Abstract
Purpose: :
Increased aqueous levels of TGF-β2 have been found in many primary open-angle glaucoma (POAG) patients. We reported that secreted protein, acidic and rich in Cysteine (SPARC) was involved in IOP regulation in SPARC-null mice. The relationship between SPARC and TGF-β2 in trabecular meshwork (TM) is unknown. We hypothesized that TGF-β2 up-regulates SPARC expression in TM.
Methods: :
Primary cultures of human TM endothelial cells were incubated with inhibitors for p38 MAP kinase, smad3 or TGF-β2 receptor for 2 hrs and then TGF-β2 (2ng/ml) was added for 24 hrs. Western blot analysis was performed to identify SPARC in CM, phosphorylation of p38a and Smad3 in CL, respectively. Lenti-viruses were constructed including shRNAs for p38 and Samd3. Western blot analysis was performed to identify the suppression of p38, Smad3 and then SPARC, respectively. Translocation of phosphorylated p38 and Smad3 were investigated using confocal microscopy.
Results: :
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) 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) and TGF-b2 receptor (-79.0 ± 11.2%, N=6, p=0.00009). In the suppression experiment using shRNA, SPARC expression was significantly suppressed by shRNA for p38 (-58.0 ± 11.3%, N=5, p=0.02) and Smad3 (-36.2 ± 1.6%, N=5, p=0.001).
Conclusions: :
We found that TGF-β2 up-regulates SPARC expression in human TM through Smad3 or p38 signaling pathways. SPARC may play an important role in TGF-β2 mediated POAG.
Keywords: trabecular meshwork • signal transduction • gene/expression