Mechanisms of endothelial cell protection by blockade of the JAK2 pathway

1 Laboratorio de Nefrología-Hipertensión, Fundación Jiménez Díaz, Madrid; and 2 Departamento de Bioquímica, Instituto de Investigaciones Biomédicas "Alberto Sols," Consejo Superior de Investigaciones Científicas-Universidad Autónoma, Madrid, Spain Submitted 27 October 2005 ; accepted in final form 6 October 2006 Inhibition of the JAK2/STAT pathway has been implicated recently in cytoprotective mechanisms in both vascular smooth muscle cells and astrocytes. The advent of JAK2-specific inhibitors provides a practical tool for the study of this pathway in different cellular types. An interest in finding methods to improve endothelial cell (EC) resistance to injury led us to examine the effect of JAK2/STAT inhibition on EC protection. Furthermore, the signaling pathways involved in JAK2/STAT inhibition-related actions were examined. Our results reveal, for the first time, that blockade of JAK2 with the tyrosine kinase inhibitor AG490 strongly protects cultured EC against cell detachment-dependent death and serum deprivation and increases reseeding efficiency. Confirmation of the specificity of the effects of JAK2 inhibition was attained by finding protective effects on transfection with a dominant negative JAK2. Furthermore, AG490 blocked serum deprivation-induced phosphorylation of JAK2. In terms of mechanism, treatment with AG490 induces several relevant responses, both in monolayer and detached cells. These mechanisms include the following: 1 ) Increase and nuclear translocation of the active, dephosphorylated form of -catenin. In functional terms, this translocation is transcriptionally active, and its protective effect is further supported by the stimulation of EC cytoprotection by transfectionally induced excess of -catenin. 2 ) Increase of platelet endothelial cell adhesion molecule (PECAM)/CD31 levels. 3 ) Increase in total and phosphorylated AKT. 4 ) Increase in phosphorylated glycogen synthase kinase (GSK)3 / . The present findings imply potential practical applications of JAK2 inhibition on EC. These applications affect not only EC in the monolayer but also circulating detached cells and involve mechanistic interactions not previously described. JAK2/STAT; cytoprotection; anoikis; AG490 Address for reprint requests and other correspondence: Mª Ángeles Castilla, Laboratorio de Nefrología-Hipertensión, Fundación Jiménez Díaz, Universidad Autónoma, Avda. Reyes Católicos 2, 28040 Madrid, Spain