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. 2007 Feb 22;580(Pt 3):937–949. doi: 10.1113/jphysiol.2007.129007

Dexamethasone induces the expression of metalloproteinase inhibitor TIMP-1 in the murine cerebral vascular endothelial cell line cEND

Carola Förster 1, Timo Kahles 2, Silke Kietz 3, Detlev Drenckhahn 1
PMCID: PMC2075456  PMID: 17317742

Abstract

In many neuroinflammatory conditions, including multiple sclerosis (MS), encephalitis, meningitis, brain tumours and cerebral ischaemia, the matrix metalloproteinases (MMPs) play an important role in disrupting the blood–brain barrier (BBB). Normally under tight regulation, increased MMP-9 cerebrospinal fluid levels and excessive proteolytic activity is detected in the blood and cerebrospinal fluid in patients with acute MS. MMP-9 is a member of the type IV collagenases, which attack components of the endothelial basal lamina, including type IV collagen. The disruption of the BBB and clinical symptoms can be reduced with different inhibitors to MMPs including activators of tissue inhibitor of metalloproteinases-1 (TIMP-1), the cognate tissue inhibitor of MMP-9. Since intravenous glucocorticoid (GC) treatment reduces the levels of MMP-9 markedly in patients, we hypothesized that GC effects might be mediated by transcriptional activation of the TIMP-1 gene in addition to reported repressive effects on MMP-9 transcription. Our results provide direct evidence that GCs increase TIMP-1 in the brain endothelial cell line cEND, prevent alterations in microvascular integrin α1 subunit expression and help maintain endothelial barrier function in response to pro-inflammatory stimuli (TNFα administration). GC-induced up-regulation of TIMP-1 expression by the CNS vascular endo-thelium may thus play a role in preservation of the endothelial basal lamina and maintain integrin α1 and tight junction protein expression important for vessel wall integrity.

The microenvironment of the central nervous system (CNS) is normally maintained by the presence of the blood–brain barrier (BBB). This is a complex cellular system comprising cerebral vascular endothelial cells sealed by tight junctions (TJs), resting on a basal lamina of collagen type IV, laminin, fibronectin and proteoglycans (Rubin & Staddon, 1999). Breakdown of the BBB is a key feature of neuroinflammatory conditions and is associated with the influx of inflammatory cells, fluid and proteins, including complement and cytokines. Mediators of BBB disruption include the matrix metalloproteinases (MMPs), a group of zinc-containing endopeptidases: MMPs are extracellular matrix remodelling neutral proteases, that are important in normal development, angiogenesis, wound repair, and a wide range of pathological processes (reviewed in: Nagase & Woessner, 1999). The involvement of MMPs in many neuroinflammatory diseases, including brain tumours, cerebral ischaemia, meningitis, encephalitis and multiple sclerosis (MS) has been demonstrated (reviewed in: MunBryce & Rosenberg, 1998; Lukes et al. 1999; Yong et al. 2001).

In inflammatory processes, MMPs attack the basal lamina macromolecules that line the blood vessels, opening the BBB. MMPs are effectors of BBB opening and invasion of the brain parenchyma by immune cells in MS. In addition they can act as enhancers of the immune response via their proteolytic release of membrane-bound cytokines and their receptors (reviewed in Leppert et al. 2001). Normally under tight regulation, excessive proteolytic activity is detected in the blood and cerebrospinal fluid in patients with acute MS (Leppert et al. 2001). Agents that block the action of the MMPs have been shown to reduce the damage to the BBB and lead to symptomatic improvement in several animal models of neuroinflammation, e.g. experimental autoimmune encephalitis (EAE) (Gijbels et al. 1994).

The MMP gene family is made up of four groups of enzymes: collagenases, stromelysins, gelatinases and membrane-type metalloproteinases (Nelson et al. 2000). Of these, a selective up-regulation of MMP-9 in MS disease activity has been described (Avolio et al. 2005). Increased MMP-9 cerebrospinal fluid levels in MS patients are associated with BBB damage as seen on enhanced-magnetic resonance imaging (MRI) scans (Rosenberg et al. 1996). MMP-9 (gelatinase B), together with MMP-2 (gelatinase A) is a member of the type IV collagenases, which attack components of the endothelial basal lamina, including type IV collagen, fibronectin, laminin and heparan sulphate (Nagase & Woessner, 1999; Nelson et al. 2000). Gelatinase B is induced during the inflammatory response secondary to factors such as the immediate early genes, c-fos and c-jun, and the cytokines, tumour necrosis factor-α (TNFα) and interleukin-1β (IL-1β) (Romanic et al. 1998). MMP-9-deficient mice are less susceptible to the development of EAE than wild-type mice (Dubois et al. 1999), consistent with the notion that MMP-9-mediated opening of the BBB allows amplification of the inflammation, as demonstrated by radioisotopes (Kermode et al. 1990).

In EAE, the disruption of the BBB and clinical symptoms have been reduced with different inhibitors to MMPs (Gijbels et al. 1994), including activators of TIMP-1, the cognate tissue inhibitor of MMP-9 (Brew et al. 2000). Tissue inhibitors of metalloproteinases (TIMPs) form complexes with either activated MMPs or with their pro-forms after their secretion thus reducing MMP activity (Brew et al. 2000; Yong et al. 2001). Levels of TIMP-1 are reduced in MS patients relative to control patients, suggesting an imbalance in MMP-9/TIMP-1 ratios in MS (Avolio et al. 2005), rendering the development of MMP inhibitors a possible avenue in the treatment of MS.

High-dose intravenous glucocorticoid (GC) treatment reduced the levels of MMP-9 markedly in patients with enhancement on MRI, which can be seen during acute exacerbations of MS (Burnham et al. 1991; Rosenberg et al. 1996). Levels of MMP-9 in the cerebrospinal fluid (CSF) correlated with the presence of enhancement with Gd-diethylenetriamine-pentaacetic acid (DTPA) on the MRI (Rosenberg et al. 1994, 1996). As an explanation for steroid action on pro-inflammatory MMP-9 secretion, glucocorticoid receptor (GR)-mediated blockade of the AP-1 site in the MMP-9 gene was presented (Harkness et al. 2000). We now show additionally, that GCs exert a positive inductive function on the expression of TIMP-1. In the present studies, TIMP-1 could be demonstrated to be a direct target gene for GR-mediated gene expression. GC-mediated elevation of TIMP-1 expression could thus counteract MMP-mediated degradation of the endothelial basal lamina and possibly MMP-9-induced disruption of integrin binding of extracellular matix (ECM) ligands in cerebral vessels. Together with previous data demonstrating GC-mediated induction of the TJ component occludin at the BBB (Förster et al. 2005, 2006), a model for GC-mediated preservation of the blood–brain barrier can be drawn, arguing for the development of improved tissue-specific GR ligands for therapeutic applications.

