Biological Studies of Glaucoma Gene Myocilin

Glaucoma is a major blinding disease. Myocilin is the first candidate gene identified for the most common form of glaucoma, primary open angle glaucoma (POAG). The human myocilin gene encodes for an acidic glycoprotein of 504 amino acids. Mutations of myocilin such as Pro370Leu (P370L) and Gln368Stop (Q368X) were found in 2-4% of POAG patients. Its structure, function and regulation are still largely unknown. We conducted new investigations on myocilin in five aspects (parts). Part I is to examine the involvement of Wnt pathway in myocilin mediated phenotypes such as loss of actin stress fibers and focal adhesions, elevated protein kinase A activity and downregulated RhoA activity. Wnt signaling is a key pathway involved in many important cellular processes. We showed that Wnt signaling is a player in the above mentioned myocilin phenotypes using Wnt specific inhibitor and activator. Blocking Wnt signaling pathway may have therapeutic potential for myocilin glaucoma. Part II is to establish 3 dimensional culture of human trabecular meshwork (TM) cells, a cell type that plays an important role in regulation of bulk flow of the aqueous humor. Several 3D platforms such as hydrogels and inserts were tested. Human TM cells were grown in multilayers when plated in QGel and Alvetex scaffold, formed cell-cell communication and showed a more in vivo like pattern of gene expression. The 3D system can be used for an effective in vitro model to help portray activities, characteristics, and stress responses of TM cells in vivo. Part III is to establish Tet-on inducible wild-type or mutated (P370L or Q368X) myocilin expressing neuronal RGC5 cell lines. The Tet-on inducible RGC5 cells provide a new tool for exploring the effects of myocilin up-regulation and mutations at cellular and molecular levels. Part IV is to detect alterations in protein expression profile and identify novel downstream pathways induced by myocilin. Cutting-edge quantitative proteomics were used. RGC5 cell lines established in part III were induced to express wild-type or mutated myocilin-GFP. The global protein profiles of these cells were compared with non-induced controls. Differentially expressed protein profiles pinpointed molecular trafficking, cytoskeleton reorganization, autophagy, microtubule dynamics, organization of filaments, apoptosis, mitochondria dysfunction and several signaling pathways. The information obtained may provide clues as to the functions of myocilin and point to new directions for myocilin investigations. Finally, part V is to identify microRNAs (miRNAs) that regulate the expression of mouse myocilin gene. MiRNAs are short RNAs functioning as posttranslational regulators (mostly in translation silencing). MiRNA array was performed and the up- or down-regulated miRNAs in induced cells were identified. Along with miRNAs predicted by computer algorithms, miRNAs that target mouse myocilin were identified and validated. Since expression of mutated myocilin is likely to be under the same miRNA control as the wild-type myocilin, results from the proposed experiments may be applied to silence myocilin mutants to abrogate mutant phenotypes in myocilin glaucoma

Tet-on inducible RGC5 cell lines express mutant P370L myocilin-GFP upon Dox induction.

<p>Clones with different expression levels of P370L myocilin-GFP (MYOC_P370L-GFP) fusion protein are presented. Without Dox induction, the green fluorescence in the cells was minimal (<b>A</b>). After Dox treatment, low (<b>B</b>), moderate (<b>C</b>) and high (<b>D</b>) levels of MYOC_P370L-GFP were seen in, respectively, low, moderate, and high expresser clones. Cytoplasmic aggregates were visible in both moderate and high expressers. Scale bar, 10 µm. <b>E.</b> Western blot analyses of cell lysates using anti-GFP and anti-GAPDH polyclonal antibodies. <b>F</b>. Western blot analyses of lysate (left panel) and media samples (right panel) using anti-myocilin monoclonal antibody. The blots confirmed that the level of MYOC_P370L-GFP relative to that of GAPDH in total cell lysates was low, moderate, and high from the various expresser clones. No MYOC_P370L-GFP protein band was seen in medium samples. −, Non-induced control; +, Induced cells.</p

Effects of sFRP1 on PKA activity in TM cells.

