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Dysregulated overproduction of parathyroid hormone (PTH), the prototypic calciotropic hormone, is a common endocrine disorder with the greatest prevalence in adult women. Hyperparathyroidism conveys increased risk for long-term cardiovascular (CV) morbidity and mortality independent of serum calcium levels. Primary hyperparathyroidism (pHPT) impairs normal arterial vasorelaxation and blood flow in response to physiological demands of the heart and kidneys. The PTH 1 receptor (PTH1R), shared by PTH and PTH-related protein, is highly expressed and biologically active in the CV system in addition to bone and kidney. Marked differences exist in the CV response to acute vs chronic PTH exposure, mediated in part via rapid tachyphylaxis to vascular PTH1R activation that normally improves arterial blood flow. A better understanding of vascular PTH/PTH1R signaling provides novel targets for therapeutic intervention CV disease including arteriosclerosis, CV fibrosis, and heart failure. Validated biomarkers are needed to establish the healthy 'set point' for CV PTH1R signaling tone to better guide the management of patients with pHPT or secondary hyperparathyroidism. Primary hyperparathyroidism (pHPT) afflicts our aging population with an incidence approaching 50 per 100 000 patient-years at a female:male ratio of ~3:1. Decisions surrounding surgical management are currently driven by age, hypercalcemia severity, presence of osteoporosis, renal insufficiency, or hypercalciuria with or without nephrolithiasis. Cardiovascular (CV) disease (CVD) is not systematically considered. This is notable since the parathyroid hormone (PTH) 1 receptor (PTH1R) is biologically active in the vasculature, and adjusted CV mortality risk is increased almost threefold in individuals with pHPT who do not meet contemporary recommendations for surgical cure. We provide an overview of epidemiology, pharmacology, and physiology that highlights the need to: (i) identify biomarkers that establish a healthy 'set point' for CV PTH1R signaling tone; (ii) better understand the pharmacokinetic–pharmacodynamic (PK-PD) relationships of PTH1R ligands in CV homeostasis; and (iii) incorporate CVD risk assessment into the management of hyperparathyroidism. [ABSTRACT FROM AUTHOR]

Parathyroid hormone-related protein (PTHrP) is an important regulatory factor in the growth, development and remodeling of bone or cartilage, and acts through its sole receptor, parathyroid hormone receptor-1 (PTH1R). The present study aimed to research the expression changes of PTHrP, PTH1R and other relevant factors in condylar cartilage during the progress of temporomandibular joint osteoarthritis (TMJOA). The animal model of TMJOA was constructed by the "resin-modified method", and Sprague Dawley (SD) rats were euthanized at 2 weeks, 4 weeks, 6 weeks and 8 weeks after occlusal elevation. The histological changes of condylar cartilage were observed by X-ray, hematoxylin-eosin (HE) and safranine O-fast green (SO-FG) staining. The expressions of PTHrP, PTH1R, Ki67, Collagen II (Col II), Collagen X (Col X) and Caspase 3 in each group were detected by quantitative real-time polymerase chain reaction (qRT-PCR) and immunohistochemistry (IHC). TMJOA progression was time-dependent. In the experimental group, PTHrP expression was unimodal with a peak at 4 weeks, but PTH1R expression showed a decreasing trend. From 2 weeks to 8 weeks in the experimental group, Col X expression rather than Caspase 3 expression was negatively related to PTHrP's, which has no positive relation to Ki67 or Col II. These results demonstrated abnormal occlusal load may be an important pathogenic factor of TMJOA. It may be one of the reasons of TMJOA progression that PTHrP can't play an effective role due to the low expression of PTH1R. PTHrP may be a direct factor regulating the hypertrophic differentiation of chondrocytes, but it does not directly regulate the proliferation and apoptosis of chondrocytes, and the realization of both regulatory effects may depend on the inhibition of hypertrophic differentiation. [ABSTRACT FROM AUTHOR]

Efficiently driving chondrogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) while avoiding undesired hypertrophy remains a challenge in the field of cartilage tissue engineering. Here, we report the sequential combined application of dimethyloxalylglycine (DMOG) and parathyroid hormone-related protein (PTHrP) to facilitate chondrogenesis and prevent hypertrophy. To support their delivery, poly(lactic- co -glycolic acid) (PLGA) microspheres were fabricated using a double emulsion method. Subsequently, these microspheres were incorporated onto a poly(l-lactic acid) (PLLA) scaffold with a highly porous structure, high interconnectivity and collagen-like nanofiber architecture to construct a microsphere-based scaffold delivery system. These functional constructs demonstrated that the spatiotemporally controlled release of DMOG and PTHrP effectively mimicked the hypoxic microenvironment to promote chondrogenic differentiation with phenotypic stability in a 3D culture system, which had a certain correlation with the interaction between hypoxia-inducible Factor 1 alpha (HIF-1α) and yes-associated protein (YAP). Subcutaneous implantation in nude mice revealed that the constructs were able to maintain cartilage formation in vivo at 4 and 8 weeks. Overall, this study indicated that DMOG and PTHrP controlled-release PLGA microspheres incorporated with PLLA nanofibrous scaffolds provided an advantageous 3D hypoxic microenvironment for efficacious and clinically relevant cartilage regeneration and is a promising treatment for cartilage injury. [ABSTRACT FROM AUTHOR]

[Display omitted] • Soluble hydrophobic support-assisted liquid-phase synthesis technology of Abaloparatide. • Conditions of removing support Tag-OH. • Improving the synthesis conditions of Fragment 4. Abaloparatide, a novel parathyroid hormone-related peptide, emerges as a leading drug for treating osteoporosis in postmenopausal women at high risk of fracture. The therapeutic agent has demonstrated efficacy in significantly enhancing bone mineral density, fortifying bone strength, and stimulating bone formation. Developing a synthetic route for Abaloparatide with high-yield and purity products is of paramount importance. This paper reported a newly developed liquid-phase synthetic method based on soluble hydrophobic support assistance for the total synthesis of Abaloparatide, which has proven to be simple, scalable, and highly effective. The optimization of reaction conditions has yielded Abaloparatide with both high yield and purity, producing satisfactory results. [ABSTRACT FROM AUTHOR]

Congenital mesoblastic nephroma is a rare pediatric renal tumor and has been reported in patients presenting with palpable abdominal mass, arterial hypertension, hematuria, polyuria, or hypercalcemia. Here we present the case of a 1-month-old neonate with suspected parathyroid hormone (PTH)-related peptide (PTH-rp)-mediated severe hypercalcemia revealing congenital mesoblastic nephroma. Preoperatively, hypercalcemia was corrected with hydration, furosemide, pamidronate, and low-calcium infant formula. Unilateral nephrectomy led to the resolution of hypercalcemia, transient hyperparathyroidism, and transient vitamin D and mineral supplementation. We conclude that congenital mesoblastic nephroma can secrete PTH-rp that can cause severe hypercalcemia. [ABSTRACT FROM AUTHOR]

[Display omitted] • A novel concept of demonstrated pyridoxal phosphate permeability in E.coli • Pyridoxal phosphate inclusion in media increases protein yield and enzymatic activity • A novel protein model system for pyridoxal phosphate linked permeation studies. PLP (Pyridoxal 5′-phosphate) dependent proteins are important drug targets and effector molecules, and thus, their heterologous overexpression is of industrial importance and has commercial value. We have predicted the docking site of PLP on O-acetylserine sulphdrylase (OASS) of H.contortus and determined that the lysine-47 is very important for the binding of PLP. We have used this protein as a model protein for testing the effect of PLP on the expression of PLP dependent proteins by E.coli. Soluble recombinant protein could be purified from each of the culture vials grown with variable amount of PLP [0 mM (Group I), 0.01 mM (Group II), 0.025 mM (Group III), 0.05 mM (Group IV) and 0.1 mM (Group V)]. There was approximately 4.2 %, 7.2 %, 10.5 % and 18 % increase in purified protein yield in Group II, III, IV and V, respectively, in comparison to group I. There was a significant quenching of tryptophan fluorescence emission in groups II, III, IV and V compared to group I. We could find a PLP concentration-dependent increase in expression and catalytic activity signifying the probable transport of PLP across the membrane. E.coli does not secrete any dephosphorylation factor for PLP in the immediate microenvironment. There was a significant reduction in levels of inorganic phosphate after PLP dephosphorylation in induced group in comparison to uninduced E.coli and BL21 groups, signifying PLP internalization in E.coli. The mechanism of transport of PLP in the light of the current experiment is still unknown and should be a subject of future studies. [ABSTRACT FROM AUTHOR]

