Parathyroid Hormone Treatment Research Articles

Parathyroid hormone promotes induction of beige adipocytes and reversibly ameliorates muscle quality and atrophy following chronic rotator cuff tear in a rat model.

Fatty infiltration (FI) and muscle atrophy (MA) in the rotator cuff muscles following rotator cuff tears (RCT) persist post repair, increasing the risk of re-tears. Brown adipocyte-like "beige adipocytes" are expected to have a therapeutic effect on intramuscular FI and MA due to their lipolytic activity and the muscle regenerative effects of their secreted factors. However, whether parathyroid hormone (PTH) ameliorates the already advanced FI and MA remains unknown. Therefore, this study aimed to clarify whether PTH promotes the expression of beige adipocytes and ameliorates advanced FI and MA following chronic RCT in rats. Supraspinatus muscles were harvested from rats with chronic RCT after 4 or 8 weeks of PTH treatment and compared to those in the control group or to those at the start of treatment. FI was assessed by Oil Red O staining, and the staining area was evaluated as a percentage of the muscle cross-sectional area. MA was evaluated by measuring muscle wet weight and cross-sectional area of muscle fiber. Beige adipocyte expression was evaluated by immunostaining for uncoupling protein 1 (UCP1). Fibro-adipogenic progenitors (FAPs) were separated from muscle-injured mice. We assessed whether PTH could diminish fat droplet accumulation by promoting the differentiation of FAPs into beige adipocytes. After four weeks, PTH reduced the area fraction of FI in the rat supraspinatus muscle following chronic RCT compared with that at the beginning of treatment (P = .028). In addition, PTH increased wet muscle mass (P < .001) and muscle fiber cross-sectional area (P < .018) compared with measurements at the start of treatment. PTH administration promoted the expression of UCP1, a beige adipocyte marker, in the supraspinatus muscle (P = .019). PTH increased gene expression of beige adipocyte-related markers and suppressed fat droplet accumulation even after adipogenic differentiation of FAPs (P = .004) but did not reduce fat droplets that had already accumulated in in vitro experiments. PTH facilitated beige adipocyte expression and reversibly ameliorated muscle quality and atrophy following chronic RCT by hindering fat droplet accumulation and facilitating muscle regeneration. Therefore, PTH may be a medicament for FI and MA following RCT, leading to expanded rotator cuff repair indications.

Gprc5a is a novel parathyroid hormone-inducible gene and negatively regulates osteoblast proliferation and differentiation.

Teriparatide is a peptide derived from a parathyroid hormone (PTH) and an osteoporosis therapeutic drug with potent bone formation-promoting activity. To identify novel druggable genes that act downstream of PTH signaling and are potentially involved in bone formation, we screened PTH target genes in mouse osteoblast-like MC3T3-E1 cells. Here we show that Gprc5a, encoding an orphan G protein-coupled receptor, is a novel PTH-inducible gene and negatively regulates osteoblast proliferation and differentiation. PTH treatment induced Gprc5a expression in MC3T3-E1 cells, rat osteosarcoma ROS17/2.8 cells, and mouse femurs. Induction of Gprc5a expression by PTH occurred in the absence of protein synthesis and was mediated primarily via the cAMP pathway, suggesting that Gprc5a is a direct target of PTH signaling. Interestingly, Gprc5a expression was induced additively by co-treatment with PTH and 1α, 25-dihydroxyvitamin D3 (calcitriol), or retinoic acid in MC3T3-E1 cells. Reporter analysis of a 1 kb fragment of human GPRC5A promoter revealed that the promoter fragment showed responsiveness to PTH via the cAMP response element, suggesting that GPRC5A is also a PTH-inducible gene in humans. Gprc5a knockdown promoted cell viability and proliferation, as demonstrated by MTT and BrdU assays. Gprc5a knockdown also promoted osteoblast differentiation, as indicated by gene expression analysis and mineralization assay. Mechanistic studies showed that Gprc5a interacted with BMPR1A and suppressed BMP signaling induced by BMP-2 and constitutively active BMP receptors, ALK2 (ACVR1) Q207D and ALK3 (BMPR1A) Q233D. Thus, our results suggest that Gprc5a is a novel gene induced by PTH that acts in an inhibitory manner on both cell proliferation and osteoblast differentiation and is a candidate for drug targets for osteoporosis.

PTH treatment before cyclic joint loading improves cartilage health and attenuates load-induced osteoarthritis development in mice.

