December 2008
Volume 49, Issue 12
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Glaucoma  |   December 2008
Oxidative Stress and TGF-β2 Increase Heat Shock Protein 27 Expression in Human Optic Nerve Head Astrocytes
Author Affiliations
  • Alice L. Yu
    From the Department of Ophthalmology, Ludwig-Maximilians-University, Munich, Germany;
  • Rudolf Fuchshofer
    Department of Anatomy, University of Regensburg, Regensburg, Germany;
  • Marco Birke
    Departments of Anatomy and
  • Anselm Kampik
    From the Department of Ophthalmology, Ludwig-Maximilians-University, Munich, Germany;
  • Hans Bloemendal
    Department of Biomolecular Chemistry NCMLS, Radboud University Njimegen, Njimegen, The Netherlands.
  • Ulrich Welge-Lüssen
    From the Department of Ophthalmology, Ludwig-Maximilians-University, Munich, Germany;
    Ophthalmology, Friedrich-Alexander-University, Erlangen, Germany; and
Investigative Ophthalmology & Visual Science December 2008, Vol.49, 5403-5411. doi:10.1167/iovs.07-1478
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      Alice L. Yu, Rudolf Fuchshofer, Marco Birke, Anselm Kampik, Hans Bloemendal, Ulrich Welge-Lüssen; Oxidative Stress and TGF-β2 Increase Heat Shock Protein 27 Expression in Human Optic Nerve Head Astrocytes. Invest. Ophthalmol. Vis. Sci. 2008;49(12):5403-5411. doi: 10.1167/iovs.07-1478.

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      © 2016 Association for Research in Vision and Ophthalmology.

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Abstract

purpose. Reactive astrocytes of glaucomatous optic nerve heads (ONHs) are characterized by an increased expression of transforming growth factor (TGF)-β2 and heat shock proteins (Hsps) such as Hsp27. The goal of the present study was to determine the effect of oxidative stress on TGF-β2 and Hsp27 expression in human ONH astrocytes.

methods. Cultured ONH astrocytes were exposed to 100, 200, and 400 μM hydrogen peroxide (H2O2) for 1 hour. Levels of TGF-β2 were analyzed by ELISA. Additionally, cells were treated with 1.0 ng/mL TGF-β2 for 12, 24, and 48 hours, respectively. Expression of Hsp27 was detected by immunohistochemistry, real-time PCR, and Western blot analyses. To investigate the role of signal transduction proteins, cells were pretreated with the specific inhibitors SB203580, PD98059, U0126, and SP600125. Furthermore, induction of p38MAP kinase and its phosphorylated form were determined by Western blot analyses.

results. H2O2 exposure increased the secretion of TGF-β2. Both H2O2 and TGF-β2 increased the Hsp27 expression. Pretreatment of cells with SB203580, an inhibitor for p38MAP kinase, reduced the H2O2- and TGF-β2-stimulated Hsp27 expression, whereas pretreatment with PD98059 and U0126, specific inhibitors of ERK1/2, and SP600125, a specific c-Jun N-terminal kinase inhibitor, had no effects. H2O2 and TGF-β2 upregulated the phosphorylated p38MAP kinase.

conclusions. Oxidative stress increased TGF-β2 secretion in cultured human ONH astrocytes. Both H2O2 and TGF-β2 increased Hsp27 expression via p38MAP kinase activation. Therefore, it is tempting to speculate that reduction of oxidative stress and TGF-β2 may help to minimize the incidence of characteristic changes in the ONH of patients with glaucoma.

