Connective Tissue Growth Factor Is Increased in Pseudoexfoliation Glaucoma

Glaucoma Connective Tissue Growth Factor Is Increased in Pseudoexfoliation Glaucoma

John G. Browne,1 Su Ling Ho,2 Rosemary Kane,1 Noelynn Oliver,3 Abbot. F. Clark,4,5 Colm J. O’Brien,1,2 and John K. Crean6

PURPOSE. Pseudoexfoliation (PXF) syndrome is a generalized seudoexfoliation syndrome (PXF) is an age-related disorder disorder of the extracellular matrix (ECM) involving the trabec- Pthat manifests with abnormal fibrillar extracellular material ular meshwork (TM), associated with raised intraocular pres- (ECM) accumulation in ocular tissues.1 Fibrillar material similar sure, glaucoma, and cataract. The purposes of this study were to that in the eyes of PXF patients has more recently been to quantify aqueous humor connective tissue growth factor detected in the skin and visceral organs of patients with PXF.2 (CTGF) in PXF glaucoma, to determine the effect of CTGF on In the eye, PXF is detected by pupil dilation and subsequent slit ECM production in TM cells, and to identify intracellular CTGF lamp examination. Deposits of fibrillar material are observed in signaling pathways. the anterior segment, primarily on the pupillary border and anterior lens capsule. The disease usually has an insidious METHODS. Aqueous humor samples were obtained from patients undergoing routine cataract surgery or trabeculectomy. onset, unless complications occur such as cataract and glau- CTGF levels were quantified by ELISA. The effect of CTGF on coma. PXF is reported to be responsible for more than half of the cases of open-angle glaucoma in Norway, Ireland, Greece, fibrillin-1 expression in TM cells was investigated by real-time 3 PCR. Western immunoblot analysis was used to investigate and Saudi Arabia. Patients with PXF have a 5.3% chance of developing glaucoma within 5 years, increasing to 15.4% CTGF signaling. c-Jun/AP-1 activation was measured in CHO 4 cells by ELISA after stimulation with CTGF. within 10 years. PXF-associated secondary open-angle glaucoma is a relatively severe and progressive type of glaucoma RESULTS. PXF with glaucoma had the highest aqueous humor with a worse prognosis and more extensive complications ϭ Ϯ Ͻ level of CTGF (n 18; 5.15 0.79 ng/mL [SEM]; P 0.01) with surgery.5 Many factors contribute to the pathologic fea- ϭ Ϯ compared with PXF without glaucoma (n 15; 2.76 0.64 tures of open-angle glaucoma in PXF, including obstruction of ϭ ng/mL), primary open-angle glaucoma (POAG; n 20; the trabecular meshwork (TM) area by the deposition of pseu- Ϯ ϭ Ϯ 3.05 0.40 ng/mL), and the control (n 21; 2.60 0.29 doexfoliative material, juxtacanalicular endothelial cell dys- ng/mL). In vitro exposure of TM cells to CTGF resulted in a function, and increased aqueous humor protein levels.6 50% upregulation of fibrillin-1, which was partially blocked The protein components of pseudoexfoliative material in- with the MEK (mitogen-activated protein extracellular ki- clude both noncollagenous basement membrane components nase) inhibitor PD098059. Western blot analysis demon- such as laminin, fibronectin,7 amyloid P, and vitronectin. Also strated increased phosphorylation of p42/44 MAPK, p38 contained in the pseudoexfoliative material are proteinaceous MAPK, and the JNK pathways in response to CTGF. c-Jun/ components of elastic fibers, such as elastin, tropoelastin, fibril- AP-1 activity was significantly increased in response to CTGF lin-1, microfibril-associated glycoprotein-1, and latent TGF-␤- treatment. binding proteins (LTBP-1 and -2).8,9 The elastic microfibril CONCLUSIONS. Increased levels of CTGF in the aqueous humor of theory of PXF pathogenesis hypothesizes that PXF is a type of PXF patients likely has pathologic significance through in- elastosis characterized by excessive synthesis of elastic micro- creased production of fibrillin-1 by TM cells through activation fibrillar components throughout the body. This theory was of p42/44 MAPK, p38 MAPK, and JNK pathways. (Invest Oph- supported by molecular biological studies confirming the over- thalmol Vis Sci. 2011;52:3660–3666) DOI:10.1167/iovs.10- expression of fibrillin-1, LTBP-1, and LTBP-2 mRNA in most of 5209 the affected tissues and cell types.8,10 In 1991 Bradham et al.11 discovered human connective tissue growth factor (CTGF/CCN2) as a protein secreted by human umbilical vascular endothelial cells. CTGF is a member From the Schools of 1Medicine and Medical Science and 6Biomo- of a family of proteins with a similar structure known as the lecular and Biomedical Science, The UCD Conway Institute of Biomo- CCN family.12 Members of the CCN family have four distinct lecular and Biomedical Research, University College Dublin, Dublin, modules in their structure. These modules are an insulin-like Ireland; the 2Mater Misericordiae University Hospital, Dublin, Ireland; 3Fibrogen, Inc., South San Francisco, California; and the 4Department growth factor (IGF)-binding protein–like module (IGF-BP), von of Cell Biology and Anatomy and the 5North Texas Eye Research Willebrand factor type C repeat (VWC), thrombospondin Institute, University of North Texas Health Science Center, Fort Worth, type-1 repeat (TSP1), and C-terminal module (CT). CTGF is a Texas. 36- to 38-kDa protein rich in cysteine13 and is expressed in a Supported by the Health Research Board and Science Foundation variety of cell types, including fibroblasts, vascular smooth Ireland (JGB, JKC). muscle cells, endothelial cells, neuronal cells, and epithelial Submitted for publication January 14, 2010; revised June 8, Sep- cells.14 CTGF and transforming growth factor (TGF)-␤ interact tember 22, and November 15, 2010, and January 14, 2011; accepted via a binding site located in the amino terminal von Willebrand January 19, 2011. factor domain of CTGF.15 CTGF expression is induced by Disclosure: J.G. Browne, Fibrogen (F); S.L. Ho, Fibrogen (F); R. TGF␤, and CTGF mediates some of the downstream effects of Kane, Fibrogen (F); N. Oliver, Fibrogen (F); A.F. Clark, Fibrogen (F); 16,17 C.J. O’Brien, Fibrogen (F); J.K. Crean, Fibrogen (F) TGF␤ on proliferation, migration, and ECM production. Corresponding author: John K. Crean, University College Dublin, Pathologic fibrosis is often attributed to uncontrolled matrix Conway Institute Belfield, Dublin 4, Ireland; [email protected]. deposition, perhaps mediated by CTGF.18