Osteoarthritis (OA) treatment is limited by the lack of effective nonsurgical interventions to slow disease progression. Here, we examined the contributions of the subchondral bone properties to OA development. We used parathyroid hormone (PTH) to modulate bone mass before OA initiation and alendronate (ALN) to inhibit bone remodeling during OA progression. We examined the spatiotemporal progression of joint damage by combining histopathological and transcriptomic analyses across joint tissues. The additive effect of PTH pretreatment before OA initiation and ALN treatment during OA progression most effectively attenuated load-induced OA pathology. Individually, PTH directly improved cartilage health and slowed the development of cartilage damage, whereas ALN primarily attenuated subchondral bone changes associated with OA progression. Joint damage reflected early transcriptomic changes. With both treatments, the structural changes were associated with early modulation of immunoregulation and immunoresponse pathways that may contribute to disease mechanisms. Overall, our results demonstrate the potential of subchondral bone-modifying therapies to slow the progression of OA.

The Effects of Local Treatment of PTH(1-34) and Whitlockite and Hydroxyapatite Graft to the Calvarial Defect in a Rat Osteoporosis Model.

With the aging population, there is a rising incidence of senile diseases, notably osteoporosis, marked by fractures, prolonged recovery, and elevated mortality rates, underscoring the urgency for effective treatments. In this study, we applied the method of absorbing parathyroid hormone (PTH), a treatment for osteoporosis, into graft materials. Two types of graft materials with different properties, whitlockite (WH) and hydroxyapatite (HAP), were used. After forming calvarial defects in osteoporotic rats, WH and HAP grafts were implanted, with PTH applied directly to the graft sites. Micro-CT analysis was employed to assess bone regeneration, while tissue sections were stained to elucidate the regeneration process and bone cell dynamics. The results showed that bone regeneration was higher in the grafts that were actively degraded by osteoclasts in the early stage of regeneration. When PTH was applied, osteoclast activity increased, leading to enhanced bone regeneration. Furthermore, the activation of osteoclasts resulted in the penetration and formation of new bone within the degraded graft, which exhibited higher osseointegration. Therefore, for osteoporotic bone defects, bone grafts that can be easily degraded by osteoclasts are more suitable. Additionally, treatment with PTH can activate osteoclasts around the bone graft in the early stages of regeneration, inducing higher bone regeneration and improving osseointegration.

Abstract 6772: A 3D model of breast cancer-bone metastasis to mimic dysregulation of bone remodeling and drug response

Abstract Introduction: Bone metastases are common among advanced stage breast cancer patients and induce dysregulation of bone remodeling. Crosstalk between cancer cells and the bone niche drives the vicious cycle of bone loss and breast cancer growth. To accelerate discovery of targeted therapies, there is a critical need for experimental models that can mimic breast cancer-induced dysregulation of bone remodeling in patients. Mouse models are the current gold standard, but are low throughput, costly, and cannot fully recapitulate human-human cell interactions. To address these limitations, our goal is to engineer a fully human 3D in vitro model of breast cancer-bone metastasis that can mimic dysregulation of bone remodeling and drug responses in patients, and enable screening of potential new drug candidates. Methods: Two human breast cancer cell lines (MDA-MB-231, MCF-7) were chosen given their well characterized clinical features and tendency to invade bone. Our 3D model comprises an outer ring of tissue engineered (TE) bone and a center of breast cancer cells to mimic invasion through the bone marrow/long bone interface. Human mesenchymal stem cells were encapsulated in 3D gelatin microribbon scaffolds and cultured in osteogenic media for 28 days to form TE bone. Human monocyte-derived osteoclasts were seeded onto the bone, and the center was replaced with GFP-labeled breast cancer cells. Denosumab and zoledronic acid were selected as they are standard treatments for reducing bone loss in patients, and were used to treat the model at 30 µg/mL and 2 µg/mL, respectively. Intermittent parathyroid hormone (iPTH) treatment was selected as it showed efficacy in animal models, and was used to treat the model at 10 µg/mL for 6 hours daily. All groups were treated with drugs for 14 days. Bone volume was assessed using microCT. Cancer cell proliferation and invasion into bone was monitored with fluorescence confocal microscopy. Results: MicroCT imaging showed both breast cancer cell lines induced bone loss in the triculture model, compared to the control with no breast cancer cells in the center. Consistent with known clinical features, the more aggressive cell line (MDA-MB-231) induced a greater amount of bone loss than the less aggressive breast cancer cell line (MCF-7). Consistent with clinical findings, both denosumab and zoledronic acid reduced bone loss in our 3D model, and zoledronic acid further reduced breast cancer cell invasion and proliferation. Finally, the 3D model recapitulated the in vivo drug response of iPTH treatment, reducing bone resorption and breast cancer invasion. Conclusions: Here we report a fully human triculture model of breast cancer-bone metastasis that can mimic key clinical features of breast cancer-induced bone resorption and drug response in patients. Such a 3D model could enable screening of drug candidates with reduced time and cost and enable identification of novel druggable targets. Citation Format: Michelle Tai, Eva C. González Díaz, Callan E. Monette, Joy Wu, Fan Yang. A 3D model of breast cancer-bone metastasis to mimic dysregulation of bone remodeling and drug response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6772.

