Effect of heparin II domain of fibronectin on actin cytoskeleton and adherens junctions in human trabecular meshwork cells.
OBJECTIVE
To determine whether the heparin II (HepII) domain of fibronectin previously shown to increase outflow facility affects the formation and assembly of actin cytoskeleton and adherens junctions in human trabecular meshwork (HTM) cells.


METHODS
Normal HTM cells and two transformed HTM cell lines were treated for 24 hours with increasing concentrations of the HepII domain. Disruptions in adherens junctions and the actin cytoskeleton were determined using immunofluorescence microscopy and Western blot analysis of immunoprecipitated cadherin/catenin complexes. Actin filaments were detected with phalloidin. Catenin (alpha and beta) and cadherin antibodies were used to detect adherens junctions.


RESULTS
Treatments of cultures with the HepII domain caused cadherin/beta-catenin complexes in adherens junctions and actin filaments to disassemble. The disruption of adherens junctions and actin filaments occurred in a dose-dependent and temporal fashion. The disassembly of actin filaments occurred first, followed by the disassembly of adherens junctions. Dissociation of adherens junctions, but not actin filaments, was reversible if the HepII domain was removed. Reassembly of actin filaments required the addition of serum. Serum, however, could not trigger the reassembly of actin filaments if the HepII domain was present, suggesting that the HepII domain acted downstream of the serum stimulated RhoA activity.


CONCLUSIONS
The exposure of HTM cells to the HepII domain triggers the disassembly of actin filaments and the subsequent destabilization of adherens junctions in HTM cells. This suggests that the HepII domain may increase outflow facility in cultured anterior segments by altering the organization of the TM cytoarchitecture.
Gonzalez
Jose M
JM
Department of Pathology and Laboratory Medicine, University of Wisconsin, Madison 53706, USA.
Faralli
Jennifer A
JA
Peters
Joanne M
JM
Newman
Jessica R
JR
Peters
Donna M
DM
D008841:Actin Cytoskeleton / Q000378:metabolism*; D022005:Adherens Junctions / Q000378:metabolism*; D015153:Blotting, Western; D015820:Cadherins / Q000378:metabolism; D018929:Cell Culture Techniques; D004305:Dose-Response Relationship, Drug; D005353:Fibronectins / Q000494:pharmacology*; D019084:Fluorescent Antibody Technique, Indirect; D006493:Heparin / Q000494:pharmacology*; D006801:Humans; D008856:Microscopy, Fluorescence; D013997:Time Factors; D014129:Trabecular Meshwork / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D051178:alpha Catenin / Q000378:metabolism; D051176:beta Catenin / Q000378:metabolism
D015820:Cadherins; D005353:Fibronectins; D051178:alpha Catenin; D051176:beta Catenin; D006493:Heparin