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CTGF was first identified as a profibrotic mediator that was graphic information were obtained from the case notes. The patients upregulated in both in vivo and in vitro models of diabetic were diagnosed and graded with pseudoexfoliation (PXF) syndrome nephropathy,19 with a suppression subtractive hybridization based on the biomicroscopy findings, and the PXF group was divided (SSH) screen and characterized the mechanism through which into two subcategories—PXF without glaucoma and PXF with glau- elevated CTGF expression leads to matrix accumulation20 (for coma—on the basis of case notes history. The diagnosis of glaucoma review, see Ref. 21). Current models of activity propose that included cupped optic disc, documented pattern of glaucomatous CTGF largely functions as a matricellular protein, modulating visual field loss, and a history of an intraocular pressure greater than 21 and integrating the role of other growth factor signaling path- mm Hg. The POAG patients had the same clinical features as the PXF ways, including those of the TGF␤ superfamily. Signaling and glaucoma group other than having no evidence of PXF, both groups regulatory insights have remained elusive with the observation having open drainage angles on slit lamp biomicroscopy. Patients with that CTGF can bind to multiple receptors on the cell surface glaucoma were maintained on their preoperative antiglaucoma medi- and activate divergent signaling pathways, including p42/44 cations. MAPK,20 PI3K,22 Rho GTPase,23 and p38 MAPK.24 Functional interplay of these signaling networks is proposed as the key Enzyme Immunoassay for CTGF mechanism controlling CTGF-mediated gene transcription. The three isoforms of TGF␤,-␤1, -␤2, and -␤3 are found in Levels of CTGF in the aqueous humor were determined by sandwich the eye.25,26 TGF␤ has been shown in numerous studies to play ELISAs developed by Fibrogen, Inc. (South San Francisco, CA), by using a role in ocular wound healing,27,28 and its role in the patho- pairs of CTGF-specific monoclonal antibodies that specifically bind to genesis of glaucoma is also well documented.29 Several studies distinct epitopes on the N-terminal half of the CTGF protein and thus have reported elevated aqueous humor levels of TGF␤2in allows for detection of both full-length CTGF and N-terminal half patients with POAG.30,31 On the other hand, the aqueous fragments of CTGF. CTGF is unstable in vivo and proteolytic fragments humor of patients with PXF glaucoma was found to have of full-length CTGF varying from 10 to 20 kDa have been found in ␤ 9 human biological fluids (serum, cerebrospinal, amniotic, follicular, and elevated levels of TGF 1. In their study, Schlotzer-Schrehardt 34 35 et al.9,32 showed that TGF␤1, in both the latent and intrinsi- peritoneal fluids). and in porcine uterine luminal secretory fluid by Western blot analysis. Biological activities from these low-mass frag- cally active forms, was significantly raised in PXF glaucoma 36–38 eyes compared with POAG eyes. In contrast, both the latent ments have also been reported. and active forms of TGF␤2 were elevated in POAG eyes but not A range-finding experiment was initially conducted to determine 9,32 ␤ the most suitable sample dilution, as per the method previously de- in PXF glaucomatous eyes. TGF 1 had a more pronounced 33 effect on ECM production on cultured human Tenon’s capsule scribed. Samples were diluted 2.5- to 5-fold in assay buffer. Briefly, from PXF subjects, whereas TGF␤2 was more effective in microtiter plates were coated overnight at 4°C with a capture mono- promoting cell migration and collagen contraction in cultures clonal antibody directed to the N-terminal of CTGF. Wells were originating from POAG patients.29 washed and blocked with a buffer containing BSA and then rinsed. Assay samples (50 ␮L), prediluted in assay buffer, were added to each In a previous study, our group found that the CTGF level in ␮ aqueous humor of patients with PXF with glaucoma was