Primary open-angle glaucoma (POAG) is an optic neuropathy associated with increased IOP. 1 2 Besides the clinically visible loss of optic nerve axons, histologic studies have shown that characteristic changes, the so-called reactivation, take place in the astrocytes of the optic nerve head (ONH). 3 4 5 6  
In the central nervous system, quiescent astrocytes transform into reactive phenotypes after stressful events. 7 8 Exposure to various stress factors is accompanied by the induction of a specific group of proteins, the so-called heat shock proteins (Hsps). 9 10 11 12 13 Hsps are molecular chaperones and are involved in protein folding and translocation. 14 15 It has been shown that increased Hsp expression correlates with the ability of cells to survive stressful conditions. 16 17 18 19 Small Hsps, such as Hsp27, have been noted to accumulate in reactive cerebral astrocytes of numerous neurodegenerative diseases. 9 20 21 22 In glaucomatous optic neuropathy, increased immunohistochemical staining of Hsp27 was demonstrated in reactive ONH astrocytes. 23 Salvador-Silva and colleagues 24 have shown that elevated Hsp27 expression in human reactive ONH astrocytes is inducible by increased hydrostatic pressure. Besides an elevated IOP, a number of other stress factors such as oxidative stress 25 26 27 and transforming growth factor (TGF)-β2 28 29 30 31 and are increasingly suggested to be involved in the pathogenesis of POAG. Oxidative stress is known to be a strong inducer of TGF-β2 secretion. 32 33 34 35 Whether oxidative stress and TGF-β2 are able to upregulate Hsp27 expression in human ONH astrocytes is still unclear and requires further investigation. 
In different cellular systems, Hsp27 expression was shown to be inducible by oxidative stress. 36 37 38 39 40 41 42 Signal transduction analyses revealed that this process is mediated by the activation of p38MAP kinase, a member of mitogen-activated protein (MAP) kinase family. 36 37 38 39 40 41 42 Induction of Hsp27 synthesis via p38MAP kinase activation has also been demonstrated after exposure to TGF-β2. 43 44 45 46 47 However, ERK1/2, another subfamily of MAP kinases, has also been shown to be involved in the signal transduction of H2O2 and TGF-β2. 37 48 49 50 In addition, recent studies on the third subfamily member of MAP kinases, c-Jun N-terminal kinase (JNK), suggested that JNK can be activated by H2O2 and TGF-β2. 51 52 In this study, we first investigated the effect of oxidative stress on TGF-β2 secretion in cultured human ONH astrocytes. Furthermore, we tested the effects of oxidative stress and TGF-β2 on Hsp27 expression in our in vitro model. By using specific inhibitors of p38MAPK, ERK1/2, and JNK, we analyzed the underlying signaling pathways of oxidative stress– and TGF-β2-mediated induction of Hsp27. These data might help to reveal unknown effects of oxidative stress and TGF-β2 in the formation of glaucomatous optic nerve changes. 
Materials and Methods
Cell Culture
Primary cell cultures of human lamina cribrosa astrocytes were obtained from the eye bank of the Ludwig-Maximilians-University, Munich, Germany. Monolayer cultures were established from eyes of five human donors between 56 and 68 years of age. These eyes, without any history of eye diseases, were obtained 4 to 12 hours postmortem. Methods of securing human tissue were humane, included proper consent and approval, complied with the Declaration of Helsinki, and were approved by the local ethics committee. Astrocytes of the ONH were prepared, grown, and classified as described previously. 6 53 54 In brief, eyes were cut equatorially behind the ora serrata and the ONH was isolated from the neighboring tissues. The ONH was sagittally dissected under a microscope and the lamina cribrosa was identified. Discs of lamina cribrosa were prepared by dissection from the pre- and postlaminar regions, subsequently cut into three to four explants and placed in Petri dishes with 2 mL Dulbecco’s modified Eagle’s medium (DMEM)/F-12 supplemented with 10% fetal bovine serum (FBS; Gibco-Life Science Technology, Karlsruhe, Germany), 5 ng/mL human basic pituitary fibroblast growth factor (bFGF; Sigma, Deisenhofen, Germany), 5 ng/mL human platelet-derived growth factor-A chain (PDGF AA; Sigma), 50 U/mL penicillin and 50 μg/mL streptomycin (Gibco-Life Science Technology) at 37°C in a 5% CO2 incubator. To isolate ONH astrocytes, the primary cell cultures were first plated in serum-free astrocytes growth medium (AGM; Cambrex Bio Science, Verviers, Belgium) for 24 hours and then changed to AGM containing 5% FBS. 55 Other cell populations, such as lamina cribrosa cells, failed to attach in serum-free medium and were removed with medium change. Subsequently, cultured ONH astrocytes were maintained in DMEM/F-12 with 10% FBS. ONH astrocytes were distinguished from adjacent cells by their morphology and immunohistochemical staining (data not shown). 6 54 Primary human ONH astrocytes were characterized by positive immunostaining for glial fibrillary acidic protein (GFAP; Sigma), neural cell adhesion molecule (NCAM; Serotec, Düsseldorf, Germany), vimentin (Sigma), desmin (Abcam, Cambridge, UK), S100 (Invitrogen, Karlsruhe, Germany), and Pax2 (Abcam), and negative immunostaining for A2B5 (Chemicon International, Hampshire, UK) and smooth muscle actin (smA; Dako, Glostrup, Denmark). 6 53 56 57 58 Only cell cultures, which were at least 95% positive for GFAP, NCAM, vimentin, desmin, S100, and Pax2, and negative for A2B5 and smA, were used in this study. 54  
To test the effects of oxidative stress on astrocytes, confluent cells of passage 3 to 5 were incubated for 24 hours in serum-free DMEM/F12 at 37°C and 5% CO2. Then, the medium was replaced by fresh serum-free DMEM/F12 medium and cells were exposed to 100, 200, and 400 μM hydrogen peroxide (H2O2) for 1 hour. After H2O2 treatment, cells were placed in serum-free DMEM/F12 medium for 24 hours. In control cultures, the medium was changed at the same time points but no H2O2 was added. 
To investigate the effects of TGF-β2, second- to fifth-passage astrocytes were grown to confluence in 35-mm Petri dishes in DMEM/F12 supplemented with 10% FBS at 37°C and 5% CO2. At confluence, cells were washed and incubated overnight in serum-free DMEM/F12 medium. After 24 hours incubation, this medium was replaced by fresh serum-free DMEM/F12 supplemented with active TGF-β2 (R&D Systems, Wiesbaden, Germany) to a final concentration of 1.0 ng/mL. Under these conditions, cells were incubated for 12, 24, and 48 hours, respectively. In control cultures, the medium was changed at the same time points but no TGF-β2 was added. 
To investigate whether H2O2- or TGF-β2-induced upregulation of Hsp27 is mediated via p38MAP kinase, ERK1/2, or JNK, the specific inhibitors SB203580 (Calbiochem, Darmstadt, Germany) for p38MAP kinase, PD98059 (Calbiochem), and U0126 (Calbiochem) for MAPK/ERK1/2 kinase (MEK), and JNK inhibitor II SP600125 (Calbiochem) for JNK were used. Confluent cells were incubated in serum-free DMEM/F12 medium for 24 hours. Subsequently, the medium was replaced by fresh serum-free DMEM/F12 medium and cells were pretreated with SB203580, PD98059, U0126, and SP600125 at 10 μM for 60 minutes in serum-free medium. After pretreatment with the inhibitors, cells were exposed to either H2O2 or TGF-β2, as described above. The medium of control cells was changed at the same time points without treatment. The tetrazolium dye-reduction assay (MTT; Sigma; 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) was used to test cell viability before and at the end of treatment, and did not reveal any signs of increased cell death in H2O2- or TGF-β2-treated cells (data not shown). All experiments were performed at least in triplicate in astrocyte cultures from three different donors. 
Analysis of TGF-β2 in Cell Supernatants
Release of TGF-β2 into culture supernatants of astrocytes was measured using corresponding ELISA kit according to the manufacturer’s instructions (R&D Systems). As the ELISA only measured levels of the active protein, additional activation of the supernatants was undertaken to measure the total (latent + active) levels of TGF-β2. Briefly, cell supernatants were activated with 1.0 N HCl and subsequently neutralized with 1.2 N NaOH/0.5 M HEPES and analyzed using ELISA. All experiments were performed at least in triplicate in astrocyte cultures from three donors. The intra-assay and interassay coefficients of variation determined in our laboratory were 9.8% and 16%, respectively. 
Immunohistochemistry
