Mechanical stretch alters the actin cytoskeletal network and signal transduction in human trabecular meshwork cells.
OBJECTIVE
Human trabecular meshwork (HTM) cells were mechanically stretched in vitro as a potential model for the distension of this tissue that can occur in vivo in response to increased pressure gradients. Cell morphology and certain components of the signal transduction pathways, including the mitogen-activated protein kinase (MAPK) and c-Jun N-terminal protein kinase (JNK) pathways, were evaluated for stretch-induced alterations.


METHODS
Primary HTM cells grown in tissue culture were subjected to a mechanical stretch lasting from 10 seconds to 4 days. The actin cytoskeletal network was visualized by phalloidin staining. Proteins phosphorylated on their tyrosine residues were isolated using an immunoaffinity system and were analyzed by gel electrophoresis and immunostaining. Mitogen-activated protein kinase activity was evaluated using an in-gel assay system, and the mRNA levels of c-fos and c-jun were determined by quantitation of competitive reverse transcription-polymerase chain reaction. In addition, the amount of c-Fos protein was estimated by chemiluminescent immunoblot analysis.


RESULTS
On stretching, the HTM cells elongated but regained their normal morphologic characteristics within 24 hours. Unstretched HTM cells displayed a diffuse F-actin microfilament network, whereas stretched cells exhibited complex geodesic patterns. Ten seconds after stretching began, the level of tyrosine phosphorylation on the six major phosphoproteins significantly decreased between 80% and 100%, whereas the level of paxillin tyrosine phosphorylation significantly increased 39%. Stretching caused MAPK activity and the amount of mRNA and protein of the immediate-early gene c-fos to decrease more than 60% within 2 minutes, but within 15 to 30 minutes they increased above or equivalent to normal levels. The level of c-jun mRNA was unchanged by stretching.


CONCLUSIONS
In response to a mechanical stretch, major cytoskeletal alterations occur in HTM cells, which involve changes in the levels of tyrosine phosphorylation. Mechanotransduction (signal transduction by mechanical stimulation) through the MAPK signaling pathway was significantly depressed immediately after stretching; however, the JNK pathway appeared to be unaffected. The data suggest that HTM cells adapt to mechanical stress by altering the cytoskeletal network and signaling cascades.
Tumminia
S J
SJ
Laboratory for Mechanisms of Ocular Diseases, National Eye Institute, National Institutes of Health, Bethesda, Maryland 20892-2735, USA.
Mitton
K P
KP
Arora
J
J
Zelenka
P
P
Epstein
D L
DL
Russell
P
P
D008841:Actin Cytoskeleton / Q000378:metabolism*; D000199:Actins / Q000378:metabolism*; D000293:Adolescent; D000328:Adult; D000368:Aged; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D002478:Cells, Cultured; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008875:Middle Aged; D010590:Phalloidine; D010766:Phosphorylation; D016760:Proto-Oncogene Proteins c-fos / Q000235:genetics / Q000378:metabolism; D016755:Proto-Oncogene Proteins c-jun / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D015398:Signal Transduction; D013314:Stress, Mechanical; D014129:Trabecular Meshwork / Q000166:cytology / Q000378:metabolism*; D014443:Tyrosine / Q000378:metabolism
D000199:Actins; D016760:Proto-Oncogene Proteins c-fos; D016755:Proto-Oncogene Proteins c-jun; D012333:RNA, Messenger; D010590:Phalloidine; D014443:Tyrosine; D017871:Calcium-Calmodulin-Dependent Protein Kinases