|Molecular Vision 2003;
Received 7 November 2002 | Accepted 3 July 2003 | Published 3 July 2003
Distribution of myocilin, a glaucoma gene product, in human corneal fibroblasts
Kelly Wentz-Hunter, Xiang Shen,
Beatrice Y. J. T. Yue
Department of Ophthalmology and Visual Sciences, University of Illinois at Chicago College of Medicine, Chicago, IL
Correspondence to: Dr. Beatrice Yue, Department of Ophthalmology and Visual Sciences, University of Illinois at Chicago, 1855 W. Taylor Street, Chicago, IL, 60612; Phone: (312) 996-6125; FAX: (312) 996-7773; email: firstname.lastname@example.org
Purpose: Myocilin is a gene linked to open-angle glaucomas. In this study, the expression and distribution of myocilin in corneal fibroblasts with or without dexamethasone (DEX) treatment were investigated.
Methods: Human corneal fibroblasts were treated with 100 nM DEX for 10-14 days. Immunofluorescence staining for myocilin was performed. Cell lysates and ultracentrifugation fractions were assessed by western blotting for distribution of myocilin and its possible association with various organelles. Staurosporine was used to induce apoptosis and apoptotic cells were detected using a monoclonal single stranded DNA antibody.
Results: By immunofluorescence, myocilin protein was found to distribute throughout the cytoplasm of corneal fibroblasts including perinuclear regions. Myocilin distribution overlapped to varying degrees with that of the Golgi complex, endoplasmic reticulum, and mitochondria. Subsequent examination by subcellular fractionation however revealed that myocilin, while co-sedimenting with the Golgi complex, lysosomes, and endoplasmic reticulum, did not fractionate or associate with mitochondria. On western blots, protein bands at approximately 66, 57, and 55 kDa were detected and the intensity of the bands was not affected by DEX treatment in corneal fibroblasts. Apoptosis was induced by staurosporine to a similar extent in both DEX-treated and untreated corneal cultures.
Conclusions: In corneal fibroblasts, myocilin expression is not enhanced by DEX treatment and the protein was not associated with mitochondria, in contrast to what were found in human trabecular meshwork (TM) cells. Such differences suggest that the expression and distribution of myocilin may be distinctive for TM cells and may explain why pathology with myocilin mutations is only evident in glaucoma even though myocilin is expressed ubiquitously in ocular and nonocular tissues.
Glaucoma, a major cause of blindness, is characterized in general by elevation of the intraocular pressure, damage to the optic nerve head, and visual loss . The myocilin gene has been directly linked to juvenile and adult-onset open-angle glaucomas, the most common forms of glaucoma [2,3]. The trabecular meshwork (TM), a specialized chamber angle tissue, is a major site for regulation of normal bulk flow of the aqueous humor , and is believed to be responsible for the development of glaucoma.
Myocilin encoded by a glaucoma gene is also known as trabecular meshwork-inducible glucocorticoid response protein. It was initially identified in TM cell cultures after treatment with dexamethasone (DEX) as a 55 kDa protein secreted into the media [5-7]. The gene was also identified by Kubota, et al.  in the retina and was termed myocilin. The protein, which also exists typically in 57 and 66 kDa and other minor isoforms [9,10], has been shown to be expressed in the TM of normal and glaucomatous eyes [11-14], in the cornea [6,15] and a variety of other ocular and nonocular cell types [8,15-20].
In cultured TM cells and TM tissues, we and others [12,13,21-23] have localized myocilin to both intra- and extra-cellular sites. Intracellularly, myocilin has been found in the Golgi complex and endoplasmic reticulum (ER) [22,24-27]. Ultrastructural and cell biological studies [13,22] from our laboratory revealed an extensive association of myocilin with mitochondria. Extracellularly, myocilin is distributed in the extracellular matrix, capable of interacting with fibronectin and microfibrillar-associated proteins including fibrillin-1 [13,21,23]. In other ocular tissues such as the cornea, myocilin was also found intracellularly in keratocytes and epithelial and endothelial cells, and extracellularly in the corneal stroma .
While myocilin is expressed in numerous cell types besides TM, glaucoma appears to be the only disease manifested with mutations in this gene. The question was thus raised whether the distribution and/or regulation of myocilin in other cell types would be dissimilar from those in TM cells. To address this issue, we undertook the current study to determine myocilin distribution in human corneal fibroblasts. Immunofluorescence staining, western blotting, and subcellular fractionation were performed. Results from corneal cells were compared with those published earlier [7,9,22,24,28,29] for TM cells. Apoptosis in corneal fibroblasts was also examined, since in a prior study  TM cells were found to become sensitized to apoptotic challenges after DEX treatment .
