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Donna M Peters - One of the best experts on this subject based on the ideXlab platform.
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Glaucomatous MYOC mutations activate the IL-1/NF-κB inflammatory stress response and the glaucoma marker SELE in trabecular meshwork cells.
Molecular vision, 2015Co-Authors: Tatsuo Itakura, Donna M Peters, M. Elizabeth FiniAbstract:Purpose Activation of the IL-1/NF-κB inflammatory stress pathway and induction of SELE expression in the trabecular meshwork (TBM) is a marker for high-tension glaucomas of diverse etiology. Pathway activation stimulates aqueous outflow and protects against oxidative stress, but may be damaging in the long-term. MYOC mutations have been causally linked to high-tension forms of primary open angle glaucoma (POAG). This study investigated a possible link between MYOC mutations and activation of the IL-1/NF-κB pathway and expression of SELE. Methods We constructed MYOC expression vectors with mutations at sites that cause POAG. Mutations (Q368X, Y437H, A427T) were selected to represent proteins with differing POAG-causing potency (Q368X > Y437H > A427T) and intracellular retention behavior (Q368X and Y437H retained, A427T released). The constructs were made in two different kinds of vectors; one a plasmid designed for transient transfection (pCMV6), and one a doxycycline-inducible lentiviral vector (pSLIK) for stable cell transduction. The immortalized human trabecular meshwork line TM-1 was used for all expression studies. Expression of IL1A mRNA was determined by reverse transcription (RT)-PCR, as well as a set of five other genes associated with signaling pathways linked to glaucoma: IL1B and IL6 (NF-κB pathway), TGFB2 and ACTA2 (TGF-β pathway) and FOXO1 (E2F1 apoptotic pathway). An ELISA was used to quantify IL1A protein released into culture media. To quantify intracellular NF-κB activity, we transiently transfected stably transduced cell lines with a luciferase expression vector under control of the IL8 promoter (containing an NF-κB response element). Results Transiently expressed wild-type MYOC was released into cell culture media, whereas mutant MYOCs Q368X and Y437H remained within cells. Both mutant MYOCs activated the IL-1/ NF-κB pathway, significantly stimulating expression of IL1A and IL1B. However Y437H, which causes a severe glaucoma phenotype, was less effective than Q368X, which causes a moderate glaucoma phenotype. In addition, the retained mutants stimulated expression of stress response genes ACTA2 and FOXO1. Unexpectedly, wild-type MYOC significantly decreased expression of IL6 and TGFB2, to approximately half of the control levels, and expression of IL1B and ACTA2 was also slightly decreased. Induction of MYOC mutants Q368X and Y437H in stably transduced cell lines significantly stimulated the level of IL1A protein released into culture media. Once again however, the effect of the severe MYOC mutant Y437H was less than the effect of the moderate MYOC mutant Q368X. In contrast, induced expression of the intracellularly retained mutant MYOC A427T or wild-type MYOC did not change the amount of IL1A protein in culture media. Induction of Y437H MYOC plus IL1A treatment increased NF-κB activity by 25% over IL1A alone. In contrast, induction of Q368X or A427T plus IL1A treatment did not significantly affect NF-κB activity over IL1A alone. However, wild-type MYOC expression inhibited IL1A-stimulated NF-κB activity. We also observed that endogenous MYOC expression was induced by IL1A in TM-1 cells and primary TBM cell cultures. SELE was co-expressed with MYOC in the primary cell lines. Conclusions These results indicate that POAG-causing MYOC mutants activate the IL-1/NF-κB pathway, with activation levels correlated with intracellular retention of the protein, but not POAG-causing potency. Unexpectedly, it was also discovered that wild-type MYOC inhibits activation of the IL-1/NF-κB pathway, and that activation of the IL-1/NF-κB pathway stimulates expression of MYOC. This is the first evidence that glaucoma-causing MYOC mutants can activate the inflammatory response and that wild-type MYOC has anti-inflammatory activity.
