Contribution of p38 MAPK, NF-κB and glucocorticoid signaling pathways to ER stress-induced increase in retinal endothelial permeability

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Abstract

Diabetic retinopathy (DR) is characterized by the development of intraretinal microvascular abnormalities. Endoplasmic reticulum (ER) stress is known to play a pathogenic role in vascular impairment in DR. The present study demonstrated that the treatment of human retinal endothelial cells with ER stress inducers such as thapsigargin (Tg) and tunicamycin (Tm) significantly increased the permeability of exogenously added FITC–dextran, accompanied by a decrease of transendothelial electrical resistance (TEER). The expression of claudin-5 among tight junction proteins was significantly decreased by the treatment with Tg or Tm. A p38 MAPK inhibitor, SB203580, and an NF-κB inhibitor, dexamethasone, significantly suppressed the Tg-induced down-regulation of claudin-5, decrease of TEER and leakage of added FITC–dextran. The translocation of NF-κB p65 subunit to the nucleus was also inhibited by the addition of SB203580 or dexamethasone. The effects of dexamethasone are thought to be due to the transrepression of the above signaling and direct regulation of claudin-5 gene.

Highlights

ER stress inducers increased retinal endothelial permeability. ► ER stress inducers down-regulated claudin-5 among tight junction proteins. ► p38 MAPK and NF-κB signaling pathways contribute to the regulation of claudin-5. ► Dexamethasone–GR complex contributes to strengthen the endothelial tight junction.

Introduction

Diabetic retinopathy (DR)1 is a common and severe complication caused by diabetes mellitus and is a leading cause of acquired blindness [1]. DR has recently been recognized as a chronic low-grade inflammatory disease and the initial clinical stage of DR is characterized by the development of intraretinal microvascular abnormalities [2]. The vascular endothelium forms a barrier between the circulation and the interstitium. Disorder of endothelial barrier function leads to abnormal extravasations of blood components and accumulation of fluid in the extravascular space, resulting in inflammation and finally organ dysfunction. Retinal microvascular endothelial cells differ from other non-barrier endothelial cells, and function in the blood–retinal barrier (BRB) composed of highly specialized tight junction (TJ) complexes. The BRB also provides a dynamic interface between the peripheral circulation and the retinal nervous system [3]. TJ is an intricate complex of transmembrane proteins such as occludin, claudins and junction adhesion molecules, as well as cytoplasmic accessory proteins linked to the actin cytoskeleton, such as zonula occludens (ZO). Breakdown of BRB occurs early in DR as an increase of retinal vascular permeability associated with changes in the TJ proteins in retinal vessels [4].

Several inflammatory factors can significantly induce endothelial permeability by changing the TJ structures and functions [5]. On the other hand, the stress-triggered inflammatory reactions are known to be mediated by the activation of transcription factor nuclear factor kappa B (NF-κB) [6], [7]. NF-κB exists in the cytoplasm in inactive form by binding to inhibitory protein kappa B (IκB). NF-κB is activated mostly through IκB kinase-dependent phosphorylation and subsequent degradation of IκB. Activated NF-κB translocates to the nucleus, and binds to promoters of a large number of genes. In addition to NF-κB, mitogen-activated protein kinases (MAPKs) are activated in response to multiple stimuli [8]. Activation of MAPKs is involved in enhancing nuclear NF-κB–DNA binding activity [9]. Recently, activations of these factors have been reported to correlate with BRB breakdown [10], [11]. Glucocorticoids are the most effective medicine for chronic inflammatory diseases because of their abilities to inhibit pro-inflammatory gene expression. Glucocorticoids exert their effects by binding to the glucocorticoid receptor (GR), a transcriptional factor that is present in the cytoplasm in an inactive form. Upon ligand binding, GR–ligand complex translocates to the nucleus and do regulation of a large number of genes including repression of NF-κB and induction of IκB [12], [13].

The endoplasmic reticulum (ER) is a critical intracellular organelle and functions in processes such as protein synthesis and transport [14]; however, it is also the earliest site of transduction, responding to cellular stresses [1]. Agents or conditions that adversely affect ER protein folding result in accumulation of unfolded or misfolded proteins in the ER, a state known as ER stress, triggering an evolutionarily conserved response termed the “unfolded protein response” (UPR). ER stress can be induced by agents that interfere with protein glycosylation, e.g., tunicamycin (Tm), calcium balance, e.g., thapsigargin (Tg), and others [15]. Moreover, ER stress is a key mediator of hyperglycemia because glucose affects unfolded protein response signaling [15]. Excess intracellular glucose is converted into glucosamine by way of the hexosamine pathway. Increased hexosamine pathway activity and corresponding elevated glucosamine promote the accumulation of unfolded proteins in the ER [16]. Recently, it has been reported that ER stress plays a pathogenic role in retinal inflammation and vascular impairment in DR [1], [14], [17].

Several studies have shown that ER stress leads to the breakdown of cellular homeostasis, and NF-κB and MAPKs contribute to this process [9]. However, very little attention has been paid to the changes in the TJ proteins by ER stress and the cellular mechanisms involved. The present study aimed to clarify the effects of ER stress on the expression of TJ proteins in human retinal endothelial cells and subsequent changes of retinal vascular permeability as an in vitro pathological condition of DR. Moreover, the contributions of p38 within MAPK, NF-κB and glucocorticoid signaling pathways to the regulation of TJ protein were also addressed.

Section snippets

Cell culture

Primary human retinal microvascular endothelial cells (HRECs) and CS-C culture medium for the cells were purchased from DS Pharma Biomedical Co. (Osaka, Japan). The cells were maintained at 37 °C in an atmosphere of 95% air and 5% CO2.

Monolayer permeability assay

HRECs (1 × 104 cells/well) were seeded onto transwell inserts (1.0 μm pore size, 0.3 cm2 membrane surface area, Millicell Hanging cell culture inserts, Japan Millipore Co., Tokyo, Japan) and cultured using CS-C culture medium. The culture medium was replaced every

Effect of ER stress on the permeability of HREC layer and expression of TJ proteins

HRECs that had reached confluence were cultured for an additional 3 days, and the barrier functions were evaluated. TEER value of the cell layer treated with Tg for 24 h was significantly lower than that of the vehicle-treated cell layer, as shown in Fig. 1A. Treatment with Tm also significantly decreased the TEER value, but less than that with Tg. Treatment of cells with Tg or Tm significantly increased the permeability of exogenously added FITC–dextran across the monolayer, in time-dependent

Discussion

Chronic and excessive hyperglycemia, a well-known cause of DR, could trigger ER stress [15], [16]. Recently, the relationship between the pathogenesis of DR and the expression of factors related to ER stress in retinal cells has been revealed in both in vivo study [1], [24] and in vitro study [1], [17]. In the present study, we demonstrated that the elevation of the permeability of HREC layer was induced by ER stress inducers accompanied with down-regulation of claudin-5.

In diabetic eyes,

Acknowledgments

This study was supported in part by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (to T.A., No. 21590169).

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