Cultured human ONH astrocytes, grown in 4-well plastic chamber slides, were treated with H2O2 or TGF-β2 as described above. After incubation, cells were washed with PBS, fixed with 4% paraformaldehyde (PFA) for 15 minutes, and subsequently washed twice with PBS containing 0.1% Triton X-100. Primary incubation with all samples was performed with a rabbit anti-human Hsp27 antibody (Stressgen Bioreagents, Hamburg, Germany) diluted 1:200 in PBS containing 5% BSA for 4 hours at room temperature (RT). Control samples were incubated with PBS and 5% BSA but without the primary antibodies. Afterward, cells were washed three times with PBS, and incubated with fluorescein-conjugated goat anti-rabbit Cy3 antibody (diluted 1:500 in PBS; Dianova, Hamburg, Germany) for 1 hour at RT. Cells were then rinsed in PBS, mounted with Kaiser’s glycerin jelly (Merck, Darmstadt, Germany), and analyzed under a fluorescence microscope (Leica DMR; Leica Microsystems, Wetzlar, Germany). Representative areas were documented (Leica IM 1000 software; Leica Microsystems, Heerbrugg, Switzerland). All experiments were performed at least in triplicate in astrocyte cultures from three different donors. 
RNA Isolation and Real-time PCR
Total RNA was isolated from 10 cm Petri dishes by the guanidium thiocyanate-phenol-chloroform extraction method (Stratagene, Heidelberg, Germany). Structural integrity of the RNA samples was confirmed by electrophoresis in 1% Tris-acetate-EDTA (TAE)-agarose gels. Yield and purity were determined photometrically. After RNA isolation, mRNA was transcribed to cDNA by reverse transcriptase. This cDNA was then used for specific real-time PCR. Quantification of human mRNA was performed during 40 cycles in the real-time PCR system (LightCycler System; Roche Diagnostics, Mannheim, Germany). The primers selected were Hsp27 forward primer 5′-TGACGGTCAAGACCAAGGA-3′ (positions 433 to 451) and reverse primer 5′-TGTAGCCATGCTCGTCCTG-3′ (positions 489 to 507); and 18S rRNA forward primer 5′-CTCAACACGGGAAACCTCAC-3′ (positions 1348 to 1367) and reverse primer 5′-CGCTCCACCAACTAAGAACG-3′ (positions 1438 to 1457). Primers and probes were found with commercial software (ProbeFinder Version 2.04; Roche, Mannheim, Germany). The standard curve was obtained from probes of three different untreated human ONH astrocyte cultures. To normalize differences of the amount of total RNA added to each reaction, 18S rRNA was simultaneously processed in the same sample as an internal control. The level of Hsp27 mRNA was determined as the relative ratio (RR), which was calculated by dividing the level of Hsp27-mRNA by the level of the 18S rRNA gene in the same samples. All experiments were performed at least in triplicate in astrocyte cultures from three different donors. 
Protein Extraction and Western Blot Analysis of Hsp27, p38MAP Kinase, and p-p38 MAP Kinase
Cells grown on 35-mm tissue culture dishes were washed twice with ice-cold PBS, collected, and lysed in radio-immunoprecipitation assay (RIPA) cell lysis buffer. After centrifugation (19,000g for 30 minutes at 4°C) in a microfuge, the supernatants were transferred to fresh tubes and stored at −70°C for future use. The protein content was measured by the bicinchoninic acid (BCA) protein assay (Pierce, Rockford, IL). Denatured proteins (2 μg) were separated under reducing conditions by electrophoresis using 10% SDS-polyacrylamide gels. Thereafter, the proteins were transferred with semidry blotting onto a polyvinyl difluoride membrane (Roche) and probed with a rabbit polyclonal antibody against Hsp27 (Stressgen Bioreagents), p38MAP kinase [p38 (C-20): sc-535; Santa Cruz Biotechnology, Heidelberg, Germany] and p-p38MAP kinase [p-p38 (Thre180/182); Cell Signaling Solutions, Temecula, CA] as described previously. 59 These antibodies were used at a dilution of 1:200, respectively. Chemiluminescence was detected with the imager (LAS-1000; RayTest, Pforzheim, Germany). Exposure times ranged between 1 and 20 minutes. Quantification was performed on a computer (AIDA software; RayTest). All experiments were performed at least in triplicate in astrocyte cultures from three different donors. 
Statistical Analysis
Results are expressed as the mean ± SD. For comparison of means between two groups, an unpaired t-test was used. Statistical significance was defined as P < 0.05. 
Results
Increases in Secretion of TGF-β2 by H2O2
In a first approach, we investigated the effect of H2O2 on the secretion of TGF-β2. By using a commercially available ELISA assay, we found a basal level of total TGF-β2 (122 ± 26 pg/mL) and of active TGF-β2 (71 ± 18 pg/mL) in the media of untreated astrocytes. Exposure of astrocytes to 100, 200, and 400 μM H2O2 increased the total TGF-β2 release to 214 ± 91 pg/mL, 332 ± 56 pg/mL, and 337 ± 140 pg/mL, respectively (Fig. 1A) . Furthermore, treatment with 100, 200, and 400 μM H2O2 increased the active form of TGF-β2 to 80 ± 34 pg/mL, 104 ± 33 pg/mL, and 136 ± 77 pg/mL (Fig. 1B)
Increases in the Expression of Hsp27 by H2O2
In a subsequent approach, human ONH astrocytes were treated with 100, 200, and 400 μM H2O2 for 1 hour followed by subsequent incubation with fresh serum-free medium for 24 hours (Fig. 2) . By immunohistochemical staining, an increased expression of Hsp27 was detected after exposure of cells to 100, 200, and 400 μM H2O2 (Figs. 2B 2C 2D)compared to untreated control cells (Fig. 2A) . For mRNA quantification, real-time PCR analysis was performed. The signals generated in untreated control cells were set to 100% (Fig. 2E) . Exposure of cells to 100, 200, and 400 μM H2O2 for 1 hour increased the Hsp27 mRNA expression by 1.4 ± 0.2 fold, 1.7 ± 0.4 fold, and 1.6 ± 0.2 fold compared to untreated control cells (Fig. 2E)
At the protein level, Western blot analysis revealed no marked effects on Hsp27 expression (1.1 ± 0.2 fold) after exposure to 100 μM H2O2, compared to untreated control cells (Fig. 2F) . In contrast, an increased upregulation of Hsp27 protein amount by 1.5 ± 0.1 fold and 1.3 ± 0.2 fold was observed after 200 μM and 400 μM H2O2 treatment compared to untreated control cells (Fig. 2F)
Increases in the Expression of Hsp27 by TGF-β2
Human ONH astrocytes were treated with 1.0 ng/mL TGF-β2 for 12, 24, and 48 hours, respectively (Fig. 3) . By immunohistochemical staining, treatment with TGF-β2 for 12, 24, and 48 hours showed an increased expression of Hsp27 (Figs. 3B 3C 3D)compared to untreated control cells (Fig. 3A) . Since immunohistochemistry is not a valid quantification method, we also performed real-time PCR and Western blot analysis. The signals generated by real-time PCR analysis in untreated control cells were set to 100% (Fig. 3E) . There was a marked upregulation of Hsp27 mRNA expression after TGF-β2 treatment for 12 hours (2.9 ± 0.9 fold), for 24 hours (3.3 ± 0.5 fold), and for 48 hours (4.1 ± 0.6 fold) compared to untreated control cells (Fig. 3E)
A less significant upregulation of Hsp27 has been observed at the protein level (Fig. 3F) . By Western blot analysis, we detected an upregulation of Hsp27 protein to 1.7 ± 0.4 fold after 12 hours of TGF-β2 treatment compared to untreated control cells (Fig. 3F) . The maximal increase of Hsp27 protein amount was observed after treatment with TGF-β2 for 24 hours (2.0 ± 0.3 fold) (Fig. 3F) . Treatment with TGF-β2 for 48 hours increased the Hsp27 protein expression 1.8 ± 0.5 fold compared to untreated control cells (Fig. 3F)
Increases in Hsp27 via p38MAP Kinase by H2O2
To investigate which signal transduction protein is involved in H2O2-stimulated Hsp27 expression, the effects of p38MAP kinase and ERK1/2 inhibitors were examined. Exposure of cells with 200 μM H2O2 for 1 hour increased the Hsp27 mRNA expression by 1.6 ± 0.3 fold and protein expression by 1.5 ± 0.2 fold (Figs. 4A 4B) . H2O2-induced upregulation of Hsp27 expression could be suppressed by pretreatment with 10 μM SB203580 to 1.1 ± 0.2 fold at the mRNA and to 1.0 ± 0.1 fold at the protein level compared to untreated control cells (Figs. 4A 4B) . In contrast, pretreatment with 10 μM PD98059 and U0126 had no significant effects on the H2O2-induced Hsp27 mRNA (1.5 ± 0.2 fold and 1.4 ± 0.1 fold) and protein upregulation (1.4 ± 0.3 fold and 1.3 ± 0.2 fold; Figs. 4A 4B ). 
Increases in Hsp27 via p38MAP Kinase by TGF-β2
To investigate whether or not p38MAP kinase is involved in TGF-β2-stimulated Hsp27 expression, the effect of p38MAP kinase inhibitor SB203580 was examined. SB203580 alone had no effects on the basal level of Hsp27 (data not shown). Treatment with 1.0 ng/mL TGF-β2 for 24 hours increased the Hsp27 mRNA and protein amount by 3.0 ± 0.6 fold and 2.1 ± 0.2 fold (Figs. 5A 5B) . Pretreatment with 10 μM SB203580 significantly reduced the TGF-β2-stimulated Hsp27 upregulation to 1.9 ± 0.2 fold at the mRNA and to 1.3 ± 0.2 fold at the protein level compared to untreated control cells (Figs. 5A 5B) . Furthermore, we investigated whether or not ERK1/2 is involved in the TGF-β2-induced Hsp27 expression by using the specific inhibitors PD98059 and U0126. Both inhibitors had no effects on the basal level of Hsp27 (data not shown). Pretreatment of cells with PD98059 and U0126 before exposure to 24 hours of TGF-β2 treatment led to no significant decrease of the TGF-β2-induced Hsp27 mRNA (2.4 ± 0.3 fold and 2.7 ± 0.4 fold) and protein expression (2.3 ± 0.5 fold and 2.5 ± 0.3 fold; Figs. 5A 5B ). 
