β Common Receptor Integrates the Erythropoietin Signaling in Activation of Endothelial Nitric Oxide Synthase

Erythropoietin (EPO), the key hormone for erythropoiesis, also increases nitric oxide (NO) bioavailability in endothelial cells (ECs), yet the definitive mechanisms are not fully understood. Increasing evidence has demonstrated that beta common receptor (beta CR) plays a crucial role in EPO-mediated non-hematopoietic effects. We investigated the role of beta CR in EPO-induced endothelial NO synthase (eNOS) activation in bovine aortic ECs (BAECs) and the molecular mechanisms involved. Results of confocal microscopy and immunoprecipitation analyses revealed that beta CR was colocalized and interacted with EPO receptor (EPOR) in ECs. Inhibition of beta CR or EPOR by neutralizing antibodies or small interfering RNA abolished the EPO-induced NO production. Additionally, blockage of beta CR abrogated the EPO-induced increase in the phosphorylation of eNOS, Akt, Src, or Janus kinase 2 (JAK2). Immunoprecipitation analysis revealed that treatment with EPO increased the interaction between beta CR and eNOS, which was suppressed by inhibition of Src, JAK2, or Akt signaling with specific pharmacological inhibitors. Furthermore, EPO-induced EC proliferation, migration, and tube formation were blocked by pretreatment with beta CR antibody and Src, JAK2, or PI3K/Akt inhibitors. Moreover, in vivo experiments showed that EPO increased the level of phosphorylated eNOS, Src, JAK2, and Akt, as well as beta CR-eNOS association in aortas and promoted the angiogenesis in Matrigel plug, which was diminished by beta CR or EPOR neutralizing antibodies. Our findings suggest that beta CR may play an integrative role in the EPO signaling-mediated activation of eNOS in ECs. J. Cell. Physiol. 226: 3330-3339, 2011. (C) 2011 Wiley Periodicals, Inc.