Hsp27 phosphorylation in experimental glaucoma.
OBJECTIVE
The role of heat shock proteins (Hsp) in injury response has been well established, but it is now becoming apparent that the phosphorylation state of Hsp27 may be a critical determinant of its ability to act in a protective capacity. In this study, the expression of Hsp27 and its phosphorylation were evaluated in an experimental glaucoma model in Brown Norway rats and in a spontaneous model of glaucoma in the DBA/2J mouse.


METHODS
The intraocular pressure (IOP) of one eye was elevated by injecting 1.9 M saline into the episcleral vein, as previously described in Brown Norway rats. IOP was measured in awake Brown Norway rats before surgery and every other day after saline injection. After 10 days of elevated IOP, the retinas were either removed for Western blot analysis or fixed for immunohistochemistry (IHC). IOP measurement in 7-month-old female DBA/2J mice was performed by direct cannulation. Retinas of eyes with and without elevated IOP were collected for Western blot analysis.


RESULTS
Western blot results showed a significant increase in total Hsp27 (3.9-fold; P < 0.05; n = 8) and phosphorylated Hsp27 (pHsp27) (2.1-fold; P < 0.05; n = 6) in high IOP eyes in the experimental rat glaucoma model, and similar increases were observed in DBA/2J mice with elevated IOP (3.1-fold and 2.2-fold for Hsp27 and pHsp27, respectively; P < 0.05; n = 5). In rats, increased Hsp27 and pHsp27 immunoreactivity were observed in the nerve fiber layer of elevated IOP eyes and colocalized with glial fibrillary acidic protein (GFAP) and with vimentin staining, suggesting that glial cells contribute to the increase in Hsp27 and pHsp27 seen in experimental glaucoma. No change in Hsp70 or Hsp90 was observed.


CONCLUSIONS
These results confirm previous reports of elevated Hsp27 in experimental glaucoma and extend them to the DBA/2J mouse. In addition, a significant increase occurred in Hsp27 phosphorylation with elevated IOP in both models of glaucoma. IHC studies show that the increases in Hsp27 and pHsp27 occur primarily in glial cells.
Huang
Wei
W
Howe Laboratory of Ophthalmology, Massachusetts Eye and Ear Infirmary, Harvard Medical School, Boston, Massachusetts 02114, USA.
Fileta
John B
JB
Filippopoulos
Theodoros
T
Ray
Arjun
A
Dobberfuhl
Adam
A
Grosskreutz
Cynthia L
CL
D000818:Animals; D015153:Blotting, Western; D005260:Female; D005901:Glaucoma / Q000378:metabolism*; D055551:HSP27 Heat-Shock Proteins; D006360:Heat-Shock Proteins / Q000378:metabolism*; D006801:Humans; D007150:Immunohistochemistry; D007429:Intraocular Pressure; D051379:Mice; D008811:Mice, Inbred DBA; D009363:Neoplasm Proteins / Q000378:metabolism*; D010766:Phosphorylation; D051381:Rats; D011914:Rats, Inbred BN; D012160:Retina / Q000378:metabolism*
D055551:HSP27 Heat-Shock Proteins; D006360:Heat-Shock Proteins; C486114:Hspb1 protein, rat; C486699:Hspb2 protein, mouse; D009363:Neoplasm Proteins