Tctex-1 augments G protein-coupled receptor-mediated Gs signaling by activating adenylyl cyclase.

Proteins interacting with G protein-coupled receptors (GPCRs) can modulate signal transduction of these receptors. However, the regulatory mechanisms of the interacting proteins are diverse and largely unknown. We have previously shown that Tctex-1 (or DYNLT1) can interact with the parathyroid hormone receptor (PTHR). In the present study, we investigated the role of Tctex-1 in the PTHR signaling and found that Tctex-1 augmented the PTHR-mediated Gs/adenylyl cyclase (AC) pathway by activating AC regardless of the binding to PTHR. Furthermore, Tctex-1 directly bound to AC type 6. These data demonstrate a novel mechanism underlying GPCR/Gs signaling regulated by Tctex-1.

Brief exposure to full length parathyroid hormone-related protein (PTHrP) causes persistent generation of cyclic AMP through an endocytosis-dependent mechanism.

Parathyroid hormone (PTH)-related protein (PTHrP) (gene name Pthlh) was discovered as the factor responsible for the humoral hypercalcemia of malignancy. It shares such sequence similarity with PTH in the amino-terminal region that the two are equally able to act through a single G protein-coupled receptor, PTH1R. A number of biological activities are ascribed to domains of PTHrP beyond the amino-terminal domain. PTH functions as a circulating hormone, but PTHrP is generated locally in many tissues including bone, where it acts as a paracrine factor on osteoblasts and osteocytes. The present study compares how PTH and PTHrP influence cyclic AMP (cAMP) formation through adenylyl cyclase, the first event in cell activation through PTH1R. Brief exposure to full length PTHrP(1-141) in several osteoblastic cell culture systems was followed by sustained adenylyl cyclase activity for more than an hour after ligand washout. This effect was dose-dependent and was not found with shorter PTHrP or PTH peptides even though they were fully able to activate adenylyl cyclase with acute treatment. The persistent activation response to PTHrP(1-141) was seen also with later events in the cAMP/PKA pathway, including persistent activation of CRE-luciferase and sustained regulation of several CREB-responsive mRNAs, up to 24 h after the initial exposure. Pharmacologic blockade of endocytosis prevented the persistent activation of cAMP and gene responses. We conclude that full length PTHrP, the likely local physiological effector in bone, differs in intracellular action to PTH by undergoing endosomal translocation to induce a prolonged adenylyl cyclase activation in its target cells.

PTHR1 May Be Involved in Progression of Osteosarcoma by Regulating miR-124-3p-AR-Tgfb1i1, miR-27a-3p-PPARG-Abca1, and miR-103/590-3p-AXIN2 Axes.

Our previous study has indicated that the parathyroid hormone type 1 receptor (PTHR1) may play important roles in development and progression of osteosarcoma (OS) by regulating Wnt, angiogenesis, and inflammation pathway genes. The goal of this study was to further illuminate the roles of PTHR1 in OS by investigating upstream regulation mechanisms (including microRNA [miRNA] and transcription factors [TFs]) of crucial genes. The microarray dataset GSE46861 was downloaded from the Gene Expression Omnibus database, in which six tumors with short hairpin RNA (shRNA) PTHR1 knockdown (PTHR1.358) and six tumors with shRNA control knockdown (Ren.1309) were collected from mice. Differentially expressed genes (DEGs) between PTHR1.358 and Ren.1309 were identified using the linear models for microarray data (LIMMA) method, and then the miRNA-TF-mRNA regulatory network was constructed using data from corresponding databases, followed by module analysis, to screen crucial regulatory relationships. OS-related human miRNAs were extracted from the curated Osteosarcoma Database. Gene ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were enriched using the Database for Annotation, Visualization, and Integrated Discovery (DAVID) tool. As a result, the miRNA-TF-mRNA regulatory network, including 1049 nodes (516 miRNA, 25 TFs, and 508 DEGs) and 15942 edges (interaction relationships, such as Pparg-Abca1 and miR-590-3p-AXIN2), was constructed, from which three significant modules were extracted and modules 2 and 3 contained interactions between miRNAs/TFs and DEGs such as miR-103-3p-AXIN2, miR-124-3p-AR-Tgfb1i1, and miR-27a-3p-PPARG-Abca1. miR-27a-3p was a known miRNA associated with OS. Abca1, AR, and miR-124-3p were hub genes in the miRNA-TF-mRNA network. Tgfb1i1 was involved in cell proliferation, Abca1 participated in the cholesterol metabolic process, and AXIN2 was associated with the canonical Wnt signaling pathway. Furthermore, we also confirmed upregulation of miR-590-3p and downregulation of AXIN2 in the mouse OS cell line K7M2-WT transfected with PTHR1 shRNA. In conclusion, PTHR1 may play important roles in progression of OS by activating miR-124-3p-AR-Tgfb1i1, miR-27a-3p-PPARG-Abca1, and miR-103/590-3p-AXIN2 axes.

