A naturally occurring isoform inhibits parathyroid hormone receptor trafficking and signaling

Parathyroid hormone (PTH) regulates calcium homeostasis and bone remodeling through its cognitive receptor (PTHR). We describe here a PTHR isoform harboring an in‐frame 42‐bp deletion of exon 14 (Δe14‐PTHR) that encodes transmembrane domain 7. Δe14‐PTHR was detected in human kidney and buccal epithelial cells. We characterized its topology, cellular localization, and signaling, as well as its interactions with PTHR. The C‐terminus of the Δe14‐PTHR is extracellular, and cell surface expression is strikingly reduced compared with the PTHR. Δe14‐PTHR displayed impaired trafficking and accumulated in endoplasmic reticulum. Signaling and activation of cAMP and ERK by Δe14‐PTHR was decreased significantly compared with PTHR. Δe14‐PTHR acts as a functional dominant‐negative by suppressing the action of PTHR. Cells cotransfected with both receptors exhibit markedly reduced PTHR cell membrane expression, colocalization with Δe14‐PTHR in endoplasmic reticulum, and diminished cAMP activation and ERK phosphorylation in response to challenge with PTH. Δe14‐PTHR forms heterodimers with PTHR, which may account for cytoplasmic retention of PTHR in the presence of Δe14‐PTHR. Analysis of the PTHR heteronuclear RNA suggests that base‐pair complementarity in introns surrounding exon 14 causes exon skipping and accounts for generation of the Δe14‐PTHR isoform. Thus Δe14‐PTHR is a poorly functional receptor that acts as a dominant‐negative of PTHR trafficking and signaling and may contribute to PTH resistance. © 2011 American Society for Bone and Mineral Research.     

Predominant role of PDGF receptor transactivation in Wnt3a‐induced osteoblastic cell proliferation

Previous studies have shown that Wnt3a enhances the proliferation and inhibits the osteogenic differentiation of human mesenchymal stem cells (hMSCs). In this study, we investigated the signaling pathways involved in Wnt3a‐induced osteoblastic cell proliferation. Experiments with DKK1, a natural antagonist of Lrp5/6, indicated that Wnt/β‐catenin did not play a major role in Wnt3a‐induced osteoblastic cell proliferation. The use of selective inhibitors of known mitogenic pathways implicates Src family kinases (SFKs) and a protein kinase C (PKC) in this cellular response. Time‐dependent analysis of signaling molecules activated by Wnt3a in MC3T3‐E1 cells revealed parallel activation of the canonical pathway and of several tyrosine kinases, including SFKs and PDGF receptors (PDGF‐Rs). Functional analysis with specific inhibitors suggested a major role of PDGF‐Rs in mediating Wnt3a‐induced cell proliferation. Further investigation with an si‐RNA approach confirmed a predominant role of this receptor in this cellular response. The use of soluble decoy PDGF‐Rs that can sequester extracellular PDGFs excluding that part of the increased PDGF receptor phosphorylation by Wnt3a was the result of autocrine production of PDGFs. A selective SFK inhibitor blunted the enhanced PDGF‐R phosphorylation and cell proliferation induced by Wnt3a. Studies of initial events involved in the regulation of this pathway suggest a role of dishevelled. In conclusion, data presented in this study indicate that cell proliferation induced by Wnt3a in osteoblastic cells is mediated by a dishevelled‐dependent and β‐catenin‐independent pathway, which involves the transactivation of PDGF receptors. © 2013 American Society for Bone and Mineral Research     

Overexpression of bradykinin type 2 receptors on glioma cells enhances bradykinin-mediated blood–brain tumor barrier permeability increase

Abstract

Variations in the expression levels of bradykinin (BK) type 2 receptors (B2R) in different brain tumors may explain variable increases in BK-mediated blood-brain tumor barrier (BTB) permeability. This study investigated whether elevation of the B2R expression levels on glioma cells enhances BK-mediated BTB permeability increases. Stable transfectants of C6 rat glioma cells overexpressing B2R were established by transfection with recombinant vectors harboring rat B2R cDNA sequence. Elevated B2R expression levels in transfectants were confirmed by quantitative real-time PCR, Western blots, and [³H]-BK binding studies. BTB permeability was quantified with autoradiography and expressed as a unidirectional transport constant, Ki, for [¹⁴C]-α-aminoisobutyric acid (AIB: Mr 103), using a rat brain tumor model. Baseline Ki values in tumors overexpressing B2R were not significantly higher than in control tumors. Ki values after BK treatment in tumors overexpressing B2R, however, were significantly higher than in control tumors. Western blots confirmed that B2R expression levels in vivo in tumors overexpressing B2R remained higher than in control tumors. These results suggested that alteration of B2R expression levels on tumor cells could modulate BK-mediated BTB permeability. Therefore, B2R expression levels in human glioma could be used to analyze the treatment results of patients undergoing treatment involving BK-modulated BTB permeability.     