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in vitro localization of tigr MYOC in trabecular meshwork extracellular matrix and binding to fibronectin
Investigative Ophthalmology & Visual Science, 2002Co-Authors: Mark S Filla, Thai Nguyen, Jon R Polansky, Curtis R Brandt, Paul L Kaufman, Donna M PetersAbstract:PURPOSE. To determine whether trabecular meshwork-inducible glucocorticoid response/MYOCilin (TIGR/MYOC) protein associates with the extracellular matrix (ECM) of human trabecular meshwork (HTM) cells. METHODS. The extracellular localization of TIGR/MYOC was examined by immunofluorescence microscopy in HTM cultures treated with and without dexamethasone and ascorbate and in a transformed HTM cell line, TM-1, transiently transfected with TIGR/MYOC cDNA. Antibodies to TIGR/MYOC, fibronectin, laminin, type IV collagen, or thrombospondin were used to determine the extracellular localization of TIGR/ MYOC. Solid phase binding assays using 125 I-recombinant TIGR/MYOC and types I and IV collagens, fibronectin, and laminin were done to examine the association of TIGR/MYOC with these proteins and to identify a specific TIGR/MYOC binding site within fibronectin. The domains of fibronectin tested were the fibrin/collagen binding domain, the RGD domain, and the Heparin II (Hep II) domain. RESULTS. TIGR/MYOC colocalized with fibronectin, laminin, and type IV collagen, but not thrombospondin in both dexamethasone and dexamethasone/ascorbate-treated HTM cultures and in TM-1 cultures transfected with TIGR/MYOC cl)NA. In solid phase binding assays, 125 I-TIGR/MYOC bound fibronectin but not laminin or type IV collagen. Binding to fibronectin could be competed with excess TIGR/MYOC or fibronectin. Specific binding was found for the Hep II domain of fibronectin. CONCLUSIONS. TIGR/MYOC can associate with components of the ECM via interactions with the Hep II domain of fibronectin. The interactions with the Hep II domain of fibronectin could alter cell-matrix interactions in the 'I'M and provides an interesting lead to explore the role(s) of TIGR/MYOC in both steroid-induced and primary open angle glaucoma.
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In vitro localization of TIGR/MYOC in trabecular meshwork extracellular matrix and binding to fibronectin.
Investigative ophthalmology & visual science, 2002Co-Authors: Mark S Filla, Thai Nguyen, Jon R Polansky, Curtis R Brandt, Paul L Kaufman, Xuyang Liu, Donna M PetersAbstract:PURPOSE. To determine whether trabecular meshwork-inducible glucocorticoid response/MYOCilin (TIGR/MYOC) protein associates with the extracellular matrix (ECM) of human trabecular meshwork (HTM) cells. METHODS. The extracellular localization of TIGR/MYOC was examined by immunofluorescence microscopy in HTM cultures treated with and without dexamethasone and ascorbate and in a transformed HTM cell line, TM-1, transiently transfected with TIGR/MYOC cDNA. Antibodies to TIGR/MYOC, fibronectin, laminin, type IV collagen, or thrombospondin were used to determine the extracellular localization of TIGR/ MYOC. Solid phase binding assays using 125 I-recombinant TIGR/MYOC and types I and IV collagens, fibronectin, and laminin were done to examine the association of TIGR/MYOC with these proteins and to identify a specific TIGR/MYOC binding site within fibronectin. The domains of fibronectin tested were the fibrin/collagen binding domain, the RGD domain, and the Heparin II (Hep II) domain. RESULTS. TIGR/MYOC colocalized with fibronectin, laminin, and type IV collagen, but not thrombospondin in both dexamethasone and dexamethasone/ascorbate-treated HTM cultures and in TM-1 cultures transfected with TIGR/MYOC cl)NA. In solid phase binding assays, 125 I-TIGR/MYOC bound fibronectin but not laminin or type IV collagen. Binding to fibronectin could be competed with excess TIGR/MYOC or fibronectin. Specific binding was found for the Hep II domain of fibronectin. CONCLUSIONS. TIGR/MYOC can associate with components of the ECM via interactions with the Hep II domain of fibronectin. The interactions with the Hep II domain of fibronectin could alter cell-matrix interactions in the 'I'M and provides an interesting lead to explore the role(s) of TIGR/MYOC in both steroid-induced and primary open angle glaucoma.
