Analysis of porcine optineurin and myocilin expression in trabecular meshwork cells and astrocytes from optic nerve head

Minoru Obazawa, Yukihiko Mashima, Naoko Sanuki, Setsuko Noda, Jun Kudo, Nobuyoshi Shimizu, Yoshihisa Oguchi, Yasuhiko Tanaka, Takeshi Iwata

Research output: Contribution to journalArticle

17 Citations (Scopus)

Abstract

PURPOSE. To determine the cDNA sequences and analyze the expression of porcine optineurin and myocilin in trabecular meshwork cells (TMCs) and astrocytes from the optic nerve head under normal and experimental conditions. METHODS. Both porcine optineurin and myocilin were cloned to determine the cDNA sequences. Porcine TMCs and astrocytes were isolated and treated with dexamethasone (500 nM) for 2 weeks, incubated under hypoxic conditions (7% O2) for 72 hours, or exposed to 33 mm Hg hydrostatic pressure for 72 hours. A 10% mechanical stretch for 24 hours was also performed on TMCs. The expression level of the optineurin and myocilin transcripts was analyzed by real-time quantitative PCR. RESULTS. The sequences of porcine optineurin and myocilin cDNA were determined, and the expression of both genes was confirmed in both TMCs and astrocytes. Amino acid sequences of porcine optineurin and myocilin were homologous to those of humans by 84% and 82%, respectively, and shared protein motifs and modification sites. The expression of myocilin mRNA by TMCs and astrocytes was increased by 8.0- and 5.5-fold, respectively, after exposure to dexamethasone. In contrast, the expression of optineurin was suppressed to 68% in TMCs and 48% in astrocytes after exposure to dexamethasone. A significant reduction of myocilin expression was observed after 72 hours of incubation under hypoxic conditions in both types of cells, whereas optineurin was not affected. Hydrostatic pressure for 72 hours and mechanical stretching for 24 hours had minimal affects on gene expression of both optineurin and myocilin. CONCLUSIONS. The high homology of porcine optineurin and myocilin to the comparable human genes indicates that pigs can be used to study changes in gene expression in hypertensive eyes. The alterations in expression of myocilin but not of optineurin under stress suggest that different mechanisms in the phenotype of glaucoma associated with the two genes are involved in development of glaucoma.

Original languageEnglish
Pages (from-to)2652-2659
Number of pages8
JournalInvestigative Ophthalmology and Visual Science
Volume45
Issue number8
DOIs
Publication statusPublished - 2004 Aug

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Trabecular Meshwork
Optic Disk
Astrocytes
Swine
Dexamethasone
Hydrostatic Pressure
Complementary DNA
Gene Expression
Glaucoma
trabecular meshwork-induced glucocorticoid response protein
Amino Acid Motifs
Genes
Sequence Analysis
Real-Time Polymerase Chain Reaction
Amino Acid Sequence
Phenotype

ASJC Scopus subject areas

  • Ophthalmology

Cite this

Analysis of porcine optineurin and myocilin expression in trabecular meshwork cells and astrocytes from optic nerve head. / Obazawa, Minoru; Mashima, Yukihiko; Sanuki, Naoko; Noda, Setsuko; Kudo, Jun; Shimizu, Nobuyoshi; Oguchi, Yoshihisa; Tanaka, Yasuhiko; Iwata, Takeshi.

In: Investigative Ophthalmology and Visual Science, Vol. 45, No. 8, 08.2004, p. 2652-2659.

Research output: Contribution to journalArticle

Obazawa, M, Mashima, Y, Sanuki, N, Noda, S, Kudo, J, Shimizu, N, Oguchi, Y, Tanaka, Y & Iwata, T 2004, 'Analysis of porcine optineurin and myocilin expression in trabecular meshwork cells and astrocytes from optic nerve head', Investigative Ophthalmology and Visual Science, vol. 45, no. 8, pp. 2652-2659. https://doi.org/10.1167/iovs.03-0572
Obazawa, Minoru ; Mashima, Yukihiko ; Sanuki, Naoko ; Noda, Setsuko ; Kudo, Jun ; Shimizu, Nobuyoshi ; Oguchi, Yoshihisa ; Tanaka, Yasuhiko ; Iwata, Takeshi. / Analysis of porcine optineurin and myocilin expression in trabecular meshwork cells and astrocytes from optic nerve head. In: Investigative Ophthalmology and Visual Science. 2004 ; Vol. 45, No. 8. pp. 2652-2659.
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title = "Analysis of porcine optineurin and myocilin expression in trabecular meshwork cells and astrocytes from optic nerve head",
abstract = "PURPOSE. To determine the cDNA sequences and analyze the expression of porcine optineurin and myocilin in trabecular meshwork cells (TMCs) and astrocytes from the optic nerve head under normal and experimental conditions. METHODS. Both porcine optineurin and myocilin were cloned to determine the cDNA sequences. Porcine TMCs and astrocytes were isolated and treated with dexamethasone (500 nM) for 2 weeks, incubated under hypoxic conditions (7{\%} O2) for 72 hours, or exposed to 33 mm Hg hydrostatic pressure for 72 hours. A 10{\%} mechanical stretch for 24 hours was also performed on TMCs. The expression level of the optineurin and myocilin transcripts was analyzed by real-time quantitative PCR. RESULTS. The sequences of porcine optineurin and myocilin cDNA were determined, and the expression of both genes was confirmed in both TMCs and astrocytes. Amino acid sequences of porcine optineurin and myocilin were homologous to those of humans by 84{\%} and 82{\%}, respectively, and shared protein motifs and modification sites. The expression of myocilin mRNA by TMCs and astrocytes was increased by 8.0- and 5.5-fold, respectively, after exposure to dexamethasone. In contrast, the expression of optineurin was suppressed to 68{\%} in TMCs and 48{\%} in astrocytes after exposure to dexamethasone. A significant reduction of myocilin expression was observed after 72 hours of incubation under hypoxic conditions in both types of cells, whereas optineurin was not affected. Hydrostatic pressure for 72 hours and mechanical stretching for 24 hours had minimal affects on gene expression of both optineurin and myocilin. CONCLUSIONS. The high homology of porcine optineurin and myocilin to the comparable human genes indicates that pigs can be used to study changes in gene expression in hypertensive eyes. The alterations in expression of myocilin but not of optineurin under stress suggest that different mechanisms in the phenotype of glaucoma associated with the two genes are involved in development of glaucoma.",
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T1 - Analysis of porcine optineurin and myocilin expression in trabecular meshwork cells and astrocytes from optic nerve head

