Figure 4
Figure 4. uPA-induced XIAP mRNA up-regulation is dependent on the NF-κB pathway. (A) XIAP mRNA up-regulation in HUMECs by uPA (100 nM) in the absence or presence of monoclonal antibodies inhibiting uPA-uPAR interaction (mAB R3, mAB R5; 10 μg/mL), benzamidine (100 μM), which inhibits uPA serine protease activity, or the LRP chaperone receptor-associated protein (200 nM), or by DFP-inactivated uPA (100 nM). uPA up-regulation of XIAP is dependent on uPAR, active uPA, and a member of the LDLR family (*P < .01; n = 4). (B) XIAP mRNA up-regulation in HUMECs by uPA (100 nM) in the absence or presence of the specific PI3 kinase inhibitor Ly294002 (10 μM) or the specific IKK inhibitor BAY11-7085 (5 μM). uPA-induced XIAP up-regulation is Ly294002 independent but BAY11-7085 dependent (*P < .01; n = 3). (C) Effect of uPA (100 nM) on phosphorylation of IKK in HUMECs in the absence or presence of the specific PI3 kinase inhibitor Ly294002 (10 μM). Phosphorylation of IKK by uPA is Ly294002 independent (one representative Western blot is shown; n = 3). (D) Quantification of phospho-IKKs in immunoprecipitates obtained with mAbs against IKKα or IKKβ from HUVECs stimulated with uPA (100 nM) or VEGF165 (1.25 nM). uPA as well as VEGF165 induces phosphorylation of IKKα but not of IKKβ (P < .01; n = 3). (E) Demonstration of the nuclear translocation of NF-κB subunits in endothelial cells induced by either 100 nM uPA or 10 nM TNF-α. Confocal laser micrographs are shown for endothelial cells stimulated by vehicle control or TNF-α, or uPA for 60 minutes. One representative picture of 3 independent experiments is shown. Bar represents 10 μm. (F) Pak1 phosphorylation in HUMECs on uPA (100 nM) treatment in the absence or presence of the specific PI3 kinase inhibitor Ly294002 (10 μM). Phosphorylation of Pak1 by uPA is Ly294002 independent (one representative Western blot is shown; n = 3). (G) Number or recovered HUMECs 72 hours after seeding in percentage of control. HUMECs were either untreated (left panel) or treated with uPA (100 nM; right panel). HUMECs were transfected with either empty vector (control) or a retrovirus carrying dnCdc42 or dnRho GTPase or an adenovirus carrying IκB (*P < .01 vs respective control; n = 3).

uPA-induced XIAP mRNA up-regulation is dependent on the NF-κB pathway. (A) XIAP mRNA up-regulation in HUMECs by uPA (100 nM) in the absence or presence of monoclonal antibodies inhibiting uPA-uPAR interaction (mAB R3, mAB R5; 10 μg/mL), benzamidine (100 μM), which inhibits uPA serine protease activity, or the LRP chaperone receptor-associated protein (200 nM), or by DFP-inactivated uPA (100 nM). uPA up-regulation of XIAP is dependent on uPAR, active uPA, and a member of the LDLR family (*P < .01; n = 4). (B) XIAP mRNA up-regulation in HUMECs by uPA (100 nM) in the absence or presence of the specific PI3 kinase inhibitor Ly294002 (10 μM) or the specific IKK inhibitor BAY11-7085 (5 μM). uPA-induced XIAP up-regulation is Ly294002 independent but BAY11-7085 dependent (*P < .01; n = 3). (C) Effect of uPA (100 nM) on phosphorylation of IKK in HUMECs in the absence or presence of the specific PI3 kinase inhibitor Ly294002 (10 μM). Phosphorylation of IKK by uPA is Ly294002 independent (one representative Western blot is shown; n = 3). (D) Quantification of phospho-IKKs in immunoprecipitates obtained with mAbs against IKKα or IKKβ from HUVECs stimulated with uPA (100 nM) or VEGF165 (1.25 nM). uPA as well as VEGF165 induces phosphorylation of IKKα but not of IKKβ (P < .01; n = 3). (E) Demonstration of the nuclear translocation of NF-κB subunits in endothelial cells induced by either 100 nM uPA or 10 nM TNF-α. Confocal laser micrographs are shown for endothelial cells stimulated by vehicle control or TNF-α, or uPA for 60 minutes. One representative picture of 3 independent experiments is shown. Bar represents 10 μm. (F) Pak1 phosphorylation in HUMECs on uPA (100 nM) treatment in the absence or presence of the specific PI3 kinase inhibitor Ly294002 (10 μM). Phosphorylation of Pak1 by uPA is Ly294002 independent (one representative Western blot is shown; n = 3). (G) Number or recovered HUMECs 72 hours after seeding in percentage of control. HUMECs were either untreated (left panel) or treated with uPA (100 nM; right panel). HUMECs were transfected with either empty vector (control) or a retrovirus carrying dnCdc42 or dnRho GTPase or an adenovirus carrying IκB (*P < .01 vs respective control; n = 3).

Close Modal
This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal