PTH/PTHrP Receptor Signaling Restricts Arterial Fibrosis in Diabetic LDLR-/- Mice by Inhibiting Myocardin-Related Transcription Factor Relays.

RATIONALE: The PTH1R (PTH [parathyroid hormone]/PTHrP [PTH-related protein] receptor) is expressed in vascular smooth muscle (VSM) and increased VSM PTH1R signaling mitigates diet-induced arteriosclerosis in LDLR-/- mice. OBJECTIVE: To study the impact of VSM PTH1R deficiency, we generated mice SM22-Cre:PTH1R(fl/fl);LDLR-/- mice (PTH1R-VKO) and Cre-negative controls. METHODS AND RESULTS: Immunofluorescence and Western blot confirmed PTH1R expression in arterial VSM that was reduced by Cre-mediated knockout. PTH1R-VKO cohorts exhibited increased aortic collagen accumulation in vivo, and VSM cultures from PTH1R-VKO mice elaborated more collagen (2.5-fold; P=0.01) with elevated Col3a1 and Col1a1 expression. To better understand these profibrotic responses, we performed mass spectrometry on nuclear proteins extracted from Cre-negative controls and PTH1R-VKO VSM. PTH1R deficiency reduced Gata6 but upregulated the MADS (MCM1, Agamous, Deficiens, and Srf DNA-binding domain)-box transcriptional co-regulator, Mkl-1 (megakaryoblastic leukemia [translocation] 1). Co-transfection assays (Col3a1 promoter-luciferase reporter) confirmed PTH1R-mediated inhibition and Mkl-1-mediated activation of Col3a1 transcription. Regulation mapped to a conserved hybrid CT(A/T)6GG MADS-box cognate in the Col3a1 promoter. Mutations of C/G in this motif markedly reduced Col3a1 transcriptional regulation by PTH1R and Mkl-1. Upregulation of Col3a1 and Col1a1 in PTH1R-VKO VSM was inhibited by small interfering RNA targeting Mkl1 and by treatment with the Mkl-1 antagonist CCG1423 or the Rock (Rho-associated coiled-coil containing protein kinase)-2 inhibitor KD025. Chromatin precipitation demonstrated that VSM PTH1R deficiency increased Mkl-1 binding to Col3a1 and Col1a1, but not TNF, promoters. Proteomic studies of plasma extracellular vesicles and VSM from PTH1R-VKO mice identified C1r (complement component 1, r) and C1s (complement component 1, s), complement proteins involved in vascular collagen metabolism, as potential biomarkers. VSM C1r protein and C1r message were increased with PTH1R deficiency, mediated by Mkl-1-dependent transcription and inhibited by CCG1423 or KD025. CONCLUSIONS: PTH1R signaling restricts collagen production in the VSM lineage, in part, via Mkl-1 regulatory circuits that control collagen gene transcription. Strategies that maintain homeostatic VSM PTH1R signaling, as reflected in extracellular vesicle biomarkers of VSM PTH1R/Mkl-1 action, may help mitigate arteriosclerosis and vascular fibrosis.

In vivo evidence for an interplay of FGF23/Klotho/PTH axis on the phosphate handling in renal proximal tubules.

Phosphate homeostasis is primarily maintained in the renal proximal tubules, where the expression of sodium/phosphate cotransporters (Npt2a and Npt2c) is modified by the endocrine actions of both fibroblast growth factor 23 (FGF23) and parathyroid hormone (PTH). However, the specific contribution of each regulatory pathway in the proximal tubules has not been fully elucidated in vivo. We have previously demonstrated that proximal tubule-specific deletion of the FGF23 coreceptor Klotho results in mild hyperphosphatemia with little to no change in serum levels of FGF23, 1,25(OH)2D3, and PTH. In the present study, we characterized mice in which the PTH receptor PTH1R was specifically deleted from the proximal tubules, either alone or in combination with Klotho ( PT-PTH1R-/- and PT-PTH1R/KL-/-, respectively). PT-PTH1R-/- mice showed significant increases in serum FGF23 and PTH levels, whereas serum phosphate levels were maintained in the normal range, and Npt2a and Npt2c expression in brush border membrane (BBM) did not change compared with control mice. In contrast, PT-PTH1R/KL-/- mice displayed hyperphosphatemia and an increased abundance of Npt2a and Npt2c in the renal BBM, along with increased circulating FGF23 levels. While serum calcium was normal, 1,25(OH)2D3 levels were significantly decreased, leading to extremely high levels of PTH. Collectively, mice with a deletion of PTH1R alone in proximal tubules results in only minor changes in phosphate regulation, whereas deletion of both PTH1R and Klotho leads to a severe disturbance, including hyperphosphatemia with increased sodium/phosphate cotransporter expression in BBM. These results suggest an important interplay between the PTH/PTH1R and FGF23/Klotho pathways to affect renal phosphate handling in the proximal tubules.