Cortical thickness adaptation to combined mechanical loading and parathyroid hormone treatments is site specific and synergistic in the mouse tibia model

In this study, we aimed to quantify the localised effects of mechanical loading (ML), low (20 μg/kg/day), moderate (40 μg/kg/day) or high (80 μg/kg/day) dosages of parathyroid hormone (PTH), and combined (PTHML) treatments on cortical bone adaptation in healthy 19-week old female C57BL/6 mice. To this end, we utilise a previously reported image analysis algorithm on μCT data of the mouse tibia published by Sugiyama et al. (2008) to measure changes in cortical area, marrow cavity area and local cortical thickness measures (ΔCt.Ar, ΔMa.Ar, ΔCt.Th respectively), evaluated at two cross-sections within the mouse tibia (proximal-middle (37 %) and middle (50 %)), and are compared to a superposed summation (P + M) of individual treatments to determine the effectiveness of combining treatments in vivo. ΔCt.Ar analysis revealed a non-linear, synergistic interactions between PTH and ML in the 37 % cross-section that saturates at higher PTH dosages, whereas the 50 % cross-section experiences an approximately linear, additive adaptation response. This coincided with an increase in ΔMa.Ar (indicating resorption of the endosteal surface), which was only counteracted by combined high dose PTH with ML in the middle cross-section. Regional analysis of ΔCt.Th changes reveal localised cortical thinning in response to low dose PTH treatment in the posteromedial region of the middle cross-section, signifying that PTH does not provide a homogeneous adaptation response around the cortical perimeter. We observe a synergistic response in the proximal-middle cross-section, with regions of compressive strain experiencing the greatest adaptation response to PTHML treatments, (peak ΔCt.Th of 189.32, 213.78 and 239.30 μm for low, moderate and high PTHML groups respectively). In contrast, PTHML treatments in the middle cross-section show a similar response to the superposed P + M group, with the exception of the combined high dose PTHML treatment which shows a synergistic interaction. These analyses suggest that, in mice, adding mechanical loading to PTH treatments leads to region specific bone responses; synergism of PTHML is only achieved in some regions experiencing high loading, while other regions respond additively to this combined treatment.

Parathyroid hormone treatment partially reverses endplate remodeling and attenuates low back pain in animal models of spine degeneration.

Low back pain (LBP) is one of the most prevalent diseases affecting quality of life, with no disease-modifying therapy. During aging and spinal degeneration, the balance between the normal endplate (EP) bilayers of cartilage and bone shifts to more bone. The aged/degenerated bony EP has increased porosity because of osteoclastic remodeling activity and may be a source of LBP due to aberrant sensory innervation within the pores. We used two mouse models of spinal degeneration to show that parathyroid hormone (PTH) treatment induced osteogenesis and angiogenesis and reduced the porosity of bony EPs. PTH increased the cartilaginous volume and improved the mechanical properties of EPs, which was accompanied by a reduction of the inflammatory factors cyclooxygenase-2 and prostaglandin E2. PTH treatment furthermore partially reversed the innervation of porous EPs and reversed LBP-related behaviors. Conditional knockout of PTH 1 receptors in the nucleus pulposus (NP) did not abolish the treatment effects of PTH, suggesting that the NP is not the primary source of LBP in our mouse models. Last, we showed that aged rhesus macaques with spontaneous spinal degeneration also had decreased EP porosity and sensory innervation when treated with PTH, demonstrating a similar mechanism of PTH action on EP sclerosis between mice and macaques. In summary, our results suggest that PTH treatment could partially reverse EP restructuring during spinal regeneration and support further investigation into this potentially disease-modifying treatment strategy for LBP.