AU - Obazawa, Minoru

AU - Mashima, Yukihiko

AU - Sanuki, Naoko

AU - Noda, Setsuko

AU - Kudo, Jun

AU - Shimizu, Nobuyoshi

AU - Oguchi, Yoshihisa

AU - Tanaka, Yasuhiko

AU - Iwata, Takeshi

PY - 2004/8

Y1 - 2004/8

N2 - PURPOSE. To determine the cDNA sequences and analyze the expression of porcine optineurin and myocilin in trabecular meshwork cells (TMCs) and astrocytes from the optic nerve head under normal and experimental conditions. METHODS. Both porcine optineurin and myocilin were cloned to determine the cDNA sequences. Porcine TMCs and astrocytes were isolated and treated with dexamethasone (500 nM) for 2 weeks, incubated under hypoxic conditions (7% O2) for 72 hours, or exposed to 33 mm Hg hydrostatic pressure for 72 hours. A 10% mechanical stretch for 24 hours was also performed on TMCs. The expression level of the optineurin and myocilin transcripts was analyzed by real-time quantitative PCR. RESULTS. The sequences of porcine optineurin and myocilin cDNA were determined, and the expression of both genes was confirmed in both TMCs and astrocytes. Amino acid sequences of porcine optineurin and myocilin were homologous to those of humans by 84% and 82%, respectively, and shared protein motifs and modification sites. The expression of myocilin mRNA by TMCs and astrocytes was increased by 8.0- and 5.5-fold, respectively, after exposure to dexamethasone. In contrast, the expression of optineurin was suppressed to 68% in TMCs and 48% in astrocytes after exposure to dexamethasone. A significant reduction of myocilin expression was observed after 72 hours of incubation under hypoxic conditions in both types of cells, whereas optineurin was not affected. Hydrostatic pressure for 72 hours and mechanical stretching for 24 hours had minimal affects on gene expression of both optineurin and myocilin. CONCLUSIONS. The high homology of porcine optineurin and myocilin to the comparable human genes indicates that pigs can be used to study changes in gene expression in hypertensive eyes. The alterations in expression of myocilin but not of optineurin under stress suggest that different mechanisms in the phenotype of glaucoma associated with the two genes are involved in development of glaucoma.

AB - PURPOSE. To determine the cDNA sequences and analyze the expression of porcine optineurin and myocilin in trabecular meshwork cells (TMCs) and astrocytes from the optic nerve head under normal and experimental conditions. METHODS. Both porcine optineurin and myocilin were cloned to determine the cDNA sequences. Porcine TMCs and astrocytes were isolated and treated with dexamethasone (500 nM) for 2 weeks, incubated under hypoxic conditions (7% O2) for 72 hours, or exposed to 33 mm Hg hydrostatic pressure for 72 hours. A 10% mechanical stretch for 24 hours was also performed on TMCs. The expression level of the optineurin and myocilin transcripts was analyzed by real-time quantitative PCR. RESULTS. The sequences of porcine optineurin and myocilin cDNA were determined, and the expression of both genes was confirmed in both TMCs and astrocytes. Amino acid sequences of porcine optineurin and myocilin were homologous to those of humans by 84% and 82%, respectively, and shared protein motifs and modification sites. The expression of myocilin mRNA by TMCs and astrocytes was increased by 8.0- and 5.5-fold, respectively, after exposure to dexamethasone. In contrast, the expression of optineurin was suppressed to 68% in TMCs and 48% in astrocytes after exposure to dexamethasone. A significant reduction of myocilin expression was observed after 72 hours of incubation under hypoxic conditions in both types of cells, whereas optineurin was not affected. Hydrostatic pressure for 72 hours and mechanical stretching for 24 hours had minimal affects on gene expression of both optineurin and myocilin. CONCLUSIONS. The high homology of porcine optineurin and myocilin to the comparable human genes indicates that pigs can be used to study changes in gene expression in hypertensive eyes. The alterations in expression of myocilin but not of optineurin under stress suggest that different mechanisms in the phenotype of glaucoma associated with the two genes are involved in development of glaucoma.

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