Prostaglandin EP receptor subtypes and gastric cytoprotection

This article reviews recent studies dealing with the relationship between the cytoprotective action of PGE₂ and the EP receptor subtypes in the gastric mucosa. Gastric cytoprotection afforded by PGE₂ was mimicked by EP1 agonists and attenuated by the EP1 antagonist. Likewise, the adaptive cytoprotection induced by a mild irritant was attenuated by the EP1 antagonist and indomethacin. By contrast, capsaicin-induced protection was mitigated by indomethacin as well as sensory deafferentation but not by the EP1 antagonist. PGE₂ failed to provide both direct and adaptive cytoprotection in EP1-receptor knockout mice, while capsaicin-induced protection was observed in the animals lacking either EP1 or EP3 receptors but disappeared in IP receptor knockout mice. We conclude that PGs, either generated endogenously or administered exogenously, exhibit gastric cytoprotection directly through activation of EP1 receptors, and endogenous PGs also contribute to the mucosal protection induced by capsaicin by sensitizing sensory neurons, probably through IP receptors.     

IGF‐I Signaling in Osterix‐Expressing Cells Regulates Secondary Ossification Center Formation,Growth Plate Maturation,and Metaphyseal Formation During Postnatal Bone Development

To investigate the role of IGF‐I signaling in osterix (OSX)‐expressing cells in the skeleton, we generated IGF‐I receptor (IGF‐IR) knockout mice (^OSXIGF‐IRKO) (floxed‐IGF‐IR mice × OSX promoter‐driven GFP‐labeled cre‐recombinase [^OSXGFPcre]), and monitored postnatal bone development. At day 2 after birth (P2), ^OSXGFP‐cre was highly expressed in the osteoblasts in the bone surface of the metaphysis and in the prehypertrophic chondrocytes (PHCs) and inner layer of perichondral cells (IPCs). From P7, ^OSXGFP‐cre was highly expressed in PHCs, IPCs, cartilage canals (CCs), and osteoblasts (OBs) in the epiphyseal secondary ossification center (SOC), but was only slightly expressed in the OBs in the metaphysis. Compared with the control mice, the IPC proliferation was decreased in the ^OSXIGF‐IRKOs. In these mice, fewer IPCs invaded into the cartilage, resulting in delayed formation of the CC and SOC. Immunohistochemistry indicated a reduction of vessel number and lower expression of VEGF and ephrin B2 in the IPCs and SOC of ^OSXIGF‐IRKOs. Quantitative real‐time PCR revealed that the mRNA levels of the matrix degradation markers, MMP‐9, 13 and 14, were decreased in the ^OSXIGF‐IRKOs compared with the controls. The ^OSXIGF‐IRKO also showed irregular morphology of the growth plate and less trabecular bone in the tibia and femur from P7 to 7 weeks, accompanied by decreased chondrocyte proliferation, altered chondrocyte differentiation, and decreased osteoblast differentiation. Our data indicate that during postnatal bone development, IGF‐I signaling in OSX‐expressing IPCs promotes IPC proliferation and cartilage matrix degradation and increases ephrin B2 production to stimulate vascular endothelial growth factor (VEGF) expression and vascularization. These processes are required for normal CC formation in the establishment of the SOC. Moreover, IGF‐I signaling in the OSX‐expressing PHC is required for growth plate maturation and osteoblast differentiation in the development of the metaphysis. © 2015 American Society for Bone and Mineral Research.     

