The murine osteoprogenitor cell line, KS483 (Percuros, The Netherlands) is a well-established model for investigation of osteoblast differentiation and bone formation processes. The mesenchymal characteristics of this cell line allow it to differentiate into either adipocytes or mature, mineralizing osteoblasts. Various phases can be distinguished during osteoblast differentiation and maturation; namely proliferation, matrix formation, matrix maturation, and mineralization.
We now performed whole genome expression analysis of mRNA in order to gain additional insight into the molecular mechanisms driving these processes with the focus on Wnt/β-catenin signaling involvement.
KS483 cells were differentiated for 28 days in two biological replicates. Total cellular RNA was isolated at eight time points during this process and cRNA was generated. Expression of over 19 100 unique genes was assessed with the use of MouseRef-8 v2.0 Expression BeadChips. On average 7000 genes displayed robust expression at each time point. Comprehensive analysis of the genes related to bone formation was performed with the focus on Wnt/β-catenin signaling. Six Wnt genes were expressed throughout the differentiation process, namely Wnt5a, Wnt5b, Wnt6, Wnt7b, Wnt10a, and Wnt10b. Expression of seven members of the LRP family of Wnt co-receptors was detected; namely Lrp1, Lrp4, Lrp5, Lrp6, Lrp10, Lrp11, and Lrp12 with Lrp10 being the most abundant. Predominant role of Lrp6 over Lrp5 can be suggested as it displays, on average, threefold higher expression. Expression of known modulators of Wnt/β-catenin pathway was also investigated. For example for the R-spondin family of Wnt activators, Rspo2 and Rspo3 expression was detected and was accompanied by Lgr5 expression in the late stages of differentiation.
In conclusion, we were able to provide a useful and informative tool to investigate the osteoblast differentiation and bone formation. We believe that it grants valuable insight into the molecular mechanisms underlying these processes.
18 - 21 May 2013
European Calcified Tissue Society