Effects of TGF-␤2, BMP-4, and in the Trabecular Meshwork: Implications for Glaucoma

Robert J. Wordinger,1,2 Debra L. Fleenor,2,3 Peggy E. Hellberg,3 Iok-Hou Pang,1,3 Tara O. Tovar,1 Gulab S. Zode,1 John A. Fuller,1 and Abbot F. Clark1,3

PURPOSE. The primary causative factor of primary open-angle CONCLUSIONS. These results are consistent with the hypothesis glaucoma (POAG) is elevated intraocular pressure (IOP) due to that, in POAG, elevated expression of Gremlin by TM cells increased aqueous humor (AH) outflow resistance, which is inhibits BMP-4 antagonism of TGF-␤2 and leads to increased associated with morphologic and biochemical changes in the ECM deposition and elevated IOP. (Invest Ophthalmol Vis Sci. trabecular meshwork (TM). Patients with glaucoma have ele- 2007;48:1191–1200) DOI:10.1167/iovs.06-0296 vated levels of transforming (TGF)-␤2 in their ␤ AH, and TGF- has been shown to increase TM extracellular laucoma is a major cause of irreversible blindness, affect- matrix (ECM) production. The bone morphogenetic protein Ging more than 70 million individuals worldwide.1 Elevated (BMP) signaling pathway modifies TGF-␤ signaling in several intraocular pressure (IOP) is a major risk factor in the devel- different tissues, and a prior study demonstrated that TM cells opment of glaucoma2 and in the progression of glaucomatous and tissues express members of the BMP family. The damage.3 Elevated IOP is due to increased aqueous humor purpose of this study was to determine whether BMPs can alter (AH) outflow resistance4 and appears to be associated with TGF-␤2 signaling in the TM and whether there are defects in several morphologic and biochemical changes in the trabecu- BMP signaling in glaucoma. lar meshwork (TM). Very little is understood about the regula- METHODS. ELISA, Western immunoblot analysis, and immuno- tion of TM function, including the pathogenic causes of in- histochemistry were used to evaluate the expression of BMP creased outflow resistance in the glaucomatous TM. However, there is an accumulation of extracellular matrix (ECM) material proteins in TM cells and tissues. ELISA was used to determine 4,5 the effects of TGF-␤2 and BMPs on TM fibronectin (FN) secre- in the glaucomatous TM, and this increase may be due to tion. was determined by gene microarrays disruption of the normal balance between ECM deposition and and quantitative (q)PCR. Perfusion-cultured human anterior degradation. segments were used to study the effects of altered BMP signal- Growth factors present in the AH bathing the TM could shift ing on IOP. this balance, resulting in the accumulation of ECM, increased outflow resistance, and, ultimately, elevated IOP. For example, RESULTS. The human TM synthesized and secreted BMP-4 as recent reports indicate that transforming growth factor well as expressed BMP receptor subtypes BMPRI and BMPRII. (TGF)-␤2 is involved in the pathogenesis of primary open-angle TM cells responded to exogenous BMP-4 by phosphorylating glaucoma (POAG).6 Initially, TGF-␤2 was reported to be ele- Smad signaling proteins. Cultured human TM cells treated with vated in the AH of eyes with POAG.7–9 These initial reports TGF-␤2 significantly increased FN levels, and BMP-4 blocked were followed by studies demonstrating that TGF-␤2 acts via this FN induction. The expression of BMP family in multiple pathways within the human TM, resulting in in- normal and glaucomatous TM cells was profiled and significant creased ECM deposition and cross-linking that impede degra- elevation of mRNA and protein levels of the BMP antagonist dation. For example, exogenous TGF-␤2 (1) increases in vitro gremlin were found in glaucomatous TM cells. In addition, synthesis of ECM molecules in cultured TM cells10 and within Gremlin was present in human aqueous humor and in the the perfused anterior eye organ culture model,11 (2) increases perfusate medium of perfusion-cultured human eyes. Gremlin expression of plasminogen activator inhibitor (PAI)-1 and pre- blocked the negative effect of BMP-4 on TGF-␤-induction of FN. vents activation of matrix metalloproteinases via tissue plas- Recombinant Gremlin added to the medium of ex vivo perfu- minogen activator (tPA) and/or urokinase (uPA),12 (3) in- sion-cultured human eye anterior segments caused the glau- creases irreversible cross-linking of ECM components by TM coma phenotype of elevated IOP. cells via tissue transglutaminase,10 and (4) inhibits TM cell proliferation.13 However, within a given tissue, the actions of most growth factors are often counterbalanced by other growth factors, so 1 From the Department of Cell Biology and Genetics, University of that, normally, only small spatial and temporal changes occur North Texas Health Science Center at Fort Worth, Fort Worth, Texas; 3 in structure and function. The bone morphogenetic proteins and Glaucoma Research, Alcon Research, Ltd., Fort Worth, Texas. ␤ 2Contributed equally to the work and therefore should be consid- (BMP) are members of the TGF- superfamily of growth fac- tors. Originally identified as osteoinductive that pro- ered equivalent authors. 14 Supported by Alcon Research, Ltd. mote bone and cartilage formation, BMPs are now known to 15 Submitted for publication March 19, 2006; revised September 4, control multiple functions in a variety of cell types. A com- 2006; accepted January 10, 2007. bination of intracellular and extracellular antagonists tightly Disclosure: R.J. Wordinger, Alcon Research, Ltd. (F); D.L. control the biological activity of BMPs. Recently, a group of Fleenor, Alcon Research, Ltd. (E); P.E. Hellberg, Alcon Research, Ltd. unique but structurally related secreted BMP antagonists have (E); Iok-Hou Pang, Alcon Research, Ltd. (E); T.O. Tovar, None; G.S. been identified.16 Examples of secreted BMP antagonists in- Zode, None; J.A. Fuller, None; A.F. Clark, Alcon Research, Ltd. (E) clude , , , and members of the DAN The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertise- (differential screening-selected gene and members aberrative ment” in accordance with 18 U.S.C. §1734 solely to indicate this fact. in neuroblastoma) family including , caronte, and Corresponding author: Abbot F. Clark, Glaucoma Research R2-41, Drm/Gremlin (down-regulated by mos; CKTSF1B1). The mech- Alcon Research, Ltd., 6201 South Freeway, Fort Worth, TX 76134; anism of inhibition appears to be direct binding to BMP by [email protected]. these antagonists, thus preventing BMP from interacting with

