Continuous PTH in Male Mice Causes Bone Loss Because It Induces Serum Amyloid A

Abstract

Increased bone resorption is considered to explain why intermittent PTH is anabolic for bone but continuous PTH is catabolic. However, when cyclooxygenase-2 (COX2) is absent in mice, continuous PTH becomes anabolic without decreased resorption. In murine bone marrow stromal cells (BMSCs), serum amyloid A (SAA)3, induced in the hematopoietic lineage by the combination of COX2-produced prostaglandin and receptor activator of nuclear factor κB ligand (RANKL), suppresses PTH-stimulated osteoblast differentiation. To determine whether SAA3 inhibits the anabolic effects of PTH in vivo, wild-type (WT) and SAA3 knockout (KO) mice were infused with PTH. In WT mice, continuous PTH induced SAA3 and was catabolic for bone. In KO mice, PTH was anabolic, increasing trabecular bone, serum markers of bone formation, and osteogenic gene expression. In contrast, PTH increased all measurements associated with bone resorption, as well as COX2 gene expression, similarly in KO and WT mice. SAA1 and SAA2 in humans are likely to have analogous functions to SAA3 in mice. RANKL induced both SAA1 and SAA2 in human bone marrow macrophages in a COX2-dependent manner. PTH stimulated osteogenesis in human BMSCs only when COX2 or RANKL was inhibited. Addition of recombinant SAA1 or SAA2 blocked PTH-stimulated osteogenesis. In summary, SAA3 suppresses the bone formation responses but not the bone resorption responses to PTH in mice, and in the absence of SAA3, continuous PTH is anabolic. In vitro studies in human bone marrow suggest that SAA may be a target for enhancing the therapeutic effects of PTH in treating osteoporosis.

Figures

Figure 1.

PTH infusion increased SAA3 expression and decreased femoral BMD in WT mice. WT and SAA3 KO male mice (3.5 mo old) were infused with vehicle (VEH) or PTH (40 µg/kg/d) for 12 d. (a) Saa3 mRNA expression in tibiae of WT mice. Both ends of each tibia were cut off to remove the growth plates and the marrow was not flushed. mRNA expression was measured by quantitative real-time PCR (qPCR) and data were reported as RQ values. (b) SAA3 protein in serum of WT and SAA3 KO mice by ELISA. Und, undetectable. (c) Change in in vivo femoral BMD calculated as: (BMD at end of infusion − BMD at start of infusion)/BMD at start of infusion. (d) Saa1, Saa2/Saa1, and (e) Ptgs2 (COX2) mRNA in WT and KO tibiae. Both ends of each tibia were cut off to remove the growth plates and the marrow was not flushed. mRNA expression was measured by qPCR and data were reported as RQ values. Bars are means ± SEM for n = 6 mice per genotype and treatment group. For (a), **P < 0.01, determined by two-tailed, unpaired t test. For (c) and (e), **P < 0.01, determined by two-way ANOVA, post hoc Bonferroni pairwise multiple comparisons.

Figure 2.

PTH infusion increased trabecular bone in SAA3 KO mice but not in WT mice. WT and SAA3 KO male mice (3.5 mo old) were infused with vehicle (VEH) or PTH (40 µg/kg/d) for 12 d. (a–d) µCT analyses of trabecular bone in distal femurs and L4 vertebrae of WT and SAA3 KO male mice. (a) Representative longitudinal images (scale bars, 400 µm) of trabecular bone in distal femurs and (b) corresponding morphometry for trabecular BV/TV, Tb.Th, Tb.N, Tb.Sp, and Conn.Dens. (c) Representative longitudinal images (scale bars, 400 µm) and (d) corresponding morphometry of trabecular bone in L4 vertebrae. Bars are means ± SEM for n = 6 mice per genotype and treatment group. *P < 0.05, **P < 0.01, determined by two-way ANOVA, post hoc Bonferroni pairwise multiple comparisons.

