Regulation of bone formation by osteoclasts involves Wnt/BMP signaling and the chemokine sphingosine-1-phosphate

Abstract

Under most conditions, resorbed bone is nearly precisely replaced in location and amount by new bone. Thus, it has long been recognized that bone loss through osteoclast-mediated bone resorption and bone replacement through osteoblast-mediated bone formation are tightly coupled processes. Abundant data conclusively demonstrate that osteoblasts direct osteoclast differentiation. Key questions remain, however, as to how osteoblasts are recruited to the resorption site and how the amount of bone produced is so precisely controlled. We hypothesized that osteoclasts play a crucial role in the promotion of bone formation. We found that osteoclast conditioned medium stimulates human mesenchymal stem (hMS) cell migration and differentiation toward the osteoblast lineage as measured by mineralized nodule formation in vitro. We identified candidate osteoclast-derived coupling factors using the Affymetrix microarray. We observed significant induction of sphingosine kinase 1 (SPHK1), which catalyzes the phosphorylation of sphingosine to form sphingosine 1-phosphate (S1P), in mature multinucleated osteoclasts as compared with preosteoclasts. S1P induces osteoblast precursor recruitment and promotes mature cell survival. Wnt10b and BMP6 also were significantly increased in mature osteoclasts, whereas sclerostin levels decreased during differentiation. Stimulation of hMS cell nodule formation by osteoclast conditioned media was attenuated by the Wnt antagonist Dkk1, a BMP6-neutralizing antibody, and by a S1P antagonist. BMP6 antibodies and the S1P antagonist, but not Dkk1, reduced osteoclast conditioned media-induced hMS chemokinesis. In summary, our findings indicate that osteoclasts may recruit osteoprogenitors to the site of bone remodeling through SIP and BMP6 and stimulate bone formation through increased activation of Wnt/BMP pathways.

Fig. 1.

Influences of osteoclast conditioned media on hMS cell mineralization. (A) Marrow-derived osteoclast precursor conditioned media or mature osteoclast conditioned media (Left) or RAW 264.7 (RAW) cell conditioned media and mature multinucleated RAW-derived osteoclast conditioned media (Right) were added to hMS cells. Cultures were fixed after 3 weeks (marrow-derived CM) or 1 week (RAW-derived CM) and stained for mineralization by von Kossa or Alizarin red as indicated. (B) Alizarin red was quantified by extraction as described in Experimental Procedures (n = 3 replicates). *, P < 0.05 comparing precursor to mature osteoclast conditioned media. These results are representative of 3 experiments. (C) Human osteoclast precursors were cultured, and CM was collected on day 7 (D7; mature osteoclasts are present after day 5). Cells were fed, and CM was collected on day 14 (D14; mature osteoclasts present throughout). CMs or feeding media were concentrated and added to hMS cells as detailed in Experimental Procedures. Cultures were fixed after 10 days and stained with Alizarin red before quantitation (n = 3 replicates). *, P < 0.05 compared with media; δ, P < 0.05 compared with D7.

Fig. 2.

Coupling factor gene expression changes during osteoclast differentiation. (A, C, E, and G) Bone marrow cells were cultured with M-CSF and RANKL for 3, 4, or 5 days as described in Experimental Procedures. Mature osteoclasts appear by day 4 and peak between days 4 and 5. (B, D, F, and H) RAW 264.7 cells were either maintained in the absence of RANKL or cultured with 200 ng/mL RANKL for the indicated number of days. RNA was harvested for real-time PCR analyses (n = 3 replicates). *, P < 0.05 compared with time 0. These results are representative of 3 experiments.

Fig. 3.

Coupling factor protein expression changes during osteoclast differentiation. (A) Bone marrow cells were cultured with M-CSF and RANKL for the indicated time. Cells were either harvested (MARROW CELL EXTRACTS) or switched to serum-free media for 3 days before media collection and 10-fold concentration (MARROW CELL CONDITIONED MEDIA) before Western blotting. (B) RAW cells with or without RANKL treatment for the indicated time were either harvested for Western blotting (RAW CELL EXTRACTS) or rinsed and maintained in serum-free media for 3 days before media collection and 10-fold concentration (RAW CONDITIONED MEDIA) before Western blotting. All samples were normalized to cell extract protein. These results are representative of 2 experiments.

Fig. 4.

Wnt and BMP responsive reporter activation by osteoclast conditioned media. hMS cells were transiently transfected with 1 μg of empty reporter vector (vector), the BMP-responsive reporter SMAD6-luc (A), or the Wnt-responsive Tcf/Lef reporter TOP-FLASH-luc (B). Forty-eight hours later, the cells were treated with the indicated conditioned media for 48 h (n = 3 replicates). *, P < 0.05 compared with precursor CM. These results are representative of 2 experiments.

Fig. 5.

Blocking coupling factors reduces osteoclast conditioned media-induced hMS cell mineralization. RAW 264.7 precursor (−RL) and multinucleated RAW 264.7-derived osteoclast (+RL) conditioned media were assayed for their effects on osteoblast mineralization. DKK1, control antibody (CONT Ab), αBMP6 antibody, vehicle for the S1P receptor 1 antagonist (VEH), or a S1P receptor 1 antagonist alone or in combination were added to the osteoclast conditioned media as indicated for 7 days before fixing, Alizarin red staining, and quantitation as detailed in Experimental Procedures (n = 3 replicates). *, P < 0.05 compared with vehicle. These results are representative of 2 experiments.

Fig. 6.

hMS cell chemokinesis increases with osteoclast conditioned media treatment. hMS cells were grown on coverslips to confluence and assayed for chemokinesis as detailed in Experimental Procedures. (A) Coverslips were fixed (zero) or incubated in the indicated medium (n = 3 per treatment). Sixteen hours later, the remaining cells were fixed and stained. Cells were counted at 200×. *, P < 0.05 compared with other treatments. (B) hMS cells were treated as outlined in A before addition(s) as indicated. Cell counts were normalized to the respective vehicle control (n = 3 replicates). *, P < 0.05 compared with controls. These results are representative of 2 experiments.