Investigative Ophthalmology & Visual Science, March 2007, Vol. 48, No. 3 Copyright © Association for Research in Vision and Ophthalmology 1191

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ing BMPs and that BMP-4 selectively counteracts the effect of TGF-␤2 in TM cells with respect to ECM-related proteins. It appears that BMP-4 plays a significant role in maintaining the normal function of the TM by modifying the action of TGF-␤2. In addition, we demonstrate that the BMP antagonist Gremlin inhibits BMP-4 activity in cultured TM cells and increases out- flow resistance in a perfusion cultured human eye anterior segment model. Significantly, we demonstrate that levels of both Gremlin mRNA and protein are elevated in glaucomatous human TM cell lines. We propose that, in POAG, elevated Gremlin expression by TM cells inhibits BMP-4 regulation of TGF-␤2 effects, leading to increased ECM deposition and ele- vated IOP.

MATERIALS AND METHODS

TM Cell Culture Human TM cells were isolated from carefully dissected human TM tissue explants derived from patients with glaucoma (GTM) or nonglaucomatous donors (NTM) and characterized as previously 13,21–26 FIGURE 1. Western blot analysis of BMP-4 secreted by human TM described. All donor tissues were obtained and managed cells. Three NTM cell lines were grown in serum-free DMEM for 48 according to the guidelines in the Declaration of Helsinki for re- hours, and the culture medium was analyzed for BMP protein expres- search involving human tissue. Glaucoma donor history consisted of sion by Western immunoblot analysis. a diagnosis of open-angle glaucoma, glaucoma therapy, and docu- mented glaucomatous visual field defects. The culture and charac- 15 terization of a transformed TM cell line (GTM-3) has been de- the receptor complex. Drm/Gremlin is a member of the 27 DAN/cerberus family of BMP antagonists and is a highly con- scribed. Isolated TM cells were grown in Dulbecco’s modified served 20.7-kDa glycoprotein.17 The Drm gene was isolated Eagle’s medium (DMEM; Invitrogen-Gibco, Grand Island, NY) con- ␮ during a screening of a transformation-resistant v-mos-trans- taining 10% fetal bovine serum (HyClone, Logan, UT) and 50 g/mL formed rat fibroblast.18 The Xenopus homologue to drm was gentamicin (Invitrogen-Gibco). We chose to perform most of our designated gremlin.17 Gremlin heterodimerizes with BMP-2, -4, experiments in serum-free medium, to allow us to dissect the and -7 to prevent functional activity. Previous reports suggest independent effects of TGF-␤2, BMP-4, and Gremlin. We have that BMP antagonists likely play an important role in regulating shown that TM cells make and respond to a variety of growth multiple cell functions both during early development and in factors,13 and that serum greatly complicates the interpretation of adult tissues.19 results because of the presence of binding proteins, proteinases, We have shown that human TM cells express BMPs, BMP and various levels of growth factors (including TGF-␤). Also, TM receptors and mRNA for selective BMP antagonists.20 The cells are grown in FBS to allow them to proliferate, but they are in present study demonstrates that TM cells are capable of secret- low serum (low protein) conditions in situ.

FIGURE 2. Immunohistochemical lo- calization of BMP receptors in human TM tissue. Normal human eyes were fixed, sectioned, and stained with anti- bodies for BMP receptors Ia, Ib, and II. Slides incubated in PBS-BSA without primary antibody or with nonimmune control IgG were used as negative con- trols. Human TM tissues stained posi- tive for BMPR-Ia, BMPR-Ib, and BMPR-II, indicating that in vivo TM tis- sues express BMP receptors.