The peripartum period is characterized by dynamic shifts in metabolic, mineral, and immune metabolism as the dairy cow adapts to the demands of lactation. Emphasis over the past decade has been placed on understanding the biology of the large shift in calcium metabolism in particular. Moreover, research has also focused on exploring the role of serotonin during the transition period and lactation and further unraveling its relationship with calcium. This review aimed to demonstrate the integration of calcium physiology during the peripartal period and throughout lactation. More specifically, we sought to discuss the knowledge gained in recent years on calcium metabolism, mammary calcium transport, serotonin metabolism, and the serotonin-calcium axis. Herein we also discuss the challenges and limitations of current research and where that leaves the present understanding of the serotonin-calcium axis as we seek to move forward and continue exploring this interesting relationship. [ABSTRACT FROM AUTHOR]

Granulomatous tubulointerstitial nephritis (GTIN) is a rare pathologic finding on kidney biopsy. GTIN can be associated with drugs, infection, systemic granulomatous disease, and tubulointerstitial nephritis with uveitis syndrome. We present a case of GTIN in a kidney transplant recipient (KTR) and a literature review of published cases of GTIN in KTRs. A 65-year-old man with a history of pulmonary and ocular tuberculosis (TB), who had undergone deceased donor kidney transplant 8 years prior, was admitted for acute kidney injury, hypercalcemia, and uveitis. His medications included rifabutin, isoniazid, and tacrolimus. Serum laboratory tests revealed creatinine of 2.65 mg/dL (baseline 1.1-1.5 mg/dL) and corrected calcium of 13.2 mg/dL. Hypercalcemia workup showed parathyroid hormone 7 pg/mL, 1,25(OH) vitamin D 54 pg/mL, parathyroid hormone–related peptide <2.0 pmol/L, and angiotensin-converting enzyme 47 U/L. Kidney biopsy showed GTIN with noncaseating granulomas. Universal polymerase chain reaction testing for acid-fast bacilli, fungus, and bacteria was negative. He was treated with prednisone, and his kidney function returned to baseline, and his hypercalcemia resolved. GTIN is a rare entity seen in less than 1% of transplanted kidney biopsies. The exactly etiology of this GTIN case remains unknown. TB could not be entirely ruled out, because the patient was receiving active anti-TB therapy. Our literature review showed infection to be the leading cause of GTIN in KTRs and that GTIN with concomitant uveitis remains exceedingly rare. Steroids may be useful in certain cases. [ABSTRACT FROM AUTHOR]

After identification of fibroblast growth factor (FGF) 23 as the pivotal regulator of chronic serum inorganic phosphate (Pi) levels, the etiology of disorders causing hypophosphatemic rickets/osteomalacia has been clarified, and measurement of intact FGF23 serves as a potent tool for differential diagnosis of chronic hypophosphatemia. Additionally, measurement of bone-specific alkaline phosphatase (BAP) is recommended to differentiate acute and subacute hypophosphatemia from chronic hypophosphatemia. This article divides the etiology of chronic hypophosphatemia into 4 groups: A. FGF23 related, B. primary tubular dysfunction, C. disturbance of vitamin D metabolism, and D. parathyroid hormone 1 receptor (PTH1R) mediated. Each group is further divided into its inherited form and acquired form. Topics for each group are described, including "ectopic FGF23 syndrome," "alcohol consumption-induced FGF23-related hypophosphatemia," "anti-mitochondrial antibody associated hypophosphatemia," and "vitamin D-dependent rickets type 3." Finally, a flowchart for differential diagnosis of chronic hypophosphatemia is introduced. [ABSTRACT FROM AUTHOR]

Introduction: Scirrhous hepatocellular carcinoma (HCC) is a rare primary liver tumor characterized by extensive fibrosis and production of parathyroid hormone-related peptide. There have been conflicting reports on patient survival in scirrhous versus non-scirrhous HCC. The objective of the present study was to define the clinical features, practice patterns, and long-term outcomes of patients with scirrhous HCC versus non-scirrhous HCC in a propensity score-matched cohort.Methods: A propensity score-matched cohort was created using data from the National Cancer Database for 2004 to 2015. A multivariable Cox proportional hazards regression analysis was performed to assess the effect of the scirrhous HCC variant on overall survival.Results: Among the 70,426 patients with a diagnosis of HCC who met the inclusion criteria, 99.8% had non-scirrhous HCC (n = 70,290) whereas a small subset had scirrhous HCC (n = 136, 0.19%). While 20,330 (28.9%) patients underwent liver-directed therapy (resection, ablation, and transplantation), the majority did not (n = 50,096, 71.1%). After propensity matching, there were no difference in 1-, 3-, or 5-year overall survival among patients with scirrhous versus non-scirrhous HCC (1-year overall survival (OS), 53.7% versus 51.0%; 3-year OS, 34.6% versus 28.7%; and 5-year OS, 18.0% versus 21.0%, respectively; p = 0.52). While the scirrhous HCC variant was not associated with survival (hazard ratio [HR] 0.93, 95% CI 0.74-1.16), non-receipt of liver-directed therapy (HR 0.24, 95% CI 0.18-0.32), advanced AJCC stage (III/IV) (HR 2.14, 95% CI 1.55-2.95), and non-academic facilities (HR 0.60, 95% CI 0.49-0.73) remained associated with worse survival.Conclusion: Patients with the scirrhous variant had a comparable overall survival compared with individuals who had non-scirrhous HCC. Failure to receive liver-directed therapy, advanced AJCC stage (III/IV), and treatment at a non-academic facility was strongly associated with a worse long-term prognosis. [ABSTRACT FROM AUTHOR]

Stocking density is an important environment factor that affects the development of poultry farming, which has caused widespread concern. This study was carried out to determine the effects of stocking density on growth performance, growth regulatory factors, and endocrine hormones in broilers under appropriate environments. A total of 144 Arbor Acres male broilers (BW 1000 ± 70 g) were randomly divided into low stocking density (LSD; 6.25 birds/m2), medium stocking density (MSD; 12.50 birds/m2), and high stocking density (HSD; 18.75 birds/m2) groups, with 6 replicates in each group, and raised in 3 environmental chambers (same size) from 29-day-old to 42-day-old, respectively. The trial period lasted for 14 D with 21 ± 1°C and 60 ± 7% relative humidity, wind speed < 0.5 m/s, ammonia level<5 ppm. The results indicated that average daily food intake and average daily gain in HSD group showed significantly lower than other 2 groups (P < 0.05). Besides, the HSD group significantly reduced breast muscle yield, tibial length, tibial width, and tibial weight of broilers (P < 0.05). The HSD group increased the mRNA expression level of myostatin, and reduced the mRNA expression levels of insulin-like growth factor 1 (IGF-1) and myogenic determination factor 1 (P < 0.05). The HSD group significantly reduced the expression of parathyroid hormone-related protein in tibial growth plate (P < 0.05). The HSD group increased the serum corticosterone levels of broilers (P < 0.05), and decreased the serum IGF-1 and thyroxine (T4) levels of broiler chickens (P < 0.05) than other stocking density groups. Moreover, the serum alkaline phosphatase levels were decreased (P < 0.05) with increasing stocking density, whereas there were no significant effects on the serum 3,5,3′-triiodothyronine (T3) concentrations in 3 groups (P > 0.05). In conclusion, under appropriate environments HSD reduced the growth performance of broilers and this negative effect was likely associated with decreased growth of muscle and bone. [ABSTRACT FROM AUTHOR]

Ectodermal appendages, such as the mammary gland (MG), are thought to have evolved from hair-associated apocrine glands to serve the function of milk secretion. Through the directed differentiation of mouse embryonic stem cells (mESCs), here, we report the generation of multilineage ESC-derived mammary organoids (MEMOs). We adapted the skin organoid model, inducing the dermal mesenchyme to transform into mammary-specific mesenchyme via the sequential activation of Bone Morphogenetic Protein 4 (BMP4) and Parathyroid Hormone-related Protein (PTHrP) and inhibition of hedgehog (HH) signaling. Using single-cell RNA sequencing, we identified gene expression profiles that demonstrate the presence of mammary-specific epithelial cells, fibroblasts, and adipocytes. MEMOs undergo ductal morphogenesis in Matrigel and can reconstitute the MG in vivo. Further, we demonstrate that the loss of function in placode regulators LEF1 and TBX3 in mESCs results in impaired skin and MEMO generation. In summary, our MEMO model is a robust tool for studying the development of ectodermal appendages, and it provides a foundation for regenerative medicine and disease modeling. [Display omitted] • Mouse ESCs can self-assemble into milk-producing MEMOs • BMP4 and PTHrP activation and hedgehog inhibition promote mammogenesis • MEMOs undergo branching morphogenesis in Matrigel and upon transplantation in mice • LEF1 and TBX3 regulate MEMO development Sahu et al. have successfully created multilineage ESC-derived mammary organoids (MEMOs), shedding light on the evolution of ectodermal appendages such as the mammary gland. Their CUSTOM approach involving the sequential activation of growth factors to generate MEMOs offers new prospects in regenerative medicine and disease modeling. [ABSTRACT FROM AUTHOR]