Effect of parathyroid hormone on early chondrogenic differentiation from mesenchymal stem cells.

BACKGROUND: Treatment of articular cartilage injuries remains a difficult challenge due to the limited capacity for intrinsic repair. Mesenchymal stem cells (MSCs) can differentiate into chondrocytes under certain culture conditions. This study focused on the modulatory effects of parathyroid hormone (PTH) on chondrogenic differentiation from MSCs. METHODS: MSCs were treated with various concentrations of PTH under chondrogenic pellet culture condition. RNA was isolated for real-time polymerase chain reaction (PCR) and gene expressions of collagen type II α1 chain (Col2a1), collagen type X α1 chain, collagen type I α1 chain, SRY-box9 (Sox9), and type 1 PTH/PTHrP receptor (PTH1R) were examined. Chondrogenic differentiation was also evaluated by histological findings. RESULTS: PTH had opposite effects on chondrogenesis, depending on the concentration. A low to moderate concentration of PTH promoted chondrogenic differentiation of MSCs with increased expression of Sox9, Col2a1, and PTH1R, whereas chondrogenesis of MSCs was inhibited rather than stimulated with a higher concentration of PTH. CONCLUSION: This study provides insights into the modulatory effect of PTH on chondrogenic differentiation from MSCs and the therapeutic potential for cartilage regeneration. Based on clinical experience regarding the efficacy and safety of PTH for bone metabolism, PTH may also be useful clinically for cartilage repair.

Endosomal GPCR signaling turned off by negative feedback actions of PKA and v-ATPase.

The PTH receptor is to our knowledge one of the first G protein-coupled receptor (GPCR) found to sustain cAMP signaling after internalization of the ligand-receptor complex in endosomes. This unexpected model is adding a new dimension on how we think about GPCR signaling, but its mechanism is incompletely understood. We report here that endosomal acidification mediated by the PKA action on the v-ATPase provides a negative feedback mechanism by which endosomal receptor signaling is turned off.

[Role for PTHrP in bone and cartilage metabolism].

PTH-related peptide (PTHrP) is a widely distributed cytokine, which shares the cognate receptor PTHR1 with PTH. Originally identified as a causal factor of humoral hypercalcemia of malignancy twenty years ago, PTHrP is now recognized as a critical physiological regulator of various biological processes, including bone and cartilage metabolism.

PTHrP is endogenous relaxant for spontaneous smooth muscle contraction in urinary bladder of female rat.

Acute bladder distension causes various morphologic and functional changes, in part through altered gene expression. We aimed to investigate the physiologic role of PTHrP, which is up-regulated in an acute bladder distension model in female rats. In the control Empty group, bladders were kept empty for 6 hours, and in the Distension group, bladders were kept distended for 3 hours after an artificial storing-voiding cycle for 3 hours. In the Distention group bladder, up-regulation of transcripts was noted for 3 genes reported to be up-regulated by stretch in the cultured bladder smooth muscle cells in vitro. Further transcriptome analysis by microarray identified PTHrP as the 22nd highest gene up-regulated in Distension group bladder, among more than 27,000 genes. Localization of PTHrP and its functional receptor, PTH/PTHrP receptor 1 (PTH1R), were analyzed in the untreated rat bladders and cultured bladder cells using real-time RT-PCR and immunoblotting, which revealed that PTH1R and PTHrP were more predominantly expressed in smooth muscle than in urothelium. Exogenous PTHrP peptide (1-34) increased intracellular cAMP level in cultured bladder smooth muscle cells. In organ bath study using bladder strips, the PTHrP peptide caused a marked reduction in the amplitude of spontaneous contraction but caused only modest suppression for carbachol-induced contraction. In in vivo functional study by cystometrogram, the PTHrP peptide decreased voiding pressure and increased bladder compliance. Thus, PTHrP is a potent endogenous relaxant of bladder contraction, and autocrine or paracrine mechanism of the PTHrP-PTH1R axis is a physiologically relevant pathway functioning in the bladder.