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from normal and glaucomatous human TM cells after a 48-hour treat- ment with serum-free medium containing 0.5 mg/mL BSA. Proteins were separated on a 12% denaturing polyacrylamide gel and trans- ferred by electrophoresis to a nitrocellulose membrane. Nonspecific binding was blocked by soaking membranes in 1ϫ TBS, 5% powdered milk, and 0.05% Tween-20 for at least 15 minutes at room temperature. Mouse anti-BMP-4 monoclonal primary antibody (Mab757, 1 ␮g/mL; R&D Systems) was used. The secondary antibody consisted of goat anti-mouse polyclonal antibody (sc-2005; Santa Cruz Biotechnology, Santa Cruz, CA). Cell Lysates. Cell lysates were used for both phosphorylated and nonphosphorylated Smad signaling proteins. Confluent normal TM and glaucomatous TM cells (2 ϫ 107) were collected in 1.0 mL of mam- malian protein extraction buffer (78501) and protease inhibitor cock- tail (78415; both from Pierce Biotechnology, Inc., Rockford, IL). Pro- tein concentration was determined by the Bradford method. Lysates (40–60 ␮g) were mixed with an equal volume of electrophoresis buffer and boiled for 90 seconds. Proteins were separated on a 12%

FIGURE 3. Western blot analysis of Smad proteins in normal and glauco- matous human TM cells. Proteins from two NTM and two GTM cell lysates were separated by polyacrylamide gel electrophoresis followed by West- ern immunoblot analysis. Both NTM and GTM cells expressed intermedi- ates of the canonical Smad signaling pathway, including receptor Smad1 and Smad5, and Co-Smad4. ␤-Actin was included as a loading control.

Immunohistochemistry of BMP Receptors in TM Tissues Three sets of normal and three sets of glaucomatous human donor eyes were obtained from regional eye banks within 6 hours of death and fixed in 10% formalin. Fixed tissues were dehydrated, embedded in paraffin, and cut in 8-␮m sagittal sections that were placed on micro- scope slides (Probe On Plus; Fisher Scientific, Hampton, NH). Sections were deparaffinized and rehydrated before placement in 0.1% Triton, followed by 0.02 M glycine for 15 minutes each. Nonspecific staining was blocked by a 30-minute incubation in 10% normal serum. Sections were washed briefly and treated with primary anti-BMP-receptor anti- bodies (R&D Systems, Inc., Minneapolis, MN) or nonimmune serum (negative controls) diluted 1:100 in 1.5% normal serum for 1 hour at room temperature. These antibodies were previously characterized by Western blot analysis of TM cells and tissues.20 After three washes in PBS, sections were incubated with appropriate secondary antibodies (Alexa Fluor 488; Invitrogen-Molecular Probes, Carlsbad, CA) for 45 minutes. Sections were treated with 4Ј,6Ј-diamino-2-phenylindole (DAPI) nuclear stain, washed, and mounted. FIGURE 4. Effect of exogenous BMP-4 on Smad protein phosphoryla- tion. Human TM cells were exposed to exogenous BMP-4 (10 ng/mL) Western Blot Analysis for various times (0, 5, 15, 30, or 60 minutes, or 24 hours) and compared to the vehicle control. Total and phosphorylated protein for Secreted BMP. BMP secretion by TM cell lines was determined pSmad1, total Smad5, and pSmad1-5-8 was measured by Western blot. by Western immunoblot analysis. Conditioned medium was collected ␤-Actin was included as a loading control.