Increase in the Phosphorylation of p38MAP Kinase by H2O2 and TGF-β2
To confirm the activation of p38MAP kinase in H2O2-stimulated Hsp27 expression, Western blot analysis on p38 and p-p38MAP kinase after treatment with 200 μM H2O2 for 1 hour was performed (Figs. 6A 6B) . We were able to show equal amounts of p38MAP kinase expression both in H2O2-treated and untreated control cells (Fig. 6A) . However, there was a marked upregulation of p-p38MAP kinase in H2O2-treated cells (2.4 ± 0.8 fold) compared to untreated control cells (Fig. 6B)
To confirm the activation of p38MAP kinase in TGF-β2-stimulated Hsp27 induction, we investigated the expression of p38 and p-p38MAP kinase after 24 hours of TGF-β2 treatment (Figs. 6A 6B) . Western blot analysis showed equal amount of p38MAP kinase expression both in TGF-β2 treated and untreated control cells (Fig. 6A) . In contrast, the expression of the p-p38MAP kinase was markedly increased in TGF-β2-treated cells (3.6 ± 1.2 fold) compared to untreated control cells (Fig. 6B)
Lack of Effects on H2O2- or TGF-β2-Induced Hsp27 Upregulation by Pretreatment with JNK Inhibitor
Lastly, we could show that pretreatment with the JNK inhibitor SP600125 (TGF-β2 + SP600125: 2.7 ± 0.2 fold; H2O2 + SP600125: 1.5 ± 0.1 fold) affected neither the H2O2- (1.6 ± 0.3 fold) nor the TGF-β2-induced Hsp27 mRNA upregulation (3.3 ± 0.2 fold) compared to untreated control cells (Figs. 7A 7B)
Discussion
Besides an elevated IOP, various stress factors, such as oxidative stress 25 26 27 and TGF-β2, 28 29 30 31 may play an important role in POAG. There is emerging evidence that reactive astrocytes in glaucomatous optic neuropathies may mediate neuroprotective effects by increased expression of heat shock proteins. 23 24 60 61 Our present study demonstrates for the first time in vitro that oxidative stress is capable to induce increased secretion of TGF-β2 in cultured human ONH astrocytes. Furthermore, both oxidative stress and TGF-β2 are able to induce Hsp27 in this in vitro model. 
Oxidative stress is associated with the pathogenesis of a variety of neurodegenerative diseases. 62 TGF-β2 has been reported to provide neuroprotective effects after injury and during neurodegeneration 63 64 and to be implicated in oxidative stress-mediated pathologies. 65 TGF-β2 is a multifunctional cytokine involved in a variety of physiological and pathophysiological processes of the brain. 63 64 Previous in vitro studies could show that increased TGF-β secretion is inducible by oxidative stress in various cellular systems. 32 33 34 35 In cultured human ONH astrocytes, we have recently shown that processes of hypoxia/reoxygenation is able to induce TGF-β release into the media. 54 In the present study, our experiments revealed that hydrogen peroxide is also able to induce an increased secretion of both the total and active TGF-β2 forms. However, the role, which TGF-β2 might play in glaucomatous diseases, is still unclear. 
In the central nervous system, one way for reactive astrocytes to protect neurons from oxidative damage is the induction of Hsps. 62 In this study, exposure to hydrogen peroxide for 1 hour increased the Hsp27 expression both at the mRNA and protein level compared to untreated control cells. An oxidative stress-induced upregulation of Hsp27 expression has also been detected in other cellular systems such as vascular endothelial cells, 36 39 ventricular myocytes, 37 38 41 and corneal epithelial cells. 42 In the ocular tissue, oxidative stress-induced expression of the small Hsp αB-crystallin has been observed in human retinal pigment epithelial cells 66 and human trabecular meshwork cells. 67 Recent investigations by our group revealed that the small Hsp αB-crystallin was also inducible by processes of hypoxia/reoxygenation in human ONH astrocytes. 54 These findings may indicate that oxidative stress may be responsible, besides other factors, for increased TGF-β2 secretion 4 and elevated Hsp27 expression 24 in the ONH of glaucomatous eyes. Therefore, our in vitro results strengthen the hypothesis that oxidative stress is a key factor in the pathogenesis of glaucomatous changes. 
In our experiments, TGF-β2 induced the maximal Hsp27 mRNA expression after 48 hours, and the maximal protein level after 24 hours, compared with untreated control cells. This observation is consistent with other in vitro studies, in which Hsp27 expression is upregulated after TGF-β exposure in human reactive cerebral astrocytes, 68 human prostate cancer cells, 43 44 and mouse osteoblasts. 45 69 Previously, we showed that an increased expression of small Hsps such as αB-crystallin is inducible by TGF-β stimulation in human ocular cell types such as in trabecular meshwork cells, 59 ciliary muscle cells, 70 and ONH astrocytes. 54 In these cell types, TGF–β2-induced αB-crystallin expression at the mRNA level was not paralleled by an identical effect at the protein level. 54 59 70 A similar discrepancy between mRNA and protein expression of Hsp27 could also be observed in our experiments. Therefore, we assume that the newly transcribed Hsp27 mRNA might not be completely translated into protein, suggesting that posttranscriptional regulatory mechanisms may be operative in the regulation of the Hsp27 protein level. 
In our experiments, we were able to show that H2O2- and TGF-β2-induced Hsp27 upregulation could be attenuated by pretreatment with the selective p38MAP kinase inhibitor SB203580 in human ONH astrocytes. In contrast, pretreatment of cells with selective ERK1/2 inhibitors had no significant effects on the H2O2- and TGF-β2-induced upregulation of Hsp27. To confirm these results, we could finally demonstrate that H2O2 and TGF-β2 actually increased the phosphorylated form of p38MAP kinase. These observations are in accordance with previous studies in other cellular systems. 36 37 38 39 40 41 42 43 44 45 71 To examine whether the last major MAP kinase subfamily member, JNK, is a potent mediator for Hsp27, we have pretreated cultured ONH astrocytes with selective JNK inhibitors. However, pretreatment with SP600125 could not affect the H2O2- and TGF-β2-induced Hsp27 expression. These results are in accordance with previous studies demonstrating that Hsp27 expression is majorly mediated via p38MAP kinase, whereas JNK is rarely implicated. 72 73 Based on these results, it is assumed that p38MAP kinase is also the main mediator for Hsp27 upregulation in cultured human ONH astrocytes. 
In the central nervous system, the induction of Hsp27 in reactive astrocytes often represents the early response to stress and, initially, help to increase neuronal survival. 23 In glaucomatous optic neuropathy, as probably in other neurodegenerative diseases, chronic stress exposure may lead to persistent reactivation of astrocytes, and thus to an imbalanced defense mechanism. The knowledge about H2O2- and TGF-β2-mediated Hsp27 upregulation via the same signal transduction pathway in ONH astrocytes may suggest the possibility that H2O2 and TGF-β2 convey their stress response via the same cellular mechanisms. Further extensive studies are required to answer this hypothesis. 
In conclusion of this study, the induction of Hsp27 expression by oxidative stress or TGF-β2 may be a protective cellular response to increased stress exposure. This phenomenon has been observed in pathologic processes of various neurodegenerative diseases. 74 75 76 In ocular tissues such as in the retinal pigment epithelium, increased resistance to oxidative stress was shown to correlate with higher steady state levels of Hsp27. 77 Induction and upregulation of Hsp27 was also reported to correlate strongly with protection of retinal cells and corneal epithelial cells from stress injury. 42 78 79 Whether or not increased Hsp27 expression in human ONH astrocytes have protective effects awaits further investigations. 
In summary, we were able to show that in cultured human ONH astrocytes, both oxidative stress and TGF-β2 increased Hsp27 levels via p38MAP kinase activation. These in vitro results may provide further insights into the role of oxidative stress and TGF-β2 in glaucomatous optic nerve changes. 
 