The parathyroid hormone (PTH) type 1 receptor (PTHR) is the canonical G protein-coupled receptor (GPCR) for PTH and PTH-related protein (PTHrP) and the key regulator of calcium homeostasis and bone turnover. PTHR function is critical for human health to maintain homeostatic control of ionized serum Ca2+ levels and has several unusual signaling features, such as endosomal cAMP signaling, that are well-studied but not structurally understood. In this review, we discuss how recently solved high resolution near-atomic structures of hormone-bound PTHR in its inactive and active signaling states and discovery of extracellular Ca2+ allosterism shed light on the structural basis for PTHR signaling and function. Resolution of the subatomic structure of the PTH receptor in complex with Gs and long acting PTH (LA-PTH). Identification of the signaling LA-PTH–PTHR–Gs complex in three conformations. Physiological and disease relevance of endosomal cAMP production as it relates to Ca2+ homeostasis. Extracellular Ca2+ and ATP as novel allosteric modulators of PTHR signaling. [ABSTRACT FROM AUTHOR]

Solid tumors frequently metastasize to bone and induce bone destruction leading to severe pain, fractures, and other skeletal-related events (SREs). Osteoclast inhibitors such as bisphosphonates delay SREs but do not prevent skeletal complications or improve overall survival. Because bisphosphonates can cause adverse side effects and are contraindicated for some patients, we sought an alternative therapy to reduce tumor-associated bone destruction. Our previous studies identified the transcription factor Gli2 as a key regulator of parathyroid hormone-related protein (PTHrP), which is produced by bone metastatic tumor cells to promote osteoclast-mediated bone destruction. In this study, we tested the treatment effect of a Gli antagonist GANT58, which inhibits Gli2 nuclear translocation and PTHrP expression in tumor cells. In initial testing, GANT58 did not have efficacy in vivo due to its low water solubility and poor bioavailability. We therefore developed a micellar nanoparticle (NP) to encapsulate and colloidally stabilize GANT58, providing a fully aqueous, intravenously injectable formulation based on the polymer poly(propylene sulfide) 135 - b -poly[(oligoethylene glycol) 9 methyl ether acrylate] 17 (PPS 135 - b -POEGA 17). POEGA forms the hydrophilic NP surface while PPS forms the hydrophobic NP core that sequesters GANT58. In response to reactive oxygen species (ROS), PPS becomes hydrophilic and degrades to enable drug release. In an intratibial model of breast cancer bone metastasis, treatment with GANT58-NPs decreased bone lesion area by 49% (p <.01) and lesion number by 38% (p <.05) and resulted in a 2.5-fold increase in trabecular bone volume (p <.001). Similar results were observed in intracardiac and intratibial models of breast and lung cancer bone metastasis, respectively. Importantly, GANT58-NPs reduced tumor cell proliferation but did not alter mesenchymal stem cell proliferation or osteoblast mineralization in vitro , nor was there evidence of cytotoxicity after repeated in vivo treatment. Thus, inhibition of Gli2 using GANT58-NPs is a potential therapy to reduce bone destruction that should be considered for further testing and development toward clinical translation. [ABSTRACT FROM AUTHOR]

Musculoskeletal disorders represent an elevated socioeconomic burden for developed aging societies. Osteoporosis (OP) has been treated with antiresorptive therapies or with teriparatide that was until recently the only anabolic therapy. However, approval of osteoporosis treatment in postmenopausal women with abaloparatide, which is an analog of parathyroid hormone-related peptide (PTHrP), has created a new alternative for OP management. The success of this new treatment is related to differential mechanisms of activation of PTH receptor type 1 (PTH1R) by abaloparatide and PTH. Here, we address the distinguishing mechanisms of PTH1R activation; the effects of PTH1R stimulation in osteoblast, osteocytes, and chondrocytes; the differences between PTH and abaloparatide actions on PTH1R; potential safety concerns; and future perspectives about abaloparatide use in other musculoskeletal disorders. Management of musculoskeletal disorders such as osteoporosis and osteoarthritis are a major challenge for developed countries associated with a high socioeconomic burden given the high prevalence of these diseases and the aging population. Treatment of osteoporosis has been addressed by anabolic or antiresorptive therapies. Abaloparatide, a peptide that signals through the parathormone 1 receptor (PTH1R) has been recently added to the armamentarium of bone anabolic therapies. Selective activation of PTH1R is the key of abaloparatide success in decreasing risk of fracture and increasing bone mineral density in postmenopausal women. Use of abaloparatide might be extended to other musculoskeletal disorders. [ABSTRACT FROM AUTHOR]

Abaloparatide is an analog of human parathyroid hormone-related protein (PTHrP) that has recently been approved for the treatment of post-menopausal osteoporosis. Abaloparatide is a stimulator of bone formation similar to teriparatide (1–34 PTH/TPTD), the first-in-class osteoanabolic drug. Both PTH and PTHrP signal via the type 1 PTH receptor (PTH1R) however, the downstream signaling varies between the two ligands. Both ligands have a similar affinity for the RG (GTPγS-sensitive) state of PTH1R, but, TPTD has a four-fold higher affinity for R0 (GTPγS-insensitive) than PTHrP that results in a prolonged cAMP signaling. Consequently, a greater production from osteoblastic cells of a potent resorption inducer, receptor activator of nuclear factor κB ligand (RANKL) is caused by TPTD than PTHrP. TPTD causes an excess formation over resorption early on producing an anabolic "window" which is lost with time due to increased RANKL production causing resorption to catch up with the formation. Although highly labile, PTHrP has an osteogenic effect accompanied by lesser resorptive and hypercalcemic effects than TPTD because of faster PTHrP-PTH1R dissociation than PTH-PTH1R complex. Engineered from PTHrP (1–34), abaloparatide was made stable and overcame the loss of the anabolic window and hypercalcemia associated with TPTD. The receptor activating domain (1–21 amino acids) of both ligands is same but multiple substitutions between amino acids 22–34 of PTHrP were made to enhance the peptide's stability. In, women with osteoporosis, abaloparatide increased BMD faster than TPTD and decreased fracture risk at both vertebral and non-vertebral sites but unlike TPTD/PTH did not increase resorption or hypercalcemia. [ABSTRACT FROM AUTHOR]

A 4-year-old, neutered male Golden Retriever was presented with a 1-week history of weight loss, polyuria, and polydipsia. The diagnostic workup showed an increased ionized calcium concentration, mild increase in serum creatinine and urea concentration, and severe hyperlipasemia. A complete abdominal ultrasound revealed multiple hepatic nodules. A cytological diagnosis of malignant epithelial neoplasia, highly suggestive of bile duct adenocarcinoma was made. In order to confirm the presumptive diagnosis of hypercalcemia of malignancy due to the presence of a hepatic neoplasia, serum parathormone-related peptide concentration was measured, and the result revealed an increased concentration. The dog was hospitalized and received supportive treatments consisting of intravenous furosemide and fluid therapy. After ruling out lymphoma and hypoadrenocorticism, oral prednisone was initiated and ionized calcium concentration decreased gradually down to normal concentration after 7 days of hospitalization. Chemotherapy with intravenous epirubicin was initiated based on the cytological diagnosis. One month after diagnosis and due to the worsening of its clinical condition, the dog was humanely euthanized. Postmortem examination confirmed a cholangiocellular carcinoma. To our knowledge, this is the first report of malignant hypercalcemia associated with cholangiocellular carcinoma in a dog. [ABSTRACT FROM AUTHOR]

Abstract Osteoporosis is accompanied by insufficient osteogenic capacity. Several lines of evidence suggested that solutions to enhance osteoblastogenesis were important strategies for osteoporotic bone defect repair. This study investigated the effect of combined treatment with vitamin K2 and PTH on bone formation in calvarial bone defect in osteoporotic rats and its influence on osteoblast in vitro. Bilateral ovariectomy was used in SPF Sprague Dawley rats to generate an osteoporosis model. Subsequently, a calvarial defect model was established and all osteoporotic rats were randomly assigned to the following groups: control, VK (vitamin K2, 30 mg/kg everyday), PTH (recombinant human PTH (1–34), 60 μg/kg, three times a week) or VK + PTH (vitamin K2, 30 mg/kg everyday plus PTH, 60 μg/kg three times a week) for 8 weeks. In vitro, bone marrow-derived stem cells (BMSCs) were cultured and treated with vitamin K2, PTH or vitamin K2+PTH. ALP staining and western blot were performed to observe the influence of combined treatment on BMSCs. Bone formation within calvarial defect were assessed by serum γ-carboxylated osteocalcin (Gla-OC), micro-CT, histological and immunofluorescent labeling. In this study, combined treatment of PTH and vitamin K2 showed positive effects on preventing bone loss in femurs in OVX rats. Combined treatment increased serum Gla-OC and promoted bone formation in osteoporotic calvarial bone defects. Immunohistochemistry showed that OCN and RUNX2 were more highly expressed in the VK + PTH group than in the control groups. In vitro studies results suggested that combined treatment with PTH and vitamin K2 increased expression of ALP, BMP2 and RUNX2 in BMSCs. Our data suggested that the combination of vitamin K2 and PTH increased differentiation of osteoblast and had a synergistic effect on bone formation in osteoporotic calvarial bone defect. Highlights • Vitamin K2 plus PTH enhance bone formation in osteoporotic bone defect. • Vitamin K2 plus PTH promote the differentiation of osteoblast. • Vitamin K2 plus PTH had a better therapeutic effect than did monotherapy. [ABSTRACT FROM AUTHOR]

