Parathyroid hormone signaling in mature osteoblasts/osteocytes protects mice from age-related bone loss

PPR ablation in mature osteoblasts/osteocytes induces severe osteopenia in 13-month-old male mice

Mice lacking PPR in mature osteoblasts/osteocytes, namely Dmp1-PPR^KO, have increased bone mineral density and bone mass by three months of age and they are resistant to both the anabolic and catabolic effects of PTH [22]. To investigate whether PPR signaling in mature osteoblasts/osteocytes is needed to maintain skeletal homeostasis during age-dependent bone loss, we analyzed the skeletons of adult and middle-aged animals. Hematoxylin and eosin (H&E) staining of the tibiae and von Kossa staining of the fifth lumbar (L5) vertebrae and the femora showed significant increase in trabecular bone volume over total tissue volume (BV/TV%) at 4 months, as previously reported for young mice [22]. The increased bone mass was followed by a dramatic bone loss in Dmp1-PPR^KO mice at 13 months, as compared to littermate controls (Figure 1A–1D, Table 1, Supplementary Figure 1A). Cortical thickness was similar between Dmp1-PPR^KO and controls at both ages (Figure 1E). Micro-computed tomography (μCT) analysis further confirmed the histological data (Figure 1F–1J, Table 2). At 4 months of age, in Dmp1-PPR^KO mice there was a significant increase in BV/TV% in L5 and distal femur (Figure 1H–1I) and a significant increase in trabecular thickness (Figure 1H) in vertebral bones. In contrast, at 13 months of age, BV/TV% in both sites (L5 and femurs) were significantly decreased in Dmp1-PPR^KO mice compared to littermate controls (Figure 1H, 1I). The significant reduction in these trabecular parameters was still present in KO animals at 16 months of age (Supplementary Figure 1B). In Dmp1-PPR^KO mice, bone volume of L5 and the distal femora decreased by 41% and 71%, respectively, from 4 to 13 months, whereas in control animals this decrease was 12% and 42%, respectively (Figure 1H, 1I). At 13 months of age, trabecular separation was also significantly increased in the L5 of Dmp1-PPR^KO mice (Figure 1H) and trabecular number (Tb.N) was significantly decreased in the distal femora (Figure 1I) of Dmp1-PPR^KO mice compared to controls. Taken together this data revealed a marked age-dependent trabecular bone loss in the absence of PPR signaling in mature osteoblasts/osteocytes.

Figure 1. Age-dependent bone loss in Dmp1-PPR^KO mice. Vertebrae and long bones of male control and KO animals were analyzed by (A) histology, (B–E) histomorphometry and (F–J) μCT. (A) Representative H&E of the proximal tibiae and (B) Von Kossa staining of the distal femora. Bar = 1.0 mm. Histomorphometric analysis of (C) the L5 and (D) trabecular and (E) cortical region in the distal and midshaft femora, respectively. N = 6–10 per group. Data are presented as mean ± SEM. Representative μCT images of (F) the distal and (G) the midshaft femora. Bars = (F) 300 μm and (G) 1.0 mm. μCT analysis of (H) the L5 and (I) the distal femur (trabecular) and (J) the midshaft femur (cortical) are shown. Data are presented as box and whisker plot. N = 6–16 per group. See Tables 1 and 2 for the full list of parameters. Analyses were performed in a blinded fashion. Unpaired Student’s t test (C–E) and Two-way ANOVA with Tukey’s post hoc test or Mann-Whitney test (H–J) was performed. ^*p < 0.05, ^**p < 0.01, ^***p < 0.001. Abbreviations: Tb: Trabecular; Cort: cortical; BV: bone volume; TV: total tissue volume; Th: thickness; N: number; Sp: separation; A: area; pMOI: polar moment of inertia; Por: porosity.

