Characterization of Functional Reprogramming during Osteoclast Development Using Quantitative Proteomics and mRNA Profiling

Abstract

In addition to forming macrophages and dendritic cells, monocytes in adult peripheral blood retain the ability to develop into osteoclasts, mature bone-resorbing cells. The extensive morphological and functional transformations that occur during osteoclast differentiation require substantial reprogramming of gene and protein expression. Here we employ -omic-scale technologies to examine in detail the molecular changes at discrete developmental stages in this process (precursor cells, intermediate osteoclasts, and multinuclear osteoclasts), quantitatively comparing their transcriptomes and proteomes. The data have been deposited to the ProteomeXchange with identifier PXD000471.Our analysis identified mitochondrial changes, along with several alterations in signaling pathways, as central to the development of mature osteoclasts, while also confirming changes in pathways previously implicated in osteoclast biology. In particular, changes in the expression of proteins involved in metabolism and redirection of energy flow from basic cellular function toward bone resorption appeared to play a key role in the switch from monocytic immune system function to specialized bone-turnover function. These findings provide new insight into the differentiation program involved in the generation of functional osteoclasts.

Comparative Characterization of Osteoclasts Derived From Murine Bone Marrow Macrophages and RAW 264.7 Cells Using Quantitative Proteomics

“…First, the previous study used clonal populations of RAW 264.7 cells deemed to possess high osteoclastogenic potential; using purified precursor cells could enrich for genes related to OC differentiation. Second, numerous studies have consistently shown that gene expression levels do not correlate well with protein abundance; this phenomenon could therefore contribute to the discrepancy between the correlation analyses based on cDNA levels versus protein levels . In line with this thought, we assessed the protein levels of housekeeping genes reported to be stable at the transcript level in OCs and found that transcript and protein levels for some of these genes were inconsistent (Fig.…”

“…In BMMs, B2m was transiently upregulated, whereas Hprt was downregulated during OC differentiation; none of these proteins, however, were significantly altered in RAW264.7 cells. Because numerous studies have identified major discrepancies in the correlation between mRNA and protein expression levels, we were interested in whether housekeeping proteins exhibited any differences in OCs. In contrast to the findings by Stephens and colleagues, we observed that Gapdh levels were consistent throughout OC differentiation in BMMs, whereas B2m and Hprt were not, indicating that these two proteins may not be reliable internal controls for protein samples.…”

“…We employed a cutting‐edge proteomics strategy that pushes the boundaries of protein identification and quantification; consequently, our study provides the most comprehensive coverage of the OC proteome to date. In two most recent proteomics studies of OC differentiation from RAW 264.7 cells, one study identified 2068 nonredundant proteins (defined as ≥2 peptides per protein for identification with a 5% FDR) and the other identified 3729 unique proteins (≥2 peptides/protein and 1% FDR) . In comparison, our study reliably identified 5566 unique proteins, a 49.2% increase in coverage, in RAW 264.7 cells using a similarly stringent criteria of ≥2 peptides per protein for identification with a 5% FDR (Fig.…”

“…Notably, this technique was recently used to map the podosome proteome and helped to advance our understanding of determinants in the macrophage multinucleation process . In other examples, proteomics analysis identified changes in the expression of proteins involved in metabolism and redirection of energy flow toward bone resorption in OCs . Proteomics can therefore provide a broad yet informative overview of the systemic changes in the differentiating OC.…”

Investigation of proteome changes in osteoclastogenesis in low serum culture system using quantitative proteomics

“…Our previous study revealed that a number of proteins and molecular signaling pathways, including electron transport chain and oxidative phosphorylation pathways, were altered in RAW264.7 cells cultured in low serum compared to those cultured in the conventional 10 % culture system [ 13 ]. An et al have performed proteomics analysis to study the changes in global protein expression during osteoclastogenesis in conventional culture system [ 14 ]. However, to the best of our knowledge, the proteomic changes that occur in RAW 264.7 cells as they differentiate into osteoclasts in a low serum culture system has not been reported.…”