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.