Occludin as direct target for glucocorticoid‐induced improvement of blood–brain barrier properties in a murinein vitrosystem

Abstract

Homeostasis of the central nervous system (CNS) microenvironment is essential for its normal function. It is maintained by the blood-brain barrier (BBB) which regulates the transport of molecules from blood into brain and backwards. The integrity of the BBB is compromised in many disorders of the human CNS; therapeutical strategies for several of these diseases include treatment with glucocorticoids, but the molecular basis of how glucocorticoids regulate BBB permeability is not understood. Here, we report the generation and characterization of a murine immortalized brain (cerebral) capillary endothelial (cEND) cell line which expresses the BBB marker occludin at intercellular tight junctions (TJ). Hydrocortisone at physiological concentrations induced upregulation of occludin, accompanied by a threefold enhancement of transendothelial electrical resistance to values up to 1000 Ωcm 2 . Insulin enhanced the glucocorticoid response. At the molecular level, hydrocortisone induces increase of occludin at protein and mRNA levels by activation of the glucocorticoid receptor (GR) and its binding to putative glucocorticoid responsive elements in the occludin promoter. At the same time, insulin potentiated the ligand-dependent GR transactivation via induction of the GR in this in vitro system. This study thus provides insights into the molecular processes of barrier genesis, and may help to elucidate mechanisms of brain pathology at the microvascular level.

A Genomic Comparison of in vivo and in vitro Brain Microvascular Endothelial Cells

“…These barrier improvements were accompanied by morphological changes and alterations in junctional structure . In addition, previous studies have noted increased tight junction protein expression in immortalized BMEC as the result of glucocorticoid induction (Forster et al, 2005). To investigate more thoroughly the effects of HC induction and its ability to promote cultured BMEC to mimic their in vivo character, puromycinpurified BMEC were treated with HC and gene expression was compared with both untreated BMEC and in vivo BMEC.…”

“…Although barrier properties are an often-used measure to assess the characteristics of in vitro BBB models, several morphological and biochemical criteria have also been employed. The quality of the tight junctions has been evaluated by monitoring the expression levels and localization of various junctional proteins, such as zonula occluden, occludin, and claudin-5 Forster et al, 2005). Enzymatic activities such as those of g-glutamyl transpeptidase and alkaline phosphatase are also sometimes evaluated.…”

Effects of prednisolone on the dystrophin-associated proteins in the blood–brain barrier and skeletal muscle of dystrophic mdx mice

“…In fact, binding of the activated glucocorticoid receptor to putative glucocorticoid-responsive elements in the occludin promoter has been described. 38,59,60 Dexamethasone prevents alteration of TJ-associated proteins in bacterially infected epithelial choroidal cells by restoring the occludin phosphorylation degree via attenuation of ERK activation and MMP-3 expression. 61 It is noteworthy that the inhibition of the ERK pathways blocks the production of MMPs, which in Prednisolone in mdx mice R Tamma et al turn cleave occludin in the epithelial cells after induced apoptosis.…”

“…In the clinical setting, glucocorticoids are used to reduce BBB permeability in many pathological conditions, highlighting the importance of a more detailed preclinical analysis of PDN action in dystrophic subjects. 38 In this study, we evaluated the effects of chronic treatment with PDN in both the brain and muscle of mdx mice, by analyzing the immunocytochemical and biochemical expression of four BBB markers, including ZO-1 and occludin in the endothelium, desmin in pericytes, and glial fibrillary acidic protein (GFAP) in glial cells, and the expression of the short dystrophin isoform Dp 71 and of the DAPs, AQP4, and a-b DG in the brain. We also evaluated the BBB integrity of the mdx and PDN-treated mdx mice by means of intravascular injection of horseradish peroxidase (HRP).…”

Glucocorticoids in the control of neuroinflammation

“…Direct effects of GCs on the BBB were demonstrated in vitro (Figure 2). In cultured brain endothelial cells dexamethasone induced the reduction of pores, the concentration of F-Actin on the cell periphery and an increased expression of ZO-1 and occludin (Förster et al, 2005;Förster et al, 2006;Romero et al, 2003). How these mechanisms impact the integrity of the BBB in vivo requires A c c e p t e d M a n u s c r i p t -10 further investigation.…”