Misdiagnosis of chronic kidney disease and parathyroid hormone testing during the past 16 years

Chronic kidney disease (CKD) is a prevalent pathological condition worldwide. Parathyroid hormone (PTH) is an important index related to bone metabolism in CKD patients and has not received enough attention. This study was performed to investigate the incidence and diagnostic rate of CKDin hospital as well as PTH testing and treatment for secondary hyperparathyroidism (SHPT) in patients with stage 3 to 5 CKD. The data of patients who visited Zhejiang Provincial People's Hospital from February 2006 to April 2022 were retrieved from the hospital database. All data were divided into three subgroups using PTH testing and SHPT treatment as major comparative indicators for analysis. The data were then analyzed for overall PTH testing, CKD incidence, and diagnostic rate. Among 5,301,391 patients, the incidence of CKD was 13.14%. The missed diagnosis rate for CKD was 65.76%. The total PTH testing rate was 1.22%, of which 15.37% of PTH testing was performed in patients with stage 3 to 5 CKD. The overall diagnosis rate of SHPT in patients with stage 3 to 5 CKD was 31.0%. The prophylactic medication rate was 7.4%, and the rate of post-diagnostic drug therapy was 22.2% in patients who underwent SHPT treatment. The high misdiagnosis rate and low PTH testing rate of CKD requires prompt attention from clinicians. SHPT treatment should be considered especially in patients with stage 3 to 5 CKD.

Early gene expression profiles of anabolic and catabolic molecules in murine bone after a single PTH injection

The current study examined the gene expression profiles of anabolic and catabolic molecules after a single parathyroid hormone (PTH) injection in mice. No significant changes were observed in alkaline phosphatase area/tissue volume, tartrate-resistant acid phosphatase–positive osteoclasts, or static bone histomorphometry parameters. However, a sudden and significant decrease in Runx2 expression occurred at 1.5 h post-injection followed by immediate elevation, while sclerostin level was initially downregulated but gradually recovered. Meanwhile, Rankl expression initially increased and then returned to baseline. The prolonged elevation of anabolic molecules and transient increase in catabolic molecules may contribute to the anabolic effect of PTH treatment.

The effect of intermittent parathyroid hormone on bone lengthening: current evidence to inform future effective interventions.

Recent studies have demonstrated the positive effects of parathyroid hormone (PTH) on bone healing, and findings support the use of PTH to accelerate bone healing following distraction osteogenesis. The goal of this review was to compile and discuss the mechanisms potentially underlying the effects of PTH on newly formed bone following a bone-lengthening procedure incorporating all relevant evidence in both animal and clinical studies. This review summarized all evidence from in vivo to clinical studies regarding the effects of PTH administration on a bone-lengthening model. In addition, a comprehensive evaluation of what is currently known regarding the potential mechanisms underlying the potential benefits of PTH in bone lengthening was presented. Some controversial findings regarding the optimal dosage and timing of administration of PTH in this model were also discussed. The findings demonstrated that the potential mechanisms associated with the action of PTH on the acceleration of bone regeneration after distraction osteogenesis are involvement in mesenchymal cell proliferation and differentiation, endochondral bone formation, membranous bone formation, and callus remodeling. In the last 20 years, a number of animal and clinical studies have indicated that there is a prospective role for PTH treatment in human bone lengthening as an anabolic agent that accelerates the mineralization and strength of the regenerated bone. Therefore, PTH treatment can be viewed as a potential treatment to increase the amount of new calcified bone and the mechanical strength of the bone in order to shorten the consolidation stage after bone lengthening.

Regional modular responses in different bone compartments to the anabolic effect of PTH (1-34) and axial loading in mice.

