Shear Stress Regulates the Flk-1/Cbl/PI3K/NF-κB Pathway Via Actin and Tyrosine Kinases

Vascular endothelial cells (ECs) are continuously exposed to mechanical stimuli (e.g., shear stress). Our previous study has shown that the shear-induced nuclear factor-κB (NF-κB) activation is mediated by integrins [Bhullar, I. S., Y. S. Li, H. Miao, E. Zandi, M. Kim, et al. J. Biol. Chem. 273:30544-30549, 1998]. The shear-activated integrins can also transactivate Flk-1 (a receptor for vascular endothelial growth factor (VEGF)) [Wang, Y., H. Miao, S. Li, K. D. Chen, Y. S. Li, et al. Am. J. Physiol. Cell Physiol. 283:C1540-C1547, 2002], which subsequently recruits Casitas B-lineage lymphoma (Cbl) to regulate inhibitor of κB protein kinase (IKK) [Wang, Y., J. Chang, Y. C. Li, Y. S. Li, J. Y. Shyy, and S. Chien. Am. J. Physiol. Heart Circ. Physiol. 286:H685-H692, 2004], an upstream molecule of NF-κB. Therefore, shear stress may likely utilize the Flk-1/Cbl pathway in regulating NF-κB. In this paper, we confirmed that the inhibition of Flk-1 by its specific inhibitor SU1498 blocked the shear-induced NF-κB translocation. The inhibition of Cbl (an adaptor protein which binds to Flk-1 upon shear) by using a negative mutant (Cbl(nm)) also blocked the promoter activity of NF-κB, and the inhibition of the Cbl-downstream molecule phosphatidylinositol-3-kinase (PI3K) abolished the NF-κB translocation. Further experiments revealed that the disruption of actin cytoskeleton inhibited the Flk-1 and Cbl interaction and NF-κB translocation. The inhibition of focal adhesion kinase (FAK) and Src family kinases, which are involved in the integrin-mediated focal adhesion complex, also blocked the shear-induced NF-κB translocation. Together with our previous findings that integrins mediate the shear-induced activation of Flk-1 and NF-κB [Bhullar, I. S., Y. S. Li, H. Miao, E. Zandi, M. Kim, et al. J. Biol. Chem. 273:30544-30549, 1998; Wang, Y., H. Miao, S. Li, K. D. Chen, Y. S. Li, et al. Am. J. Physiol. Cell Physiol. 283:C1540-C1547, 2002], the present results suggest that Flk-1, Cbl, and PI3K act upstream to NF-κB in response to shear stress. This Flk-1/Cbl/PI3K/NF-κB signaling pathway may be originated from integrins and transmitted by key tyrosine kinases and actin cytoskeleton. These results shed new lights on the molecular mechanism by which mechanical shear stress activates the NF-κB signaling pathway, which is critical for vascular inflammatory responses and atherosclerosis.