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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.

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Rap2 antagonizes Rap1 by an own signalling module.(A, B) The barrier resistance of HUVECs transfected with siRNAs targeting the Rap1 proteins, the Rap2 proteins or all five Rap proteins was analysed. Different colors represent individual independent experiments (n = 4). Averages are indicated by black lines. (B) The Western blot shows Rap1 and Rap2 protein depletion. (C, D) The barrier resistance of HUVECs transfected with siRNAs targeting the Rap2 proteins, the Cadherins VE-cadherin and P-cadherin or all five proteins was analysed. Different colors represent individual independent experiments (n = 3). Averages are indicated by black lines. (D) The Western blot shows Rap2 and VE-cadherin protein depletion. (E) The barrier resistance of HUVECs transfected with siRNAs targeting RasGEF1A, RasGEF1B and RasGEF1C or (F) the Ste20 kinases MINK, TNIK or MAP4K4. All ECIS experiments were performed as in Figure 1. Different colors represent individual independent experiments (E: n = 4 for siRasGEF1A and siRasGEF1B, n = 5 for siRasGEF1C, F: n = 9 for siMAP4K4 and n = 3 for siTNIK and siMINK). Averages are indicated by black lines.
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pone-0057903-g004: Rap2 antagonizes Rap1 by an own signalling module.(A, B) The barrier resistance of HUVECs transfected with siRNAs targeting the Rap1 proteins, the Rap2 proteins or all five Rap proteins was analysed. Different colors represent individual independent experiments (n = 4). Averages are indicated by black lines. (B) The Western blot shows Rap1 and Rap2 protein depletion. (C, D) The barrier resistance of HUVECs transfected with siRNAs targeting the Rap2 proteins, the Cadherins VE-cadherin and P-cadherin or all five proteins was analysed. Different colors represent individual independent experiments (n = 3). Averages are indicated by black lines. (D) The Western blot shows Rap2 and VE-cadherin protein depletion. (E) The barrier resistance of HUVECs transfected with siRNAs targeting RasGEF1A, RasGEF1B and RasGEF1C or (F) the Ste20 kinases MINK, TNIK or MAP4K4. All ECIS experiments were performed as in Figure 1. Different colors represent individual independent experiments (E: n = 4 for siRasGEF1A and siRasGEF1B, n = 5 for siRasGEF1C, F: n = 9 for siMAP4K4 and n = 3 for siTNIK and siMINK). Averages are indicated by black lines.

Mentions: The observation that Rap2 depletion increased barrier resistance suggests that Rap1 and Rap2 can antagonize each other. Indeed, when HUVECs are depleted of all five Rap proteins, the barrier resistance was comparable to control levels (Fig. 4A, B). Similar to Rap1, the effect of Rap2 on the barrier resistance depends on Cadherins (Fig. 4C, D). In this context it is interesting to notice that RapGEFs regulating endothelial junctions, PDZ-GEF and Epac1, are activating Rap1 and Rap2 [27]–[29] and that many effectors of Rap1 interact with Rap2 as well [42]. Since Rap2 antagonizes the effect of Rap1 on barrier resistance, Rap2 should utilize at least partially its own unique GEFs and effectors. Specific activation of Rap2 proteins has been demonstrated for the RasGEF1 family of RapGEFs, which consists of three closely related proteins, two of which where characterised in vitro and found to display GEF activity towards Rap2 but not Rap1 [31]. Depletion of RasGEF1C in particular increased the barrier resistance (Fig. 4E). To identify a potential Rap2 effector protein, the members of the GCK-IV subgroup of Ste20 kinases were knocked down. Similar to depletion of Rap2 and RasGEF1, depletion of MAP4K4 caused a marked increase in barrier resistance (Fig. 4F). No effect of siMINK or siTNIK was observed. In HUVECs the RasGEF1 family and MAP4K4 therefore form a Rap2 specific signalling route, which mediates the Rap1 antagonising effect of Rap2.


Rap1 and Rap2 antagonistically control endothelial barrier resistance.

