PTH1R-CaSR Cross Talk: New Treatment Options for Breast Cancer Osteolytic Bone Metastases.

Metastatic breast cancer (BrCa) is currently incurable despite great improvements in treatment of primary BrCa. The incidence of skeletal metastases in advanced BrCa occurs up to 70%. Recent findings have established that the distribution of BrCa metastases to the skeleton is not a random process but due to the favorable microenvironment for tumor invasion and growth. The complex interplay among BrCa cells, stromal/osteoblastic cells, and osteoclasts in the osseous microenvironment creates a bone-tumor vicious cycle (a feed-forward loop) that results in excessive bone destruction and progressive tumor growth. Both the type 1 PTH receptor (PTH1R) and extracellular calcium-sensing receptor (CaSR) participate in the vicious cycle and influence the skeletal metastatic niche. Thus, this review focuses on how the PTH1R and CaSR signaling pathways interact and contribute to the pathogenesis of BrCa bone metastases. The effects of intermittent PTH and allosteric modulators of CaSR for the use of bone-anabolic agents and prevention of BrCa bone metastases constitute a proof of principle for therapeutic consideration. Understanding the interplay between PTH1R and CaSR signaling in the development of BrCa bone metastases could lead to a novel therapeutic approach to control both osteolysis and tumor burden in the bone

Global Gene Expression Analysis Identifies Age-Related Differences in Knee Joint Transcriptome during the Development of Post-Traumatic Osteoarthritis in Mice.

Aging and injury are two major risk factors for osteoarthritis (OA). Yet, very little is known about how aging and injury interact and contribute to OA pathogenesis. In the present study, we examined age- and injury-related molecular changes in mouse knee joints that could contribute to OA. Using RNA-seq, first we profiled the knee joint transcriptome of 10-week-old, 62-week-old, and 95-week-old mice and found that the expression of several inflammatory-response related genes increased as a result of aging, whereas the expression of several genes involved in cartilage metabolism decreased with age. To determine how aging impacts post-traumatic arthritis (PTOA) development, the right knee joints of 10-week-old and 62-week-old mice were injured using a non-invasive tibial compression injury model and injury-induced structural and molecular changes were assessed. At six-week post-injury, 62-week-old mice displayed significantly more cartilage degeneration and osteophyte formation compared with young mice. Although both age groups elicited similar transcriptional responses to injury, 62-week-old mice had higher activation of inflammatory cytokines than 10-week-old mice, whereas cartilage/bone metabolism genes had higher expression in 10-week-old mice, suggesting that the differential expression of these genes might contribute to the differences in PTOA severity observed between these age groups

Pseudohypoparathyroidism type I-b with neurological involvement is associated with a homozygous PTH1R mutation

Pseudohypoparathyroidism type 1b (PHP1b) is characterized by hypocalcemia, hyperphosphatemia, increased levels of circulating parathyroid hormone (PTH), and no skeletal or developmental abnormalities. The goal of this study was to perform a full characterization of a familial case of PHP1b with neurological involvement and to identify the genetic cause of disease. The initial laboratory profile of the proband showed severe hypocalcemia, hyperphosphatemia and normal levels of PTH, which was considered to be compatible with primary hypoparathyroidism. With disease progression the patient developed cognitive disturbance, PTH levels were found to be slightly elevated and a picture of PTH resistance syndrome seemed more probable. The diagnosis of PHP1b was established after the study of family members and blunted urinary cAMP results were obtained in a PTH stimulation test. Integration of whole genome genotyping and exome sequencing data supported this diagnosis by revealing a novel homozygous missense mutation in PTH1R (p.Arg186His) completely segregating with the disease. Here, we demonstrate segregation of a novel mutation in PTH1R with a phenotype of PHP1b presenting with neurological symptoms, but no bone defects. This case represents the extreme end of the spectrum of cognitive impairment in PTH dysfunction and defines a possible novel form of PHP1b resulting from the impaired interaction between PTH and PTH1R

Functional characterization and evolution ofPTH/PTHrP receptors: insights from the chicken