sig- well, together with 50 L of biotinylated, N-terminal, half-detection nificantly higher than in normal control subjects undergoing CTGF monoclonal antibody solution. After the plate was incubated for 33 1.5 hours at 37°C, it was washed three times with wash buffer, and 50 cataract surgery or patients with PXF without glaucoma, ␮ raising the possibility that CTGF plays a pathogenic role. We L of streptavidin conjugated alkaline phosphatase was added to each well. The plate was reincubated for 1 hour at room temperature and wanted to expand this work to include a group of POAG ␮ patients for comparison with a PXF glaucoma group. Second, again washed three times with wash buffer. Substrate solution (100 L) we wanted to study the molecular effects of CTGF stimulation containing para-nitrophenyl phosphate was then added to each well. After 20 minutes of color development, the enzyme activity was of TM cells, looking in particular at expression of ECM com- ␮ ponents (fibrillin-1) and the intracellular signaling pathways stopped by the addition of 50 L of 4 M NaOH, and the color absor- affected by CTGF in TM cells. bance was then read at 405 nm. Cyr61 (cysteine rich 61) and Nov (nephroblastoma overexpressed) proteins are not detected in these assays, as the CTGF ELISA antibodies do not cross-react with other CCN MATERIALS AND METHODS gene family members. Purified human recombinant (rh)CTGF was used for calibration, and standard curves were generated. Controls were Patient Samples included to ensure that plate variation was Ͻ20%, and triplicate assays were performed for each sample. The CTGF values described in this Patients with the following conditions were recruited for the study: study represent the total value of both whole CTGF and N-terminal PXF (mean age, 78.5 Ϯ 5.6 [SD] years), PXF with glaucoma (mean age, fragment CTGF in nanograms per milliliter. The assay’s minimum 79.5 Ϯ 6.3 years), POAG (mean age, 73.6 Ϯ 8.8), and cataract (mean detectable concentration was determined to be 2 ng/mL. age, 73.6 Ϯ 10.9; control group). After informed consent was obtained, aqueous humor was collected from patients who were under- Cell Culture going routine cataract or trabeculectomy surgery. This study was approved by the Research Ethics Committee of the Mater Misericordiae Human TM cells were isolated from carefully dissected TM tissue Hospital and adhered to the tenets of the Declaration of Helsinki. The explants derived from nonglaucomatous (NTM) donors, as described removal of aqueous humor was performed via a clear corneal paracen- elsewhere,39 and cultured in DMEM (Invitrogen) supplemented with 2 tesis as the first intraocular maneuver at the beginning of the operation. mM L-glutamine, sodium pyruvate, 10% fetal bovine serum, 50 U/mL In each case,a1mLinsulin syringe attached to a Rycroft cannula penicillin, and 50 ␮g/mL streptomycin (passages 6–9). The three (Maersk Medical Ltd., Redditch, UK) was used to aspirate approxi- donors were a 77 year-old Caucasian man with no history of glaucoma, mately 50 to 100 ␮L aqueous humor from the anterior chamber. The a 92-year-old Caucasian woman with no history of glaucoma, and an aqueous humor sample was immediately stored at 4°C and transferred 87-year-old Caucasian man with no history of glaucoma. Cells were to Ϫ80°C freezer within 2 hours. provided at passage 6 and were generally used up to passage 9 only. A total of 74 aqueous humor samples were obtained; 39 samples The culture medium was replenished every 3 days. The cells were were from women and 35 were from men. On the day of aqueous serum starved 24 hours before treatment. CTGF was used at a concen- harvest, all patients were assessed before surgery by the same exam- tration of 25 ng/mL for the times indicated in the relevant figures. Cells iner (SLH), with slitlamp biomicroscopy, before and after mydriasis. were also preincubated in 10 ng/mL of PD098059 for 30 minutes Data on ophthalmic history, medical history, medications, and demo- before the addition of the CTGF. PD098059 is a specific p42/p44

