Basement membrane-dependent modification of phenotype and gene expression in human retinal pigment epithelial ARPE-19 cells.
OBJECTIVE
To use porcine lens capsule (PLC) as basement membrane for ARPE-19 cells and to characterize its effects on cell differentiation and gene expression.


METHODS
Postconfluent cultures of ARPE-19 cells were established on either porous polyester filters or PLC membranes and characterized by electron microscopy, immunocytochemistry, and transepithelial electrical resistance measurements. Metabolic activity was assessed by measuring phagocytosis of rod outer segments. mRNA populations of ARPE-19 cells grown on polyester and PLC membranes were compared by suppressive subtractive hybridization. Differentially regulated messages were subsequently identified by DNA sequencing and their altered expression confirmed by Northern or virtual Northern blot analysis.


RESULTS
Culture of ARPE-19 cells on PLC membrane induced the formation of apical microvilli and the ability to phagocytose rod outer segments. These culture conditions also led to enhanced junctional distribution of ZO-1 and occludin, the formation of polarized membrane domains, and a significant increase in transepithelial resistance. Gene expression was significantly altered by growth on PLC membranes and 29 differentially expressed transcripts were identified.


CONCLUSIONS
Culture of ARPE-19 cells on PLC membranes resulted in a more differentiated phenotype and in expression of a specific set of transcripts encoding protein products that may affect epithelial differentiation, polarity and survival.
Division of Cell Biology, Institute of Ophthalmology, University College London, London, United Kingdom.
Patric
P
Turowski
Peter
P
Adamson
Julie
J
Sathia
Jin Jun
JJ
Zhang
Stephen E
SE
Moss
G William
GW
Aylward
Matthew J
MJ
Hayes
Naheed
N
Kanuga
John
J
Greenwood
D000818:Animals; D001485:Basement Membrane / Q000502:physiology*; D015152:Blotting, Northern; D018929:Cell Culture Techniques / Q000379:methods; D002454:Cell Differentiation / Q000502:physiology; D002460:Cell Line; D016764:Cell Polarity / Q000502:physiology; D002470:Cell Survival / Q000502:physiology; D004553:Electric Conductivity; D005136:Eye Proteins / Q000235:genetics* / Q000378:metabolism; D005786:Gene Expression Regulation / Q000502:physiology*; D015723:Gene Library; D006801:Humans; D007150:Immunohistochemistry; D007903:Lens Capsule, Crystalline / Q000502:physiology*; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D018613:Microscopy, Confocal; D008854:Microscopy, Electron; D062793:Occludin; D010587:Phagocytosis / Q000502:physiology; D010641:Phenotype; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism; D010857:Pigment Epithelium of Eye / Q000166:cytology* / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D012374:Rod Cell Outer Segment / Q000502:physiology; D013552:Swine; D062826:Zonula Occludens-1 Protein
D005136:Eye Proteins; D008565:Membrane Proteins; C568985:OCLN protein, human; D062793:Occludin; D010750:Phosphoproteins; D012333:RNA, Messenger; C569042:TJP1 protein, human; D062826:Zonula Occludens-1 Protein