Figure 3.

Histomorphometric analyses of trabecular bone in distal femurs were similar to µCT analyses. WT and SAA3 KO male mice (3.5 mo old) were infused with vehicle (VEH) or PTH (40 µg/kg/d) for 12 d. (a) Histomorphometric parameters for trabecular BV/TV, Tb.Th, Tb.N, and Tb.Sp. (b) Representative hematoxylin-stained ×20 original magnification (scale bars, 500 µm) microscopic images of femurs. Bars are means ± SEM for n = 6 mice per genotype and treatment group. *P < 0.05, **P < 0.01, determined by two-way ANOVA, post hoc Bonferroni pairwise multiple comparisons.

Figure 4.

PTH infusion increased trabecular bone formation parameters only in SAA3 KO mice but increased resorption parameters similarly in both WT and SAA3 KO mice. WT and SAA3 KO male mice were infused with vehicle (VEH) or PTH. (a) Static histomorphometry of trabecular bone in distal femurs for Ob.S/BS, Oc.S/BS, and eroded surface per bone surface (ES/BS). (b) Representative TRAP-stained (red) and counterstained hematoxylin ×200 original magnification (scale bars, 100 µm) microscopic images of trabecular bone in distal femurs. Plump cuboidal osteoblasts around trabeculae are marked with yellow arrows. (c) Dynamic histomorphometry of trabecular bone in distal femurs for MS/BS, MAR, and BFR/BS. (c) Representative calcein-labeled (green) and demeclocycline-labeled (orange-brown) ×400 original magnification (scale bars, 25 µm) microscopic images of trabeculae. (e and f) Measurement of (e) serum bone formation markers, PINP, and BGLAP (osteocalcin) and (f) serum bone resorption markers, CTX and TRAcP5b, measured by ELISA. (g and h) mRNA expression of Runx2, Bglap (osteocalcin), Tnfsf11 (RANKL), and Tnfrsf11b (OPG) in the tibiae measured by quantitative real-time PCR and reported as RQ values. Both ends of each tibia were cut off to remove the growth plates and the marrow was not flushed. Bars are means ± SEM for n = 6 mice per genotype and treatment group. *P < 0.05, **P < 0.01, determined by two-way ANOVA, post hoc Bonferroni pairwise multiple comparisons.

Figure 5.

PTH infusion increased expression of genes that mediate anabolic effects and decreased expression of genes that inhibit anabolic effects in SAA3 KO but not WT mice. mRNA expression measured in tibiae of WT and SAA3 KO male mice infused with vehicle (VEH) or PTH. Both ends of each tibia were cut off to remove the growth plates and the marrow was not flushed. mRNA expression was measured by quantitative real-time PCR and reported as RQ values. mRNA expression of (a) cAMP-regulated genes, Pthlh (PTHrP) and Ramp3, (b) growth factor genes, Bmp2 and Igf1, (c) genes promoting Wnt signaling, Ctnnb1 (catenin, β1), Wnt10b, Wnt7b, and Wnt4, and (d) genes inhibiting Wnt signaling, Dkk1 and Sost. Bars are means ± SEM for n = 6 mice per genotype and treatment group. *P < 0.05, **P < 0.01, determined by two-way ANOVA, post hoc Bonferroni pairwise multiple comparisons.

Figure 6.