Calcium fluxes at the bone/plasma interface: Acute effects of parathyroid hormone (PTH) and targeted deletion of PTH/PTH-related peptide (PTHrP) receptor in the osteocytes.

Calcium ion concentration ([Ca2+]) in the systemic extracellular fluid, ECF-[Ca2+], is maintained around a genetically predetermined set-point, which combines the operational level of the kidney and bone/ECF interfaces. The ECF-[Ca2+] is maintained within a narrow oscillation range by the regulatory action of Parathyroid Hormone (PTH), Calcitonin, FGF-23, and 1,25(OH)2D3. This model implies two correction mechanisms, i.e. tubular Ca2+ reabsorption and osteoclast Ca2+ resorption. Although their alterations have an effect on the ECF-[Ca2+] maintenance, they cannot fully account for rapid correction of the continuing perturbations of plasma [Ca2+], which occur daily in life. The existence of Ca2+ fluxes at quiescent bone surfaces fulfills the role of a short-term error correction mechanism in Ca2+ homeostasis. To explore the hypothesis that PTH regulates the cell system responsible for the fast Ca2+ fluxes at the bone/ECF interface, we have performed direct real-time measurements of Ca2+ fluxes at the surface of ex-vivo metatarsal bones maintained in physiological conditions mimicking ECF, and exposed to PTH. To further characterize whether the PTH receptor on osteocytes is a critical component of the minute-to-minute ECF-[Ca2+] regulation, metatarsal bones from mice lacking the PTH receptor in these cells were tested ex vivo for rapid Ca2+ exchange. We performed direct real-time measurements of Ca2+ fluxes and concentration gradients by a scanning ion-selective electrode technique (SIET). To validate ex vivo measurements, we also evaluated acute calcemic response to PTH in vivo in mice lacking PTH receptors in osteocytes vs littermate controls. Our data demonstrated that Ca2+ fluxes at the bone-ECF interface in excised bones as well as acute calcemic response in the short-term were unaffected by PTH exposure and its signaling through its receptor in osteocytes. Rapid minute-to-minute regulation of the ECF-[Ca2+] was found to be independent of PTH actions on osteocytes. Similarly, mice lacking PTH receptor in osteocytes, responded to PTH challenge with similar calcemic increases.

Transcriptomic analyses reveal the underlying pro-malignant functions of PTHR1 for osteosarcoma via activation of Wnt and angiogenesis pathways.

BACKGROUND: Increasing evidence has indicated parathyroid hormone type 1 receptor (PTHR1) plays important roles for the development and progression of osteosarcoma (OS). However, its function mechanisms remain unclear. The goal of this study was to further illuminate the roles of PTHR1 in OS using microarray data. METHODS: Microarray data were available from the Gene Expression Omnibus database under the accession number GSE46861, including six tumors from mice with PTHR1 knockdown (PTHR1.358) and six tumors from mice with control knockdown (Ren.1309). Differentially expressed genes (DEGs) between PTHR1.358 and Ren.1309 were identified using the LIMMA method, and then, protein-protein interaction (PPI) network was constructed using data from STRING database to screen crucial genes associated with PTHR1. KEGG pathway enrichment analysis was performed to investigate the underlying functions of DEGs using DAVID tool. RESULTS: A total of 1163 genes were identified as DEGs, including 617 downregulated (Lef1, lymphoid enhancer-binding factor 1) and 546 upregulated genes (Dkk1, Dickkopf-related protein 1). KEGG enrichment analysis indicated upregulated DEGs were involved in Renin-angiotensin system (e.g., Agt, angiotensinogen) and Wnt signaling pathway (e.g., Dkk1), while downregulated DEGs participated in Basal cell carcinoma (e.g., Lef1). A PPI network (534 nodes and 2830 edges) was constructed, in which Agt gene was demonstrated to be the hub gene and its interactive genes (e.g., CCR3, CC chemokine receptor 3; and CCL9, chemokine CC chemokine ligand 9) were inflammation related. CONCLUSIONS: Our present study preliminarily reveals the pro-malignant effects of PTHR1 in OS cells may be mediated by activating Wnt, angiogenesis, and inflammation pathways via changing the expressions of the crucial enriched genes (Dkk1, Lef1, Agt-CCR3, and Agt-CCL9).

