Effect of overexpressing fibroblast growth factor 2 protein isoforms in osteoblastic ROS 17/2.8 cells.
Fibroblast growth factor-2 (FGF-2) is made by osteoblasts and modulates their function. There are high molecular weight (HMW) protein isoforms of FGF-2 that have nuclear localization sequences and a low molecular weight (LMW) 18 kDa FGF-2 protein that is exported from cells. Since FGF-2 is a trophic factor and potent mitogen for osteoblasts, the goal of this study was to utilize targeted overexpression of FGF-2 as a novel means of assessing different FGF-2 isoforms on osteoblastic cell viability and proliferation. Either LMW or HMW human Fgf2 cDNAs were cloned downstream of 3.6 kb alpha1(I)-collagen 5' regulatory elements (Col 3.6). A set of expression vectors, called Col3.6-Fgf2 isoforms-IRES-GFPsaph, capable of concurrently overexpressing either LMW or HMW FGF-2 isoforms concomitant with GFPsaph from a single bicistronic mRNA were built. Viable cell number in ROS 17/2.8 cells stably transfected with Vector (Col3.6-IRES-GFPsaph) versus each of the Col3.6-Fgf2-IRES-GFPsaph constructs were compared. In the presence of 1 or 10% serum, DNA synthesis was increased in cells expressing any isoform of FGF-2 compared with vector. However, cells transfected with HMW isoform had augmented DNA synthesis in 1 or 10% serum compared with cells expressing either ALL or LMW FGF-2 isoforms. A neutralizing FGF-2 antibody significantly reduced the mitogenic response in cells harboring ALL or the LMW FGF-2 isoforms but did not block the mitogenic effect of cells harboring the HMW isoforms. In summary, overexpression of any isoform of FGF-2 protein increased viable cell number and OB proliferation in the presence of low or high concentrations of serum. However, the HMW/nuclear isoforms preferentially mediate augmented OB proliferation. We conclude that differential expression of FGF-2 proteins isoforms is important in modulating OB function.
Xiao
L
L
Department of Medicine, University of Connecticut Health Center, Farmington, Connecticut 06030, USA.
Liu
P
P
Sobue
T
T
Lichtler
A
A
Coffin
J D
JD
Hurley
M M
MM
D000818:Animals; D015152:Blotting, Northern; D015153:Blotting, Western; D002455:Cell Division / Q000235:genetics; D002470:Cell Survival; D003001:Cloning, Molecular; D016222:Fibroblast Growth Factor 2 / Q000096:biosynthesis* / Q000235:genetics; D005455:Fluorescent Antibody Technique; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins; D008856:Microscopy, Fluorescence; D010006:Osteoblasts / Q000378:metabolism*; D020033:Protein Isoforms / Q000096:biosynthesis* / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D013997:Time Factors; D014162:Transfection; D019076:Transgenes
D008164:Luminescent Proteins; D020033:Protein Isoforms; D012333:RNA, Messenger; D016222:Fibroblast Growth Factor 2; D049452:Green Fluorescent Proteins