Trabecular cell expression of fibronectin and MMP-3 is modulated by aqueous humor growth factors.
We investigated the mRNA and protein expression of fibronectin and stromelysin-1 (matrix metalloproteinase-3, MMP-3) by trabecular cells treated with growth factors present in primary and secondary aqueous humors. Serum-deprived trabecular cells were incubated for 48 hr or 7 days in medium containing either primary or secondary aqueous humor growth factors or in serum-free medium. We extracted total RNA, performed reverse transcription-polymerase chain reaction using primer pairs for fibronectin, stromelysin-1 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and quantified the products. We utilized Western blotting to detect and quantify fibronectin and stromelysin-1 protein. Compared to controls, expression of fibronectin mRNA by trabecular cells was increased by 50 and 100% after incubation in primary aqueous humor growth factors for 48 hr or 7 days, respectively, and 50 and 130% after incubation in secondary aqueous humor growth factors. Stromelysin-1 mRNA expression was decreased by 25 and 50% after incubation in primary aqueous humor growth factors for 48 hr or 7 days, respectively, and 80 and 85% after incubation for 48 hr or 7 days, respectively, in secondary aqueous humor growth factors. Fibronectin protein increased 3.5-fold and 6-fold after incubation for 48 hr with primary or secondary aqueous humor growth factors, respectively; after 7 days, the level increased 4- and 7-folds, respectively. Stromelysin-1 protein was not detectable by western blotting. The up-regulation of fibronectin mRNA by trabecular cells exposed to growth factors present in secondary aqueous humor augmented by the down-regulation of stromelysin-1 mRNA contributed to the accumulation of fibronectin. Our findings open the possibility that induction of stromelysin-1 gene expression in the trabecular meshwork of glaucomatous eyes could effectively reduce buildup of fibronectin in the aqueous outflow pathway to decrease outflow resistance in glaucomatous states of the eye.
Tripathi
Brenda J
BJ
Department of Pathology and Microbiology, University of South Carolina School of Medicine, 6439 Garners Ferry Road, Columbia, SC 29209, USA. brenda@med.sc.edu
Tripathi
Ramesh C
RC
Chen
Jianguang
J
Gotsis
Stelios
S
Li
Junping
J
D000818:Animals; D001082:Aqueous Humor / Q000737:chemistry*; D002478:Cells, Cultured; D016895:Culture Media, Serum-Free; D005353:Fibronectins / Q000235:genetics / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000187:drug effects; D006133:Growth Substances / Q000494:pharmacology*; D019278:Matrix Metalloproteinase 3 / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013552:Swine; D014129:Trabecular Meshwork / Q000187:drug effects* / Q000378:metabolism
D016895:Culture Media, Serum-Free; D005353:Fibronectins; D006133:Growth Substances; D012333:RNA, Messenger; D019278:Matrix Metalloproteinase 3