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MAPK pathway inhibitor that binds to inactive MEK and prevents its spectrophotometer (Synergy HT; BioTek, Winooski, VT) within 5 min- phosphorylation and activation by Ras. utes at 450 nm, with a reference wavelength of 655 nm.

Western Blot Analysis Statistics Data of the immunoassays are expressed as the mean Ϯ SEM. The TM cells were serum starved for 24 hours and exposed to various group means were compared and analyzed by using Fisher’s protected agents as indicated. Lysates were harvested in RIPA lysis buffer (20 mM least-significant difference (PLSD) test and the Bonferroni/Dunn test, Tris-HCl [pH 7.4], 50 mM NaCl, 5 mM EDTA, 1% Nonidet P-40, 0.5% after one-way analysis of variance (ANOVA) identified statistically sig- sodium deoxycholate, 0.1% SDS, 5 mM NaF, 1 mM PMSF, 1 mM nificant differences (StatVew; SAS Institute, Cary, NC). P Ͻ 0.05 was Na VO ,1␮M leupeptin, and 0.3 ␮M aprotinin). The lysates were 3 4 considered to be statistically significant for Fisher’s PLSD test, and clarified by centrifugation at 14,000 rpm for 20 minutes and the likewise P Ͻ 0.01 for the Bonferroni/Dunn test. Data for the c-Jun/AP-1 samples were normalized for total protein. Protein concentration in activity assay were expressed as the normalized change ratio Ϯ SEM. the cell lysates was determined with a Bradford Assay (Bio-Rad, Hemel The groups were compared using a Student’s t-test. P Ͻ 0.05 was Hempstead, UK). For Western blot analysis, 10 ␮g of TM protein considered to be significant. extract was loaded onto each lane and separated by SDS-PAGE. The proteins were electrophoretically transferred to polyvinylidene difluo- ride (PVDF) membranes (Millipore, Watford, UK), blocked with 5% RESULTS (wt/vol) nonfat dried milk in TBST for 1 hour at room temperature, and probed overnight at 4°C with antibodies raised against ␤-actin (1: Levels of CTGF in the Aqueous of Patients with 10,000;Sigma-Aldrich), phospho p42/44 (1:1,000), total p42/44 (1: PXF Glaucoma 1,000), phospho p38 (1:1,000), total p38 (1:1,000), and phospho JNK The PXF-with-glaucoma group had the highest aqueous humor (1:1,000) all Cell Signaling Technology, Danvers, MA). Membranes mean CTGF level (5.15 Ϯ 0.79 [SEM] ng/mL), and the result were incubated with HRP-conjugated secondary Abs (1:2000) for 1 was statistically significant (Fig. 1, P Ͻ 0.01; Table 1), when hour at room temperature, and proteins were visualized by chemilu- compared with PXF without glaucoma (2.76 Ϯ 0.64 ng/mL), minescence. Unless otherwise stated, the antibodies used were sup- POAG (3.05 Ϯ 0.40 ng/mL), and controls (2.60 Ϯ 0.29 ng/mL). plied by Cell Signaling Technology. Images of developed photographic There was no significant correlation between CTGF levels and film were acquired with a CCD camera and captured with Image- age or sex within the study group, combining PXF, PXF glau- acquisition and -analysis software (Visionworks LS; UVP, Upland, CA). coma, and POAG patients (Table 1). Densitometric analysis of band intensity was performed (Image, ver. 4.0; Scion Corp., Frederick MD). Effect of CTGF on ECM Expression in TM Cells Quantitative RT-PCR Human primary TM cells of passages 6 to 9 were growth arrested in serum-free medium before the start of any experi- A real-time PCR genetic analysis assay (TaqMan; Applied Biosystems, ment, and all exposures to CTGF were performed under serum- Inc. [ABI], Foster City, CA) was used to quantify the relative gene free conditions. The cells were exposed to 25 ng/mL CTGF for expression levels of fibrillin-1. The primers and probe for fibrillin-1 6, 24, and 48 hours. RNA was extracted and cDNA was ob- (product number Hs00171191_ml) were supplied as a preoptimized tained by reverse transcription. Real-time PCR (TaqMan; ABI) single tube primer/probe (Gene Expression Assay; ABI). Probe design was performed to examine the effect of CTGF on the expres- is based on Homo sapiens FBN1 sequences NM_000138.3. The probes sion of the ECM component fibrillin-1. CTGF stimulated ap- for the target genes were labeled with the fluorescent dye, FAM proximately a 1.5-fold increase in fibrillin-1 mRNA as early as 6 (amine-reactive succinimidyl ester of carboxyfluorescein) on the 5Ј hours after treatment (Fig. 2, P Ͻ 0.05, Student’s t-test). This end and a nonfluorescent quencher on the 3Ј end. 18S rRNA was used increase was sustained at 24 hours, but had returned to basal as an endogenous control for normalization of the target genes. Its by 48 hours. Results are representative of those in three sepa- primers and probe were similarly supplied as a PDAR (predeveloped rate experiments. assay reagent) from ABI with the probe labeled with VIC at the 5Ј end and TAMRA (6-carboxytetramethylrhodamine) on the 3Ј end. PCR reactions were set up with master mix (TaqMan Universal PCR Master 16 Mix; ABI). cDNA was amplified on a sequence-detection system 14 (7900HT; ABI) at default thermal cycling conditions: 2 minutes at 50°C, 10 minutes at 95°C for enzyme activation, and then 40 cycles of 15 12 seconds at 95°C for denaturation and 1 minute at 60°C for annealing 10 and extension. The results were analyzed using the relative standard