Normal human corneas from donors ages 19, 23, 27, 36, 37, and two at 47 years were obtained from either the Illinois Eye Bank (Chicago, IL) or the National Disease Research Interchange (Philadelphia, PA). The procurement of tissue was approved by the IRB Committee at the University of Illinois at Chicago in compliance with the Declaration of Helsinki. The endothelial and epithelial layers were removed from the corneas and the stromas were used as explants to initiate corneal fibroblast cultures. The cells were maintained in Dulbecco's modified Eagle's minimum essential medium (MEM) supplemented with glutamine, 10% fetal calf serum, 5% calf serum, nonessential and essential amino acids, and antibiotics as previously described . For comparison purpose, parallel experiments were also performed using TM cells established  from eyes of donors aged 17, 21, 40, and 43 years.
Immunofluorescence and mitochondrial staining
First- to third-passage corneal fibroblasts were plated (2,000 cells/well) onto 8 well glass chamber slides and treated with 100 nM of DEX for 10-14 days. This treatment has been shown previously to induce myocilin expression in TM cells [5-7,22,27,30].
Cells were washed with phosphate buffered saline (PBS), fixed for 15 min in ice cold methanol, and permeabilized for 4 min in 100 mM phosphate buffer, 1 mg/ml bovine serum albumin, and 0.2% Triton X-100. After quenching the endogenous peroxidase activity with 3% H2O2, the slides were blocked for 30 min in 0.5% blocking reagent from the Tyramide Signal Amplification (TSA)-Direct Kit TSA-Direct kit (New England Nuclear Life Science, Boston, MA). The cells were incubated in primary antibody for 1 h diluted in blocking buffer. Primary antibodies used were polyclonal anti-myocilin peptide antibody (1:200), monoclonal anti-Golgi complex (1:20, BioGenex, San Ramon, CA), anti-calreticulin (1:2000 Calbiochem, San Diego, CA), and anti-β-tubulin (1:100, BioGenex). Polyclonal anti-myocilin has been characterized and described previously [21,22]. It was generated in rabbits against a synthetic polypeptide corresponding to amino acid residues 33-43 (RTAQLRKANDQ). The synthetic peptide was made, and the antibody was raised and affinity purified by Alpha Diagnostic International (San Antonio, TX). In place of primary antibody, preimmune rabbit serum, peptide-absorbed anti-myocilin, or normal mouse IgG was used as a negative control.
Subsequently, the cells were incubated for 45 min with horseradish peroxidase-conjugated goat anti-rabbit IgG (1:200, Cappel, West Chester, PA) for myocilin detection or Cy3-conjugated goat anti-mouse IgG (1:200, Jackson Immuno Research Laboratories, Inc., West Grove, PA) for monoclonal antibody detection. Cells were then incubated with the FITC-tyramide solution (1:50, TSA-Direct kit) for 5 min, washed, and mounted in Vectashield with DAPI (Vector Laboratories, Burlingame, CA). The staining was examined under either a Zeiss 100M microscope (Carl Zeiss Jena GmbH, Jena, Germany) or a Lieca TCS SP2 confocal microscope (Lieca Microsystems, Wetzler, Germany).
For mitochondrial staining, cells were incubated with 50 nM of MitoTracker Red (Molecular Probes, Portland, OR) for 30 min in complete medium prior to fixation and immunostaining .
Complete media was changed to mimimal essential media the day before collection. Media was collected and cultured corneal fibroblasts and TM cells were lysed on ice in 10 mM Tris-HCl (pH 8.0), 150 mM NaCl, 0.5% NP-40, 2 mM phenylmethylsulfonyl fluoride, and 1X cocktail protease inhibitors (Roche Molecular Biochemicals, Indianapolis, IN). Nuclei and cellular debris were pelleted, and the lysate was collected. Proteins were quantified by Bradford Protein assay. Equal amount of total protein (20 μg) from each sample or 20 μl of media was loaded and resolved on 10% sodium dodecyl sulfate (SDS)-polyacrylamide gels under reducing conditions. The proteins were electroblotted onto Protran nitrocellulose membranes (Midwest Scientific, St. Louis, MO). After blocking with 5% non-fat dry milk, the membranes were incubated with anti-myocilin (1:2000), anti-glyceraldehyde 3-phosphate dehydrogenase (G3PDH, 1:4,000; Trevigen, Gaithersburg, MD) or anti-fibronectin (1:10,000, BD Biosciences, Bedford, MA) and subsequently with horseradish peroxidase-conjugated goat anti-rabbit IgG or anti-mouse IgG (1:10,000, Cappel, Irvine, CA). Protein bands were detected using SuperSignal Substrate from Pierce (Rockford, IL). Densitometric analysis was performed to measure the intensity of myocilin and G3PDH bands in each lane with the use of a 1D Image Analysis software from Kodak Digital Imaging (Eastman Kodak Company, New Haven, CT).
