Abstract Title

Transforming Growth Factor-β2 Regulated Expression of Bone Morphogenetic Protein 1 (BMP-1), Tissue Transglutaminase (TGM2), Lysyl Oxidase (LOX), Procollagen C-endopeptidase Enhancer 1 (PCOLCE1), and Periostin (POSTN) in Human Optic Nerve Head Cells

Presenter Name

Tara Tovar-Vidales

Abstract

Transforming Growth Factor-β2 Regulated Expression of Bone Morphogenetic Protein 1 (BMP-1), Tissue Transglutaminase (TGM2), Lysyl Oxidase (LOX), Procollagen C-endopeptidase Enhancer 1 (PCOLCE1), and Periostin (POSTN) in Human Optic Nerve Head Cells

T. Tovar-Vidales, A.F. Clark, and R. J. Wordinger

Dept. Cell Biology & Immunology, North Texas Eye Research Institute, U. North Texas Health Science Center, Ft. Worth, TX

Purpose: Transforming growth factor-β2 (TGF-β2) increases the expression of BMP1 (an enzyme responsible for the cleavage and maturation of ECM proteins) as well as TGM2 and LOX enzymatic activity to promote cross-linking. TGF-β2 has been implicated in glaucoma damage to the optic nerve head (ONH). Other factors associated with the ECM remodeling include POSTN and PCOLCE1. The purpose of this study was to determine (a) whether human ONH cells express BMP1, TGM2, LOX, POSTN and PCOLCE1, (b) whether expression of BMP1, TGM2, LOX, POSTN and PCOLCE1 are regulated by TGF-β2, and (c) if TGF-β2 regulates TGM2 and LOX activity.

Methods: Primary human ONH cells were obtained from Alcon Laboratories (Fort Worth, Texas). Human ONH cells were isolated and subjected to qPCR (n=3) and Western immunoblotting (WB; n=6) for BMP1, TGM2, LOX, POSTN and PCOLCE1. qPCR was used to determine whether expression of BMP1, TGM2, LOX, POSTN and PCOLCE1 in ONH cells are regulated by TGF-β2 (5ng/ml). WB results were used to determine the effects of TGF-β2 on BMP1, TGM2, LOX, POSTN and PCOLCE1 protein expression in cell lysates of ONH cells treated for 48 hours. TGM2 and LOX activity assays were used to determine differences between non-treated and TGF-β2 treated in ONH cells.

Results: Human ONH cells expressed mRNA and protein for BMP1, TGM2, LOX, POSTN and PCOLCE1. Exogenous TGF-β2 statistically increased BMP1, TGM2, LOX, POSTN mRNA expression at 24 hours compared to their controls. TGF-β2 statistically decreased PCOLCE1 mRNA expression compared to their controls at 24hours. WB analysis showed induced BMP1, TGM2, and POSTN levels in cell lysates after TGF-β2 treatment compared to controls. However, WB analysis showed that TGF-β2 decreased PCOLCE1 levels in cell lysates. TGF-β2 increased both TGM2 and LOX enzyme activity in the ONH.

Conclusions: BMP1, TGM2, LOX, POSTN and PCOLCE1 are expressed in human ONH cells. These molecules may be involved in the normal function of the ONH. Altered expression of BMP1, TGM2, LOX, POSTN and PCOLCE1 by TGF-β2 may lead to functional changes and ECM remodeling within the ONH.

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Transforming Growth Factor-β2 Regulated Expression of Bone Morphogenetic Protein 1 (BMP-1), Tissue Transglutaminase (TGM2), Lysyl Oxidase (LOX), Procollagen C-endopeptidase Enhancer 1 (PCOLCE1), and Periostin (POSTN) in Human Optic Nerve Head Cells

Transforming Growth Factor-β2 Regulated Expression of Bone Morphogenetic Protein 1 (BMP-1), Tissue Transglutaminase (TGM2), Lysyl Oxidase (LOX), Procollagen C-endopeptidase Enhancer 1 (PCOLCE1), and Periostin (POSTN) in Human Optic Nerve Head Cells

T. Tovar-Vidales, A.F. Clark, and R. J. Wordinger

Dept. Cell Biology & Immunology, North Texas Eye Research Institute, U. North Texas Health Science Center, Ft. Worth, TX

Purpose: Transforming growth factor-β2 (TGF-β2) increases the expression of BMP1 (an enzyme responsible for the cleavage and maturation of ECM proteins) as well as TGM2 and LOX enzymatic activity to promote cross-linking. TGF-β2 has been implicated in glaucoma damage to the optic nerve head (ONH). Other factors associated with the ECM remodeling include POSTN and PCOLCE1. The purpose of this study was to determine (a) whether human ONH cells express BMP1, TGM2, LOX, POSTN and PCOLCE1, (b) whether expression of BMP1, TGM2, LOX, POSTN and PCOLCE1 are regulated by TGF-β2, and (c) if TGF-β2 regulates TGM2 and LOX activity.

Methods: Primary human ONH cells were obtained from Alcon Laboratories (Fort Worth, Texas). Human ONH cells were isolated and subjected to qPCR (n=3) and Western immunoblotting (WB; n=6) for BMP1, TGM2, LOX, POSTN and PCOLCE1. qPCR was used to determine whether expression of BMP1, TGM2, LOX, POSTN and PCOLCE1 in ONH cells are regulated by TGF-β2 (5ng/ml). WB results were used to determine the effects of TGF-β2 on BMP1, TGM2, LOX, POSTN and PCOLCE1 protein expression in cell lysates of ONH cells treated for 48 hours. TGM2 and LOX activity assays were used to determine differences between non-treated and TGF-β2 treated in ONH cells.

Results: Human ONH cells expressed mRNA and protein for BMP1, TGM2, LOX, POSTN and PCOLCE1. Exogenous TGF-β2 statistically increased BMP1, TGM2, LOX, POSTN mRNA expression at 24 hours compared to their controls. TGF-β2 statistically decreased PCOLCE1 mRNA expression compared to their controls at 24hours. WB analysis showed induced BMP1, TGM2, and POSTN levels in cell lysates after TGF-β2 treatment compared to controls. However, WB analysis showed that TGF-β2 decreased PCOLCE1 levels in cell lysates. TGF-β2 increased both TGM2 and LOX enzyme activity in the ONH.

Conclusions: BMP1, TGM2, LOX, POSTN and PCOLCE1 are expressed in human ONH cells. These molecules may be involved in the normal function of the ONH. Altered expression of BMP1, TGM2, LOX, POSTN and PCOLCE1 by TGF-β2 may lead to functional changes and ECM remodeling within the ONH.