Characterization of TNSALP expression, localization, and activity in ovine utero-placental tissues†.

Tissue-nonspecific alkaline phosphatase (TNSALP; encoded by ALPL gene) has a critical role in the regulation of phosphate homeostasis postnatally. However, the utero-placental expression of TNSALP and the role in phosphate transport in pregnancy is poorly understood. Estrous cycles of ewes were synchronized, and ewes were euthanized and hysterectomized on Days 1, 9, or 14 of the estrous cycle or bred to fertile rams and euthanized and hysterectomized on Days 9, 12, 17, 30, 50, 70, 90, 110, or 125 of pregnancy. The expression of ALPL mRNA, immunolocalization of TNSALP protein, and quantification and localization of TNSALP enzymatic activity was performed on ovine endometria and placentomes. Day of the estrous cycle did not alter ALPL mRNA expression or enzymatic activity of TNSALP. TNSALP protein localized to uterine epithelial and stromal cells, blood vessels, myometrium, caruncular, and cotyledonary stroma. TNSALP activity was localized to uterine epithelia, blood vessels, caruncular stroma (from Day 70 of gestation), and the apical surface of chorionic epithelia (from Day 50 of gestation). TNSALP protein and activity localized to the apical surface of uterine epithelia during the estrous cycle and in early pregnancy. Endometrial TNSALP enzymatic activity was downregulated on Days 17 and 30 of gestation (P < 0.05). Expression of ALPL mRNA decreased in late gestation in endometria and placentomes (P < 0.05). TNSALP activity peaked in placentomes on Days 70 and 90 of gestation. Collectively, these results suggest a potential role of TNSALP in the regulation of phosphate transport and homeostasis at the maternal-conceptus interface in ruminants.

Phosphate, calcium, and vitamin D signaling, transport, and metabolism in the endometria of cyclic ewes.

BACKGROUND: Recent evidence suggests important roles for progesterone (P4) and interferon tau in the regulation of calcium, phosphate, and vitamin D signaling in the uteri of pregnant sheep. However, the effects of P4 and estradiol (E2), with respect to the expression of their receptors PGR and ESR1, respectively, in uterine epithelia on mineral signaling during the estrous cycle has not been investigated. Estrous cycles of mature Suffolk ewes were synchronized, prostaglandin F2α was administered, and ewes were observed for estrus (designated as Day 0) in the presence of vasectomized rams. On Days 1, 9, or 14 of the estrous cycle, hysterectomies were performed. RESULTS: 25-hydroxyvitamin D was more abundant in plasma from ewes on Day 14 than Day 1 (P < 0.05). Expression of fibroblast growth factor receptor 2 (FGFR2), a disintegrin and metalloprotease 17 (ADAM17), and parathyroid hormone-related protein (PTHrP) mRNAs was greater in endometria on Day 9 compared to Days 1 and 14 (P < 0.01). Similarly, expression of transient receptor potential cation channel subfamily V member 6 (TRPV6) mRNA was greater in endometria on Day 9 than Day 1 (P < 0.05). ATPase plasma membrane Ca2+ transporting 4 (ATP2B4) and S100 calcium binding protein G (S100G) mRNA expression was greater in endometria on Day 14 than on Days 1 and 9 (P < 0.01). In contrast, endometrial expression of vitamin D receptor (VDR) mRNA was lower on Days 9 and 14 than Day 1 (P < 0.01). Expression of klotho (KL) (P < 0.05) and cytochrome P450 family 24 subfamily A member 1 (CYP24) (P < 0.01) mRNAs was lower on Day 14 than Days 1 and 9. ADAM17, FGF23, CYP2R1, CYP27B1, KL, and VDR proteins immunolocalized to the uterine myometrium, blood vessels, and uterine luminal (LE), superficial glandular (sGE), and glandular (GE) epithelia. S100A9 protein was weakly expressed in the uterine myometrium, LE, sGE, and GE. Immunoreactivity of CYP2R1 and KL proteins in uterine LE and sGE was less on Day 1 than on Days 9 and 14. In contrast, S100G protein was expressed exclusively by GE, and immunoreactive S100G protein was less on Day 9. S100A12 protein localized to stromal cells of the uterine stratum spongiosum and blood vessels, but not by uterine epithelial cells. CONCLUSION: Collectively, these results implicate E2, P4, and PGR in the regulation of phosphate, calcium, and vitamin D signaling in cyclic ewes.