Cortical area, as assessed by μCT analysis of the midshaft femur, was significantly increased at 4 months in Dmp1-PPR^KO mice (Figure 1G, 1J, Table 2). Similarly, other cortical parameters, including polar moment of inertia, were also significantly increased in the mutant mice at 4 months of age, compared to controls whereas they were indistinguishable between the two genotypes at 13 months, indicating a differential temporal regulation of trabecular and cortical bone by PPR signaling in mature osteoblasts/osteocytes with age.

PPR deletion in mature osteoblasts/osteocytes increases osteoclast, but decreases osteoblast, activity in 13-month-old mice

To delineate the cellular mechanism of the age-related osteopenia in the Dmp1-PPR^KO animals, we performed histomorphometric analysis on the L5 and the femora of adult and middle-aged mice. As shown in Figure 1C–1E, histomorphometric analysis confirmed the decrease in trabecular BV/TV% and Tb.N in male Dmp1-PPR^KO mice at 13 months, both in axial and appendicular sites. Tartrate resistant acid phosphatase (TRAP) staining on the distal femora of these mice showed relatively fewer osteoclasts per bone perimeter at 4 months, but strikingly more TRAP-positive cells at 13 months in Dmp1-PPR^KO animals as compared with controls (Figure 2A–2B). The significant increase in the number of TRAP-positive osteoclasts was also present in the proximal tibiae of 20-month-old Dmp1-PPR^KO female mice compared with control littermates (Supplementary Figure 2A). These findings were further supported by a significant reduction in osteoclast activity, such as erosion and osteoclast surface per bone surface, in 4-month-old Dmp1-PPR^KO mice but a significant increase of all these parameters at 13 months of age (Figure 2B, Table 1). Bone formation rate (BFR) over bone volume (BFR/BV) was significantly decreased in Dmp1-PPR^KO at 4 months and mineral apposition rate (MAR) was significantly reduced in 13-month-old Dmp1-PPR^KO mice compared to controls (Figure 2C–2D, Table 1). These results indicate that, in the absence of PPR signaling in mature osteoblasts/osteocytes, there is an age-dependent increase in osteoclast numbers and activity and age-independent decrease in osteoblast activity resulting in increased bone resorption and bone loss.

Figure 2. Histomorphometric analysis of trabecular bones of Dmp1-PPR^KO mice. (A, B) Representative TRAP staining images and bone resorption parameters of the distal femora from male control and KO animals. The inset shows a closeup displaying the TRAP-positive osteoclasts on the trabecular bone surface. Bar = 200 μm. (C) Representative images of calcein double-staining on the distal femora of these mice. Bone formation within 7 days was visualized by double calcein labeling. Bar = 50 μm. (D) Representative bone-formation parameters of the distal femora are shown. See Table 1 for the full list of resorption and formation parameters. N = 6–10 per group. Analyses were performed in a blinded fashion. Unpaired Student’s t test was performed. ^*p < 0.05, ^**p < 0.01. Data are presented as mean ± SEM. Abbreviations: ES: Erosion surface; BS: bone surface; Oc.S: osteoclast surface; N.Oc: number of osteoclasts; B.Pm: bone perimeter; MAR: mineral apposition rate; N.Ob: number of osteoblasts; T.Ar: tissue area; BFR: bone formation rate; BV: bone volume.