<p><b>A</b>. Cells were transfected with pTarget and pTarget-myocilin_WT (pTarget-MYOC_WT) for 48 h. One set of samples was treated overnight with sFRP1 and the other was untreated. Lysates collected were subjected to PKA assay as described in Fig. 2B. <b>B</b>. Cells transfected with pTarget, pTarget-myocilin_P370L (pTarget-MYOC_P370L) and pTarget-myocilin_Q368X (pTarget-MYOC_Q368X) were untreated or treated overnight with sFRP1. Lysates were subjected to PKA assays. The PKA activity (arrowhead) was determined by densitometric analyses. Results were expressed as ratios relative to the pTarget control. Experiments were repeated 3 times. Data from one representative experiment are presented.</p

Western blotting and immunostaining for occluding levels.

<p><b>A</b>. Lysates from non-induced (−) and induced (+) moderate expressers were examined by Western blotting. Ratios between occludin and GAPDH levels, relative to those in non-induced controls are presented for each of the myocilin wild type, Q368X, and P370L (MYOC_WT, MYOC_Q368X, and MYOC_P370L) pairs. <b>B</b>. Non-induced RGC5 cells (left panel) and induced low expressers of wild type myocilin (right panel) were immunostained for occludin. Positive occludin staining in red was seen between cells around the border. The staining in induced cells was reduced compared to non-induced controls. Inset in the left panel shows negative control in which the primary antibody was replaced with normal rabbit IgG during the staining procedure. Inset in the right panel shows the same view to indicate the induced cells in green. C. Induced cells in a mixed culture of low (L), moderate (M), and high (H) expressers of wild type myocilin were stained for occludin. Compared to low expressers, moderate and high expressers with stronger green fluorescence had lower red fluorescence intensity for occludin. Scale bar, 10 µm.</p

Establishment of Inducible Wild Type and Mutant Myocilin-GFP-Expressing RGC5 Cell Lines

<div><h3>Background</h3><p>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.</p> <h3>Methodology/Principal Findings</h3><p>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.</p> <h3>Conclusions/Significance</h3><p>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.</p> </div

A. Actin (green), vinculin (red), and β-catenin (red) staining in normal human TM cells without (control) or with overnight treatment with 2 and 10 µM SB216763.

<p>Bar, 20 µm. <b>B</b>. PKA activity in TM cells without (control) or with treatment with SB216763. Equal amounts of protein lysates were subjected to PKA assays. Positive (+) and negative (-) controls were included. The non-phosphorylated (upper band) and the phosphorylated (lower band, arrowhead) substrates were resolved on agarose gels. The PKA activity, judged by the level of the phosphorylated substrate, in 2 (1.76±0.14, mean ± SD, n = 3) and 10 (1.85±0.15, n = 3) µM SB216763 treated cells (P<0.006 compared to control) was determined by densitometric analyses and normalized to that in untreated controls. <b>C</b>. RhoA activity in TM cells treated with 2 and 10 µM SB216763 or 2 µM SB216763 plus 10 nM H89, a PKA inhibitor. Cells untreated served as control. Active RhoA, measured by pull down assays, was normalized to total RhoA. The level of active RhoA was reduced by approximately 30% by treatment of SB216763. Its level however returned to normal when H89 was included in the treatment. <b>D</b>. RhoA activity as measured by G-LISA. The activity was lower in TM cells treated with 10 µM SB216763 (0.227±0.022 vs. 0.318±0.004 in control, mean ± SEM, n = 3). H89 treatment elevated the RhoA back to the control range (0.293±0.016, n = 3). *, P<0.001 compared to untreated control.</p

Figure 5

<p><b>A. </b><i>In vitro</i> scratch assays. Cell migration was inhibited when moderate expressers were induced to express myocilin wild type (WT), Q368X, or P370L-GFP (lower panel) compared with non-induced controls (top panel). Inset shows the same view under a fluorescence microscope. The induced cells are in green. Scale bar, 50 µm. <b>B.</b> Bar graph to show the percent area (mean ± SD) covered by myocilin (MYOC)_WT-, MYOC_Q368X-, or MYOC_P370L-GFP-expressing cells that were migrated into the scratched area. N, Non-induced control; I, Induced cells. *, P<0.0001 compared to non-induced control.</p

A. Actin (red) staining in pEGFP-N1 (EGFP-N1)-, pMyocilin_P370L-EGFP (MYOC_P370L-GFP)-, and pMyocilin_Q368X-EGFP (MYOC_Q368X-GFP)-transfected TM cells without or with subsequent overnight treatment of sFRP1.

