Scribble scrambles parathyroid hormone receptor interactions to regulate phosphate and vitamin D homeostasis.

G protein-coupled receptors, including PTHR, are pivotal for controlling metabolic processes ranging from serum phosphate and vitamin D levels to glucose uptake, and cytoplasmic interactors may modulate their signaling, trafficking, and function. We now show that direct interaction with Scribble, a cell polarity-regulating adaptor protein, modulates PTHR activity. Scribble is a crucial regulator for establishing and developing tissue architecture, and its dysregulation is involved in various disease conditions, including tumor expansion and viral infections. Scribble co-localizes with PTHR at basal and lateral surfaces in polarized cells. Using X-ray crystallography, we show that colocalization is mediated by engaging a short sequence motif at the PTHR C-terminus using Scribble PDZ1 and PDZ3 domain, with binding affinities of 31.7 and 13.4 µM, respectively. Since PTHR controls metabolic functions by actions on renal proximal tubules, we engineered mice to selectively knockout Scribble in proximal tubules. The loss of Scribble impacted serum phosphate and vitamin D levels and caused significant plasma phosphate elevation and increased aggregate vitamin D3 levels, whereas blood glucose levels remained unchanged. Collectively these results identify Scribble as a vital regulator of PTHR-mediated signaling and function. Our findings reveal an unexpected link between renal metabolism and cell polarity signaling.

Actions of Parathyroid Hormone Ligand Analogues in Humanized PTH1R Knockin Mice.

Rodent models are commonly used to evaluate parathyroid hormone (PTH) and PTH-related protein (PTHrP) ligands and analogues for their pharmacologic activities and potential therapeutic utility toward diseases of bone and mineral ion metabolism. Divergence, however, in the amino acid sequences of rodent and human PTH receptors (rat and mouse PTH1Rs are 91% identical to the human PTH1R) can lead to differences in receptor-binding and signaling potencies for such ligands when assessed on rodent vs human PTH1Rs, as shown by cell-based assays in vitro. This introduces an element of uncertainty in the accuracy of rodent models for performing such preclinical evaluations. To overcome this potential uncertainty, we used a homologous recombination-based knockin (KI) approach to generate a mouse (in-host strain C57Bl/6N) in which complementary DNA encoding the human PTH1R replaces a segment (exon 4) of the murine PTH1R gene so that the human and not the mouse PTH1R protein is expressed. Expression is directed by the endogenous mouse promoter and hence occurs in all biologically relevant cells and tissues and at appropriate levels. The resulting homozygous hPTH1R-KI (humanized) mice were healthy over at least 10 generations and showed functional responses to injected PTH analog peptides that are consistent with a fully functional human PTH1R in target bone and kidney cells. The initial evaluation of these mice and their potential utility for predicting behavior of PTH analogues in humans is reported here.

Serum PTH, PTH1R/ATF4 pathway, and the sRANKL/OPG system in bone as a new link between bone growth, cross-sectional geometry, and strength in young rats with experimental chronic kidney disease.

The progression of chronic kidney disease (CKD) in children is associated with deregulated parathyroid hormone (PTH), growth retardation, and low bone accrual. PTH can cause both catabolic and anabolic impact on bone, and the activating transcription factor 4 (ATF4), a downstream target gene of PTH, is related to its anabolic effect. Osteoprotegerin (OPG) and receptor activator of NF-κB ligand (RANKL) are PTH-dependent cytokines, which may play an important role in the regulation of bone remodeling. This study aimed to evaluate the impact of endogenous PTH and the bone RANKL/OPG system on bone growth, cross-sectional geometry and strength utilizing young, nephrectomized rats. The parameters of cross-sectional geometry were significantly elevated in rats with CKD during the three-month experimental period compared with the controls, and they were strongly associated with serum PTH levels and the expression of parathyroid hormone 1 receptor (PTH1R)/ATF4 genes in bone. Low bone soluble RANKL (sRANKL) levels and sRANKL/OPG ratios were also positively correlated with cross-sectional bone geometry and femoral length. Moreover, the analyzed geometric parameters were strongly related to the biomechanical properties of femoral diaphysis. In summary, the mild increase in endogenous PTH, its anabolic PTH1R/ATF4 axis and PTH-dependent alterations in the bone RANKL/OPG system may be one of the possible mechanisms responsible for the favorable impact on bone growth, cross-sectional geometry and strength in young rats with experimental CKD.

A new neuropeptide insect parathyroid hormone iPTH in the red flour beetle Tribolium castaneum.