Abstract Although PTHrP is implicated in several cancers, its role in chemoresistance is not fully elucidated. We found that in CRC cells, PTHrP exerts proliferative and protective effects and induces cell migration. The aim of this work was to further study the effects of PTHrP in CRC cells. Herein we evidenced, for the first time, that PTHrP induces resistance to CPT-11 in Caco-2 and HCT116 cells; although both cell lines responded to the drug through different molecular mechanisms, the chemoresistance by PTHrP in these models is mediated through ERK, which in turn is activated by PCK, Src and Akt. Moreover, continue administration of PTHrP in nude mice xenografts increased the protein levels of this MAPK and of other markers related to tumorigenic events. The understanding of the molecular mechanisms leading to ERK 1/2 activation and the study of ERK targets may facilitate the development of new therapeutic strategies for CRC treatment. Highlights • PTHrP activates ERK pathway through PKC, Scr and Akt in CRC cells. • PTHrP induces resistance to Irinotecan in Caco-2 and HCT116 cells. • The chemoresistance by PTHrP in CRC cells is mediated through ERK. • PTHrP administration increases ERK protein levels in nude mice xenografts. • In vivo PTHrP modulates markers related to tumorigenic events of CRC. [ABSTRACT FROM AUTHOR]

Parathyroid hormone (PTH) activates the PTH/PTH-related peptide receptor (PTH1R) on osteoblasts and other target cells. Mechanical stimulation of cells, including osteoblasts, causes release of nucleotides such as ATP into the extracellular fluid. In addition to its role as an energy source, ATP serves as an agonist at P2 receptors and an allosteric regulator of many proteins. We investigated the effects of concentrations of extracellular ATP, comparable to those that activate low affinity P2X7 receptors, on PTH1R signaling. Cyclic AMP levels were monitored in real-time using a bioluminescence reporter and β-arrestin recruitment to PTH1R was followed using a complementation-based luminescence assay. ATP markedly enhanced cyclic AMP and β-arrestin signaling as well as downstream activation of CREB. CMP – a nucleotide that lacks a high energy bond and does not activate P2 receptors – mimicked this effect of ATP. Moreover, potentiation was not inhibited by P2 receptor antagonists, including a specific blocker of P2X7. Thus, nucleotide-induced potentiation of signaling pathways was independent of P2 receptor signaling. ATP and CMP reduced the concentration of PTH (1–34) required to produce a half-maximal cyclic AMP or β-arrestin response, with no evident change in maximal receptor activity. Increased potency was similarly apparent with PTH1R agonists PTH (1–14) and PTH-related peptide (1–34). These observations suggest that extracellular nucleotides increase agonist affinity, efficacy or both, and are consistent with modulation of signaling at the level of the receptor or a closely associated protein. Taken together, our findings establish that ATP enhances PTH1R signaling through a heretofore unrecognized allosteric mechanism. [ABSTRACT FROM AUTHOR]

Background Parathyroid Hormone-Related Protein (PTHrP) is involved in intracellular calcium (Ca) regulation, and has been demonstrated to participate in regulation of Ca in brain cells, activation of neurons, and modulation of pain. However, there are conflicting reports regarding the presence of PTHrP in CSF. Design and methods PTHrP and Ca were quantified in paired CSF and serum samples using mass spectrometry-based methods. Associations between PTHrP and Ca concentrations with age, sex and concentrations of nine CSF diagnostic markers in a set of 140 paired serum and CSF patient samples were evaluated. Results The observed median PTHrP concentration in CSF was 51 times higher than in serum; the median concentration of Ca in CSF was 1.8 times lower than in serum. We observed positive correlation between concentrations of PTHrP in CSF and serum (p = 0.013). Distribution of PTHrP concentrations in serum was associated with age (p = 0.0068) and the concentrations were higher in women. In samples with serum calcium concentrations within the reference intervals (n = 118), central 95% distribution of concentrations for Ca-CSF, PTHrP-serum and PTHrP-CSF were 5.4 (4.5–6.1) mg/dL, 1.2 (0.5–2.5) pmol/L, 62 (22–125) pmol/L, respectively. Conclusions Our data demonstrate that PTHrP is a normal constituent of human CSF with median concentrations 51 fold higher than in serum. Elevated serum PTHrP concentrations were positively correlated with age and significantly higher in women. Our data suggest that CSF could be a significant source of circulating PTHrP. [ABSTRACT FROM AUTHOR]

Introduction Parathyroid hormone–related protein plays an important role in bone remodeling. Its N-terminal domain parathyroid hormone–related protein (107-111) is called osteostatin (OST). OST has demonstrated osteogenic potential when combined with biomaterials such as hydroxyapatite or bioceramics. However, the odontogenic potential of OST has not yet been reported. Therefore, the aim of this study was to determine whether OST has an odontogenic effect or a synergistic effect with mineral trioxide aggregate (MTA) in human dental pulp cells (hDPCs) and to examine the underlying signaling mechanisms involved in OST-mediated odontogenic differentiation. Methods Viability of hDPCs on stimulation with OST or MTA was measured. Real-time polymerase chain reaction and Western blot analyses were performed to evaluate the expression levels of odontogenic markers and the activation of extracellular signal-regulated kinase (ERK). To evaluate mineralized nodule formation, alkaline phosphatase (ALP) staining and alizarin red S staining were performed. Combined effects of OST and MTA were evaluated. Results OST promoted odontogenic differentiation, as evidenced by the formation of mineralized nodules, induction of ALP activity, and upregulation of odontogenic markers (dentin sialophosphoprotein, dentin matrix protein-1, and ALP). Phosphorylation of ERK was increased by OST. However, ERK inhibitor (U0126) inhibited the increase in dentin sialophosphoprotein and dentin matrix protein-1 expression and mineralization induced by OST. A combination of MTA and OST upregulated odontogenic differentiation-associated gene expression and calcium nodule mineralization in hDPCs compared with MTA alone. Conclusions The present study revealed that OST can promote odontogenic differentiation and mineralization through activating the ERK signaling pathway. A combination of MTA and OST showed a synergistic effect compared with MTA alone in hDPCs. [ABSTRACT FROM AUTHOR]

The parathyroid hormone (PTH) 1 receptor (PTH1R) is a G protein-coupled receptor (GPCR) that regulates skeletal development and calcium homeostasis. Here, we describe cryo-EM structures of the PTH1R in complex with fragments of the two hormones, PTH and PTH-related protein, the drug abaloparatide, as well as the engineered tool compounds, long-acting PTH (LA-PTH) and the truncated peptide, M-PTH(1–14). We found that the critical N terminus of each agonist engages the transmembrane bundle in a topologically similar fashion, reflecting similarities in measures of Gαs activation. The full-length peptides induce subtly different extracellular domain (ECD) orientations relative to the transmembrane domain. In the structure bound to M-PTH, the ECD is unresolved, demonstrating that the ECD is highly dynamic when unconstrained by a peptide. High resolutions enabled identification of water molecules near peptide and G protein binding sites. Our results illuminate the action of orthosteric agonists of the PTH1R. [Display omitted] • Cryo-EM structures of the PTH1R bound to native hormones and a therapeutic • The PTH1R extracellular domain is highly dynamic when bound to a truncated agonist • Structural water networks stabilize the binding of PTH The parathyroid hormone 1 receptor is differentially activated by peptide hormones, therapeutics, and tool compounds. Cary et al. determine cryo-EM structures of the parathyroid hormone 1 receptor and a coupling partner bound to five peptides, which revealed subtle differences in agonist engagement. [ABSTRACT FROM AUTHOR]

