ELMO1 Regulates RANKL-Stimulated Differentiation and Bone Resorption of Osteoclasts

Abstract

Bone homeostasis is a metabolic balance between the new bone formation by osteoblasts and old bone resorption by osteoclasts. Excessive osteoclastic bone resorption results in low bone mass, which is the major cause of bone diseases such as rheumatoid arthritis. Small GTPases Rac1 is a key regulator of osteoclast differentiation, but its exact mechanism is not fully understood. ELMO and DOCK proteins form complexes that function as guanine nucleotide exchange factors for Rac activation. Here, we report that ELMO1 plays an important role in differentiation and bone resorption of osteoclasts. Osteoclast precursors derived from bone marrow monocytes (BMMs) of Elmo1^-/- mice display defective adhesion and migration during differentiation. The cells also have a reduced activation of Rac1, p38, JNK, and AKT in response to RANKL stimulation. Importantly, we show that bone erosion is alleviated in Elmo1^-/- mice in a rheumatoid arthritis mouse model. Taken together, our results suggest that ELMO1, as a regulator of Rac1, regulates osteoclast differentiation and bone resorption both in vitro and in vivo.

Keywords: ELMO1; adhesion; bone resorption; migration; osteoclast differentiation.

FIGURE 1

Elmo1-deficient osteoclasts show impaired bone resorption activity and differentiation. (A) Pit formation assay. BMMs were cultured with M-CSF alone or M-CSF and RANKL on inorganic crystalline calcium phosphate plates. Attached cells were removed and resorption lacunae were visualized by bright-field microscopy. One representative photograph is shown. Scale bar, 500 μm. (B) Pit areas were quantified using ImageJ and graphed. Data are indicated as means ± SEM (n = 3). (C) RANKL-induced osteoclast differentiation of BMMs. Mouse BMMs were cultured in the presence of M-CSF and RANKL for 4 days. The cells were fixed and stained for TRAP staining. One representative picture is shown. Scale bar, 100 μm. (D) The numbers of TRAP-positive cells were counted and graphed. Data are indicated as means ± SEM (n = 13). (E–G) Relative mRNA level of TRAP, NFATc1, and DC-STAMP. Osteoclast precursors were treated with M-CSF and RANKL for 4 days, then relative mRNA level of TRAP (n = 6), NFATc1 (n = 4), and DC-STAMP (n = 3) were determined by qRT-PCR. Data are indicated as means ± SEM. Statistical significance was assessed by t-test, *P < 0.05 and ***P < 0.001.

FIGURE 2

Effect of Elmo1 deficiency on apoptosis, adhesion and migration of osteoclast precursors. (A) Apoptosis of osteoclast precursors. RANKL-induced osteoclasts were stained with Annexin V-FITC and PI to detect the apoptosis by flow cytometry. One representative result is shown. (B) The percentage of Annexin V-FITC positive cells were quantified with FlowJo software and the data was indicated as means ± SEM (n = 4). (C) Osteoclast precursors adhesion assay. Cells were incubated at 37°C for 0 or 60 min, and stained with crystal violet followed by DMSO dissolving for measuring absorbance at 570 nm. The data was indicated as means ± SEM (n = 4). (D) Relative mRNA level of integrin β3 in osteoclast precursors. Cells were treated with RANKL for 4 days in the presence of M-CSF, then relative mRNA level of integrin β3 was determined by qRT-PCR. The data was indicated as means ± SEM (n = 3). (E) Osteoclast precursors migration assay. Cells were allowed to migrate under stimulation of M-CSF for 24 h. Migrated cell numbers in the bottom chamber were counted. The data was indicated as means ± SEM (n = 3). Statistical significance was assessed by t-test, *P < 0.05, **P < 0.01, ***P < 0.001, and ^nsP > 0.05.

FIGURE 3

Elmo1 upregulates RANKL-induced activation of Rac1, MAPKs, and AKT. (A) RANKL-induced Rac1 activation assay. Osteoclast precursors were lysed after RANKL stimulation for 15 and 30 min. The lysates were incubated with PAK-PBD beads, and proteins complexed to the beads were subjected to SDS-PAGE and analyzed by immunoblotting using anti-Rac1. (B) The ratio of Rac1-GTP to total Rac1 was quantified and graphed. Data are indicated as means ± SEM (n = 3). (C) Osteoclast precursors were stimulated by RANKL for the indicated time. Cell lysates were subjected to SDS-PAGE and analyzed by immunoblotting for detecting phosphorylation form and total p38, JNK, ERK, and AKT. (D–G) The intensity of phosphorylated p38 (n = 3), JNK (n = 4), ERK (n = 3), and AKT (n = 3) was quantified by densitometry using ImageJ software and expressed as the ratio of phosphorylated form to total protein. Data are indicated as means ± SEM. Statistical significance was assessed by t-test, *P < 0.05, **P < 0.01, ***P < 0.001, and ^nsP > 0.05.

FIGURE 4

Bone erosion of Elmo1^–/– mice is alleviated in rheumatoid arthritis. Generation of serum transfer mouse model of arthritis was performed as mentioned in the materials and methods. (A) Arthritis was monitored by clinical index. Data are indicated as means ± SEM (n = 6). (B) Measurement of ankle thickness over time. Data are indicated as means ± SEM (n = 6). (C) Representative micro-CT images of ankle joints are shown. (D) Bone volume as a fraction of total bone volume (BV/TV) of ankle joints cortical bone. Data are indicated as means ± SEM (n = 6). (E) H&E-stained corresponding histological joint sections. Yellow arrow indicates site of bone erosion. Scale bar, 200 μm. Statistical significance was assessed by t-test, *P < 0.05, **P < 0.01, and ***P < 0.001.