In the postgenomics era, comparative genomics have advanced the understanding of evolutionary processes of neuropeptidergic signaling systems. The evolutionary origin of many neuropeptidergic signaling systems can be traced date back to early metazoan evolution based on the conserved sequences. Insect parathyroid hormone receptor (iPTHR) was previously described as an ortholog of vertebrate PTHR that has a well-known function in controlling bone remodeling. However, there was no sequence homologous to PTH sequence in insect genomes, leaving the iPTHR as an orphan receptor. Here, we identified the authentic ligand insect PTH (iPTH) for the iPTHR. The taxonomic distribution of iPTHR, which is lacking in Diptera and Lepidoptera, provided a lead for identifying the authentic ligand. We found that a previously described orphan ligand known as PXXXamide (where X is any amino acid) described in the cuttlefish Sepia officinalis has a similar taxonomic distribution pattern as iPTHR. Tests of this peptide, iPTH, in functional reporter assays confirmed the interaction of the ligand-receptor pair. Study of a model beetle, Tribolium castaneum, was used to investigate the function of the iPTH signaling system by RNA interference followed by RNA sequencing and phenotyping. The results suggested that the iPTH system is likely involved in the regulation of cuticle formation that culminates with a phenotype of defects in wing exoskeleton maturation at the time of adult eclosion. Moreover, RNAi of iPTHRs also led to significant reductions in egg numbers and hatching rates after parental RNAi.

Investigational anabolic agents for the treatment of osteoporosis: an update on recent developments.

INTRODUCTION: Teriparatide, a PTH analogue, was the first anabolic agent to be approved for the treatment of osteoporosis in 2002. Abaloparatide was also recently approved by the FDA. The need for other anabolic agents is still unmet. Areas covered: In this review, we discuss target molecules and recent advances in the field of anabolic therapy for osteoporosis. PTH and PTHrP analogues binding to the PTH receptor and different routes of administration of teriparatide to avoid the burden of daily subcutaneous injections are discussed. We also review antibodies targeting suppressors of the Wnt pathway such as sclerostin and Dickopff-1. Expert opinion: The development of alternative ways of administering PTH receptor ligands is a promising field, especially via the transdermal route. Other more promising molecules are still at very early stages of development. FDA recently requested more data on Romosozumab.

Oxidation inhibits PTH receptor signaling and trafficking.

Reactive Oxygen Species (ROS) increase during aging, potentially affecting many tissues including brain, heart, and bone. ROS alter signaling pathways and constitute potential therapeutic targets to limit oxidative damaging effects in aging-associated diseases. Parathyroid hormone receptors (PTHR) are widely expressed and PTH is the only anabolic therapy for osteoporosis. The effects of oxidative stress on PTHR signaling and trafficking have not been elucidated. Here, we used Fluorescence Resonance Energy Transfer (FRET)-based cAMP, ERK, and calcium fluorescent biosensors to analyze the effects of ROS on PTHR signaling and trafficking by live-cell imaging. PTHR internalization and recycling were measured in HEK-293 cells stably transfected with HA-PTHR. PTH increased cAMP production, ERK phosphorylation, and elevated intracellular calcium. Pre-incubation with H2O2 reduced all PTH-dependent signaling pathways. These inhibitory effects were not a result of PTH oxidation since PTH incubated with H2O2 triggered similar responses. PTH promoted internalization and recycling of the PTHR. Both events were significantly reduced by H2O2 pre-incubation. These findings highlight the role of oxidation on PTHR signaling and trafficking, and suggest the relevance of ROS as a putative target in diseases associated with oxidative stress such as age-related osteoporosis.

PTH and Vitamin D.

PTH and Vitamin D are two major regulators of mineral metabolism. They play critical roles in the maintenance of calcium and phosphate homeostasis as well as the development and maintenance of bone health. PTH and Vitamin D form a tightly controlled feedback cycle, PTH being a major stimulator of vitamin D synthesis in the kidney while vitamin D exerts negative feedback on PTH secretion. The major function of PTH and major physiologic regulator is circulating ionized calcium. The effects of PTH on gut, kidney, and bone serve to maintain serum calcium within a tight range. PTH has a reciprocal effect on phosphate metabolism. In contrast, vitamin D has a stimulatory effect on both calcium and phosphate homeostasis, playing a key role in providing adequate mineral for normal bone formation. Both hormones act in concert with the more recently discovered FGF23 and klotho, hormones involved predominantly in phosphate metabolism, which also participate in this closely knit feedback circuit. Of great interest are recent studies demonstrating effects of both PTH and vitamin D on the cardiovascular system. Hyperparathyroidism and vitamin D deficiency have been implicated in a variety of cardiovascular disorders including hypertension, atherosclerosis, vascular calcification, and kidney failure. Both hormones have direct effects on the endothelium, heart, and other vascular structures. How these effects of PTH and vitamin D interface with the regulation of bone formation are the subject of intense investigation.