PTH receptor-1 signalling-mechanistic insights and therapeutic prospects.

Parathyroid hormone/parathyroid hormone-related protein receptor (PTH/PTHrP type 1 receptor; commonly known as PTHR1) is a family B G-protein-coupled receptor (GPCR) that regulates skeletal development, bone turnover and mineral ion homeostasis. PTHR1 transduces stimuli from PTH and PTHrP into the interior of target cells to promote diverse biochemical responses. Evaluation of the signalling properties of structurally modified PTHR1 ligands has helped to elucidate determinants of receptor function and mechanisms of downstream cellular and physiological responses. Analysis of PTHR1 responses induced by structurally modified ligands suggests that PTHR1 can continue to signal through a G-protein-mediated pathway within endosomes. Such findings challenge the longstanding paradigm in GPCR biology that the receptor is transiently activated at the cell membrane, followed by rapid deactivation and receptor internalization. Evaluation of structurally modified PTHR1 ligands has further led to the identification of ligand analogues that differ from PTH or PTHrP in the type, strength and duration of responses induced at the receptor, cellular and organism levels. These modified ligands, and the biochemical principles revealed through their use, might facilitate an improved understanding of PTHR1 function in vivo and enable the treatment of disorders resulting from defects in PTHR1 signalling. This Review discusses current understanding of PTHR1 modes of action and how these findings might be applied in future therapeutic agents.

PTH receptor signaling in osteoblasts regulates endochondral vascularization in maintenance of postnatal growth plate.

Longitudinal growth of postnatal bone requires precise control of growth plate cartilage chondrocytes and subsequent osteogenesis and bone formation. Little is known about the role of angiogenesis and bone remodeling in maintenance of cartilaginous growth plate. Parathyroid hormone (PTH) stimulates bone remodeling by activating PTH receptor (PTH1R). Mice with conditional deletion of PTH1R in osteoblasts showed disrupted trabecular bone formation. The mice also exhibited postnatal growth retardation with profound defects in growth plate cartilage, ascribable predominantly to a decrease in number of hypertrophic chondrocytes, resulting in premature fusion of the growth plate and shortened long bones. Further characterization of hypertrophic zone and primary spongiosa revealed that endochondral angiogenesis and vascular invasion of the cartilage were impaired, which was associated with aberrant chondrocyte maturation and cartilage development. These studies reveal that PTH1R signaling in osteoblasts regulates cartilaginous growth plate for postnatal growth of bone.

PTH1 receptor is involved in mediating cellular response to long-chain polyunsaturated fatty acids.

The molecular pathways by which long chain polyunsaturated fatty acids (LCPUFA) influence skeletal health remain elusive. Both LCPUFA and parathyroid hormone type 1 receptor (PTH1R) are known to be involved in bone metabolism while any direct link between the two is yet to be established. Here we report that LCPUFA are capable of direct, PTH1R dependent activation of extracellular ligand-regulated kinases (ERK). From a wide range of fatty acids studied, varying in chain length, saturation, and position of double bonds, eicosapentaenoic (EPA) and docosahexaenoic fatty acids (DHA) caused the highest ERK phosphorylation. Moreover, EPA potentiated the effect of parathyroid hormone (PTH(1-34)) in a superagonistic manner. EPA or DHA dependent ERK phosphorylation was inhibited by the PTH1R antagonist and by knockdown of PTH1R. Inhibition of PTH1R downstream signaling molecules, protein kinases A (PKA) and C (PKC), reduced EPA and DHA dependent ERK phosphorylation indicating that fatty acids predominantly activate G-protein pathway and not the ß-arrestin pathway. Using picosecond time-resolved fluorescence microscopy and a genetically engineered PTH1R sensor (PTH-CC), we detected conformational responses to EPA similar to those caused by PTH(1-34). PTH1R antagonist blocked the EPA induced conformational response of the PTH-CC. Competitive binding studies using fluorescence anisotropy technique showed that EPA and DHA competitively bind to and alter the affinity of PTH1 receptor to PTH(1-34) leading to a superagonistic response. Finally, we showed that EPA stimulates protein kinase B (Akt) phosphorylation in a PTH1R-dependent manner and affects the osteoblast survival pathway, by inhibiting glucocorticoid-induced cell death. Our findings demonstrate for the first time that LCPUFAs, EPA and DHA, can activate PTH1R receptor at nanomolar concentrations and consequently provide a putative molecular mechanism for the action of fatty acids in bone.