Critical role of parathyroid hormone (PTH) receptor-1 phosphorylation in regulating acute responses to PTH.

Agonist-induced phosphorylation of the parathyroid hormone (PTH) receptor 1 (PTHR1) regulates receptor signaling in vitro, but the role of this phosphorylation in vivo is uncertain. We investigated this role by injecting "knock-in" mice expressing a phosphorylation-deficient (PD) PTHR1 with PTH ligands and assessing acute biologic responses. Following injection with PTH (1-34), or with a unique, long-acting PTH analog, PD mice, compared with WT mice, exhibited enhanced increases in cAMP levels in the blood, as well as enhanced cAMP production and gene expression responses in bone and kidney tissue. Surprisingly, however, the hallmark hypercalcemic and hypophosphatemic responses were markedly absent in the PD mice, such that paradoxical hypocalcemic and hyperphosphatemic responses were observed, quite strikingly with the long-acting PTH analog. Spot urine analyses revealed a marked defect in the capacity of the PD mice to excrete phosphate, as well as cAMP, into the urine in response to PTH injection. This defect in renal excretion was associated with a severe, PTH-induced impairment in glomerular filtration, as assessed by the rate of FITC-inulin clearance from the blood, which, in turn, was explainable by an overly exuberant systemic hypotensive response. The overall findings demonstrate the importance in vivo of PTH-induced phosphorylation of the PTHR1 in regulating acute ligand responses, and they serve to focus attention on mechanisms that underlie the acute calcemic response to PTH and factors, such as blood phosphate levels, that influence it.

Activation of a non-cAMP/PKA signaling pathway downstream of the PTH/PTHrP receptor is essential for a sustained hypophosphatemic response to PTH infusion in male mice.

PTH increases urinary Pi excretion by reducing expression of two renal cotransporters [NaPi-IIa (Npt2a) and NaPi-IIc (Npt2c)]. In contrast to acute transporter regulation that is cAMP/protein kinase A dependent, long-term effects require phospholipase C (PLC) signaling by the PTH/PTHrP receptor (PPR). To determine whether the latter pathway regulates Pi through Npt2a and/or Npt2c, wild-type mice (Wt) and animals expressing a mutant PPR incapable of PLC activation (DD) were tested in the absence of one (Npt2a(-/-) or Npt2c(-/-)) or both phosphate transporters (2a/2c-dko). PTH infusion for 8 days caused a rapid and persistent decrease in serum Pi in Wt mice, whereas serum Pi in DD mice fell only transiently for the first 2 days. Consistent with these findings, fractional Pi excretion index was increased initially in both animals, but this increase persisted only when the PPR Wt was present. The hypophosphatemic response to PTH infusion was impaired only slightly in PPR Wt/Npt2c(-/-) or DD/Npt2c(-/-) mice. Despite lower baselines, PTH infusion in PPR Wt/Npt2a(-/-) mice decreased serum Pi further, an effect that was attenuated in DD/Npt2a(-/-) mice. Continuous PTH had no effect on serum Pi in 2a/2c-dko mice. PTH administration increased serum 1,25 dihydroxyvitamin D3 levels in Wt and DD mice and increased levels above the elevated baseline with ablation of either but not of both transporters. Continuous PTH elevated serum fibroblast growth factor 23 and blood Ca(2+) equivalently in all groups of mice. Our data indicate that PLC signaling at the PPR contributes to the long-term effect of PTH on Pi homeostasis but not to the regulation of 1,25 dihydroxyvitamin D3, fibroblast growth factor 23, or blood Ca(2+).