Guanylyl Cyclase-B Dependent Bone Formation in Mice is Associated with Youth, Increased Osteoblasts, and Decreased Osteoclasts.

C-type natriuretic peptide (CNP) activation of guanylyl cyclase-B (GC-B) catalyzes the synthesis of cGMP in chondrocytes and osteoblasts. Elevated cGMP stimulates long bone growth, and inactivating mutations in CNP or GC-B reduce cGMP, which causes dwarfism. GC-B7E/7E mice that express a GC-B mutant that cannot be inactivated by dephosphorylation exhibit increased CNP-dependent GC-B activity, which increases bone length, as well as bone mass and strength. Importantly, how GC-B increases bone mass is not known. Here, we injected 12-week-old, wild type mice once daily for 28 days with or without BMN-111 (Vosoritide), a proteolytically resistant CNP analog. We found that BMN-111 treated mice had elevated levels of osteocalcin and collagen 1 C-terminal telopeptide (CTX) as well as increased osteoblasts and osteoclasts. In BMN-111 injected mice, tibial mRNAs for Rank ligand and osteoprotegrin were increased and decreased, respectively, whereas sclerostin mRNA was elevated 400-fold, consistent with increased osteoclast activity and decreased osteoblast activity. Mineral apposition rates and trabecular bone mass were not elevated in response to BMN-111. Because 9-week-old male GC-B7E/7E mice have increased bone mass but do not exhibit increased mineral apposition rates, we examined 4-week-old male GC-B7E/7E mice and found that these animals had increased serum osteocalcin, but not CTX. Importantly, tibias from these mice had 37% more osteoblasts, 26% fewer osteoclasts as well as 36% and 40% higher mineral apposition and bone formation rates, respectively. We conclude that GC-B-dependent bone formation is coupled to an early juvenile process that requires both increased osteoblasts and decreased osteoclasts.

Structural pharmacology of PTH and PTHrP.

Parathyroid hormone (PTH) and PTH-related peptide (PTHrP) regulate extracellular phosphate and calcium homeostasis as well as bone remodeling. PTH is a classic endocrine peptide hormone whose synthesis and negative feedback by multiple factors control release from the parathyroid glands. PTHrP is ubiquitously expressed (pre- and postnatally) and acts in an autocrine/paracrine manner. This review considers the structural pharmacology and actions of PTH and PTHrP, biological consequences of inherited mutations, engineered analogs that illuminate similarities and differences in physiologic actions, and targeted therapeutic opportunities.

Uptake of Phosphate, Calcium, and Vitamin D by the Pregnant Uterus of Sheep in Late Gestation: Regulation by Chorionic Somatomammotropin Hormone.

Minerals are required for the establishment and maintenance of pregnancy and regulation of fetal growth in mammals. Lentiviral-mediated RNA interference (RNAi) of chorionic somatomammotropin hormone (CSH) results in both an intrauterine growth restriction (IUGR) and a non-IUGR phenotype in sheep. This study determined the effects of CSH RNAi on the concentration and uptake of calcium, phosphate, and vitamin D, and the expression of candidate mRNAs known to mediate mineral signaling in caruncles (maternal component of placentome) and cotyledons (fetal component of placentome) on gestational day 132. CSH RNAi Non-IUGR pregnancies had a lower umbilical vein−umbilical artery calcium gradient (p < 0.05) and less cotyledonary calcium (p < 0.05) and phosphate (p < 0.05) compared to Control RNAi pregnancies. CSH RNAi IUGR pregnancies had less umbilical calcium uptake (p < 0.05), lower uterine arterial and venous concentrations of 25(OH)D (p < 0.05), and trends for lower umbilical 25(OH)D uptake (p = 0.059) compared to Control RNAi pregnancies. Furthermore, CSH RNAi IUGR pregnancies had decreased umbilical uptake of calcium (p < 0.05), less uterine venous 25(OH)D (vitamin D metabolite; p = 0.055), lower caruncular expression of SLC20A2 (sodium-dependent phosphate transporter; p < 0.05) mRNA, and lower cotyledonary expression of KL (klotho; p < 0.01), FGFR1 (fibroblast growth factor receptor 1; p < 0.05), FGFR2 (p < 0.05), and TRPV6 (transient receptor potential vanilloid member 6; p < 0.05) mRNAs compared to CSH RNAi Non-IUGR pregnancies. This study has provided novel insights into the regulatory role of CSH for calcium, phosphate, and vitamin D utilization in late gestation.

