J Li1, B J Tripathi, R C Tripathi. 1. Departments of Ophthalmology and. Pathology, University of South Carolina School of Medicine, Columbia 29209, USA.
Abstract
PURPOSE: To determine the effect of transforming growth factor (TGF)-beta2 on the pre-mRNA splicing pattern of fibronectin, as well as on the synthesis and secretion of this glycoprotein by porcine trabecular cells. METHODS: First-passage porcine trabecular cells were rendered quiescent and incubated in culture medium containing 15% newborn calf serum, in serum-free culture medium containing either activated TGF-beta2 (concentration range: 0.2-2.7 ng/ml) or activated TGF-beta1 (1 ng/ml), or in serum-free medium alone (untreated control samples). For investigation of alternative splicing, total RNA was extracted, and reverse transcription-polymerase chain reaction (RT-PCR) was performed with primer pairs located in exons flanking the exon (extra domain [ED]A, or EDB) that undergoes alternative splicing. The polymerase chain reaction (PCR) products were verified by Southern hybridization and quantified by using laser densitometry. The percentage of EDA-positive (+) isoforms was compared with that of the EDB+ isoforms among the groups. To study the effect of TGF-beta2 on the synthesis and secretion of fibronectin, total protein was extracted from both cultured cells and conditioned medium, Western blot analysis was performed using an anti-fibronectin antibody, and the products were quantified by laser densitometry. Immunocytochemical analysis was also performed on cultured trabecular cells to detect fibronectin. RESULTS: Fibronectin mRNA that was detected in untreated serum-starved control cells was EDA and EDB negative. Incubation of trabecular cells in medium containing 1 ng/ml TGF-beta2, 1 ng/ml TGF-beta1, or 15% newborn calf serum induced the expression of EDA+ and EDB+ mRNA to varying degrees. At concentrations of 0.2, 0.5, 1.5, and 2.7 ng/ml, TGF-beta2 increased the concentration of fibronectin by 2-, 3-, 3.8-, and 5-fold in the conditioned medium, and by 3-, 3.7-, 4-, and 4.3-fold in the cell extracts, respectively. The trabecular cells treated with TGF-beta2 exhibited strong immunoreaction for fibronectin, whereas the cells incubated in serum-free medium showed only minimal immunoreactivity. CONCLUSIONS: Our results demonstrate that TGF-beta2 and TGF-beta1 modified the alternative splicing pattern of fibronectin pre-mRNA and enhanced the synthesis and secretion of this extracellular matrix molecule by trabecular cells in a dose-dependent fashion. These findings indicate a mechanism whereby TGF-beta2, the concentration of which is elevated in aqueous humor of patients with primary open-angle glaucoma, contributes to the increased deposition of extracellular matrix molecules in the outflow pathway.
PURPOSE: To determine the effect of transforming growth factor (TGF)-beta2 on the pre-mRNA splicing pattern of fibronectin, as well as on the synthesis and secretion of this glycoprotein by porcine trabecular cells. METHODS: First-passage porcine trabecular cells were rendered quiescent and incubated in culture medium containing 15% newborn calf serum, in serum-free culture medium containing either activated TGF-beta2 (concentration range: 0.2-2.7 ng/ml) or activated TGF-beta1 (1 ng/ml), or in serum-free medium alone (untreated control samples). For investigation of alternative splicing, total RNA was extracted, and reverse transcription-polymerase chain reaction (RT-PCR) was performed with primer pairs located in exons flanking the exon (extra domain [ED]A, or EDB) that undergoes alternative splicing. The polymerase chain reaction (PCR) products were verified by Southern hybridization and quantified by using laser densitometry. The percentage of EDA-positive (+) isoforms was compared with that of the EDB+ isoforms among the groups. To study the effect of TGF-beta2 on the synthesis and secretion of fibronectin, total protein was extracted from both cultured cells and conditioned medium, Western blot analysis was performed using an anti-fibronectin antibody, and the products were quantified by laser densitometry. Immunocytochemical analysis was also performed on cultured trabecular cells to detect fibronectin. RESULTS:Fibronectin mRNA that was detected in untreated serum-starved control cells was EDA and EDB negative. Incubation of trabecular cells in medium containing 1 ng/ml TGF-beta2, 1 ng/ml TGF-beta1, or 15% newborn calf serum induced the expression of EDA+ and EDB+ mRNA to varying degrees. At concentrations of 0.2, 0.5, 1.5, and 2.7 ng/ml, TGF-beta2 increased the concentration of fibronectin by 2-, 3-, 3.8-, and 5-fold in the conditioned medium, and by 3-, 3.7-, 4-, and 4.3-fold in the cell extracts, respectively. The trabecular cells treated with TGF-beta2 exhibited strong immunoreaction for fibronectin, whereas the cells incubated in serum-free medium showed only minimal immunoreactivity. CONCLUSIONS: Our results demonstrate that TGF-beta2 and TGF-beta1 modified the alternative splicing pattern of fibronectin pre-mRNA and enhanced the synthesis and secretion of this extracellular matrix molecule by trabecular cells in a dose-dependent fashion. These findings indicate a mechanism whereby TGF-beta2, the concentration of which is elevated in aqueous humor of patients with primary open-angle glaucoma, contributes to the increased deposition of extracellular matrix molecules in the outflow pathway.
Authors: Swarup S Swaminathan; Dong-Jin Oh; Min Hyung Kang; Allan R Shepard; Iok-Hou Pang; Douglas J Rhee Journal: Invest Ophthalmol Vis Sci Date: 2014-06-06 Impact factor: 4.799