Figure 1.
 
(A) Total and (B) active TGF-β2 protein secretion by cultured ONH astrocytes exposed to 100, 200, and 400 μM H2O2 for 1 hour. Results are given as mean ± SD of nine experiments with three different cell cultures from different donors (*P < 0.05). Co, control.
Figure 1.
 
(A) Total and (B) active TGF-β2 protein secretion by cultured ONH astrocytes exposed to 100, 200, and 400 μM H2O2 for 1 hour. Results are given as mean ± SD of nine experiments with three different cell cultures from different donors (*P < 0.05). Co, control.
Figure 2.
 
H2O2 increased the expression of Hsp27. (A) By immunohistochemistry, basal levels of Hsp27 staining were observed in untreated ONH astrocytes incubated in serum-free medium for 48 hours. Treatment with (B) 100 μM, (C) 200 μM, and (D) 400 μM H2O2 for 1 hour increased the expression of Hsp27 compared to untreated control cells. Scale bar: 100 μm. (E) Real-time PCR analysis of H2O2-induced Hsp27 mRNA expression. Results were normalized to 18S rRNA as reference. The steady state mRNA level of Hsp27 in untreated control cells was considered as 100%. Results are given as mean ± SD of nine experiments with three different cell cultures from different donors. (B) For Western blot analysis of Hsp27 protein expression, lysates containing approximately equal amounts of protein (2 μg) were separated by SDS-PAGE and blotted for immunochemical detection of Hsp27 content. Data are expressed as x-fold changes compared to untreated control cells and represent the mean ± SD of results of nine experiments with three different cell cultures from different donors (*P < 0.05).
Figure 2.
 
H2O2 increased the expression of Hsp27. (A) By immunohistochemistry, basal levels of Hsp27 staining were observed in untreated ONH astrocytes incubated in serum-free medium for 48 hours. Treatment with (B) 100 μM, (C) 200 μM, and (D) 400 μM H2O2 for 1 hour increased the expression of Hsp27 compared to untreated control cells. Scale bar: 100 μm. (E) Real-time PCR analysis of H2O2-induced Hsp27 mRNA expression. Results were normalized to 18S rRNA as reference. The steady state mRNA level of Hsp27 in untreated control cells was considered as 100%. Results are given as mean ± SD of nine experiments with three different cell cultures from different donors. (B) For Western blot analysis of Hsp27 protein expression, lysates containing approximately equal amounts of protein (2 μg) were separated by SDS-PAGE and blotted for immunochemical detection of Hsp27 content. Data are expressed as x-fold changes compared to untreated control cells and represent the mean ± SD of results of nine experiments with three different cell cultures from different donors (*P < 0.05).
Figure 3.
 
