Oxidative stress and TGF-beta2 increase heat shock protein 27 expression in human optic nerve head astrocytes.
OBJECTIVE
Reactive astrocytes of glaucomatous optic nerve heads (ONHs) are characterized by an increased expression of transforming growth factor (TGF)-beta2 and heat shock proteins (Hsps) such as Hsp27. The goal of the present study was to determine the effect of oxidative stress on TGF-beta2 and Hsp27 expression in human ONH astrocytes.


METHODS
Cultured ONH astrocytes were exposed to 100, 200, and 400 muM hydrogen peroxide (H(2)O(2)) for 1 hour. Levels of TGF-beta2 were analyzed by ELISA. Additionally, cells were treated with 1.0 ng/mL TGF-beta2 for 12, 24, and 48 hours, respectively. Expression of Hsp27 was detected by immunohistochemistry, real-time PCR, and Western blot analyses. To investigate the role of signal transduction proteins, cells were pretreated with the specific inhibitors SB203580, PD98059, U0126, and SP600125. Furthermore, induction of p38MAP kinase and its phosphorylated form were determined by Western blot analyses.


RESULTS
H(2)O(2) exposure increased the secretion of TGF-beta2. Both H(2)O(2) and TGF-beta2 increased the Hsp27 expression. Pretreatment of cells with SB203580, an inhibitor for p38MAP kinase, reduced the H(2)O(2)- and TGF-beta2-stimulated Hsp27 expression, whereas pretreatment with PD98059 and U0126, specific inhibitors of ERK1/2, and SP600125, a specific c-Jun N-terminal kinase inhibitor, had no effects. H(2)O(2) and TGF-beta2 upregulated the phosphorylated p38MAP kinase.


CONCLUSIONS
Oxidative stress increased TGF-beta2 secretion in cultured human ONH astrocytes. Both H(2)O(2) and TGF-beta2 increased Hsp27 expression via p38MAP kinase activation. Therefore, it is tempting to speculate that reduction of oxidative stress and TGF-beta2 may help to minimize the incidence of characteristic changes in the ONH of patients with glaucoma.
Department of Ophthalmology, Ludwig-Maximilians-University, Munich, Germany.
Alice L
AL
Yu
Rudolf
R
Fuchshofer
Marco
M
Birke
Anselm
A
Kampik
Hans
H
Bloemendal
Ulrich
U
Welge-Lüssen
D000368:Aged; D001253:Astrocytes / Q000187:drug effects* / Q000378:metabolism; D015153:Blotting, Western; D002478:Cells, Cultured; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004797:Enzyme-Linked Immunosorbent Assay; D019084:Fluorescent Antibody Technique, Indirect; D055551:HSP27 Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism*; D006360:Heat-Shock Proteins; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D048031:JNK Mitogen-Activated Protein Kinases; D008875:Middle Aged; D019950:Mitogen-Activated Protein Kinase 1 / Q000037:antagonists & inhibitors / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000037:antagonists & inhibitors / Q000378:metabolism; D018832:Molecular Chaperones; D009898:Optic Disk / Q000187:drug effects* / Q000378:metabolism; D018384:Oxidative Stress; D010766:Phosphorylation; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D053781:Transforming Growth Factor beta2 / Q000378:metabolism / Q000494:pharmacology*; D015854:Up-Regulation; D048051:p38 Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism
D004791:Enzyme Inhibitors; D055551:HSP27 Heat-Shock Proteins; C057725:HSPB1 protein, human; D006360:Heat-Shock Proteins; D018832:Molecular Chaperones; D012333:RNA, Messenger; D053781:Transforming Growth Factor beta2; D006861:Hydrogen Peroxide; D048031:JNK Mitogen-Activated Protein Kinases; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D048051:p38 Mitogen-Activated Protein Kinases