<p>Insets show the same field in black and white. Transfected cells are marked by green fluorescence, or white or black (in insets) asterisks. Bar, 20 µm. <b>B</b>. The trypsinization time (mean ± SEM) needed for pEGFP-N1 (EGFP-N1)-, pMyocilin_P370L-EGFP (MYOC_P370L-GFP)-, and pMyocilin_Q368X-EGFP (MYOC_Q368X-GFP)-transfected TM cells to become refractile. Asterisk indicates that the trypsinization time for myocilin mutant transfectants was significantly (P<0.0001, n = 30) lower than that of GFP controls. <b>C</b>. GTP-bound active RhoA in pTarget-, pTarget-myocilin_WT (MYOC_WT)-, pTarget-myocilin_P370L (MYOC_P370L)- and pTarget-myocilin_Q368X (MYOC_Q368X)-transfected TM cells. Pull down assays were performed in duplicates to determine the RhoA activity. The amount of the active or GTP-bound RhoA was normalized against the total amount in cell lysates and expressed as mean ± SD relative to that in pTarget control. The RhoA activity upon myocilin wild type (0.41±0.04, n = 2) and mutant transfection (0.51±0.03 for P370L; 0.45±0.04 for Q368X, n = 2) was found to be significantly reduced (P<0.0001) compared to controls. Experiments were repeated two times with similar results. <b>D.</b> Top−/Fop-Flash assays. Caco-2 cells were co-transfected with TOP- or FOP-Flash reporter construct, pTarget-myocilin_P370L, pTarget-myocilin_Q368X, or pTarget, as well as pEGFP-N1. Luciferase activity (mean ± SEM, n = 8) was measured post transfection and was normalized to the GFP level. *, P<0.001 compared to the pTarget control.</p

A. Myocilin expression activates Tcf/Lef-dependent transcription in Caco-2 cells.

<p>Caco-2 cells were co-transfected with TOP- or FOP-Flash reporter construct, and pTarget-myocilin_WT (pTarget-MYOC_WT) or pTarget plasmid for 48 h. A pEGFP-N1 vector was also co-transfected for standardization. Luciferase activity measured was normalized to the GFP reading. Three experiments were performed in triplicate. Results (mean ± SEM) from a representative experiment are shown. *, P<0.001 compared to pTarget control. <b>B</b>. β-catenin (red) staining (top panel) in human TM cells after transfection with pEGFP-N1 (EGFP-N1, mock control) or pMyocilin_WT-EGFP (MYOC_WT-GFP). Minimal staining was observed when normal mouse IgG was used in place of primary anti-β-catenin (bottom panel) in the procedure. Insets highlight the green transfected cells (white asterisks) in the same fields. Nuclei were stained by DAPI in blue. Bar, 20 µm.</p

Treatment of sFRP1 averts the myocilin actin and focal adhesion phenotypes.

<p>TM cells were transfected with pEGFP-N1 (EGFP-N1, mock control) or pMyocilin_WT-EGFP (MYOC_WT-GFP) for 48 h, treated overnight with 50 nM sFRP1 and stained for actin (in red, <b>A</b>) or vinculin (in red, <b>B</b>). The transfected cells were marked by green fluorescence and/or white asterisks. Myocilin overexpression induced a loss of actin stress fibers (<b>A</b>) and vinculin-positive focal adhesions (<b>B</b>). The loss was averted by treatment of sFRP1. The staining was visualized using a Zeiss 100 M microscope. Insets in <b>A</b> show the actin stress fibers in same fields in black and white. The same transfected cells are indicated by black asterisks. Insets in <b>B</b> show the green transfected cells (white asterisks). Bar, 10 µm.</p