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in vitro localization of tigr MYOC in trabecular meshwork extracellular matrix and binding to fibronectin
Investigative Ophthalmology & Visual Science, 2002Co-Authors: Mark S Filla, Thai Nguyen, Jon R Polansky, Curtis R Brandt, Paul L Kaufman, Xuyang Liu, Donna M PetersAbstract:PURPOSE. To determine whether trabecular meshwork-inducible glucocorticoid response/MYOCilin (TIGR/MYOC) protein associates with the extracellular matrix (ECM) of human trabecular meshwork (HTM) cells. METHODS. The extracellular localization of TIGR/MYOC was examined by immunofluorescence microscopy in HTM cultures treated with and without dexamethasone and ascorbate and in a transformed HTM cell line, TM-1, transiently transfected with TIGR/MYOC cDNA. Antibodies to TIGR/MYOC, fibronectin, laminin, type IV collagen, or thrombospondin were used to determine the extracellular localization of TIGR/ MYOC. Solid phase binding assays using 125 I-recombinant TIGR/MYOC and types I and IV collagens, fibronectin, and laminin were done to examine the association of TIGR/MYOC with these proteins and to identify a specific TIGR/MYOC binding site within fibronectin. The domains of fibronectin tested were the fibrin/collagen binding domain, the RGD domain, and the Heparin II (Hep II) domain. RESULTS. TIGR/MYOC colocalized with fibronectin, laminin, and type IV collagen, but not thrombospondin in both dexamethasone and dexamethasone/ascorbate-treated HTM cultures and in TM-1 cultures transfected with TIGR/MYOC cl)NA. In solid phase binding assays, 125 I-TIGR/MYOC bound fibronectin but not laminin or type IV collagen. Binding to fibronectin could be competed with excess TIGR/MYOC or fibronectin. Specific binding was found for the Hep II domain of fibronectin. CONCLUSIONS. TIGR/MYOC can associate with components of the ECM via interactions with the Hep II domain of fibronectin. The interactions with the Hep II domain of fibronectin could alter cell-matrix interactions in the 'I'M and provides an interesting lead to explore the role(s) of TIGR/MYOC in both steroid-induced and primary open angle glaucoma.
Mark S Filla - One of the best experts on this subject based on the ideXlab platform.
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in vitro localization of tigr MYOC in trabecular meshwork extracellular matrix and binding to fibronectin
Investigative Ophthalmology & Visual Science, 2002Co-Authors: Mark S Filla, Thai Nguyen, Jon R Polansky, Curtis R Brandt, Paul L Kaufman, Donna M PetersAbstract:PURPOSE. To determine whether trabecular meshwork-inducible glucocorticoid response/MYOCilin (TIGR/MYOC) protein associates with the extracellular matrix (ECM) of human trabecular meshwork (HTM) cells. METHODS. The extracellular localization of TIGR/MYOC was examined by immunofluorescence microscopy in HTM cultures treated with and without dexamethasone and ascorbate and in a transformed HTM cell line, TM-1, transiently transfected with TIGR/MYOC cDNA. Antibodies to TIGR/MYOC, fibronectin, laminin, type IV collagen, or thrombospondin were used to determine the extracellular localization of TIGR/ MYOC. Solid phase binding assays using 125 I-recombinant TIGR/MYOC and types I and IV collagens, fibronectin, and laminin were done to examine the association of TIGR/MYOC with these proteins and to identify a specific TIGR/MYOC binding site within fibronectin. The domains of fibronectin tested were the fibrin/collagen binding domain, the RGD domain, and the Heparin II (Hep II) domain. RESULTS. TIGR/MYOC colocalized with fibronectin, laminin, and type IV collagen, but not thrombospondin in both dexamethasone and dexamethasone/ascorbate-treated HTM cultures and in TM-1 cultures transfected with TIGR/MYOC cl)NA. In solid phase binding assays, 125 I-TIGR/MYOC bound fibronectin but not laminin or type IV collagen. Binding to fibronectin could be competed with excess TIGR/MYOC or fibronectin. Specific binding was found for the Hep II domain of fibronectin. CONCLUSIONS. TIGR/MYOC can associate with components of the ECM via interactions with the Hep II domain of fibronectin. The interactions with the Hep II domain of fibronectin could alter cell-matrix interactions in the 'I'M and provides an interesting lead to explore the role(s) of TIGR/MYOC in both steroid-induced and primary open angle glaucoma.
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In vitro localization of TIGR/MYOC in trabecular meshwork extracellular matrix and binding to fibronectin.