The guanine nucleotide exchange factor Vav2 is a negative regulator of parathyroid hormone receptor/Gq signaling.

The parathyroid hormone receptor (PTHR) is a class B G protein-coupled receptor (GPCR) that mediates the endocrine and paracrine effects of parathyroid hormone and related peptides through the activation of phospholipase Cß-, adenylyl cyclase-, mitogen-activated protein kinase-, and ß-arrestin-initiated signaling pathways. It is currently not clear how specificity among these downstream signaling pathways is achieved. A possible mechanism involves adaptor proteins that affect receptor/effector coupling. In a proteomic screen with the PTHR C terminus, we identified vav2, a guanine nucleotide exchange factor (GEF) for Rho GTPases, as a PTHR-interacting protein. The core domains of vav2 bound to the intracellular domains of the PTHR independent of receptor activation. In addition, vav2 specifically interacted with activated Gα(q) but not with Gα(s) subunits, and it competed with PTHR for coupling to Gα(q). Consistent with its specific interaction with Gα(q), vav2 impaired G(q)-mediated inositol phosphate generation but not G(s)-mediated cAMP generation. This inhibition of G(q) signaling was specific for PTHR signaling, compared with other G(q)-coupled GPCRs. Moreover, the benefit for PTHR-mediated inositol phosphate generation in the absence of vav2 required the ezrin binding domain of Na(+)/H(+)-exchanger regulatory factor 1. Our results show that a RhoA GEF can specifically interact with a GPCR and modulate its G protein signaling specificity.

[Anabolic action of PTH under the loading/unloading condition].

Parathyroid hormone (PTH) is available for the treatment of osteoporosis in Japan. PTH is administered subcutaneously daily or weekly. According to the results of animal experiments, the anabolic action of PTH on bone depends on dosage, condition of loading, and region of bone. Administration of PTH preserved trabecular bone formation rate reduced by immobilization or unloading at the same level of that under the normal mobilization or loading condition, but at low dose did not preserve periosteal bone formation rate reduced by unloading. PTH at high dose (40µg/kg BW) completely preserved trabecular bone volume reduced by immobilization in mice. Thus, we consider that PTH could be clinically effective for immobilization- and/or unloading-induced bone loss.

Osteoblastic expansion induced by parathyroid hormone receptor signaling in murine osteocytes is not sufficient to increase hematopoietic stem cells.

Microenvironmental expansion of hematopoietic stem cells (HSCs) is induced by treatment with parathyroid hormone (PTH) or activation of the PTH receptor (PTH1R) in osteoblastic cells; however, the osteoblastic subset mediating this action of PTH is unknown. Osteocytes are terminally differentiated osteoblasts embedded in mineralized bone matrix but are connected with the BM. Activation of PTH1R in osteocytes increases osteoblastic number and bone mass. To establish whether osteocyte-mediated PTH1R signaling expands HSCs, we studied mice expressing a constitutively active PTH1R in osteocytes (TG mice). Osteoblasts, osteoclasts, and trabecular bone were increased in TG mice without changes in BM phenotypic HSCs or HSC function. TG mice had progressively increased trabecular bone but decreased HSC function. In severely affected TG mice, phenotypic HSCs were decreased in the BM but increased in the spleen. TG osteocytes had no increase in signals associated with microenvironmental HSC support, and the spindle-shaped osteoblastic cells that increased with PTH treatment were not present in TG bones. These findings demonstrate that activation of PTH1R signaling in osteocytes does not expand BM HSCs, which are instead decreased in TG mice. Therefore, osteocytes do not mediate the HSC expansion induced by PTH1R signaling. Further, osteoblastic expansion is not sufficient to increase HSCs.

The parathyroid hormone receptorsome and the potential for therapeutic intervention.

