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

Rap1 controls the barrier resistance via Cadherins.(A) HUVECs transfected with siScrambled or both siVE-Cadherin and siP-cadherin were transduced with control or V12Rap1A containing lentivirus and analysed before (pre) and after (post) stimulation with 007-AM as in Figure 1. Different colours represent individual independent experiments (n = 4). Averages are indicated by black lines. (B) Total cell lysates of an experiment shown in A, subjected to SDS-page and subsequent Western blotting to show V12Rap1A and VE-cadherin protein expression.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3585282&req=5

pone-0057903-g002: Rap1 controls the barrier resistance via Cadherins.(A) HUVECs transfected with siScrambled or both siVE-Cadherin and siP-cadherin were transduced with control or V12Rap1A containing lentivirus and analysed before (pre) and after (post) stimulation with 007-AM as in Figure 1. Different colours represent individual independent experiments (n = 4). Averages are indicated by black lines. (B) Total cell lysates of an experiment shown in A, subjected to SDS-page and subsequent Western blotting to show V12Rap1A and VE-cadherin protein expression.

Mentions: To confirm that modulation of the barrier resistance by Rap1 reflects an effect of Rap1 on cell-cell junctions, VE-cadherin and P-cadherin were depleted from HUVEC. Under these conditions the barrier resistance was close to zero, whereas α was hardly affected (Fig. 2). Cm was strongly reduced, but variable, partially due to the limitation of data acquisition of frequencies below 40000 Hz (data not shown). Proper analysis requires an intact monolayer [40]. Depletion of Cadherins is approaching this limit as the barrier resistance was close to zero, but is not breaking this limit as α stayed hardly affected (Fig. 2). This confirmed the robustness of the model. Over-expression of the constitutively active V12Rap1A mutant was not able to rescue cadherin depletion (Fig. 2). In contrast to V12Rap1A overexpression, stimulation with 007-AM did induce a minor increase in the barrier resistance when Cadherins are depleted (Fig. 2). This barrier tightening effect of 007-AM was also apparent in monolayers transduced with V12Rap1A, possibly caused by incomplete transduction efficiency of V12Rap1A, remaining GEF sensitivity of the V12Rap1A, GEF activity towards endogenous Rap1 or the previously reported Rap1- and Cadherin-independent effect of Epac on permeability [11], [13]. In conclusion, ECIS analysis allows the direct correlation of Rap signalling to junctional permeability.


Rap1 and Rap2 antagonistically control endothelial barrier resistance.

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

Rap1 controls the barrier resistance via Cadherins.(A) HUVECs transfected with siScrambled or both siVE-Cadherin and siP-cadherin were transduced with control or V12Rap1A containing lentivirus and analysed before (pre) and after (post) stimulation with 007-AM as in Figure 1. Different colours represent individual independent experiments (n = 4). Averages are indicated by black lines. (B) Total cell lysates of an experiment shown in A, subjected to SDS-page and subsequent Western blotting to show V12Rap1A and VE-cadherin protein expression.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0057903-g002: Rap1 controls the barrier resistance via Cadherins.(A) HUVECs transfected with siScrambled or both siVE-Cadherin and siP-cadherin were transduced with control or V12Rap1A containing lentivirus and analysed before (pre) and after (post) stimulation with 007-AM as in Figure 1. Different colours represent individual independent experiments (n = 4). Averages are indicated by black lines. (B) Total cell lysates of an experiment shown in A, subjected to SDS-page and subsequent Western blotting to show V12Rap1A and VE-cadherin protein expression.
Mentions: To confirm that modulation of the barrier resistance by Rap1 reflects an effect of Rap1 on cell-cell junctions, VE-cadherin and P-cadherin were depleted from HUVEC. Under these conditions the barrier resistance was close to zero, whereas α was hardly affected (Fig. 2). Cm was strongly reduced, but variable, partially due to the limitation of data acquisition of frequencies below 40000 Hz (data not shown). Proper analysis requires an intact monolayer [40]. Depletion of Cadherins is approaching this limit as the barrier resistance was close to zero, but is not breaking this limit as α stayed hardly affected (Fig. 2). This confirmed the robustness of the model. Over-expression of the constitutively active V12Rap1A mutant was not able to rescue cadherin depletion (Fig. 2). In contrast to V12Rap1A overexpression, stimulation with 007-AM did induce a minor increase in the barrier resistance when Cadherins are depleted (Fig. 2). This barrier tightening effect of 007-AM was also apparent in monolayers transduced with V12Rap1A, possibly caused by incomplete transduction efficiency of V12Rap1A, remaining GEF sensitivity of the V12Rap1A, GEF activity towards endogenous Rap1 or the previously reported Rap1- and Cadherin-independent effect of Epac on permeability [11], [13]. In conclusion, ECIS analysis allows the direct correlation of Rap signalling to junctional permeability.

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