The Hyaluronan Receptor RHAMM is Necessary for the Activation of Src Kinase and the Signaling Pathway for Endothelial Nitric Oxide Production

Abstract

RHAMM (Receptor for Hyaluronan‐Mediated Motility) is necessary for endothelial cell (EC) migration in vitro and angiogenesis in vivo, but the mechanisms are incompletely understood. Nitric oxide (NO) production by endothelial nitric oxide synthase (eNOS) involves a signaling cascade through Src kinase and Akt, and results in eNOS phosphorylation. A number of diverse agonists, including VEGF, HDL, estradiol, acetylcholine and insulin, activate eNOS to produce NO. We hypothesized that RHAMM is necessary for the activation of Src kinase and eNOS to produce NO. To determine the molecular mechanism by which RHAMM regulates eNOS activation, we used cultured EC and RHAMM KO mice.RHAMM siRNA knockdown in HUVEC, BAEC or HPAEC inhibited the increased cell migration after single scratch wounding and eNOS activity observed with treatment with all EC agonists, and the exogenous NO donor NONOate rescued the migratory defect, suggesting that RHAMM is universally required for eNOS activation and NO production. RHAMM siRNA‐mediated decrease in cell migration and eNOS activity correlated with decreased Akt and eNOS phosphorylation but not Erk phosphorylation. In RHAMM KO mice, normal alveolarization and re‐endothelialization after arterial injury, processes that require NO, were delayed. RHAMM KO mice also did not respond to exogenous VEGF, but treatment with either inhaled NO (iNO) or Molsidomine normalized lung alveolarization and restored re‐endothelialization after arterial injury respectively.Since activation of Src is a proximal step in the signaling cascade, we examined the effect of RHAMM knockdown on Src activation. RHAMM siRNA treatment of EC was associated with hyperphosphorylation of the inhibitory site of Src (Y527) and an inhibition of the phosphorylation of the activation site of Src (Y416). In this process, the phosphatase Shp2 is critical in the dephosphorylation of Y527 and Src activation, and associates with VEGFR2. Interestingly, Shp2 and Src both co‐immunoprecipitated with RHAMM in vivo as well as in vitro. Compared to EC treated with scrambled siRNA, co‐immunoprecipitation of Shp2 and VEGFR2 after VEGF stimulation was significantly reduced in RHAMM siRNA‐treated EC. These data suggest that the RHAMM‐Shp2 complex facilitates Shp2 tethering to VEGFR2, which is necessary for the dephosphorylation of Y527 in Src and the downstream activation of the signaling pathway for NO production in endothelial cells. We speculate that RHAMM is a novel target for the regulation of the wide array of physiologic and pathological functions that endothelial NO regulates.Support or Funding InformationNIH: HL093535, HL079090, HL075930; Children's Medical Center Foundation; William Buchanan Chair in Pediatrics.