Limits...
Rap1 and Rap2 antagonistically control endothelial barrier resistance.

Pannekoek WJ, Linnemann JR, Brouwer PM, Bos JL, Rehmann H - PLoS ONE (2013)

Bottom Line: In line with its well-established effect on cell-cell junctions, depletion of Rap1 decreases, whereas activation of Rap1 increases barrier resistance.Despite its high sequence homology with Rap1, depletion of Rap2 has an opposite, enhancing, effect on barrier resistance.This Rap1-antagonizing effect of Rap2 is established independent of junctional actin formation.

View Article: PubMed Central - PubMed

Affiliation: Molecular Cancer Research, University Medical Center Utrecht, Utrecht, The Netherlands.

ABSTRACT
Rap1 and Rap2 are closely related proteins of the Ras family of small G-proteins. Rap1 is well known to regulate cell-cell adhesion. Here, we have analysed the effect of Rap-mediated signalling on endothelial permeability using electrical impedance measurements of HUVEC monolayers and subsequent determination of the barrier resistance, which is a measure for the ease with which ions can pass cell junctions. In line with its well-established effect on cell-cell junctions, depletion of Rap1 decreases, whereas activation of Rap1 increases barrier resistance. Despite its high sequence homology with Rap1, depletion of Rap2 has an opposite, enhancing, effect on barrier resistance. This effect can be mimicked by depletion of the Rap2 specific activator RasGEF1C and the Rap2 effector MAP4K4, establishing Rap2 signalling as an independent pathway controlling barrier resistance. As simultaneous depletion or activation of both Rap1 and Rap2 results in a barrier resistance comparable to control cells, Rap1 and Rap2 control barrier resistance in a reciprocal manner. This Rap1-antagonizing effect of Rap2 is established independent of junctional actin formation. These data establish that endothelial barrier resistance is determined by the combined antagonistic actions of Rap1 and Rap2.

Show MeSH

Related in: MedlinePlus

Rap2 antagonizes Rap1 downstream of junctional actin formation.HUVECs transfected with siRNAs targeting either all three Rap2 proteins (A, B) or the RapGAPs RapGAP1 or RapGAP2 (C, D) were plated onto coverslips, stimulated with or without 007-AM for 10 minutes and stained for F-actin (phalloidin, green) and β-catenin (red). Quantifications of the amount of signal correlation between β-catenin and F-actin (0 = random, 1 = perfect correlation) are shown in (B) and (D). Different colors represent individual independent experiments (B: n = 3, D: n = 4, E: n = 4 for siRapGAP1 and n = 5 for siRapGAP2). Averages are indicated by black lines. (E) The effect of RapGAP1 or RapGAP2 depletion on the barrier resistance was determined as described in Figure 1.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3585282&req=5

pone-0057903-g005: Rap2 antagonizes Rap1 downstream of junctional actin formation.HUVECs transfected with siRNAs targeting either all three Rap2 proteins (A, B) or the RapGAPs RapGAP1 or RapGAP2 (C, D) were plated onto coverslips, stimulated with or without 007-AM for 10 minutes and stained for F-actin (phalloidin, green) and β-catenin (red). Quantifications of the amount of signal correlation between β-catenin and F-actin (0 = random, 1 = perfect correlation) are shown in (B) and (D). Different colors represent individual independent experiments (B: n = 3, D: n = 4, E: n = 4 for siRapGAP1 and n = 5 for siRapGAP2). Averages are indicated by black lines. (E) The effect of RapGAP1 or RapGAP2 depletion on the barrier resistance was determined as described in Figure 1.

Mentions: Rap1 has been suggested to regulate endothelial permeability by inducing junctional actin [7], [8], [11], [21]. Conversely, Rap2 has been proposed to disrupt filamentous actin via members of the GCK-IV subgroup of Ste20 kinases, which controls the activity of the actin-severing-enzyme Gelsolin [35], [43]. As Rap1 and Rap2 both impinge on Cadherins, this might occur by the opposite regulation of junctional actin formation. Indeed, HUVECs stained for F-actin and the junction marker β-catenin showed a transverse actin fiber pattern that changes to junctional actin upon activation of Rap1 by addition of 007-AM (Fig. 5A). Depletion of Rap2 did not affect junction morphology as β-catenin staining was indiscernible from control cells. Intriguingly, Rap2 depletion did not induce the typical junctional actin formation that would normally correlate with its permeability-reducing effect. Instead, actin fibres ran in transverse stress fibres, which did relocate to the cell-cell junction upon activation of Rap1 by addition of 007-AM, which was quantified to be similar to control cells (Fig. 5B). Hence, both basal and Rap1-induced actin morphology were similar in control and Rap2-depleted cells, suggesting that Rap2 does not utilize its actin-severing capacity to decrease the barrier resistance and therefore antagonizes Rap1 independent of junctional actin. To corroborate this result further, we hypothesized simultaneous activation of Rap1 and Rap2 to increase junctional actin while leaving the barrier resistance unaffected. Here, we were intrigued by a recent report showing that RapGAP is required for, rather than antagonizing, epithelial cell-cell adhesion, as these RapGAP-depleted epithelial cells show enhanced activation of both Rap1 and Rap2 [18]. Concomitant with its effect on Rap1 activity, depletion of either RapGAP1 or RapGAP2 strongly induced the formation of junctional actin (Fig. 5C, D). Nevertheless, depletion of RapGAP1 or RapGAP2 had no effect on the barrier resistance (Fig. 5E). This result suggests that Rap2 antagonizes Rap1 independent of junctional actin formation and that Rap1 activity and barrier resistance can be uncoupled by Rap2 activation. Furthermore, these data establish that the relative ratio between Rap1 and Rap2 activity controls the barrier resistance.