Beneficial effects of intermittent parathyroid hormone (PTH) on bone mass and architecture are described to either simply add to, or to synergise with those of mechanical loading. We evaluate whether interaction with in vivo loading is reinforced by PTH dosing regimen and exhibits compartment-specific sensitivities. Female 12-week-old C57Bl6 mice received daily (7/7) or interrupted 5day/week (5/7) PTH for 3weeks (two vehicle groups). All mice had six loading episodes (12N) applied to right tibia (left, non-loaded) for the last 2weeks. Micro-CT scans were used to evaluate mass and architecture in almost the entire cortical and proximal trabecular regions. Epiphyseal cortical, trabecular and marrow space volumes, and bony growth-plate bridge incidence were evaluated. Statistical analyses employed a linear mixed-effects model at each percentile and 2-way ANOVA with post-hoc test for epiphyses and bridging. We found that daily PTH enhances cortical mass and modifies shape along almost the entire tibia and that these effects are partly mitigated by brief interruption in treatment. Mechanical loading alone augments cortical mass and modifies shape but only in a region proximal to the tibiofibular junction. The effect of combining load and daily PTH dosing is solely additive for cortical bone mass with no significant load: PTH interaction, but exhibits clear synergy with interrupted PTH treatment. Daily, not interrupted PTH stimulates trabecular bone gains, yet load:PTH interaction is present at limited regions with both daily and interrupted treatment. PTH treatment, but not loading, modifies epiphyseal bone but, in contrast, only loading modifies bridge number and areal density. Our findings demonstrate impressive local effects on tibial mass and shape of combined loading and PTH that are sensitive to dosing regimen and exert their effects modularly. These findings emphasise a need to clarify PTH dosing regimens and that advantages could be accrued by aligning treatment accordingly to patient requirements and life-style.

A Review of Potential Therapies to Attenuate Bone Mineral Density Loss in Obese Individuals Prior to Total Joint Replacement Surgery.

The focus of this review is to examine therapeutic interventions which may be used to increase bone mineral density (BMD), reduce bone loss, and ultimately reduce complications in obese patients prior to total joint replacement (TJR). It is recommended that obese patients lose weight prior to surgery to reduce post-surgical complications, but weight loss can also increase bone loss and fracture risk in older individuals. In this review, we investigate potential therapies to improve bone density and reduce bone loss including exercise therapy, parathyroid hormone (PTH), estrogen, bisphosphonate, and calcitonin treatment in obese patients prior to TJR. Our review of existing literature found that treatment with PTH increased total body BMD in both men and women with osteoporosis; exercise therapy in combination with weight loss prevents the weight loss-induced increase in bone turnover and attenuates the weight loss-induced decrease in BMD; and estrogen, bisphosphonate, and calcitonin reduce bone resorption.

β-Arrestin 2 knockout prevents bone loss in response to continuous parathyroid hormone stimulation in male and female mice

ABSTRACT Background β-Arrestin 2 (β-arr2) binds activated parathyroid hormone (PTH) receptors stimulating internalization. PTH stimulates both anabolic and catabolic effect on bone depending on the way it is administered. Intermittent PTH stimulation increases trabecular bone formation in mice, but this is decreased in mice lacking β-arr 2, suggesting a role for β-arr 2 in the anabolic effects of PTH. The role of β-arr 2 in the catabolic effects of continuous PTH (cPTH) treatment is not known. Objective To assess the effects of cPTH administration on bone in mice lacking β-arr 2 compared to wild-type (WT). Methods Groups of male and female WT or β-arr2 knockout (KO) mice were administered either PTH or phosphate-buffered saline by osmotic pumps for 2 weeks. Following treatment, serum calcium and phosphate levels were measured, bone structure and mineral density were measured by microcomputed tomography, and bone cells measured by static and dynamic histomorphometry. Results β-arr2 KO had no effects on skeletal development in mice of either sex. PTH treatment caused hypercalcemia and hypophosphatemia and decreased trabecular and cortical bone only in male WT mice. β-arr2 KO in male mice completely abrogated the effects of PTH on bone, while in female β-arr2 KO mice, PTH treatment increased trabecular bone with no effects on cortical bone. Conclusions These results demonstrate a profound sex effect on skeletal responses to cPTH treatment, suggesting a protective effect of estrogen on bone loss. β-arr2 plays a role in restraining the anabolic effects of PTH in both male and female mice.

Mitochondrial β-oxidation of adipose-derived fatty acids by osteoblasts fuels parathyroid hormone-induced bone formation.

The energetic costs of bone formation require osteoblasts to coordinate their activities with tissues, like adipose, that can supply energy-dense macronutrients. In the case of intermittent parathyroid hormone (PTH) treatment, a strategy used to reduce fracture risk, bone formation is preceded by a change in systemic lipid homeostasis. To investigate the requirement for fatty acid oxidation by osteoblasts during PTH-induced bone formation, we subjected mice with osteoblast-specific deficiency of mitochondrial long-chain β-oxidation as well as mice with adipocyte-specific deficiency for the PTH receptor or adipose triglyceride lipase to an anabolic treatment regimen. PTH increased the release of fatty acids from adipocytes and β-oxidation by osteoblasts, while the genetic mouse models were resistant to the hormone's anabolic effect. Collectively, these data suggest that PTH's anabolic actions require coordinated signaling between bone and adipose, wherein a lipolytic response liberates fatty acids that are oxidized by osteoblasts to fuel bone formation.

