Establishment of inducible wild type and mutant myocilin-GFP-expressing RGC5 cell lines.
BACKGROUND
Myocilin is a gene linked directly to juvenile- and adult-onset open angle glaucoma. Mutations including Gln368stop (Q368X) and Pro370Leu (P370L) have been identified in patients. The exact role of myocilin and its functional association with glaucoma are still unclear. In the present study, we established tetracycline-inducible (Tet-on) wild type and mutant myocilin-green fluorescence protein (GFP) expressing RGC5 stable cell lines and studied the changes in cell migration and barrier function upon induction.


RESULTS
After several rounds of selection, clones that displayed low, moderate, or high expression of wild type, Q368X or P370L myocilin-GFP upon doxycycline (Dox) induction were obtained. The levels of wild type and mutant myocilin-GFP in various clones were confirmed by Western blotting. Compared to non-induced controls, the cell migration was retarded, the actin stress fibers were fewer and shorter, and the trypsinization time needed for cells to round up was reduced when wild type or mutant myocilin was expressed. The barrier function was in addition aberrant following induced expression of wild type, Q368X or P370L myocilin. Immunoblotting further showed that tight junction protein occludin was downregulated in induced cells.


CONCLUSIONS
Tet-on inducible, stable RGC5 cell lines were established. These cell lines, expressing wild type or mutant (Q368X or P370L) myocilin-GFP upon Dox induction, are valuable in facilitating studies such as proteomics, as well as functional and pathogenesis investigations of disease-associated myocilin mutants. The barrier function was found impaired and the migration of cells was hindered with induced expression of wild type and mutant myocilin in RGC5 cell lines. The reduction in barrier function might be related to the declined level of occludin. The retarded cell migration was consistent with demonstrated myocilin phenotypes including the loss of actin stress fibers, lowered RhoA activities and compromised cell-matrix adhesiveness.
Department of Ophthalmology and Visual Sciences, University of Illinois at Chicago, College of Medicine, Chicago, Illinois, USA.
Hongyu
H
Ying
Xiang
X
Shen
Beatrice Y J T
BY
Yue
D000199:Actins / Q000378:metabolism; D000818:Animals; D015153:Blotting, Western; D018929:Cell Culture Techniques / Q000379:methods*; D002460:Cell Line; D002465:Cell Movement / Q000187:drug effects; D003598:Cytoskeletal Proteins / Q000378:metabolism*; D004318:Doxycycline / Q000494:pharmacology; D005136:Eye Proteins / Q000378:metabolism*; D006023:Glycoproteins / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000378:metabolism*; D006801:Humans; D050505:Mutant Proteins / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D013194:Staining and Labeling; D022502:Stress Fibers / Q000187:drug effects / Q000378:metabolism; D013752:Tetracycline / Q000494:pharmacology; D014357:Trypsin / Q000378:metabolism; D020742:rhoA GTP-Binding Protein / Q000378:metabolism; D000099179:Myocilin
D000199:Actins; D003598:Cytoskeletal Proteins; D004318:Doxycycline; D005136:Eye Proteins; D006023:Glycoproteins; D049452:Green Fluorescent Proteins; D050505:Mutant Proteins; D011993:Recombinant Fusion Proteins; D013752:Tetracycline; D014357:Trypsin; D020742:rhoA GTP-Binding Protein; D000099179:Myocilin