Objective To investigate the role of parathyroid hormone related protein (PTHrP) in diabetic periodontitis. Methods After injected with 55 mg/kg streptozotocin, diabetic rats were treated subcutaneously with low-dose (40 μg/kg, once daily for 5 days per week), middle-dose (80 μg/kg) or high-dose (160 μg/kg) PTHrP(1–34) peptide. Treatment continued for 12 weeks. Changes in periodontal tissues were confirmed by micro-computerized tomography assay and H&E analysis. We used tartrate resistant acid phosphatase (TRAP) staining to identify osteoclast cells. The expression of TNF-α, IL-1β and IL-6 was assessed by immunohistochemistry and Western blot. Results Tooth-supporting structure loss was observed in periodontal tissues of diabetic rats. PTHrP (1–34) attenuated alveolar bone loss, especially in the middle-dose and high-dose group. Whereas TNF-α, IL-1β and IL-6 protein levels were increased in the diabetic gingival tissues, PTHrP (1–34) treatment inhibited the increase of IL-1β and IL-6, but had no effect on TNF-α. Conclusion Type 1 diabetes increased the susceptibility to periodontal disease. Intermittent administration of PTHrP (1–34) exhibited an inhibitory effect on alveolar bone resorption and the gingival inflammation in periodontal tissues of diabetic rats. [ABSTRACT FROM AUTHOR]

Parathyroid hormone-related protein (PTHrP), a ubiquitously expressed protein, is composed of four functional domains including N-terminus, mid region, nuclear localization signal (NLS) and C-terminus. Under the direction of NLS, PTHrP can enter cell nucleus from cytoplasm and stimulate mitogenesis. Although PTHrP is considered to have important developmental roles, the role of PTHrP NLS and C-terminus in developmental process remains unknown, especially in T-cell development. Here, we used a knock-in mouse model, which expresses a truncated form of PTHrP missing the NLS (87–107) and C-terminus (108–139) of the protein, to examine the role of PTHrP NLS and C-terminus in T-cell development. Our results showed that the truncated PTHrP (1-84) led to abnormal subpopulations, impaired proliferation and increased apoptosis in the thymus, indicating that PTHrP is involved in the development of T cells, and the NLS and C-terminus part is necessary for the normal role of PTHrP in T-cell development. [ABSTRACT FROM AUTHOR]

The treatment of osteoporosis is generally either by inhibition of bone resorption with antiresorptive agents or by stimulation of bone formation with anabolic agents. Currently, teriparatide (recombinant human parathyroid hormone 1-34 [rhPTH (1-34)]) is the only available approved anabolic agent in the U.S. Other anabolic agents are under investigation however. Abaloparatide is recombinant human parathyroid hormone-related peptide 1-34. This agent is an anabolic agent that appears more potent than teriparatide, and it may have more rapid onset of fracture reduction than teriparatide. It is currently undergoing FDA review, with approval expected in 2017. [ABSTRACT FROM AUTHOR]

The osteocyte expressed gene SOST encodes sclerostin, a potent negative regulator of bone formation and inducer of bone resorption. We have recently demonstrated that the human SOST gene is positively regulated in response to 1α,25-dihydroxyvitamin D 3 (1,25D). Responsiveness may be mediated at least in part by a single classical DR3-type vitamin D response element (VDRE). In this study we examined the early responsiveness of the SOST gene to both 1,25D and to parathyroid hormone (PTH), a known repressor of SOST expression, in SaOS2 cells differentiated to an osteocyte-like stage of cell maturation. Both SOST mRNA levels and sclerostin protein levels increased in these cultures as early as 3 h post-treatment with 1,25D and declined in response to PTH in the same timeframe. For 1,25D, the level of induced SOST appeared dependent on the extent, to which the degradative enzyme 1,25-dihydroxyvitamin D 24-hydroxylase ( CYP24A1 ) was induced. Together with the observed rapid decrease in SOST /sclerostin levels in response to PTH, endocrine regulation of sclerostin production appears to be an important determinant of sclerostin levels. These findings confirm that the human SOST gene and sclerostin expression can be considered to be directly 1,25D-responsive in osteocytes. [ABSTRACT FROM AUTHOR]

The dental follicle is involved in tooth eruption and it expresses a great amount of the parathyroid hormone-related protein (PTHrP). PTHrP as an extracellular protein is required for a multitude of different regulations of enchondral bone development and differentiation of bone precursor cells and of the development of craniofacial tissues. The dental follicle contains also precursor cells (DFCs) of the periodontium. Isolated DFCs differentiate into periodontal ligament cells, alveolar osteoblast and cementoblasts. However, the role of PTHrP during the human periodontal development remains elusive. Our study evaluated the influence of PTHrP on the osteogenic differentiation of DFCs under in vitro conditions for the first time. The PTHrP protein was highly secreted after 4 days of the induction of the osteogenic differentiation of DFCs with dexamethasone (2160.5 pg/ml ± 345.7 SD. in osteogenic differentiation medium vs. 315.7 pg/ml ± 156.2 SD. in standard cell culture medium; Student’s t Test: p < 0.05 (n = 3)). We showed that the supplementation of the osteogenic differentiation medium with PTHrP inhibited the alkaline phosphatase activity and the expression of the transcription factor DLX3, but the depletion of PTHrP did not support the differentiation of DFCs. Previous studies have shown that Indian Hedgehog (IHH) induces PTHrP and that PTHrP, in turn, inhibits IHH via a negative feedback loop. We showed that SUFU (Suppressor Of Fused Homolog) was not regulated during the osteogenic differentiation in DFCs. So, neither the hedgehog signaling pathway induced PTHrP nor PTHrP suppressed the hedgehog signaling pathway during the osteogenic differentiation in DFCs. In conclusion, our results suggest that PTHrP regulates independently of the hedgehog signaling pathway the osteogenic differentiated in DFCs. [ABSTRACT FROM AUTHOR]

Efficient therapies are available for the treatment of osteoporosis, however, there are still unmet needs. Anti-resorptive therapies only increase bone mineral density to a certain extent and reduce the risk of non-vertebral fractures by 20%, only one anabolic option is available — the effect of which levels off over time, and the evidence for combination therapy targeting both resorption and formation is limited. The current review will focus on emerging treatments of osteoporosis with the potential of enhanced anabolic effects (romosozumab and abaloparatide) or uncoupling of resorption and formation (odanacatib and romosozumab) as well as the effect of combination therapy. [ABSTRACT FROM AUTHOR]

The placenta actively transports minerals whereas the intestines and kidneys may be nonessential for fetal mineral homeostasis. Mineral concentrations are higher in fetal blood than in adults in order for the developing skeleton to accrete adequate mineral content. Fetal bone development and serum mineral regulation are dependent upon parathyroid hormone (PTH) and PTH-related protein (PTHrP), but not calcitriol, fibroblast growth factor-23, calcitonin, or the sex steroids. After birth, a switch from fetal to neonatal regulatory mechanisms is triggered by loss of the placental calcium infusion, onset of a breathing, and a postnatal fall in serum calcium and rise in phosphorus. This is followed by an increase in PTH, then a rise in calcitriol, and developmental changes in kidneys and intestines. Serum calcium increases and phosphorus declines over days. The intestines become the main source of mineral, while kidneys reabsorb mineral, and bone turnover contributes additional mineral to the circulation. [ABSTRACT FROM AUTHOR]

Cancer patients frequently develop skeletal metastases that significantly impact quality of life. Since bone metastases remain incurable, a clearer understanding of molecular mechanisms regulating skeletal metastases is required to develop new therapeutics that block establishment of tumors in bone. While many studies have suggested that the microenvironment contributes to bone metastases, the factors mediating tumors to progress from a quiescent to a bone-destructive state remain unclear. In this study, we hypothesized that the “soil” of the bone microenvironment, specifically the rigid mineralized extracellular matrix, stimulates the transition of the tumor cells to a bone-destructive phenotype. To test this hypothesis, we synthesized 2D polyurethane (PUR) films with elastic moduli ranging from the basement membrane (70 MPa) to cortical bone (3800 MPa) and measured expression of genes associated with mechanotransduction and bone metastases. We found that expression of Integrin β3 ( Iβ3 ), as well as tumor-produced factors associated with bone destruction ( Gli2 and parathyroid hormone related protein ( PTHrP )), significantly increased with matrix rigidity, and that blocking Iβ3 reduced Gli2 and PTHrP expression. To identify the mechanism by which Iβ3 regulates Gli2 and PTHrP (both are also known to be regulated by TGF-β), we performed Förster resonance energy transfer (FRET) and immunoprecipitation, which indicated that Iβ3 co-localized with TGF-β Receptor Type II (TGF-β RII) on rigid but not compliant films. Finally, transplantation of tumor cells expressing Iβ3 shRNA into the tibiae of athymic nude mice significantly reduced PTHrP and Gli2 expression, as well as bone destruction, suggesting a crucial role for tumor-produced Iβ3 in disease progression. This study demonstrates that the rigid mineralized bone matrix can alter gene expression and bone destruction in an Iβ3/TGF-β-dependent manner, and suggests that Iβ3 inhibitors are a potential therapeutic approach for blocking tumor transition to a bone destructive phenotype. [ABSTRACT FROM AUTHOR]