Effects of exogenous progesterone on the expression of mineral regulatory molecules by ovine endometrium and placentomes†.

This study aimed to determine whether the acceleration of conceptus development induced by the administration of exogenous progesterone (P4) during the preimplantation period of pregnancy alters calcium, phosphate, and vitamin D signaling at the maternal-conceptus interface. Suffolk ewes (n = 48) were mated to fertile rams and received daily intramuscular injections of either corn oil (CO) vehicle or 25 mg of progesterone in CO (P4) for the first 8 days of pregnancy and hysterectomized on either Day 9 (CO, n = 5; P4, n = 6), 12 (CO, n = 9; P4, n = 4) or 125 (CO, n = 14; P4, n = 10) of gestation. The expression of S100A12 (P < 0.05) and fibroblast growth factor receptor (FGFR2) (P < 0.01) messenger RNAs (mRNAs) was lower in endometria from P4-treated ewes on Day 12. The expression of ADAM10 (P < 0.05) mRNA was greater in endometria from P4-treated ewes on Day 125. The expression of ADAM10 (P < 0.01), FGFR2 (P < 0.05), solute carrier (SLC)20A1 (P < 0.05), TRPV5 (P < 0.05), and TRPV6 (P < 0.01) mRNAs was greater, but KL mRNA expression was lower (P < 0.05) in placentomes from P4-treated ewes at Day 125. There was lower endometrial and greater placentomal expression of mRNAs involved in mineral metabolism and transport in twin compared to singleton pregnancies. Further, the expression of mRNAs involved in mineral metabolism and transport was greater in P4-treated twin placentomes. KL, FGF23, vitamin D receptor (VDR), S100A9, S100A12, S100G, and CYP27B1 proteins were immunolocalized in endometria and placentomes. Exogenous P4 in early pregnancy altered the expression of regulators of calcium, phosphate, and vitamin D on Day 125 of pregnancy indicating a novel effect of P4 on mineral transport at the maternal-conceptus interface.

Effects of progesterone and interferon tau on ovine endometrial phosphate, calcium, and vitamin D signaling†.

Given recent reports of expression of postnatal mineral transport regulators at the maternal-conceptus interface during the peri-implantation period, this study tested the hypothesis that progesterone (P4) and interferon tau (IFNT) regulate phosphate, calcium, and vitamin D signaling in the ovine endometrium. Mature Rambouillet ewes (n = 24) were surgically fitted with intrauterine catheters on day 7 of the estrous cycle. Ewes received daily intramuscular injections of 50 mg of P4 in corn oil vehicle and 75 mg of progesterone receptor antagonist (RU486) in corn oil from days 8 to 15, and twice-daily intrauterine injections of either control proteins (CX) or IFNT (25 µg/uterine horn/day) from days 11 to 15 resulting in four treatment groups: P4 + CX; P4 + IFNT; RU486 + P4 + CX; and RU486 + P4 + IFNT. On day 16, ewes were hysterectomized. RU486 + P4 + CX treated ewes had lower concentrations of 25 (OH) D in plasma than P4 + CX treated ewes (P < 0.05). Endometria from ewes treated with IFNT had greater expression of FGF23 (P < 0.01), S100A9 (P < 0.05), and S100A12 (P = 0.05) mRNAs and lower expression of ADAM10 mRNA (P < 0.01) than of ewes treated with CX proteins. Expression of FGF23 mRNA was greater in endometria of ewes that received RU486 + P4 + IFNT than in ewes that received RU486 + P4 + CX (hormone × protein interaction, P < 0.05). The expression of S100G mRNA was greater in endometria of ewes that received P4 + IFNT compared to ewes that received RU486 + P4 + IFNT (P < 0.05; hormone × protein interaction, P < 0.01). These data implicate P4 and IFNT in the regulation of phosphate, calcium, and vitamin D signaling during the peri-implantation period of pregnancy and provide a platform for continued mechanistic investigations.

