Modulation of pre-mRNA splicing and protein production of fibronectin by TGF-beta2 in porcine trabecular cells.
OBJECTIVE
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.
Li
J
J
Departments of Ophthalmology and. Pathology, University of South Carolina School of Medicine, Columbia 29209, USA.
Tripathi
B J
BJ
Tripathi
R C
RC
D017398:Alternative Splicing / Q000187:drug effects*; D000818:Animals; D015153:Blotting, Western; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D005353:Fibronectins / Q000096:biosynthesis* / Q000235:genetics*; D007124:Immunoenzyme Techniques; D012333:RNA, Messenger / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013552:Swine; D014129:Trabecular Meshwork / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D016212:Transforming Growth Factor beta / Q000494:pharmacology*
D005353:Fibronectins; D012333:RNA, Messenger; D016212:Transforming Growth Factor beta