Molecular Cloning of the Bovine MYOC and Induction of Its Expression in Trabecular Meshwork Cells

Abstract

purpose. Myocilin gene (MYOC) was identified as one of the disease-causing genes of primary open-angle glaucoma. This study was conducted to establish a system for the investigation of the biological role of MYOC in vitro by using bovine eyes, which are easy to obtain and have been widely used to examine the aqueous outflow system. The cDNA sequence of the bovine MYOC was determined and its expression in bovine eyes was examined with a quantitative polymerase chain reaction (PCR) assay.

methods. Bovine MYOC cDNA was obtained from cultured bovine trabecular meshwork cells, and part of its sequence was determined using a primer pair designed based on the known sequence of the human MYOC gene. The 3′ and 5′ ends of this sequence were determined using the method of 3′ and 5′ rapid amplification of cDNA ends. The induction of the MYOC gene in cultured bovine trabecular meshwork cells after exposure to dexamethasone was quantitatively examined with real-time quantitative PCR using a probe designed according to the sequence of the determined bovine MYOC gene.

results. Bovine MYOC protein was composed of 490 amino acids, which was 81.6% identical with that of human MYOC protein. Most of the amino acid residues of which mutation was reported to cause glaucoma were conserved in the bovine MYOC protein. After 2 weeks of treatment with 500 nM dexamethasone, expression of bovine MYOC mRNA was amplified 14-fold (14.1 ± 5.1-fold, mean ± SEM) measured by real-time quantitative PCR.

conclusions. The cDNA sequence of the bovine MYOC gene had a high degree of similarity to that of the human MYOC gene. Investigation of the function of bovine MYOC may contribute to identifying the role of MYOC protein in the aqueous outflow system.

In 1997 Stone et al. ¹ reported that mutations in the trabecular-meshwork inducible glucocorticoid response (TIGR) gene, later designated as the myocilin gene (MYOC), cause primary open-angle glaucoma in 4.4% of patients with familial glaucoma and 2.9% of unselected patients with glaucoma. Since then, many disease-causing mutations of the MYOC gene have been reported. However, the role of the MYOC protein remains unknown. This protein is amplified by stimulation with glucocorticoids in vivo, but no patients with steriod responder glaucoma reportedly carry the mutation in the coding region of this gene. Morissette et al. ² reported that only heterozygotic individuals with a mutation of this gene manifest glaucoma, whereas homozygotic individuals were asymptomatic. ²  

The expression of MYOC mRNA or protein has been investigated in human and mouse eyes and in extraocular tissues. It has been reported that the MYOC protein is expressed in the trabecular meshwork (TM) and in Schlemm’s canal, leading to the hypothesis that the MYOC protein obstructs the aqueous outflow and elevates intraocular pressure (IOP). ^{3 4} However, human or mouse TM tissue is rather difficult to obtain, and experiments in vitro are limited in these species. By contrast, bovine eyes are easy to obtain for TM cell culture. Bovine eyes have been used as a model to study the aqueous outflow system and the regulation of IOP. ^{5 6 7} To facilitate the functional study of the MYOC protein and the roles of the mutated form of the protein in the aqueous outflow system, it is important to determine the sequence of bovine MYOC and to clarify the characteristics of its product. We cloned the sequence of bovine MYOC and subsequently investigated its expression using quantitative polymerase chain reaction (PCR). 

Materials and Methods

Cell Culture

Bovine TM cells were established in tissue culture according to the methods described by Grierson et al. ⁸ Three-year-old bovine eyes were obtained from an abattoir. Trabecular tissue was dissected carefully and placed in Dulbecco’s modified Eagle’s medium (Gibco, Grand Island, NY) with 15% fetal bovine serum and 50 μg/ml gentamicin (Gibco). Within 1 to 2 weeks of incubation at 37°C under a 5% CO₂ atmosphere, TM cells migrated from the tissue explant. Then the explant was removed, and cells were passaged with trypsin-EDTA (Gibco). The cells grew to form a monolayer TM cell sheet consisting of a homogenous population of trabecular cells. 

