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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 decreased the barrier resistance.Simultaneous depletion of all three Rap2 isoforms (A, B) increases the barrier resistance of HUVEC monolayers, whereas individual depletion of Rap2A, Rap2B and Rap2C showed no effect (C, D). Analysis was performed as in Figure 1. Different colors represent individual independent experiments (A: n = 6, C: n = 3 for siRap2A and siRap2C, n = 6 for siRap2B). Averages are indicated by black lines. (B, D) The Western blots show Rap2 protein depletion. (E) HUVECs transfected with siScrambled, siRap2A, siRap2B or siRap2C were grown to confluency, after which RNA was extracted and Rap2 mRNA levels were assessed by QPCR. The histogram shows averages of three independent experiments which were related to one identical reference mRNA extraction of untreated cells. Error bars indicate standard deviation.
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pone-0057903-g003: Rap2 decreased the barrier resistance.Simultaneous depletion of all three Rap2 isoforms (A, B) increases the barrier resistance of HUVEC monolayers, whereas individual depletion of Rap2A, Rap2B and Rap2C showed no effect (C, D). Analysis was performed as in Figure 1. Different colors represent individual independent experiments (A: n = 6, C: n = 3 for siRap2A and siRap2C, n = 6 for siRap2B). Averages are indicated by black lines. (B, D) The Western blots show Rap2 protein depletion. (E) HUVECs transfected with siScrambled, siRap2A, siRap2B or siRap2C were grown to confluency, after which RNA was extracted and Rap2 mRNA levels were assessed by QPCR. The histogram shows averages of three independent experiments which were related to one identical reference mRNA extraction of untreated cells. Error bars indicate standard deviation.

Mentions: Whereas the effect of Rap1 on junctional permeability is extensively described, the function of the highly homologous Rap2 proteins in this process has remained unexplored. To analyse a putative contribution of Rap2A, Rap2B and Rap2C, these proteins were depleted from HUVEC and assayed for their effect on the barrier resistance (Fig. 3A, B). Depletion of the three Rap2 proteins increased the barrier resistance twofold. This is opposite to the effect of Rap1 depletion (Fig. 1) and indicates that Rap2 increases junctional permeability. Individual depletion of Rap2A, Rap2B or Rap2C resulted in no significant effect (Fig. 3C). On Western blots, a Rap2 antibody detected two bands which were predominantly sensitive to depletion of Rap2B (lower band) and Rap2C (upper band) (Fig. 3D). The expression of all three Rap2 isoforms was detected by RT-QPCR in HUVECs, but the used set-up did not allow determining and comparing absolute expression levels (Fig. 3E). The used RNAi oligos showed no significant effect on the respective non targeted isoforms (Fig. 3E). As the Rap2 antibody detected recombinant version of the three Rap2 isoforms equally well (data not shown), western blot analysis would suggests that Rap2B and Rap2C are the major isoforms in HUVECs (Fig. 3D) and act redundantly to control barrier resistance.


Rap1 and Rap2 antagonistically control endothelial barrier resistance.

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

Rap2 decreased the barrier resistance.Simultaneous depletion of all three Rap2 isoforms (A, B) increases the barrier resistance of HUVEC monolayers, whereas individual depletion of Rap2A, Rap2B and Rap2C showed no effect (C, D). Analysis was performed as in Figure 1. Different colors represent individual independent experiments (A: n = 6, C: n = 3 for siRap2A and siRap2C, n = 6 for siRap2B). Averages are indicated by black lines. (B, D) The Western blots show Rap2 protein depletion. (E) HUVECs transfected with siScrambled, siRap2A, siRap2B or siRap2C were grown to confluency, after which RNA was extracted and Rap2 mRNA levels were assessed by QPCR. The histogram shows averages of three independent experiments which were related to one identical reference mRNA extraction of untreated cells. Error bars indicate standard deviation.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0057903-g003: Rap2 decreased the barrier resistance.Simultaneous depletion of all three Rap2 isoforms (A, B) increases the barrier resistance of HUVEC monolayers, whereas individual depletion of Rap2A, Rap2B and Rap2C showed no effect (C, D). Analysis was performed as in Figure 1. Different colors represent individual independent experiments (A: n = 6, C: n = 3 for siRap2A and siRap2C, n = 6 for siRap2B). Averages are indicated by black lines. (B, D) The Western blots show Rap2 protein depletion. (E) HUVECs transfected with siScrambled, siRap2A, siRap2B or siRap2C were grown to confluency, after which RNA was extracted and Rap2 mRNA levels were assessed by QPCR. The histogram shows averages of three independent experiments which were related to one identical reference mRNA extraction of untreated cells. Error bars indicate standard deviation.
Mentions: Whereas the effect of Rap1 on junctional permeability is extensively described, the function of the highly homologous Rap2 proteins in this process has remained unexplored. To analyse a putative contribution of Rap2A, Rap2B and Rap2C, these proteins were depleted from HUVEC and assayed for their effect on the barrier resistance (Fig. 3A, B). Depletion of the three Rap2 proteins increased the barrier resistance twofold. This is opposite to the effect of Rap1 depletion (Fig. 1) and indicates that Rap2 increases junctional permeability. Individual depletion of Rap2A, Rap2B or Rap2C resulted in no significant effect (Fig. 3C). On Western blots, a Rap2 antibody detected two bands which were predominantly sensitive to depletion of Rap2B (lower band) and Rap2C (upper band) (Fig. 3D). The expression of all three Rap2 isoforms was detected by RT-QPCR in HUVECs, but the used set-up did not allow determining and comparing absolute expression levels (Fig. 3E). The used RNAi oligos showed no significant effect on the respective non targeted isoforms (Fig. 3E). As the Rap2 antibody detected recombinant version of the three Rap2 isoforms equally well (data not shown), western blot analysis would suggests that Rap2B and Rap2C are the major isoforms in HUVECs (Fig. 3D) and act redundantly to control 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