J. Ignacio Aguirre

A novel ligand-independent function of the estrogen receptor is essential for osteocyte and osteoblast mechanotransduction.

Bone senses and adapts to meet mechanical needs by means of an extensive mechanotransduction network comprising osteocytes (former osteoblasts entrapped in mineral) and their cytoplasmic projections through which osteocytes communicate with osteoblasts and osteoclasts on the bone surface. Mechanical stimulation promotes osteocyte (and osteoblast) survival by activating the extracellular signal-regulated kinases, ERKs. Estrogens have similar effects and, intriguingly, the adaptive response of bone to mechanical forces is defective in mice lacking estrogen receptor (ER) α or ERβ. We report that ERKs are not activated by stretching in osteocytic and osteoblastic cells in which both ERα and ERβ have been knocked out or knocked down and this is reversed partially by transfection of either one of the two human ERs and fully by transfection of both receptors. ERK activation in response to stretching is also recovered by transfecting the ligand-binding domain (E) of either receptor or an ERα mutant that does not bind estrogens. Furthermore, mechano-responsiveness is restored by transfecting the Eα targeted to the plasma membrane, but not to the nucleus, whereas ERα mutants with impaired plasma membrane localization or binding to caveolin-1 fail to confer ERK activation in response to stretching. Lastly, the ER antagonist ICI 182,780 abrogates ERK activation and the anti-apoptotic effect of mechanical stimulation. We conclude that in addition to their role as ligand-dependent mediators of the effects of estrogens, the ERs participate in the transduction of mechanical forces into pro-survival signaling in bone cells, albeit in a ligand-independent manner.

Oncologic doses of zoledronic acid induce osteonecrosis of the jaw-like lesions in rice rats (Oryzomys palustris) with periodontitis

Though osteonecrosis of the jaw (ONJ) is temporally‐associated with the use of nitrogen‐containing bisphosphonates (N‐BPs), a cause‐and‐effect relationship has not yet been established. We hypothesize that ONJ is a two‐stage process in which: (1) risk factors initiate pathologic processes in the oral cavity that lead to a supranormal rate of hard tissue necrosis; and (2) powerful antiresorptives reduce the rate of removal of necrotic bone sufficiently to allow its net accumulation in the jaw. To test this hypothesis, we used the rice rat model of periodontitis. At age 28 days, rats (n = 15/group) were placed on a high‐sucrose and casein diet to exacerbate the development of periodontitis. Animals were injected subcutaneously (SC) biweekly with vehicle or alendronate (ALN, 15 µg/kg), or IV once monthly with vehicle, a low dose (LD) of zoledronic acid (ZOL), or a high dose (HD) of ZOL and sacrificed after 6, 12, 18, and 24 weeks. Mandibles and maxillae were analyzed to determine the effects on the: (1) progression of periodontitis; (2) integrity of alveolar bone; (3) status of bone resorption and formation; (4) vascularity; and (5) osteocyte viability. We found that only HD‐ZOL induced ONJ‐like lesions in mandibles of rice rats after 18 and 24 weeks of treatment. These lesions were characterized by areas of exposed necrotic alveolar bone, osteolysis, a honeycomb‐like appearance of the alveolar bone, presence of bacterial colonies, and periodontal tissue destruction. In addition, inhibition of bone formation, a paradoxical abolition of the antiresorptive effect of only HD‐ZOL, increased osteocyte necrosis/apoptosis, and decreased blood vessel number were found after 18 and/or 24 weeks. Our study suggests that only HD‐ZOL exacerbates the inflammatory response and periodontal tissue damage in rice rats, inducing bone lesions that resemble ONJ.