In this review we analyse structure/sequence–function relationships for the superfamily of PLP-dependent enzymes with special emphasis on class III transaminases. Amine transaminases are highly important for applications in biocatalysis in the synthesis of chiral amines. In addition, other enzyme activities such as racemases or decarboxylases are also discussed. The substrate scope and the ability to accept chemically different types of substrates are shown to be reflected in conserved patterns of amino acids around the active site. These findings are condensed in a sequence–function matrix, which facilitates annotation and identification of biocatalytically relevant enzymes and protein engineering thereof. [ABSTRACT FROM AUTHOR]

Wnt/β-catenin, Indian hedgehog (Ihh)/Parathyroid-related peptide (PTHrP) and retinoid signaling pathways regulate cartilage differentiation, growth, and function during development and play a key role in endochondral ossification. The objective of this study was to elucidate the gene and protein expression of signaling molecules of these regulatory pathways in chondrocytes surrounding cartilage canals and the osteochondral junction during neonatal and pre-adolescent development. This study revealed cell-specific and age-related differences in gene and protein expression of signaling molecules of these regulatory pathways. A trend for higher gene expression of PTHrP along the cartilage canals and Ihh along the osteochondral junction suggests the presence of paracrine feedback in articular–epiphyseal cartilage. Differential expression of canonical (β-catenin, Wnt-4, Lrp4, Lrp6) and noncanonical Wnt signaling (Wnt-5b, Wnt-11) and their inhibitors (Dkk1, Axin1, sFRP3, sFRP5, Wif-1) surrounding the cartilage canals and osteochondral junction provides evidence of the complex interactions occurring during endochondral ossification. [ABSTRACT FROM AUTHOR]

Homeostasis during lactation is a special case in which the unit for regulation is a dyad comprising the mother and her currently nursing offspring (the mother–offspring dyad). This arrangement is not a trivial. A litter of mice can have a mass greater than the mother and nutrient demands that far exceed her. Homeostasis for milk secretion, appetite, and calcium metabolism must come under integrated control, responding seamlessly to the needs of the mother and the offspring. Serotonin (5-HT) is a primary local regulator of mammary homeostasis. 5-HT synthesis in the mammary epithelium is high during lactation and increases during milk stasis. Two important functions are attributed to the 5-HT system. Firstly, when alveolar spaces are filled with milk 5-HT inhibits milk secretion and opens tight junctions. This feedback induces early phases of involution. Secondly, 5-HT induces synthesis and secretion of parathyroid hormone-related peptide (PTHrP). • Homeostasis during lactation is governed by the mother and the offspring, unlike homeostasis in a single individual. • Prolactin is the main systemic regulator of lactation, and serotonin is the main local (intramammary) regulator of lactation. • During sustained milk stasis serotonin inhibits lactation and initiates involution acting via 5-HT7 receptors. • Durin lactation serotonin acts via 5-HT2B receptors to stimulate PTHrP secretion, thereby controlling calcium homeostasis. [ABSTRACT FROM AUTHOR]

The aim of this study was to investigate the mechanisms of how protein kinase A (PKA) is activated during bone morphogenetic protein 2 (BMP2)-induced osteogenic differentiation in dental follicle stem cells. Human dental follicle stem cells were cultured and treated with a BMP2-containing osteogenic differentiation medium or differentiation medium without BMP2. Specific siRNAs and substances/proteins were used to modulate pathways. PKA activity and activity of alkaline phosphatase were determined. Expression of targets was measured by Western Blots and reverse transcription-quantitative polymerase chain reaction, while protein interactions were investigated by immunoprecipitation. Immunofluorescence staining was used for subcellular target localization. PKA activity is stimulated after osteogenic induction by BMP2. Differentiation medium without BMP2 strongly induces BMP2 gene expression, which correlates with downstream target expression. Elevation of cAMP levels does not affect alkaline phosphatase activity and PKA does not directly interact with Smad 4. However, PKA activation requires expression of parathyroid hormone-related protein (PTHrP), which is stimulated after BMP2-induced differentiation. Furthermore, neither supplementation with PTHrP nor with the receptor antagonist parathyroid hormone (7−34) affects PKA activity. Thus, endogenous PTHrP expression is required for PKA activation and immunofluorescence staining shows that PTHrP is mainly located in the nucleus of dental follicle stem cells. Beyond, knockdown of PKA stimulates the BMP2 signaling pathway and down-stream expression of PTHrP. BMP2-induced osteogenic differentiation activates PKA in dental follicle stem cells via endogenous expression of PTHrP. Additionally, PKA inhibits BMP2 signaling and expression of PTHrP in a negative feedback loop. • Protein kinase A is activated by osteogenic induction in dental follicle cells. • A medium containing bone morphogenetic protein 2 was used to induce osteogenesis. • The kinase activation occurs via endogenous parathyroid hormone-related protein. • Parathyroid hormone-related protein is induced by bone morphogenetic protein 2. • Protein kinase A inhibits bone morphogenetic protein 2 signaling. [ABSTRACT FROM AUTHOR]

The signal transducer and activator of transcription 5a (Stat5a) modulates genes involved in proliferation and survival and plays pivotal roles in regulating the function of the mammary gland during pregnancy, lactation, and involution. However, there is little information about the effects of Stat5a on apoptosis of goat mammary gland epithelial cells (GMECs). In addition, parathyroid hormone-related protein (PTHrP) is a key regulator in cellular calcium transport, mammary gland development and breast tumor biology. This study aimed to explore the interaction of Stat5a and PTHrP in GMEC apoptosis. Quantitative real time PCR (qRT-PCR) suggested that Stat5a was predominantly expressed in the mammary gland, lung, liver and spleen of goats. Treating the GMECs with pimozide, an inhibitor of Stat5a that decreases Stat5a tyrosine phosphorylation, increased PTHrP levels in GMECs in a dose-dependent manner and simultaneously promoted apoptosis of the GMECs. We also demonstrated that PTHrP inhibition induced GMEC apoptosis and restrained cell proliferation. In contrast, PTHrP overexpression protected GMECs from pimozide- and calcium-induced apoptosis, and promoted cell proliferation. Furthermore, pimozide and CaCl 2 downregulated the antiapoptotic protein Bcl-2 mRNA expression, respectively, and these effects were protected by PTHrP overexpression. Interestingly, we also found that Stat5a suppressed the expression of matrix metalloproteinase 9 (MMP-9) which can induce goat mammary epithelial cell migration, but PTHrP increased MMP-9 mRNA level. Thus, Stat5a may regulate GMEC survival by regulating the expression of PTHrP. [ABSTRACT FROM AUTHOR]

Based on the known fact that the parathyroid hormone (PTH) might be extended at its C-terminus with biotechnological protein cargoes, a vector directing the secretion of PTH 1–84 C-terminally fused with the antigenic epitope myc (PTH-myc) was exploited. The functional properties and potential of this analog for imaging PTH 1 R-expressing cells were examined. The PTH-myc construct was recombinantly produced as a conditioned medium (CM) of transfected HEK 293a cells (typical concentrations of 187 nM estimated with ELISAs for PTH). PTH-myc CM induced cyclic AMP formations (10 min), with a minor loss of potency relative to authentic PTH 1–84 , and c-Fos expression (1–3 h). Treatment of recipient HEK 293a cells transiently expressing PTH 1 R with PTH-myc CM (supplemented with a fluorescent monoclonal anti-myc tag antibody, either 4A6 or 9E10) allowed the labeling of endosomal structures positive for Rab5 and/or for β-arrestin 1 (microscopy, cytofluorometry). Authentic PTH was inactive in this respect, ruling out a non-specific form of endocytosis like pinocytosis. Using a horseradish peroxidase-conjugated secondary antibody, the endocytosis of the PTH-myc-based antibody complex by endogenous PTH 1 R was evidenced in MG-63 osteoblastoid cells. The secreted construct PTH-myc represents a bona fide agonist that supports the feasibility of transporting cargoes of considerable molecular weight inside cells using arrestin and Rab5-mediated PTH 1 R endocytosis. PTH-myc is also transported into cells that express PTH 1 R at a physiological level. Such tagged peptide hormones may be part of a cancer chemotherapy scheme exploiting a modular cytotoxic secondary antibody and the receptor repertoire expressed in a given tumor. [ABSTRACT FROM AUTHOR]