Rap1 and Rap2 antagonistically control endothelial barrier resistance.

Pannekoek WJ, Linnemann JR, Brouwer PM, Bos JL, Rehmann H - PLoS ONE (2013)

Rap2 antagonizes Rap1 downstream of junctional actin formation.HUVECs transfected with siRNAs targeting either all three Rap2 proteins (A, B) or the RapGAPs RapGAP1 or RapGAP2 (C, D) were plated onto coverslips, stimulated with or without 007-AM for 10 minutes and stained for F-actin (phalloidin, green) and β-catenin (red). Quantifications of the amount of signal correlation between β-catenin and F-actin (0 = random, 1 = perfect correlation) are shown in (B) and (D). Different colors represent individual independent experiments (B: n = 3, D: n = 4, E: n = 4 for siRapGAP1 and n = 5 for siRapGAP2). Averages are indicated by black lines. (E) The effect of RapGAP1 or RapGAP2 depletion on the barrier resistance was determined as described in Figure 1.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3585282&req=5

pone-0057903-g005: Rap2 antagonizes Rap1 downstream of junctional actin formation.HUVECs transfected with siRNAs targeting either all three Rap2 proteins (A, B) or the RapGAPs RapGAP1 or RapGAP2 (C, D) were plated onto coverslips, stimulated with or without 007-AM for 10 minutes and stained for F-actin (phalloidin, green) and β-catenin (red). Quantifications of the amount of signal correlation between β-catenin and F-actin (0 = random, 1 = perfect correlation) are shown in (B) and (D). Different colors represent individual independent experiments (B: n = 3, D: n = 4, E: n = 4 for siRapGAP1 and n = 5 for siRapGAP2). Averages are indicated by black lines. (E) The effect of RapGAP1 or RapGAP2 depletion on the barrier resistance was determined as described in Figure 1.
Mentions: Rap1 has been suggested to regulate endothelial permeability by inducing junctional actin [7], [8], [11], [21]. Conversely, Rap2 has been proposed to disrupt filamentous actin via members of the GCK-IV subgroup of Ste20 kinases, which controls the activity of the actin-severing-enzyme Gelsolin [35], [43]. As Rap1 and Rap2 both impinge on Cadherins, this might occur by the opposite regulation of junctional actin formation. Indeed, HUVECs stained for F-actin and the junction marker β-catenin showed a transverse actin fiber pattern that changes to junctional actin upon activation of Rap1 by addition of 007-AM (Fig. 5A). Depletion of Rap2 did not affect junction morphology as β-catenin staining was indiscernible from control cells. Intriguingly, Rap2 depletion did not induce the typical junctional actin formation that would normally correlate with its permeability-reducing effect. Instead, actin fibres ran in transverse stress fibres, which did relocate to the cell-cell junction upon activation of Rap1 by addition of 007-AM, which was quantified to be similar to control cells (Fig. 5B). Hence, both basal and Rap1-induced actin morphology were similar in control and Rap2-depleted cells, suggesting that Rap2 does not utilize its actin-severing capacity to decrease the barrier resistance and therefore antagonizes Rap1 independent of junctional actin. To corroborate this result further, we hypothesized simultaneous activation of Rap1 and Rap2 to increase junctional actin while leaving the barrier resistance unaffected. Here, we were intrigued by a recent report showing that RapGAP is required for, rather than antagonizing, epithelial cell-cell adhesion, as these RapGAP-depleted epithelial cells show enhanced activation of both Rap1 and Rap2 [18]. Concomitant with its effect on Rap1 activity, depletion of either RapGAP1 or RapGAP2 strongly induced the formation of junctional actin (Fig. 5C, D). Nevertheless, depletion of RapGAP1 or RapGAP2 had no effect on the barrier resistance (Fig. 5E). This result suggests that Rap2 antagonizes Rap1 independent of junctional actin formation and that Rap1 activity and barrier resistance can be uncoupled by Rap2 activation. Furthermore, these data establish that the relative ratio between Rap1 and Rap2 activity controls the barrier resistance.

Bottom Line: In line with its well-established effect on cell-cell junctions, depletion of Rap1 decreases, whereas activation of Rap1 increases barrier resistance.Despite its high sequence homology with Rap1, depletion of Rap2 has an opposite, enhancing, effect on barrier resistance.This Rap1-antagonizing effect of Rap2 is established independent of junctional actin formation.

View Article: PubMed Central - PubMed

Affiliation: Molecular Cancer Research, University Medical Center Utrecht, Utrecht, The Netherlands.

ABSTRACT
Rap1 and Rap2 are closely related proteins of the Ras family of small G-proteins. Rap1 is well known to regulate cell-cell adhesion. Here, we have analysed the effect of Rap-mediated signalling on endothelial permeability using electrical impedance measurements of HUVEC monolayers and subsequent determination of the barrier resistance, which is a measure for the ease with which ions can pass cell junctions. In line with its well-established effect on cell-cell junctions, depletion of Rap1 decreases, whereas activation of Rap1 increases barrier resistance. Despite its high sequence homology with Rap1, depletion of Rap2 has an opposite, enhancing, effect on barrier resistance. This effect can be mimicked by depletion of the Rap2 specific activator RasGEF1C and the Rap2 effector MAP4K4, establishing Rap2 signalling as an independent pathway controlling barrier resistance. As simultaneous depletion or activation of both Rap1 and Rap2 results in a barrier resistance comparable to control cells, Rap1 and Rap2 control barrier resistance in a reciprocal manner. This Rap1-antagonizing effect of Rap2 is established independent of junctional actin formation. These data establish that endothelial barrier resistance is determined by the combined antagonistic actions of Rap1 and Rap2.

Show MeSH
Related in: MedlinePlus