The parathyroid hormone (PTH)-family consists of a group of structurally related factors that regulate calcium and bone homeostasis and are also involved in development of organs such as the heart, mammary gland and immune system. They interact with specific members of family 2 B1 G-protein coupled receptors (GPCRs), which have been characterised in teleosts and mammals. Two PTH/PTHrP receptors, PTH1R and PTH2R exist in mammals and in teleost fish a further receptor PTH3R has also been identified. Recently in chicken, PTH-family members involved in calcium transport were characterized and specific PTHRs are suggested to exist although they have not yet been isolated or functionally characterized. The aim of this study is to further explore the evolution and function of the vertebrate PTH/PTHrP system through the isolation, phylogenetic analysis and functional characterization of the chicken receptors. Results Two PTHRs were isolated in chicken and sequence comparison and phylogenetic analysis indicate that the chicken receptors correspond to PTH1R and PTH3R, which emerged prior to the teleost/tetrapod divergence since they are present in cartilaginous fish. The vertebrate PTH2R receptor and its ligand TIP39 have been lost from bird genomes. Chicken PTH1R and PTH3R have a divergent and widespread tissue expression and are also evident in very early embryonic stages of development. Receptor stimulation studies using HEK293 cells stably expressing the chicken PTH1R and PTH3R and monitoring cAMP production revealed they are activated by chicken 1–34 N-terminal PTH-family peptides in a dose dependent manner. PTH-L and PTHrP were the most effective peptides in activating PTH1R (EC50 = 7.7 nM and EC50 = 22.7 nM, respectively). In contrast, PTH-L (100 nM) produced a small cAMP accumulation on activation of PTH3R but PTHrP and PTH (EC50 = 2.5 nM and EC50 = 22.1 nM, respectively) readily activated the receptor. PTHrP also stimulated intracellular Ca2+ accumulation on activation of PTH1R but not PTH3R. Conclusion Two PTHR homologues of the vertebrate PTH1R and PTH3R were isolated and functionally characterized in chicken. Their distinct pattern of expression during embryo development and in adult tissues, together with their ligand preference, suggests that they have acquired specific functions, which have contributed to their maintenance in the genome. PTH2R and its activating ligand, TIP39, are absent from bird genomes. Nonetheless identification of putative PTH2R and TIP39 in the genome of an ancient agnathan, lamprey, suggests the PTH/PTHrP ligand and receptor family was already present in an early basal paraphyletic group of vertebrates and during the vertebrate radiation diverged via gene/genome duplication and deletion events. Knowledge of the role PTH/PTHrP system in early vertebrates will help to establish evolution of function.Peer Reviewe

Evolution of Parathyroid Hormone Receptor Family and Their Ligands in Vertebrate

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A transgenic mouse model for studying the role of the parathyroid hormone-related protein system in renal injury

Parathyroid hormone- (PTH-) related protein (PTHrP) and its receptor, the PTH1 receptor (PTH1R), are widely expressed in the kidney, where PTHrP exerts a modulatory action on renal function. PTHrP is known to be upregulated in several experimental nephropathies such as acute renal failure (ARF), obstructive nephropathy (ON) as well as diabetic nephropathy (DN). In this paper, we will discuss the functional consequences of chronic PTHrP overexpression in the damaged kidney using a transgenic mouse strain overexpressing PTHrP in the renal proximal tubule. In both ARF and ON, PTHrP displays proinflammatory and profibrogenic actions including the induction of epithelia to mesenquima transition. Moreover, PTHrP participates in the mechanisms of renal hypertrophy as well as proteinuria in experimental DN. Angiotensin II (Ang II), a critical factor in the progression of renal injury, appears to be, at least in part, responsible for endogenous PTHrP upregulation in these pathophysiological settings. These findings provide novel insights into the well-known protective effects of Ang II antagonists in renal diseases, paving the way for new therapeutic approaches.The studies included in this paper were supported in part by Grants from Ministerio de Educación y Cultura of Spain (SAF2002-04356-C03-01, -02, and -03),Ministerio de Ciencia e Innovación (SAF2009- 12009-C02-01), Comunidad Autónoma de Madrid (CAM 08.6/0038.1/2000-2 and GR/SAL/0415/2004), Instituto de Salud Carlos III (RETICEF RD06/0013/1002), Spanish Society of Nephrology, Eugenio Rodríguez Pascual Foundation, and Conchita Rábago de Jiménez Díaz Foundation

Novel role of parathyroid hormone-related protein in the pathophysiology of the diabetic kidney: Evidence from experimental and human diabetic nephropathy

Parathyroid hormone-related protein (PTHrP) and its receptor type 1 (PTH1R) are extensively expressed in the kidney, where they are able to modulate renal function. Renal PTHrP is known to be overexpressed in acute renal injury. Recently, we hypothesized that PTHrP involvement in the mechanisms of renal injury might not be limited to conditions with predominant damage of the renal tubulointerstitium and might be extended to glomerular diseases, such as diabetic nephropathy (DN). In experimental DN, the overexpression of both PTHrP and the PTH1R contributes to the development of renal hypertrophy as well as proteinuria. More recent data have shown, for the first time, that PTHrP is upregulated in the kidney from patients with DN. Collectively, animal and human studies have shown that PTHrP acts as an important mediator of diabetic renal cell hypertrophy by a mechanism which involves the modulation of cell cycle regulatory proteins and TGF-β1. Furthermore, angiotensin II (Ang II), a critical factor in the progression of renal injury, appears to be responsible for PTHrP upregulation in these conditions. These findings provide novel insights into the well-known protective effects of Ang II antagonists in renal diseases, paving the way for new therapeutic approaches.N. Olea is the recipient of a Research Contract from the Comunidad Autónoma de Madrid (S-BIO-2083-2006) and the University of Alcalá. A. Izquierdo is currently Assistant Professor at the Rey Juan Carlos University, Alcorc´on, Madrid. This work was supported in part by Grants from Ministerio de Educaci´on y Cultura of Spain (SAF2002- 04356-C03-01, -02, and -03) Ministerio de Ciencia e Innovación (SAF2009-12009-C02-01), Instituto de Salud Carlos III (RETICEF RD06/0013/1002 and RD12/0043/0008 and PI12/02825), the Spanish Society of Nephrology, and the Eugenio Rodr´ıguez Pascual Foundation