Hormone resistance at the clinical level.

This presentation introduces clinical aspects of hormone resistance through a number of case studies that illustrate how molecular defects at various steps in hormone production, signaling, or responsiveness can produce disease in humans. The presentation also shows how careful phenotyping of such patients has triggered decades of translational research.

Disruption of PTH receptor 1 in T cells protects against PTH-induced bone loss.

BACKGROUND: Hyperparathyroidism in humans and continuous parathyroid hormone (cPTH) treatment in mice cause bone loss by regulating the production of RANKL and OPG by stromal cells (SCs) and osteoblasts (OBs). Recently, it has been reported that T cells are required for cPTH to induce bone loss as the binding of the T cell costimulatory molecule CD40L to SC receptor CD40 augments SC sensitivity to cPTH. However it is unknown whether direct PTH stimulation of T cells is required for cPTH to induce bone loss, and whether T cells contribute to the bone catabolic activity of PTH with mechanisms other than induction of CD40 signaling in SCs. METHODOLOGY/PRINCIPAL FINDINGS: Here we show that silencing of PTH receptor 1 (PPR) in T cells blocks the bone loss and the osteoclastic expansion induced by cPTH, thus demonstrating that PPR signaling in T cells is central for PTH-induced reduction of bone mass. Mechanistic studies revealed that PTH activation of the T cell PPR stimulates T cell production of the osteoclastogenic cytokine tumor necrosis factor alpha (TNF). Attesting to the relevance of this effect, disruption of T cell TNF production prevents PTH-induced bone loss. We also show that a novel mechanism by which TNF mediates PTH induced osteoclast formation is upregulation of CD40 expression in SCs, which increases their RANKL/OPG production ratio. CONCLUSIONS/SIGNIFICANCE: These findings demonstrate that PPR signaling in T cells plays an essential role in PTH induced bone loss by promoting T cell production of TNF. A previously unknown effect of TNF is to increase SC expression of CD40, which in turn increases SC osteoclastogenic activity by upregulating their RANKL/OPG production ratio. PPR-dependent stimulation of TNF production by T cells and the resulting TNF regulation of CD40 signaling in SCs are potential new therapeutic targets for the bone loss of hyperparathyroidism.

Indian hedgehog stimulates periarticular chondrocyte differentiation to regulate growth plate length independently of PTHrP.

In the developing growth plate, periarticular chondrocytes proliferate, differentiate into columnar chondrocytes, and then further differentiate into postmitotic hypertrophic chondrocytes. Parathyroid hormone-related (PTH-related) protein (PTHrP), regulated by Indian hedgehog (Ihh), prevents premature hypertrophic differentiation, thereby maintaining the length of columns. Ihh regulates cartilage development through PTHrP-independent pathways as well. Here we show that Ihh stimulates differentiation of periarticular to columnar chondrocytes (periarticular chondrocyte differentiation) and thereby regulates the length of columns independently of PTHrP. Mosaic ablation of the PTH/PTHrP receptor in the growth plate caused upregulation of Ihh action, PTHrP upregulation, acceleration of periarticular chondrocyte differentiation, and elongation of the columnar region. Decreasing Ihh action in these mice reduced elongation of columns, whereas decreasing PTHrP showed only a modest effect on column length. Overexpression of Ihh caused PTHrP upregulation, elongation of columns, and acceleration of periarticular chondrocyte differentiation. PTHrP heterozygosity in this model had a minimal effect on the elongation of columns. Moreover, the elongation of columns and stimulation of periarticular chondrocyte differentiation in these models were still observed when PTHrP signaling was maintained so that it remained constant. These results demonstrate that Ihh acts on periarticular chondrocytes to stimulate their differentiation, thereby regulating the columnar cell mass independently of PTHrP.

