A distinctive role of the leukotriene B4 receptor BLT1 in osteoclastic activity during bone loss

Abstract

Although leukotriene B(4) (LTB(4)) is produced in various inflammatory diseases, its functions in bone metabolism remain unknown. Using mice deficient in the high-affinity LTB(4) receptor BLT1, we evaluated the roles of BLT1 in the development of two bone resorption models, namely bone loss induced by ovariectomy and lipopolysaccharide. Through observations of bone mineral contents and bone morphometric parameters, we found that bone resorption in both models was significantly attenuated in BLT1-deficient mice. Furthermore, osteoclasts from BLT1-deficient mice showed reduced calcium resorption activities compared with wild-type osteoclasts. Osteoclasts expressed BLT1, but not the low-affinity LTB(4) receptor BLT2, and produced LTB(4). LTB(4) changed the cell morphology of osteoclasts through the BLT1-Gi protein-Rac1 signaling pathway. Given the causal relationship between osteoclast morphology and osteoclastic activity, these findings suggest that autocrine/paracrine LTB(4) increases the osteoclastic activity through the BLT1-Gi protein-Rac1 signaling pathway. Inhibition of BLT1 functions may represent a strategy for preventing bone resorption diseases.

Fig. 1.

Radiographic analysis of hindlimb bones. (A) Areal bone mineral density (BMD) of the metaphyseal region of the femur measured by DXA. Left graph, female mice were ovariectomized (OVX) or sham-operated (Sham). *, P < 0.05 vs. WT-OVX, as determined by ANOVA with Tukey's multiple comparison test (n = 8 animals per group). Right graph, male mice were injected with LPS or saline. *, P < 0.05 vs. WT-LPS, as determined by ANOVA with Tukey's multiple comparison test (n = 9–10 animals per group). (B) Trabecular bone mineral content per tissue volume (BMC/TV) of the metaphyseal region of the femur measured by microCT. Data are shown as described for A. *, P < 0.001 vs. WT-OVX or WT-LPS, as determined by ANOVA with Tukey's multiple comparison test (n = 8 and 9–10 animals per group for ovariectomy and LPS injection, respectively). (C) Representative microCT photographs of the metaphyseal regions of femurs. Note the highly porous inside of the bone (transparent regions) in the ovariectomized and LPS-injected WT mice. (Scale bar, 1 mm.)

Fig. 2.

Computerized morphometry of femurs. (A) Trabecular bone volume per tissue volume (BV/TV), trabecular number (Tb.N), and trabecular separation (Tb.Sp) of the metaphyseal region of the femur from ovariectomized mice. These bone mass indices were quantified based on analyses of three-dimensional microCT images of the metaphyseal region of the femur. *, P < 0.01 vs. WT-OVX by ANOVA with Tukey's multiple comparison test (n = 8 animals per group). (B) Trabecular BV/TV, Tb.N, and Tb.Sp of the metaphyseal region of the femur from LPS-injected mice quantified as described for A. *, P < 0.01 vs. WT-LPS by ANOVA with Tukey's multiple comparison test (n = 9–10 animals per group).

Fig. 3.

Expression of LTB₄-related molecules and production of LTB₄ in primary osteoclasts. (A) mRNA expression of BLT1 and BLT2 in osteoclasts differentiated from bone marrow cells in the presence of RANKL (30 ng/mL) and M-CSF (50 ng/mL) for 5 days. RT, reverse transcription. (B) mRNA expression of 5-lipoxygenase (5-LO) and LTA₄ hydrolase (LTA4H) in the osteoclasts. Data from two independent primary osteoclast cultures (#1 and #2) are shown. (C) Western blot analysis for 5-lipoxygenase (5-LO) protein expression in the osteoclasts. (D) Production of LTB₄ by the osteoclasts upon stimulation with 1 μM A23187 (n = 4 per group).

Fig. 4.

Morphological changes of osteoclasts through the BLT1-Gi protein-Rac1 signaling pathway. (A) Images of rhodamine-phalloidin staining of primary osteoclasts. WT osteoclasts alter their contours from round shapes to irregular shapes after 30 min of treatment with 100 nM LTB₄, while BLT1-KO osteoclasts do not change their morphology. (Scale bar, 50 μm.) (B) Inhibition of the LTB₄-induced morphological changes of WT osteoclasts by an antagonist of BLT1 and inhibitors of Gi protein and Rac1. Primary osteoclasts were incubated with 1 μM CP105696 (BLT1 antagonist) for 5 min, 10 ng/mL PTX (Gi protein inhibitor) for 2 h, or 50 μM NSC23766 (Rac1 inhibitor) for 10 min. The cells were then treated with 100 nM LTB₄ for 30 min. (Scale bar, 50 μm.)

Fig. 5.

Roles of BLT1, Gi and Rac1 in the calcium resorption activity and osteoclast number. (A) Calcium resorption by primary osteoclasts. Osteoclasts were cultured on calcium phosphate-coated dishes for 6 days with or without 1 μM CP105696 (BLT1 antagonist), 10 ng/mL PTX (Gi protein inhibitor), or 50 μM NSC23766 (Rac1 inhibitor). *, P < 0.05 vs. WT osteoclasts without drug treatment, as determined by ANOVA with Dunnett's multiple comparison test (n = 3 per group). (B) Number of osteoclasts. Primary osteoclasts were cultured in 96-well dishes for 5 days. The numbers of TRAP-positive multinucleated (greater than or equal to three nuclei) cells per well were counted. *, P < 0.05 vs. WT osteoclasts without drug treatment, as determined by ANOVA with Tukey's multiple comparison test (n = 6 per group).