Multiple hormonal resistances: Diagnosis, evaluation and therapy.

Molecular alterations of cAMP-mediated signaling affect primarily the signaling of the PTH/PTHrp receptor, and, with different severities the signaling of other hormones, including TSH. The identification of PTH and other hormonal resistances implies to look for the genetic disorder supporting the metabolic disorder.

Synergistic effects of high dietary calcium and exogenous parathyroid hormone in promoting osteoblastic bone formation in mice.

In the present study, we investigated whether high dietary Ca and exogenous parathyroid hormone 1-34 fragments (PTH 1-34) have synergistic effects on bone formation in adult mice, and explored the related mechanisms. Adult male mice were fed a normal diet, a high-Ca diet, a PTH-treated diet, or a high-Ca diet combined with subcutaneously injected PTH 1-34 (80 µg/kg per d) for 4 weeks. Bone mineral density, trabecular bone volume, osteoblast number, alkaline phosphatase (ALP)- and type I collagen-positive areas, and the expression levels of osteoblastic bone formation-related genes and proteins were increased significantly in mice fed the high-Ca diet, the PTH-treated diet, and, even more dramatically, the high-Ca diet combined with PTH. Osteoclast number and surface and the ratio of receptor activator for nuclear factor-κB ligand (RANKL):osteoprotegerin (OPG) were decreased in the high-Ca diet treatment group, increased in the PTH treatment group, but not in the combined treatment group. Furthermore, third-passage osteoblasts were treated with high Ca (5 mM), PTH 1-34 (10⁻8 M) or high Ca combined with PTH 1-34. Osteoblast viability and ALP activity were increased in either the high Ca-treated or PTH-treated cultures and, even more dramatically, in the cultures treated with high Ca plus PTH, with consistent up-regulation of the expression levels of osteoblast proliferation and differentiation-related genes and proteins. These results indicate that dietary Ca and PTH play synergistic roles in promoting osteoblastic bone formation by stimulating osteoblast proliferation and differentiation.

International Union of Basic and Clinical Pharmacology. XCIII. The parathyroid hormone receptors--family B G protein-coupled receptors.

The type-1 parathyroid hormone receptor (PTHR1) is a family B G protein-coupled receptor (GPCR) that mediates the actions of two polypeptide ligands; parathyroid hormone (PTH), an endocrine hormone that regulates the levels of calcium and inorganic phosphate in the blood by acting on bone and kidney, and PTH-related protein (PTHrP), a paracrine-factor that regulates cell differentiation and proliferation programs in developing bone and other tissues. The type-2 parathyroid hormone receptor (PTHR2) binds a peptide ligand, called tuberoinfundibular peptide-39 (TIP39), and while the biologic role of the PTHR2/TIP39 system is not as defined as that of the PTHR1, it likely plays a role in the central nervous system as well as in spermatogenesis. Mechanisms of action at these receptors have been explored through a variety of pharmacological and biochemical approaches, and the data obtained support a basic "two-site" mode of ligand binding now thought to be used by each of the family B peptide hormone GPCRs. Recent crystallographic studies on the family B GPCRs are providing new insights that help to further refine the specifics of the overall receptor architecture and modes of ligand docking. One intriguing pharmacological finding for the PTHR1 is that it can form surprisingly stable complexes with certain PTH/PTHrP ligand analogs and thereby mediate markedly prolonged cell signaling responses that persist even when the bulk of the complexes are found in internalized vesicles. The PTHR1 thus appears to be able to activate the Gα(s)/cAMP pathway not only from the plasma membrane but also from the endosomal domain. The cumulative findings could have an impact on efforts to develop new drug therapies for the PTH receptors.

Bone-remodeling transcript levels are independent of perching in end-of-lay white leghorn chickens.

Osteoporosis is a bone disease that commonly results in a 30% incidence of fracture in hens used to produce eggs for human consumption. One of the causes of osteoporosis is the lack of mechanical strain placed on weight-bearing bones. In conventionally-caged hens, there is inadequate space for chickens to exercise and induce mechanical strain on their bones. One approach is to encourage mechanical stress on bones by the addition of perches to conventional cages. Our study focuses on the molecular mechanism of bone remodeling in end-of-lay hens (71 weeks) with access to perches. We examined bone-specific transcripts that are actively involved during development and remodeling. Using real-time quantitative PCR, we examined seven transcripts (COL2A1 (collagen, type II, alpha 1), RANKL (receptor activator of nuclear factor kappa-B ligand), OPG (osteoprotegerin), PTHLH (PTH-like hormone), PTH1R (PTH/PTHLH type-1 receptor), PTH3R (PTH/PTHLH type-3 receptor), and SOX9 (Sry-related high mobility group box)) in phalange, tibia and femur. Our results indicate that the only significant effect was a difference among bones for COL2A1 (femur > phalange). Therefore, we conclude that access to a perch did not alter transcript expression. Furthermore, because hens have been used as a model for human bone metabolism and osteoporosis, the results indicate that bone remodeling due to mechanical loading in chickens may be a product of different pathways than those involved in the mammalian model.

