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. 2018 Feb 26:6:4.
doi: 10.1038/s41413-017-0006-3. eCollection 2018.

Lrp1 in osteoblasts controls osteoclast activity and protects against osteoporosis by limiting PDGF-RANKL signaling

Alexander Bartelt  1   2   3 , Friederike Behler-Janbeck  1   2 , F Timo Beil  1   4 , Till Koehne  4   5 , Brigitte Müller  1   2 , Tobias Schmidt  1   2 , Markus Heine  2   6 , Laura Ochs  1   2 , Tayfun Yilmaz  4 , Martin Dietrich  7 , Jan P Tuckermann  8 , Michael Amling  4 , Joachim Herz  7 , Thorsten Schinke  4 , Joerg Heeren  2 , Andreas Niemeier  1   2
Affiliations

Lrp1 in osteoblasts controls osteoclast activity and protects against osteoporosis by limiting PDGF-RANKL signaling

Alexander Bartelt et al. Bone Res. .

Abstract

Skeletal health relies on architectural integrity and sufficient bone mass, which are maintained through a tightly regulated equilibrium of bone resorption by osteoclasts and bone formation by osteoblasts. Genetic studies have linked the gene coding for low-density lipoprotein receptor-related protein1 (Lrp1) to bone traits but whether these associations are based on a causal molecular relationship is unknown. Here, we show that Lrp1 in osteoblasts is a novel regulator of osteoclast activity and bone mass. Mice lacking Lrp1 specifically in the osteoblast lineage displayed normal osteoblast function but severe osteoporosis due to highly increased osteoclast numbers and bone resorption. Osteoblast Lrp1 limited receptor activator of NF-κB ligand (RANKL) expression in vivo and in vitro through attenuation of platelet-derived growth factor (PDGF-BB) signaling. In co-culture, Lrp1-deficient osteoblasts stimulated osteoclastogenesis in a PDGFRβ-dependent manner and in vivo treatment with the PDGFR tyrosine kinase inhibitor imatinib mesylate limited RANKL production and led to complete remission of the osteoporotic phenotype. These results identify osteoblast Lrp1 as a key regulator of osteoblast-to-osteoclast communication and bone mass through a PDGF-RANKL signaling axis in osteoblasts and open perspectives to further explore the potential of PDGF signaling inhibitors in counteracting bone loss as well as to evaluate the importance of functional LRP1 gene variants in the control of bone mass in humans.

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Conflict of interest statement

The authors declare no competing financial interests.

