Increasing cell density down-regulates the expression of acidic FGF by human RPE cells in vitro.
Previous studies have reported the expression of acidic fibroblast growth factor (aFGF) by rat, bovine, and human retinal pigment epithelium (RPE) in vivo. To critically examine the expression of aFGF by RPE cells, we studied the density dependence of steady-state levels of mRNA and protein expression in vitro. Northern blot analysis demonstrated 5 transcripts ranging from 4.5 kB to 1 kB. Steady-state levels of all the transcripts decreased as a function of culture density. A polyclonal antibody was raised against recombinant human aFGF and affinity purified on aFGF coupled to AffiGel-10. The resulting antibody crossreacted with bFGF but not FGF-5, but this crossreactivity could be eliminated by absorption of the antibody on bFGF coupled to AffiGel-10. The final antibody preparation recognized only a single band at approximately 18.5 kD in lysates of RPE. Immunohistochemical staining with this antibody preparation demonstrated a marked dependence on cell density after 3 days in culture. Low culture density yielded cells staining moderately for aFGF, while confluent cells exhibited little or no staining. The reduction of aFGF from RPE cells in culture in a density-dependent fashion could also be demonstrated by Western blot analysis.
Kitaoka
T
T
Department of Ophthalmology, University of California, Davis 95616.
Bost
L M
LM
Ishigooka
H
H
Aotaki-Keen
A E
AE
Hjelmeland
L M
LM
D000818:Animals; D015152:Blotting, Northern; D002452:Cell Count; D002478:Cells, Cultured; D002846:Chromatography, Affinity; D003429:Cross Reactions / Q000276:immunology; D015536:Down-Regulation / Q000502:physiology; D016220:Fibroblast Growth Factor 1 / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D015870:Gene Expression; D006801:Humans; D007124:Immunoenzyme Techniques; D010857:Pigment Epithelium of Eye / Q000166:cytology* / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D011817:Rabbits; D011994:Recombinant Proteins / Q000276:immunology
D012333:RNA, Messenger; D011994:Recombinant Proteins; D016220:Fibroblast Growth Factor 1