Methods

Chemicals

Dexamethasone was purchased from Sigma, Taufkirchen, Germany.

Animals and collection of tissues

Neonatal mice (strain 129Sv) of either sex (3 days old) were killed with a rising concentration of CO2. The brains were immediately removed and transferred into a dissection chamber containing the following solution (hereafter referred to as buffer A): 15 mm Hepes (pH 7.4), containing 153 mm NaCl, 5.6 mm KCl, 2.3 mm CaCl2.2H2O, 2.6 mm MgCl2.6H2O, 1% (w/v) bovine serum albumin (BSA). All experiments were approved by the local Animal Care Committee (Tierschutzbeauftragter).

Isolation and culture of cerebral endothelial cells

The immortalized mouse brain capillary endothelial cell line cEND was generated as described (Förster et al. 2005). Briefly, brains (cerebrum without cerebellum and brain stem) were isolated from neonatal mice (3 days post partum) as described above, and after removal of the meninges and capillary fragments, the tissue was minced in buffer A using a sterile cutter. Fragments were digested in 0.75% (w/v) collagenase A (Roche, Mannheim, Germany) for 30 min at 37°C in a water bath (occasionally shaking). Digestion was stopped by addition of 10 vol. ice-cold buffer A. To remove myelin, centrifugation through a 25% (w/v) BSA gradient was carried out for 20 min at 1000 g. The resulting endothelial cell pellet was washed twice with buffer A to remove myelin and BSA. Primary cells were then resuspended in Dulbecco's modified Eagle's medium (DMEM) (Sigma, Taufkirchen, Germany) growth medium [10% heat-inactivated fetal calf serum (FCS)] and plated on 24-well plates (Greiner, Frickenhausen, Germany), freshly coated with collagen IV (Fluka, Taufkirchen, Germany) and transfected with polyoma middle T antigen of murine polyomavirus (PymT) (Sabapathy et al. 1997) after 24 and 48 h, as previously described (Aumailley et al. 1991; Golenhofen et al. 2002; Förster et al. 2005).

Cell cultures

cEND cells were cultivated as described above (DMEM, 10% FCS) until confluence. At confluence, the medium was changed to differentiation medium (DMEM, 2% FCS). MCF-7 breast cancer cells were cultivated in RPMI medium, pH 7.4, containing 10% FCS. All cultures were supplemented with 100 i.u. ml−1 penicillin and 100 mg ml−1 streptomycin (1% PEST (Sigma, Taufkirchen, Germany)). Cells were maintained in an atmosphere of 5.0% CO2–95% O2 and at 37°C.

Electrophoresis and immunoblotting

Cells were plated at a density of 1.1 × 105 cells per 3.5 cm2 dish and grown to confluence. At confluence, cells were maintained at 2% FCS and treated with TNFα and glucocorticoids as indicated in the figure legends.

For determination of deposited collagen IV protein, cEND cells were plated on uncoated Petri dishes. For determination of tight junction proteins, cEND cells were plated on Petri dishes coated with collagen IV (Fluka). For Western blot analyses, cells were then dissolved in Laemmli sample buffer (Laemmli, 1970) and subjected to sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE, 15% gels).

For determination of secreted MMP-9 protein, cell culture supernatants (2 ml) were concentrated using Microcon YM-30 filters (Millipore, Bedford, MA, USA) to a volume of 20 μl and stored at −20°C for future usage. Freshly defrosted culture supernatants were then suspended in sample buffer (10% SDS, 0.1% bromophenol blue, 0.5 m Tris-HCl, pH 6.9) at a ratio of 2: 1. A total volume of 30 μl was resolved by SDS-PAGE.

Protein contents were quantified by protein estimation directly from SDS-PAGE loading buffer using 0.1% (w/v) Amidoschwarz (AppliChem, Darmstadt, Germany) in 25% (v/v) methanol–5% (v/v) acetic acid. For immunoblotting, proteins were transferred in Kyhse-Andersen transfer buffer (Kyhse-Andersen, 1984) to Hybond nitrocellulose membranes (Amersham, Braunschweig, Germany) which were blocked with 10% (w/v) low fat milk in phosphate-buffered saline (PBS, pH 7.4) and incubated overnight at 4°C with the respective primary antibody (in PBS plus 10% low fat milk). Rabbit polyclonal anti-mouse MMP-9, full-length, antibody was obtained from Chemicon, Hofheim, Germany (AB 19047). The polyclonal rabbit antibodies against occludin, claudin-5 and ZO-1 were were purchased from Zymed Laboratories (CA, USA). Rabbit polyclonal collagen IV antibody was kindly provided by S. Gay (Department of Medicine, University of Alabama at Birmingham, USA). The polyclonal rabbit antibodies against collagen IV, MMP-9, occludin, claudin-5 and ZO-1 were used at a dilution of 1: 1000. As secondary antibody, horseradish peroxidase-labelled goat anti-rabbit IgG (Jackson Immuno Res. Laboratory, West Grove, PA, USA) was used diluted 1: 3000 with PBS. Bound immunoglobulins were visualized by the enhanced chemiluminescence technique (ECL, Amersham). Densitometric analysis using Scion Image Beta 4.02 (Scion Corp., MD, USA) was performed for quantification.