Figures

Fig. 1
Fig. 1. Osteoblast-specific disruption of Lrp 1.
a PCR detection of cre-mediated recombination of floxed Lrp1 alleles in Lrp1Runx2Cre mice and Lrp1flox/flox controls. WAT white adipose tissue. b Tissue Lrp1 mRNA levels quantified by real-time PCR. c Immunoblot of calvarial Lrp1 protein. d Primary cells Lrp1 mRNA levels quantified by real-time PCR. e Primary osteoblast Lrp1 immunoblot as well as f in vivo immunohistochemistry of Lrp1 protein in sections from distal femur trabecular bone of Lrp1Runx2Cre mice and Lrp1flox/flox controls. Scale bar, 25 µm. Means ± s.e.m. n > 5 per group (*P < 0.05 as determined by two-tailed, unpaired T-test)
Fig. 2
Fig. 2. Loss of Lrp1 in osteoblasts results in an age-dependent osteoporotic phenotype.
a Histology of vertebra and b tibia in female Lrp1Runx2Cre mice compared to Lrp1flox/flox controls. Scale bar, 1 mm. c Histomorphometric analysis of lumbar vertebra with bone volume per total volume (BT/TV), d trabecular number (TbN), e trabecular thickness (TbTh), f trabecular spacing (TbSp). Means ± s.e.m. n > 5 per group (*€P < 0.05 as determined by two-tailed, unpaired T-test)
Fig. 3
Fig. 3. Loss of Lrp1 in osteoblasts results in deteriorated bone architecture.
µCT analysis of L5 vertebra from 26-week-old female Lrp1Runx2Cre mice compared to Lrp1flox/flox controls for a bone volume per total volume (BT/TV), b trabecular number (TbN), c trabecular thickness (TbTh), d trabecular spacing (TbSp), and e bone mineral density (BMD), and for distal femoral trabecular bone (f–j) as well as femoral midshaft cortical bone (k) cortical area per total area (Ct.Ar per Tt.Ar), (l) cortical thickness (Ct.Th), and (m) BMD. Means ± s.e.m. n = 4–6 per group (*P < 0.05 as determined by two-tailed, unpaired T-test)
Fig. 4
Fig. 4. Loss of Lrp1 in osteoblasts results in increased osteoclastogenesis in vivo.
Histomorphometric analysis of lumbar vertebra for a osteoblast number (ObN) per bone perimeter (BPm), b osteoblast surface (ObS) per bone surface (BS), c bone formation rate (BFR), and d BFR assessed by double-calcein labeling. e Osteoclast number (OcN) per BPm and f osteoclast surface (OcS) per BS as well as g urinary bone resorption product deoxypyridinoline (DPD) per creatinine in female Lrp1Runx2Cre mice compared to Lrp1flox/flox controls. Means ± s.e.m., n > 5 per group. (*P < 0.05 as determined by two-tailed, unpaired T-test)
Fig. 5
Fig. 5. Loss of Lrp1 in osteoblasts results in increased RANKL expression in vivo and in vitro.
a Serum OPG levels. b, c In vivo bone expression of Tnfrsf11b (encoding OPG) quantified by real-time PCR. Nd not detectable. d Serum RANKL levels. e In vivo bone expression of Tnfsf11 (encoding RANKL) and (f) in parts of the femur quantified by real-time PCR. Means ± s.e.m., n = 6 per group. g Tnfrsf11b expression and h Tnfsf11 expression in primary calvarial osteoblasts at days 6 and 21 in differentiation, two independent experiment in triplicates; a–h, *P < 0.05 as determined by two-tailed, unpaired T-test
Fig. 6
Fig. 6. Lrp1 modulates RANKL expression and osteoclastogenesis by limiting PDGF signaling.
a Immunoblot detection of PDGFRβ in primary calvarial osteoblasts at days 6 and 12 of differentiation. Ob osteoblast, Oc osteoclast. b Lrp1 and Tnfsf11 (encoding RANKL) expression levels quantified by real-time PCR in control and PDGF-BB-stimulated primary calvarial osteoblasts. c Representative images and quantification of osteoblast–osteoclast co-culture experiments. Co-culture of primary wild-type osteoclasts with primary Lrp1-deficient osteoblasts compared to co-culture with primary wild-type osteoblasts increased osteoclastogenesis, which was blocked by an anti-PDGFRβ antibody (Two independent experiment in triplicates). Bar: 500 μmol·L-1 (*P < 0.05 as determined by 2-way ANOVA followed by Tukey's Test on data from two independent experiments in triplicates)
Fig. 7
Fig. 7. An Lrp1–PDGF–RANKL axis in osteoblasts controls osteoporosis.
a, b Female Lrp1Runx2Cre mice and Lrp1flox/flox controls were fed an Imatinib-containing or mock diet from the age of 4 to 26 weeks. µCT analysis of calvaria for imaging and quantification of osteoclast resorption pits. Scale bar, 2 mm. c–f Histomorphometric analysis of lumbar vertebrae. g Osteoblast number, h osteoblast surface, i osteoclast number, j osteoclast surface, and k urinary DPD bone resorption levels. l Real-time PCR quantification of Tnfsf11 (encoding RANKL) in femoral diaphysis and m distal femur. Means ± s.e.m., n > 5 per group. Statistically significant differences between genotypes are indicated in black symbols and between diets in red symbols (*&€ P < 0.05 as determined by 2-way ANOVA followed by Tukey's Test)
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