Investigative ophthalmology & visual science, 2002Co-Authors: Mark S Filla, Thai Nguyen, Jon R Polansky, Curtis R Brandt, Paul L Kaufman, Xuyang Liu, Donna M PetersAbstract:PURPOSE. To determine whether trabecular meshwork-inducible glucocorticoid response/MYOCilin (TIGR/MYOC) protein associates with the extracellular matrix (ECM) of human trabecular meshwork (HTM) cells. METHODS. The extracellular localization of TIGR/MYOC was examined by immunofluorescence microscopy in HTM cultures treated with and without dexamethasone and ascorbate and in a transformed HTM cell line, TM-1, transiently transfected with TIGR/MYOC cDNA. Antibodies to TIGR/MYOC, fibronectin, laminin, type IV collagen, or thrombospondin were used to determine the extracellular localization of TIGR/ MYOC. Solid phase binding assays using 125 I-recombinant TIGR/MYOC and types I and IV collagens, fibronectin, and laminin were done to examine the association of TIGR/MYOC with these proteins and to identify a specific TIGR/MYOC binding site within fibronectin. The domains of fibronectin tested were the fibrin/collagen binding domain, the RGD domain, and the Heparin II (Hep II) domain. RESULTS. TIGR/MYOC colocalized with fibronectin, laminin, and type IV collagen, but not thrombospondin in both dexamethasone and dexamethasone/ascorbate-treated HTM cultures and in TM-1 cultures transfected with TIGR/MYOC cl)NA. In solid phase binding assays, 125 I-TIGR/MYOC bound fibronectin but not laminin or type IV collagen. Binding to fibronectin could be competed with excess TIGR/MYOC or fibronectin. Specific binding was found for the Hep II domain of fibronectin. CONCLUSIONS. TIGR/MYOC can associate with components of the ECM via interactions with the Hep II domain of fibronectin. The interactions with the Hep II domain of fibronectin could alter cell-matrix interactions in the 'I'M and provides an interesting lead to explore the role(s) of TIGR/MYOC in both steroid-induced and primary open angle glaucoma.
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in vitro localization of tigr MYOC in trabecular meshwork extracellular matrix and binding to fibronectin
Investigative Ophthalmology & Visual Science, 2002Co-Authors: Mark S Filla, Thai Nguyen, Jon R Polansky, Curtis R Brandt, Paul L Kaufman, Xuyang Liu, Donna M PetersAbstract:PURPOSE. To determine whether trabecular meshwork-inducible glucocorticoid response/MYOCilin (TIGR/MYOC) protein associates with the extracellular matrix (ECM) of human trabecular meshwork (HTM) cells. METHODS. The extracellular localization of TIGR/MYOC was examined by immunofluorescence microscopy in HTM cultures treated with and without dexamethasone and ascorbate and in a transformed HTM cell line, TM-1, transiently transfected with TIGR/MYOC cDNA. Antibodies to TIGR/MYOC, fibronectin, laminin, type IV collagen, or thrombospondin were used to determine the extracellular localization of TIGR/ MYOC. Solid phase binding assays using 125 I-recombinant TIGR/MYOC and types I and IV collagens, fibronectin, and laminin were done to examine the association of TIGR/MYOC with these proteins and to identify a specific TIGR/MYOC binding site within fibronectin. The domains of fibronectin tested were the fibrin/collagen binding domain, the RGD domain, and the Heparin II (Hep II) domain. RESULTS. TIGR/MYOC colocalized with fibronectin, laminin, and type IV collagen, but not thrombospondin in both dexamethasone and dexamethasone/ascorbate-treated HTM cultures and in TM-1 cultures transfected with TIGR/MYOC cl)NA. In solid phase binding assays, 125 I-TIGR/MYOC bound fibronectin but not laminin or type IV collagen. Binding to fibronectin could be competed with excess TIGR/MYOC or fibronectin. Specific binding was found for the Hep II domain of fibronectin. CONCLUSIONS. TIGR/MYOC can associate with components of the ECM via interactions with the Hep II domain of fibronectin. The interactions with the Hep II domain of fibronectin could alter cell-matrix interactions in the 'I'M and provides an interesting lead to explore the role(s) of TIGR/MYOC in both steroid-induced and primary open angle glaucoma.
Daniel W Stamer - One of the best experts on this subject based on the ideXlab platform.