TGF-β2 increased the expression of Hsp27. (A) By immunohistochemistry, basal levels of Hsp27 staining was observed in untreated ONH astrocytes incubated in serum-free medium for 48 hours. Treatment with 1.0 ng/mL TGF-β2 for (B) 12, (C) 24, and (D) 48 hours increased the expression of Hsp27 compared to untreated control cells. Scale bar: 100 μm. (E) Real-time PCR analysis of TGF-β2-induced Hsp27 mRNA expression. Results were normalized to 18S rRNA as reference. The steady state mRNA level of Hsp27 in untreated control cells after 12 hours was considered as 100%. Results are given as mean ± SD of nine experiments with three different cell cultures from different donors. (F) For Western blot analysis of Hsp27 protein expression, lysates containing approximately equal amounts of protein (2 μg) were separated by SDS-PAGE and blotted for immunochemical detection of Hsp27 content. Data are expressed as x-fold changes compared to untreated control cells and represent the mean ± SD of results of nine experiments with three different cell cultures from different donors (*P < 0.05).
Figure 3.
 
TGF-β2 increased the expression of Hsp27. (A) By immunohistochemistry, basal levels of Hsp27 staining was observed in untreated ONH astrocytes incubated in serum-free medium for 48 hours. Treatment with 1.0 ng/mL TGF-β2 for (B) 12, (C) 24, and (D) 48 hours increased the expression of Hsp27 compared to untreated control cells. Scale bar: 100 μm. (E) Real-time PCR analysis of TGF-β2-induced Hsp27 mRNA expression. Results were normalized to 18S rRNA as reference. The steady state mRNA level of Hsp27 in untreated control cells after 12 hours was considered as 100%. Results are given as mean ± SD of nine experiments with three different cell cultures from different donors. (F) For Western blot analysis of Hsp27 protein expression, lysates containing approximately equal amounts of protein (2 μg) were separated by SDS-PAGE and blotted for immunochemical detection of Hsp27 content. Data are expressed as x-fold changes compared to untreated control cells and represent the mean ± SD of results of nine experiments with three different cell cultures from different donors (*P < 0.05).
Figure 4.
 
Effects of signal transduction protein inhibitors on the H2O2-induced Hsp27 expression. Cultured human ONH astrocytes were pretreated with 10 μM SB203580, PD98059 and U0126 for 60 minutes and then exposed to 200 μM H2O2 for 1 hour. (A) Hsp27 mRNA expression was analyzed by real-time PCR. Results were normalized to 18S rRNA as reference. The steady state mRNA level of Hsp27 in untreated control cells was considered as 100%. Results are given as mean ± SD of nine experiments with three different cell cultures from different donors. (B) For Western blot analysis of Hsp27 protein expression, lysates containing approximately equal amounts of protein (2 μg) were separated by SDS-PAGE and blotted for immunochemical detection of Hsp27 content. Data are expressed as x-fold changes compared to untreated control cells and represent the mean ± SD of results of nine experiments with three different cell cultures from different donors (*P < 0.05).
Figure 4.
 
Effects of signal transduction protein inhibitors on the H2O2-induced Hsp27 expression. Cultured human ONH astrocytes were pretreated with 10 μM SB203580, PD98059 and U0126 for 60 minutes and then exposed to 200 μM H2O2 for 1 hour. (A) Hsp27 mRNA expression was analyzed by real-time PCR. Results were normalized to 18S rRNA as reference. The steady state mRNA level of Hsp27 in untreated control cells was considered as 100%. Results are given as mean ± SD of nine experiments with three different cell cultures from different donors. (B) For Western blot analysis of Hsp27 protein expression, lysates containing approximately equal amounts of protein (2 μg) were separated by SDS-PAGE and blotted for immunochemical detection of Hsp27 content. Data are expressed as x-fold changes compared to untreated control cells and represent the mean ± SD of results of nine experiments with three different cell cultures from different donors (*P < 0.05).
Figure 5.
 
Effects of signal transduction protein inhibitors on the TGF-β2-induced Hsp27 expression. Cultured human ONH astrocytes were pretreated with 10 μM SB203580, PD98059 and U0126 for 60 minutes and then exposed to 1.0 ng/mL TGF-β2 for 24 hours. (A) Hsp27 mRNA expression was analyzed by real-time PCR. Results were normalized to 18S rRNA as reference. The steady state mRNA level of Hsp27 in untreated control cells after 24 hours was considered as 100%. Results are given as mean ± SD of nine experiments with three different cell cultures from different donors. (B) For Western blot analysis of Hsp27 protein expression, lysates containing approximately equal amounts of protein (2 μg) were separated by SDS-PAGE and blotted for immunochemical detection of Hsp27 content. Data are expressed as x-fold changes compared to untreated control cells and represent the mean ± SD of results of nine experiments with three different cell cultures from different donors (*P < 0.05).
Figure 5.
 
Effects of signal transduction protein inhibitors on the TGF-β2-induced Hsp27 expression. Cultured human ONH astrocytes were pretreated with 10 μM SB203580, PD98059 and U0126 for 60 minutes and then exposed to 1.0 ng/mL TGF-β2 for 24 hours. (A) Hsp27 mRNA expression was analyzed by real-time PCR. Results were normalized to 18S rRNA as reference. The steady state mRNA level of Hsp27 in untreated control cells after 24 hours was considered as 100%. Results are given as mean ± SD of nine experiments with three different cell cultures from different donors. (B) For Western blot analysis of Hsp27 protein expression, lysates containing approximately equal amounts of protein (2 μg) were separated by SDS-PAGE and blotted for immunochemical detection of Hsp27 content. Data are expressed as x-fold changes compared to untreated control cells and represent the mean ± SD of results of nine experiments with three different cell cultures from different donors (*P < 0.05).
Figure 6.
 
Effects of TGF-β2 and H2O2 on the expression of p38MAP kinase (p38) and phosphorylated p38MAP kinase (p-p38). (A) Western blot analysis of p38MAP kinase expression was performed after treatment of cultured human ONH astrocytes with either 1.0 ng/mL TGF-β2 for 24 hours or 200 μM H2O2 for 1 hour. Lysates containing approximately equal amounts of protein (2 μg) were separated by SDS-PAGE and blotted for immunochemical detection of p38MAP kinase content. Data are expressed as x-fold changes compared to untreated control cells and represent the mean ± SD of results of nine experiments with three different cell cultures from different donors. (B) Western blot analysis of p-p38MAPK kinase expression was performed and analyzed as described in (A) (*P < 0.05).
Figure 6.
 