PTHrP-Derived Peptides Restore Bone Mass and Strength in Diabetic Mice: Additive Effect of Mechanical Loading

There is an unmet need to understand the mechanisms underlying skeletal deterioration in diabetes mellitus (DM) and to develop therapeutic approaches to treat bone fragility in diabetic patients. We demonstrate herein that mice with type 1 DM induced by streptozotocin exhibited low bone mass, inferior mechanical and material properties, increased bone resorption, decreased bone formation, increased apoptosis of osteocytes, and increased expression of the osteocyte-derived bone formation inhibitor Sost/sclerostin. Further, short treatment of diabetic mice with parathyroid hormone related protein (PTHrP)-derived peptides corrected these changes to levels undistinguishable from non-diabetic mice. In addition, diabetic mice exhibited reduced bone formation in response to mechanical stimulation, which was corrected by treatment with the PTHrP peptides, and higher prevalence of apoptotic osteocytes, which was reduced by loading or by the PTHrP peptides alone and reversed by a combination of loading and PTHrP peptide treatment. In vitro experiments demonstrated that the PTHrP peptides or mechanical stimulation by fluid flow activated the survival kinases ERKs and induced nuclear translocation of the canonical Wnt signaling mediator β-catenin, and prevented the increase in osteocytic cell apoptosis induced by high glucose. Thus, PTHrP-derived peptides cross-talk with mechanical signaling pathways to reverse skeletal deterioration induced by DM in mice. These findings suggest a crucial role of osteocytes in the harmful effects of diabetes on bone and raise the possibility of targeting these cells as a novel approach to treat skeletal deterioration in diabetes. Moreover, our study suggests the potential therapeutic efficacy of combined pharmacological and mechanical stimuli to promote bone accrual and maintenance in diabetic subjects

Amphiregulin-EGFR Signaling Mediates the Migration of Bone Marrow Mesenchymal Progenitors toward PTH-Stimulated Osteoblasts and Osteocytes

Intermittent administration of parathyroid hormone (PTH) dramatically increases bone mass and currently is one of the most effective treatments for osteoporosis. However, the detailed mechanisms are still largely unknown. Here we demonstrate that conditioned media from PTH-treated osteoblastic and osteocytic cells contain soluble chemotactic factors for bone marrow mesenchymal progenitors, which express a low amount of PTH receptor (PTH1R) and do not respond to PTH stimulation by increasing cAMP production or migrating toward PTH alone. Conditioned media from PTH-treated osteoblasts elevated phosphorylated Akt and p38MAPK amounts in mesenchymal progenitors and inhibition of these pathways blocked the migration of these progenitors toward conditioned media. Our previous and current studies revealed that PTH stimulates the expression of amphiregulin, an epidermal growth factor (EGF)-like ligand that signals through the EGF receptor (EGFR), in both osteoblasts and osteocytes. Interestingly, conditioned media from PTH-treated osteoblasts increased EGFR phosphorylation in mesenchymal progenitors. Using several different approaches, including inhibitor, neutralizing antibody, and siRNA, we demonstrate that PTH increases the release of amphiregulin from osteoblastic cells, which acts on the EGFRs expressed on mesenchymal progenitors to stimulate the Akt and p38MAPK pathways and subsequently promote their migration in vitro. Furthermore, inactivation of EGFR signaling specifically in osteoprogenitors/osteoblasts attenuated the anabolic actions of PTH on bone formation. Taken together, these results suggest a novel mechanism for the therapeutic effect of PTH on osteoporosis and an important role of EGFR signaling in mediating PTH's anabolic actions on bone

Intranuclear Signaling Cascades Triggered by Nuclear GPCRs

G protein-couped receptors (GPCRs) play a key role on cellular membranes, where they respond to a broad array of extracellular signals such as lipids, peptides, proteins and sensory agents. Intracellular biological responses triggered by these receptors include hormone secretion, muscle contraction, cellular metabolism a tyrosine kinase receptors transactivation. Recent results indicate that GPCRs localize to and signal also at nuclear level, thus regulating distinct signaling pathways which can also result from the integration of extracellular and intracellular stimuli. Nuclear GPCRs play a central role in many cellular processes, including regulation of gene transcription, cellular proliferation, neovascularization and RNA synthesis. On nuclear membranes and in nucleoplasm are present all the downstream signal transduction components of GPCRs, including G proteins, adenylyl cyclase, and second messengers such as Ca++, ERKs, p38MAPK and other protein kinases. Nuclear GPCRs may be constitutively active or may be activated by ligands internalized from the extracellular space or synthesized within the cell. The translocation of membrane receptors to the nucleus could be attributed to the presence of a Nuclear Localization Signal, which is present in the eighth helix or in the third intracellular loop of a limited number of GPCRs. However, several sequence motifs that do not resemble classical Nuclear Localization Signals can promote import of GPCRs. In this review we discuss the most recent results on nuclear localization and signaling of several GPCRS