Increasing cell density down-regulates the expression of acidic FGF by human RPE cells in vitro

Takashi Kitaoka, Laurie M. Bost, Hitoshi Ishigooka, Amy E. Aotaki-Keen, Leonard M Hjelmeland

Research output: Contribution to journalArticle

17 Scopus citations

Abstract

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.

Original languageEnglish (US)
Pages (from-to)993-999
Number of pages7
JournalCurrent Eye Research
Volume12
Issue number11
DOIs
StatePublished - 1993

Keywords

  • Acidic fibroblast growth factor (aFGF)
  • Cell culture
  • Gene expression
  • Growth factors
  • Human
  • Retinal pigment epithelium

ASJC Scopus subject areas

  • Ophthalmology
  • Sensory Systems
  • Cellular and Molecular Neuroscience

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