Role for β1 integrins in cortical osteocytes during acute musculoskeletal disuse

The mammalian skeleton adjusts bone structure and strength in response to changes in mechanical loading, however the molecular and cellular mechanisms governing this process in vivo are unknown. Terminally differentiated osteoblasts, the osteocytes, are presumptive mechanosensory cells for bone, and cell culture studies demonstrate that beta1 integrins participate in mechanical signaling. To determine the role of beta1 integrins in osteoblasts in vivo, we used the Cre-lox system to delete beta1 integrin from cells committed to the osteoblast lineage. While pCol2.3 Cre-mediated recombination was widespread in bones from Colalpha1(I)-Cre+/beta1fl/fl conditional knockout mice (cKO), beta1 integrin protein was depleted from cortical osteocytes, but not from cancellous osteocytes or cells lining bone surfaces in adults. Bones from cKO mice that were normally loaded were similar in structure to WT littermates. However, hindlimb unloading of adult cKO mice for one week intended to cause bone loss (disuse osteopenia), resulted in unexpected, rapid changes in the geometry of cortical bone; hindlimb unloading increased the cross-sectional area, marrow area, and moments of inertia in cKO, but not WT mice. Furthermore, these hindlimb unloading-induced geometric changes in cortical bone of cKO mice resulted in increased whole bone bending stiffness and strength of the femur. Together, these results confirmed the concept that osteocytes are mechanosensory cells and showed beta1 integrins in cortical osteocytes limited changes in cortical geometry in response to disuse, thus providing the first in vivo evidence that beta1 integrins on osteocytes mediate specific aspects of mechanotransduction.

Time-dependent cellular and transcriptional changes in the osteoblast lineage associated with sclerostin antibody treatment in ovariectomized rats

Inhibition of sclerostin with sclerostin antibody (Scl-Ab) has been shown to stimulate bone formation, decrease bone resorption, and increase bone mass in both animals and humans. To obtain insight into the temporal cellular and transcriptional changes in the osteoblast (OB) lineage associated with long-term Scl-Ab treatment, stereological and transcriptional analyses of the OB lineage were performed on lumbar vertebrae from aged ovariectomized rats. Animals were administered Scl-Ab 3 or 50mg/kg/wk or vehicle (VEH) for up to 26weeks (d183), followed by a treatment-free period (TFP). At 50mg/kg/wk, bone volume (BV/total volume [TV]) increased through d183 and declined during the TFP. Bone formation rate (BFR/bone surface [BS]) and total OB number increased through d29, then progressively declined, coincident with a decrease in total osteoprogenitor (OP) numbers from d29 through d183. Analysis of differentially expressed genes (DEGs) from microarray analysis of mRNA isolated from laser capture microdissection samples enriched for OB, lining cells, and osteocytes (OCy) revealed modules of genes that correlated with BFR/BS, BV/TV, and osteoblastic surface (Ob.S)/BS. Expression change of canonical Wnt target genes was similar in all three cell types at d8, including upregulation of Twist1 and Wisp1. At d29, the pattern of Wnt target gene expression changed in the OCy, with Twist1 returning to VEH level, sustained upregulation of Wisp1, and upregulation of several other Wnt targets that continued into the TFP. Predicted activation of pathways recognized to integrate with and regulate canonical Wnt signaling were also activated at d29 in the OCy. The most significantly affected pathways represented transcription factor signaling known to inhibit cell cycle progression (notably p53) and mitogenesis (notably c-Myc). These changes occurred at the time of peak BFR/BS and continued as BFR/BS declined during treatment, then trended toward VEH level in the TFP. Concurrent with this transcriptional switch was a reduction in OP numbers, an effect that would ultimately limit bone formation. This study confirms that the initial transcriptional response in response to Scl-Ab is activation of canonical Wnt signaling and the data demonstrate that there is induction of additional regulatory pathways in OCy with long-term treatment. The interactions between Wnt and p53/c-Myc signaling may be key in limiting OP populations, thus contributing to self-regulation of bone formation with continued Scl-Ab administration.

Effects of Basic Fibroblast Growth Factor and a Prostaglandin E2 Receptor Subtype 4 Agonist on Osteoblastogenesis and Adipogenesis in Aged Ovariectomized Rats

bFGF stimulates osteo‐ and adipogenesis concurrently at skeletal sites with red but not with fatty marrow, whereas a PGE2 receptor subtype 4 agonist has bone anabolic effects at both skeletal sites and decreases adipose tissue within red and fatty marrow.