Age-dependent changes in serum markers and skeletal genes in Dmp1-PPR^KO mice

The main function of PTH is to maintain mineral homeostasis and it is still unclear whether mature osteoblasts/osteocytes directly contribute to mineral-ion homeostasis. To investigate if lack of PPR signaling in mature osteoblasts/osteocytes impaired mineral ions homeostasis, we measured serum levels of calcium, phosphate and PTH in adult and middle-aged mice. Biochemical analysis of male Dmp1-PPR^KO and littermate control mice showed normal calcemia and phosphatemia at 4 and 13 months of age (Figure 3A), demonstrating that PPR in mature osteoblasts/osteocytes is not required to maintain mineral-ion homeostasis. In both control and Dmp1-PPR^KO, with age, there was a significant decrease in serum calcium and an increase in serum PTH, similar to what has been observed in older adult mice. PTH levels were similar between Dmp1-PPR^KO and controls at 4 months of age (Figure 3A), whereas they were significantly increased in Dmp1-PPR^KO mice at 13 months of age (Figure 3A), indicating a possible resistance to PTH. This increase in serum PTH in Dmp1-PPR^KO was not observed in 13-month-old female mice (Supplementary Figure 3B), suggesting the sex-dependent difference. Interestingly, phosphate serum levels significantly increased with age in Dmp1-PPR^KO mice but not in controls (Figure 3A). Serum markers of bone formation, procollagen type 1 N-terminal propeptide (P1NP), were significantly reduced in both male Dmp1-PPR^KO and controls at 13 months whereas markers of bone resorption, C-terminal telopeptide of type I collagen (CTX), were unchanged in both mice groups at both ages (Figure 3B), despite the significant increase in osteoclast numbers and activities present in Dmp1-PPR^KO mice at 13 months of age, as shown in Table 1 and Figure 2B.

Figure 3. Serum markers and skeletal gene expression in Dmp1-PPR^KO mice. (A) Serum calcium, phosphate, PTH and sclerostin in male control and Dmp1-PPR^KO mice was measured by ELISA. PTH levels were normalized to 13-month-old control due to a high intra-assay variation (>9.8%). (B) Serum levels of bone formation (P1NP) and resorption marker (CTX) were also measured by ELISA. N = 7–19 per group. Data are presented as mean ± SEM. (C) Gene expression in marrow-removed long bones of male mice was analyzed with qPCR. N = 4–6 per group. Two-way ANOVA with Tukey’s post hoc test, unpaired Welch’s t test, or Mann-Whitney test was performed. ^*p < 0.05, ^**p < 0.01, ^***p < 0.001. Each gene expression was relative to 3 housekeeping genes (Actb, B2m, and Gapdh), which were selected by GeNorm. Expression data are presented as mean ± SEM.

To investigate the molecular mechanism leading to the increased osteoclasts in 13-month-old Dmp1-PPR^KO mice, we assessed the expression of PPR (encoded by Pth1r) and other osteocytic markers in bone marrow-deprived long bones of adult and middle-aged Dmp1-PPR^KO and control mice. Receptor ablation in osteocytes was still present in both male and female 13-month-old KO animals, as demonstrated by a significant decrease in PPR expression (Figure 3C, Supplementary Figure 4A). Although middle-aged male Dmp1-PPR^KO mice showed a trend of increase (2.0-fold, p = 0.06) in Sost expression compared to controls, overall Sost expression was unaffected by genotypes (Figure 3C). Interestingly, serum sclerostin was significantly increased in the mutant mice as compared to controls, which can contribute to the suppression of bone formation present in these animals (Figure 3A). Other osteocytic genes, namely RANKL and osteoprotegerin (OPG), were unchanged, suggesting that other factors might be driving the age-dependent increase in osteoclast numbers and activity present in middle-aged male Dmp1-PPR^KO mice. In the female mice, OPG expression was significantly downregulated, while the RANKL/OPG ratio was significantly increased, in KO mice at 13 months of age (Supplementary Figure 4A), demonstrating sex-dependent differences.

Tumor necrosis factor α (TNFα) promotes osteoclastogenesis independently of RANKL [23]; therefore, we performed immunofluorescence staining for TNFα on the tibias of 13-month-old male mice and control littermates. In Dmp1-PPR^KO mice, the number of TNFα-expressing osteocytes was significantly decreased as compared to control littermates (Supplementary Figure 5), suggesting that other factors might drive osteoclastogenesis. In addition, we found a trend of increase (1.3-fold, p = 0.06) in M-CSF expression in the bone marrow of Dmp1-PPR^KO mice at 13 months of age compared to control littermates Figure 4B), and this upregulation of M-CSF in KO mice was also observed in 16-month-old mice (1.5-fold, p < 0.001, data not shown). Sphingosine kinase (Sphk1) mediates TNFα-induced arthritis and osteoclastogenesis via TNFα receptor activating factor 2 (TRAF2) [24]. We analyzed the expression of Sphk1 in the bone marrow of Dmp1-PPR^KO mice and found that Sphk1 was significantly upregulated as compared to control littermates (Supplementary Figure 6B), suggesting a potential involvement of TNFα-expressing osteocytes in the increase in M-CSF and Sphk1 expression in the bone marrow of Dmp1-PPR^KO mice. Next we measured serum levels of cytokines, previously reported to be regulated by PTH or by aging, including TNFα, monocyte chemoattractant protein 1 (MCP-1/CCL2), and interleukin (IL)-6 and -10 in serum of 13-month-old male mice (Supplementary Figure 3A). While there was a trend of increase (2.2-fold, p = 0.06) in serum IL-10 in Dmp1-PPR^KO compared to controls, serum concentration of other cytokines was unchanged.