The P2Y13 receptor regulates extracellular ATP metabolism and the osteogenic response to mechanical loading

ATP release and subsequent activation of purinergic receptors has been suggested to be one of the key transduction pathways activated by mechanical stimulation of bone. The P2Y₁₃ receptor, recently found to be expressed by osteoblasts, has been suggested to provide a negative feedback pathway for ATP release in different cell types. Therefore, we hypothesized that the P2Y₁₃ receptor may contribute to the mediation of osteogenic responses to mechanical stimulation by regulating ATP metabolism by osteoblasts. To test this hypothesis, wild‐type (WT) and P2Y₁₃ receptor knockout (P2Y₁₃R^?/?) mice were subject to non‐invasive axial mechanical loading of the left tibiae to induce an osteogenic response. Micro‐computed tomography analysis showed mechanical loading induced an osteogenic response in both strains of mice in terms of increased total bone volume and cortical bone volume, with the P2Y₁₃R^?/? mice having a significantly greater response. The extent of the increased osteogenic response was defined by dynamic histomorphometry data showing dramatically increased bone formation and mineral apposition rates in P2Y₁₃R^?/? mice compared with controls. In vitro, primary P2Y₁₃R^?/? osteoblasts had an accumulation of mechanically induced extracellular ATP and reduced levels of hydrolysis. In addition, P2Y₁₃R^?/? osteoblasts also had a reduction in their maximal alkaline phosphatase (ALP) activity, one of the main ecto‐enzymes expressed by osteoblasts, which hydrolyzes extracellular ATP. In conclusion, deletion of the P2Y₁₃ receptor leads to an enhanced osteogenic response to mechanical loading in vivo, possibly because of the reduced extracellular ATP degradation by ALP. The augmented osteogenic response to mechanical stimulation, combined with suppressed bone remodeling activities and protection from OVX‐induced bone loss after P2Y₁₃ receptor depletion as previously described, suggests a potential role for P2Y₁₃ receptor antagonist‐based therapy, possibly in combination with mechanical loading, for the treatment of osteoporosis.     

Calcitonin: Physiology or fantasy?

Calcitonin, a potent hypocalcemic hormone produced by the C‐cells of the thyroid, was first discovered by Harold Copp in 1962. The physiological significance of calcitonin has been questioned, but recent studies using genetically modified mouse models have uncovered additional actions of calcitonin acting through its receptor (CTR) that are of particular significance to the regulation of bone and calcium homeostasis. Mice in which the CTR is deleted in osteoclasts are more susceptible to induced hypercalcemia and exogenous calcitonin is able to lower serum calcium in younger animals. These data are consistent with the hypothesis that calcitonin can regulate serum calcium by inhibiting the efflux of calcium from bone, and that this action is most important when bone turnover is high. Calcitonin has also been implicated in protecting the skeleton from excessive loss of bone mineral during times of high calcium demand, such as lactation. This action may be linked to an intriguing and as yet unexplained observation that calcitonin inhibits bone formation, because deletion of the CTR leads to increased bone formation. We propose several mechanisms by which calcitonin could protect the skeleton by regulating bone turnover, acting within the bone and/or centrally. A new more holistic notion of the physiological role of calcitonin in bone and calcium homeostasis is required and we have highlighted some important knowledge gaps so that future calcitonin research will help to achieve such an understanding. © 2013 American Society for Bone and Mineral Research.     

Diet‐Derived Phenolic Acids Regulate Osteoblast and Adipocyte Lineage Commitment and Differentiation in Young Mice

A blueberry (BB)‐supplemented diet has been previously shown to significantly stimulate bone formation in rapidly growing male and female rodents. Phenolic acids (PAs) are metabolites derived from polyphenols found in fruits and vegetables as a result of the actions of gut bacteria, and they were found in the serum of rats fed BB‐containing diet. We conducted in vitro studies with PAs and demonstrated stimulation of osteoblast differentiation and proliferation. On the other hand, adipogenesis was inhibited. To more fully understand the mechanistic actions of PAs on bone formation, we administered hippuric acid, one of the major metabolites found in animal circulation after BB consumption, to prepubertal female mice for 2 weeks. We found that hippuric acid was able to stimulate bone‐forming gene expression but suppress PPARγ expression, leading to increased bone mass dose‐dependently. Cellular signaling studies further suggested that the skeletal effects of PAs appeared to be mediated through activation of G‐protein‐coupled receptor 109A and downstream p38 MAP kinase and osterix. In conclusion, PAs are capable of altering the mesenchymal stem cell differentiation program and merit investigation as potential dietary therapeutic alternatives to drugs for degenerative bone disorders. © 2014 American Society for Bone and Mineral Research.