Suppression of PKC-α attenuates TNF-α-evoked cerebral barrier breakdown via regulations of MMP-2 and plasminogen-plasmin system.
Ischaemic stroke, accompanied by neuroinflammation, impairs blood-brain barrier integrity through a complex mechanism involving both protein kinase C (PKC) and urokinase. Using an in vitro model of human blood-brain barrier (BBB) composed of brain microvascular endothelial cells (HBMEC) and astrocytes, this study assessed the putative roles of these elements in BBB damage evoked by enhanced availability of pro-inflammatory cytokine, TNF-α. Treatment of HBMEC with TNF-α significantly increased the mRNA and protein expressions of all plasminogen-plasmin system (PPS) components, namely tissue plasminogen activator, urokinase, urokinase plasminogen activator receptor and plasminogen activator inhibitor-1 and also the activities of urokinase, total PKC and extracellular MMP-2. Inhibition of urokinase by amiloride abated the effects of TNF-α on BBB integrity and MMP-2 activity without affecting that of total PKC. Conversely, pharmacological inhibition of conventional PKC isoforms dramatically suppressed TNF-α-induced overactivation of urokinase. Knockdown of PKC-α gene via specific siRNA in HBMEC suppressed the stimulatory effects of TNF-α on protein expression of all PPS components, MMP-2 activity, DNA fragmentation rates and pro-apoptotic caspase-3/7 activities. Establishment of co-cultures with BMEC transfected with PKC-α siRNA attenuated the disruptive effects of TNF-α on BBB integrity and function. This was partly due to elevations observed in expression of a tight junction protein, claudin-5 and partly to prevention of stress fibre formation. In conclusion, specific inhibition of PKC-α in cerebral conditions associated with exaggerated release of pro-inflammatory cytokines, notably TNF-α may be of considerable therapeutic value and help maintain endothelial cell viability, appropriate cytoskeletal structure and basement membrane.
Stroke, Division of Clinical Neuroscience, Clinical Sciences Building, School of Medicine, Hucknall Road, Nottingham NG5 1PB, UK.
Zuraidah
Z
Abdullah
Stroke, Division of Clinical Neuroscience, Clinical Sciences Building, School of Medicine, Hucknall Road, Nottingham NG5 1PB, UK. Electronic address: ulvi.bayraktutan@nottingham.ac.uk.
Ulvi
U
Bayraktutan
D017209:Apoptosis; D001812:Blood-Brain Barrier / Q000276:immunology / Q000473:pathology*; D002460:Cell Line; D005341:Fibrinolysin / Q000276:immunology*; D020868:Gene Silencing; D006801:Humans; D007249:Inflammation / Q000276:immunology / Q000473:pathology*; D020778:Matrix Metalloproteinase 2 / Q000276:immunology*; D010958:Plasminogen / Q000276:immunology*; D051571:Protein Kinase C-alpha / Q000235:genetics / Q000276:immunology*; D014409:Tumor Necrosis Factor-alpha / Q000276:immunology*; D014568:Urokinase-Type Plasminogen Activator / Q000276:immunology
D014409:Tumor Necrosis Factor-alpha; D010958:Plasminogen; C496628:PRKCA protein, human; D051571:Protein Kinase C-alpha; D005341:Fibrinolysin; D014568:Urokinase-Type Plasminogen Activator; D020778:Matrix Metalloproteinase 2
Apoptosis; Blood–brain barrier; Claudin-5; MMP-2; PKC-α; PPS components; Stress fibres; TNF-α