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extracellular trafficking of MYOCilin in human trabecular meshwork cells
Journal of Biological Chemistry, 2005Co-Authors: Katharine M. Hardy, Brian S Mckay, Pedro Gonzalez, E.a. Hoffman, Daniel W StamerAbstract:Abstract MYOCilin (MYOC) is a protein with a broad expression pattern, but unknown function. MYOC associates with intracellular structures that are consistent with secretory vesicles, however, in most cell types studied, MYOC is limited to the intracellular compartment. In the trabecular meshwork, MYOC associates with intracellular vesicles, but is also found in the extracellular space. The purpose of the present study was to better understand the mechanism of extracellular transport of MYOC in trabecular meshwork cells. Using a biochemical approach, we found that MYOC localizes intracellularly to both the cytosolic and particulate fractions. When intracellular membranes were separated over a linear sucrose gradient, MYOC equilibrated in a fraction less dense than traditional secretory vesicles and lysosomes. In pulse-labeling experiments that followed nascent MYOC over time, the characteristic doublet observed for MYOC by SDS-PAGE did not change, even in the presence of brefeldin A; indicating that MYOC is not glycosylated and is not released via a traditional secretory mechanism. When conditioned media from human trabecular meshwork cells were examined, both native and recombinant MYOC associated with an extracellular membrane population having biochemical characteristics of exosomes, and containing the major histocompatibility complex class II antigen, HLA-DR. The association of MYOC with exosome-like membranes appeared to be specific, on the extracellular face, and reversible. Taken together, data suggest that MYOC appears in the extracellular space of trabecular meshwork cells by an unconventional mechanism, likely associated with exosome-like vesicles.
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extracellular trafficking of MYOCilin in human trabecular meshwork cells
Journal of Biological Chemistry, 2005Co-Authors: Katharine M. Hardy, Brian S Mckay, Pedro Gonzalez, E.a. Hoffman, Daniel W StamerAbstract:Abstract MYOCilin (MYOC) is a protein with a broad expression pattern, but unknown function. MYOC associates with intracellular structures that are consistent with secretory vesicles, however, in most cell types studied, MYOC is limited to the intracellular compartment. In the trabecular meshwork, MYOC associates with intracellular vesicles, but is also found in the extracellular space. The purpose of the present study was to better understand the mechanism of extracellular transport of MYOC in trabecular meshwork cells. Using a biochemical approach, we found that MYOC localizes intracellularly to both the cytosolic and particulate fractions. When intracellular membranes were separated over a linear sucrose gradient, MYOC equilibrated in a fraction less dense than traditional secretory vesicles and lysosomes. In pulse-labeling experiments that followed nascent MYOC over time, the characteristic doublet observed for MYOC by SDS-PAGE did not change, even in the presence of brefeldin A; indicating that MYOC is not glycosylated and is not released via a traditional secretory mechanism. When conditioned media from human trabecular meshwork cells were examined, both native and recombinant MYOC associated with an extracellular membrane population having biochemical characteristics of exosomes, and containing the major histocompatibility complex class II antigen, HLA-DR. The association of MYOC with exosome-like membranes appeared to be specific, on the extracellular face, and reversible. Taken together, data suggest that MYOC appears in the extracellular space of trabecular meshwork cells by an unconventional mechanism, likely associated with exosome-like vesicles.
Jon R Polansky - One of the best experts on this subject based on the ideXlab platform.
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current perspectives on the tigr MYOC gene MYOCilin and glaucoma
Ophthalmology Clinics of North America, 2003Co-Authors: Jon R PolanskyAbstract:Over the past several years, many groups worldwide have confirmed the presence of probable disease-causing mutations in the coding region of the (TIGR/MYOC) gene associated with glaucoma. Disease-associated point mutations are often found in the third exon of TIGR/MYOC and are predicted to exert a substantial influence on protein structure. Although there has been speculation as to the mechanisms involved in the pathogenic effects for a number of the mutations, the processes leading to the development of glaucoma involving TIGR/MYOC remain to be elucidated. In addition to ongoing mutation studies, efforts are underway to follow up on TIGR/MYOC gene regulation studies in human trabecular meshwork cells and other possibly relevant cell types. Potentially by altering gene regulation, a major variant (-1000 G/C), present in 15-20% of individuals, appears to be associated with a more rapid progression of glaucomatous disease. This article addresses several of these areas of research on the TIGR/MYOC gene and glaucoma, briefly presenting currently available evidence and considering or updating information presented previously.
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Current perspectives on the TIGR/MYOC gene (MYOCilin) and glaucoma.