Effects of Cinacalcet on Post-transplantation Hypercalcemia and Hyperparathyroidism in Adult Kidney Transplant Patients: A Single-Center Experience.

Secondary hyperparathyroidism may manifest as hypercalcemia in the post-transplant period. The classical treatment method is parathyroidectomy and the alternative is oral cinacalcet, a calcimimetic agent therapy. We retrospectively investigated the effect of cinacalcet therapy on kidney and patient survival in these patients. In our single-center, retrospective, observational study, files of 934 patients who underwent renal transplantation in our unit between 2008 and 2022 were reviewed. A total of 23 patients were started on cinacalcet for the treatment of hypercalcemia (calcium > 10.3 mg/dl) and parathyroid hormone (PTH) elevation (>65 pg/ml). Patients with calcium < 10.3 mg/dl and PTH > 700 pg/ml at any time in the follow-up after renal transplantation were included in the study. In addition, the demographic data of the patients,baseline creatine, calcium, phosphorus, and PTH levels at the time of hypercalcemia, parathyroid ultrasonography, parathyroid scintigraphy, creatinine, calcium, phosphorus,and PTH levels in the last controls, and survival status were evaluated. The mean age of 23 patients included in the study was 52.7 ± 11 years (minimum: 32; maximum: 66). Of the patients, 16 (69.6%) were male, and 15 (65.2%) were transplanted from a living donor. Parathyroid scintigraphic revealed adenoma in three (13%) patients, hyperplasia in five patients (21.7%), and no involvement in 15 patients (65.2%). Cinacalcet treatment was initiated at a median of 33 months (interquartile range (IQR) = 13-96) after the kidney transplant operation. There was no graft loss in the patients during the follow-up period. Twenty-two patients (95.7%) were alive, and one patient died. The calcium level of the patients decreased from 11.3 ± 0.64 mg/dl to 9.98 ± 0.78 mg/dl (p = 0.001) after cinacalcet treatment. Phosphorus values increased from 2.7 ± 0.65 mg/dl to 3.10 ± 0.65 mg/dl (p = 0.004). On the other hand, there was no significant difference in PTH levels between the initial and final controls (285 (IQR = 150-573) vs. 260 pg/ml (IQR = 175-411), p = 0.650). Also, creatinine levels were similar (1.2 ± 0.38 vs. 1.24 ± 0.48 mg/dl, p = 0.43). Despite cinacalcet treatment, calcium levels did not decrease in eight patients. Complications such as renal dysfunction and pathological fracture did not develop in these patients. It seems that cinacalcet treatment is a suitable option for patients with hypercalcemia and/or hyperparathyroidism with low drug interactions and good biochemical control after renal transplantation.

Low-intensity pulsed ultrasound and parathyroid hormone improve muscle atrophy in estrogen deficiency mice

Due to aging and long-term estrogen deficiency, postmenopausal women suffer muscle atrophy (MA), which is characterized by decreased muscle mass and muscle quality. Low-intensity pulsed ultrasound (LIPUS) is an acoustic wave inducing biological effects mainly by the mechanical stimulation and used as a non-invasive physical therapy for muscle repair. Parathyroid hormone (PTH) is an 84-amino-acid polypeptide, and its bioactive fragment [PTH (1–34)] has potential application in the treatment of MA. We speculate that the combination of physical therapy (i.e., the LIPUS) and regulatory hormone (i.e., the PTH) would be more effective in the treatment of MA. The objective of this study was to evaluate the individual and combined effects of LIPUS and PTH therapy on MA in estrogen deficiency mice. Seventy 8-week-old female C57BL/6J mice were used in this study and the MA model was induced by an intraperitoneal injection of 4-vinylcyclohexene diepoxide (VCD) for 20 consecutive days. The VCD-induced MA mice were randomly divided into MA, LIPUS, PTH and LIPUS + PTH (Combined) groups (n = 10/group). In the LIPUS group, the mice were treated by LIPUS in bilateral quadriceps muscles for 20 min, five times a week for 6 weeks. In the PTH group, the mice received subcutaneous injection of PTH (1–34) (80 ug/kg/d) five times a week, for 6 weeks. In the Combined group, the PTH was administrated 30 min before each LIPUS session. Hematoxylin-eosin (H&E) staining, serum biochemical analysis and quantitative real-time polymerase chain reaction (qRT-PCR) were applied to evaluate the therapeutic effects of related treatments. The results showed that the MA mice had a disordered estrus cycle, significantly decreased muscle mass and myofibers cross-sectional area (CSA). After treatments, LIPUS, PTH and Combined groups had a significantly increased CSA, compared with the MA mice without treatment. In addition, Combined group had a significantly increased mRNA expression of Pax7, MyoD and MyoG, compared with LIPUS and PTH monotherapy groups. Our findings indicated that the combination of LIPUS and PTH treatment improves muscle regeneration ability, which might have potential for treating MA in postmenopausal women.