Effects of TGF-β2 and H2O2 on the expression of p38MAP kinase (p38) and phosphorylated p38MAP kinase (p-p38). (A) Western blot analysis of p38MAP kinase expression was performed after treatment of cultured human ONH astrocytes with either 1.0 ng/mL TGF-β2 for 24 hours or 200 μM H2O2 for 1 hour. Lysates containing approximately equal amounts of protein (2 μg) were separated by SDS-PAGE and blotted for immunochemical detection of p38MAP kinase content. Data are expressed as x-fold changes compared to untreated control cells and represent the mean ± SD of results of nine experiments with three different cell cultures from different donors. (B) Western blot analysis of p-p38MAPK kinase expression was performed and analyzed as described in (A) (*P < 0.05).
Figure 7.
 
Effects of the JNK inhibitor SP600125 on the H2O2- and TGF-β2-induced Hsp27 expression. Cultured human ONH astrocytes were pretreated with 10 μM SP600125 for 60 minutes and then exposed to (A) 200 μM H2O2 for 1 hour or (B) 1.0 ng/mL TGF-β2 for 24 hours. Hsp27 mRNA expression was analyzed by real-time PCR. Results were normalized to 18S rRNA as reference. The steady state mRNA level of Hsp27 in untreated control cells was considered as 100%. Results are given as mean ± SD of nine experiments with three different cell cultures from different donors (*P < 0.05).
Figure 7.
 
Effects of the JNK inhibitor SP600125 on the H2O2- and TGF-β2-induced Hsp27 expression. Cultured human ONH astrocytes were pretreated with 10 μM SP600125 for 60 minutes and then exposed to (A) 200 μM H2O2 for 1 hour or (B) 1.0 ng/mL TGF-β2 for 24 hours. Hsp27 mRNA expression was analyzed by real-time PCR. Results were normalized to 18S rRNA as reference. The steady state mRNA level of Hsp27 in untreated control cells was considered as 100%. Results are given as mean ± SD of nine experiments with three different cell cultures from different donors (*P < 0.05).
The authors thank Jerome Moriniere and Katja Obholzer for excellent technical assistance. 
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Figure 1.
 
(A) Total and (B) active TGF-β2 protein secretion by cultured ONH astrocytes exposed to 100, 200, and 400 μM H2O2 for 1 hour. Results are given as mean ± SD of nine experiments with three different cell cultures from different donors (*P < 0.05). Co, control.
Figure 1.
 
(A) Total and (B) active TGF-β2 protein secretion by cultured ONH astrocytes exposed to 100, 200, and 400 μM H2O2 for 1 hour. Results are given as mean ± SD of nine experiments with three different cell cultures from different donors (*P < 0.05). Co, control.
Figure 2.
 
H2O2 increased the expression of Hsp27. (A) By immunohistochemistry, basal levels of Hsp27 staining were observed in untreated ONH astrocytes incubated in serum-free medium for 48 hours. Treatment with (B) 100 μM, (C) 200 μM, and (D) 400 μM H2O2 for 1 hour increased the expression of Hsp27 compared to untreated control cells. Scale bar: 100 μm. (E) Real-time PCR analysis of H2O2-induced Hsp27 mRNA expression. Results were normalized to 18S rRNA as reference. The steady state mRNA level of Hsp27 in untreated control cells was considered as 100%. Results are given as mean ± SD of nine experiments with three different cell cultures from different donors. (B) For Western blot analysis of Hsp27 protein expression, lysates containing approximately equal amounts of protein (2 μg) were separated by SDS-PAGE and blotted for immunochemical detection of Hsp27 content. Data are expressed as x-fold changes compared to untreated control cells and represent the mean ± SD of results of nine experiments with three different cell cultures from different donors (*P < 0.05).
Figure 2.
 
H2O2 increased the expression of Hsp27. (A) By immunohistochemistry, basal levels of Hsp27 staining were observed in untreated ONH astrocytes incubated in serum-free medium for 48 hours. Treatment with (B) 100 μM, (C) 200 μM, and (D) 400 μM H2O2 for 1 hour increased the expression of Hsp27 compared to untreated control cells. Scale bar: 100 μm. (E) Real-time PCR analysis of H2O2-induced Hsp27 mRNA expression. Results were normalized to 18S rRNA as reference. The steady state mRNA level of Hsp27 in untreated control cells was considered as 100%. Results are given as mean ± SD of nine experiments with three different cell cultures from different donors. (B) For Western blot analysis of Hsp27 protein expression, lysates containing approximately equal amounts of protein (2 μg) were separated by SDS-PAGE and blotted for immunochemical detection of Hsp27 content. Data are expressed as x-fold changes compared to untreated control cells and represent the mean ± SD of results of nine experiments with three different cell cultures from different donors (*P < 0.05).
Figure 3.
 
TGF-β2 increased the expression of Hsp27. (A) By immunohistochemistry, basal levels of Hsp27 staining was observed in untreated ONH astrocytes incubated in serum-free medium for 48 hours. Treatment with 1.0 ng/mL TGF-β2 for (B) 12, (C) 24, and (D) 48 hours increased the expression of Hsp27 compared to untreated control cells. Scale bar: 100 μm. (E) Real-time PCR analysis of TGF-β2-induced Hsp27 mRNA expression. Results were normalized to 18S rRNA as reference. The steady state mRNA level of Hsp27 in untreated control cells after 12 hours was considered as 100%. Results are given as mean ± SD of nine experiments with three different cell cultures from different donors. (F) For Western blot analysis of Hsp27 protein expression, lysates containing approximately equal amounts of protein (2 μg) were separated by SDS-PAGE and blotted for immunochemical detection of Hsp27 content. Data are expressed as x-fold changes compared to untreated control cells and represent the mean ± SD of results of nine experiments with three different cell cultures from different donors (*P < 0.05).
Figure 3.
 
TGF-β2 increased the expression of Hsp27. (A) By immunohistochemistry, basal levels of Hsp27 staining was observed in untreated ONH astrocytes incubated in serum-free medium for 48 hours. Treatment with 1.0 ng/mL TGF-β2 for (B) 12, (C) 24, and (D) 48 hours increased the expression of Hsp27 compared to untreated control cells. Scale bar: 100 μm. (E) Real-time PCR analysis of TGF-β2-induced Hsp27 mRNA expression. Results were normalized to 18S rRNA as reference. The steady state mRNA level of Hsp27 in untreated control cells after 12 hours was considered as 100%. Results are given as mean ± SD of nine experiments with three different cell cultures from different donors. (F) For Western blot analysis of Hsp27 protein expression, lysates containing approximately equal amounts of protein (2 μg) were separated by SDS-PAGE and blotted for immunochemical detection of Hsp27 content. Data are expressed as x-fold changes compared to untreated control cells and represent the mean ± SD of results of nine experiments with three different cell cultures from different donors (*P < 0.05).
Figure 4.
 