Ophthalmology clinics of North America, 2003Co-Authors: Jon R PolanskyAbstract:Over the past several years, many groups worldwide have confirmed the presence of probable disease-causing mutations in the coding region of the (TIGR/MYOC) gene associated with glaucoma. Disease-associated point mutations are often found in the third exon of TIGR/MYOC and are predicted to exert a substantial influence on protein structure. Although there has been speculation as to the mechanisms involved in the pathogenic effects for a number of the mutations, the processes leading to the development of glaucoma involving TIGR/MYOC remain to be elucidated. In addition to ongoing mutation studies, efforts are underway to follow up on TIGR/MYOC gene regulation studies in human trabecular meshwork cells and other possibly relevant cell types. Potentially by altering gene regulation, a major variant (-1000 G/C), present in 15-20% of individuals, appears to be associated with a more rapid progression of glaucomatous disease. This article addresses several of these areas of research on the TIGR/MYOC gene and glaucoma, briefly presenting currently available evidence and considering or updating information presented previously.
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in vitro localization of tigr MYOC in trabecular meshwork extracellular matrix and binding to fibronectin
Investigative Ophthalmology & Visual Science, 2002Co-Authors: Mark S Filla, Thai Nguyen, Jon R Polansky, Curtis R Brandt, Paul L Kaufman, Donna M PetersAbstract:PURPOSE. To determine whether trabecular meshwork-inducible glucocorticoid response/MYOCilin (TIGR/MYOC) protein associates with the extracellular matrix (ECM) of human trabecular meshwork (HTM) cells. METHODS. The extracellular localization of TIGR/MYOC was examined by immunofluorescence microscopy in HTM cultures treated with and without dexamethasone and ascorbate and in a transformed HTM cell line, TM-1, transiently transfected with TIGR/MYOC cDNA. Antibodies to TIGR/MYOC, fibronectin, laminin, type IV collagen, or thrombospondin were used to determine the extracellular localization of TIGR/ MYOC. Solid phase binding assays using 125 I-recombinant TIGR/MYOC and types I and IV collagens, fibronectin, and laminin were done to examine the association of TIGR/MYOC with these proteins and to identify a specific TIGR/MYOC binding site within fibronectin. The domains of fibronectin tested were the fibrin/collagen binding domain, the RGD domain, and the Heparin II (Hep II) domain. RESULTS. TIGR/MYOC colocalized with fibronectin, laminin, and type IV collagen, but not thrombospondin in both dexamethasone and dexamethasone/ascorbate-treated HTM cultures and in TM-1 cultures transfected with TIGR/MYOC cl)NA. In solid phase binding assays, 125 I-TIGR/MYOC bound fibronectin but not laminin or type IV collagen. Binding to fibronectin could be competed with excess TIGR/MYOC or fibronectin. Specific binding was found for the Hep II domain of fibronectin. CONCLUSIONS. TIGR/MYOC can associate with components of the ECM via interactions with the Hep II domain of fibronectin. The interactions with the Hep II domain of fibronectin could alter cell-matrix interactions in the 'I'M and provides an interesting lead to explore the role(s) of TIGR/MYOC in both steroid-induced and primary open angle glaucoma.
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In vitro localization of TIGR/MYOC in trabecular meshwork extracellular matrix and binding to fibronectin.
Investigative ophthalmology & visual science, 2002Co-Authors: Mark S Filla, Thai Nguyen, Jon R Polansky, Curtis R Brandt, Paul L Kaufman, Xuyang Liu, Donna M PetersAbstract:PURPOSE. To determine whether trabecular meshwork-inducible glucocorticoid response/MYOCilin (TIGR/MYOC) protein associates with the extracellular matrix (ECM) of human trabecular meshwork (HTM) cells. METHODS. The extracellular localization of TIGR/MYOC was examined by immunofluorescence microscopy in HTM cultures treated with and without dexamethasone and ascorbate and in a transformed HTM cell line, TM-1, transiently transfected with TIGR/MYOC cDNA. Antibodies to TIGR/MYOC, fibronectin, laminin, type IV collagen, or thrombospondin were used to determine the extracellular localization of TIGR/ MYOC. Solid phase binding assays using 125 I-recombinant TIGR/MYOC and types I and IV collagens, fibronectin, and laminin were done to examine the association of TIGR/MYOC with these proteins and to identify a specific TIGR/MYOC binding site within fibronectin. The domains of fibronectin tested were the fibrin/collagen binding domain, the RGD domain, and the Heparin II (Hep II) domain. RESULTS. TIGR/MYOC colocalized with fibronectin, laminin, and type IV collagen, but not thrombospondin in both dexamethasone and dexamethasone/ascorbate-treated HTM cultures and in TM-1 cultures transfected with TIGR/MYOC cl)NA. In solid phase binding assays, 125 I-TIGR/MYOC bound fibronectin but not laminin or type IV collagen. Binding to fibronectin could be competed with excess TIGR/MYOC or fibronectin. Specific binding was found for the Hep II domain of fibronectin. CONCLUSIONS. TIGR/MYOC can associate with components of the ECM via interactions with the Hep II domain of fibronectin. The interactions with the Hep II domain of fibronectin could alter cell-matrix interactions in the 'I'M and provides an interesting lead to explore the role(s) of TIGR/MYOC in both steroid-induced and primary open angle glaucoma.