Quantitative real-time RT-PCR

For real-time RT-PCR, cDNA was synthesized using iSCRIPT cDNA synthesis kit (Bio-Rad) and 1 μg of RNA from cEND cells treated or untreated with TNFα and dexamethasone. TIMP-1, integrin α1 and integrin αv and primers were designed using the Primer Express Software (Applied Biosystems) and obtained from MWG Biotech. TIMP-1, integrin α1 and integrin αv real-time RT-PCR was performed using the SYBRGreen PCR Master Mix (Applied Biosystems). Primers TIMP-1 forward (5′-CCAGAACCGCAGTGAAGAGT-3′), TIMP-1 reverse (5′-TCTCCAAGTGCACAAGCCTA-3′), GAPDH forward (5′-CAA GAC GGA CCA GAG CGA AAG C-3′), GAPDH reverse (5′-CAA TCT CGG GTG GCT GAA CGC-3′), integrin α1 forward (5′-CAC CAA GAT GAA CGA GCC TCT-3′), integrin α1 reverse (5′-ACC TTG CCC TGT TCC TCT TTC-3′), integrin αv forward (5′-AGC GCA ATC CTG TAC GTG AA-3′), integrin αv reverse (5′-ACG TTT GAA AAA GCC CAT CC-3′). MMP-9 real-time RT-PCR was performed using the TaqMan PCR Master Mix (Applied Biosystems). Assays for MMP-9 (Mm01240564_g1) and GAPDH (Mm99999915_g1) were obtained from Applied Biosystems.

Claudin-12 real-time RT-PCR was performed using the SYBRGreen PCR Master Mix (Applied Biosystems). Primers claudin-12 reverse (5′-TGT CGA TTT CAA TGG CAG AG-3′), GAPDH forward (5′-CAA GAC GGA CCA GAG CGA AAG C-3′), GAPDH reverse (5′-CAA TCT CGG GTG GCT GAA CGC-3′). Occludin, claudin-5 and claudin-1 real-time RT-PCR was performed using the TaqMan PCR Master Mix (Applied Biosystems). Assays for occludin (Mm00500912_m1), claudin-1 (Mm00516701_m1), claudin-5 (Mm00727012_s1) and GAPDH (Mm99999915_g1) were obtained from Applied Biosystems.

The ABI PRISM 7300 SDS software (relative quantification study) was used to determine the cycle threshold (CT) for each reaction and gene expression was normalized to expression of the endogenous housekeeping gene, glyceraldehyde phosphate dehydrogenase (GAPDH). The mean fold change in expression of the target gene was calculated using the following equation (where CT means threshold cycle):

graphic file with name tjp0580-0937-m1.jpg

Localization of GC-responsive elements in the human TIMP-1 promoter

Several studies have identified GR binding sites (glucocorticoid-responsive elements, GREs) in a number of target genes (Nakabayashi et al. 2001; Chen et al. 2003; Pascussi et al. 2003). These GREs are not always identical, but show some variability in several nucleotide positions. Nevertheless, Nakabayashi et al. (2001) determined the GRE consensus sequence to six nucleotides in a palindromic repeat separated by three unspecific nucleotides: TCY TGT nnn ACA RGA (Nakabayashi et al. 2001). We further reconstituted a degenerate full-site GRE from various consensus and imperfect GREs described in the literature (Falkner et al. 1999; Kraus et al. 1999; Adcock, 2001; Nakabayashi et al. 2001; Gerbal-Chaloin et al. 2002; Chen et al. 2003; Hao et al. 2003; Pascussi et al. 2003) and were able to deduce the sequence HHNKGHnnnHCMNNW (H = A/C/T; W = A/T; K = T/G; M = A/C) as a putative degenerate consensus motiv. Screening the −1718/+95 fragment of the human TIMP-1 promoter for the presence of consensus or degenerate GREs, we were able to identify one consensus GRE half-site located 1018–1012 bp upstream of the 5′ flanking region and several putative degenerate full-site GREs located 1061–1047, 933–919, 559–545, 453–439 and 206–192 bp upstream of the 5′ flanking region, which might belong to imperfect GREs.

Transfection and luciferase assay

Essentially, transfection and luciferase assays were carried out as described (Förster et al. 2005). Briefly, MCF-7 and COS-1 cells were seeded on 6-well cell culture plates 24 h before transfection in RPMI or DMEM medium, respectively, containing 10% dextran-coated charcoal (DCC)-treated FCS (Fagart et al. 1998), and 1% PEST at a density of 2 × 106 cells per well. Transient transfection experiments utilizing the Effectene reagent (Qiagen, Germany) were performed as described by the manufacturer, using 2 μg of the TIMP-1 promoter constructs −1718/+95 and −102/+95 (Clark et al. 1997), respectively, and 1 μg of the internal control reporter pTRL-TK (Lorenz et al. 1991) (Promega) and, in the case of COS-7 cells, 0.4 μg of GR expression vector pCMVhGRα (Almlof et al. 1995), in the absence or presence of ligands (as indicated in the figure legends).

To assess dexamethasone effects on TIMP-1 promoter transactivation, after addition of the DNA–Effectene mixture, cells were incubated overnight at 37°C and 5% CO2. After this, 4 ml fresh RPMI containing 10% DCC-treated FCS–1% PEST and ligands or vehicle alone (as indicated in the figure legends) were added. After 24 h, cells were washed once with PBS and harvested with 500 μl lysis buffer. Thereafter, cellular extracts were prepared and analysed for luciferase activity. Measurement of both firefly and Renilla luciferase activity was performed with the Dual-Luciferase assay kit (Promega, Madison, USA) according to the manufacturer's instructions. Protein concentration was estimated by standard Bradford protein assay (Bradford, 1976). Enzymatic activities were assayed in 20 μl of cell lysate using a LB9507 luminometer with dual injector (Berthold, Bad Wildbad, Germany). Each lysate was measured twice. Promoter activities were expressed as relative light units (RLU), normalized for the protein content and the activity of Renilla luciferase in each extract. The data were calculated as the mean of five identical setups.

Analysis and statistics

Values for densitometry, gene expression and promoter transactivation were averaged to establish a single value for control cells and treated cells as indicated. Throughout, averaged values were reported as means ± standard deviation (s.d.). The indicated statistical test (Mann–Whitney U test) was performed assuming significance for P < 0.05 (*) and high statistical significance at P < 0.001 (**).