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denaturing polyacrylamide gel and transferred by electrophoresis to a on fibronectin production using a commercially available ELISA kit nitrocellulose membrane. Nonspecific binding was blocked by soaking (AssayMax; AssayPro LLC, Winfield, MO). We had already demon- membranes in 1ϫ TBS, 5% powdered milk, and 0.05% Tween-20 for at strated that treatment of cultured human TM cells with TGF-␤2 signif- least 15 minutes at room temperature. Membranes were incubated icantly increases fibronectin levels in the culture medium.28 with the following primary antibodies for 45 minutes: nonphosphory- lated Smad1 (9512 at 1:500; Technology, Beverly, MA), Smad4 (MAB1132 at 1 ␮g/mL; Chemicon International, Temecula, CA), Gene Analysis of Normal and Glaucomatous Smad 5 (51-3700 at 1:1000; Zymed, San Francisco, CA), Smad6 (51- Trabecular Meshwork Cells ␮ 0900 at 1 g/mL; Zymed); phosphorylated pSmad1 (566411 at 1 Total RNA was isolated from four normal TM cell lines and four mg/mL; Calbiochem, La Jolla, CA), pSmad1-5-8 (AB3848 at 1:500; glaucomatous TM cell lines. Aliquots of normal TM RNA and glauco- ␮ Chemicon International); and Gremlin (AP6133a at 0.5 g/mL; Abgent, matous TM RNA were each pooled. Total RNA from normal and San Diego, CA). After the membranes were washed three times with glaucomatous TM cells were reverse transcribed, and second-strand TBS plus 0.05% Tween-20 for 30 minutes, they were incubated with cDNA synthesis and biotin-labeled amplified RNA were performed horseradish peroxidase (HRP)-conjugated secondary goat anti-rabbit according to standard procedures. gene chips antibody (2030; Santa Cruz Biotechnology) diluted 1:500 to 1:5000, (U133Plus2; Affymetrix Inc., Santa Clara, CA) were hybridized, followed by three washes with TBS plus 0.05% Tween-20 and one washed, and scanned (Gene Array scanner; Agilent Technologies, wash in TBS alone. Detection was performed with chemiluminescence Englewood, CO), and raw data were collected and analyzed (Microar- detection reagents (ECL; GE Healthcare, Piscataway, NJ). Blots were ray Suite; Affymetrix). Only genes flagged as “present” on the gene exposed to autoradiograph film (Hyperfilm-ECL; GE Healthcare) for 1 chip were analyzed. minute to 1 hour, depending on the amount of target protein present. Electrophoresis buffer alone and neutralization of primary antibodies by control peptides served as negative controls. Primary antibodies Quantitative PCR were neutralized with a 10-fold (by weight) excess of control peptide Real-time PCR was performed (Mx3000P Real-Time System; Stratagene in PBS overnight at 4°C. Western blot analysis for each protein was La Jolla, CA) with PCR master mix (SYBR Green; Stratagene). Each repeated twice, to confirm the results. reaction contained 12.5 ␮Lof2ϫ master mix, 500 nM Gremlin forward Gremlin Protein Expression and reverse primers, and 2.5 ng cDNA from well-established, normal (n ϭ 5) or glaucomatous (n ϭ 6) TM cell lines. PCR primers were Gremlin protein expression was determined in TM cell lysates, TM designed with the Primer3 design program (Whitehead Institute; MIT, tissue lysates, human AH (obtained after informed consent from nor- Cambridge, MA) to span an exon–intron boundary and tested negative mal patients undergoing cataract surgery), and perfusate medium from for genomic DNA amplification with the in silico PCR program (Uni- perfusion-cultured human eyes by Western immunoblot analysis, as versity of California, Santa Cruz): Gremlin 5Ј: GTCACACTCAACTGC- just described. CCTGA, Gremlin 3Ј: ATGCAACGACACTGCTTCAC, product size: 77 bp; and TBP 5Ј: GAAACGCCGAATATAATCCCA, TBP 3Ј: GCTG- ELISA Assay for Fibronectin GAAAACCCAACTTCTG, product size: 181 bp. Cultured normal and glaucomatous human TM cells were treated with Cycle threshold (Ct) values were normalized to the housekeeper commercially available recombinant BMP-4 (0–100 ng/mL; 314-BP; TATA binding protein (TBP) and were analyzed (MxPro software; R&D Systems) and/or TGF-␤2 (5 ng/mL; Sigma-Aldrich, St. Louis, MO) Stratagene). Standard curves for Gremlin and TBP were Ͼ0.994. Com- for 24 hours. Conditioned medium was evaluated for BMP’s effects parative quantification was performed based on the ⌬⌬Ct method.

TABLE 1. Effect of BMP4, TGF-␤2, and Gremlin on TM Cell Fibronectin

Fibronectin (␮g/well) Donor Age/ Culture Cell Strain Gender Method Vehicle BMP4 TGF␤2 TGF␤2 ؉ BMP4 TGF␤2 ؉ BMP4 ؉ Gremlin

