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

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.

Signals for stress erythropoiesis are integrated via an erythropoietin receptor-phosphotyrosine-343-Stat5 axis.

Anemia due to chronic disease or chemotherapy often is ameliorated by erythropoietin (Epo). Present studies reveal that, unlike steady-state erythropoiesis, erythropoiesis during anemia depends sharply on an Epo receptor-phosphotyrosine-343-Stat5 signaling axis. In mice expressing a phosphotyrosine-null (PY-null) Epo receptor allele (EpoR-HM), severe and persistent anemia was induced by hemolysis or 5-fluorouracil. In short-term transplantation experiments, donor EpoR-HM bone marrow cells also failed to efficiently repopulate the erythroid compartment. In each context, stress erythropoiesis was rescued to WT levels upon the selective restoration of an EpoR PY343 Stat5-binding site (EpoR-H allele). As studied using a unique primary culture system, EpoR-HM erythroblasts exhibited marked stage-specific losses in Epo-dependent growth and survival. EpoR-H PY343 signals restored efficient erythroblast expansion, and the selective Epo induction of the Stat5 target genes proviral integration site-1 (Pim-1) and oncostatin-M. Bcl2-like 1 (Bcl-x), in contrast, was not significantly induced via WT-EpoR, EpoR-HM, or EpoR-H alleles. In Kit+ CD71+ erythroblasts, EpoR-PY343 signals furthermore enhanced SCF growth effects, and SCF modulation of Pim-1 kinase and oncostatin-M expression. In maturing Kit- CD71+ erythroblasts, oncostatin-M exerted antiapoptotic effects that likewise depended on EpoR PY343-mediated events. Stress erythropoiesis, therefore, requires stage-specific EpoR-PY343-Stat5 signals, some of which selectively bolster SCF and oncostatin-M action.