curve/delta Ct method of analysis. 8

c-Jun/AP-1 Transcription Factor ELISA 6 CTGF Level (ng/ml) 4 The c-Jun/AP-1 activation assay from Active Motif (cat. no. 46096; Carlsbad, CA) was performed according to the manufacturer’s instruc- 2 tions, with Chinese hamster ovary (CHO) cells, as these provide a 0 readily available model for cells of a fibroblastic phenotype, and TM Controls (n=21) POAG (n=20) PXF (n=15) PXF Glaucoma (n=18) cells are not readily transfectable. Briefly, oligonucleotides correspond- Mean=2.6 Mean=3.05 Mean=2.76 Mean=5.15 ing to the consensus binding site specific for the transcription factor(s) of interest were coated on a 96-well plate, and nuclear extracts from FIGURE 1. CTGF levels in aqueous humor (ELISA). The PXF with CTGF and/or control treated cells were added to the wells. Activated glaucoma group had the highest aqueous humor mean CTGF level of 5.15 Ϯ 0.79 (SEM) ng/mL and this is statistically significant (P Ͻ 0.01) transcription factors bind the oligonucleotide sequence at its consen- when compared with PXF without glaucoma (2.76 Ϯ 0.64 ng/mL), sus site and are then quantified by ELISA; a primary antibody specific POAG (3.05 Ϯ 0.40 ng/mL), and controls (2.60 Ϯ 0.29 ng/mL). The for an epitope on the bound and active form of the transcription factor group means were compared and analyzed using Fisher’s (PLSD) test is then added followed by subsequent incubation with secondary and Bonferroni/Dunn test after one-way analysis of variance (ANOVA) antibody and developing solution. Absorbance was then read on a identified statistically significant differences.