In vitro, the presence of SAA3 blocked PTH-stimulated osteoblast differentiation and cAMP production but had no effect on PTH-stimulated osteoclast-like cell differentiation. (a) Measurement of mRNA (Bglap) for the osteoblast marker, osteocalcin, and alizarin red staining for mineralized nodules at day 21 in BMSCs following treatment with vehicle (VEH) or continuous PTH (10 nM). (b) PTH-stimulated cAMP production and Bglap mRNA in POBs. CM from M-CSF plus RANKL (30 ng/mL each)–treated WT and SAA3 KO BMMs was added to POBs (3 parts CM and 1 part differentiation medium). OPG (50 ng/mL) was added to prevent osteoclastogenesis by any marrow remaining in POB cultures. For cAMP, POBs were grown for 5 d before treatment with CM, OPG, and 0.5 mM IBMX to block phosphodiesterase activity. One hour later, VEH or PTH was added for 20 min. For Bglap, POBs were treated with CM, OPG and VEH, or PTH for 14 d. (c) WT and SAA3 KO bone marrow cultures were treated with VEH or PTH. TRAP+ MNCs were counted per well. Gene expression for Tnfsf11 (RANKL) and Tnfrsf11b (OPG) was measured by quantitative real-time PCR on day 4 and reported as RQ values. Data are means ± SEM for n = 3 to 4 independent samples. For osteoclast count in (c), **P < 0.01, significantly different from day 3, determined by one-way ANOVA, and for (a), (b), and the rest of (c), *P < 0.05, **P < 0.01, determined by two-way ANOVA, post hoc Bonferroni pairwise multiple comparisons.

Figure 7.

Continuous PTH increased osteoblast differentiation in hBMSCs only when COX2 or RANKL activity was blocked. hBMSCs were cultured with vehicle (VEH) or PTH (10 nM), with or without NS398 (100 nM), a selective inhibitor of COX2, or OPG (100 ng/mL), which blocks RANKL binding to its receptor. (a) Measurement of accumulated medium PGE2 by ELISA. (b) Markers of osteoblast differentiation, ALPL (alkaline phosphatase) and BGLAP (osteocalcin), and alizarin red staining for mineralization at day 21. (c) TNFSF11 (RANKL) mRNA at day 7 and TRAP staining at day 8. Scale bar, 100 µm. (d) BGLAP mRNA and alizarin red staining at day 21. (e) RUNX2 mRNA at day 7 and IGF1 and BMP2 mRNA at day 21. (f) WNT10B and DKK1 mRNA at day 7. mRNA was measured by quantitative real-time PCR and data are reported as RQ values. Bars are means ± SEM for n = 3 independent samples. For (a), **P < 0.01, determined by one-way ANOVA, post hoc Bonferroni pairwise multiple comparisons. For (c), **P < 0.01, determined by two-tailed unpaired t test. For (d)–(f), *P < 0.05, **P < 0.01, determined by two-way ANOVA, post hoc Bonferroni pairwise multiple comparisons. Und, undetectable.

Figure 8.

NS398 blocked the RANKL induction of SAA1 and SAA2 expression in hBMMs but did not affect osteoclast-like cell numbers, and SAA1 and SAA2 inhibited PTH-stimulated osteoblastic differentiation in hBMSCs. (a–c) hBMMs were treated with M-CSF (30 ng/mL) plus vehicle (VEH) or M-CSF plus RANKL (30 ng/mL each) with/without NS398 (100 nM). (a) SAA1 and SAA2 protein measurement in the culture medium of hBMMs measured by ELISA. (b) SAA1 and SAA2 mRNA at day 3. (c) TRAP-stained microscopic images at ×100 original magnification (scale bars, 200 µm) and TRAP+ MNC counts per well. (d) ALPL (alkaline phosphatase) and BGLAP (osteocalcin) mRNA expression in hBMSCs at day 21 treated with VEH or PTH in presence of OPG (100 ng/mL) to prevent osteoclastogenesis with/without rhSAA1 (10 µg/mL) or rhSAA2 (10 to 50 ng/mL). Data are means ± SEM for n = 3 independent samples. For (a), **P < 0.01, significantly different from VEH treated at same time point, determined by two-tailed unpaired t test. For (c), **P < 0.01, significantly different from day 4, determined by one-way ANOVA, post hoc Bonferroni pairwise multiple comparisons. For (b) and (d), *P < 0.05, **P < 0.01, determined by two-way ANOVA, post hoc Bonferroni pairwise multiple comparisons.