Figure 2.
IL-1β–induced and Gab2-dependent mobilization of P-selectin and VWF was Rho-dependent and independent of TAK1 and NF-κB. (A) IL-1β activates Rho kinase in endothelial cells. HUVECs were treated with a control vehicle or Rho-specific inhibitor, rhosin, for 1 hour. After that, the cells were stimulated with IL-1β for indicated periods. Rho activation was measured as GTP-bound Rho using the Rho activation assay kit (left panel). Rho-GTP signals were quantified by densitometric analysis, and the signal intensity in cells not treated with IL-1β or rhosin was taken as 1. (B) Gab2 silencing blocks IL-1β–induced Rho activation in endothelial cells. HUVECs were transfected with 200 nM of scRNA or Gab2 siRNA. After 48 hours, the transfected cells were treated with IL-1β for indicated time periods. Rho activation was measured and quantified as described in (A). (C) Pharmacological inhibition of Rho activation attenuates IL-1β–induced translocation of P-selectin to the cell surface and VWF secretion. HUVECs were treated with a control vehicle or Rho-specific inhibitors, rhosin or Y27632 (10 µM), for 1 hour. Then, the cells were stimulated with IL-1β. After 1 hour, the cell supernatants were collected, and cell lysates were harvested. Cell lysates were probed for total P-selectin, VWF, and GAPDH. Cell supernatants were precipitated with TCA to concentrate proteins and probed for VWF by immunoblot analysis to assess secreted (S) VWF (left panel). The translocation of P-selectin to the cell surface was measured by cell surface ELISA (middle panel). VWF secretion was quantified by densitometric analysis of VWF (S) immunoblots (right panel). (D) TAK1 silencing does not affect IL-1β–induced translocation of P-selectin to the cell surface or VWF secretion. HUVECs were transfected with scRNA or TAK1 siRNA (200 nM) for 48 hours. The transfected cells were stimulated with IL-1β for 1 hour. P-selectin translocation to the cell surface and VWF secretion were evaluated as described in (C). (E) Pharmacological inhibition of NF-κB does not curtail IL-1β–induced translocation of P-selectin to the cell surface or VWF secretion. HUVECs were treated with an NF-κB–specific inhibitor, BAY117082 (20 µM), or a control vehicle for 1 hour. Thereafter, the cells were stimulated with IL-1β for 1 hour, and P-selectin translocation to the cell surface and VWF secretion were evaluated as described in (C). *P < .05, **P < .01, and ***P < .001. ns, no statistically significant difference.

IL-1β–induced and Gab2-dependent mobilization of P-selectin and VWF was Rho-dependent and independent of TAK1 and NF-κB. (A) IL-1β activates Rho kinase in endothelial cells. HUVECs were treated with a control vehicle or Rho-specific inhibitor, rhosin, for 1 hour. After that, the cells were stimulated with IL-1β for indicated periods. Rho activation was measured as GTP-bound Rho using the Rho activation assay kit (left panel). Rho-GTP signals were quantified by densitometric analysis, and the signal intensity in cells not treated with IL-1β or rhosin was taken as 1. (B) Gab2 silencing blocks IL-1β–induced Rho activation in endothelial cells. HUVECs were transfected with 200 nM of scRNA or Gab2 siRNA. After 48 hours, the transfected cells were treated with IL-1β for indicated time periods. Rho activation was measured and quantified as described in (A). (C) Pharmacological inhibition of Rho activation attenuates IL-1β–induced translocation of P-selectin to the cell surface and VWF secretion. HUVECs were treated with a control vehicle or Rho-specific inhibitors, rhosin or Y27632 (10 µM), for 1 hour. Then, the cells were stimulated with IL-1β. After 1 hour, the cell supernatants were collected, and cell lysates were harvested. Cell lysates were probed for total P-selectin, VWF, and GAPDH. Cell supernatants were precipitated with TCA to concentrate proteins and probed for VWF by immunoblot analysis to assess secreted (S) VWF (left panel). The translocation of P-selectin to the cell surface was measured by cell surface ELISA (middle panel). VWF secretion was quantified by densitometric analysis of VWF (S) immunoblots (right panel). (D) TAK1 silencing does not affect IL-1β–induced translocation of P-selectin to the cell surface or VWF secretion. HUVECs were transfected with scRNA or TAK1 siRNA (200 nM) for 48 hours. The transfected cells were stimulated with IL-1β for 1 hour. P-selectin translocation to the cell surface and VWF secretion were evaluated as described in (C). (E) Pharmacological inhibition of NF-κB does not curtail IL-1β–induced translocation of P-selectin to the cell surface or VWF secretion. HUVECs were treated with an NF-κB–specific inhibitor, BAY117082 (20 µM), or a control vehicle for 1 hour. Thereafter, the cells were stimulated with IL-1β for 1 hour, and P-selectin translocation to the cell surface and VWF secretion were evaluated as described in (C). *P < .05, **P < .01, and ***P < .001. ns, no statistically significant difference.

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