Figure 1.
Treatment of sFRP1 averts the myocilin actin and focal adhesion phenotypes.
TM cells were transfected with pEGFP-N1 (EGFP-N1, mock control) or pMyocilinWT-EGFP (MYOCWT-GFP) for 48 h, treated overnight with 50 nM sFRP1 and stained for actin (in red, A) or vinculin (in red, B). The transfected cells were marked by green fluorescence and/or white asterisks. Myocilin overexpression induced a loss of actin stress fibers (A) and vinculin-positive focal adhesions (B). The loss was averted by treatment of sFRP1. The staining was visualized using a Zeiss 100 M microscope. Insets in A show the actin stress fibers in same fields in black and white. The same transfected cells are indicated by black asterisks. Insets in B show the green transfected cells (white asterisks). Bar, 10 µm.
Figure 2.
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.
Bar, 20 µm. 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. C. 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. D. 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.
Figure 3.
A. Myocilin expression activates Tcf/Lef-dependent transcription in Caco-2 cells.
Caco-2 cells were co-transfected with TOP- or FOP-Flash reporter construct, and pTarget-myocilinWT (pTarget-MYOCWT) 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. β-catenin (red) staining (top panel) in human TM cells after transfection with pEGFP-N1 (EGFP-N1, mock control) or pMyocilinWT-EGFP (MYOCWT-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.
Figure 4.
A. Actin (red) staining in pEGFP-N1 (EGFP-N1)-, pMyocilinP370L-EGFP (MYOCP370L-GFP)-, and pMyocilinQ368X-EGFP (MYOCQ368X-GFP)-transfected TM cells without or with subsequent overnight treatment of sFRP1.
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. The trypsinization time (mean ± SEM) needed for pEGFP-N1 (EGFP-N1)-, pMyocilinP370L-EGFP (MYOCP370L-GFP)-, and pMyocilinQ368X-EGFP (MYOCQ368X-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. C. GTP-bound active RhoA in pTarget-, pTarget-myocilinWT (MYOCWT)-, pTarget-myocilinP370L (MYOCP370L)- and pTarget-myocilinQ368X (MYOCQ368X)-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. D. Top−/Fop-Flash assays. Caco-2 cells were co-transfected with TOP- or FOP-Flash reporter construct, pTarget-myocilinP370L, pTarget-myocilinQ368X, 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.
Figure 5.
Effects of sFRP1 on PKA activity in TM cells.
A. Cells were transfected with pTarget and pTarget-myocilinWT (pTarget-MYOCWT) 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. Cells transfected with pTarget, pTarget-myocilinP370L (pTarget-MYOCP370L) and pTarget-myocilinQ368X (pTarget-MYOCQ368X) 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.
Figure 6.
A schematic model of possible events in human TM cells triggered by upregulation of myocilin.
Myocilin, when moderately upregulated, induces Wnt activation, and the downstream cAMP/PKA activation and RhoA inactivation, resulting in a loss of actin stress fibers and focal adhesions and disassembly of matrix network. These changes or other pathways may affect the integrity of TM cells, rendering them more susceptible to additional stress or challenge on a chronic, long-term basis, leading to pathologic consequences. Other intermediate mediators or pathways are yet to be identified. Dotted lines indicate steps that have not been investigated in the present work or our previous investigation [19]–[21].