Figure 4. Increased bone marrow adipocytes in middle-aged Dmp1-PPR^KO mice. (A) The area of bone marrow adipose tissue (BMAT) over the total bone marrow (BM) space within 300-μm from the epiphyseal plate was analyzed on H&E-stained tibiae sections of male control and Dmp1-PPR^KO mice at 13 months of age. Representative images are shown in Figure 1A. N = 4 per group. (B) Expression of M-CSF in the BM isolated from the femora of middle-aged male animals (13 months old) was analyzed by qPCR. N = 4 per group. (C) Immunofluorescence staining of perilipin-1, RANKL and DAPI was performed on the tibiae of middle-aged (13 months) male control and Dmp1-PPR^KO mice. Representative images of a tibia from Dmp1-PPR^KO mouse are shown. In the merged image, DAPI, perilipin-1 and RANKL staining is shown in blue, red, and green, respectively. The orange arrowheads indicate RANKL⁺ BMAT (identified as perilipin-1⁺). The number of BMAT (left) and the number (middle) and frequency (right) of RANKL⁺ BMAT in the BM space were analyzed. N = 4 per group. Unpaired student’s t test was performed. ^*p < 0.05. Data are presented as mean ± SEM.

Serum from middle-aged Dmp1-PPR^KO male animals increases the number of osteoclasts in vitro

To explore the molecular mechanism(s) driving the osteopenia and the increase in osteoclast numbers and activity in middle-aged male Dmp1-PPR^KO mice, we treated bone marrow mononuclear cells (BMMCs) isolated from wild type mice (3–4 months old) with serum obtained from 13-month-old male mice (Dmp1-PPR^KO and littermate controls) and analyzed osteoclastogenesis by TRAP staining and activity. BMMCs treated with serum from KO mice significantly increased the number of TRAP⁺ osteoclasts compared to the control group (Figure 5A), whereas TRAP activity was similar between the two groups (Supplementary Figure 3C). These findings suggest that, in the absence of PTH signaling, osteocytes secrete factors that contribute, in part, to the increase in osteoclasts present in middle-aged Dmp1-PPR^KO mice.

Figure 5. Serum from Dmp1-PPR^KO mice increases osteoclastogenesis and adipogenesis. (A) Representative TRAP staining images of BMMCs isolated from 3-month-old male control mice under osteoclastic differentiation in the presence of serum from 13-month old male control and KO mice. The total number (middle) and size distribution (right) of TRAP⁺ osteoclasts (OCs) per field were quantified. N = 13 per group. Data are presented as mean ± SEM. (B) CFU assay for osteoblasts was performed on BMSCs isolated from 13-month-old male control and Dmp1-PPR^KO animals. Data are presented as mean ± SEM. (C) Proliferation assay (left) and ALP activity assay (right) were performed on BMSCs isolated from 3-month-old male control mice under osteogenic differentiation in the presence of serum from male control and Dmp1-PPR^KO mice at 13 months of age. ALP activity assay was performed on day 6 of the osteogenic differentiation. N = 13–15 per group. (D) Representative Oil-red-O staining images of BMSCs isolated from 3-month-old male control mice under adipogenic differentiation in the presence of serum from male control and KO mice at 13 months of age. Quantification of lipid was performed by elution of Oil-red-O stain. N = 18 per group. Mann-Whitney test (A) or paired or unpaired t test (B–D) was performed. ^*p < 0.05, ^**p < 0.01, ^***p < 0.001. Data are presented as mean ± SEM.

