Expression of Porcine Myocilin and Optineurin in Trabecular Meshwork Cells and Astrocytes from Optic Nerve Head

Abstract

Abstract: : Purpose: Recently, optineurin (OPTN), a new gene responsible for primary open-angle glaucoma and normal tension glaucoma was identified (Sarfarazi et al. Science 2002). To compare the expression of OPTN with myocilin (MYOC) we determined the complete sequence of porcine OPTN cDNA and performed quantitative-PCR analysis on two porcine primary cells, trabecular meshwork cells (TMC) and astrocytes, cultured under various stimuli and stresses, which mimic the glaucoma cellular conditions. Methods: Coding region of porcine OPTN cDNA was amplified by RT-PCR from porcine TMC total RNA using primers designed from conserved sequence between human, mouse, and rat. The lacking 5' and 3' cDNA ends were amplified by 5' and 3' RACE method respectively. Total RNA was isolated from cultured cells treated with dexamethasone (500nM, for 2weeks), hydrostatic pressure (+30mmHg, 72h) or hypoxia (7% O₂, 72h). The expression level of the OPTN and MYOC in TMC and astrocytes were analyzed by real-time quantitative PCR (GeneAmp5700, PE Biosystems, Inc.). Total RNA used for quantitation was treated with DNase prier to measurement. Results: Porcine OPTN protein was composed of 574 amino acids, which was 84% identical with that of human OPTN protein. Treatment with dexamethasone increased MYOC expression by 8.2-fold and 5.5-fold in TMC and astrocytes respectively, while OPTN was suppressed to 48% in astrocytes and 68% in TMC. Incubation under hypoxia showed significant decrease for MYOC in both cells, while OPTN transcript was not affected. Hydrostatic pressure did not affect both genes in both cells. Conclusions: Nucleotide sequence of full-length porcine OPTN cDNA was determined and showed high degree of homology with human. OPTN expression was compared with MYOC under stimuli or stress in two porcine primary cells. Expression of OPTN was suppressed by dexamethasone but no effect was observed under hypoxia or hydrostatic pressure.

Keywords: gene/expression • outflow: trabecular meshwork • optic disc