HUMAN AND MONKEY TRABECULAR MESHWORK ACCUMULATE ALPHA-B-CRYSTALLIN INRESPONSE TO HEAT-SHOCK AND OXIDATIVE STRESS

Purpose. Oxidative stress and other forms of injury to trabecular mesh work (TM) cells may contribute to changes seen with age and primary op en-angle glaucoma. This study was designed to investigate if TM expres ses alpha B-crystallin, a small heat-shock protein with chaperone acti vity, and whether it might be overexpressed under stress conditions. M ethods. The TM from human and monkey eyes, as well as organ and primar y cell cultures derived from these eyes, were investigated for alpha B -crystallin by immunohistochemistry, two-dimensional gel electrophores is, Northern and Western blot analysis. The TM cell cultures were stre ssed by heat shock (44 degrees C for 15 minutes) or hydrogen peroxide (200 mu mol for 1 hour). Semiquantitation of alpha B-crystallin messen ger RNA (mRNA) or protein was obtained by densitometry. Results. In bo th species, alpha B-crystallin could be detected in fresh and cultured TM by two-dimensional gel electrophoresis in conjunction with Western blot analysis. Immunohistochemistry of fresh samples showed that alph a B-crystallin was expressed predominantly in the cribriform area. Pro tein expression was enhanced in 4- to 7-day organ cultures. Primary cu ltures from human TM cells expressed two sizes (approximately 0.8 and 1.1 kb) of alpha B-crystallin mRNA in Northern blots. In monkey TM cul tures, a 0.8-kb band was observed, which comigrated with lens alpha B- crystallin. In both species, heat shuck caused a significant increase in alpha B-crystallin mRNA with a peak after 4 hours. An increase in a lpha B-crystallin mRNA also was observed after oxidative stress; howev er, the onset of mRNA induction was slower. After heat shock, but not after oxidative stress, a transient change in mRNA mobility was observ ed. Western dot blot analysis showed a 3.4-fold increase in protein 24 hours after heat shock and a 20-fold increase after 48 hours. No cons titutive mRNA expression and only a minimal increase 4 hours after hea t shock could be observed in simian virus 40 transformed cell lines fr om human TM. Conclusions Overexpression of alpha B-crystallin might be an important mechanism for TM to prevent cellular damage associated w ith various stress conditions.