Osteoprogenitors are decreased in middle-aged Dmp1-PPR^KO animals

Next we assessed if lack of PPR signaling in mature osteoblasts/osteocytes influenced the commitment or frequency of osteoprogenitor cells, and therefore their osteogenic potential. As shown in Figure 5B, colony forming unit osteoblasts (CFU-Ob) were significantly reduced in bone marrow of male Dmp1-PPR^KO animals compared to controls at 13 months, demonstrating a progressive reduction in osteoprogenitors. Similarly, middle-aged female KO animals showed a marked reduction in CFU-Ob in the bone marrow (Supplementary Figure 3D).

Serum from middle-aged Dmp1-PPR^KO male animals promotes adipogenic differentiation of BMSCs in vitro

Since circulating factors, including bone morphogenic proteins, are involved in osteolineage commitment of bone marrow stromal cells (BMSCs) [25], we examined the effect of serum from middle-aged mice on osteogenic differentiation of BMSCs. We treated BMSCs isolated from wild type mice (3–4 months old) with serum from 13-month-old male Dmp1-PPR^KO mice and littermate controls. Treatment with serum from control and KO mice showed no difference in proliferation and alkaline phosphatase (ALP) activity of BMSCs (Figure 5C), while BMSCs treated with serum from KO mice significantly increased adipogenic differentiation, as assessed by oil-red-O staining (Figure 5D). These findings demonstrate that circulating factors promote lineage commitment of BMSCs. To investigate if the secreted factors are osteocyte-derived, we treated BMSCs with conditioned medium from ex vivo culture of osteocyte-enrichment bone explants (OEBEs) from 13-month-old control and Dmp1-PPR^KO mice. Similarly, there was no difference in both BMSC proliferation and osteogenic differentiation between both treatment groups (Supplementary Figure 3E). These results indicate that factors secreted from osteocytes are not the major contributor to the reduced osteoprogenitors in Dmp1-PPR^KO mice.

Marrow adipocytes are the source of RANKL

As demonstrated in Figure 5D, treatment with serum from 13-month-old Dmp1-PPR^KO mice promoted adipogenic differentiation of BMSCs in vitro. To examine if there was any change in marrow adiposity in these mice, we performed histological analysis on bone marrow. In Dmp1-PPR^KO male animals, at 13 months of age, there was a significant increase in marrow adiposity (Figures 1A, 4A). Since marrow adipocytes have been reported as a source of RANKL [26, 27], we analyzed RANKL expression in marrow adipocytes by immunofluorescence staining. Staining of RANKL along with an adipocyte marker, perilipin-1, revealed that the number of RANKL⁺ marrow adipocytes was markedly increased in Dmp1-PPR^KO compared to control male mice (Figure 4C). However, the ratio of RANKL⁺ adipocytes over total adipocytes was unchanged between control and mutant animals, suggesting that the increase in the number, but not the frequency, of RANKL⁺ adipocytes contributed to the increased osteoclast numbers in KO mice. It has also been reported that bone marrow adipocytes express M-CSF [28]. Interestingly, we found that M-CSF expression was upregulated (1.3-fold, p = 0.06) in the bone marrow of male Dmp1-PPR^KO mice as compared to controls (Figure 4B). This may indicate that the increased marrow adiposity also contributed to the upregulation of M-CSF in the middle-aged mutant mice. Expression of RANKL was unchanged in the bone marrow of 13-month-old male mice (Supplementary Figure 6A). Interestingly, the marrow adiposity was unchanged between female control and Dmp1-PPR^KO at 20 months of age, suggesting sexual dimorphism (Supplementary Figure 2B).