Results

Loss of extracellular matrix component collagen IV in TNFα-activated endothelium

The influence of the pro-inflammatory cytokine TNFα on extracellular matrix was assessed in cEND brain endothelial cells and analysed by Western blot analysis (Fig. 1). An 8 h treatment of confluent cEND cells with TNFα reduced the extracellular matrix component collagen IV to 66.31 ± 2.33% of control cells as estimated by Western blot (Fig. 1A) and densitometric quantification (Fig. 1B). This effect could be reduced by simultaneous administration of TNFα and 100 nm dexamethasone: collagen levels amounted to 83.18 ± 2.9% of control cells, pointing to an role for GCs in reducing TNFα-induced gelatinase activity.

Figure 1. Dexamethasone treatment reduces collagen IV reduction in response to pro-inflammatory stimuli.

Figure 1

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A, influence of TNFα treatment on the extracellular matrix component collagen IV could be shown by Western blot. For this, cells were seeded on plastic cell culture flasks without further coating, treated as indicated and subjected to SDS-gel electrophoresis and Western blotting. An 8 h treatment with TNFα reduced the extracellular matrix component collagen IV to 66.31 ± 2.33% of control cells as estimated by densitometric quantification. This effect could be reduced by simultaneous administration of TNFα and 100 nm dexamethasone (dex): collagen levels amounted to 83.18 ± 2.9% of control cells pointing to an role for GCs in reducing TNFα-induced gelatinase activity. Data are representative of 3 independent experiments. B, densitometric evaluation of A. Measurements were performed with Scion Image Beta 4.02 and data are shown as mean ± s.d. of 3 independent experiments.

Effect of TNFα and glucocorticoids on MMP-9 secretion in vascular endothelial cells cEND

MMP-9 Western blot analysis showed increased secretion of MMP-9 into cell culture supernatants from cEND cells 8 h after treatment with TNFα compared with untreated cells (Fig. 2A and B): while MMP-9 protein levels were below the detection limit in cell culture supernatants from untreated cEND cells, treatment with TNFα caused a strong up-regulation in the supernatants. Addition of 100 nm of the synthetic GC dexamethasone inhibited this up-regulation significantly: estimated MMP-9 secretion amounted only to 15.03 ± 2.16% of the value determined for TNFα-treated cEND cells as determined by Western blot (Fig. 2A) and densitometric quantification (Fig. 2B). Administration of 100 nm of the natural GC hydrocortisone reduced MMP-9 secretion less efficiently to 56.4 ± 5.26% of the value determined for TNFα-treated cEND cells (Fig. 2A and B).

Figure 2. MMP-9 secretion from vascular endothelial cells cEND.

Figure 2

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A, MMP-9 Western blot analysis showed increased secretion of MMP-9 into cell culture supernatants from cEND cells 8 h after treatment with TNFα; while MMP-9 protein could not be detected in cell culture supernatants from untreated cells, treatment with TNFα caused a significant secretion of MMP-9 into supernatants. Addition of 100 nm dexamethasone reduced MMP-9 secretion to 15.03 ± 2.16% of the value determined for TNFα-treated cells. Addition of hydrocortisone reduced MMP-9 secretion to 56.4 ± 5.26% of TNFα-treated cells. B, densitometric evaluation of A. Measurements were performed with Scion Image Beta 4.02 and data are shown as mean ± s.d. of 3 independent experiments.

Effects of dexamethasone and TNFα on MMP-9 and TIMP gene expression in cEND cells

We previously described loss of barrier properties in response to inflammatory stimuli (TNFα administration) in cEND cells (Silwedel & Förster, 2006). TNFα-treated cEND cells exhibited increased permeability and decreased synthesis of TJ components concomitant with an increase in VCAM-1 and ICAM-1 cell surface adhesion molecules as previously reported (Silwedel & Förster, 2006). To initially determine whether MMP-9 could play a role in this inflammatory breakdown of barrier properties, we compared its expression in cEND cells treated with TNFα with control cEND cells by quantitative real-time RT-PCR. We were able to show a 1.62 ± 0.2-fold overexpression of MMP-9 in response to TNFα treatment, which could be reversed by steroid treatment (0.41 ± 0.18-fold expression for TNFα–dexamethasone treated cells and 0.76 ± 0.13-fold expression for TNFα–hydrocortisone-treated cells) as compared to control cEND cells (Fig. 3A).

Figure 3. Selective effects of dexamethasone upon the expression of MMP-9 and TIMP-1 in cytokine-activated endothelium.

Figure 3

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A, quantitative real-time RT-PCR analysis of MMP-9 gene expression after TNFα stimulation and dexamethasone administration in cEND cells. PCR analysis for MMP-9 showed a 1.62 ± 0.2-fold overexpression of MMP-9 in response to TNFα treatment, which could be reversed by steroid treatment (0.41 ± 0.18-fold expression for TNFα–dexamethasone-treated cells and 0.76 ± 0.13-fold expression for TNFα–hydrocortisone-treated cells). B, quantitative real-time RT-PCR analysis of TIMP-1 gene expression after TNFα stimulation and dexamethasone administration in cEND cells. Real-time RT-PCR analysis for TIMP-1 showed a 1.91 ± 0.08-fold increase in TIMP-1 expression for cells treated with TNFα–dexamethasone. Dexamethasone treatment only led to a 2.2 ± 0.09-fold increase of TIMP-1 expression, while co-treatment with dexamethasone and the antagonist RU486 abolished transcription of the TIMP-1 mRNA. C, quantitative real-time RT-PCR analysis of TIMP-2 gene expression after TNFα stimulation and dexamethasone administration in cEND cells. Analysis for TIMP-2 showed no effect on TIMP-2 gene expression for treatments with TNFα or dexamethasone or a combination thereof. Co-treatment with dexamethasone and the antagonist RU486 had no effect on TIMP-2 transcript levels. Data are shown as mean ± s.d. of 3 independent experiments.

