Expression of CD44 and variant isoforms in cultured human retinal pigment epithelial cells.
OBJECTIVE
CD44 is a major hyaluronic acid receptor that exists as a number of isoforms, generated by alternative splicing of 9 "variant" exons in humans (v2 to v10) and 10 exons in rodents. Little is known about the expression and function of CD44 in human retinal pigment epithelium (RPE) cells. Therefore, the authors determined whether human RPE cells express CD44, and whether the expression differs depending on the proliferative status of the cells.


METHODS
Human RPE cells were harvested from normal donor eyes and propagated in culture. Total RNA was extracted from cultured cells. mRNA expression of CD44 was determined by reverse transcription-polymerase chain reaction (RT-PCR), followed by cloning and sequencing of the PCR products, and by Southern hybridization. CD44 cell surface expression was measured by flow cytometry. Western hybridization and immunohistochemistry were used to determine the CD44 immunoreactivity of cultured human RPE cells and normal human RPE cells in situ.


RESULTS
The standard form of CD44 mRNA and variant isoforms containing exon v6 or v10 were expressed in cultured human RPE cells. CD44 mRNA and protein levels were increased in proliferating human RPE cells compared with density-arrested counterparts. Addition of 1 microM retinoic acid enhanced the cell density-induced downregulation of CD44 mRNA, but did not significantly affect the CD44 cell surface protein expression. As previously reported, CD44 immunoreactivity was not detected in normal human RPE cells in situ.


CONCLUSIONS
Cultured human RPE cells express CD44 standard form and variant isoforms containing exon v6 or v10, which are preferentially expressed by proliferating human RPE cells.
Liu
N P
NP
Department of Ophthalmology, Duke University Medical Center, Durham, North Carolina 27710, USA.
Roberts
W L
WL
Hale
L P
LP
Levesque
M C
MC
Patel
D D
DD
Lu
C L
CL
Jaffe
G J
GJ
D015153:Blotting, Western; D002452:Cell Count / Q000187:drug effects; D002455:Cell Division; D002478:Cells, Cultured; D017931:DNA Primers / Q000737:chemistry; D005434:Flow Cytometry; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D018960:Hyaluronan Receptors / Q000235:genetics / Q000378:metabolism*; D010857:Pigment Epithelium of Eye / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D012313:RNA / Q000302:isolation & purification; D012333:RNA, Messenger / Q000378:metabolism; D014212:Tretinoin / Q000494:pharmacology
D017931:DNA Primers; D018960:Hyaluronan Receptors; D012333:RNA, Messenger; D014212:Tretinoin; D012313:RNA