Subcellular fractionation of human corneal fibroblasts
Cells grown to confluence were treated with DEX as described above. Subcellular fractionation was performed using Optiprep (Accurate Chemical and Scientific Company, Westbury, NY) and ultracentrifugation . A discontinuous gradient was prepared using 30%, 25%, 20%, 15%, and 10% Optiprep solution. The gradient was allowed to equilibrate vertically at room temperature for 30 min. Corneal fibroblasts, after washing with PBS, were harvested in homogenization buffer (0.25 M sucrose, 10 mM Hepes-NaOH, pH 7.4, 1 mM EDTA) and broken open by repeated strokes in a Dounce homogenizer. Cell debris and nuclei were pelleted by centrifugation at 1,000x g for 10 min. The post-nuclear supernatant was overlaid onto the discontinuous gradient and centrifuged at 100,000x g for 3 h at 4 °C. Equal fractions were collected from the top of the gradient. Western blot analysis as above was completed using anti-myocilin (1:2000), anti-human cytochrome C oxidase subunit II (1 μg/ml, Molecular Probes), anti-calreticulin (1:2000 Calbiochem, San Diego, CA), anti-Golgi 58K protein (1:1000, Sigma, St Loius, MO), or anti-Lamp 2 (1:5000, BD PharMingen, San Diego, CA). Horseradish peroxidase-conjugated goat anti-rabbit or mouse IgG (1:10,000, Cappel) was used as the secondary antibody and protein bands were detected with SuperSignal Substrate. For repeated probing of blots, the blots were stripped at room temperature with ImmunoPure IgG Elution Buffer (Pierce) overnight and washed extensively. The blots were treated with SuperSignal Substrate and exposed to confirm that all signals had been stripped.
Induction of apoptosis and immunostaining for ssDNA
Corneal fibroblasts plated on chamber slides (2,000 cells/well) were grown to confluence and treated with DEX as above. Both DEX-treated and untreated cells were exposed to staurosporine (0.5 μM, Sigma) for 2 h to induce apoptosis [32-34]. Cells were then fixed with 4% paraformaldehyde at 4 °C for 1 h, and were incubated with formamide for 5 min at room temperature followed by heating at 75 °C for 10 min. After washing and blocking, they were allowed to react with anti-single-stranded (ss) DNA (10 μg/ml, Alexis Biochemicals, San Diego, CA) and Cy3-conjugated goat anti-mouse IgM (1:100, Jackson Immno Research), and were mounted with Vectashield with DAPI for counterstaining of nuclei.
Total number of cells and the number of ssDNA-positive or apoptotic ones in each 20x field were determined. The percentage of apoptotic cells was calculated and a minimum of 20 fields (>100 total cells per field) was analyzed for each culture specimen. All experiments were repeated three times. Statistical analysis was performed using Student's t-test.
By immunofluorescence, modest staining for myocilin was found dispersed throughout the cytoplasm (Figure 1A), including the perinuclear regions of human corneal fibroblasts. The staining intensity in DEX-treated cells (Figure 1B) was similar to that in untreated control cells (Figure 1A). Prior incubation of anti-myocilin with the synthetic peptide used for antibody production (Figure 1C) or replacing the antibody with preimmune rabbit serum (data not shown) abrogated the staining to nonspecific background level. A parallel experiment using human TM cells showed a prominent perinuclear staining pattern in human TM cells (photographs not shown) as was reported in our earlier study . The myocilin staining in TM cells was enhanced after DEX treatment (photographs not shown).
Double labeling experiments performed indicated that in both control (Figure 2) and DEX-treated corneal fibroblasts, myocilin staining overlapped partially with Golgi complex (Figure 2C), ER (Figure 2F), and mitochondria (Figure 2I). The fibrillar staining pattern of b-tubulin (Figure 2J), a major component of microtubules, was quite different from that of myocilin.
Western blot analysis showed that lysates from both untreated control and DEX-treated corneal fibroblasts contained protein bands of approximately 66, 57, and 55 kDa molecular sizes which were immunoreactive to anti-myocilin (Figure 3A, top panel, lanes 1 and 2). The blots were reprobed for G3PDH (Figure 3A, bottom panel) for calibration of the protein loading. No difference in the ratio of myocilin/G3PDH band intensities (0.38 versus 0.37) was noted between control and DEX-treated samples. The lack of myocilin induction by DEX further confirmed the immunofluorescence data. DEX nevertheless did promote by 2.3 fold the level of fibronectin that was secreted into the culture media (Figure 3B), a well-documented DEX-mediated response of fibroblasts [35,36]. In human TM samples examined in parallel, the typical 66, 57, and 55 kDa myocilin bands (Figure 3A, top panel, lanes 3 and 4) were seen. Consistent with previous demonstrations [5-7,9,22,31,37], the myocilin level in TM cells (Figure 3A, lanes 3 and 4, myocilin/G3PDH ratio: 0.23 versus 1.30) and the secreted fibronectin in TM cultures (Figure 3B, lanes 3 and 4) were both enhanced by DEX treatment (Figure 3).