GTM-3 72 y/F Explant 2.4 Ϯ 0.4 3.2 Ϯ 0.4 17.7 Ϯ 1.8* 5.1 Ϯ 0.6 18.6 Ϯ 1.3* n ϭ 59 n ϭ 43 n ϭ 59 n ϭ 47 n ϭ 9 NTM-35D 6 mo/M Explant 3.2 Ϯ 1.8 2.1 Ϯ 1.2 13.0 Ϯ 2.7* 7.6 Ϯ 3.0 NT n ϭ 7 n ϭ 7 n ϭ 7 n ϭ 7 NTM25D-91 2 y/F Explant 3.1 Ϯ 0.4 1.8 Ϯ 0.2 19.3 Ϯ 1.5* 4.0 Ϯ 0.2 NT n ϭ 4 n ϭ 4 n ϭ 4 n ϭ 4 NTM553-02 87 y/F Explant 1.4 Ϯ 0.2 1.7 Ϯ 0.2 11.6 Ϯ 1.0* 3.0 Ϯ 0.4* 3.0 Ϯ 0.1 n ϭ 11 n ϭ 11 n ϭ 11 n ϭ 11 n ϭ 3 NTM875-03 77 y/M Explant 0.7 Ϯ 0.3 1.1 Ϯ 0.5 13.3 Ϯ 4.7* 1.0 Ϯ 0.4 0.7 Ϯ 0.4 n ϭ 7 n ϭ 7 n ϭ 7 n ϭ 7 n ϭ 3 NTM974-03 87 y/M Explant 3.9 Ϯ 1.1 3.9 Ϯ 1.2 8.9 Ϯ 1.5* 2.6 Ϯ 0.4 9.8 Ϯ 2.3* n ϭ 9 n ϭ 9 n ϭ 9 n ϭ 9 n ϭ 9 GTM29A-01 72 y/F Explant 1.4 Ϯ 0.2 1.1 Ϯ 0.2 12.8 Ϯ 2.7* 2.7 Ϯ 0.4* 5.3 Ϯ 0.2* n ϭ 7 n ϭ 7 n ϭ 7 n ϭ 7 n ϭ 3 GTM686-03 61 y/F Explant 0.22 Ϯ 0.02 0.26 Ϯ 0.02 25.2 Ϯ 9.6* 0.16 Ϯ 0.02 NT n ϭ 3 n ϭ 3 n ϭ 3 n ϭ 3 GTM730-03 88 y/M Explant 0.8 Ϯ 0.1 1.0 Ϯ 0.1 26.2 Ϯ 1.6* 6.2 Ϯ 0.6* NT n ϭ 4 n ϭ 4 n ϭ 4 n ϭ 4 SGTM1233-99 78 y/M Enzymatic 4.5 Ϯ 1.2 5.8 Ϯ 1.6 14.0 Ϯ 2.9* 2.0 Ϯ 0.4 12.1 Ϯ 4.1* n ϭ 6 n ϭ 6 n ϭ 6 n ϭ 6 n ϭ 6 SGTM2697 72 y/M Enzymatic 8.4 Ϯ 1.5 7.4 Ϯ 1.8 21.0 Ϯ 2.1 14.1 Ϯ 5.6 NT n ϭ 8 n ϭ 8 n ϭ 8 n ϭ 8

Concentrations of compounds used: TGF-␤2; 5 ng/mL; BMP4, 10 ng/mL; gremlin, 10 ␮g/mL. NT, not tested. * P Ͻ 0.05 vs. vehicle control group by one-way ANOVA with the Dunnett test.

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Statistical analysis was performed using an unpaired two-tailed Stu- proteins were present in both normal and glaucomatous hu- dent’s t-test assuming unequal variances. man TM cells (Fig. 3). A major difference in protein levels was not apparent between normal and glaucomatous TM cell lines. Human Eye Anterior Segment Perfusion Organ These data indicate that human TM cells should be capable of Culture Model responding to BMPs via the Smad signaling pathway. Human ocular perfusion organ culture was performed as de- scribed.24,25,29–33 Human donor eyes (with an average age of 81.9 Ϯ Effect of Exogenous BMP-4 on Phosphorylated 10.9 years; age range, 56–96 years), none of which were known to Receptor Smad Proteins Expressed in Human have glaucoma, were obtained at 16 to 20 hours after death and TM Cells dissected at the equator, and the iris, lens, most of the ciliary body, and We had shown that TM cells express BMP receptors and vitreous were removed. The anterior segment of the eye, including contain Smad signaling proteins, so we next sought to deter- cornea and sclera ring containing the TM, was placed in a custom-made mine whether exogenous BMP activates Smad signaling in Plexiglas culture dish and sealed in place with a Plexiglas O-ring. cultured TM cells. Binding of BMPs to the BMPRI/BMPRII DMEM was perfused through a central cannula in the bottom of the receptor complex initiated downstream signaling via phos- ␮ dish at a flow rate of 2.5 L/min with a perfusion pump (Harvard phorylation of receptor Smads. Treating human TM cells with Apparatus, South Natick, MA). The IOP was monitored via a second BMP-4 increased phosphorylated Smad1-5-8 protein (Fig. 4), cannula attached to a pressure transducer (model P23XL; GE Health- with protein levels that were increased by 60 minutes and then care). The IOP was recorded every 5 minutes, and hourly averages declined over 24 hours. Therefore, human TM cells are capable were calculated.

The anterior segment, cultured at 37°C in 5% CO2, was allowed to equilibrate for 2 to 4 days before the start of the study. Tissues that did not reach a stable IOP baseline were discarded (ϳ40% of the eyes). Acceptable tissues were perfused with medium containing recombi- nant mouse Gremlin (10 ␮g/mL; R&D Systems) for 2 to 4 days. At the end of each study, the tissues were perfusion fixed at 15 mm Hg constant pressure, dissected into four quadrants, and processed for light microscopy and transmission electron microscopy, as previously described.24,30–33 The viability of the outflow pathway tissue, espe- cially the TM, was evaluated in a masked fashion. Studies were re- garded as invalid and the data discarded if more than one quadrant per eye had unacceptable morphologic findings such as: excessive TM cell loss, denudation of trabecular beams, an excess in cellular debris in the TM region, loss of Schlemm’s canal endothelial cells, or breaks in the Schlemm’s canal inner wall lining. Based on these criteria, none of the perfused tissues were rejected in the study.

