Culture-induced increase in alpha integrin subunit expression in retinal pigment epithelium is important for improved resurfacing of aged human Bruch's membrane.
The purpose of this study was to examine the change in integrin expression in adult human retinal pigment epithelium (RPE) after culturing and to characterize the role of integrins in RPE adhesion to aged submacular human Bruch's membrane. Expression of alpha integrin subunits 1 through 6 in adult RPE cells, cultured or uncultured, was examined by reverse transcription/real-time polymerase chain reaction (PCR) and Western blotting. RPE was cultured on bovine corneal endothelial cell-secreted extracellular matrix (BCE-ECM). The role of alpha integrin subunits in RPE attachment was examined by immunofluorescent localization of these subunits at sites of focal adhesions in cultured adult RPE attached to laminin or collagen-I-coated culture dishes. Additionally, the effect of function-blocking antibodies to alpha integrin subunits on RPE attachment to laminin, collagen I, and aged submacular human Bruch's membrane was determined. Cultured adult RPE had increased expression of alpha1-5 integrin subunits by PCR compared to uncultured RPE. Western blots showed that alpha2, 3, and 5 subunit levels were low or absent in uncultured adult RPE. Cultured adult RPE had a substantially higher expression of these integrins. Alpha 1-3 subunits co-localized with phosphorylated focal adhesion kinase (FAK) at focal adhesions in RPE cells spread on laminin. Only alpha2 and alpha3 co-localized with phosphorylated FAK in focal adhesions of RPE on collagen I. Using function blocking antibodies, blocking alpha1 subunit singly or in combination with alpha2 and/or alpha3 significantly decreased RPE adhesion to laminin. Blocking alpha1 and alpha2 or blocking alpha1, alpha2, and alpha3 subunits significantly decreased RPE adhesion to collagen I. Compared to controls, significantly fewer RPE cells were able to spread on aged submacular human Bruch's membrane when alpha1-6 integrin subunits were blocked. These results indicate that alpha 1-5 subunits that are upregulated by culturing on BCE-ECM are necessary for RPE attachment to aged submacular human Bruch's membrane. Relative lack of these integrin subunits in uncultured adult RPE may be responsible for poor resurfacing of aged submacular human Bruch's membrane by these cells.
The Institute of Ophthalmology and Visual Science, New Jersey Medical School, University of Medicine and Dentistry of New Jersey, Newark, NJ 07103, USA.
Vamsi K
VK
Gullapalli
Ilene K
IK
Sugino
Marco A
MA
Zarbin
D000368:Aged; D000369:Aged, 80 and over; D000375:Aging / Q000378:metabolism / Q000473:pathology; D015153:Blotting, Western / Q000379:methods; D016570:Bruch Membrane / Q000166:cytology / Q000378:metabolism*; D002448:Cell Adhesion / Q000502:physiology; D002478:Cells, Cultured; D024042:Collagen Type I / Q000378:metabolism; D006801:Humans; D039001:Integrin alpha Chains / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D007797:Laminin / Q000378:metabolism; D008856:Microscopy, Fluorescence / Q000379:methods; D008875:Middle Aged; D010857:Pigment Epithelium of Eye / Q000166:cytology / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods
D024042:Collagen Type I; D039001:Integrin alpha Chains; D007797:Laminin; D012333:RNA, Messenger