FAK activation and the role of epithelial membrane protein 2 (EMP2) in collagen gel contraction.
OBJECTIVE
Proliferative vitreoretinopathy (PVR) occurs in approximately 10% of patients after retinal detachment. PVR results from a multiphase process that leads to an aberrant wound-healing strategy with contractile cellular forces and tractional retinal detachment (TRD). Epithelial membrane protein (EMP) 2 controls cell surface expression and function of integrin isoforms associated with cellular contraction in many cell types. Since EMP2 is highly expressed in retinal pigment epithelium, this study investigates the role of EMP2 in collagen gel contraction.


METHODS
EMP2 expression was recombinantly modified in the ARPE-19 cell line. Cell surface integrin expression was assessed by flow cytometry. Collagen gel contraction was assessed by using an in vitro assay and the percentage of contraction was quantified. Proliferation and migration were measured by BrdU incorporation and a wound-healing assay, respectively. Cellular invasion was investigated with polycarbonate membranes coated with collagen.


RESULTS
EMP2 expression levels correlated positively with the ability to contract collagen gels. Compared with wild-type ARPE-19 cells, the cells with increased EMP2 expression exhibited enhanced contraction (P = 0.02), and decreased EMP2 expression concomitantly resulted in decreased contraction (P = 0.002). EMP2 overexpression resulted in reduced proliferation, migration, and integrin alpha1 and alpha2 integrin expression. EMP2 overexpression was associated with a 70% increase in FAK activation (P = 0.0003) and relative resistance of gel contraction to inhibitors of FAK/Src activation.


CONCLUSIONS
ARPE-19-mediated collagen gel contraction is a multistep process that requires integrin ligation and activation of the FAK/Src complex. EMP2 positively modulates collagen gel contraction by ARPE-19 cells through increased FAK activation.
Morales
Shawn A
SA
Department of Pathology and Laboratory Medicine, University of California, Los Angeles, California, USA.
Mareninov
Sergey
S
Wadehra
Madhuri
M
Zhang
Lily
L
Goodglick
Lee
L
Braun
Jonathan
J
Gordon
Lynn K
LK
D000911:Antibodies, Monoclonal; D015153:Blotting, Western; D002460:Cell Line; D002465:Cell Movement; D049109:Cell Proliferation; D024042:Collagen Type I / Q000378:metabolism*; D005434:Flow Cytometry; D051417:Focal Adhesion Kinase 1 / Q000378:metabolism*; D006801:Humans; D007124:Immunoenzyme Techniques; D007211:Indoles / Q000494:pharmacology; D039423:Integrin alpha1 / Q000378:metabolism; D039421:Integrin alpha2 / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000502:physiology*; D011743:Pyrimidines / Q000494:pharmacology; D055213:Retinal Pigment Epithelium / Q000166:cytology / Q000378:metabolism*; D013449:Sulfonamides / Q000494:pharmacology; D019061:src-Family Kinases / Q000037:antagonists & inhibitors
C412373:AG 1879; D000911:Antibodies, Monoclonal; D024042:Collagen Type I; C486403:EMP2 protein, human; D007211:Indoles; D039423:Integrin alpha1; D039421:Integrin alpha2; D008562:Membrane Glycoproteins; D011743:Pyrimidines; C416927:SU 6656; D013449:Sulfonamides; D051417:Focal Adhesion Kinase 1; C496050:PTK2 protein, human; D019061:src-Family Kinases