Bone remineralization of lytic lesions in multiple myeloma - The Arkansas experience.

Multiple myeloma (MM) patients frequently present with extensive osteolytic bone lesions. However, the impact of myeloma treatment on focal lytic lesion remineralization has not been extensively studied. In this study, the effect of anti-myeloma treatment on the extent of bone remineralization was examined and potential mediators identified. Newly diagnosed MM patients enrolled in the Total Therapy 4 and 5 (TT4; n = 231, TT5; n = 64) protocols were longitudinally evaluated for changes in radiological parameters for a median of 6.1 years. Bone remineralization was defined as a sclerotic CT change within the lytic lesion and quantified as a percentage of remineralization, using the initial lesion size as a reference. Such changes were correlated to clinical and biochemical parameters, and the gene expression profile of bone marrow biopsy. Overall, remineralization occurred in 72% of patients (213/295). Of those patients that experienced remineralization, 36% (107/295) achieved at least 25% of bone remineralization. Patients with high-risk disease defined by gene expression profile signature (GEP70 ≥ 0.66) experienced significant remineralization compared to low-risk MM. Female patients were also more likely to experience bone remineralization and in a shorter median time (2.0 vs. 3.3 y). Factors such as serum alkaline phosphatase along with high levels of RUNX2 and SOX4 gene expression correlated with increasing extent of bone remineralization. This analysis demonstrated significant remineralization of lytic lesions in MM patients treated on TT clinical trials. While the underlying mechanism remains elusive these findings support the hypothesis that patient baseline bone-related factors play a fundamental role in the skeletal repair of bone lesions in MM that provide new opportunities for improving patient outcomes.

Omega-3 fatty acid modulation of serum and osteocyte tumor necrosis factor-α in adult mice exposed to ionizing radiation.

Chronic inflammation leads to bone loss and fragility. Proinflammatory cytokines such as tumor necrosis factor-alpha (TNF-α) consistently promote bone resorption. Dietary modulation of proinflammatory cytokines is an accepted therapeutic approach to treat chronic inflammation, including that induced by space-relevant radiation exposure. As such, these studies were designed to determine whether an anti-inflammatory diet, high in omega-3 fatty acids, could reduce radiation-mediated bone damage via reductions in the levels of inflammatory cytokines in osteocytes and serum. Lgr5-EGFP C57BL/6 mice were randomized to receive diets containing fish oil and pectin (FOP; high in omega-3 fatty acids) or corn oil and cellulose (COC; high in omega-6 fatty acids) and then acutely exposed to 0.5-Gy 56Fe or 2.0-Gy gamma-radiation. Mice fed the FOP diet exhibited consistent reductions in serum TNF-α in the 56Fe experiment but not the gamma-experiment. The percentage osteocytes (%Ot) positive for TNF-α increased in gamma-exposed COC, but not FOP, mice. Minimal changes in %Ot positive for sclerostin were observed. FOP mice exhibited modest improvements in several measures of cancellous microarchitecture and volumetric bone mineral density (BMD) postexposure to 56Fe and gamma-radiation. Reduced serum TNF-α in FOP mice exposed to 56Fe was associated with either neutral or modestly positive changes in bone structural integrity. Collectively, these data are generally consistent with previous findings that dietary intake of omega-3 fatty acids may effectively mitigate systemic inflammation after acute radiation exposure and facilitate maintenance of BMD during spaceflight in humans.NEW & NOTEWORTHY This is the first investigation, to our knowledge, to test the impact of a diet high in omega-3 fatty acids on multiple bone structural and biological outcomes following space-relevant radiation exposure. Novel in biological outcomes is the assessment of osteocyte responses to this stressor. These data also add to the growing evidence that low-dose exposures to even high-energy ion species like 56Fe may have neutral or even small positive impacts on bone.

