Rap1 GTPase has been implicated as a signaling protein involved in regulating cell-cell adhesion events involved in barrier function in many cell types, such as endothelia.
25 –27 We set out to test whether this is true for RPE cells, which do express Rap1 protein (data not shown). As an initial means to inhibit Rap1 activity, we exogenously expressed the protein RapGAP,
37 a GTPase activating protein (GAP) that acts as a negative regulator of Rap1 activity by promoting the hydrolysis of GTP to GDP.
38 Within 24 hours of transduction with an adenoviral expression vector, Rap1 activity assays revealed that RPE expressing RapGAP had reduced Rap1 activity as shown by decreased pull-down of Rap1-GTP compared with cells expressing GFP only (
Fig. 1A). In ARPE-19 cells, adenoviral infection efficiency of RapGAP approaches 100% as determined by visualization of a cocistronically expressed GFP marker (
Fig. 1B). We then measured the TER of ARPE-19 cells grown on Transwell filters after expression of either GFP-RapGAP or GFP alone for 72 hours.
Figure 1C shows that 72 hours after expression and Transwell plating, the average TER of GFP control cells is 35 ohm/cm
2, while the TER of RapGAP-expressing cells is significantly lower, at 24.67 ± 0.58 ohms per cm
2 (*
P = 0.0000032). As a second means of assessing barrier function in cultured RPE, we took advantage of real-time cell analysis (RTCA), an electrical impedance-based system that can be setup to take automatic, noninvasive measurements of barrier function in real-time.
36 Cells pretreated so as to begin expressing either GFP alone, or RapGAP for 24 hours before the experiment were replated at equal cell density into microelectrode coated wells of the RTCA E-Plate. Impedance measurements were recorded every 15 minutes for up to 22 hours. A representative trace demonstrating decreased impedance with RapGAP expression is shown in
Figure 2A. The average Cell Index of GFP or RapGAP-expressing cells from 3 independent experiments at the 22-hour time point is presented in
Figure 2B. Expression of RapGAP significantly reduced the plateau Cell Index values (*
P = 0.0093) compared with GFP control cells (average Cell Index of GFP control, 10.43 ± 2.02 vs. RapGAP, 4.23 ± 1.95). A confirmatory experiment was performed using hfRPE cells, and a similar pattern of reduced Cell Index on expression of RapGAP was observed (data not shown). Taken together,
Figures 1 and
2 show two independent experimental methods (TER, RTCA) to indicate that activation of Rap1 is important for the acquisition of RPE barrier function.