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CCM proteins control endothelial β1 integrin dependent response to shear stress.

Macek Jilkova Z, Lisowska J, Manet S, Verdier C, Deplano V, Geindreau C, Faurobert E, Albigès-Rizo C, Duperray A - Biol Open (2014)

Bottom Line: Indeed, we show that overexpression of the CCM complex, an inhibitor of β1 integrin activation, blocks endothelial actin rearrangement and cell reorientation in response to shear stress similarly to β1 integrin silencing.Conversely, depletion of CCM2 protein leads to an elongated "shear-stress-like" phenotype even in the absence of flow.Taken together, our findings reveal the existence of a balance between positive extracellular and negative intracellular signals, i.e. shear stress and CCM complex, for the control of β1 integrin activation and subsequent adaptation of vascular endothelial cells to mechanostimulation by fluid shear stress.

View Article: PubMed Central - PubMed

Affiliation: INSERM, Institut Albert Bonniot, F-38000 Grenoble, France Université Grenoble Alpes, Institut Albert Bonniot, F-38000 Grenoble, France.

No MeSH data available.


Related in: MedlinePlus

CCM2 negatively regulates β1-integrin mediated cytoskeletal reinforcement and reorientation after shear stress stimulation.HUVEC, EA.hy926 and CCM2-silenced EA.hy926 were exposed to fluid shear stress at 2.0 Pa for 18 h. Cells were stained by (A) paxillin and activated-state-specific β1-integrin antibody (Clone:9EG7) and by (B) F-actin and zyxin antibody. White arrow: indicates the direction of flow. Scale bars: 10 µm.
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f04: CCM2 negatively regulates β1-integrin mediated cytoskeletal reinforcement and reorientation after shear stress stimulation.HUVEC, EA.hy926 and CCM2-silenced EA.hy926 were exposed to fluid shear stress at 2.0 Pa for 18 h. Cells were stained by (A) paxillin and activated-state-specific β1-integrin antibody (Clone:9EG7) and by (B) F-actin and zyxin antibody. White arrow: indicates the direction of flow. Scale bars: 10 µm.

Mentions: To investigate whether the high levels of CCM proteins are indeed the reason for the inability of EA.hy926 to respond to shear stress, we analyzed the response of CCM2-silenced EA.hy926 to shear stress. As we have previously shown, CCM2 silencing induces the destabilization of its partner proteins CCM1 and ICAP-1 resulting in the loss of the entire complex (Faurobert et al., 2013). Silencing efficiency of CCM2 siRNA on EA.hy926 cell line was determined by western blot (Fig. 3D). Remarkably, CCM2 deficiency led to elongation of the EA.hy926 cells (Fig. 3E) and restored their capacity to orient toward the flow (Fig. 3C,E) similarly to HUVEC. This was correlated with the appearance of aligned β1 integrin-containing focal adhesions on the ventral face of CCM2-depleted EA.hy926 (Fig. 4A). Paxillin was recruited to these focal adhesions (Fig. 4A) showing that release of β1 integrin inhibition upon CCM2 loss led to the generation of functional β1 integrin-dependent signaling scaffolds in response to shear stress. Transversal actin fibers were polymerized from these β1 integrin scaffolds and generated internal tension as suggested by the recruitment of zyxin (Fig. 4B), a typical component of focal adhesions which accumulates at force-bearing sites (Beningo et al., 2001; Colombelli et al., 2009) and is mobilized by mechanical forces from focal adhesions to actin filament (Yoshigi et al., 2005). Therefore, contrary to EA.hy926, shear stress increased internal cell tension in CCM2-depleted EA.hy926 in a similar way as it did in HUVEC. These results confirm that overexpression of CCM/ICAP-1 proteins is responsible for the absence of morphological response of EA.hy926 most likely by preventing shear-stress-induced β1 integrin activation.


CCM proteins control endothelial β1 integrin dependent response to shear stress.

Macek Jilkova Z, Lisowska J, Manet S, Verdier C, Deplano V, Geindreau C, Faurobert E, Albigès-Rizo C, Duperray A - Biol Open (2014)

CCM2 negatively regulates β1-integrin mediated cytoskeletal reinforcement and reorientation after shear stress stimulation.HUVEC, EA.hy926 and CCM2-silenced EA.hy926 were exposed to fluid shear stress at 2.0 Pa for 18 h. Cells were stained by (A) paxillin and activated-state-specific β1-integrin antibody (Clone:9EG7) and by (B) F-actin and zyxin antibody. White arrow: indicates the direction of flow. Scale bars: 10 µm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4265761&req=5

f04: CCM2 negatively regulates β1-integrin mediated cytoskeletal reinforcement and reorientation after shear stress stimulation.HUVEC, EA.hy926 and CCM2-silenced EA.hy926 were exposed to fluid shear stress at 2.0 Pa for 18 h. Cells were stained by (A) paxillin and activated-state-specific β1-integrin antibody (Clone:9EG7) and by (B) F-actin and zyxin antibody. White arrow: indicates the direction of flow. Scale bars: 10 µm.
Mentions: To investigate whether the high levels of CCM proteins are indeed the reason for the inability of EA.hy926 to respond to shear stress, we analyzed the response of CCM2-silenced EA.hy926 to shear stress. As we have previously shown, CCM2 silencing induces the destabilization of its partner proteins CCM1 and ICAP-1 resulting in the loss of the entire complex (Faurobert et al., 2013). Silencing efficiency of CCM2 siRNA on EA.hy926 cell line was determined by western blot (Fig. 3D). Remarkably, CCM2 deficiency led to elongation of the EA.hy926 cells (Fig. 3E) and restored their capacity to orient toward the flow (Fig. 3C,E) similarly to HUVEC. This was correlated with the appearance of aligned β1 integrin-containing focal adhesions on the ventral face of CCM2-depleted EA.hy926 (Fig. 4A). Paxillin was recruited to these focal adhesions (Fig. 4A) showing that release of β1 integrin inhibition upon CCM2 loss led to the generation of functional β1 integrin-dependent signaling scaffolds in response to shear stress. Transversal actin fibers were polymerized from these β1 integrin scaffolds and generated internal tension as suggested by the recruitment of zyxin (Fig. 4B), a typical component of focal adhesions which accumulates at force-bearing sites (Beningo et al., 2001; Colombelli et al., 2009) and is mobilized by mechanical forces from focal adhesions to actin filament (Yoshigi et al., 2005). Therefore, contrary to EA.hy926, shear stress increased internal cell tension in CCM2-depleted EA.hy926 in a similar way as it did in HUVEC. These results confirm that overexpression of CCM/ICAP-1 proteins is responsible for the absence of morphological response of EA.hy926 most likely by preventing shear-stress-induced β1 integrin activation.

Bottom Line: Indeed, we show that overexpression of the CCM complex, an inhibitor of β1 integrin activation, blocks endothelial actin rearrangement and cell reorientation in response to shear stress similarly to β1 integrin silencing.Conversely, depletion of CCM2 protein leads to an elongated "shear-stress-like" phenotype even in the absence of flow.Taken together, our findings reveal the existence of a balance between positive extracellular and negative intracellular signals, i.e. shear stress and CCM complex, for the control of β1 integrin activation and subsequent adaptation of vascular endothelial cells to mechanostimulation by fluid shear stress.

View Article: PubMed Central - PubMed

Affiliation: INSERM, Institut Albert Bonniot, F-38000 Grenoble, France Université Grenoble Alpes, Institut Albert Bonniot, F-38000 Grenoble, France.

No MeSH data available.


Related in: MedlinePlus