Bisphosphonates in veterinary medicine: The new horizon for use.

Bisphosphonates (BPs) are characterized by their ability to bind strongly to bone mineral and inhibit bone resorption. However, BPs exert a wide range of pharmacological activities beyond the inhibition of bone resorption, including the inhibition of cancer cell metastases and angiogenesis and the inhibition of proliferation and apoptosis in vitro. Additionally, the inhibition of matrix metalloproteinase activity, altered cytokine and growth factor expression, as well as reductions in parameters of pain have also been reported. In humans, clinical BP use has transformed the treatment of post-menopausal osteoporosis, rare bone diseases such as osteogenesis imperfecta, as well as multiple myeloma and metastatic breast and prostate cancer, albeit not without infrequent but significant adverse events. Despite the well-characterized health benefits of BP use in humans, the evidence-base for the therapeutic efficacy of BPs in veterinary medicine is, by comparison, limited. Notwithstanding, BPs are used widely in small animal veterinary practice for the medical management of hyperparathyroidism, idiopathic hypercalcemia in cats, as well as for the palliative care of bone tumors which are common in dogs, and in particular, primary bone tumors such as osteosarcoma. Palliative BP treatment has also recently increased in veterinary oncology to alleviate tumor-associated bone pain. In equine veterinary practice, non-nitrogen-containing BPs are FDA-approved to control clinical signs associated with navicular syndrome in adult horses. However, there are growing concerns regarding the off-label use of BPs in juvenile horses. Here we discuss the current understanding of the strengths, weaknesses and current controversies surrounding BP use in veterinary medicine to highlight the future utility of these potentially beneficial drugs.

PTH and PTHrP Actions on Bone.

Parathyroid hormone (PTH), PTH-related peptide (PTHrP), PTHR, and their cognate G protein-coupled receptor play defining roles in the regulation of extracellular calcium and phosphate metabolism and in controlling skeletal growth and repair. Acting through complex signaling mechanisms that in many instances proceed in a tissue-specific manner, precise control of these processes is achieved. A variety of direct and indirect disease processes, along with genetic anomalies, can cause these schemes to become dysfunctional. Here, we review the basic components of this regulatory network and present both the well-established elements and emerging findings and concepts with the overall objective to provide a framework for understanding the elementary aspects of how PTH and PTHrP behave and as a call to encourage further investigation that will yield more comprehensive understanding of the physiological and pathological steps at play, with a goal toward novel therapeutic interventions.

Interaction of Brucella abortus with Osteoclasts: a Step toward Understanding Osteoarticular Brucellosis and Vaccine Safety.

Osteoarticular disease is a frequent complication of human brucellosis. Vaccination remains a critical component of brucellosis control, but there are currently no vaccines for use in humans, and no in vitro models for assessing the safety of candidate vaccines in reference to the development of bone lesions currently exist. While the effect of Brucella infection on osteoblasts has been extensively evaluated, little is known about the consequences of osteoclast infection. Murine bone marrow-derived macrophages were derived into mature osteoclasts and infected with B. abortus 2308, the vaccine strain S19, and attenuated mutants S19vjbR and B. abortusΔvirB2 While B. abortus 2308 and S19 replicated inside mature osteoclasts, the attenuated mutants were progressively killed, behavior that mimics infection kinetics in macrophages. Interestingly, B. abortus 2308 impaired the growth of osteoclasts without reducing resorptive activity, while osteoclasts infected with B. abortus S19 and S19vjbR were significantly larger and exhibited enhanced resorption. None of the Brucella strains induced apoptosis or stimulated nitric oxide or lactose dehydrogenase production in mature osteoclasts. Finally, infection of macrophages or osteoclast precursors with B. abortus 2308 resulted in generation of smaller osteoclasts with decreased resorptive activity. Overall, Brucella exhibits similar growth characteristics in mature osteoclasts compared to the primary target cell, the macrophage, but is able to impair the maturation and alter the resorptive capacity of these cells. These results suggest that osteoclasts play an important role in osteoarticular brucellosis and could serve as a useful in vitro model for both analyzing host-pathogen interactions and assessing vaccine safety.