Stimulatory G protein directly regulates hypertrophic differentiation of growth plate cartilage in vivo.

Stimulatory heterotrimeric G protein (Gs) transduces signals from various cell-surface receptors to adenylyl cyclases, which generate cAMP. The alpha subunit of Gs (Gsalpha) is encoded by GNAS (Gnas in mice), and heterozygous Gsalpha inactivating mutations lead to Albright hereditary osteodystrophy. The in vivo role of Gsalpha in skeletogenesis is largely unknown, because of early embryonic lethality of mice with disruption of Gnas exon 2 (Gnas(E2-/E2-)) and the absence of easily detectable phenotypes in growth plate chondrocytes of heterozygous mutant mice (Gnas(+/E2-)). We generated chimeric mice containing wild-type cells and either Gnas(E2-/E2-) or Gnas(+/E2-) cells. Gnas(E2-/E2-) chondrocytes phenocopied PTH/PTHrP receptor (PPR)(-/-) cells by prematurely undergoing hypertrophy. Introduction of a transgene expressing Gsalpha, one of several gene products that include Gnas exon 2, into Gnas(E2-/E2-) cells prevented premature hypertrophy. Gsalpha mRNA expression detected by real-time RT-PCR analysis was reduced to approximately half that of the wild-type in both paternal and maternal Gnas(+/E2-) growth plate chondrocytes, indicating biallelic expression of Gsalpha in these cells. Hypertrophy of Gnas(+/E2-) chondrocytes was modestly but significantly premature in chimeric growth plates of mice containing wild-type and Gnas(+/E2-) cells. These data suggest that Gsalpha is the primary mediator of the actions of PPR in growth plate chondrocytes and that there is haploinsufficiency of Gsalpha signaling in Gnas(+/E2-) chondrocytes.

Characterization of PTH/PTHrP receptor in rat duodenum: effects of ageing.

In rat enterocytes, signaling through the parathyroid hormone (PTH)/PTH-related peptide receptor type 1(PTHR1) includes stimulation of adenylyl cyclase, increases of intracellular calcium, activation of phospholipase C, and the MAP kinase pathway, mechanisms that suffer alterations with ageing. The purpose of this study was to evaluate whether an alteration at the level of the PTH receptor (PTHR1) is the basis for impaired PTH signaling in aged rat enterocytes. Western Blot analysis with a specific monoclonal anti-PTHR1 antibody revealed that a 85 kDa PTH binding component, the size expected for the mature PTH/PTHrP receptor, localizes in the basolateral (BLM) and brush border (BBM) membranes of the enterocyte, being the protein expression about 7-fold higher in the BLM. Two other bands of 105 kDa (corresponding to highly glycosylated, incompletely processed receptor form) and 65 kDa (proteolytic fragment) were also seen. BLM PTHR1 protein expression significantly decreases with ageing, while no substantial decrease was observed in the BBM from old rats. PTHR1 immunoreactivity was also present in the nucleus where PTHR1 protein levels were similar in enterocytes from young and aged rats. Immunohistochemical analysis of rat duodenal sections showed localization of PTHR1 in epithelial cells all along the villus with intense staining of BBM, BLM, and cytoplasm. The nuclei of these cells were reactive to the PTHR1 antiserum, but not all cells showed the same nuclear staining. The receptor was also detected in the mucosae lamina propria cells, but was absent in globets cells from epithelia. In aged rats, PTHR1 immunoreactivity was diffused in both membranes and cytoplasm and again, PTH receptor expression was lower than in young animals, while the cell nuclei showed a similar staining pattern than in young rats. Ligand binding to PTHR1 was performed in purified BLM. rPTH(1-34) displaced [I(125)]PTH(1-34) binding to PTHR1 in a concentration-dependent fashion. In both, aged (24 months) and young (3 months) rats, binding of [I(125)]PTH was characterized by a single class of high-affinity binding sites. The affinity of the receptor for PTH was not affected by age. The maximum number of specific PTHR1 binding sites was decreased by 30% in old animals. The results of this study suggest that age-related declines in PTH regulation of signal transduction pathways in rat enterocytes may be due, in part, to the loss of hormone receptors.