Inhibition of Glycogen Synthase Kinase 3β (GSK3β) Decreases Inflammatory Responses in Brain Endothelial Cells

Immune mediators and leukocyte engagement of brain microvascular endothelial cells (BMVECs) contribute to blood–brain barrier impairment during neuroinflammation. Glycogen synthase kinase 3β (GSK3β) was recently identified as a potent regulator of immune responses in in vitro systems and animal models. However, the role of GSK3β in regulation of immune endothelial functions remains undetermined. Here we evaluated the effect of GSK3β inhibition on the regulation of inflammatory responses in BMVECs. A focused PCR gene array of 84 genes was performed to identify the cytokine and chemokine gene expression profile in tumor necrosis factor (TNF) α-stimulated BMVECs after GSK3β inactivation by specific inhibitors. Fifteen of 39 genes induced by TNFα stimulation were down-regulated after GSK3β inhibition. Genes known to contribute to neuroinflammation that were most negatively affected by GSK3β inactivation included IP-10/CXCL10, MCP-1/CCL2, IL-8/CXCL8, RANTES/CCL5, and Groα/CXCL1. GSK3β suppression resulted in diminished secretion of these proinflammatory mediators by inflamed BMVECs detected by ELISA. GSK3β inhibition in BMVECs reduced adhesion molecule expression as well as monocyte adhesion to and migration across cytokine stimulated BMVEC monolayers. Interactions of monocytes with TNFα-activated BMVECs led to barrier disruption, and GSK3β suppression in the endothelium restored barrier integrity. GSK3β inhibition in vivo substantially decreased leukocyte adhesion to brain endothelium under inflammatory conditions. In summary, inhibition of GSK3β emerges as an important target for stabilization of the blood–brain barrier in neuroinflammation.The blood–brain barrier (BBB) is composed of endothelial cells with a unique phenotype. Compared with endothelial cells from other vascular beds, brain microvascular endothelial cells (BMVECs) characteristically have very low permeability to solutes, high electrical resistance, complex tight junctions, and an array of transport systems that both supply the brain with nutrients and eliminates byproducts of brain metabolism.¹ Low permeability is thought to be important in protecting the brain from toxins circulating in the blood as well as restricting the migration of leukocytes into the neuropil. Neuroinflammation can lead to a loss of barrier function, which is manifested by an increase in permeability. This breach of the barrier results in accumulation of serum neurotoxins and proteins exacerbating brain inflammatory response and neuronal injury.² The triggers of BBB permeability occurring during the course of neuroinflammation (ie, multiple sclerosis and HIV-1 encephalitis [HIVE]) include proinflammatory mediators and leukocyte engagement of the BMVECs.³ As a consequence of immune and endothelial cell interactions, the BBB could be further compromised because of enhanced and continuous passage of immune cells across the endothelium. It is this combination of immune cells and immune mediators, such as proinflammatory cytokines and chemokines, which contributes to the disruption of neuronal homeostasis.³Glycogen synthase kinase 3β (GSK3β) is a ubiquitous serine/threonine protein kinase, which is involved in numerous and diverse biological functions including: glycogen metabolism, regulation of cell division, differentiation, and apoptosis.⁴ Unlike most kinases, GSK3β is constitutively active in cells, and a wide range of extracellular stimuli exerts their effects by inhibiting GSK3β activity.⁵ GSK3β activity is regulated by signals originating from numerous signaling pathways (for example, the phosphoinositide 3-kinase-AKT pathway, protein kinase A, protein kinase C, and the WNT pathway) which lead to inhibition of the kinase by phosphorylation of the Ser 9 residue in the N-terminal domain of GSK3β (inactive GSK3β).⁶ However, phosphorylation at the tyrosine 216 residue of GSK3β either by autophosphorylation or by other kinases increases the activity of the kinase (active GSK3β).⁷Recently, GSK3β has been implicated as a key regulator of the inflammatory response. The anti-inflammatory effects of GSK3β inhibition have been shown in vitro and in several in vivo models of acute and chronic inflammation.^4,8,9 In the endotoxin shock model, GSK3β inhibition attenuated multiorgan injury, improved survival rates, and decreased proinflammatory cytokine production before and after the administration of lethal doses of Escherichia coli (E. coli) lipopolysaccharide (LPS).^8,10 These anti-inflammatory effects of GSK3β inhibitors are in part attributable to suppression of the inflammatory response in the vascular endothelium. GSK3β inactivation prevented vascular cell adhesion molecule-1 (VCAM-1) upregulation by tumor necrosis factor (TNF) α in aortic endothelial cells¹¹ and E-selectin expression in umbilical vein endothelium.¹² GSK3β inhibition has also been shown to promote endothelial barrier properties in pulmonary arterial endothelial cells.¹³ Anti-inflammatory and neuroprotective effects of GSK3β inhibition have been shown in models of stroke¹⁴ and spinal cord injury.¹⁵ Although it has been shown that GSK3β inhibition can attenuate inflammation globally and in many cell types, the role of GSK3β in regulating inflammatory responses in the brain endothelium remains largely unexplored.Using primary human BMVECs we investigated the effects of GSK3β inhibition under inflammatory conditions. Suppression of GSK3β activity in inflamed brain endothelium prevented the enhanced adhesion of leukocytes in vitro and in vivo, down-regulated expression of adhesion molecule, protected barrier function, and diminished migration of monocytes across BMVEC monolayers in response to relevant proinflammatory factors. Furthermore, GSK3β inhibition down-regulated secretion of inflammatory factors in TNFα-stimulated BMVECs. Relevance of these observations was further confirmed by the presence of active GSK3β in brain endothelium in human brain tissue affected by neuroinflammation. These results suggest that GSK3β could be a potential target for the treatment of BBB injury associated with proinflammatory insult and leukocyte infiltration of the central nervous system, thus providing both an anti-inflammatory and neuroprotective effect.     

Sun Protection Intervention for Highway Workers: Long-Term Efficacy of UV Photography and Skin Cancer Information on Men’s Protective Cognitions and Behavior

Background  

The risk for skin cancer is increased among older males and outdoor workers who have high levels of ultraviolet (UV) exposure.