Abstract 326: Syndecan-4 Knockout Mice Exhibit Decreased eNOS Activity And Increased Blood Pressure Via Disruption Of mTOR Complex Assembly In Raft Domains
Background: Growth factor-induced NO release requires PI3K- and mTOR-dependent Akt activation that in turn controls eNOS phosphorylation. Several studies have shown that syndecan-4, a heparan sulfate proteoglycan expressed on the surface of virtually all adherent cells plays a role in regulation of eNOS activity and angiogenesis. In this paper we analyzed the role of syndecan-4 in regulation of mTOR-Akt signaling.
Methods: Endothelial cells were isolated from lungs and heart of syndecan-4 knockout mice (S4−/−) and their wild type littermates (S4+/+). Proteins were immunoblotted with phosphospecific antibodies. Raft domains were isolated by sucrose gradient followed by ultracentrifugation. Blood pressure and heart rate were measured in anesthetized mice using a Millar transducer inserted into the right carotid artery.
Results: We find that growth factor-induced phosphorylation and activation of Akt was markedly reduced in syndecan-4 deficient endothelial cells. While there were no overall differences in the expression levels of Akt, mTOR or rictor between S4−/− and S4+/+ endothelial cells, the levels of rictor, mLST8 as well as PKCα were markedly decreased in S4−/− raft membrane domains where Akt is phosphorylated. Since it is known that syndecan-4 cytoplasmic domain binds PKCα and regulates PKCα cellular distribution and activity, we expressed a myristoylated-PKCα in S4−/− cells to compensate for the loss of this potential membrane PKCα anchor. Remarkably, expression of myristoylated-PKCα fully rescued growth factor-induced Akt phosphorylation while also restoring the presence of rictor and mLST8 in the rafts. The reduced Akt activity in absence of syndecan-4 was associated with a significant decrease in nitric oxide release from endothelial cells, a marked reduction in cGMP content of heart and lung tissues and a 20% increase in the mean arterial pressure of S4−/− mice.
Conclusion: Our data demonstrate that syndecan-4 regulates Akt/eNOS activation via PKCα-dependent localization of rictor and mLST8, two components of mTOR complex 2 into membrane raft domains. The decreased eNOS activity in syndecan-4 deficient mice is associated with a significant increase in their mean arterial pressure. .