Atypical femoral fractures from bisphosphonate in cancer patients - Review.

Bisphosphonates are commonly used in patients with metastatic bone disease to prevent skeletal related events. Atypical femur fracture is a known complication of long-term bisphosphonate use but the incidence in cancer patients and pathogenesis are not well known. Several mechanisms of pathogenesis have been proposed including altered angiogenesis, altered bone mechanical properties, micro damage and bone remodeling suppression. Atypical femur fractures are atraumatic or minimally traumatic fractures in the sub trochanteric region or the femoral shaft. Awareness of atypical femur fractures is critical to diagnose and treat them in a timely manner. There is a paucity of data regarding the management of atypical femur fracture in patients with malignancy. Management options of atypical femur fractures include stopping bisphosphonates, initiating calcium/vitamin D supplementation and either surgery with internal fixation or conservative management. In the future, it will be important to explore the effect of continuous vs. intermittent exposure, cumulative dose and length of exposure on the incidence of this complication. Herein, we review the epidemiology, risk factors, management options and proposed mechanisms of pathogenesis of atypical femur fractures.

Bisphosphonate use in the horse: what is good and what is not?

BACKGROUND: Bisphosphonates (BPs) are a family of molecules characterized by two key properties: their ability to bind strongly to bone mineral and their inhibitory effects on mature osteoclasts and thus bone resorption. Chemically two groups of BPs are recognized, non-nitrogen-containing and nitrogen-containing BPs. Non-nitrogen-containing BPs incorporate into the energy pathways of the osteoclast, resulting in disrupted cellular energy metabolism leading to cytotoxic effects and osteoclast apoptosis. Nitrogen-containing BPs primarily inhibit cholesterol biosynthesis resulting in the disruption of intracellular signaling, and other cellular processes in the osteoclast. BODY: BPs also exert a wide range of physiologic activities beyond merely the inhibition of bone resorption. Indeed, the breadth of reported activities include inhibition of cancer cell metastases, proliferation and apoptosis in vitro. In addition, the inhibition of angiogenesis, matrix metalloproteinase activity, altered cytokine and growth factor expression, and reductions in pain have been reported. In humans, clinical BP use has transformed the treatment of both post-menopausal osteoporosis and metastatic breast and prostate cancer. However, BP use has also resulted in significant adverse events including acute-phase reactions, esophagitis, gastritis, and an association with very infrequent atypical femoral fractures (AFF) and osteonecrosis of the jaw (ONJ). CONCLUSION: Despite the well-characterized health benefits of BP use in humans, little is known regarding the effects of BPs in the horse. In the equine setting, only non-nitrogen-containing BPs are FDA-approved primarily for the treatment of navicular syndrome. The focus here is to discuss the current understanding of the strengths and weaknesses of BPs in equine veterinary medicine and highlight the future utility of these potentially highly beneficial drugs.

FRAX is a robust predictor of baseline vertebral fractures in multiple myeloma patients.

