ER-HOXB8 cell line, a new tool for the study of osteoclasts in osteoarthritis.

Purpose: Osteoclasts are specialised bone resorbing cells which are recruited to subchondral bone in osteoarthritis and may contribute to bone and cartilage degradation. To understand the mechanism by which osteoclasts degrade bone, in vivo studies are performed using mouse models and in vitro tests based on bone marrow derived cells. Disadvantages of this approach are the large use of animals, limitation in the number of cells available and heterogeneity in the bone marrow starting population. The use of a well-defined cell line could allow to overcome these limitations and simplify the study of the complex biology of these cells. ER-Hoxb8 cells are conditionally immortalized monocyte/ macrophage murine progenitor cell line, with estrogen-dependent production of Hoxb8, a crucial transcription factor blocking myeloid differentiation. Hoxb8 cells have been already described to efficiently differentiate into functional macrophages upon estrogen withdrawal. In the present study we investigated whether the Hoxb8 cell line can also differentiate into functional osteoclasts comparable to bone marrow derived osteoclasts. Methods: Immortalized ER-Hoxb8 cell lines were generated from BM cells of C57Bl/6 wild type or TLR4 -/- mice and expanded in presence of estrogen. Upon estrogen withdrawal, ER-Hoxb8 cells were differentiated with M-CSF and RANK-L towards osteoclasts on plastic, bone and dentin slices and on calcium phosphate coated plates. BM cells were cultured in the same conditions. Expression of osteoclast differentiation markers was determined by quantitative RT-PCR analysis. Staining for TRAP activity was performed to prove the differentiation into osteoclasts. Presence of actin rings was determined through F-Actin staining. Coomassie Brillant Blue or Von Kossa staining were used to monitor resorption activity. Results: ER-Hoxb8 cells differentiated with high efficiency into multinucleated osteoclasts, positive for TRAP activity and with a morphology that was not different from BM derived osteoclasts. Osteoclast differentiation markers (c-Fms, RANK, NFATc1, DC - STAMP, TRAP, CatK, CTR) were strongly upregulated in ER-Hoxb8 derived osteoclasts compared to undifferentiated ER- Hoxb8 and ER-Hoxb8 derived macrophages. Moreover also during the differentiation process ER- Hoxb8 osteoclasts showed comparable kinetic and expression levels of differentiation markers. Confocal analysis showed that ER-Hoxb8 derived osteoclasts formed actin-rings, a crucial prerequisite for bone resorption. Development of resorption pits on bone and dentin slices and on calcium phosphate coated plate demonstrated that ER-Hoxb8 derived osteoclasts are functionally active in bone degradation. Next we developed ER - Hoxb8 cells from bone marrow cells from TLR4 - / - mice. A major advantage of the ER-hoxb8 approach is to generate immortalized myeloid precursor cells from bone marrow of animals with different genetic backgrounds, allowing study of the function of disregulated genes in osteoclast differentiation and activity. ER - Hoxb8 cells deficient for TLR4 differentiate into mature osteoclasts not different from WT controls. TLR4-/- osteoclasts efficiently resorbed bone but lacked the ability to respond to LPS when compared to WT ER-Hoxb8. Conclusions: ER-Hoxb8 derived osteoclasts form a useful tool to study osteoclasts that allow the reduction in the number of animal used for in vitro experimentation and constitute a potentially unlimited source of osteoclasts with different genetic background.