncovalently associated with latency-associated peptide (LAP). Activation is the extracellular process in which TGF-Beta is released from LAP, and is considered to be a primary regulatory control. We recently reported rapid and persistent changes in TGF-Beta immunoreactivity in conjunction with extracellular matrix remodeling in gamma-irradiated mouse mammary gland. Our hypothesis is that these specific changes in immunoreactivity are indicative of latent TGF-Beta Activation. In the present study, we determined the radiation dose response and tested whether a functional relationship exists between radiation-induced TGF-Beta and collagen type III remodeling. After radiation exposures as low as 0.1 Gy, we detected increased TGF-Beta immunoreactivity in the mammary epithelium concomitant with decreased LAP immunostaining, which are events consistent with Activation. Quantitative image analysis demonstrated a significant (P=0.0005) response at 0.1 Gy without an apparent threshold and a linear dose response to 5 Gy. However, in the adipose stroma, loss of LAP demonstrated a qualitative threshold at 0.5 Gy. Loss of LAP paralleled induction of collagen III immunoreactivity in this tissue compartment. We tested whether TGF-Beta mediates collagen III expression by treating animals with TGF-Beta panspecific monoclonal antibody, 1D11.16, administered i.p. shortly before irradiation. Radiation-induced collagen III staining in the adipose stroma was blocked in an antibody dose-dependent manner, which persisted through 7 days postirradiation. RNase protection assay revealed that radiation-induced elevation of total gland collagen III mRNA was also blocked by neutralizing antibody treatment. These data provide functional confirmation of the hypothesis that radiation exposure leads to latent TGF-Beta Activation, support our interpretation of the reciprocal shift in immunoreactivity as evidence of Activation, and implicate TGF-Beta as a mediator of tissue response to ionizing radiation. The sensitivity of Activation to low radiation doses points to a potential role for TGF-Beta in orchestrating tissue response to oxidative stress. As such, radiation may be useful as a probe to delineate the consequences of latent TGF-Beta Activation in situ.
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Immunohistochemical detection of active transforming growth factor-Beta in situ using engineered tissue
The American journal of pathology, 1995Co-Authors: Mary Helen Barcellos-hoff, E J Ehrhart, M. Kalia, R. Jirtle, K. Flanders, M. L.-s. TsangAbstract:The biological activity of transforming growth factor-Beta 1 (TGF-Beta) is governed by dissociation from its latent complex. Immunohistochemical discrimination of active and latent TGF-Beta could provide insight into TGF-Beta Activation in physiological and pathological processes. However, evaluation of immunoreactivity specificity in situ has been hindered by the lack of tissue in which TGF-Beta status is known. To provide in situ analysis of antibodies to differentiate between these functional forms, we used xenografts of human tumor cells modified by transfection to overexpress latent TGF-Beta or constitutively active TGF-Beta. This comparison revealed that, whereas most antibodies did not differentiate between TGF-Beta Activation status, the immunoreactivity of some antibodies was Activation dependent. Two widely used peptide antibodies to the amino-terminus of TGF-Beta, LC(1-30) and CC(1-30) showed marked preferential immunoreactivity with active TGF-Beta versus latent TGF-Beta in cryosections. However, in formalin-fixed, paraffin-embedded tissue, discrimination of active TGF-Beta by CC(1-30) was lost and immunoreactivity was distinctly extracellular, as previously reported for this antibody. Similar processing-dependent extracellular localization was found with a neutralizing antibody raised to recombinant TGF-Beta. Antigen retrieval recovered cell-associated immunoreactivity of both antibodies. Two antibodies to peptides 78-109 showed mild to moderate preferential immunoreactivity with active TGF-Beta only in paraffin sections. LC(1-30) was the only antibody tested that discriminated active from latent TGF-Beta in both frozen and paraffin-embedded tissue. Thus, in situ discrimination of active versus latent TGF-Beta depends on both the antibody and tissue preparation. We propose that tissues engineered to express a specific form of a given protein provide a physiological setting in which to evaluate antibody reactivity with specific functional forms of a protein.
Cay M Kielty - One of the best experts on this subject based on the ideXlab platform.
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expression of latent TGF Beta binding proteins and association with TGF Beta1 and fibrillin 1 following arterial injury
Cardiovascular Research, 2002Co-Authors: Sanjay Sinha, A M Heagerty, Adrian C Shuttleworth, Cay M KieltyAbstract:OBJECTIVES: Transforming growth factor-Beta (TGF-Beta), a potent regulator of wound healing and scar formation, is thought to have a key role in the response to arterial injury. Latent TGF-Beta binding proteins (LTBPs), members of the fibrillin superfamily, govern TGF-Beta1 release, targeting and Activation in vitro and also play a role as structural components of fibrillin-rich microfibrils. Despite the potential of LTBPs to modulate the response to arterial injury through either or both of these mechanisms, as yet their expression and function in the injured vasculature remain poorly defined. METHODS: In this study, a porcine model of coronary angioplasty was used to investigate LTBP-1 and LTBP-2 synthesis and their association with TGF-Beta 1 and fibrillin-1. RESULTS: After angioplasty, increased LTBP-1 and LTBP-2 immunostaining was detected in a similar distribution to increased TGF-Beta 1 expression in the neointima and in the neoadventitia. Overnight organ cultures revealed the formation of large latent TGF-Beta 1 complexes containing LTBP-1. Increased LTBP-1 proteolysis after arterial injury correlated with increased active and latent TGF-Beta levels. LTBP-2 synthesis increased in response to arterial injury but was neither present in large latent complexes nor proteolytically processed. LTBP-1 and LTBP-2 both co-localised to fibrillin-rich fibrillar structures in the neointima and adventitia. CONCLUSIONS: These data suggest that LTBP-1 may have a TGF-Beta 1 binding role in the arterial response to injury, and that LTBP-1 and LTBP-2 may have a structural role in association with microfibrils within the developing neointimal lesion. LTBP-1 proteolysis is potentially an important regulatory step for TGF-Beta Activation in the vasculature and inhibition of proteolysis could represent a novel therapeutic modality for controlling the arterial injury response.
Mary Helen Barcelloshoff - One of the best experts on this subject based on the ideXlab platform.
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latent transforming growth factor Beta1 Activation in situ quantitative and functional evidence after low dose gamma irradiation
The FASEB Journal, 1997Co-Authors: E J Ehrhart, Monica L.-s. Tsang, Patricia Segarini, Alan G Carroll, Mary Helen BarcelloshoffAbstract:The biological activity of transforming growth factor Beta1 (TGF-Beta) is controlled by its secretion as a latent complex in which it is noncovalently associated with latency-associated peptide (LAP). Activation is the extracellular process in which TGF-Beta is released from LAP, and is considered to be a primary regulatory control. We recently reported rapid and persistent changes in TGF-Beta immunoreactivity in conjunction with extracellular matrix remodeling in gamma-irradiated mouse mammary gland. Our hypothesis is that these specific changes in immunoreactivity are indicative of latent TGF-Beta Activation. In the present study, we determined the radiation dose response and tested whether a functional relationship exists between radiation-induced TGF-Beta and collagen type III remodeling. After radiation exposures as low as 0.1 Gy, we detected increased TGF-Beta immunoreactivity in the mammary epithelium concomitant with decreased LAP immunostaining, which are events consistent with Activation. Quanti...