Human and monkey trabecular meshwork accumulate alpha B-crystallin in response to heat shock and oxidative stress

Abstract

Purpose: Oxidative stress and other forms of injury to trabecular meshwork (TM) cells may contribute to changes seen with age and primary open-angle glaucoma. This study was designed to investigate if TM expresses alpha B-crystallin, a small heat-shock protein with chaperone activity, and whether it might be overexpressed under stress conditions.

Methods: The TM from human and monkey eyes, as well as organ and primary cell cultures derived from these eyes, were investigated for alpha B-crystallin by immunohistochemistry, two-dimensional gel electrophoresis, Northern and Western blot analysis. The TM cell cultures were stressed by heat shock (44 degrees C for 15 minutes) or hydrogen peroxide (200 mumol for 1 hour). Semiquantitation of alpha B-crystallin messenger RNA (mRNA) or protein was obtained by densitometry.

Results: In both 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 alpha B-crystallin was expressed predominantly in the cribriform area. Protein expression was enhanced in 4- to 7-day organ cultures. Primary cultures 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 cultures, a 0.8-kb band was observed, which comigrated with lens alpha B-crystallin. In both species, heat shock caused a significant increase in alpha B-crystallin mRNA with a peak after 4 hours. An increase in alpha B-crystallin mRNA also was observed after oxidative stress; however, the onset of mRNA induction was slower. After heat shock, but not after oxidative stress, a transient change in mRNA mobility was observed. 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 constitutive mRNA expression and only a minimal increase 4 hours after heat shock could be observed in simian virus 40 transformed cell lines from human TM.

Conclusions: Overexpression of alpha B-crystallin might be an important mechanism for TM to prevent cellular damage associated with various stress conditions.