FRAX is a commonly used tool to evaluate patient fracture risk based on individual patient models that integrate the risks associated with clinical risk factors with or without bone mineral density (BMD) at the femoral neck. Retrospectively, factors identified by the FRAX scoring algorithm were used to predict the risk for vertebral compression fractures at baseline in newly diagnosed multiple myeloma patients. The data were derived from myeloma patients enrolled in Total Therapy Protocols (TT4 & TT5) between 8/2008 and 9/2017. FRAX scores were calculated and baseline PET and MRI imaging obtained. Univariate and multivariate logistic regression analyses determined the association between FRAX components and the existence of vertebral compression fractures, both pathologic and osteoporotic. The patient population had a median age of 61 years (43-76), 37% female, and 87% white. The median major osteoporotic score (MOS) and Hip fracture scores (HFS) for TT4 patients (low-risk myeloma) were 5.6 and 0.5, respectively, while median MOS and HFS for TT5 (high risk myeloma) patients were 6.2 and 0.7, respectively. The odds ratio for fracture at diagnosis in patients with elevated MOS (>2), and HFS (>4.5) was significant OR (1.48, 95% confidence interval (1.35,1.62)) and OR (1.61, 95% confidence interval (1.42, 1.81)), respectively. In sum, an elevated baseline FRAX score was highly predictive of baseline vertebral fractures in MM patients at presentation. In addition, patients with higher FRAX scores had significantly shorter survival in the low-risk (TT4) group but this survival effect was not seen in the high-risk (TT5) group. These findings suggest that FRAX assessment of baseline fracture risk is beneficial in MM patients to identify an individual patients' risk of vertebral fracture.

Surgical thyroparathyroidectomy prevents progression of 5TGM1 murine multiple myeloma in vivo.

The 5TGM1 multiple myeloma transplanted C57BL6/KaLwRij model recapitulates many disease features including monoclonal paraprotein production as well as the development of osteolytic bone lesions. Since a significant association between serum parathyroid hormone PTH variations, bone anabolism and myeloma progression in patients receiving proteasome inhibitors exists, this study investigated the effect of the PTH axis on murine myeloma development in vivo. C57BL6/KaLwRij myeloma-bearing mice underwent thyroparathyroidectomy (TPTX) before and after 5TGM1 cell transplantation. TPTX significantly and permanently inhibited 5TGM1 myeloma cell engraftment and prevented multiple myeloma growth and progression. These data support the hypothesis that the PTH axis is an important mediator of myeloma bone disease.

Thymic PTH Increases After Thyroparathyroidectomy in C57BL/KaLwRij Mice.

We previously reported a substantial correlation between serum parathyroid hormone (PTH) levels and the myeloma response to proteasome inhibition that suggests a crucial role for the PTH receptor 1 system in the control of myeloma tumor growth. While investigating the role of PTH in the antimyeloma effect, we observed the recovery of serum PTH levels after thyroparathyroidectomy (TPTX). Although the presence of thymus-derived PTH has been reported previously, the existence or role of thymic PTH in the serum remains controversial. Here, TPTX was performed in 8- to 12-week-old C57BL/KaLwRij mice to delineate the potential source(s) for the recovery of serum PTH. Immediately after TPTX, the expected loss of measurable serum PTH was observed. Serum PTH levels recovered 3 to 4 weeks after TPTX. Thirteen endocrine organs from mice with recovered serum PTH were examined. The thymus from control mice expressed measurable and detectable Pth transcripts; however, the Pth transcript level was substantially elevated in tissue from TPTX mice. Western blot analysis of the thymus demonstrated a reproducible and distinct PTH band in thymus tissue that was significantly increased after TPTX. To directly confirm the identity of the distinct PTH band, immunoprecipitated proteins were isolated and subjected to tandem mass spectrometry. After fragmentation and direct peptide sequencing, PTH peptides PTH(1-13) and PTH(54-70), diagnostic for PTH, were identified. These data demonstrate that the murine thymus produces PTH and that after TPTX the thymus becomes the major source of serum PTH, compensating for the loss of the parathyroid glands and returning circulating PTH levels to normal.

Hallmarks of Bone Metastasis.

Breast cancer bone metastasis develops as the result of a series of complex interactions between tumor cells, bone marrow cells, and resident bone cells. The net effect of these interactions are the disruption of normal bone homeostasis, often with significantly increased osteoclast and osteoblast activity, which has provided a rational target for controlling tumor progression, with little or no emphasis on tumor eradication. Indeed, the clinical course of metastatic breast cancer is relatively long, with patients likely to experience sequential skeletal-related events (SREs), often over lengthy periods of time, even up to decades. These SREs include bone pain, fractures, and spinal cord compression, all of which may profoundly impair a patient's quality-of-life. Our understanding of the contributions of the host bone and bone marrow cells to the control of tumor progression has grown over the years, yet the focus of virtually all available treatments remains on the control of resident bone cells, primarily osteoclasts. In this perspective, our focus is to move away from the current emphasis on the control of bone cells and focus our attention on the hallmarks of bone metastatic tumor cells and how these differ from primary tumor cells and normal host cells. In our opinion, there remains a largely unmet medical need to develop and utilize therapies that impede metastatic tumor cells while sparing normal host bone and bone marrow cells. This perspective examines the impact of metastatic tumor cells on the bone microenvironment and proposes potential new directions for uncovering the important mechanisms driving metastatic progression in bone based on the hallmarks of bone metastasis.

