Suppression of PKC-α attenuates TNF-α-evoked cerebral barrier breakdown via regulations of MMP-2 and plasminogen–plasmin system

Abdullah, Zuraidah; Bayraktutan, Ulvi

Abstract

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- 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- may be of considerable therapeutic value and help maintain endothelial cell viability, appropriate cytoskeletal structure and basement membrane.

Citation

Abdullah, Z., & Bayraktutan, U. (2016). Suppression of PKC-α attenuates TNF-α-evoked cerebral barrier breakdown via regulations of MMP-2 and plasminogen–plasmin system. BBA - Molecular Basis of Disease, 1862(7), 1354-1366. https://doi.org/10.1016/j.bbadis.2016.03.014

• Journal Article Type: Article
• Acceptance Date: Mar 30, 2016
• Online Publication Date: Apr 17, 2016
• Publication Date: 2016-07
• Deposit Date: Oct 26, 2016
• Publicly Available Date: Oct 26, 2016
• Journal: Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease
• Print ISSN: 0925-4439
• Electronic ISSN: 0006-3002
• Publisher: Elsevier
• Peer Reviewed: Peer Reviewed
• Volume: 1862
• Issue: 7
• Pages: 1354-1366
• DOI: https://doi.org/10.1016/j.bbadis.2016.03.014
• Keywords: TNF-α; blood-brain barrier; PKC-α; PPS components; MMP-2; claudin-5; apoptosis; stress fibres
• Public URL: https://nottingham-repository.worktribe.com/output/798190
• Publisher URL: http://www.sciencedirect.com/science/article/pii/S0925443916300710
• Additional Information: This article is maintained by: Elsevier; Article Title: Suppression of PKC-α attenuates TNF-α-evoked cerebral barrier breakdown via regulations of MMP-2 and plasminogen–plasmin system; Journal Title: Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease; CrossRef DOI link to publisher maintained version: https://doi.org/10.1016/j.bbadis.2016.03.014; Content Type: article; Copyright: © 2016 Elsevier B.V.