Abstract
Purpose::
The purpose of this study was to investigate whether there are molecular interactions between two glaucoma genes, myocilin (MYOC) and optineurin (OPTN).
Methods::
MYOC or OPTN in pTargeT expression vector was transfected into human trabecular meshwork (TM) cells, and other cell types including RPE, corneal stromal fibroblasts (CSF), RGC-5, HeLa, HEK and PC12. The expression level of MYOC and OPTN was examined by reverse transcriptase-relative quantitative polymerase chain reaction (RT-RQPCR). The expression level of MYOC and OPTN was investigated in PC12 cells after neurite induction. The turnover rate of MYOC mRNA was measured by RT-RQPCR in cells treated with actinomycin D. Promoter constructs containing up to 5.3 kb 5’-flanking region of the human MYOC gene were created. Activities of these promoter fragments were examined by secreted alkaline phosphatase reporter assays. Localization of OPTN was assessed by immunofluorescence staining and by Western blotting of cell extracts from nuclear and cytosolic fractions.
Results::
MYOC overexpression did not affect the OPTN expression level, whereas OPTN overexpression dramatically induced MYOC expression in human TM cells. This upregulation was also observed in RPE, CSF and RGC-5 cells as well as in nonocular cell types such as HeLa, HEK and PC12 cells. RT-RQPCR analyses also showed that the expression level of MYOC and OPTN genes was elevated in PC12 cells after treatment of nerve growth factor (NGF). OPTN, when overexpressed, resulted in a prolonged turnover rate of MYOC mRNA, but elicited little effect on the activity of MYOC promoter fragments. The overexpressed OPTN was found in the cytosol, not in the nucleus.
Conclusions::
The current study documents molecular interactions between MYOC and OPTN. The MYOC expression is upregulated by forced expression of OPTN or upon induced differentiation in PC12 cells. The upregulation is mediated primarily by an increase in the MYOC mRNA stability. OPTN appears to have a regulatory role in MYOC expression at the posttranscriptional level.
Keywords: gene/expression • transcription • trabecular meshwork