Objective Jaw movement is an important mechanical factor for prenatal development of the condylar cartilage of mandible. Fetal jaw movement restriction has been shown to cause deformity of the mandibular condyle. We hypothesized that this treatment affects the expression of mechanosensitive molecules, namely Indian hedgehog ( Ihh ) and Parathyroid hormone related protein ( PTHrP ) in the condyle. Experimental methods We restrained jaw movement by suturing the jaw of E15.5 mouse embryos and allowed them to develop until E18.5 using exo utero system, and analyzed them by immunohistochemistry and in situ hybridization methods. Results Morphological, histomorphometric and immunohistochemical study showed that the mandibular condylar cartilage was reduced and deformed, the volume and total cell numbers in the condylar cartilage were also reduced, and number and/or distribution of 5-bromo-2′-deoxyuridine-positive cells, Ihh-positive cells in the mesenchymal and pre-hypertrophic zones were significantly and correspondingly decreased in the sutured group. Using in situ hybridization , reduced expression of Ihh , PTHrP and their related receptors were observed in condylar cartilage of the sutured embryos. Conclusions Our results revealed that the altered mechanical stress induced by prenatal jaw movement restriction decreased proliferating cells, the amount of cartilage, and altered expression of the Ihh and PTHrP , suggesting that Ihh act as mechanotransduction mediators in the development of mandibular condylar cartilage. [ABSTRACT FROM AUTHOR]

Objective The aim of this study was to explore the mutual communication of the parathyroid hormone-related peptide (PTHrP) and phosphatidylinositol 3-kinase/threonine protein kinase (PI3K/Akt) pathway on the proliferation and differentiation of condylar chondrocytes from Sprague-Dawley (SD) rats. Methods Condylar chondrocytes from the condylar cartilage were cultured and an organ culture system of mandibular condyles was employed. The distribution of PI3K, phospho-Akt (p-Akt), and PTHrP in condylar cartilage was detected by either immunohistochemistry or immunofluorescence. The second passage chondrocytes and condyle specimens in the organ culture system were treated with PTHrP, LY294002, PTHrP and LY294002 in combination, or dimethyl sulfoxide (DMSO), separately. The mRNA and protein levels of type II (Col II) and type X collagen (Col X) were investigated by real-time polymerase chain reaction (PCR) and Western blot analysis. The condyle growth in organ culture system was analysed by haematoxylin–eosin staining. Results PTHrP, PI3K, and p-Akt were mainly located in the proliferative and hypertrophic zones. PTHrP promoted the proliferation of condylar chondrocytes, while LY294002 limited this effect. The mRNA and protein levels of Col II and Col X in these cells were reduced by PTHrP and enhanced by LY294002. Organ culture showed a significant enhancement of condyle elongation with PTHrP treatment or a combination of PTHrP and LY294002 treatment. After treatment with LY294002, the length of condyles was reduced compared with the samples treated with DMSO. Conclusions We conclude that the PI3K/Akt pathway plays an essential role in the proliferation and differentiation of condylar chondrocytes and is a potential target for PTHrP in regulating chondrocyte differentiation at condylar cartilage. [ABSTRACT FROM AUTHOR]

Prostate cancer frequently metastasizes to the skeleton but the underlying mechanism remains largely undefined. Discoidin domain receptor 2 (DDR2) is a member of receptor tyrosine kinase (RTK) family and is activated by collagen binding. This study aimed to investigate the function and detailed mechanism of DDR2 in prostate cancer bone dissemination. Herein we found that DDR2 was strongly expressed in bone-metastatic prostate cancer cells and tissues compared to that in normal controls. Enhanced expression of constitutively activated DDR2 led to elevation in motility and invasiveness of prostate cancer cells, whereas knockdown of DDR2 through specific shRNA caused a dramatic repression. Knockdown of DDR2 in prostate cancer cells resulted in significant decrease in the proliferation, differentiation and function of osteoblast. Over-expression of DDR2 in prostate cancer cells resulted in notable acceleration of osteoclast differentiation and bone resorption, whereas knockdown of DDR2 exhibited the opposite effects. An intrabone injection bone metastasis animal model demonstrated that DDR2 promoted osteolytic metastasis in vivo . Molecular evidence demonstrated that DDR2 regulated the expression, secretion, and promoter activity of parathyroid hormone-related protein (PTHrP), via modulating Runx2 phosphorylation and transactivity. DDR2 was responsive to TGF-β and involved in TGF-β-mediated osteoclast activation and bone resorption. In addition, DDR2 facilitated prostate cancer cells adhere to type I collagen. This study reveals for the first time that DDR2 plays an essential role in prostate cancer bone metastasis. The mechanism disclosure may provide therapeutic targets for the treatment of prostate cancer. [ABSTRACT FROM AUTHOR]

Parathyroid hormone-related protein (PTHrP) has been identified to play a crucial role in osteolysis formation and stromal cell (GCTSC) proliferation in giant cell tumor (GCT). MiR-126-5p is an intronic miRNA identified as tumor suppressor in many tumors, but its role in GCT is poorly understood. We found that miR-126-5p was decreased in GCT and could directly regulate PTHrP expression. Furthermore, miR-126-5p could control osteoclast (OC) differentiation, GCTSC proliferation and osteolysis formation in GCT through negative regulation of PTHrP. Thus, these results suggest that miR-126-5p could directly target PTHrP and have a tumor suppressor function in GCT. [ABSTRACT FROM AUTHOR]

Abstract: Biopolymer-coated nanocrystalline hydroxyapatite (HA) made as macroporous foams which are degradable and flexible are promising candidates as orthopaedic implants. The C-terminal (107-111) epitope of parathyroid hormone-related protein (PTHrP) exhibits osteogenic properties. The main aim of this study was to evaluate whether PTHrP (107-111) loading into gelatin–glutaraldehyde biopolymer-coated HA (HAGlu) scaffolds would produce an optimal biomaterial for tissue engineering applications. HAGlu scaffolds with and without PTHrP (107-111) were implanted into a cavitary defect performed in both distal tibial metaphysis of adult rats. Animals were sacrificed after 4weeks for histological, microcomputerized tomography and gene expression analysis of the callus. At this time, bone healing occurred only in the presence of PTHrP (107-111)-containing HAGlu implant, related to an increase in bone volume/tissue volume and trabecular thickness, cortical thickness and gene expression of osteocalcin and vascular cell adhesion molecule 1, but a decreased gene expression of Wnt inhibitors, SOST and dickkopf homolog 1. The autonomous osteogenic effect of the PTHrP (107-111)-loaded HAGlu scaffolds was confirmed in mouse and human osteoblastic cell cultures. Our findings demonstrate the advantage of loading PTHrP (107-111) into degradable HAGlu scaffolds for achieving an optimal biomaterial that is promising for low load bearing clinical applications. [Copyright &y& Elsevier]

The concentration of parathyroid hormone-related protein (PTHrP) in the blood of healthy animals is extremely low. However, milk contains a relatively large amount of PTHrP, and the changes in its levels in the early stages of lactation in cows remain unclear. To understand the characteristics of parturient changes in milk PTHrP content and the biological implications thereof, changes in milk PTHrP concentrations were measured at 7 time intervals between parturition and 21 days postpartum in 8 primiparous and 8 multiparous Holstein cows. Based on these results, milk samples were collected from 47 primiparous and 66 multiparous Holstein cows at 3 days postpartum to investigate the relationship between milk PTHrP concentration and the variables of cow age, milk yield, and milk calcium concentration. Milk PTHrP concentration in both parity groups was found to be lowest on the day of parturition (primiparous, 3.1±0.5nM and multiparous, 1.6±0.3nM) but to significantly increase on day 14 of lactation in primiparous cows (6.2±0.8nM) and day 7 of lactation in multiparous cows (4.1±0.2nM). Comparison of the 2 groups revealed that milk PTHrP concentrations in primiparous cows were higher than those of multiparous cows in the first 3 days of lactation. Although a significant negative relationship was found between milk PTHrP concentration and both age (r=−0.65) and milk calcium concentration (r=−0.19) at 3 days postpartum, no significant correlation was found between milk PTHrP concentration and milk yield. The study thus identified 3 unique characteristics of milk PTHrP concentration in the early stages of lactation: milk PTHrP concentration is higher in primiparous than multiparous cows, milk PTHrP concentration is lower in colostrum than later milk, and the difference in milk PTHrP concentration between primiparous and multiparous cows at 3 days postpartum is more strongly influenced by age than milk yield. [ABSTRACT FROM AUTHOR]