Levels of MMP-9 in the CSF of MS patients have been reported to correlate with the presence of enhancement of Gd-DTPA signals on the MRI (Rosenberg et al. 1994, 1996). Intravenous treatment with the GC dexamethasone has been shown to reduce the levels of MMP-9 markedly in patients with enhancement on MRI. We thus further pursued this observation and tested the effect of dexamethasone on the expression of various TIMPs. Analysis demonstrated a small but statistically significant positive effect of dexamethasone on the expression of TIMP-1 (Fig. 3B) while no effect on TIMP-2 was observed (Fig. 3C). Our results show a 2.2 ± 0.08-fold increase of TIMP-1 expression by dexamethasone treatment. For cells treated synergistically with TNFα–dexamethasone, a 1.91 ± 0.09-fold increase in TIMP-1 expression in response to inflammatory stimulus and dexamethasone co-treatment was determined. TNFα treatment alone did not change TIMP-1 expression levels (Fig. 3B). Transcriptional up-regulation of TIMP-1 mRNA by dexamethasone was almost completely abolished in the presence of the GR antagonist RU486 which interferes with the binding of the steroid–receptor complex to the major groove in the DNA (Agarwai, 1996). An effect of RU486 on TIMP-2 gene expression was not observed.

Taken together, treatment with the GC dexamethasone showed reciprocal effects on MMP-9 and TIMP-1 expression: while the exposure of cEND cells to dexamethasone represses the expression of the MMP-9 gene, it synergistically induces the expression of TIMP-1 mRNA.

TIMP-1 promoter transactivation by dexamethasone

It has been demonstrated previously that dexamethasone selectively represses the induction of MMP-9–gelatinase B (Harkness et al. 2000). Here we report the induction of expression of TIMP-1 by the glucocorticoid dexamethasone. Since control of TIMP-1 gene expression has been reported to occur at the level of transcription in connective tissue cells (Edwards et al. 1987, 1992; Clark et al. 1997), we analysed the human TIMP-1 promoter (Clark et al. 1997) for transactivation by the glucocorticoid dexamethasone. For this, the −1718/+95 fragment of the human TIMP-1 promoter was transfected into GC-responsive MCF-7 cells to test GC responsivity. Transient transfection experiments with the full-length TIMP-1 promoter region (−1718/+95; Clark et al. 1997) encompassing five putative degenerate full-site GREs demonstrated responsiveness to dexamethasone induction in MCF-7 cells: treatment of −1718/+95-transfected MCF-7 cells with 100 nm dexamethasone stimulated reporter gene expression about 2.4 + 0.18-fold (Fig. 4A). In contrast, treatment with 100 nm dexamethasone did not stimulate reporter gene expression in MCF-7 cells transfected with the TIMP-1 minimal promoter −102/+95 (Clark et al. 1997), not containing a putative degenerate GRE (0.7 + 0.28-fold) (Fig. 4A).

Figure 4. Transcriptional activation of the TIMP-1 promoter by dexamethasone.

Figure 4

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A, the −1718/+95 fragment of the human TIMP-1 promoter was transfected into MCF-7 cells to test GC responsivity. Treatment of the full-length TIMP-1 promoter region −1718/+95 transfected in MCF-7 cells with 100 nm dexamethasone stimulated reporter gene expression about 2.4 + 0.18-fold. In contrast, treatment with 100 nm dexamethasone did not stimulate reporter gene expression in MCF-7 cells transfected with the TIMP-1 minimal promoter −102/+95, not containing a putative degenerate GRE (0.7 + 0.28-fold). Data are shown as mean ± s.d. of 3 independent experiments. B, TIMP-1 promoter transactivation was verified to be mediated specifically via the glucocorticoid receptor (GR): treatment of COS-1 cells cotransfected with hGRα with 100 nm dexamethasone stimulated reporter gene expression about 3.2 + 0.26-fold, while a negative control, where cotransfection of COS-1 cells with hGRα was omitted, did not show glucocorticoid responsivity. These data indicate that the observed transcriptional activation by dexamethasone is mediated via the GR and is detectable at physiological glucocorticoid concentrations. Data are shown as mean ± s.d. of three independent experiments.

This effect was verified to be mediated specifically via the GR: COS-1 cells, which do not contain endogenous functional GR (Hoeck & Groner, 1990), were cotransfected with an expression plasmid of the human GRα [hGRα] (Almlof et al. 1995) (Fig. 4B). Treatment of COS-1 cells cotransfected with hGRα and the full-length TIMP-1 promoter region −1718/+95 with 100 nm dexamethasone stimulated reporter gene expression about 3.2 + 0.26-fold, while a negative control, where cotransfection with hGRα was omitted, did not show GC responsivity. These data indicate that the observed transcriptional activation by dexamethasone is mediated via the GR and is detectable at therapeutic dexamethasone concentrations (Fig. 4B).

Alteration of integrin α1 and integrin αv expression following treatment of cEND cells with TNFα and dexamethasone

We speculated that TNFα-mediated down-regulation of collagen type IV could lead to reduced expression of integrin α1, the α-subunit of the major collagen receptor integrin α1β1, on microvascular endothelium responsible for endothelial cell adhesion to the matrix ligand collagen type IV (DeFilippi et al. 1991). The expression levels of integrin α1 were assessed by quantitative real-time RT-PCR (Fig. 5). An 8 h administration of TNFα led to a down-regulation of integrin α1 expression (0.78 ± 0.13-fold of control cells) (Fig. 5, light bars) while elevated levels of the αv integrin subunit of the vitronectin receptor αvβ3 were observed (1.45 ± 0.13-fold of control cells) (Fig. 5, dark bars). In the presence of dexamethasone, integrin α1 was not reduced by TNFα but instead up-regulated (1.78 ± 0.25-fold of control cells), while the expression levels of αv integrin remained elevated (1.6 ± 0.23-fold of control cells).

Figure 5. Differential effects of TNFα and dexamethasone on integrin expression.

Figure 5

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The modulation of expression levels of the integrin α1 and αv subunits were assessed by quantitative real-time RT-PCR. An 8 h administration of TNFα led to a down-regulation of integrin α1 expression (0.78 ± 0.13-fold of control cells) while elevated levels of the αv integrin subunit of the vitronectin receptor αvβ3 were observed (1.45 ± 0.13-fold of control cells). In the presence of dexamethasone, integrin α1 was not reduced by TNFα but instead up-regulated (1.78 ± 0.25-fold of control cells). The expression levels of αv integrin remained elevated (1.6 ± 0.23-fold of control cells). Data are shown as mean ± s.d. of 3 independent experiments.