PPR signaling protects osteocytes from early onset of oxidative stress in vivo

Aging is accompanied by an accumulation of oxidative stress due to an imbalance between pro-oxidants and antioxidants [29, 30]. PTH protects osteoblasts from oxidative stress-induced cell death [31]; therefore we hypothesize that a similar effect was also present in osteocytes. To test the hypothesis, we analyzed the expression of 4-hydroxynonenal (4-HNE), a biomarker for oxidative stress-induced lipid peroxidation, in the L3/4 vertebrae of 4- and 13-month-old Dmp1-PPR^KO and control littermates. As shown in Figure 6A, Dmp1-PPR^KO mice exhibited a significant increase in 4-HNE-positive osteocytes at 4 months of age, suggesting the protective effect of PPR signaling in osteocytes from early onset of oxidative stress.

Figure 6. PTH protects osteocytes from oxidative stress-induced cell death and senescence. (A) Representative images of immunohistochemistry for 4-HNE on the L3/4 vertebrae from male animals are shown. The frequency of 4-HNE⁺ osteocytes per image field was analyzed. Mean ± SEM is shown. (B) Expression of p16^Ink4a in the tibiae of male control and Dmp1-PPR^KO mice was analyzed by qPCR. N = 4–6 per group. Mean ± SEM is shown. (C–E) Control and 12H-PPR^KO osteocytic cell line was pretreated with either 10 nM hPTH(1–34) or 10 μM forskolin (FSK) for 18-22 hrs prior to H₂O₂ exposure. (C) After H₂O₂ exposure (1 mM, overnight), cell viability was measured by resazurin-based assays. (D) After H₂O₂ exposure (1 mM, 4 h) intracellular ROS levels were measured using a fluorescent probe (DCFDA). Data are presented as relative fluorescence unit (RFU). (E) After continuous exposure to H₂O₂ (150 μM, 14 d), cells were stained for SA β-gal. Representative SA β-gal staining images and the quantification of SA β-gal⁺ cells (red arrowheads) are shown. Bar = 100 μm. (F) Representative TRAP staining images are shown of BMMCs isolated from 3–4-month-old male control mice under osteoclastic differentiation in the presence of conditioned medium from H₂O₂-treated control osteocytic cell line (–H₂O₂ or +H₂O₂, 150 μM for 7 days). Bar = 400 μm. N = 18 per group. (G) After continuous exposure to H₂O₂ (100 or 150 μM, 7 or 14 d), cells were harvested for RNA isolation. mRNA expression of p16^Ink4a, p21, Sost, and RANKL were analyzed by qPCR. N = 3 per group. Kruskal-Wallis test with Dunn’s post hoc test, two-way ANOVA with Tukey’s post hoc test, one-way ANOVA with Sidak’s post hoc test or Mann-Whitney test were performed. ^*p < 0.05, ^**p < 0.01, ^***p < 0.001. Same letter indicates n.s. Data are presented as mean ± SD.

Oxidative stress is one of the causes of cellular senescence and it has been previously shown that senescent osteocytes contribute to age-related bone loss [32]. We investigated if the age-dependent bone loss in Dmp1-PPR^KO mice was driven by senescent osteocytes. The expression of the senescence marker p16^Ink4a was significantly increased in Dmp1-PPR^KO mice at 13 months of age compared to 4 months, whereas it was unchanged in controls (Figure 6B). Other markers of cellular senescence were unchanged (Supplementary Figure 4C).

PTH protects osteocytes from oxidative stress-induced cell death and intracellular ROS accumulation in vitro

To study the effects of PTH in oxidative stress, we used an in vitro model in which PPR expression was knocked-out by CRISPR/Cas9 technique. In the absence of PPR expression in Ocy454-12H cells [33] (12H PPR^KO) (Supplementary Figure 7A–7C), there was a significant decrease in the basal RANKL and RANKL/OPG expression (Supplementary Figure 7C), which is similar to the phenotype present in 3-month-old Dmp1-PPR^KO animals [22], whereas Sost expression was unchanged compared to control cells. 12H-PPR^KO cells were treated with hPTH(1–34) or forskolin for 18–22 hrs and then exposed to a high dose of H₂O₂ (1 mM). As shown in Figure 6C–6D, PTH treatment significantly suppressed oxidative stress-induced cell death and intracellular accumulation of ROS in control cells, whereas this effect was lost in 12H-PPR^KO cells, demonstrating that PTH protects osteocytes from oxidative stress-induced cell death.