[Rare abnormalities of parathyroid gland function and parathyroid hormone receptor action]. / Rzadkie zaburzenia funkcji przytarczyc i dzialania receptorowego parathormonu.

The parathyroid glands, located near or within the posterior surface of the thyroid gland and secreting parathyroid hormone, are essential organs for the regulation of calcium and phosphate metabolism. As they are necessary to sustain life and maintain homeostasis, undetected or misdiagnosed parathyroid disorders may pose a significant threat to health outcomes, as their presence may increase morbidity and mortality in affected individuals. The clinical picture of some disorders associated with abnormal parathyroid hormone secretion and receptor action is sometimes complicated by coexisting abnormalities, and in these cases establishing the correct diagnosis is challenging. The remarkable progress of recent years in the area of hormonal assessment, imaging procedures and molecular biology, has resulted in a great improvement in the identification, differentiation and treatment of various parathyroid disorders and has made it possible to identify several new clinical entities. In this paper, we discuss the present state-of-art on the etiopathogenesis, clinical manifestations, diagnosis and treatment of chosen rare abnormalities of parathyroid gland function and parathyroid hormone receptor action.

[Establishment of HEK293 cell lines stably expressing human parathyroid hormone receptors].

OBJECTIVE: To establish HEK293 cell lines with stable expression of human parathyroid hormone (PTH) receptors. METHODS: The purified gene fragments of PTH-related peptide receptor (PTHR) and its mutant form (DSEL) were cloned separately into pcDNA3.1(+) vector after digestion with EcoR I and Not I, and the resulted pcDNA3.1(+)-PTHR and pcDNA3.1(+)-DSEL plasmids were verified by restriction enzyme digestion and DNA sequencing. HEK293 cells were transfected with these plasmids and the expression of PTHR and DSEL in the cells were examined by RT-PCR and ELSIA. RESULTS: Sequencing and restriction enzyme digestion analysis showed that PTHR and DSEL cDNAs were correctly cloned into pcDNA3.1(+)vector. After a 48-h transfection of HEK293 cells with the recombinant plasmids and G418 selection, the positive cell clones stably expressing the constructs were obtained, which showed expressions of PTHR and DSEL mRNAs detected by RT-PCR. These positive cells showed high levels of PLC and aAMP production in response to PTH stimulation. CONCLUSION: The HEK293 cell lines with stable expression of PTH1R or DSEL gene established in this study provide useful cell models for studying the physiological functions of PTH peptides.

Kinetics and dynamics in the G protein-coupled receptor signaling cascade.

We describe optical and microscopy methods based on Förster resonance energy transfer, fluorescence recovery after photobleaching, and imaging cross-correlation spectroscopy that permit to determine kinetic and dynamic properties of key reactions involved G protein-coupled receptor (GPCR) signaling from the initial ligand binding step to the generation of the second messenger, cAMP. Well suited to determine rate-limiting reactions taking place along a GPCR signaling cascade in live cells, these techniques have also uncovered new concepts in GPCR signaling as well as many interesting mechanistic subtleties by which GPCRs transmit neurotransmitter and hormone signals into cells.

Effects of PTH on osteocyte function.

Osteocytes are ideally positioned to detect and respond to mechanical and hormonal stimuli and to coordinate the function of osteoblasts and osteoclasts. However, evidence supporting the involvement of osteocytes in specific aspects of skeletal biology has been limited mainly due to the lack of suitable experimental approaches. Few crucial advances in the field in the past several years have markedly increased our understanding of the function of osteocytes. The development of osteocytic cell lines initiated a plethora of in vitro studies that have provided insights into the unique biology of osteocytes and continue to generate novel hypotheses. Genetic approaches using promoter fragments that direct gene expression to osteocytes allowed the generation of mice with gain or loss of function of particular genes revealing their role in osteocyte function. Furthermore, evidence that Sost/sclerostin is expressed primarily in osteocytes and inhibits bone formation by osteoblasts, fueled research attempting to identify regulators of this gene as well as other osteocyte products that impact the function of osteoblasts and osteoclasts. The discovery that parathyroid hormone (PTH), a central regulator of bone homeostasis, inhibits sclerostin expression generated a cascade of studies that revealed that osteocytes are crucial target cells of the actions of PTH. This review highlights these investigations and discusses their significance for advancing our understanding of the mechanisms by which osteocytes regulate bone homeostasis and for developing therapies for bone diseases targeting osteocytes.