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

Rap2 antagonizes Rap1 by an own signalling module.(A, B) The barrier resistance of HUVECs transfected with siRNAs targeting the Rap1 proteins, the Rap2 proteins or all five Rap proteins was analysed. Different colors represent individual independent experiments (n = 4). Averages are indicated by black lines. (B) The Western blot shows Rap1 and Rap2 protein depletion. (C, D) The barrier resistance of HUVECs transfected with siRNAs targeting the Rap2 proteins, the Cadherins VE-cadherin and P-cadherin or all five proteins was analysed. Different colors represent individual independent experiments (n = 3). Averages are indicated by black lines. (D) The Western blot shows Rap2 and VE-cadherin protein depletion. (E) The barrier resistance of HUVECs transfected with siRNAs targeting RasGEF1A, RasGEF1B and RasGEF1C or (F) the Ste20 kinases MINK, TNIK or MAP4K4. All ECIS experiments were performed as in Figure 1. Different colors represent individual independent experiments (E: n = 4 for siRasGEF1A and siRasGEF1B, n = 5 for siRasGEF1C, F: n = 9 for siMAP4K4 and n = 3 for siTNIK and siMINK). Averages are indicated by black lines.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3585282&req=5

pone-0057903-g004: Rap2 antagonizes Rap1 by an own signalling module.(A, B) The barrier resistance of HUVECs transfected with siRNAs targeting the Rap1 proteins, the Rap2 proteins or all five Rap proteins was analysed. Different colors represent individual independent experiments (n = 4). Averages are indicated by black lines. (B) The Western blot shows Rap1 and Rap2 protein depletion. (C, D) The barrier resistance of HUVECs transfected with siRNAs targeting the Rap2 proteins, the Cadherins VE-cadherin and P-cadherin or all five proteins was analysed. Different colors represent individual independent experiments (n = 3). Averages are indicated by black lines. (D) The Western blot shows Rap2 and VE-cadherin protein depletion. (E) The barrier resistance of HUVECs transfected with siRNAs targeting RasGEF1A, RasGEF1B and RasGEF1C or (F) the Ste20 kinases MINK, TNIK or MAP4K4. All ECIS experiments were performed as in Figure 1. Different colors represent individual independent experiments (E: n = 4 for siRasGEF1A and siRasGEF1B, n = 5 for siRasGEF1C, F: n = 9 for siMAP4K4 and n = 3 for siTNIK and siMINK). Averages are indicated by black lines.
Mentions: The observation that Rap2 depletion increased barrier resistance suggests that Rap1 and Rap2 can antagonize each other. Indeed, when HUVECs are depleted of all five Rap proteins, the barrier resistance was comparable to control levels (Fig. 4A, B). Similar to Rap1, the effect of Rap2 on the barrier resistance depends on Cadherins (Fig. 4C, D). In this context it is interesting to notice that RapGEFs regulating endothelial junctions, PDZ-GEF and Epac1, are activating Rap1 and Rap2 [27]–[29] and that many effectors of Rap1 interact with Rap2 as well [42]. Since Rap2 antagonizes the effect of Rap1 on barrier resistance, Rap2 should utilize at least partially its own unique GEFs and effectors. Specific activation of Rap2 proteins has been demonstrated for the RasGEF1 family of RapGEFs, which consists of three closely related proteins, two of which where characterised in vitro and found to display GEF activity towards Rap2 but not Rap1 [31]. Depletion of RasGEF1C in particular increased the barrier resistance (Fig. 4E). To identify a potential Rap2 effector protein, the members of the GCK-IV subgroup of Ste20 kinases were knocked down. Similar to depletion of Rap2 and RasGEF1, depletion of MAP4K4 caused a marked increase in barrier resistance (Fig. 4F). No effect of siMINK or siTNIK was observed. In HUVECs the RasGEF1 family and MAP4K4 therefore form a Rap2 specific signalling route, which mediates the Rap1 antagonising effect of Rap2.

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