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TABLE 1. Patient Characteristics and Mean CTGF Levels A * 1.8 Patients* Mean CTGF Characteristic n (%) (ng/mL) P 1.6 1.4 Ϯ Mean age, y 76 8.04 3.38 0.98 1.2 Sex 0.20 Male 47.3 (35) 3.77 1 Female 52.7 (39) 3.03 0.8 Fold / Basal PXF 0.002 0.6 With glaucoma 24.4 (18) 5.15 0.4 Without glaucoma 20.2 (15) 2.76 POAG 27 (20) 3.05 0.365 0.2 Control 28.4 (21) 2.60 0 6 Hour CTGF 6 Hour CTGF + PD 24 Hour CTGF 24 Hour CTGF + PD 98059 98059 * n ϭ 74. † Univariate ANOVA. B CTGF (25ng/ml) 0 10 30 180 Time (Minutes) Role of p42/44 MAPK in CTGF Induction of 50 kDa Phospho p42/44/ MAPK Fibrillin-1 in Human TM Cells 37 kDa Total p42/44 MAPK

Having demonstrated that treatment of TM cells with CTGF 50 kDa Phospho p38 MAPK resulted in upregulation of fibrillin-1, we wished to deter- 37 kDa mine which signaling pathways are involved. Preincubation Total p38 MAPK for 30 minutes with PD098059 (10 ng/mL) was used to 62.5 kDa investigate the role played by MAPK signaling in the CTGF Phospho JNK 50 kDa induction of fibrillin-1 mRNA levels (Fig. 3A). A statistically Total JNK significant downregulation of fibrillin-1 mRNA levels was observed after pretreatment with PD098059 (P Ͻ 0.05, 2.5 *** Student’s t-test). The effect of CTGF treatment on the intra- C ** cellular-mediated p42/44 MAPK pathway in TM cells was determined with phospho-specific antibodies. Lysates taken 2 * from TM cells exposed to CTGF (25 ng/mL for 0, 10, 30, and Phospho p42/44 MAPK/Total 180 minutes) resulted in time-dependent effects on p42/44 1.5 p42/44 MAPK Phospho p38 MAPK/Total p38 MAPK phosphorylation. The results showed a transient in- MAPK crease in phosphorylation of p42/P44 MAPK, reaching a Arbitrary Units 1 Phospho JNK/Total JNK maximum at 10 minutes and declining to below the back- *p<0.05 ground levels within 180 minutes. Representative Western 0.5 **p<0.01 blot and densitometric analysis of phosphorylation status ***p<0.05

(phosphorylated/total) are shown in Figures 3B and 3C. 0 Treatment of the TM cells with CTGF (25 ng/mL for 0, 10, 0 10 30 180 30, and 180 minutes) did not substantially alter total cellular Time (minutes) content of p42/44 MAPK. FIGURE 3. CTGF stimulates the expression of fibrillin-1 and the activation of p42/44 MAPK, p38 MAPK and c-Jun-N-terminal kinase The Effect of CTGF on the p38 Signaling Pathway pathways. (A) Real-time PCR for fibrillin-1 after CTGF treatment and The JNK and p38 pathways comprise other components of addition of PD098059. Real-time PCR was performed to investigate the MAPK cascade. The effect of CTGF treatment on the JNK changes in the levels of fibrillin-1 mRNA in TM cells treated with CTGF (25 ng/mL). Preincubation for 30 minutes with PD098059 (10 and p38 pathways in TM cells was determined using phos- ng/mL) was used to investigate the role played by MAP kinase signaling in CTGF-driven changes in fibrillin-1 mRNA levels (P Ͻ t n ϭ * 0.05, Student’s -test; 3). (B) Western blot analysis of p42/44 2 MAPK, p38 MAPK, and JNK pathways. TM cells were treated with 1.8 * CTGF for up to 3 hours, and the phosphorylation status of p42/44

1.6 MAPK, p38 MAPK, and JNK determined by Western blot. Results are representative of three independent experiments. (C) Densitomet- 1.4 ric analysis of Western blots. Images were acquired using a CCD 1.2 camera and captured with image-acquisition and -analysis software. Densitometric analysis of band intensity was then performed. Re- 1 sults are shown as arbitrary units Ϯ SE. n ϭ 3, *P Ͻ 0.05, **P Ͻ 0.01,

Fold / Basal Fold 0.8 ***P Ͻ 0.05; Students t-test.