Glucocorticoid preservation of the brain endothelial barrier function

In order to identify functional changes in the brain endothelium, we assessed transendothelial electrical resistance, and barrier-constituting tight junction protein and mRNA levels in response to TNFα and TNFα–glucocorticoid treatment (Fig. 6, Table 1). In order to test whether glucocorticoid treatment prevents a compromise of BBB function in response to TNFα administration, we assessed transendothelial electrical resistance (TER) of untreated cells and compared the values with cells treated with hydrocortisone and dexamethasone alone or a combination of TNFα with either glucocorticoid (Fig. 6A). We were able to show that 8 h TNFα treatment led to a reduction in TER to 65 ± 8% of control values, while treatment with hydrocortisone or dexamethasone increased TER values to 160 ± 7% and 163 ± 6% of control values, respectively. Simultaneous administration of TNFα with hydrocortisone effectively prevented barrier breakdown, TER values amounted to 96 ± 7% of control values, while simultaneous administration of TNFα with dexamethasone even led to increased tightness of the barrier (TER = 145 ± 9% of control values) (Fig. 6A).

Figure 6. Glucocorticoid preservation of endothelial barrier function under pro-inflammatory conditions.

Figure 6

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A, glucocorticoid treatment prevents a compromise of BBB function in response to TNFα administration: 8 h TNFα treatment led to a reduction in TER to 65 ± 8% of control values, while treatment with hydrocortisone or dexamethasone increased TER values to 160 ± 7% and 163 ± 6% of control values, respectively. Simultaneous administration of TNFα with hydrocortisone effectively prevented barrier breakdown, TER values amounted to 96 ± 7% of control values, while simultaneous administration of TNFα with dexamethasone even led to increased tightness of the barrier (TER = 145 ± 9% of control values). B, confluent monolayers of cEND cells were grown in gelatine-coated cell culture flasks to confluence for 5 days and maintained in differentiation medium containing 2% FCS for an additional 3 days. On day 3, cells were treated with 10 nm TNFα for 8 h and cell lysates prepared. Cell lysates were analysed by Western blot for occludin, claudin-5 and ZO-1. Eight hours of TNFα treatment caused a decrease in occludin protein of 21 ± 3% as compared to the untreated control. Simultaneous administration of TNFα and hydrocortisone or dexamethasone prevented occludin loss; we were even able to detect an increase in 17 ± 5% and 49 ± 7%, respectively, as compared to untreated cells. When treated with hydrocortisone and dexamethasone alone, occludin contents were increased by 22 ± 2% and 57 ± 4%, respectively. Eight hours of TNFα treatment caused a decrease in claudin-5 protein of 24 ± 4% as compared to the untreated control. Simultaneous administration of TNFα and hydrocortisone or dexamethasone, however, did not significantly prevent claudin-5 loss, claudin-5 protein levels were reduced by 26 ± 5% and 13 ± 3%, respectively, as compared to untreated cells. When treated with hydrocortisone and dexamethasone alone, claudin-5 contents did not change significantly (88 ± 7% and 93 ± 4%, respectively). Protein levels in ZO-1 were not changed significantly by either treatment.

Table 1.

Modulation of tight junction gene expression in cEND cells by the inflammatory mediator TNFα and glucocorticoids

Occludin ZO-1 Claudin-1 Claudin-5 Claudin-12
TNFα 0.65 ± 0.03  ** 1 ± 0.05 * 1.17 ± 0.9 * 0.86 ± 0.1 * 1.1 ± 0.13 *
HC 1.8 ± 0.12 ** 0.99 ± 0.17 n.s. 2.3 ± 0.14 * 0.98 ± 0.17 * 1.2 ± 0.15 n.s
TNFα/HC 1.2 ± 0.07 ** 1.52 ± 0.36 n.s. 1.98 ± 0.31 n.s. 1.02 ± 0.1 * 1.3 ± 0.05 n.s.
Dex 2.4 ± 0.28 * 1.98 ± 0.2 * 4.36 ± 0.27 ** 1.03 ± 0.13 * 2.6 ± 0.23 **
TNFα/Dex 1.54 ± 0.18 ** 2.04 ± 0.07 ** 1.85 ± 0.15 * 0.90 ± 0.16 * 2.3 ± 0.17 *
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Fold expression versus untreated cells (means ± s.d.), followed by statistical significance.

*

P < 0.05

**

P < 0.001.

n.s., not significant. Values for untreated cells are set = 1. HC, hydrocortisone; Dex, dexamethasone.

Tight junction proteins are known to be key mediators of blood–brain barrier sealing and maintenance. In order to identify potential mechanisms of glucocorticoid preservation of the blood–brain barrier we therefore assessed changes in tight junction protein levels and gene expression. The negative effect of TNFα on barrier properties of the cells could be attributed to a reduction in tight junction protein levels by Western blot (Fig. 6B) and densitometric quantification. We could show a decreased amount of claudin-5 and occludin in cEND cells treated with the inflammatory mediator: 8 h of TNFα treatment caused a decrease in occludin protein of 21 ± 3% as compared to the untreated control. Simultaneous administration of TNFα and hydrocortisone or dexamethasone prevented occludin loss; we were even able to detect an increase in 17 ± 5% and 49 ± 7%, respectively, as compared to untreated cells. When treated with hydrocortisone and dexamethasone alone, occludin contents were increased by 22 ± 2% and 57 ± 4%, respectively. Comparably, 8 h of TNFα treatment caused a decrease in claudin-5 protein of 24 ± 4% as compared to the untreated control. Simultaneous administration of TNFα and hydrocortisone or dexamethasone, however, did not significantly prevent claudin-5 loss; we were still able to detect claudin-5 protein levels reduced by 26 ± 5% and 13 ± 3%, respectively, as compared to untreated cells. When treated with hydrocortisone and dexamethasone alone, claudin-5 content did not change significantly as compared to untreated cells (88 ± 7% and 93 ± 4%, respectively) (Fig. 6B). We also assessed whether the inflammatory cytokine or the glucocorticoids would influence levels of the tight junction plaque protein ZO-1; linking occludin to the actin cytoskeleton, however, could not detect significant changes in either case (Fig. 6B).

Also by real-time RT-PCR analysis we could show that treatment with the inflammatory mediator TNFα alone or simultaneous treatment with glucocorticoids influenced gene expression of tight junction components differently (Table 1).