RESULTS Western Blot Analysis of Secreted BMP-4 by Human TM Cells In order for BMPs to counterbalance the effects of TGF-␤2 within the human TM, secretion of BMPs by TM cells would be required. Normal TM cells were cultured for 48 hours in serum-free medium, and the medium was concentrated 10-fold before analysis. Western immunoblot analysis showed that normal TM cell lines secrete BMP-4 (Fig. 1). Immunohistochemical Localization of BMP Receptors in Human TM Tissue We examined the expression of BMP receptor subtypes BMPRIa, BMPRIb, and BMPRII in three normal and three glau- comatous TM tissues from human donors. Each of the three BMP receptors was present in normal and glaucomatous hu- man TM tissue, and there were no obvious differences be- tween normal and glaucomatous donor eyes (Fig. 2). Human TM cells should therefore be capable of responding to BMPs in IGURE Effect of BMP-4 on TGF-␤2 stimulation of FN content in the AH (e.g., ) and/or to BMPs secreted F 5. culture medium of normal and glaucomatous human TM cell lines. (A) directly by TM itself (e.g., autocrine signaling), because they Two NTM and three GTM cell lines were treated for 24 hours, and FN express BMP receptors. levels in the culture medium were assayed by ELISA. TGF-␤2 alone (5 ng/mL) caused a significant increase in FN secretion by all cell lines (P Western Blot Analysis of Smad Proteins in Ͻ 0.05). BMP-4 alone (10 ng/mL) had no significant effect on FN Human TM Cells secretion. However, a combination of TGF-␤2 and BMP-4 caused a significant reduction in TGF-␤2-stimulated FN secretion (P Ͻ 0.05). The canonical downstream signaling pathway for BMPs utilizes Mean Ϯ SEM; P determined by one-way ANOVA with the Dunnett test. intracellular Smad proteins, so we examined the expression of (B) GTM3 cells were exposed to various concentrations of BMP-4 receptor Smad (R-Smad1, R-Smad5) and co-Smad (Co-Smad4) (pg/mL) in combination with TGF-␤2 (5 ng/mL). BMP-4 dose-depen- proteins in human TM cells. R-Smad1, R-Smad5, and Co-Smad4 dently inhibited TGF-␤2 stimulation of FN secretion by these cells.

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of responding to BMPs via phosphorylation of receptor Smads, Expression of Gremlin mRNA and Protein in indicating that the canonical Smad signaling pathway can be Glaucomatous Human TM Cell Lines activated via exogenous BMPs. We have shown that human TM cells and tissues express the mRNA of four BMPs, all three BMP receptors, and several BMP Effect of BMP-4 on TGF-␤2 Stimulation of antagonists, including Gremlin.20 To examine whether defects Fibronectin Secretion by Normal and in BMP signaling could be responsible for the elevated IOP Glaucomatous Human TM Cell Lines associated with glaucoma, we compared the expression of genes involved in BMP signaling pathways in normal and glau- ECM metabolism in the TM is altered by TGF-␤.11,12 We have comatous TM cells. Messenger RNA from four normal and four shown that TGF-␤2 increased fibronectin (FN) content in the glaucomatous TM cell lines was pooled, and gene expression medium of cultured TM cells.28 Previous studies have demon- was profiled by using gene chips (Affymetrix). Gremlin was strated that BMPs can modify TGF-␤ signaling pathways,34,35 expressed 16-fold higher in the pooled glaucomatous TM cell and so we tested whether BMP-4 regulates TGF-␤2-induced FN lines in comparison to the pooled normal TM cell lines. In- secretion by TM cells. Exogenous TGF-␤2 (5 ng/mL) increased creased expression of Gremlin in glaucomatous TM cells was FN secretion in both normal and glaucomatous TM cell lines confirmed by quantitative (q)PCR. Gremlin mRNA expression (Table 1; Fig. 5A). Overall, exposure of TM cell lines to exog- was 11.6-fold higher in GTM than in NTM cells (P ϭ 0.05). Cell enous BMP-4 (10 ng/mL) alone did not alter FN secretion when lysates from both normal and glaucomatous cell lines were compared with vehicle control. However, BMP-4 significantly examined for Gremlin protein via Western immunoblot analy- reduced the TGF-␤2-stimulated secretion of FN (Table 1, Fig. sis to confirm these Gremlin mRNA results. Gremlin was 5A). This effect of BMP-4 on TGF-␤2-induced fibronectin secre- present as a doublet at approximately 25 to 28 kDa (Fig. 6B) as tion was dose dependent (Fig. 5B). previously described.18 Gremlin protein levels were signifi-

FIGURE 6. Expression of Gremlin mRNA and protein in normal and glaucomatous human TM cells. (A) qPCR of Gremlin mRNA in NTM (n ϭ 5) and GTM (n ϭ 6) cells. Gremlin mRNA expression was increased 11.6-fold in GTM cells (P ϭ 0.05). (B) Western blot analysis for Gremlin in four NTM and five GTM cell lines. (C) GTM cells had significantly elevated levels of Gremlin protein (P ϭ 0.029). (D) Gremlin Western immunoblot analysis of human AH samples (AQ), human TM tissue, and perfusate medium for perfusion organ-cultured human eyes (POC).

