Cellular adhesion regulates bFGF gene expression in human retinal pigment epithelial cells.

It has previously been shown that cell density has a profound effect on the expression of the basic fibroblast growth factor (bFGF) gene by human retinal pigment epithelial (RPE) cells in vitro. This study was designed to examine cellular mechanisms which may be responsible for the density-dependent expression of bFGF. Examination of RPE labeling index with respect to cell density suggests that cell cycle progression is not responsible for changes in steady-state mRNA levels. Media switch experiments between sparse and dense RPE cell cultures also rule out a soluble mediator. Extracellular matrix deposited within the 3-day plating period could also not account for the observed changes in bFGF gene expression. However, the synchronous loss of cell contact induced by Ca2+ chelation caused a transient elevation in steady-state mRNA levels. These results suggest that changes in cell adhesion may be responsible for the density-dependent regulation of bFGF mRNA levels in RPE cells in vitro.

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.

Developmental expression of bFGF in the bovine retina.

PURPOSE: To examine the expression of bFGF in the developing bovine retina. METHODS: Fetal bovine eyes at 90, 120, 150, and 180 days gestational age, as well as adult bovine eyes, were immunohistochemically stained for the presence of bFGF. Detailed characterization of the anti-bFGF antibodies by immunoblot and Western blot analysis against pure FGF gene family standards and crude extracts of bovine retina were also performed. RESULTS: Expression of bFGF occurs beginning at 150 days of gestation, a period when photoreceptor development and secondary capillary network development is in process. No bFGF expression was found at 90 days, but primary capillaries were already apparent at this stage of development. CONCLUSIONS: Expression of bFGF in the developing bovine retina may play a functional role in outer retinal development.

Cell density regulates differential production of bFGF transcripts.

In vitro cultures of human retinal pigment epithelial (RPE) cells were used to study the regulation of basic fibroblast growth factor (bFGF) gene expression. Four transcripts of 7.0, 3.7, 2.2, and 1.2 kB are produced from the bFGF gene. Increasing cell density has a profound effect on the expression of the 7.0 kB transcript relative to the 3.7 kB transcript. Here, evidence is presented suggesting that posttranscriptional processing events are responsible for differential expression of the 7.0 and 3.7 kB bFGF transcripts as a function of cell density. Primer extension analysis demonstrates that these two transcripts originate from a single transcription initiation site. Determination of the half-lives of the 7.0 and 3.7 kB transcripts at confluent cell density did not explain the relative expression of these mRNAs. These differences may arise from the use of alternative polyadenylation sites in the 3' untranslated region (UTR) as a function of cell density. Polysomal analysis indicates no selective translation of any of the four bFGF transcripts in RPE cells.

Coexpression of FGF-5 and bFGF by the retinal pigment epithelium in vitro.

Members of the fibroblast growth factor (FGF) gene family have been proposed to play critical roles in the biology of the outer retina. In this study, in vitro cultures of human retinal pigment epithelial (RPE) cells were surveyed for the expression of FGF gene family members. Northern analysis provided evidence for the expression of the previously reported 7.0 and 3.7 kb basic FGF (bFGF) transcripts and for the 4.0 and 2.1 kb FGF-5 transcripts. Western analysis demonstrated the presence of three bFGF proteins ranging in size from 18 to 26 kDa and three FGF-5 proteins of molecular weights 28.5, 34, and 36 kDa. We were particularly interested in cellular mechanisms which might regulate the steady-state mRNA levels of these genes. It was determined that bFGF message levels decreased with increasing culture density, increased upon serum stimulation and when placed in contact with matrix components found in the extracellular matrix of RPE cells in vivo. In a similar fashion, the steady state mRNA levels for FGF-5 decreased with increasing culture density, increased upon serum stimulation, but appeared to be unaffected by matrix contact.