The Role Of ENH In PKA Pathway And Vascular Remodeling

1.Aims and SignificanceENH,also known as PDLIM5,is a member of PDZ-LIM domain protein family.As an adaptor protein,ENH is involved in cytoskeleton through binding to alpha-actinin.In addition,ENH interacts with PKC and PKD,two important cellular protein kinases,helping them to phosphorylate their substrates.By analyzing the amino-acid sequence,we found Ser228 as an potential phosphorylation site for PKA.Thus we speculated ENH may be a new AKAP.Recent study showed that ENH deficiency enhanced hypoxia-mediated vascular remodeling and promoted pulmonary hypertension.The phosphorylation of ENH was also reported to inhibit the proliferation of vascular smooth muscle cells,which was closely related to vascular remodeling.This study about "The role of ENH in vascular remodeling as an AKAP" may helps to broaden our understanding of the pathogenesis of vascular remodeling related diseases,and also introduced a potential new target for the treatment of these diseases.2.MethodsIn vitro,ENH and PKA regulatory subunits or ID2 are co-expressed in HEK 293T cells.With the method of truncating mutation,we identify the region of ENH which interacts with ID2.In ID2,there are three potential loci that might be phosphorylated by PKA while in ENH there is one.We mutate serine of the possible PKA phosphorylation loci into alanine.Mutants are expressed in HEK 293T cells,and stimulated by PKA agonist.Finally,we use antibodies of the tagged protein to conduct co-immunoprecipitation,and then check the level of PKA phosphorylation of mutants.In vivo,we establish the vascular remodeling model by ligating the common carotid artery on ENH deficiency mice,endothelial specific knockout ENH mice and smooth muscle specific knockout ENH mice,in order to study the role of ENH in vascular remodeling.3.Results1)ENH interacted with R?? regulatory subunit of PKA and can be phosphorylated by PKA.The single mutation S228A completely eliminated the phosphorylation of ENH in forskolin-stimulated HEK 293T cell.2)ID2 can be phosphorylated by PKA.The single mutation S14A eliminated the phosphorylation of ID2.3)ENH interacted with ID2 and facilitated PKA to phosphorylate ID2.4)ENH interacted with the C-teminal of STAT3 through LIM domain.5)The ENH deficiency mice showed defective aorta with aortic dissection.6)Deficiency of ENH suppressed neointima formation after carotid artery ligation.7)ENH specific ablation in smooth muscle or endothelium also attenuated vascular remodeling.