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cantly higher in glaucomatous TM cell lines compared with the on dose–response studies in cultured TM cells. Continuous normal TM cell lines (P ϭ 0.029; Figs. 6B, 6C). exposure to Gremlin for 4 days caused a significant increase in IOP compared to vehicle-treated contralateral eyes (n ϭ 6, P Ͻ Expression of Gremlin in TM Tissue, Human AH, 0.05; Fig. 8A). In a second study, perfusion with Gremlin also and Perfusates of Cultured Human significantly elevated IOP compared with contralateral control Anterior Segments eyes (P Ͻ 0.05, n ϭ 5), and this IOP elevation was reversed by removal of Gremlin from the perfusion medium after 2 days To determine whether Gremlin is also expressed in vivo, Grem- (Fig. 8B). lin Western immunoblots were performed on lysates of TM tissue, human AH, and perfusate medium from perfusion-cul- tured human anterior segments (Fig. 6D). Gremlin was present as a doublet in AH and perfusate medium (Fig. 6D), similar to DISCUSSION that in cultured TM cell lysates (Fig. 6B). However, the lysate of TM tissue appeared to have several additional bands, including Glaucomatous elevated IOP is caused by increased AH outflow a major higher-molecular-weight band, which may be a pro- resistance4 and is closely associated with morphologic and form of Gremlin. biochemical changes that occur within the TM. For example, in patients with glaucoma, there is an accumulation of ECM Gremlin Antagonized the BMP-4 Inhibition of proteins in the TM.4,5 Little is known about the cellular control TGF␤-2-Stimulated Fibronectin Secretion by of ECM synthesis, secretion, and degradation within the human Human TM Cells TM. However, growth factors in AH or produced locally by TM Increased expression of Gremlin in glaucomatous TM cells cells may be of great importance, in that we have reported that human TM cells express numerous growth factor receptors could possibly antagonize the positive effect of BMP-4 in block- 13 ing TGF-␤2 induction of FN in TM cells. We evaluated the and respond to exogenous growth factors. Thus, the accu- effect of Gremlin on the regulation of FN in TM cells by TGF-␤2 mulation of ECM proteins in the human TM may be directly and BMP-4. TGF-␤2 caused a threefold increase in fibronectin controlled by growth factors. ␤ secretion when compared with untreated TM cells (P Ͻ 0.05), Members of the TGF- superfamily of growth factors appear ␤ to be involved in the pathogenesis of glaucoma.6 TGF-␤2 levels whereas a combination of TGF- 2 and BMP-4 inhibited fi- 7–9,36 bronectin secretion (P Ͻ 0.05; Fig. 7, Table 1). Gremlin antag- are elevated in AH of patients with POAG, and exogenous TGF-␤2 increases ECM protein synthesis in cultured TM onized the inhibitory action of BMP-4 on fibronectin secretion, 10,28 restoring FN levels to those of TGF-␤2 alone. cells and in perfusion-cultured anterior eye organ cul- tures.11 In addition, increased cross-linking of TM ECM pro- Gremlin Elevated IOP in the Perfused Anterior teins by tissue transglutaminase and increased expression of 10,12 Eye Organ Model PAI-1 are correlated with exogenous TGF-␤2 treatment. These reports indicate that TGF-␤2 is an important mediator of Elevated Gremlin levels in the glaucomatous TM may be re- the synthesis, secretion, and degradation of ECM within the sponsible for the ocular hypertension associated with glau- human TM. However, within a given tissue, the action of most coma. To test this hypothesis, we studied the effect of Gremlin growth factors is often counterbalanced by other growth fac- in an ex vivo model of perfusion-cultured human eye anterior tors so that normally, only small changes in tissue structure and segments. Ten micrograms Gremlin per milliliter was added to function occur. Our data indicate that BMP-4 may counteract the perfusion medium based on the reported ED50 for Gremlin the effect of TGF-␤2 with respect to metabolism of ECM pro- to inhibit BMP-4 activity in cultured cells (R&D Systems) and teins within the human TM. Several BMPs and their receptors are expressed and play a significant role in ocular development.37,38 Knockout studies have shown that BMP-4 and -7 are essential in the early mor- phogenesis of the eye.39–43 A recent report by Chang et al.44 showed that a heterozygous deficiency of BMP-4 results in anterior segment dysgenesis and elevated IOP, implicating the BMP pathway in glaucoma’s pathogenesis. The abnormalities were similar to those in patients with developmental glau- coma. Thus BMP-4 may be involved in developmental condi- tions associated with human glaucoma. Although BMPs, BMP receptors, and BMP antagonists have been reported to have significant roles in ocular development, their role in normal or diseased adult tissues is not well understood. Our laboratory has shown that human TM cells express BMPs, BMP receptors, and mRNA for several BMP antago- nists.20 In this study, we demonstrated that TM cells secrete BMP-4 and are capable of responding to exogenous BMP via the canonical Smad signaling pathway. We further demon- strated that BMP-4 inhibits the stimulation of FN secretion by TGF-␤2. It thus appears that BMP-4 may act normally to coun- FIGURE 7. Effect of Gremlin on BMP-4 inhibition of TGF-␤2 induced teract the effects of TGF-␤2 on ECM proteins in the human TM. FN secretion by human TM cells. GTM-3 cells were treated for 24 hours ␤ ␤ ␤ Inhibition of TGF- signaling via BMP may not be a unique with TGF- 2 alone (5 ng/mL); TGF- 2 and BMP-4 (1 ng/mL); or TGF- feature of the human TM. TGF-␤ is known to be involved in the ␤2, BMP-4, and Gremlin (10 ␮g/mL). Untreated cells served as a initiation and progression of renal disease, including renal control. FN levels were determined by ELISA immunoassay. BMP-4 34 significantly inhibited TGF-␤2 stimulation of FN (*P Ͻ 0.05 vs. vehicle fibrosis. Zeisberg et al. demonstrated that BMP-7 can reverse control group by one-way ANOVA with the Dunnett test). The addition the epithelial-to-mesenchymal transition by directly counteract- of Gremlin blocked the effect of BMP-4. ing TGF-␤-induced cell signaling. In a subsequent report,35

