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M. Kang, D.-J. Oh, J.-H. Kang, D. J. Rhee; The Expression of Secreted Protein, Acidic and Rich in Cysteine (SPARC) is Up-Regulated by Transforming Growth Factor β-2 in Both of Smad2/3-Dependent and Independent Signaling Pathways in Human Trabecular Meshwork. Invest. Ophthalmol. Vis. Sci. 2010;51(13):6100.
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Increased aqueous levels of TGF-β2 have been found in many primary open-angle glaucoma (POAG) patients. Experimentally, TGF-β2 increases ECM deposition and cross-linking in the human trabecular meshwork (TM).1 We reported that secreted protein, acidic and rich in Cysteine (SPARC) was involved in IOP regulation in SPARC-null mice.2 The relationship between SPARC and TGF-β2 in trabecular meshwork (TM) is unknown. We hypothesized that TGF-β2 regulates SPARC expression in TM.
Primary cultures of human TM endothelial cells were incubated with inhibitors for p38 MAP kinase, smad3, JNK or TGF-β2 receptor for 2 hrs and then TGF-β2 was added for 24 hrs. Total RNA and protein were isolated. Quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) and Western blot analysis were performed to identify SPARC mRNA and protein level, respectively.
Western blot analysis showed that SPARC was highly up-regulated by TGF-β2 in the human TM cells (3.8 ± 1.7 fold, N=6, p=0.01). SPARC expression by TGF-β2 was significantly inhibited by inhibitors for p38 MAP kinase (-41.1 ± 7.1%, N=6, p=0.003, smad3 (-76.7± 10.6%, N=6, p=0.002), JNK (-73.9 ± 19.7%, N=6, p=0.006) and TGF-β2 receptor (-83.6 ± 14.4%, N=6, p=0.003) as compared with the treatment of TGF-β2 and no inhibitor (N=6). In qRT-PCR experiments, SPARC mRNA was also highly up-regulated (7.1 ± 3.7 fold, N=6, p=0.01) by TGF-β2. Furthermore, SPARC was transcriptionally inhibited by inhibitors for p38 (-43.3 ± 12.6%, N=6, p=0.001), Smad3 (-38.4 ± 19.5%, N=6, p=0.01), JNK (-41.8 ± 4.6%, N=6, p=0.00003) and TGF-b2 receptor (-79.0 ± 11.2%, N=6, p=0.00009).
We found that TGF-β2 up-regulates SPARC expression in human TM through both Smad-dependent (Smad2/3) and independent (p38 or JNK) signaling pathway. SPARC may play an important role in TGF-β2 mediated POAG.
1. Wordinger RJ et al. Effects of TGF-β2, BMP-4, and Gremlin in the Trabecular Meshwork: Implications for Glaucoma. Invest Ophtalmol Vis Sci. 2007;48:1191-1200.2. Haddadin RI et al. SPARC-null mice exhibit lower intraocular pressures. Invest Ophthalmol Vis Sci. 2009;50:3771-3777.Acknowledgement: This research was supported by Massachusetts Lions Eye Research Fund, RPB physician Scientist Award and American Glaucoma Society
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