The parathyroid hormone 1 receptor (PTH1R) is activated by parathyroid hormone (PTH) and parathyroid hormone related protein (PTHrP), hormones that mediate mineral ion homeostasis and tissue development, respectively. These diverse actions mediated by one receptor are likely due to the formation of cell-specific receptorsome complexes with cytosolic constituents. Through the second and third intracellular loops, the PTH1R couples to several G protein subclasses, including Gs, Gq/11, Gi/o and G12/13, resulting in the activation of many pathways. The PTH1R carboxy-terminal tail directs interactions with a plethora of binding partners. The WD1 and WD7 repeats of the G protein ß subunit directly bind to a novel interaction domain located near the amino-terminal end of the PTH1R carboxy-terminal tail. This Gßγ binding site likely contributes to the promiscuous G protein coupling displayed by the PTH1R. Partially overlapping this site is an EF-hand binding domain that directs interactions with calpain, a calcium-activated protease, and calmodulin, a ubiquitous calcium sensor. A lysine-arginine-lysine motif located on the juxtamembrane region of the carboxy-terminal tail mediates interactions with ezrin, an actin-membrane cross-linking protein. The C-terminus of the PTH1R binds to the sodium-hydrogen regulatory factors (NHERFs) via a PDZ domain-mediated interaction, an association that influences signaling and membrane anchoring. Through direct interactions with ezrin and NHERF-1, a PTH1R receptorsome complex exists on apical membranes of the proximal tubule, an assembly that directs PTH-mediated regulation of phosphate transport. Targeting the PTH1R receptorsome will likely enhance therapies directed towards the treatment of osteoporosis and enhancing the hematopoietic stem cell niche.

[Tooth and bone research using genetically modified mice].

Teeth and bone are both hard tissues and composed of hydroxyapatite. Tooth development initiates with the invasination of oral epithelium, followed by aggregation of supporting ectomesenchymal cells. From mouse study, numbers of molecules have been discovered to relate tooth development. These discoveries have helped to clarify the responsible genes of human genetic disorders with abnormal tooth number and structure. During tooth development, teeth erupt into the outer environment, oral cavity. From this point, teeth are completely different from bone which is always covered by soft tissues. Tooth eruption is composed of two different processes, that is, eruption pathway formation and vertical tooth movement. In this review, mutant mice with abnormal tooth development and eruption are introduced, and molecular mechanism required for this process is discussed.

[Morphological analysis of bone dynamics and metabolic bone disease. Effect of loading on bone tissue].

We developed a voluntarily climbing animal model to investigate the effect of skeletal loading on bone tissue. At the cross section of the mid-femur, climbing exercise increases outer diameter and area of cortical bone. The mechanical strength of the femur is increased. This change of cortical volume and structure is more marked in anti-gravity exercise, such as climbing and jumping, than aerobic exercise. At the bone marrow area, climbing exercise increases trabecular bone volume and osteoblast number, while it decreases fat volume and adipocyte number. Skeletal loading promotes differentiation from mesenchymal stem cells to osteoblasts and suppresses that to adipocytes by facilitating the signal through PTH÷PTHrP receptor.

Molecular basis of parathyroid hormone receptor signaling and trafficking: a family B GPCR paradigm.

The parathyroid hormone (PTH) receptor type 1 (PTHR), a G protein-coupled receptor (GPCR), transmits signals to two hormone systems-PTH, endocrine and homeostatic, and PTH-related peptide (PTHrP), paracrine-to regulate different biological processes. PTHR responds to these hormonal stimuli by activating heterotrimeric G proteins, such as G(S) that stimulates cAMP production. It was thought that the PTHR, as for all other GPCRs, is only active and signals through G proteins on the cell membrane, and internalizes into a cell to be desensitized and eventually degraded or recycled. Recent studies with cultured cell and animal models reveal a new pathway that involves sustained cAMP signaling from intracellular domains. Not only do these studies challenge the paradigm that cAMP production triggered by activated GPCRs originates exclusively at the cell membrane but they also advance a comprehensive model to account for the functional differences between PTH and PTHrP acting through the same receptor.

[Cytokines in bone diseases. Genetic defects of PTH/PTHrP receptor in chondrodysplasia].