Isolation and Cloning of the Bovine MYOC Gene

Total RNA was isolated from the cultured bovine TM cells by using Isogen (Nippon Gene, Toyama, Japan). Reverse transcription of 10 μg RNA was performed with Superscript II (Gibco). A primer pair was designed based on the sequence of human MYOC. The sense primer had the sequence 5-′AGCACGGGTGCTGTGGTGTAC-3′ (which corresponds to nucleotides 970–990 of the human MYOC gene) and the antisense primer 5′-AAGGTGCCACAGATGATGAA-3′ (nucleotides 1288–1307). cDNA was amplified using the PCR method with this primer pair under the condition of 40 cycles of 30 seconds at 94°C, 30 seconds at 61°C, and 1 minute at 72°C. PCR products were subcloned using TA vectors (Invitrogen, San Diego, CA), and the sequences of the obtained clones were determined using fluorescent dideoxynucleotides on an automated sequencer (model 310; Applied Biosystems, Foster City, CA). Nested PCR was performed with a primer pair 5′-CGGGGCAGCCTCTACTTCCA-3′ (nucleotides 991–1010) and 5′-GCCGCCTCGGTGCTGTAGAT-3′ (nucleotides 1171–1190) that was designed based on the sequence of human MYOC. A DNA fragment of 200 bp was obtained, the sequence of which had a high degree of similarity (80.9%) to the nucleotide sequence of human MYOC. The 3′ and 5′ ends of this sequence were determined using the method of 3′ and 5′ rapid amplification of cDNA ends (RACE) using a 5′/3′ RACE kit (Boehringer Mannheim, Mannheim, Germany). 

Induction of MYOC mRNA with Corticosteroid and Analysis of Its Expression in Cultured Bovine TM Cells by RT-PCR

Third- or fourth-passage bovine TM cells were used for reverse transcribed–polymerase chain reaction (RT-PCR). After the cells grew to confluence, they were exposed to 50 to 500 nM dexamethasone for 2 weeks. The cells were collected, and total RNA was isolated using Isogen (Nippon Gene). The extracted total RNA was treated with RNase-free DNase I (Stratagene, La Jolla, CA) for 30 minutes. RT of 10 μg RNA was performed using Superscript r2 (Gibco) and T₁₂VN primer. 

The obtained cDNA samples were standardized according to the expression level of internal control GAPDH (glyceraldehyde 3-phosphate dehydrogenase) mRNA. One nanogram of cDNA sample was amplified in a buffer containing 1.5 mM MgCl₂ (Perkin–Elmer, Oceanport, NJ), 0.2 mM of each dNTP (Perkin Elmer), 0.5% DNA polymerase (AmpliTaq Gold; Perkin Elmer), and 0.2 μM of primer pair 5′-GCTCACTGGCATGGCC-3′ and 5′-CAGGTCAGATCCACAACAGACA-3′. The conditions for PCR were as follows: an initial 10-minute denaturing step at 95°C and 37 cycles of 15 seconds at 95°C, 1 minute at 55°C, and 1 minute at 72°C. The PCR products were examined by 1% agarose gel electrophoresis with ethidium bromide staining. According to the expression level of GAPDH mRNA, the amount of sample template was normalized, and the cDNA of each sample was amplified to examine the level of MYOC mRNA expression with the same PCR condition as used for GAPDH and the primer pair 5′-CAACATCCGTAAGCAGTCCGTC-3′ and 5′-CATAGGCGAAGTTGACAGTGGC-3′. 

Quantitative analysis was also performed to examine the expression of MYOC mRNA using the real-time quantitative RT-PCR method described by Heid et al. ⁹ A hybridization probe was labeled with a reporter dye, FAM (6-carboxyfluorescein), on the 5′ nucleotide and a quenching dye, TAMRA (6-carboxy-tetramethyl-rhodamine), on the 3′ nucleotide. When the probe is intact, the reporter dye emission is quenched. During the extension phase of the PCR cycle, the DNA polymerase cleaves the hybridization probe and releases the reporter dye from the probe. The increase in fluorescence emission of the reporter dye is proportional to the amount of the PCR product, which in turn is proportional to the beginning target concentration. Fluorescence intensity was monitored and analyzed on a sequence detector (model 7700; Applied Biosystems). The expression level of GAPDH mRNA was used as an internal control. 