Differential temporal effects of sclerostin antibody and parathyroid hormone on cancellous and cortical bone and quantitative differences in effects on the osteoblast lineage in young intact rats

Sclerostin antibody (Scl-Ab) and parathyroid hormone (PTH) are bone-forming agents that have different modes of action on bone, although a study directly comparing their effects has not been conducted. The present study investigated the comparative quantitative effects of these two bone-forming agents over time on bone at the organ, tissue, and cellular level; specifically, at the level of the osteoblast (Ob) lineage in adolescent male and female rats. Briefly, eight-week old male and female Sprague-Dawley rats were administered either vehicle, Scl-Ab (3 or 50mg/kg/week subcutaneously), or human PTH (1-34) (75 μg/kg/day subcutaneously) for 4 or 26 weeks. The 50mg/kg Scl-Ab and the PTH dose were those used in the respective rat lifetime pharmacology studies. Using robust stereological methods, we compared the effects of these agents specifically at the level of the Ob lineage in vertebrae from female rats. Using RUNX2 or nestin immunostaining, location, and morphology, the total number of osteoprogenitor subpopulations, Ob, and lining cells were estimated using the fractionator or proportionator estimators. Density estimates were also calculated referent to total bone surface, total Ob surface, or total marrow volume. Scl-Ab generally effected greater increases in cancellous and cortical bone mass than PTH, correlating with higher bone formation rates (BFR) at 4 weeks in the spine and mid-femur without corresponding increases in bone resorption indices. The increases in vertebral BFR/BS at 4 weeks attenuated with continued treatment to a greater extent with Scl-Ab than with PTH. At 4 weeks, both Scl-Ab and PTH effected equivalent increases in total Ob number (Ob.N). Ob density on the formative surfaces (Ob.N/Ob.S) remained similar across groups while mineral apposition rate (MAR) was significantly higher with Scl-Ab at week 4, reflecting an increase in individual Ob vigor relative to vehicle and PTH. After 26 weeks, Scl-Ab maintained BFR/BS with fewer Ob and lower Ob.N/Ob.S by increasing the Ob footprint (bone surface area occupied by an Ob) and increasing MAR, compared with PTH. The lower Ob.N and Ob.N/Ob.S with Scl-Ab at 26 weeks were associated with decreased osteoprogenitor numbers compared with both vehicle and PTH, an effect not evident at week 4. Osteoprogenitor numbers were generally positively correlated with Ob.N across groups and timepoints, suggesting dynamic coordination between the progenitor and Ob populations. The time-dependent reductions in subpopulations of the Ob lineage with Scl-Ab may be integral to the greater attenuation or self-regulation of bone formation observed at the vertebra, as PTH required more Ob at the formative site with correlative increased numbers of progenitors compared with Scl-Ab indicating potentially greater stimulus for progenitor pool proliferation or differentiation.

Teriparatide [rhPTH (1-34)], But Not Strontium Ranelate, Demonstrated Bone Anabolic Efficacy in Mature, Osteopenic, Ovariectomized Rats

We compared teriparatide (TPTD) and strontium ranelate (SR) efficacy on bone formation activity in a mature rat model of estrogen-deficiency bone loss. Rats were ovariectomized (OVX) at age 6 months and permitted to lose bone for 2 months to establish osteopenia before initiation of treatment with TPTD (5 or 15 μg/kg · d sc) or SR (150 or 450 mg/kg · d oral gavage). After 3 wk, RT-PCR analyses of bone formation genes in the distal femur metaphysis showed significant elevation of collagen 1α2, osteocalcin, bone sialoprotein, alkaline phosphatase, and Runx2 gene expression at both TPTD doses, relative to OVX controls. SR had no significant effect on expression of these genes. TPTD treatment for 12 wk dose dependently increased lumbar vertebral (LV) and femoral midshaft bone mineral content (BMC) and bone mineral density over pretreatment and age-matched OVX controls. SR 150 increased BMC, and SR 450 increased BMC and bone mineral density of femoral midshaft and LV over OVX controls. There were significant dose-dependent TPTD increases of LV and femoral neck strength, and TPTD 15 also increased midshaft strength compared with pretreatment and age-matched OVX controls. SR did not enhance bone strength relative to pretreatment or age-matched OVX controls. Histomorphometry of the proximal tibial metaphysis showed dose-dependent effects of TPTD on trabecular area, number, width, and osteoblast surface, bone mineralizing surface, and bone formation rate relative to pretreatment and age-matched OVX controls, whereas SR had no effect on these parameters. These findings confirmed the bone anabolic efficacy of teriparatide, but not SR in mature, osteopenic, OVX rats.

