Osteoclasts control osteoblast chemotaxis via PDGF-BB/PDGF receptor beta signaling

Abstract

Background: Bone remodeling relies on the tightly regulated interplay between bone forming osteoblasts and bone digesting osteoclasts. Several studies have now described the molecular mechanisms by which osteoblasts control osteoclastogenesis and bone degradation. It is currently unclear whether osteoclasts can influence bone rebuilding.

Methodology/principal findings: Using in vitro cell systems, we show here that mature osteoclasts, but not their precursors, secrete chemotactic factors recognized by both mature osteoblasts and their precursors. Several growth factors whose expression is upregulated during osteoclastogenesis were identified by DNA microarrays as candidates mediating osteoblast chemotaxis. Our subsequent functional analyses demonstrate that mature osteoclasts, whose platelet-derived growth factor bb (PDGF-bb) expression is reduced by siRNAs, exhibit a reduced capability of attracting osteoblasts. Conversely, osteoblasts whose platelet-derived growth factor receptor beta (PDGFR-beta) expression is reduced by siRNAs exhibit a lower capability of responding to chemotactic factors secreted by osteoclasts.

Conclusions/significance: We conclude that, in vitro mature osteoclasts control osteoblast chemotaxis via PDGF-bb/PDGFR-beta signaling. This may provide one key mechanism by which osteoclasts control bone formation in vivo.

Figure 1. Chemotaxis response of osteoblasts and precursors to factors secreted by osteoclasts.

Raw264.7 cells were grown in the presence or absence of RANKL. Conditioned media were collected every day. The chemotactic activity of the corresponding conditioned media towards pre-osteoblastic MC3T3-E1 cells and derived osteoblasts was measured as described under materials and methods. Migration Index of mouse pre-osteoblastic MC3T3-E1 cells (A) and derived osteoblasts (diff MC3T3-E1) (C) in response to conditioned media of Raw264,7 cells (□) and derived osteoclasts (•). Chemotaxis of pre-osteoblastic MC3T3-E1 cells (B) and derived osteoblasts (D) by conditioned media of primary osteoclasts and their precursors. For comparison, the chemotactic activity of conditioned media from Raw264, 7 cells and derived osteoclasts collected after 2 and 4 days of differentiation are shown. Shown are mean values±S.D. of four independent experiments performed in triplicates. P values from ANOVA tests equal or less than 0.05 were considered significant and are marked with an asterisk (*).

Figure 2. Gene expression levels during osteoclastogenesis.

Shown are the expression levels of (▪) VEGFc, (•) LIF, (♦) IL1ra, (▴) PDGF-bb, (+) CCL9 and (×) Twgs1 during the RANKL-induced differentiation of Raw264,7 cells. Quantitative RT-PCR analyses were performed as described under materials and methods. Data are expressed in variations of ΔCt.

Figure 3. PDGF-bb secreted by osteoclasts triggers osteoblast chemotaxis.

Genes encoding PDGF-bb, VEGFc, LIF were silenced in Raw264,7 cell-derived osteoclasts as described under materials and methods. Conditioned media of treated osteoclasts were collected and different dilutions were tested for their chemotactic activity towards pre-osteoblastic MC3T3 cells. Knockdown efficiencies of PDGF-bb (A), VEGFc (C) and LIF (E) in Raw264,7 cell-derived osteoclasts were determined by quantitative RT-PCR. The knockdown efficiencies were 74%, 71% and 70% respectively (p<0,00001, ANOVA). Chemoattraction of pre-osteoblastic MC3T3 cells by different dilutions of conditioned media of Raw264,7 cells (•) or Raw264,7 cell-derived osteoclasts (□) in which the expression of PDGF-bb (B), VEGFc (D) and LIF (F) were silenced (p<0,0001, ANOVA). (▪ in B) Rescue of PDGF-bb knockdown in osteoclasts: conditioned media of siRNA-treated osteoclasts were supplemented with 10 ng/ml recombinant human PDGF-bb. Data points represent the average of 5 experiments.

Figure 4. PDGFR-β of pre-osteoblastic MC3T3-E1 cells and derived osteoblasts relays osteoclast signaling.

PDGFR-β and PDGFR-α genes were silenced in MC3T3-E1 cells (A, B) and derived osteoblasts (F, H) as described under materials and methods. The knockdown efficiencies were 85%, 80% (p<0.0001), 82% and 50% (p<0.000001 and p = 0.01), respectively. (B, D, F, H) The siRNA-treated cells were tested for their chemotactic activities towards conditioned media of Raw264, 7 cells and derived osteoclasts after 2 or 4 days of RANLK induction (p<0.001 and p<0.0001 respectively). Shown are mean values±S.D. of three independent experiments performed in triplicates.