0.6 0.4 pho-speciﬁc antibodies. Lysates taken from TM cells ex- 0.2 posed to CTGF (25 ng/mL for 0, 10, 30, and 180 minutes)

0 resulted in time-dependent increases in p38 and JNK phos- 0 Hour Control 6 Hour Control 6 Hour CTGF 24 Hour Control 24 Hour CTGF phorylation. There were increases in phosphorylation of both p38 and JNK, reaching a maximum at 30 minutes and FIGURE 2. Relative quantitation of ﬁbrillin-1 by real-time PCR after declining thereafter, whereas levels of total p38 MAP and CTGF treatment. TM cells were treated with CTGF (25 ng/mL) after 24 hours of serum starvation. Real-time PCR was performed to investigate JNK remained essentially unchanged. Representative West- changes in the levels of ﬁbrillin-1 mRNA in TM cells treated with CTGF ern blot and densitometric analysis of phosphorylation sta- (25 ng/mL). P Ͻ 0.05, Student’s t-test; n ϭ 3. tus (phosphorylated/total) are shown in Figures 3B and 3C.

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The Effect of CTGF on Activity of c-Jun/AP-1 in human TM cells, as evidenced by increased phosphorylation of the p42/44 MAPK. The consequences of this increase are To study the role of CTGF on the parallel activity of the JNK not clear; however, it is likely that the mechanism through target c-Jun/AP-1, CHO cells were treated with CTGF (25 ng/ which CTGF results in increased fibrillin expression in TM cells mL) for 10, 30, and 60 minutes. CTGF stimulated a 1.38-fold is similar to that of other cell types. The CTGF-induced phos- increase at 10 minutes after treatment (P Ͻ 0.05 vs. 10 minute phorylation of p42/44 MAPK was blocked by pretreating the control, Student’s t-test; Fig. 4) indicating a marked increase in cells with the MEK inhibitor PD098059. Signaling pathway transcription from the c-Jun/AP-1 promoter. These results sug- cross talk is a common feature of matricellular growth and gest that that phosphorylation of the JNK pathway down- adhesion factors, including the CNN family, of which CTGF is stream of the MAPK signaling cascade forms the basis of a the most prominent member.20 Previous work in our labora- potential intracellular signaling response to CTGF. tory suggested transactivation of the stress-activated protein kinase cascade (SAPK) by CTGF signaling through the MAPK DISCUSSION cascade.22 We investigated the phosphorylation status of the SAPK effector, c-Jun NH(2)-terminal kinase (JNKs), which Previous studies have examined the aqueous humor CTGF phosphorylates and activates the ATF transcription factor and levels in glaucoma patients compared with those in nonglau- 33 other cellular factors implicated in regulating altered gene comatous individuals. The levels of CTGF were higher in PXF expression. CTGF stimulated both phosphorylation of JNK and patients than in the controls, and the increase in CTGF was the activity of c-Jun/AP-1. related to the severity of the disease. The amount of PXF The sequence of events on binding of CTGF to multiple cell material in the juxtacanalicular tissue and the outflow filtration surface receptors remains somewhat controversial. Clearly, the area correlated with intraocular pressure and optic nerve dam- 40 activation and interplay of signaling pathways and networks age, suggesting that increased levels of CTGF are pathologi- ultimately has consequences for cell behavior and transcrip- cally significant. tional activity. The timecourse of signaling events suggests a CTGF is a potent inducer of ECM protein expression, in- 41 transient activation of pathways, perhaps reflecting the rela- cluding fibrillar and basement membrane collagens. There tively short half-life of CTGF. Studies performed by our group appears to be a direct correlation between increased expres- and others have shown that much higher concentrations of sion of CTGF and excessive accumulation and deposition of CTGF lead to prolonged ERK signaling but ultimately have no type-1 collagen in areas of fibrosis in diseased tissue from 42 bearing on outcomes such as cell migration and cell adhesion human and clinical specimens and animal models of fibrosis. or, indeed, ECM production.20,22,24,44 The apparent increase in Fibrillin-containing fibrils increase in the extracellular compo- 8 c-Jun/AP-1 activity after 10 minutes of treatment with CTGF nent in PXF, and fibrillin is a component of the elastic micro- reflects the view that transcription factor activation is regu- fibrils of the pseudoexfoliation material (PXM). Therefore, lated by these cascades, but artificially suggests that gene CTGF may increase deposition of the elastic microfibrillar com- expression would be activated at these early time points, when ponents in PXM, resulting in an excessive ECM accumulation. the experimental reality is that increased fibrillin expression To elucidate the role of CTGF in the fibrotic process in PXF in was only significantly increased at 6 hours. The addition of the vitro, we introduced rhCTGF into cultured primary human TM MEK1 inhibitor PD098059 reduced fibrillin levels to a nonsig- cells, and the induction stimulated an increase in the mRNA nificant ratio to basal at all time points and was significantly levels of fibrillin-1. Previous studies have shown increased different from CTGF treated only at 24 hours when induction extracellular deposition of fibrillin-containing fibrils in PXF, of fibrillin-1 was at maximum. Many transcription factors con- suggesting that enhanced expression of fibrillin or abnormal trol multiple genes, which are often expressed at different time aggregation of fibrillin-containing microfibrils is involved in the points and levels, differing in induction threshold and expres- pathogenesis of PXF.8 ␤ sion capacity. The key interaction between transcription fac- Previous work has demonstrated that CTGF can bind 3- tors and DNA is dependent on both DNA sequence and a integrin and activate signaling cascades that ultimately result in change in the accessibility of the DNA. It is likely that these the upregulation of expression of extracellular matrix compo- 20,43 nucleosome-mediated alterations in DNA are necessary for nents. In our study, rhCTGF activated the MAPK cascade transcription binding, and hence, availability of the active transcription factor itself is not sufficient for activation of gene expression. We currently do not clearly understand how inter- 1.8 * action between transcription factors and promoters translates 1.6 into quantitative gene expression profiles.45

