title: Protein kinase C-α signals P115RhoGEF phosphorylation and RhoA activation in TNF-α-induced mouse brain microvascular endothelial cell barrier dysfunction.
abstract: BACKGROUND
  Tumor necrosis factor-α (TNF-α), a proinflammatory cytokine, is capable of activating the small GTPase RhoA, which in turn contributes to endothelial barrier dysfunction. However, the underlying signaling mechanisms remained undefined. Therefore, we aimed to determine the role of protein kinase C (PKC) isozymes in the mechanism of RhoA activation and in signaling TNF-α-induced mouse brain microvascular endothelial cell (BMEC) barrier dysfunction.
  
  
  METHODS
  Bend.3 cells, an immortalized mouse brain endothelial cell line, were exposed to TNF-α (10 ng/mL). RhoA activity was assessed by pull down assay. PKC-α activity was measured using enzyme assasy. BMEC barrier function was measured by transendothelial electrical resistance (TER). p115RhoGEF phosphorylation was detected by autoradiography followed by western blotting. F-actin organization was observed by rhodamine-phalloidin staining. Both pharmacological inhibitors and knockdown approaches were employed to investigate the role of PKC and p115RhoGEF in TNF-α-induced RhoA activation and BMEC permeability.
  
  
  RESULTS
  We observed that TNF-α induces a rapid phosphorylation of p115RhoGEF, activation of PKC and RhoA in BMECs. Inhibition of conventional PKC by Gö6976 mitigated the TNF-α-induced p115RhoGEF phosphorylation and RhoA activation. Subsequently, we found that these events are regulated by PKC-α rather than PKC-β by using shRNA. In addition, P115-shRNA and n19RhoA (dominant negative mutant of RhoA) transfections had no effect on mediating TNF-α-induced PKC-α activation. These data suggest that PKC-α but not PKC-β acts as an upstream regulator of p115RhoGEF phosphorylation and RhoA activation in response to TNF-α. Moreover, depletion of PKC-α, of p115RhoGEF, and inhibition of RhoA activation also prevented TNF-α-induced stress fiber formation and a decrease in TER.
  
  
  CONCLUSIONS
  Taken together, our results show that PKC-α phosphorylation of p115RhoGEF mediates TNF-α signaling to RhoA, and that this plays a critical role in signaling F-actin rearrangement and barrier dysfunction in BMECs.
authors.affiliation: Department of Pediatrics, Xiangya Hospital of Central South University, No,87 Xiangya Road, Changsha, Hunan 410008, China.
authors.forename: Jing
authors.initials: J
authors.lastname: Peng
authors.forename: Fang
authors.initials: F
authors.lastname: He
authors.forename: Ciliu
authors.initials: C
authors.lastname: Zhang
authors.forename: Xiaolu
authors.initials: X
authors.lastname: Deng
authors.forename: Fei
authors.initials: F
authors.lastname: Yin
mesh_terms: D000199:Actins / Q000378:metabolism; D000818:Animals; D002199:Capillary Permeability / Q000187:drug effects; D002460:Cell Line; D042783:Endothelial Cells / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects / Q000503:physiopathology; D004789:Enzyme Activation; D020662:Guanine Nucleotide Exchange Factors / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D010766:Phosphorylation; D051571:Protein Kinase C-alpha / Q000235:genetics / Q000378:metabolism*; D064067:Rho Guanine Nucleotide Exchange Factors; D015398:Signal Transduction / Q000187:drug effects; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*; D020742:rhoA GTP-Binding Protein / Q000235:genetics / Q000378:metabolism*
chemical_list: D000199:Actins; D020662:Guanine Nucleotide Exchange Factors; D064067:Rho Guanine Nucleotide Exchange Factors; D014409:Tumor Necrosis Factor-alpha; D051571:Protein Kinase C-alpha; D020742:rhoA GTP-Binding Protein