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in vitro localization of tigr MYOC in trabecular meshwork extracellular matrix and binding to fibronectin
Investigative Ophthalmology & Visual Science, 2002Co-Authors: Mark S Filla, Thai Nguyen, Jon R Polansky, Curtis R Brandt, Paul L Kaufman, Xuyang Liu, Donna M PetersAbstract:PURPOSE. To determine whether trabecular meshwork-inducible glucocorticoid response/MYOCilin (TIGR/MYOC) protein associates with the extracellular matrix (ECM) of human trabecular meshwork (HTM) cells. METHODS. The extracellular localization of TIGR/MYOC was examined by immunofluorescence microscopy in HTM cultures treated with and without dexamethasone and ascorbate and in a transformed HTM cell line, TM-1, transiently transfected with TIGR/MYOC cDNA. Antibodies to TIGR/MYOC, fibronectin, laminin, type IV collagen, or thrombospondin were used to determine the extracellular localization of TIGR/ MYOC. Solid phase binding assays using 125 I-recombinant TIGR/MYOC and types I and IV collagens, fibronectin, and laminin were done to examine the association of TIGR/MYOC with these proteins and to identify a specific TIGR/MYOC binding site within fibronectin. The domains of fibronectin tested were the fibrin/collagen binding domain, the RGD domain, and the Heparin II (Hep II) domain. RESULTS. TIGR/MYOC colocalized with fibronectin, laminin, and type IV collagen, but not thrombospondin in both dexamethasone and dexamethasone/ascorbate-treated HTM cultures and in TM-1 cultures transfected with TIGR/MYOC cl)NA. In solid phase binding assays, 125 I-TIGR/MYOC bound fibronectin but not laminin or type IV collagen. Binding to fibronectin could be competed with excess TIGR/MYOC or fibronectin. Specific binding was found for the Hep II domain of fibronectin. CONCLUSIONS. TIGR/MYOC can associate with components of the ECM via interactions with the Hep II domain of fibronectin. The interactions with the Hep II domain of fibronectin could alter cell-matrix interactions in the 'I'M and provides an interesting lead to explore the role(s) of TIGR/MYOC in both steroid-induced and primary open angle glaucoma.
Teresa Borrás - One of the best experts on this subject based on the ideXlab platform.
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first look at the effect of overexpression of tigr MYOC on the transcriptome of the human trabecular meshwork
Experimental Eye Research, 2006Co-Authors: Teresa Borrás, Paul A. Bryant, Sarah S. ChisolmAbstract:Abstract Wild-type TIGR/MYOC is a secreted protein implicated in the development of steroid glaucoma. Mutations in TIGR/MYOC have been linked to some patients who develop elevated intraocular pressure (IOP) and glaucoma. Because there is evidence of some other factors contributing to the TIGR/MYOC causative role in glaucoma, and because substantial increased levels of a particular cellular mRNA and protein might alter expression of other host genes, we began to investigate the effect of TIGR/MYOC overexpression on the transcriptome of human trabecular meshwork cells. We used a recombinant adenovirus carrying wild-type TIGR/MYOC cDNA, primary HTM cells, 300 viral particles per cell and U133 Affymetrix GeneChips. Our results indicate that 2361 out of the 22 284 genes (10.6%) were altered more than two-fold ( p ≤0.005) by the overexpression of TIGR/MYOC. A higher proportion of the altered genes were downregulated (1412 vs. 949). Potentially relevant upregulated genes include angiopoietin 2, matrix metalloproteinase 1 (MMP1) and thrombomodulin; among those downregulated we observed growth arrest specific 1, proteins involved in the ubiquitination pathway and vascular cell adhesion molecule 1. In addition, collagen type 1, one of the MMP1 substrates, was also downregulated. Genes affected by wild-type TIGR/MYOC might prove to be candidate mediators for future studies of the mechanisms of glaucoma.
