title: BMP-2 differentially modulates FGF-2 isoform effects in osteoblasts from newborn transgenic mice.
abstract: We previously generated separate lines of transgenic mice that specifically overexpress either the Fibroblast growth factor (FGF)-2 low-molecular-mass isoform (Tg(LMW)) or the high-mass isoforms (Tg(HMW)) in the osteoblast lineage. Vector/control (Tg(Vector)) mice were also made. Here we report the use of isolated calvarial osteoblasts (COBs) from those mice to investigate whether the FGF-2 protein isoforms differentially modulate bone formation in vitro. Our hypothesis states that FGF-2 isoforms specifically modulate bone morphogenetic protein 2 (BMP-2) function and subsequently bone differentiation genes and their related signaling pathways. We found a significant increase in alkaline phosphatase-positive colonies in Tg(LMW) COBs compared with Tg(Vector) controls. BMP-2 treatment significantly increased mineralized colonies in Tg(Vector) and Tg(LMW) COBs. BMP-2 caused a further significant increase in mineralized colonies in Tg(LMW) COBs compared with Tg(Vector) COBs but did not increase alkaline phosphatase-positive colonies in Tg(HMW) COBs. Time-course studies showed that BMP-2 caused a sustained increase in phosphorylated mothers against decapentaplegic-1/5/8 (Smad/1/5/8), runt-related transcription factor-2 (Runx-2), and osterix protein in Tg(LMW) COBs. BMP-2 caused a sustained increase in phospho-p38 MAPK in Tg(Vector) but only a transient increase in Tg(LMW) and Tg(HMW) COBs. BMP-2 caused a transient increase in phospho-p44/42 MAPK in Tg(Vector) COBs and no increase in Tg(LMW) COBs, but a sustained increase was found in Tg(HMW) COBs. Basal expression of FGF receptor 1 protein was significantly increased in Tg(LMW) COBs relative to Tg(Vector) COBs, and although BMP-2 caused a transient increase in FGF receptor 1 expression in Tg(Vector) COBs and Tg(HMW) COBs, there was no further increase Tg(LMW) COBs. Interestingly, although basal expression of FGF receptor 2 was similar in COBs from all genotypes, BMP-2 treatment caused a sustained increase in Tg(LMW) COBs but decreased FGF receptor 2 in Tg(Vector) COBs and Tg(HMW) COBs.
authors.affiliation: School of Biosciences and Biotechnology, University of Camerino, Camerino, MC, 62032 Italy. giovanna.sabbieti@unicam.it
authors.forename: Maria Giovanna
authors.initials: MG
authors.lastname: Sabbieti
authors.forename: Dimitrios
authors.initials: D
authors.lastname: Agas
authors.forename: Luigi
authors.initials: L
authors.lastname: Marchetti
authors.forename: J Douglas
authors.initials: JD
authors.lastname: Coffin
authors.forename: Liping
authors.initials: L
authors.lastname: Xiao
authors.forename: Marja M
authors.initials: MM
authors.lastname: Hurley
mesh_terms: D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D000831:Animals, Newborn; D015153:Blotting, Western; D055396:Bone Morphogenetic Protein 2 / Q000494:pharmacology*; D002478:Cells, Cultured; D050659:Core Binding Factor Alpha 1 Subunit / Q000378:metabolism; D005260:Female; D016222:Fibroblast Growth Factor 2 / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008297:Male; D051379:Mice; D008822:Mice, Transgenic; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D010006:Osteoblasts / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D034622:RNA Interference; D051496:Receptor, Fibroblast Growth Factor, Type 1 / Q000235:genetics / Q000378:metabolism; D051497:Receptor, Fibroblast Growth Factor, Type 2 / Q000235:genetics / Q000378:metabolism; D012886:Skull / Q000166:cytology; D051785:Smad Proteins / Q000378:metabolism; D000076182:Sp7 Transcription Factor; D014157:Transcription Factors / Q000378:metabolism
chemical_list: D055396:Bone Morphogenetic Protein 2; D050659:Core Binding Factor Alpha 1 Subunit; D020033:Protein Isoforms; C493716:Runx2 protein, mouse; D051785:Smad Proteins; D000076182:Sp7 Transcription Factor; C479466:Sp7 protein, mouse; D014157:Transcription Factors; D016222:Fibroblast Growth Factor 2; C496349:Fgfr1 protein, mouse; C496354:Fgfr2 protein, mouse; D051496:Receptor, Fibroblast Growth Factor, Type 1; D051497:Receptor, Fibroblast Growth Factor, Type 2; D020928:Mitogen-Activated Protein Kinases; D000469:Alkaline Phosphatase