All the endothelial cells studied expressed occludin. Levels were, however, significantly reduced in response to TNFα treatment. Co-treatment with TNFα–hydrocortisone or TNFα–dexamethasone maintained or increased occludin gene expression, respectively, as compared to the control and prevented down-regulation of occludin gene expression. As previously described (Förster et al. 2005, 2006), treatment with the glucocortiocoids alone induced occludin gene expression even more strongly. The claudins 1, 12 and 5 showed different responses to the different treatment regimens: while claudin-5 gene expression decreased in response to TNFα treatment, glucocorticoid treatment did not lead to an increase in claudin-5 levels as compared to untreated cells. However, concomitant TNFα–glucocorticoid treatment prevented a down-regulation of gene expression, but the amounts of claudin-5 transcript did not exceed the levels measured in control cells. The situation differed from observations in the case of claudin-12: for claudin-12, a down-regulation of gene expression in response to TNFα was not observed as reported previously (Silwedel & Förster, 2006), while treatment with the glucocorticoid dexamethasone (with or without simultaneous TNFα treatment) significantly increased gene expression levels.

For claudin-1, a down-regulation of gene expression in response to TNFα was equally not observed, while treatment with both glucocorticoids, dexamethasone and hydrocortisone (with or without simultaneous TNFα treatment), significantly increased gene expression levels.

Gene expression of the tight junctional plaque protein ZO-1 linking occludin to the actin cytoskeleton was significantly induced in response to dexamethasone treatment, alone as well as in combination with TNFα, while TNFα administration or supplementation with the glucocorticoid hydrocortisone showed no effect on gene expression (Table 1). Dexamethasone effects at mRNA level, however, did not match observations at protein level, where no increased amounts in response to dexamethasone treatment had been observed (cf. Fig. 6B).

The influence of TNFα on cEND cells was observed from 0.5 to 24 h after the beginning of the treatment. We received the most pronounced differences in gene expression after a duration of 8 h; a longer treatment did not lead to any differences.

Discussion

The microenvironment of the central nervous system is normally maintained by the presence of the BBB. Breakdown of the BBB is a key feature of neuroinflammatory conditions, such as multiple sclerosis (MS), encephalitis, meningitis, brain tumours and cerebral ischaemia (Hamann et al. 1995; Rosenberg, 2002; Sellner & Leib, 2006).

However, the opening of the BBB is not only due to degradation and decreased synthesis of tight junction proteins (Harkness et al. 2000; Chang & Werb, 2001; Silwedel & Förster, 2006; Yang et al. 2006), but also to degradation of the basal lamina macromolecules that line the blood vessels, such as collagen type IV and laminin (Hamann et al. 1995; Chang & Werb, 2001; Rosenberg, 2002). Disintegration of the structural and functional vascular unit, made up of endothelial cells of the capillary wall, the extracellular matrix of the basal lamina and the endfeet of astrocytes plus adjacent neurons, is associated with weakening of the vessels which predispose to leakage and rupture. In addition to vascular rupture, disruption of the homeostatic cell–matrix interactions may directly lead to cell death by loss of integrins, which mediate signals essential for cell survival (Lee & Lo, 2004).

Mediators of BBB disruption include the MMPs, a group of zinc-containing endopeptidases (Rosenberg, 2002). Amongst these, it has been demonstrated that, selectively, MMP-9 (gelatinase B) is increased in CSF in MS patients while increased serum MMP-9/TIMP-1 ratios have been reported during disease activity during IFN-β-1α treatment (Avolio et al. 2005). In the pathology of MS, MMP-9 thus appears to be associated with BBB damage as seen on enhanced-magnetic resonance imaging (MRI) scans (Rosenberg et al. 1996). MMP-9 can degrade collagen type IV, which constitutes up to 90% of the total protein of the basal lamina and confers the structural integrity of the vessel wall (Hamann et al. 1995). Besides this, MMPs have been shown to act on non-matrix substrates, including the inter-endothelial tight junctions, cell-surface and matrix-bound growth regulators, releasing them from stores (Nagase & Woessner, 1999). The physiological and pharmacological inhibition of MMP-9 activity thus represents a major level of control of MMP activity rendering it a therapeutic target (Vincenti et al. 1994).

Using the murine immortalized brain endothelial cell line cEND, which maintains characteristic qualities of the BBB in vivo after immortalization, we previously demonstrated (Förster et al. 2005; Silwedel & Förster, 2006) that barrier properties can be improved by GCs through the induction of occludin (Förster et al. 2005, 2006). We further validated our cell culture model suitable to study the barrier-compromising effects of pro-inflammatory stimuli (Silwedel & Förster, 2006), demonstrating the TNFα-induced down-regulation of tight junction proteins in cEND cells. In this study, we expand our characterization of GC-mediated effects on barrier integrity by investigating the effects on cytokine-provoked matrix degradation. We show that TNFα administration leads to increased secretion of, specifically, MMP-9 into cEND cell culture supernatants, indicating that the CNS vascular endo-thelium plays an active part in the breakdown of the BBB through altered expression of MMPs. In contrast to MMP-9, MMP-2 is secreted constitutively by cEND cells and is not further increased by administration of TNFα (data not shown). A similar finding has also been observed in rat microvascular endothelial cells, GP8/3.9 (Harkness et al. 2000), where treatment with TNFα led to selective up-regulation of MMP-9, while levels of MMP-2 remained unchanged, consistent with in vivo observations (Avolio et al. 2005), further supporting the use of the cEND cell culture model for investigation of potential protective effects of GCs on the extracellular matrix.