Effects of signal transduction protein inhibitors on the H2O2-induced Hsp27 expression. Cultured human ONH astrocytes were pretreated with 10 μM SB203580, PD98059 and U0126 for 60 minutes and then exposed to 200 μM H2O2 for 1 hour. (A) Hsp27 mRNA expression was analyzed by real-time PCR. Results were normalized to 18S rRNA as reference. The steady state mRNA level of Hsp27 in untreated control cells was considered as 100%. Results are given as mean ± SD of nine experiments with three different cell cultures from different donors. (B) For Western blot analysis of Hsp27 protein expression, lysates containing approximately equal amounts of protein (2 μg) were separated by SDS-PAGE and blotted for immunochemical detection of Hsp27 content. Data are expressed as x-fold changes compared to untreated control cells and represent the mean ± SD of results of nine experiments with three different cell cultures from different donors (*P < 0.05).
Figure 4.
 
Effects of signal transduction protein inhibitors on the H2O2-induced Hsp27 expression. Cultured human ONH astrocytes were pretreated with 10 μM SB203580, PD98059 and U0126 for 60 minutes and then exposed to 200 μM H2O2 for 1 hour. (A) Hsp27 mRNA expression was analyzed by real-time PCR. Results were normalized to 18S rRNA as reference. The steady state mRNA level of Hsp27 in untreated control cells was considered as 100%. Results are given as mean ± SD of nine experiments with three different cell cultures from different donors. (B) For Western blot analysis of Hsp27 protein expression, lysates containing approximately equal amounts of protein (2 μg) were separated by SDS-PAGE and blotted for immunochemical detection of Hsp27 content. Data are expressed as x-fold changes compared to untreated control cells and represent the mean ± SD of results of nine experiments with three different cell cultures from different donors (*P < 0.05).
Figure 5.
 
Effects of signal transduction protein inhibitors on the TGF-β2-induced Hsp27 expression. Cultured human ONH astrocytes were pretreated with 10 μM SB203580, PD98059 and U0126 for 60 minutes and then exposed to 1.0 ng/mL TGF-β2 for 24 hours. (A) Hsp27 mRNA expression was analyzed by real-time PCR. Results were normalized to 18S rRNA as reference. The steady state mRNA level of Hsp27 in untreated control cells after 24 hours was considered as 100%. Results are given as mean ± SD of nine experiments with three different cell cultures from different donors. (B) For Western blot analysis of Hsp27 protein expression, lysates containing approximately equal amounts of protein (2 μg) were separated by SDS-PAGE and blotted for immunochemical detection of Hsp27 content. Data are expressed as x-fold changes compared to untreated control cells and represent the mean ± SD of results of nine experiments with three different cell cultures from different donors (*P < 0.05).
Figure 5.
 
Effects of signal transduction protein inhibitors on the TGF-β2-induced Hsp27 expression. Cultured human ONH astrocytes were pretreated with 10 μM SB203580, PD98059 and U0126 for 60 minutes and then exposed to 1.0 ng/mL TGF-β2 for 24 hours. (A) Hsp27 mRNA expression was analyzed by real-time PCR. Results were normalized to 18S rRNA as reference. The steady state mRNA level of Hsp27 in untreated control cells after 24 hours was considered as 100%. Results are given as mean ± SD of nine experiments with three different cell cultures from different donors. (B) For Western blot analysis of Hsp27 protein expression, lysates containing approximately equal amounts of protein (2 μg) were separated by SDS-PAGE and blotted for immunochemical detection of Hsp27 content. Data are expressed as x-fold changes compared to untreated control cells and represent the mean ± SD of results of nine experiments with three different cell cultures from different donors (*P < 0.05).
Figure 6.
 
Effects of TGF-β2 and H2O2 on the expression of p38MAP kinase (p38) and phosphorylated p38MAP kinase (p-p38). (A) Western blot analysis of p38MAP kinase expression was performed after treatment of cultured human ONH astrocytes with either 1.0 ng/mL TGF-β2 for 24 hours or 200 μM H2O2 for 1 hour. Lysates containing approximately equal amounts of protein (2 μg) were separated by SDS-PAGE and blotted for immunochemical detection of p38MAP kinase content. Data are expressed as x-fold changes compared to untreated control cells and represent the mean ± SD of results of nine experiments with three different cell cultures from different donors. (B) Western blot analysis of p-p38MAPK kinase expression was performed and analyzed as described in (A) (*P < 0.05).
Figure 6.
 
Effects of TGF-β2 and H2O2 on the expression of p38MAP kinase (p38) and phosphorylated p38MAP kinase (p-p38). (A) Western blot analysis of p38MAP kinase expression was performed after treatment of cultured human ONH astrocytes with either 1.0 ng/mL TGF-β2 for 24 hours or 200 μM H2O2 for 1 hour. Lysates containing approximately equal amounts of protein (2 μg) were separated by SDS-PAGE and blotted for immunochemical detection of p38MAP kinase content. Data are expressed as x-fold changes compared to untreated control cells and represent the mean ± SD of results of nine experiments with three different cell cultures from different donors. (B) Western blot analysis of p-p38MAPK kinase expression was performed and analyzed as described in (A) (*P < 0.05).
Figure 7.
 
Effects of the JNK inhibitor SP600125 on the H2O2- and TGF-β2-induced Hsp27 expression. Cultured human ONH astrocytes were pretreated with 10 μM SP600125 for 60 minutes and then exposed to (A) 200 μM H2O2 for 1 hour or (B) 1.0 ng/mL TGF-β2 for 24 hours. Hsp27 mRNA expression was analyzed by real-time PCR. Results were normalized to 18S rRNA as reference. The steady state mRNA level of Hsp27 in untreated control cells was considered as 100%. Results are given as mean ± SD of nine experiments with three different cell cultures from different donors (*P < 0.05).
Figure 7.
 
Effects of the JNK inhibitor SP600125 on the H2O2- and TGF-β2-induced Hsp27 expression. Cultured human ONH astrocytes were pretreated with 10 μM SP600125 for 60 minutes and then exposed to (A) 200 μM H2O2 for 1 hour or (B) 1.0 ng/mL TGF-β2 for 24 hours. Hsp27 mRNA expression was analyzed by real-time PCR. Results were normalized to 18S rRNA as reference. The steady state mRNA level of Hsp27 in untreated control cells was considered as 100%. Results are given as mean ± SD of nine experiments with three different cell cultures from different donors (*P < 0.05).
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