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FIGURE 8. Effect of Gremlin on IOP in the perfused anterior segment or- gan culture model. Anterior seg- ments from paired human donor eyes were placed in an ex vivo per- fusion organ culture system. (A) One anterior segment from each pair was perfused with recombinant Gremlin (10 ␮g/mL perfusion medium) for 4 days. Gremlin caused a significant in- crease in IOP (n ϭ 6; *P Ͻ 0.05). (B) One anterior segment from each pair was perfused with recombinant Gremlin (10 ␮g/mL perfusion me- dium) for 2 days followed by 2 days of perfusion with medium without Gremlin. Gremlin also significantly raised IOP when perfused for 2 days, and this effect was reversible on re- moval of Gremlin from the medium (n ϭ 5; *P Ͻ 0.05 compared with vehicle-treated contralateral eyes).

they indicated that direct counteraction between TGF-␤ and expression or BMP signaling may contribute to the develop- BMP activation was unique, because it did not appear to in- ment of elevated IOP and glaucoma. We profiled gene expres- volve either extracellular or intracellular BMP antagonists. sion of members of the BMP signaling gene family in normal Smad5-mediated BMP-7 signaling directly counteracted Smad3- and glaucomatous TM cells and found significantly increased dependent TGF-␤ induced epithelial-mesenchymal transition in expression of the BMP antagonist Gremlin in the glaucomatous kidney tubules and mammary epithelial cells. In addition, TM cells. A statistically significant increase in both Gremlin Wang and Hirschberg45 demonstrated that BMP-7 inhibits mRNA and protein expression was shown by qPCR and West- TGF-␤ driven fibrogenesis by mesangial cells. Inhibition of ern immunoblot analyses. We also showed that Gremlin was fibrogenesis occurred primarily by preventing TGF-␤-depen- present in vivo in TM tissue as well as in human AH and dent downregulation of matrix degradation and upregulation perfusate medium from perfusion-cultured human eyes. Grem- of PAI-1. Finally, Izumi et al.46 reported that BMP-7 opposes lin is a secreted BMP antagonist that binds BMPs and prevents TGF-␤1-mediated collagen induction in mouse pulmonary myo- their interaction with the BMP receptor complex, thus reduc- fibroblasts. They demonstrated that BMP-7 suppresses TGF-␤ ing BMP biological activity.18 Gremlin was able to block the action via the upregulation of Id2. Although BMP-4 caused positive effects of BMP-4, which suppressed the TGF-␤2-induc- phosphorylation of Smad proteins in the TM, we do not yet tion of fibronectin in cultured TM cells. Even more important, know whether the BMP-4 effect on TGF-␤2-induction of fi- the addition of recombinant Gremlin to the perfusate of ex bronectin in TM cells is mediated through the canonical BMP vivo cultured human eyes caused the glaucomatous phenotype Smad signaling pathway. BMP also has been shown to signal via of elevated IOP, which was reversed on removal of Gremlin Smad-independent pathways, including MAP kinases.47–48 from the perfusate. We did not introduce exogenous BMP-4 or If BMP-4 acts to counterbalance the effect of TGF-␤2 and TGF-␤2 to the perfusion medium of the ex vivo perfusion regulate normal TM function, then any alteration in BMP-4 organ-cultured eyes, yet Gremlin still was able to increase IOP.

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Acknowledgments

The authors thank Paula Billman for acquisition of the donor eyes, Mitch McCartney and Karen Stropki for technical assistance on assess- ment of TM tissue viability in the perfusion culture experiments, and Robin Chambers and Debbie Lane for providing the cultured TM cells used in the study.

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