The active forms of all MMPs are inhibited by a family of specific inhibitors, the tissue inhibitors of metalloproteinases (TIMPs). MMP-9 has in this context been demonstrated to be preferentially inhibited by TIMP-1 (Brew et al. 2000). The restoration of the normal MMP-9/TIMP-1 balance would represent a tempting therapeutical perspective. We pursued this notion and investigated glucocorticoid effects on TIMP-1 expression. While we could clearly demonstrate that TIMP-2 expression is not regulated by dexamethasone, we were able to show a significant increase in TIMP-1 expression mediated by dexamethasone. Even more, a simultaneous treatment of cEND cells with the pro-inflammatory cytokine TNFα and dexamethasone led to reciprocal effects on MMP-9 and TIMP-1 expression: while MMP-9 activity and secretion was reduced by dexamethasone, administration of dexamethasone led to an induction of TIMP-1 expression in cEND cells, indicating that GC-mediated reduction of MMP-9 levels not only originates from repression of MMP-9 transcription, but also from induction of its cognate inhibitor, TIMP-1 (Yong et al. 2001). Our findings demonstrating selective up-regulation of TIMP-1 but not TIMP-2 are in accordance with studies in rat (Harkness et al. 2000) and mouse (Pagenstecher et al. 1998), where constitutive TIMP-2 expression was reported with no significant alteration in response to dexamethasone administration. These data point to a glucocorticoid-mediated up-regulation of TIMP-1, leading to decreased MMP-9 activity, besides the inhibition activation of MMP-9 previously described by Harkness et al. (2000). An increase in a complex of MMP-9 and, at that point, a non-specified TIMP after glucocoricoid treatment has additionally already been observed by Rosenberg et al. in the past (Rosenberg et al. 1996).

In an attempt to further elucidate the molecular mechanisms of GC-mediated induction of TIMP-1, we were able to show that GC signals can directly act at the transcriptional level by putative interaction with specific cis-acting DNA sequence elements in the TIMP-1 gene promoter: in the present study, dexamethasone increased transcription of TIMP-1 mRNA in murine cEND cells. This effect was verified to be mediated specifically via the GR: we demonstrated, using mifepristone (RU-486), a potent and selective GR antagonist, that the effect of dexamethasone on TIMP-1 mRNA expression requires interaction of the GC with its intracellular receptor.

We furthermore hypothesized that alterations in endo-thelial cell–matrix adhesion mediated by integrins in response to pro-inflammatory stimuli may be crucially involved in disruption of the permeability barrier of the brain and a potential target of glucocorticoid action: the matrix-binding integrin α1β1 receptor has been demonstrated to be the major collagen receptor on microvascular endothelial cells responsible for binding of the ECM ligand collagen IV (Defilippi et al. 1991). We therefore analysed putative consequences of MMP-9-mediated degradation of collagen IV on expression of the α chain of its integrin receptor. Our data demonstrate reduction in the expression levels of integrin α1 in response to TNFα treatment. Concomitantly, an up-regulation of the α subunit of the vitronectin receptor αvβ3 was observed as reported for MS lesions (Sobel et al. 1998a). Simultaneous administration of TNFα and dexamethasone prevented down-regulation of integrin α1 but surprisingly did not prevent up-regulation of integrin αv. Matching data supporting the important role of functional integrin–ECM binding have been demonstrated in vivo, breakdown of the BBB has been observed in mice lacking selected integrins (in Del Zoppo & Milner, 2006), and altered expression patterns of, specifically, integrin α1 and integrin αv have been observed in MS lesions (Sobel et al. 1998b). The influence of altered integrin expression pattern on cellular differentiation has been described in integrin knock-out mice (Sheppard, 2000) and for epithelial cells in vitro (Gumbiner, 1996), and, specifically, a role in barrier formation has been described in mammary epithelial cells (Förster et al. 2002), supporting the notion that ECM integrin receptors like integrin α1 should participate in the maintenance of cerebrovascular integrity. Whether the observed maintainance of integrin α1 levels by dexamethasone is due to preservation of its ECM ligand collagen VI by elevation of TIMP-1, or whether integrin α1 itself might represent yet another target gene for the glucocorticoid receptor and its ligand dexamethasone needs further investigation. Similarly, the function of the potentially compensatory up-regulation of integrin αv remains to be elucidated.

Our study further demonstrates that the administration of the glucocorticoids dexamethasone and hydrocortisone preserves the functional integrity of tight junctions under pro-inflammatory conditions, chiefly by maintaining the levels of the tight junction components occludin, claudin-1, claudin-12 and ZO-1, while effects on claudin-5 were negligible. The same physiological insult without simultaneous glucocorticoid administration significantly reduced BBB tightness and tight junction protein expression. Previously, we showed that GCs directly induce the formation of the endothelial TJ protein occludin (Förster et al. 2005, 2006), thus providing a pro-barrier effect. Whether ZO-1 and the claudins 1 and 12 might represent further glucocorticoid receptor target genes remains to be elucidated in the future, including analysis of the exact time and concentration dependency of gene induction and protein synthesis for these tight junction components.

Taken together, our observations support the assumption that up-regulation of MMP-9 during inflammation in endothelial cells leads to the degradation of occludin and could affect tight junction structural integrity, while glucocorticoid treatment is an effective way to prevent this. The use of GCs to prevent pathological matrix degradation appears to be rewarding, since currently available synthetic MMP inhibitors show little target specificity within the MMP family and administration frequently leads to side-effects due to interference with physiological functions of MMPs. Work has been published reporting disappointing results of clinical trials for synthetic MMP inhibitors in inflammatory diseases, i.e. failure to inhibit effects in humans as opposed to animal models or, even worse, little or no specificity within the MMP family and therefore detrimental inhibition of MMPs whose function is required for necessary physiological functions (reviewed in Leppert et al. 2001). Although reversible after discontinuation of the drug, such side-effects preclude their long term use in chronic diseases, e.g. in MS. The restoration of the normal MMP-9/TIMP-1 balance by GCs in contrast would not cause these side-effects but limit the excess activity of MMP-9 in inflammation to control levels. Consequently, improved GR ligands might thus be useful in the targeted increase in TIMP-1 expression and thus limit BBB breakdown, concomitant with a reinforcement of the TJ barrier (Romero et al. 2003; Förster et al. 2005, 2006).

Acknowledgments

This research was supported by grant SFB688 from the Deutsche Forschungsgemeinschaft to C.F. and grant SFB487 from the Deutsche Forschungsgemeinschaft to D.D. TIMP-1 promoter-reporter constructs −1718/+95 and −102/+95 were a generous gift from Dr Ian M. Clark, Biomedical Research Centre, School of Biological Sciences, University of East Anglia, Norfolk, UK. The authors are grateful to Eva-Maria Klute for excellent technical assistance.

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