1.4 Our previous ﬁndings suggested that CTGF contributes to ECM regulation in ﬁbrotic diseases via interaction with integ- 1.2 rins and subsequent recruitment and activation of Src kinase.20 1 We further characterized the divergent early signaling events 0.8 including activation of p42/44 MAPK, PI3Kinase, and p38 24

Fold Induction MAPK, and proposed that the functional interplay of these 0.6 networks is key to the regulation of CTGF-mediated gene 0.4 transcription. These in vitro studies support this hypothesis by 0.2 indicating activation of p42/44 MAPK, p38 MAPK, and JNK by CTGF in TM cells. Moreover, increased expression of fibrillin-1 0 0 Min 10 Min 10 Min 30 Min 30 Min 60 Min 60 Min in response to CTGF could be partially abrogated with the MEK Control Control CTGF Control CTGF Control CTGF inhibitor PD098059, suggesting a crucial role for p42/44 MAPK. Results of a recent study from the laboratory of Kolch FIGURE 4. c-Jun/AP-1 activation assay. c-Jun/AP-1 activity levels in 46 CHO cells was seen to increase significantly (1.38-fold) at 10 minutes (von Kriegsheim et al. ) propose a key role for growth factor– after treatment with CTGF (25 ng/mL), when compared with a time- mediated ERK signaling in the determination of cell fate deci- matched, untreated control. *P Ͻ 0.05 vs. 10-minute control (Student’s- sions, such as migration and matrix production. In these cir- test). Detection and quantification of c-Jun/AP-1 activation was per- cumstances, the consequences of ERK activation in TM cells, formed using a c-Jun/AP-1 ELISA-based assay (n ϭ 6). because of increased levels of CTGF are likely to be profound.

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In conclusion, we show raised CTGF concentrations in the 17. Kothapalli D, Grotendorst GR. CTGF modulates cell cycle progres- aqueous of PXF glaucoma patients compared with POAG, PXF sion in cAMP-arrested NRK fibroblasts. J Cell Physiol. 2000;182(1): without glaucoma, and normal cataracts. This finding raises the 119–126. intriguing possibility that stresses associated with increased 18. Hinton DR, He S, Jin ML, Barron E, Ryan SJ. Novel growth factors intraocular pressure in glaucoma in combination with PXF involved in the pathogenesis of proliferative vitreoretinopathy. result in altered pathophysiology. Given the established role Eye. 2002;16(4):422–428. for CTGF as a modulator of matrix production in many fibrotic 19. Murphy M, Godson C, Cannon S, et al. Suppression subtractive diseases, we propose a pathophysiological significance for in- hybridization identifies high glucose levels as a stimulus for ex- creased CTGF levels in PXF glaucoma. 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