PTHrP(12-48) Modulates the Bone Marrow Microenvironment and Suppresses Human Osteoclast Differentiation and Lifespan.

Bone is a common site for metastasis in breast cancer patients and is associated with a series of complications that significantly compromise patient survival, partially due to the advanced stage of disease at the time of detection. Currently, no clinically-approved biomarkers can identify or predict the development of bone metastasis. We recently identified a unique peptide fragment of parathyroid hormone-related protein (PTHrP), PTHrP(12-48), as a validated serum biomarker in breast cancer patients that correlates with and predicts the presence of bone metastases. In this study, the biological activity and mode of action of PTHrP(12-48) was investigated. Sequence-based and structure-based bioinformatics techniques predicted that the PTHrP(12-48) fragment formed an alpha helical core followed by an unstructured region after residue 40 or 42. Thereafter, detailed structure alignment and molecular docking simulations predicted a lack of interaction between PTHrP(12-48) and the cognate PTH1 receptor (PTHR1). The in silico prediction was confirmed by the lack of PTHrP(12-48)-stimulated cAMP accumulation in PTHR1-expressing human SaOS2 cells. Using a specific human PTHrP(12-48) antibody that we developed, PTHrP(12-48) was immunolocalized in primary and bone metastatic human breast cancer cells, as well as within human osteoclasts (OCLs) in bone metastasis biopsies, with little or no localization in other resident bone or bone marrow cells. In vitro, PTHrP(12-48) was internalized into cultured primary human OCLs and their precursors within 60 min. Interestingly, PTHrP(12-48) treatment dose-dependently suppressed osteoclastogenesis, via the induction of apoptosis in both OCL precursors as well as in mature OCLs, as measured by the activation of cleaved caspase 3. Collectively, these data suggest that PTHrP(12-48) is a bioactive breast cancer-derived peptide that locally regulates the differentiation of hematopoietic cells and the activity of osteoclasts within the tumor-bone marrow microenvironment, perhaps to facilitate tumor control of bone. © 2017 American Society for Bone and Mineral Research.

Extensive Remineralization of Large Pelvic Lytic Lesions Following Total Therapy Treatment in Patients With Multiple Myeloma.

Osteolytic bone lesions are a hallmark of multiple myeloma (MM) bone disease. Bone destruction is associated with severely imbalanced bone remodeling, secondary to increased osteoclastogenesis and significant osteoblast suppression. Lytic lesions of the pelvis are relatively common in MM patients and are known to contribute to the increased morbidity because of the high risk of fracture, which frequently demands extensive surgical intervention. After observing unexpected radiological improvement in serial large pelvic CT assessment in a patient treated in a total therapy protocol, the radiographic changes of pelvic osteolytic lesions by PET/CT scanning in patients who received Total Therapy 4 (TT4) treatment for myeloma were retrospectively analyzed. Sixty-two (62) patients with lytic pelvic lesions >1 cm in diameter were identified at baseline PET/CT scanning. Follow-up CT studies showed that 27 of 62 patients (43%) with large baseline pelvic lesions achieved significant reaccumulation of radiodense mineralization at the lytic cortical site. The average size of lytic lesions in which remineralization occurred was 4 cm (range, 1.3 to 10 cm). This study clearly demonstrates that mineral deposition in large pelvic lesions occurs in a significant proportion of MM patients treated with TT4, potentially affecting patient outcomes, quality of life, and future treatment strategies. © 2017 American Society for Bone and Mineral Research.