Expression and Activity of p-Glycoprotein Elevated by Dexamethasone in Cultured Retinal Pigment Epithelium Involve Glucocorticoid Receptor and Pregnane X Receptor

Abstract

PURPOSE. To investigate whether dexamethasone has an effect on functional expression of p-glycoprotein in cultured human RPE and, if so, whether this occurs through interaction with glucocorticoid receptor (GR) and pregnane X receptor (PXR).METHODS. The human RPE D407 was treated with increasing concentrations of dexamethasone and/or RU486 for various time periods up to 24 hours. Treated cells were collected for cell viability, expressions of p-glycoprotein and PXR, and rhodamine 123 accumulation assays. GR expression plasmid and rifampicin were chosen to investigate the relationship of GR/PXR activation and p-glycoprotein expression.RESULTS. Significant increases in p-glycoprotein, as indicated by mRNA and protein levels, as well as by functional activity, were induced within 12 hours of dexamethasone treatment, persisted as long as 24 hours, and were dose-dependent and attenuable with coculture of RU486. In parallel, a dosedependent upregulation of PXR was notable at both mRNA and protein levels by 24 hours of dexamethasone treatment, and was partially reversible with RU486 coculture. Additionally, transfection of GR expression plasmid increased the transitional expressions of PXR and p-glycoprotein in untreated cells, and enhanced PXR transcriptional expression in dexamethasone-treated cells. Further, PXR silencing inhibited the dexamethasone-induced p-glycoprotein alterations; however, rifampicin had no apparent effects on the dexamethasoneinduced p-glycoprotein alterations. CONCLUSIONS.Our results suggest for the first time that expression and activation of p-glycoprotein involve GR and PXR in human RPE. (Invest Ophthalmol Vis Sci. 2012;53:3508-3515) DOI:10.1167/iovs.11-9337 R PE plays an essential role in protecting neural tissues from toxic materials and in maintaining vision and neural function in the retina by forming the outer blood-retinal barrier (BRB). It has been demonstrated that the outer BRB not only regulates the ionic environment of the subretinal space, secretes factors for structural integrity of the retina, phagocytizes shed outer segments of photoreceptors, and participates in the visual cycle, 1 but also limits vitreal penetration of drugs administered by the systemic and transscleral routes. 2-4 Efflux transport systems provide further barriers for the retina, by actively removing cytotoxic drugs and specific xenobiotic compounds from the retina and transferring them back into the systemic circulation.5,6 P-glycoprotein (P-gp), a 170-kDa protein encoded by the multiple drug resistance human MDR1 gene, is a member of the ABC superfamily of energy-dependent transport systems.7 As a well-characterized efflux transporter, P-gp is strongly expressed by retinal vascular endothelial cells 8 and has recently been identified in human RPE. 9 P-gp displays broad specificity, accepting many structurally, functionally, and mechanistically unrelated compounds, 10 and its role in limiting drug penetration across biological barriers is well established. Although efflux pumps such as P-gp are best known ...

Characterization of ABC transporters in human skin

“…These results are in agreement with those of previous studies, which showed that treatment with dexamethasone caused increased expression of ABCB1 in rat brain microvessels [22], in human RPE cell line [23], and in human T-lymphocyte cell line [24]. Furthermore, dexamethasone can increase expression of MDR1 transporter at the blood-brain barrier and in retinal pigment epithelium through the pathway involving pregnane xenobiotic receptor (PXR) [22,23]. The PXR belongs to a superfamily of nuclear receptors; it is also recognized as a key regulator for the induction of a large number of genes in drug metabolism and transport in a liganddependent manner [30].…”

“…Moreover, the substrate specificities of CYP3A4 often overlap those of MDR1. Previous studies also demonstrated that treatment with dexamethasone caused increased expression of ABCB1 in rat brain microvessels [22], in human retinal pigment epithelium RPE cell line [23] and in human T-lymphocyte cell line [24], while treatment with rifampicin decreased expression of MRP1 in HepG2 cells [25]. In order to determine whether dexamethasone and rifampicin treatment modified the expression of drug efflux transporters in human skin, skin samples from two different donors were treated with dexamethasone or rifampicin for 72 h. On the one hand, dexamethasone markedly increased transcript levels of ABCB1 (MDR1) and slightly decreased expression of ABCC1 (Figure 3).…”

Functional characterization of a full length pregnane X receptor, expression in vivo, and identification of PXR alleles, in Zebrafish (Danio rerio)

“…Tissue distribution pattern of zebrafish pxr in four organs including liver, intestine, kidney and heart was similar to that in juvenile rainbow trout (Wassmur et al, 2010). While earlier reports noted PXR expression principally in liver or gastrointestinal tract in mammals, including humans (Kliewer et al, 1998; Lehmann et al, 1998; Zhang et al, 1999), more recently expression of PXR and the induction of ABC transporters in brain and retina indicate that this receptor is expressed in those organs in mammals as well (Bauer et al, 2004; Lamba et al, 2004; Zhang et al, 2012). Bertrand et al (2007) reported spatiotemporal expression patterns of a full suite of nuclear receptors in developing zebrafish.…”

Progesterone treatment shows greater protection in brain vs. retina in a rat model of middle cerebral artery occlusion: Progesterone receptor levels may play an important role

“…PXR activates p-Glycoprotein (P-gp), an efflux pump implicated in the removal of cytotoxic and xenobiotic substances in both the blood-brain and blood-retina barriers (Bauer et al, 2004; Zhang, Lu, et al, 2012). PXR has previously been identified in brain capillaries (Bauer et al, 2004) and retinal pigmented epithelium (Zhang, Li, et al, 2012; Zhang, Lu, et al, 2012). The literature on PXR expression after injury is not consistent (Chen et al, 2011; Hartz et al, 2010; Souidi et al, 2005).…”