Parathyroid Hormone Reduces Persistent Proton Induced DNA Double-Strand Breaks in Osteocytes

<h3>Purpose/Objective(s)</h3> Healthy bone is commonly irradiated during radiotherapy for many cancers, and bone fractures can be a late complication. A recent study revealed a higher incidence of rib fractures after breast irradiation with protons (7%) compared to photons (1%) at the same dose, but the underlying biological mechanisms are unknown. Here we used <i>in vitro</i> experiments, supported by Monte Carlo track structure simulations, to investigate differences in bone cell responses to proton and photon radiation, and to identify potential therapeutic interventions. <h3>Materials/Methods</h3> We used TOPAS-nBio, a radiobiology simulation toolkit, to simulate a segment of bone using geometric models of bone cells (osteoblasts, osteoclasts, and osteocytes). Photon and proton tracks were simulated through bone matrix to investigate energy distributions received by each cell. For <i>in vitro</i> experiments, we utilized the murine osteocytic cell line Ocy454. Cells were irradiated with different doses of protons or photons. Surviving fraction as a function of dose was determined using colony formation assays. DNA double-strand breaks were assessed by 53BP1 foci assay. For PTH treatments, cells were treated with 10 nM human PTH(1-34) for 24 hours prior to irradiation. All experiments were repeated independently three or more times. Statistical analysis was performed by unpaired t-test with Bonferroni correction. <h3>Results</h3> Monte Carlo track simulations showed osteocytes are the bone cell most likely to receive radiation damage, particularly with protons. In the case of large multi-nucleated osteoclasts, both photons and protons spared some of the nuclei, suggesting functional sparing of osteoclasts from radiation damage. We therefore focused our <i>in vitro</i> experiments on Ocy454 osteocytes. Interestingly, the surviving fraction of osteocytes was significantly lower after protons compared to photons for all investigated doses of 1 to 8 Gy. The dose for 10% surviving fraction was 5.38 Gy for protons and 7.46 Gy for photons (Relative Biological Effect 1.39). Although protons and photons induced similar numbers of 53BP1 foci at 1 hour (79% vs. 72% cells with foci), a significantly higher number of foci was observed at 24 hours after protons compared to photons (22% vs. 6.5%, p=0.0078), suggestive of persistent DNA damage after protons. Remarkably, we found that pre-treatment with parathyroid hormone (PTH) decreased 53BP1 foci at 24 hours in proton-irradiated cells to levels comparable to that of non-irradiated cells (22% without PTH vs. 6.3% with PTH, p=0.014). <h3>Conclusion</h3> Surviving fraction of osteocytes was lower after proton compared to photon irradiation, suggesting a greater sensitivity of osteocytes to protons. Protons result in greater DNA damage in osteocytes, but this damage may be mitigated with the addition of PTH. Further study is needed to investigate the potential of PTH to reduce bone damage induced by proton irradiation.

Lower Bone Turnover and Skeletal PTH Responsiveness in Japanese Compared to European Patients on Hemodialysis.

