Erk1 Plays Critical Role in Macrophage Development

Abstract 517

Extracellular signal-regulated kinase (ERK 1 and 2) are widely expressed and are involved in the regulation of meiosis, mitosis, and postmitotic functions in multiple cell lineages, including T cells, B cells and osteoblasts. Macrophages are capable of differentiating into osteoclasts, which resorb bone. Abnormal osteoclast development and functions underlie certain diseases, especially skeletal defects. Altered ERK1/2 signaling has been found in several genetic diseases with skeletal phenotypes, including Noonan syndrome, polycystic kidney disease and serious developmental disorders such as cardio-facio-cutaneous syndrome. These clinical findings suggest the importance of the ERK MAPK pathway in human skeletal development. In the present study, we examined the consequence of Erk1 and Erk2 disruption in modulating macrophage development in the murine system. We found that deletion of Erk1 reduced macrophage progenitor numbers. Erk1^−/− bone marrow mononuclear cells (BMMNCs) had significant reduction in osteoclast formation as compared to wildtype BMMNCs. In addition, Erk1^−/− macrophages; the osteoclast progenitors, had a two-three fold reduction in migration and a two-fold reduction in αv ß3 mediated adhesion as compared to WT macrophages as evaluated by transwell and adhesion assay, respectively. These in vitro data demonstrate that Erk1 positively regulates macrophage differentiation into osteoclasts.

To evaluate the impact of deficiency of Erk1 in vivo, we examined bone mineral density and trabecular microarchitecture in the distal femoral metaphysis by dual-energy X-ray absorptiometry (DEXA) with a Lunar Piximus densitometer and a high-resolution desktop microcomputed tomography imaging system (μCT-20; Scanco Medical AG, Basserdorf, Switzerland), respectively. Erk1^−/− mice displayed elevated bone mineral density and increased trabecular bone formation as compared to WT mice. Histomorphometric analysis indicated that the Erk1^−/− femur had significant reduction in osteoclast numbers as determined by tartrate resistant acid phosphatase staining, an osteoclast specific staining, as compared to femur of wildtype and Erk2^−/− mice. Most importantly, Erk1^−/− plasma had reduced C-terminal telopeptide of type I collagen, indicating less bone resorption in vivo. These data suggest that the impaired macrophage differentiation and osteoclast bone resorptive activity play an important role in increased bone mass in Erk1^−/− mice.

Finally, to verify that the macrophage-osteoclast lineage is a key cell lineage for the phenotypic changes in vivo in Erk1^−/− mice, we performed bone marrow transplantation. WT mice reconstituted long-term with Erk1^−/− hematopoietic stem cells demonstrated increased bone mineral density as compared to WT and Erk2^−/− stem cell recipients, implicating marrow autonomous, Erk1-dependent macrophage differentiation and osteoclast bioactivity in vivo. Collectively, our in vitro and in vivo data demonstrate isoform-specific Erk function in macrophage while providing rationale for the development of a specific inhibitor for Erk1 that might be used for the treatment of dysplastic and erosive bone diseases.