The distribution of myocilin in corneal fibroblasts was further analyzed by subcellular fractionation. Seventeen equal fractions were collected from the top of the Optiprep discontinuous gradient after ultracentrifugation. Western blot analysis was performed (Figure 4) using anti-myocilin, and organelle-specific antibodies including anti-58K protein (marker for Golgi apparatus), anti-calreticulin (marker for ER), anti-Lamp 2 (marker for lysosomes), and anti-cytochrome oxidase subunit II (marker for mitochondria). In DEX-treated cells, the Golgi complex was found mainly in fractions 4-9 while the ER had a wide range of distribution, enriched in fractions 1-3 and 7-13. The lysosomes were concentrated in fractions 3-10 and mitochondria were predominantly in the heavier fractions 10-12. The major portion of myocilin was noted in fractions 3-7, coinciding with the distribution of Golgi apparatus and lysosomes and to a lesser extent ER, but by and large not with that of mitochondria. The distribution pattern was essentially identical in control corneal fibroblasts (data not shown).
Staurosporine is a general protein kinase inhibitor that has been shown to induce apoptosis in a variety of cell types [32-34]. This agent was used to induce apoptosis in corneal fibroblasts. The apoptosis level, as detected by a monoclonal antibody for ssDNA, was approximately 4-5% in cells with or without the DEX treatment (Figure 5). The level was significantly (p<0.001) increased upon induction with staurosporine and the extent of increase was similar in the control and DEX-treated samples. Quantitative data obtained from cell counting are summarized in Table 1.
The present study demonstrates that myocilin, the product encoded by a glaucoma gene, is expressed in cultured human corneal fibroblasts. Immunofluorescence staining showed that myocilin is dispersed throughout the cytoplasm. Its staining appeared to overlap to some degree with that of Golgi complex, ER, and mitochondria. Since the overlap of staining at the light microscopic level does not necessarily indicate co-distribution of myocilin with any of the organelles, subcellular fractionation experiments were subsequently performed. The results disclosed that myocilin, while co-sedimenting at least partially with Golgi complex, lysosomes, and ER, did not fractionate together with mitochondria. Myocilin is thus likely to be present in compartments separated from mitochondria in corneal fibroblasts. This is in contrast to that found in TM cells, in which myocilin has been shown to co-fractionate and associate extensively with mitochondria [13,22].
The typical myocilin isoforms of 66, 57, and 55 kDa identified in TM cells [9,22] are also found in corneal fibroblasts. This is in accordance with a previous report  that myocilin extracted from human cornea is of identical size as full length TM myocilin. Unlike that found in TM cells [6,27,29] however, the myocilin level was comparable in fibroblasts with or without DEX treatment. It appears that DEX did not elicit any induction effect on myocilin expression in corneal fibroblasts even though the level of fibronectin secreted was enhanced as expected [35,36].
The lack of myocilin induction by DEX in corneal fibroblasts demonstrated in this current study agreed with a previous observation . The corneal fibroblasts can thus be added to a list of cell types including skin and scleral fibroblasts [24,28] and retinal pigmented epithelial cells  that do not upregulate myocilin in response to DEX. Even when myocilin is induced by DEX, such as in nonpigmented ciliary epithelial and Schlemm's canal cells, the induction level is far lower than that in TM cells [24-26,28]. These data suggest that the vast induction of myocilin by DEX is a cell type-specific property of TM cells. Furthermore, the mitochondrial association was observed only in TM cells and astrocytes in the optic nerve head [13,18,22], not in corneal fibroblasts. The regulation and distribution of myocilin may therefore be distinctive in TM cells.
In a prior study, we showed that when TM cells were exposed to DEX, the cells became sensitized to apoptotic challenges . We hypothesized that this vulnerability may be the key for the eventual development of glaucoma. The increased susceptibility to secondary challenges observed after DEX treatment may be related to either the induction of myocilin or the mitochondrial connection. Lacking myocilin induction and with little mitochondrial association, it was not surprising to discover in this study that in corneal fibroblasts, treatment with staurosporine did not cause further apoptosis in DEX-treated cultures. This finding is once again consistent with the notion that myocilin regulation, distribution, and/or function(s) may be TM cell specific. The specificity may explain why no other ocular or systemic abnormalities except glaucoma is manifested in patients with mutations of myocilin, despite its ubiquitous distribution throughout ocular and nonocular tissues .
The authors would like to thank E. Lillian Cheng for maintenance and treatment of cell cultures, and Kira Lathrop and Ruth Zelkha for expert imaging. National Eye Institute EY 05628, EY 03890, and EY 01792; Senior Investigator Award, Research to Prevent Blindness, Inc., New York, NY.
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