Comparative study of protein and mRNA expression during osteoclastogenesis

Abstract

Osteoclasts, the bone-digesting cells, are key players in bone remodeling. To identify proteins potentially involved in osteoclast function, we analyzed the patterns of protein expression during osteoclastogenesis by2-D DIGE. As a model system we used the mouse myeloid Raw 264.7 cell line that differentiates in vitro into osteoclasts upon treatment with specific growth factors. In 2-D DIGE, we identified 86 up- and 34 down-regulated proteins including known osteoclast differentiation markers as well as proteins regulating key cellular functions of osteoclasts such as energy production, cytoskeleton dynamics, and digestion of organic and inorganic bone matrix. Comparison of protein expression using 2-D DIGE techniques with mRNA expression analyzed by DNA microarrays revealed essentially two groups of genes. The first group comprises genes for which differences in both mRNA and protein expressions were found. A second group covers genes whose expression was not altered at the mRNA level but whose corresponding gene products exhibited different electrophoretic mobilities, thereby revealing potential changes in post-transcriptional processing and PTM. Thus, these combined approaches identify new potential therapeutic targets for treatment of bone diseases and provide complementary information on regulatory processes that might affect osteoclastogenesis.

Systems level analysis of osteoclastogenesis reveals intrinsic and extrinsic regulatory interactions

“…Several previous studies on osteoclastogenesis have used RAW264.7, a transformed monocytic cell line (Ishida et al, 2002;Czupalla et al, 2005;Nomiyama et al, 2005). Curiously, while RANKL induces RAW264.7 cells to become osteoclastlike, many of the processes observed here, including regulation of apoptosis, secretion of pro-angiogenic factors, the regulation of and by cholesterol levels, have not been described for the cell lines.…”

microRNA Expression in Rat Apical Periodontitis Bone Lesion

“…Bone resorption is a multistep process initiated by the proliferation of immature osteoclast precursors, the commitment of these cells to the osteoclast phenotype, and finally, degradation of the organic and inorganic phases of bone by the mature resorptive cells (45). Each stage of osteoclast differentiation requires regulatory factors, that also serve as markers of osteoclast function, and bone resorbing activity (46)(47)(48), and could be regulated by microRNAs through the specific targeting with the protein-coding gene mRNAs.…”

Src-dependent repression of ARF6 is required to maintain podosome-rich sealing zones in bone-digesting osteoclasts

“…To elucidate this regulatory mechanism, it will be important to clearly identify the effector molecules coordinating Rho and ARF6 signaling. We took complementary approaches to identify genes whose expression is drastically modified during osteoclastogenesis (35). Besides Vav proteins, this analysis highlighted several other regulators of Rho GTPases.…”

“…Production of RANKL, cultivation of Raw 264.7 cells (American Type Culture Collection), and differentiation toward osteoclasts were performed as previously described (35). At day 4 of differentiation, cells were transferred to BD BioCoat osteologic discs (BD Bioscience) for microscopy.…”