Abstract: Human PTHrP gene displays a complex organization with nine exons producing diverse mRNA variants due to alternative splicing at 5′ and 3′ ends and the existence of three different transcriptional promoters (P1, P2 and P3), two of which (P2 and P3) contain CpG islands. It is known that the expression of PTHrP isoforms may be differentially regulated in a developmental stage- and tissue-specific manner. To search for novel molecular markers of stemness/differentiation, here we have examined isoform expression in fat-derived mesenchymal stem cells both maintained in stem conditions and induced toward adipo- and osteogenesis. In addition, the expression of the splicing isoforms derived from P2 and P3 promoters was correlated to the state of methylation of the latter. Moreover, we also performed a quantitative evaluation of intracellular and secreted PTHrP protein product in undifferentiated stem cells and in parallel cultures at various differentiation stages. The data obtained indicate that from the stemness condition to that of osteo- and adipo-genic differentiated cells, the expression of isoforms becomes increasingly selective, thereby being a potential gene signature for the monitoring of cell stem or committed/differentiating state and that the switching-off of PTHrP isoform expression is mostly promoter methylation-dependent. Moreover, PTHrP intracellular retention is down-regulated in osteo-differentiating cells whereas the secretion of the protein in the extracellular medium is up-regulated with respect to stem cells, thereby suggesting that these variations of the intracellular and extracellular levels of PTHrP could potentially be enclosed in the list of the available protein signature of osteogenic differentiation. [Copyright &y& Elsevier]

Abstract: Mechanical loading has been widely considered to be a crucial regulatory factor for growth plate development, but the exact mechanisms of this regulation are still not completely understood. In the growth plate, parathyroid hormone-related protein (PTHrP) regulates chondrocyte differentiation and longitudinal growth. Cyclic mechanical strain has been demonstrated to influence growth plate chondrocyte differentiation and metabolism, whereas the relationship between cyclic mechanical strain and PTHrP expression is not clear. The objective of this study was to investigate whether short-term cyclic tensile strain regulates PTHrP expression in postnatal growth plate chondrocytes in vitro and to explore whether the organization of cytoskeletal F-actin microfilaments is involved in this process. To this end, we obtained growth plate chondrocytes from 2-week-old Sprague–Dawley rats and sorted prehypertrophic and hypertrophic chondrocytes using immunomagnetic beads coated with anti-CD200 antibody. The sorted chondrocytes were subjected to cyclic tensile strain of varying magnitude and duration at a frequency of 0.5Hz. We found that cyclic strain regulates PTHrP expression in a magnitude- and time-dependent manner. Incubation of chondrocytes with cytochalasin D, an actin microfilament-disrupting reagent, blocked the induction of PTHrP expression in response to strain. The results suggest that short-term cyclic tensile strain induces PTHrP expression in postnatal growth plate prehypertrophic and hypertrophic chondrocytes and that PTHrP expression by these chondrocytes may subsequently affect growth plate development. The results also support the idea that the organization of cytoskeletal F-actin microfilaments plays an important role in mechanotransduction. [Copyright &y& Elsevier]

Abstract: The repair of osteochondral defects can be enhanced with scaffolds but is often accompanied with undesirable terminal differentiation of bone marrow-derived mesenchymal stem cells (BMSCs). Parathyroid hormone-related protein (PTHrP) has been shown to inhibit aberrant differentiation, but administration at inappropriate time points would have adverse effects on chondrogenesis. This study aims to develop an effective tissue engineering strategy by combining PTHrP and collagen-silk scaffold for osteochondral defect repair. The underlying mechanisms of the synergistic effect of combining PTHrP administration with collagen-silk scaffold implantation for rabbit knee joint osteochondral defect repair were investigated. In vitro studies showed that PTHrP treatment significantly reduced Alizarin Red staining and expression of terminal differentiation-related markers. This is achieved in part through blocking activation of the canonical Wnt/β-catenin signaling pathway. For the in vivo repair study, intra-articular injection of PTHrP was carried out at three different time windows (4–6, 7–9 and 10–12 weeks) together with implantation of a bi-layer collagen-silk scaffold. Defects treated with PTHrP at the 4–6 weeks time window exhibited better regeneration (reconstitution of cartilage and subchondral bone) with minimal terminal differentiation (hypertrophy, ossification and matrix degradation), as well as enhanced chondrogenesis (cell shape, Col2 and GAG accumulation) compared with treatment at other time windows. Furthermore, the timing of PTHrP administration also influenced PTHrP receptor expression, thus affecting the treatment outcome. Our results demonstrated that intra-articular injection of PTHrP at 4–6 weeks post-injury together with collagen-silk scaffold implantation is an effective strategy for inhibiting terminal differentiation and enhancing chondrogenesis, thus improving cartilage repair and regeneration in a rabbit model. [Copyright &y& Elsevier]

Abstract: Excessive secretion of parathyroid hormone-related protein (PTHrP) by tumors stimulates bone resorption and increases renal tubular reabsorption of calcium, resulting in hypercalcemia of malignancy. We investigated the ability of cinacalcet, an allosteric modulator of the calcium-sensing receptor, to attenuate hypercalcemia by assessing its effects on blood ionized calcium, serum PTHrP, and calcium-sensing receptor mRNA in mice bearing either Rice H-500 Leydig cell or C26-DCT colon tumors. Cinacalcet effectively decreased hypercalcemia in a dose- and enantiomer-dependent manner; furthermore, cinacalcet normalized phosphorus levels, but did not affect serum PTHrP. Ribonuclease protection assay results demonstrated presence of PTHrP receptor, but not calcium-sensing receptor mRNA in C26-DCT tumors. The mechanism by which cinacalcet lowered serum calcium was investigated in parathyroidectomized rats (i.e., without PTH) made hypercalcemic by PTHrP. Cinacalcet attenuated PTHrP-mediated elevations in blood ionized calcium, which were accompanied by increased plasma calcitonin. Taken together these results suggest that the cinacalcet-mediated decrease in serum calcium is not the result of a direct effect on tumor cells, but rather is the result of increased calcitonin release. In summary, cinacalcet effectively reduced tumor-mediated hypercalcemia and corrected hypophosphatemia in mice. Further investigation of cinacalcet for treatment of hypercalcemia of malignancy is warranted. [Copyright &y& Elsevier]

Abstract: Parathyroid-hormone-related protein (PTHrP) has been shown to be an important factor in osteolysis in the setting of metastatic carcinoma to the bone. However, PTHrP may also be central in the setting of primary bone tumors. Giant cell tumor of bone (GCT) is an aggressive osteolytic bone tumor characterized by osteoclast-like giant cells that are recruited by osteoblast-like stromal cells. The stromal cells of GCT are well established as the only neoplastic element of the tumor, and we have previously shown that PTHrP is highly expressed by these cells both in vitro and in vivo. We have also found that the stromal cells exposed to a monoclonal antibody to PTHrP exhibited rapid plate detachment and quickly died in vitro. Therefore, PTHrP may serve in an autocrine manner to increase cell proliferation and promote invasive properties in GCT. The purpose of this study was to use transcriptomic microarrays and functional assays to examine the effects of PTHrP neutralization on cell adhesion, migration and invasion. Microarray and proteomics data identified genes that were differentially expressed in GCT stromal cells under various PTHrP treatment conditions. Treatment of GCT stromal cells with anti-PTHrP antibodies showed a change in the expression of 13 genes from the integrin family relative to the IgG control. Neutralization of PTHrP reduced cell migration and invasion as evidenced by functional assays. Adhesion and anoikis assays demonstrated that although PTHrP neutralization inhibits cell adhesion properties, cell detachment related to PTHrP neutralization did not result in associated cell death, as expected in mesenchymal stromal cells. Based on the data presented herein, we conclude that PTHrP excreted by GCT stromal cells increases bone tumor cell local invasiveness and migration. [Copyright &y& Elsevier]

Abstract: Parathyroid hormone-related protein (PTHrP) is a polyhormone secretory protein that plays fundamental roles in the development and function of various tissues. Transforming growth factor (TGF)-β is an important tumor suppressor that induces cell cycle arrest and apoptosis. Increased PTHrP expression has been implicated in TGF-β-induced growth inhibition in human hepatocellular carcinoma cells. However, whether PTHrP is involved in TGF-β-induced apoptosis remains unknown. Using Hep3B and HuH-7, two human hepatocellular carcinoma cell lines, the current study examined the hypothesis that TGF-β-induced apoptosis is mediated by the induction of PTHrP expression. We found that (1) TGF-β induces PTHrP mRNA expression, protein expression and secretion in a time-dependent fashion; (2) knockdown of PTHrP gene expression or neutralization of secreted PTHrP isoforms blocks TGF-β-induced apoptosis; and (3) TGF-β-induced PTHrP expression is Smad3-dependent. Thus, we have identified PTHrP as a novel mediator for TGF-β-induced apoptosis in Hep3B cells. Our findings provide further insights into the mechanisms through which TGF-β conveys tumor suppression activity. [Copyright &y& Elsevier]