Longitudinal Examination of Bone Loss in Male Rats After Moderate–Severe Contusion Spinal Cord Injury

To elucidate mechanisms of bone loss after spinal cord injury (SCI), we evaluated the time-course of cancellous and cortical bone microarchitectural deterioration via microcomputed tomography, measured histomorphometric and circulating bone turnover indices, and characterized the development of whole bone mechanical deficits in a clinically relevant experimental SCI model. 16-weeks-old male Sprague–Dawley rats received T9 laminectomy (SHAM, n = 50) or moderate–severe contusion SCI (n = 52). Outcomes were assessed at 2-weeks, 1-month, 2-months, and 3-months post-surgery. SCI produced immediate sublesional paralysis and persistent hindlimb locomotor impairment. Higher circulating tartrate-resistant acid phosphatase 5b (bone resorption marker) and lower osteoblast bone surface and histomorphometric cancellous bone formation indices were present in SCI animals at 2-weeks post-surgery, suggesting uncoupled cancellous bone turnover. Distal femoral and proximal tibial cancellous bone volume, trabecular thickness, and trabecular number were markedly lower after SCI, with the residual cancellous network exhibiting less trabecular connectivity. Periosteal bone formation indices were lower at 2-weeks and 1-month post-SCI, preceding femoral cortical bone loss and the development of bone mechanical deficits at the distal femur and femoral diaphysis. SCI animals also exhibited lower serum testosterone than SHAM, until 2-months post-surgery, and lower serum leptin throughout. Our moderate–severe contusion SCI model displayed rapid cancellous bone deterioration and more gradual cortical bone loss and development of whole bone mechanical deficits, which likely resulted from a temporal uncoupling of bone turnover, similar to the sequalae observed in the motor-complete SCI population. Low testosterone and/or leptin may contribute to the molecular mechanisms underlying bone deterioration after SCI.

Treatment With a Soluble Bone Morphogenetic Protein Type 1A Receptor (BMPR1A) Fusion Protein Increases Bone Mass and Bone Formation in Mice Subjected to Hindlimb Unloading: BMPR1A-mFc INCREASES BONE MASS/FORMATION IN MICE SUBJECTED TO HLU

Previous work has shown that the soluble murine BMPR1A–fusion protein (mBMPR1A‐mFc) binds to BMP2 and BMP4 with high affinity, preventing downstream signaling. Further, treatment of intact and ovariectomized mice with mBMPR1A‐mFc leads to increased bone mass, and improved bone microarchitecture and strength, via increased bone formation and reduced resorption. In this study, we tested the effects of mBMPR1A‐mFc on disuse‐induced bone loss caused by 21 days of hindlimb unloading (HLU) via tail suspension versus cage controls (CONs). Adult female C57BL/6J mice (12 weeks old) were assigned to one of four groups (n = 10 each): CON‐VEH; CON‐mBMPR1A‐mFc; HLU‐VEH; and HLU‐mBMPR1A‐mFc. Mice were injected subcutaneously with VEH or mBMPR1A‐mFc (4.5 mg/kg, 2×/week). Leg BMD declined in the HLU‐VEH group (–5.3% ± 1.3%), whereas it was unchanged in HLU‐mBMPR1A‐mFc (–0.3% ± 0.9%, p < 0.05 versus HLU‐VEH). Leg BMD increased significantly more in CON‐mBMPR1A‐mFc than CON‐VEH (10.2% ± 0.6% versus 4.4% ± 0.8%). In the femur, trabecular, and cortical bone microarchitecture was worse in the HLU‐VEH compared to CON‐VEH mice, whereas mBMPR1A‐mFc treatment for 3 weeks led to greater Tb.BV/TV, Tb.Th, and midshaft Ct.Th in both the HLU and CON groups compared to comparable VEH‐treated counterparts (p < 0.05). HLU‐mBMPR1A‐mFc mice also had 21% greater failure load (p < 0.05) compared to their VEH‐treated counterparts. Dynamic histomorphometry indicated that treatment with mBMPR1A‐mFc led to significantly greater mineralizing surface and mineral apposition rate, resulting in a 3.5‐fold and fivefold higher bone formation rate in the mBMPR1A‐mFc‐treated CON and HLU animals versus VEH groups, respectively. mBMPR1A‐mFc‐treated mice had a similar osteoblast surface but significantly lower osteoclast surface than VEH‐treated animals in both the CON and HLU groups. Altogether, these findings suggest that treatment with the soluble BMPR1A fusion protein may be useful for maintenance of skeletal integrity in the setting of disuse‐induced bone loss.