TY - JOUR
T1 - A mechanism for trabecular meshwork cell retraction
T2 - Ethacrynic acid initiates the dephosphorylation of focal adhesion proteins
AU - O'Brien, E. Timothy
AU - Kinch, Michael
AU - Harding, Timothy W.
AU - Epstein, David L.
N1 - Funding Information:
Supported by NIH grant EY01894, P30EY05722 (a core research grant from the National Eye Institute), and a National Glaucoma Research grant from the American Health Assistance Foundation.
PY - 1997/10
Y1 - 1997/10
N2 - Ethacrynic acid (ECA) increases aqueous humor outflow facility in human and animal model systems, and causes cellular retraction in cultured trabecular meshwork (TM) cells. ECA-induced retraction, a possible correlate to the opening of spaces in the outflow pathway in vivo, takes place coincident with disruption of cell-cell attachments and actin stress fibers. Tyrosine phosphorylated proteins are located predominantly where actin filaments terminate at sites of cell-to-cell and cell-to-substrate adhesion, and are understood to regulate cellular adhesions and filamentous (F) actin organization in many cell types. In the present study we investigated whether ECA might affect cell adhesions and F-actin in TM cells by altering levels of phosphotyrosine. We analysed levels of phosphotyrosine in cultured human TM and calf pulmonary artery endothelial cells after exposure to ECA. Using immunofluorescence microscopy and antibodies to phosphotyrosinated proteins we found a rapid decrease in phosphotyrosine levels at the focal contacts of cells treated with ECA. Immunoblots of whole cell extracts showed a decrease in phosphotyrosine predominantly in a band running at about 120 kD, with a more subtle decrease in a band about 65 kD. Reprobing the blot with antibodies to pp120 focal adhesion kinase (FAK) or paxillin indicated that the 120 kD band was FAK and the 65 kD band was likely paxillin. Immunoprecipitation of FAK or paxillin and probing the resulting blot with antibodies to phosphotyrosine confirmed that these proteins were rapidly dephosphorylated after ECA addition. Loss of FAK and paxillin proteins in cells was then confirmed using immunofluorescence microscopy. Dephosphorylation of these proteins was detected before the onset of retraction, stress fiber disruption, or complete disruption of focal adhesions. A pure microtubule inhibitor (colchicine), did not cause stress fiber disruption or decrease focal adhesion phosphorylation. We postulate that dephosphorylation of FAK and paxillin by ECA disrupts signaling pathways that normally maintain the stability of the actin cytoskeleton and cellular adhesions, and that this action leads both to cell shape change in culture, and to facility changes in vivo.
AB - Ethacrynic acid (ECA) increases aqueous humor outflow facility in human and animal model systems, and causes cellular retraction in cultured trabecular meshwork (TM) cells. ECA-induced retraction, a possible correlate to the opening of spaces in the outflow pathway in vivo, takes place coincident with disruption of cell-cell attachments and actin stress fibers. Tyrosine phosphorylated proteins are located predominantly where actin filaments terminate at sites of cell-to-cell and cell-to-substrate adhesion, and are understood to regulate cellular adhesions and filamentous (F) actin organization in many cell types. In the present study we investigated whether ECA might affect cell adhesions and F-actin in TM cells by altering levels of phosphotyrosine. We analysed levels of phosphotyrosine in cultured human TM and calf pulmonary artery endothelial cells after exposure to ECA. Using immunofluorescence microscopy and antibodies to phosphotyrosinated proteins we found a rapid decrease in phosphotyrosine levels at the focal contacts of cells treated with ECA. Immunoblots of whole cell extracts showed a decrease in phosphotyrosine predominantly in a band running at about 120 kD, with a more subtle decrease in a band about 65 kD. Reprobing the blot with antibodies to pp120 focal adhesion kinase (FAK) or paxillin indicated that the 120 kD band was FAK and the 65 kD band was likely paxillin. Immunoprecipitation of FAK or paxillin and probing the resulting blot with antibodies to phosphotyrosine confirmed that these proteins were rapidly dephosphorylated after ECA addition. Loss of FAK and paxillin proteins in cells was then confirmed using immunofluorescence microscopy. Dephosphorylation of these proteins was detected before the onset of retraction, stress fiber disruption, or complete disruption of focal adhesions. A pure microtubule inhibitor (colchicine), did not cause stress fiber disruption or decrease focal adhesion phosphorylation. We postulate that dephosphorylation of FAK and paxillin by ECA disrupts signaling pathways that normally maintain the stability of the actin cytoskeleton and cellular adhesions, and that this action leads both to cell shape change in culture, and to facility changes in vivo.
KW - Actin
KW - Aqueous humor outflow
KW - Colchicine
KW - Cytochalasin B
KW - Endothelial cells
KW - Ethacrynic acid
KW - Focal adhesion kinase
KW - Focal adhesions
KW - Glaucoma
KW - Paxillin, cell signalling
KW - Phosphotyrosine
KW - Trabecular meshwork cells
UR - http://www.scopus.com/inward/record.url?scp=0030687577&partnerID=8YFLogxK
U2 - 10.1006/exer.1997.0357
DO - 10.1006/exer.1997.0357
M3 - Article
C2 - 9464181
AN - SCOPUS:0030687577
SN - 0014-4835
VL - 65
SP - 471
EP - 483
JO - Experimental eye research
JF - Experimental eye research
IS - 4
ER -