PCR was performed in buffer containing 1.5 mM MgCl₂ (Perkin–Elmer) with 1 to 10 ng cDNA samples, 0.2 mM each dNTP, 0.2 μM probe (5′-CCGCGTCCCCACTCCCAAC-3′ for GAPDH and 5′-CAACGCCTTCATCATCTGTGGCACCT-3′ for MYOC), 0.2 μM of each primer pair (5′-GCTCACTGGCATGGCC-3′ and 5′-CAGGTCAGATCCACAACAGACA-3′ for GAPDH and 5′-CAACATCCGTAAGCAGTCCGTC-3′ and 5′-CATAGGCGAAGTTGACAGTGGC-3′ for MYOC), and 0.5% DNA polymerase (AmpliTaq Gold; Perkin–Elmer). The conditions for PCR were as follows: an initial 10-minute denaturing step at 95°C and 40 cycles of 15 seconds at 95°C, and 1 minute at 55°C. 

Results

Cloning of the Bovine MYOC Gene

The nucleotides and deduced amino acid sequences of the bovine, human, rat and mouse MYOC cDNA are shown in Figure 1 . The bovine MYOC protein is composed of 490 amino acids, which is 14 amino acids shorter at the N terminus than that of the human and is the same size as that of the mouse. Bovine MYOC protein also contains a leucine zipper motif ranging from 103 to 152 aa, like human MYOC protein. Bovine MYOC protein is hydrophilic and has a partial hydrophobic region between amino acids 1 and 18 and 412 and 426. Human and mouse MYOC protein have an N-linked glycosylation site (human: 57–59 aa and mouse: 43–45 aa), but bovine MYOC protein does not have this site. Most of the amino acid sequences in which change was reported to be responsible for the development of glaucoma are conserved in bovine MYOC except for the Ala445Val and 396INS mutations. 

The homology of the bovine MYOC gene to human, rat, and mouse MYOC at the amino acid level is 81.6%, 78.8%, and 78.2%, respectively, and 83.1%, 80.2%, and 80.6% at the nucleotide level. (The nucleotide sequence data reported in this article has been submitted to DDBJ/EMBL/GenBank nucleotide sequence databases with the accession number AB027758.) 

Induction of MYOC mRNA Expression by Dexamethasone

Bovine TM cells were exposed to 50 to 500 nM dexamethasone for 2 weeks, and the expression of MYOC mRNA was estimated. Figure 2 shows that MYOC mRNA was induced by 50 and 500 nM dexamethasone, with the latter inducing more abundant MYOC mRNA. Quantitative data from five independent cell lines are shown in Figure 3 . After 2 weeks of treatment with 50 nM dexamethasone, bovine MYOC mRNA expression was amplified approximately 7-fold (7.1 ± 2.5-fold, mean ± SEM), and that treated with 500 nM dexamethasone was amplified 14-fold (14.1 ± 5.1-fold), compared with that of the untreated control MYOC gene. 

Discussion

Bovine MYOC cDNA encodes a protein of 490 amino acids with a leucine zipper motif. Most of the sequence variants that change amino acids in patients with glaucoma are located in exon 3, and bovine MYOC is well conserved in the portion that corresponds to the exon 3 of human MYOC. (The amino acids of exons 1, 2, and 3 of human MYOC are 75.9%, 71.4%, and 87.4% identical with that of bovine MYOC.) These data suggest that the amino acid sequences encoded by exon 3 may play important roles in MYOC gene function. The leucine zipper motif is conserved between species. This leads to the hypothesis that the MYOC protein may function as a dimer or oligomer, and the mutated molecules may inhibit the formation of the MYOC complex. This interpretation could account for the fact that homozygotes with the Lys423Glu mutation of this gene do not manifest glaucoma. ²  

