Analysis of porcine optineurin and myocilin expression in trabecular meshwork cells and astrocytes from optic nerve head.
OBJECTIVE
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% O(2)) 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.
Obazawa
Minoru
M
National Institute of Sensory Organs, National Tokyo Medical Center, Tokyo, Japan.
Mashima
Yukihiko
Y
Sanuki
Naoko
N
Noda
Setsuko
S
Kudoh
Jun
J
Shimizu
Nobuyoshi
N
Oguchi
Yoshihisa
Y
Tanaka
Yasuhiko
Y
Iwata
Takeshi
T
D000595:Amino Acid Sequence; D000818:Animals; D001253:Astrocytes / Q000187:drug effects / Q000378:metabolism*; D001483:Base Sequence; D015687:Cell Hypoxia; D003001:Cloning, Molecular; D003598:Cytoskeletal Proteins; D018076:DNA, Complementary / Q000032:analysis; D003907:Dexamethasone / Q000494:pharmacology; D005136:Eye Proteins / Q000235:genetics* / Q000378:metabolism; D005786:Gene Expression Regulation; D005938:Glucocorticoids / Q000494:pharmacology; D006023:Glycoproteins / Q000235:genetics* / Q000378:metabolism; D006874:Hydrostatic Pressure; D008969:Molecular Sequence Data; D009898:Optic Disk; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D013314:Stress, Mechanical; D013552:Swine; D014129:Trabecular Meshwork / Q000187:drug effects / Q000378:metabolism*; D000099179:Myocilin
D003598:Cytoskeletal Proteins; D018076:DNA, Complementary; D003907:Dexamethasone; D005136:Eye Proteins; D005938:Glucocorticoids; D006023:Glycoproteins; D012333:RNA, Messenger; D000099179:Myocilin