Abstract 5803: Interaction of Docking Protein Gab1 With Tyrosine Phosphatase SHP2 Regulates Hepatocyte Growth Factor-dependent ERK5 Activation and Upregulation of KLF2 in the Vascular Endothelium
Background: Docking protein Gab1 has been reported to have crucial roles for activation of both mitogen-acitvated protein kinase and AKT downstream of various receptor tyrosine kinases. In the last scientific session, we reported that endothelium-specific Gab1 knockout mice (Gab1ECKO) displayed severe limb necrosis after operatively-induced hindlimb ischemia (HLI) due to the defect of angiogenic response. We also reported that Gab1 was essential for activation of both extracellular signal-regulated kinase 1/2 (ERK1/2) via tyrosine phosphatase SHP2 and AKT via PI3-kinase p85 subunit in human umbilical vein endothelial cells (HUVECs) in response to hepatocyte growth factor (HGF), respectively. Here, we investigated the downstream target of the HGF/Met/Gab1-dependent signaling pathway responsible for the maintenance of endothelium.
Methods and Results: We examined the effect of adenovirus-mediated overexpression of β-galactosidase (β-gal), wild-type Gab1 (Gab1WT), or mutated Gab1 which can’t bind with SHP2 (Gab1ΔSHP2) or p85 (Gab1Δp85) on the activation of downstream signaling pathways in HUVECs. HGF-induced activation of both ERK1/2 and ERK5 was enhanced in the HUVECs expressing Gab1WT or Gab1Δp85, but abrogated in those expressing Gab1ΔSHP2, compared with control cells expressing β-gal. HGF-induced AKT activation was specifically attenuated in cells expressing Gab1Δp85, compared with other three groups. Using DNA microarray analysis, we found that the stimulation with HGF upregulated the expression of Kruppel-like factor 2 (KLF2), a crucial transcriptional factor for endothelial maintenance, and thrombomodulin, a well-known target gene of KLF2, through interaction of Gab1 with SHP2 in HUVECs. Next, we examined the involvement of MEK5-ERK5 pathway for the KLF2 gene expression. HGF-dependent upregulation of KLF2 was almost abrogated by the adenoviral overexpression of either dominant-negative MEK5 or dominant-negative ERK5 in HUVECs. Furthermore, the expression of KLF2 and TM in the endothelium was significantly decreased in the Gab1ECKO, compared with control after HLI.
Conclusion: Interaction of Gab1 with SHP2 is essential for both activation of ERK5 and upregulation of KLF2 in response to HGF in the endothelium.