Parathyroid hormone-related protein (PTHrP) signaling plays important roles in regulating the differentiation of chondrocytes in endochondral bone development. PTHrP signaling functions as an inhibitory effect on chondrocyte hypertrophy which is a terminal stage of differentiation at a growth plate. Mutations of the PTH÷PTHrP receptor have been identified in Jansen metaphyseal chondrodysplasia, Blomstrand's lethal chondrodysplasia, and enchondromatosis. Furthermore, genetic manipulations of the PTHrP and its receptor genes in mice have demonstrated the critical roles of these proteins in regulating both the switch between proliferation and differentiation of chondrocytes.

Generation of human PTH1R construct with FLAG epitope located internally: comparison of two-fragment assembly by using PCR overlap extension or ligase.

Parathyroid hormone (PTH) regulates bone remodeling and calcium and phosphate homeostasis. PTH actions are mediated by type I PTH/PTH-related peptide receptor (PTH1R). There has been no commercially available, specific antibody to detect human PTH1R expression so far. Flag-tagged human PTH1R construct, converting the sequence DKEAPTGS (residues 94-101) in the exon E2 region of PTH1R to DYKDDDDK of Flag epitope, was generated by using PCR overlap extension or ligase enzyme for two-fragment assembly. We found that Flag-tagged PTH1R assembled by ligase was easy to be manipulated, but its efficiency was lower than that of PCR overlap extension. The PTH1R plasmids generated by both techniques were expressed successfully in vitro and in vivo and possessed the same physiological function as wild-type PTH1R. The Flag-tagged PTH1R construct will provide invaluable tools for study of PTH1R signaling and trafficking.

Sustained cyclic AMP production by parathyroid hormone receptor endocytosis.

Cell signaling mediated by the G protein-coupled parathyroid hormone receptor type 1 (PTHR) is fundamental to bone and kidney physiology. It has been unclear how the two ligand systems--PTH, endocrine and homeostatic, and PTH-related peptide (PTHrP), paracrine--can effectively operate with only one receptor and trigger different durations of the cAMP responses. Here we analyze the ligand response by measuring the kinetics of activation and deactivation for each individual reaction step along the PTHR signaling cascade. We found that during the time frame of G protein coupling and cAMP production, PTHrP(1-36) action was restricted to the cell surface, whereas PTH(1-34) had moved to internalized compartments where it remained associated with the PTHR and Galpha(s), potentially as a persistent and active ternary complex. Such marked differences suggest a mechanism by which PTH and PTHrP induce differential responses, and these results indicate that the central tenet that cAMP production originates exclusively at the cell membrane must be revised.

Phytoestrogen modulation of bone-related cytokines and its impact on cell viability in human prostate cancer cells.

AIMS: Prostate cancer (PCa) has a high propensity to metastasize to the bone. PCa cells produce several bone-related factors, namely parathyroid hormone related protein (PTHrP), its PTH type 1 receptor (PTH1R), osteoprotegerin (OPG), and receptor activator of NF-kappa B ligand (RANKL). The effects of these factors might explain, at least in part, the ability of PCa cells to grow in and interact with bone. MAIN METHODS: We first analyzed the expression of the aforementioned factors (by western blot and flow cytometry), and their modulation by the phytoestrogens genistein and daidzein (as potential anti-tumoral agents), in human PCa cells in vitro. We also assessed the impact of these osteomimetic factors on PCa cell viability (by propidium iodide staining and flow cytometry, and trypan blue staining). KEY FINDINGS: Genistein and daidzein, at nM range, increased both the PTHrP/PTH1R system and the OPG/RANKL protein ratio, while genistein and, to a lesser extent, daidzein, at >microM doses, inhibited cell viability in PCa cells. Both N- and C-terminal domains of PTHrP inhibited genistein-induced cell death by modulating transcription factor Runx-2 and the Bcl-2/Bax protein ratio in PCa cells. SIGNIFICANCE: Our findings indicate that high doses of genistein and daidzein cause PCa cell death. On the other hand, low doses of these phytoestrogens induce some osteomimetic features in PCa cells with putative impact on PCa development.