Non-canonical signaling of the PTH receptor.

The classical model of arrestin-mediated desensitization of cell-surface G-protein-coupled receptors (GPCRs) is thought to be universal. However, this paradigm is incompatible with recent reports that the parathyroid hormone (PTH) receptor (PTHR), a crucial GPCR for bone and mineral ion metabolism, sustains G(S) activity and continues to generate cAMP for prolonged periods after ligand washout; during these periods the receptor is observed mainly in endosomes, associated with the bound ligand, G(S) and ß-arrestins. In this review we discuss possible molecular mechanisms underlying sustained signaling by the PTHR, including modes of signal generation and attenuation within endosomes, as well as the biological relevance of such non-canonical signaling.

Acute and chronic regulation of circulating PTH: significance in health and in disease.

Circulating human parathyroid hormone (PTH) is immunoheterogenous. It is composed of 80% carboxyl-terminal (C) fragments and of 20% PTH(1-84). This composition contrasts with the biological activity of the hormone, which is only related to PTH(1-84), creating a paradox between circulating PTH composition and PTH bioactivity. PTH molecular forms are either secreted by the parathyroid glands or generated by the peripheral metabolism of PTH(1-84) in the liver. The kidney has a major role in the disposal of C-PTH fragments. Secretion of PTH molecular forms by the parathyroid glands is highly regulated under a variety of clinical conditions, suggesting that C-PTH fragments could exert some biological effects of their own. Recent data suggest that C-PTH fragments can exert biological actions opposite to those of PTH(1-84) by acting on a C-PTH receptor not yet cloned. They can decrease calcium concentration, phosphate excretion, bone resorption and 1,25(OH)2 synthesis. The clinical implications of this new concept are reviewed.

Phenotypical and functional characteristics of mesenchymal stem cells from bone marrow: comparison of culture using different media supplemented with human platelet lysate or fetal bovine serum.

INTRODUCTION: Mesenchymal stem cells (MSCs) are multipotent cells able to differentiate into several mesenchymal lineages, classically derived from bone marrow (BM) but potentially from umbilical cord blood (UCB). Although they are becoming a good tool for regenerative medicine, they usually need to be expanded in fetal bovine serum (FBS)-supplemented media. Human platelet lysate (HPL) has recently been proposed as substitute for safety reasons, but it is not yet clear how this supplement influences the properties of expanded MSCs. METHODS: In the present study, we compared the effect of various media combining autologous HPL with or without FBS on phenotypic, proliferative and functional (differentiation, cytokine secretion profile) characteristics of human BM-derived MSCs. RESULTS: Despite less expression of adipogenic and osteogenic markers, MSCs cultured in HPL-supplemented media fully differentiated along osteoblastic, adipogenic, chondrogenic and vascular smooth muscle lineages. The analyses of particular specific proteins expressed during osteogenic differentiation (calcium-sensing receptor (CaSR) and parathormone receptor (PTHR)) showed their decrease at D0 before any induction for MSC cultured with HPL mostly at high percentage (10%HPL). The cytokine dosage showed a clear increase of proliferation capacity and interleukin (IL)-6 and IL-8 secretion. CONCLUSIONS: This study shows that MSCs can be expanded in media supplemented with HPL that can totally replace FBS. HPL-supplemented media not only preserves their phenotype as well as their differentiation capacity, but also shortens culture time by increasing their growth rate.

Parathyroid hormone and its effects on dental tissues.

In the current era, various pharmacological agents exist for osteoporosis management, and synthetic parathyroid hormone (PTH) (Teriparatide, Forteo) is one of the treatment options. Depending on the timing of administration, PTH has a unique ability to cause both bone apposition and bone resorption. This review focuses on the effects of PTH on the bone, specifically the jaw bones mandible and maxilla. The article briefly describes the fundamental mechanism of PTH action at the molecular level, as well as in experimental animals and in humans. It differentiates intermittent administration of PTH, especially at doses tolerated by humans that increase bone strength and prevent bone fractures, from continuous use that may lead to bone loss. In particular, it shows how intermittent administration of PTH can play a significant role in periodontal repair and implant success via stimulation of bone mineral content especially in the pre-alveolar region.