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First look at the effect of overexpression of TIGR/MYOC on the transcriptome of the human trabecular meshwork.
Experimental eye research, 2006Co-Authors: Teresa Borrás, Paul A. Bryant, Sarah S. ChisolmAbstract:Abstract Wild-type TIGR/MYOC is a secreted protein implicated in the development of steroid glaucoma. Mutations in TIGR/MYOC have been linked to some patients who develop elevated intraocular pressure (IOP) and glaucoma. Because there is evidence of some other factors contributing to the TIGR/MYOC causative role in glaucoma, and because substantial increased levels of a particular cellular mRNA and protein might alter expression of other host genes, we began to investigate the effect of TIGR/MYOC overexpression on the transcriptome of human trabecular meshwork cells. We used a recombinant adenovirus carrying wild-type TIGR/MYOC cDNA, primary HTM cells, 300 viral particles per cell and U133 Affymetrix GeneChips. Our results indicate that 2361 out of the 22 284 genes (10.6%) were altered more than two-fold ( p ≤0.005) by the overexpression of TIGR/MYOC. A higher proportion of the altered genes were downregulated (1412 vs. 949). Potentially relevant upregulated genes include angiopoietin 2, matrix metalloproteinase 1 (MMP1) and thrombomodulin; among those downregulated we observed growth arrest specific 1, proteins involved in the ubiquitination pathway and vascular cell adhesion molecule 1. In addition, collagen type 1, one of the MMP1 substrates, was also downregulated. Genes affected by wild-type TIGR/MYOC might prove to be candidate mediators for future studies of the mechanisms of glaucoma.
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tissue differential microarray analysis of dexamethasone induction reveals potential mechanisms of steroid glaucoma
Investigative Ophthalmology & Visual Science, 2003Co-Authors: Laura Leigh S Rowlette, M Caballero, Ping Yang, Rosario M Hernandez, Teresa BorrásAbstract:PURPOSE To identify MYOCilin (TIGR/MYOC) properties that are specific to the human trabecular meshwork (HTM). To search for genes highly expressed in dexamethasone (DEX)-induced HTM cells that are barely expressed or absent in DEX-induced cells from other tissues. METHODS TIGR/MYOC induction by DEX (10(-7) M for 8-10 days) was analyzed by Northern and Western blot analyses in HTM, human umbilical vein endothelial cells, HeLa cells, and human embryonic skeletal muscle cells and optic nerve head (ONH) astrocytes at confluence. Processing and secretion were analyzed after the cells were infected with adenoviruses overexpressing wild-type and mutant forms of TIGR/MYOC. Affymetrix U95Av2 GeneChips (n = 6) and software were used to compare paired expression profiles of HTM, HTM-DEX, ONH astrocytes, and ONH astrocytes-DEX. Identification of HTM-DEX-specific genes (compared with ONH astrocytes-DEX) was performed by selecting genes with the highest fold change values (>/=20). Genes with fold change values of four or more were matched with loci linked to glaucoma, by using gene databases. RESULTS TIGR/MYOC induction by DEX occurred only in HTM cells. Secretory and glycosylation characteristics remained the same across cell types. Expression profile analysis revealed multiple genes differentially upregulated in HTM-DEX including, in addition to TIGR/MYOC, a serine protease inhibitor (alpha1-antichymotrypsin), a neuroprotective factor (pigment epithelium-derived factor), an antiangiogenesis factor (cornea-derived transcript 6), and a prostaglandin synthase (prostaglandin D(2) synthase). Fifteen of the 249 genes with fold change values of four or more mapped to glaucoma-linked loci. CONCLUSIONS The induction of TIGR/MYOC by DEX is HTM-specific, whereas its secretory and glycosylation characteristics are ubiquitous. The known functions of HTM-DEX-specific genes reveal the presence of protective and damaging mechanisms for regulation of IOP during DEX treatment. Besides TIGR/MYOC, other HTM-DEX-specific genes may be good candidates for linkage to glaucoma.
