Transient loss of alphaB-crystallin: an early cellular response to mechanical stretch.
Human trabecular meshwork (HTM) is distended and stretched with increases in intraocular pressure. During this stretching, there is a rearrangement of actin filaments. The HTM cells express alpha B-crystallin, a small heat shock protein that may have a role in the stabilization and regulation of the cytoskeleton in mammalian cells. The levels of alpha B-crystallin were examined in trabecular meshwork cells after mechanical stretch. Human TM primary cell cultures, plated onto silicone sheets, were subjected to a single 10% linear stretch and samples were prepared at various times after stretch for immunoblotting or Northern blotting. Immunoblots of total protein extracts with antibody specific for alpha B-crystallin detected a 26% decrease of cellular alpha B-crystallin levels within 2 minutes. After 1 hour alpha B-crystallin levels had decreased 90% compared to control cells. The levels of alpha B-crystallin began to recover in cells stretched for 2 hours and returned to initial levels by 24 hours. Northern blots probed with alpha B-crystallin exon III cDNA detected a transcript of 0.65 kb in human TM cells and the levels of the alpha B mRNA remained constant during alpha B-crystallin protein decrease. Later, levels of the 0.65 kb transcript of alpha B-crystallin increased during the cellular recovery. These results suggest that decreased levels of alpha B-crystallin after mechanical stretch were probably not due to transcriptional changes but rather to increased degradation of alpha B-crystallin protein. An increase in mRNA levels may play a role in the recovery of alpha B-crystallin during reorganization of the cytoskeleton and attachment to the substratum. These data raise the possibility of a specific proteolysis of alpha B-crystallin protein in cells after a physiological challenge.
Mitton
K P
KP
Laboratory for Mechanisms of Ocular Diseases, National Eye Institute, National Institutes of Health, Bethesda, Maryland 20892, USA.
Tumminia
S J
SJ
Arora
J
J
Zelenka
P
P
Epstein
D L
DL
Russell
P
P
D015152:Blotting, Northern; D015153:Blotting, Western; D002478:Cells, Cultured; D003459:Crystallins / Q000235:genetics / Q000378:metabolism*; D003599:Cytoskeleton / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D005786:Gene Expression Regulation; D006360:Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007525:Isoelectric Focusing; D010766:Phosphorylation; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D013314:Stress, Mechanical; D014129:Trabecular Meshwork / Q000166:cytology / Q000378:metabolism*; D014158:Transcription, Genetic
D003459:Crystallins; D006360:Heat-Shock Proteins; D012333:RNA, Messenger