PTH protects osteocytes from oxidative stress

ROS accumulation promotes oxidative stress and cellular senescence, therefore we evaluated the effects of PTH during oxidative stress. Control and 12H-PPR^KO cells were pretreated with hPTH(1–34) prior to 7 and 14 days of continuous exposure to a low dose of H₂O₂ (150 μM). In the non-pretreated groups, H₂O₂ exposure markedly increased the number of senescence-associated β-galactosidase-positive (SA β-gal⁺) cells in both control and 12H-PPR^KO cells, while PTH-pretreatment significantly reduced the number of SA β-gal⁺ cells in control, but not in 12H-PPR^KO cells (Figure 6E, Supplementary Figure 8A). Gene expression in these cells after 7 and 14 days of H₂O₂ (100–200 μM) exposure (Figure 6G) demonstrated that PTH had a long-lasting effect on Sost and RANKL expression. Importantly, pretreatment of control, but not 12H-PPR^KO, cells with PTH significantly suppressed p21 expression at day 7, demonstrating a potential molecular mechanism by which PTH protects osteocytes from oxidative stress-induced senescence. Expression of p16^Ink4a was significantly upregulated in control cells exposed to H₂O₂ alone (–PTH) at day 14 (Figure 6G).

Next we assessed whether factors secreted by osteocytes under oxidative stress promote osteoclastogenesis. Conditioned medium (CM) from cells treated with H₂O₂ (+H₂O₂ CM, 7 days) (both without PTH pretreatment) was used to treat BMMCs. The BMMCs treated with +H₂O₂ CM increased osteoclast numbers compared to CM control (–H₂O₂ CM) (Figure 6F), demonstrating that, under oxidative stress, osteocytes secrete osteoclastogenic factors, which might contribute to the increased osteoclasts in Dmp1-PPR^KO mice. Treatment with H₂O₂ medium alone did not increase osteoclast numbers compared to CM control (data not shown).

Interestingly, when 12H-PPR^KO cells were pre-treated with PTH and then exposed to γ-irradiation (5 Gy) or busulfan (50 μM, 7 days), an alkylating agent [34], PTH had no effect (Supplementary Figure 8B).

Taken together these results demonstrate that, in the absence of PPR signaling in mature osteoblasts/osteocytes, there is an age-dependent trabecular bone loss associated with increased bone resorption driven by a significant increase in osteoclast numbers and activity and impaired bone formation. Mechanistically, PPR signaling in mature osteoblasts/osteocytes regulates osteoblast formation through serum sclerostin and osteoclastogenesis via secreted factors other than RANKL and OPG. At the molecular level, PPR signaling protects, in vitro, osteocytes from oxidative stress-induced cell death through a cAMP-mediated mechanism. Further studies will be needed to elucidate the downstream effectors.

Mice

Dmp1-PPR^KO (Dmp1-Cre⁺; PPR^fl/fl) mice under the C57BL/6 genetic background were generated as described previously [22]. Littermates homozygous for floxed PPR gene, but lacking Cre-recombinase expression, were used as control (Dmp1-Cre^–;PPR^fl/fl). Details are provided in Supplementary Methods. Institutional Animal Care and Use Committee and the Subcommittee on Research Animal Care at Massachusetts General Hospital and Boston University Medical Center approved all animal protocols.