With the use of dot-blot analysis, it has been reported that human MYOC mRNA was progressively increased after exposure to glucocorticoids from insignificant amounts in the untreated control to 0.5%, 0.75%, 1.75%, and 2.7% of total cellular mRNA after 1, 4, 7, and 10 days in cultured human TM cells. ¹⁰ Our study quantitatively showed that MYOC mRNA expression was induced with glucocorticoid in bovine TM cells, with similar time course and dose dependency. In general, the expression level of mRNA and that of the protein are correlative, and it is known that human MYOC is induced by glucocorticoid, both at the mRNA level and at the protein level. However, the antibody against bovine MYOC was not available in our laboratory, and we could not estimate the expression of bovine MYOC protein in bovine eyes. 

Primary open-angle glaucoma is a chronic and progressive disease that threatens the sight of otherwise healthy people. Little is known about the pathogenesis of the disease, and the method of treatment has not been completely established. Elucidation of the function of MYOC may give an important clue to the regulatory mechanism of IOP and may help to clarify the pathogenesis of glaucoma. 

Bovine eyes are useful, because they could be readily and freshly available for primary TM cell culture. The bovine angle chamber has a structure similar to that of the human. It consists of a trabecular zone adjacent to the ciliary cleft and nontrabecular connective tissue deeper within the outflow tissues. The aqueous humor passes through these structures into the adjacent vessels, which is considered to be the functional equivalent of Schlemm’s canal. This outflow tissue of the bovine eye has often been used as a model system to investigate the mechanism of the glaucoma development. Studies that have been reported using an anterior segment perfusion system or cultured TM cells of bovine eyes include the investigation of the outflow facility, the morphologic or synthetic characteristics of TM cells, and effects of the reagents, such as dexamethasone, prostaglandins, adrenergic agents, and bradykinins, on the regulation of the outflow facility. ^{5 6 7} An investigative system using bovine eyes would be a useful model to facilitate the study of MYOC.  

This is the first report to show the full sequence of the bovine MYOC gene and its inducible characteristics with corticosteroids. Further investigation is needed to clarify the function of both normal and mutated forms of the MYOC protein in the regulation of IOP and in the development of glaucoma. 

 Submitted for publication August 4, 1999; revised January 7, 2000; accepted January 31, 2000.

 Commercial relationships policy: N.

 

Figure 1.

 

(A) The nucleotides and deduced amino acid sequence of bovine MYOC cDNA. The bovine MYOC protein is composed of 490 amino acids. The leucine zipper motif is underlined. The mutant residues reported to be responsible for the development of glaucoma are presented in bold above the wild-type amino acid residues. Most of these mutations occur at amino acids that are conserved between bovine and human MYOC, except for the Ala445Val and 396INS mutations. (B) The comparison of the predicted amino acid sequences of bovine, human, rat, and mouse MYOC proteins. Dots indicate the same amino acid residues as bovine MYOC.

Figure 2.

 

Induction of MYOC mRNA expression in bovine TM cells examined by 1% agarose gel electrophoresis. The amount of MYOC mRNA was examined after the PCR templates of each sample were normalized according to the level of GAPDH mRNA. With ethidium bromide staining, the density of the bands gradually amplified as PCR progressed. MYOC mRNA was induced more with 500 nM dexamethasone than with 50 nM dexamethasone.

Figure 3.

 

Quantitative analysis of MYOC mRNA expression in bovine TM cells. The relative MYOC mRNA expression levels were calculated dividing the MYOC mRNA levels by the GAPDH mRNA levels measured in the same RNA preparation. Values are means ± SEM. MYOC mRNA was markedly amplified after the TM cells were exposed to dexamethasone. (*P = 0.08, †P = 0.016; Wilcoxon rank sum test.)

The authors thank Kaori Suzuki for technical assistance.