Parathyroid hormone (PTH) treatment targets for patients receiving hemodialysis (HD) are lower in Japan than in Europe. Whether this translates to lower bone turnover is unknown and could depend on skeletal PTH responsiveness. This study investigates whether skeletal PTH responsiveness is better preserved in Japanese vs European patients receiving HD. This is a post hoc analysis of data from 2 prospective cohort studies, using a case-control design. Patients receiving chronic intermittent HD therapy were eligible for inclusion. Participating Belgian and Japanese patients (n = 374) were matched 1:1 by age (59 ± 12 years), sex (66% male), diabetes (34%), and dialysis duration (39 months [22-63 months]). PTH, bone-specific alkaline phosphatase (BALP), and tartrate-resistant acid phosphatase isoform 5b (TRAP5b) were measured centrally in Liège, Belgium. Japanese patients had lower levels of iPTH (207 vs 268 pg/mL; P < .001), BALP (15.3 vs 24.5 μg/L; P < .001), and TRAP5b (3.35 vs 5.79 U/L; P < .001). Linear regression analyses revealed lower levels of bone turnover markers for any given level of PTH in Japanese vs Belgian patients, indicating lower skeletal PTH responsiveness. Consistently, bone turnover markers were significantly lower in Japanese vs Belgian patients when stratifying or matching according to PTH levels. Male sex, obesity, and hyperphosphatemia were the main determinants of the bone turnover marker/PTH ratios. Japanese patients receiving HD have lower bone turnover than their European counterparts, even at similar PTH levels. The rationale for the current regional differences in PTH treatment targets remains obscure and deserves further attention.

Ano5 modulates calcium signaling during bone homeostasis in gnathodiaphyseal dysplasia

ANO5 encodes transmembrane protein 16E (TMEM16E), an intracellular calcium-activated chloride channel in the endoplasmic reticulum. Mutations in ANO5 are associated with gnathodiaphyseal dysplasia (GDD), a skeletal disorder causing the jaw deformity and long bone fractures. However, the coordinated mechanism by which ANO5 mediates bone homeostasis in GDD remains poorly defined. Here, we show that ablation of Ano5 reduced intracellular calcium transients, leading to defects in osteogenesis and osteoclastogenesis and thus bone dysplasia. We found a causative de novo ANO5 frameshift insertion mutation (p.L370_A371insDYWRLNSTCL) in a GDD family with osteopenia, accompanied by a decrease in TMEM16E expression and impaired RANKL-induced intracellular calcium ([Ca2+]i) oscillations in osteoclasts. Moreover, using Ano5 knockout (KO) mice, we found that they exhibited low bone volume, abnormal calcium deposits, and defective osteoblast and osteoclast differentiation. We also showed that Ano5 deletion in mice significantly diminished [Ca2+]i oscillations in both osteoblasts and osteoclasts, which resulted in reduced WNT/β-Catenin and RANKL-NFATc1 signaling, respectively. Osteoanabolic treatment of parathyroid hormone was effective in enhancing bone strength in Ano5 KO mice. Consequently, these data demonstrate that Ano5 positively modulates bone homeostasis via calcium signaling in GDD.

Maintenance of increased bone mass after PTH withdrawal by sequential medicarpin treatment via augmentation of cAMP-PKA pathway.

Osteoporosis is a metabolic bone disorder associated with impaired bone microarchitecture leading to fragility fractures. Long-term usage of parathyroid hormone (PTH) enhances bone resorption and leads to osteosarcoma in rats which limits its exposure to maximum 2 years in human. Notably, the anabolic effects of PTH do not endure in the absence of sustained administration. Studies in our lab identified osteogenic and antiresorptive activity in medicarpin, a phytoestrogen belonging to the pterocarpan class. Considering dual-acting property of medicarpin and limitations of PTH therapy, we envisaged that medicarpin sequential treatment after PTH withdrawal could serve as promising therapeutic approach for osteoporosis treatment. As PTH exerts its bone anabolic effect by increasing osteoblast survival, our study aims to determine whether medicarpin amplifies this effect of PTH. Our results show that PTH withdrawal led to reduced bone mineral density and bone parameters, while sequential treatment of medicarpin after PTH withdrawal significantly enhanced these parameters. Remarkably, these effects were more pronounced than 8-week PTH treatment. Sequential therapy also significantly increased P1NP levels and decreased CTX levels and TRAP positive cells compared to PTH 8W group where CTX levels were quite high due to bone resorptive action of PTH. Protein expression studies revealed that medicarpin along with PTH betters the antiapoptotic potential compared to PTH alone, through augmentation of cyclic adenosine monophosphate-PKA-CREB pathway. These results proclaim that medicarpin sequential treatment prevented the reduction in bone accrual and strength accompanying PTH withdrawal and also aided in antiapoptotic role of PTH. The study points toward the potential use of medicarpin as a replacement therapeutic option postdiscontinuation of PTH.