Cell lines

Ocy454-12H (or 12H) cells, a derivative of the conditionally immortalized osteocytic cell line Ocy454, were used for all the in vitro experiments [33, 53, 54]. CRISPR/Cas9 genome editing technique was used to knockout PPR expression in these cells (12H-PPR^KO). Both control and 12H-PPR^KO cells were routinely maintained at 33°C (permissive temperature) with 5% CO₂ and cultured in growth medium (α minimum essential medium (Gibco, Thermo Fisher Scientific) containing 10% heat-inactivated fetal bovine serum (Gibco) and 1% antibiotic-antimycotic (Gibco)). Upon proliferation, cells were transferred to 37°C with 5% CO₂ to be fully differentiated into mature osteocytes and incubated for the number of days required for each experiment. Fresh growth medium was added every 3–4 days. Details are provided in Supplementary Methods.

Immunofluorescent staining

Immunofluorescent staining for RANKL and perilipin-1 was performed on paraffin sections of the left tibiae from middle-aged (13 months old) male mice. See Supplementary Methods for details. The following antibodies were used: anti-mouse perilipin-1 (#9349, Cell Signaling Technology, Danvers, MA, USA) (1:100), anti-mouse RANKL (#sc-7628, Santa Cruz Biotechnology, Dallas, TX, USA) (1:50), Alexa flour 546 donkey anti-rabbit IgG (#A10040, Invitrogen, Carlsbad, CA, USA) (1:100) and Alexa flour 488 donkey anti-goat IgG (#A11055, Invitrogen) (1:100).

Senescence-associated β-gal staining

Cells were plated at a density of 1.0 × 10⁵ cells/ml in growth medium on a 6- or 12-well plate (Biolite™) under 37°C with 5% CO₂ followed by treatment, on the next day, with 10 nM hPTH(1–34) for 18–22 hrs. Cells were then exposed to oxidative stress by adding an equal volume of 300 μM H₂O₂ medium (final concentration: 150 μM) on top of the well and cultured for an additional 7 or 14 days. Medium was replaced once on day 7. On day 7 and 14, conditioned medium was collected and the cells were either harvested for RNA isolation or fixed with 4% paraformaldehyde (Acros Organics, Fair Lawn, NJ, USA) in PBS for 15 min at room temperature for SA β-gal staining. Cells were then washed with PBS and incubated with SA β-gal staining solution (pH 6.0) overnight at 37°C without CO₂ injection. The SA β-gal staining solution was prepared by following the protocol from Chen et al. [55]. Cell viability was measured using PrestoBlue cell viability reagent (Invitrogen) as per manufacturer’s instruction. Bright field images were acquired under the microscope with a 4X objective (Keyence). The number of SA β-gal positive osteocytes was counted in a blinded fashion using ImageJ.

Oxidative stress-induced cell death and reactive oxygen species accumulation

Cells were plated at a density of 2.6 × 10⁴ cells/cm² in growth medium on a 24-well plate and grown for 3 days at 33°C. The growth medium was replaced and the cells were cultured for an additional 2 days under 37°C and treated with either 10 nM hPTH(1–34) or 10 μM forskolin for 18–22 hrs followed by an exposure to 1 mM H₂O₂ for 4 hrs or overnight. Cell viability was measured using PrestoBlue cell viability reagent as per manufacturer’s instruction. Intracellular levels of ROS were determined upon cell staining with 14 μM of 2′, 7′-dichlorofluorescin diacetate (DCFDA) (Sigma) for 30 min at 37°C with 5% CO₂. The cells were then trypsinized and transferred into wells of a 96-well plate and fluorescence (485 nm excitation and 535 nm emission) was measured using a spectrophotometer (TriStar LB941, Berthold Technologies, Oak Ridge, TN, USA). The fluorescent intensity was normalized with cell viability.

Statistics

Normal distribution and heteroscedasticity of the data was tested using D'Agostino-Pearson or Anderson-Darling test and Spearman's test, respectively. Equality of variance between two datasets was analyzed with the F test. Parametrical statistics were used for the data that follow normal distribution and/or equal variance. Otherwise, non-parametrical statistics were used. Statistical significance was defined as p values less than 0.05 (two-tailed). Statistical test used for each analysis is described in the legends of each figure. All statistical analysis was performed on GraphPad Prism (GraphPad Software, San Diego, CA, USA).