Cloning and characterization of osteoclast precursors from the RAW264.7 cell line - PubMed (original) (raw)

Cloning and characterization of osteoclast precursors from the RAW264.7 cell line

Bethany L V Cuetara et al. In Vitro Cell Dev Biol Anim. 2006 Jul-Aug.

Abstract

Osteoclasts are bone-resorbing cells that differentiate from macrophage precursors in response to receptor activator of NF-kappaB ligand (RANKL). In vitro models of osteoclast differentiation are principally based on primary cell cultures, which are poorly suited to molecular and transgene studies because of the limitations associated with the use of primary macrophage. RAW264.7 is a transfectable macrophage cell line with the capacity to form osteoclast-like cells. In the present study, we have identified osteoclast precursors among clones of RAW264.7 cells. RAW264.7 cell were cloned by limiting dilution and induced to osteoclast differentiation by treatment with recombinant RANKL. Individual RAW264.7 cell clones formed tartrate resistant acid phosphatase (TRAP)-positive multinuclear cells to various degrees with RANKL treatment. All clones tested expressed the RANKL receptor RANK. Each of the clones expressed the osteoclast marker genes TRAP and cathepsin-K mRNA with RANKL treatment. However, we noted that only select clones were able to form large, well-spread, TRAP-positive multinuclear cells. Clones capable of forming large TRAP-positive multinuclear cells also expressed beta3 integrin and calcitonin receptor mRNAs and were capable of resorbing a mineralized matrix. All clones tested activated NF-kappaB with RANKL treatment. cDNA expression profiling of osteoclast precursor RAW264.7 cell clones demonstrates appropriate expression of a large number of genes before and after osteoclastic differentiation. These osteoclast precursor RAW264.7 cell clones provide a valuable model for dissecting the cellular and molecular regulation of osteoclast differentiation and activation.

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Figures

Figure 1

Osteoclast formation in RAW264.7 cell clones. RAW264.7 cell clones were plated in 6 well dishes at 1X105 cells / well and were grown for 5 days with RANKL (20ng/ml) or without (control). Cells were fixed and stained for TRAP activity. Clones displaying a representative range of morphologies are shown (original magnification 40X).

Figure 2

Osteoclast marker gene expression by RAW264.7 clones. Total RNA was purified from each of 12 clones, grown with (+) or without (-) 20ng/ml RANKL, as described in figure 1. Total RNA was used as template for RT-PCR reactions with primers for osteoclast gene markers, TRAP, CTR, Cath-K, β3. Controls included RT-PCR with RNA from primary bone marrow macrophage (Mφ) and osteoclasts derived from them by RANKL treatment (Oc).

Figure 3

Hydroxyapatite resorption by RAW264.7 cell clones. RAW264.7 cell clones C1-C12 (numbered 1 - 12) grown on Osteologic slides (synthetic calcium phosphate thin films) with 20 ng/ml RANKL or without RANKL (not shown). Cells were removed and mineral was stained with Von Kossa's stain. Clones phenotypically similar to osteoclasts (figure 1 and figure 2) are capable of resorption (original magnification 10X).

Figure 4

NF-κB activation by RANKL in RAW264.7 cell clones. EMSA analysis of nuclear extracts from clones C1-C12 without (-) or with (+) RANKL treatment (100ng/ml for 15 min). Equal volumes of nuclear extract from treated and control samples were incubated with radiolabeled NF-κB oligonucleotide probe for 30 min on ice. Bound complexes were separated by electorphoresis on 5% polyacrylamide in 1X TBE. The arrow indicates the position of the NF-κB containing complex. All clones are capable of RANKL-dependent NF-κB activation.

Figure 5

Gene expression in primary cells vs RAW267.4 cell clones. (A) Dense arrays were hybridized with cDNA probes from RNAs of RAW264.7 clones and mouse primary BMMs. Autoradiographs were scanned and relative gene expression levels were determined. The relative expression levels from 3 RAW264.7 clones (C3, C6, & C11) were averaged. The primary macrophage and RAW cell genes with relative expression levels above 100 were plotted (approximately 712 genes). The circled point corresponds to c-myc. (B) cDNA probes from primary osteoclasts and RAW264.7 cell-derived osteoclasts. Arrays were hybridized and analyzed as above. The relative expression levels from 3 clones (C3, C6, & C11) were averaged and the primary osteoclast and RAW264.7 osteoclast genes with relative expression levels above 100 were plotted (approximately 405 genes).

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Cloning and characterization of osteoclast precursors from the RAW264.7 cell line - PubMed (original) (raw)