RESUMO
Parathyroid hormone 1 receptor (PTH1R) plays a key role in mediating calcium homeostasis and bone development, and aberrant PTH1R activity underlies several human diseases. Peptidic PTH1R antagonists and inverse agonists have therapeutic potential in treating these diseases, but their poor pharmacokinetics and pharmacodynamics undermine their in vivo efficacy. Herein, we report the use of a backbone-modification strategy to design a peptidic PTH1R inhibitor that displays prolonged activity as an antagonist of wild-type PTH1R and an inverse agonist of the constitutively active PTH1R-H223R mutant both in vitro and in vivo. This peptide may be of interest for the future development of therapeutic agents that ameliorate PTH1R malfunction.
Assuntos
Agonismo Inverso de Drogas , Receptor Tipo 1 de Hormônio Paratireóideo , Humanos , Peptídeos , Hormônio Paratireóideo/farmacologiaRESUMO
BACKGROUND: Peptide YY (PYY) is an anorexigenic gut hormone that also has anti-osteogenic effects, inhibiting osteoblastic activity and inducing catabolic effects. It has been postulated that increases in PYY after Roux-en-Y gastric bypass (RYGB) contribute to declines in bone mineral density (BMD) and increases in bone turnover. The aim of this study is to determine the role of the PYY Y2-receptor in mediating bone loss post-RYGB in mice. METHODS: We compared adult male wildtype (WT) and PYY Y2 receptor-deficient (KO) C57BL/6 mice that received RYGB (WT: n = 8; KO: n = 9), with sham-operated mice (Sham; WT: n = 9; KO: n = 10) and mice that were food-restricted to match the weights of the RYGB-treated group (Weight-Matched, WM; WT: n = 7; KO: n = 5). RYGB or sham surgery was performed at 15-16 weeks of age, and mice sacrificed 21 weeks later. We characterized bone microarchitecture with micro-computed tomography (µCT) at the distal femur (trabecular) and femoral midshaft (cortical). Differences in body weight, bone microarchitecture and biochemical bone markers (parathyroid hormone, PTH; C-telopeptide, CTX; and type 1 procollagen, P1NP) were compared using 2-factor ANOVA with Tukey's adjustments for multiple comparisons. RESULTS: Body weights were similar in the WT-RYGB, WT-WM, KO-RYGB, and KO-WM: 41-44 g; these groups weighed significantly less than the Sham surgery groups: 55-57 g. Trabecular BMD was 31-43 % lower in RYGB mice than either Sham or WM in WT and KO groups. This deficiency in trabecular bone was accompanied by a lower trabecular number (19 %-23 %), thickness (22 %-30 %) and increased trabecular spacing (25 %-34 %) in WT and KO groups (p < 0.001 for all comparisons vs. RYGB). RYGB led to lower cortical thickness, cortical tissue mineral density, and cortical bone area fraction as compared to Sham and WM in WT and KO groups (p ≤ 0.004 for all). There were no interactions between genotype and bone microarchitecture, with patterns of response to RYGB similar in both WT and KO groups. CTX and P1NP were significantly higher in RYGB mice than WM in WT and KO groups. PTH did not differ among groups. CONCLUSIONS: RYGB induced greater trabecular and cortical deficits and high bone turnover than observed in weight-matched mice, with a similar pattern in the WT and Y2RKO mice. Thus, skeletal effects of RYGB are independent of weight loss, and furthermore, PYY signaling through Y2R is not a key mediator of bone loss post-RYGB.
Assuntos
Doenças Ósseas Metabólicas , Derivação Gástrica , Animais , Masculino , Camundongos , Densidade Óssea/fisiologia , Camundongos Endogâmicos C57BL , Peptídeo YY , Microtomografia por Raio-XRESUMO
The parathyroid hormone type 1 receptor (PTHR1), a Class B GPCR, is activated by long polypeptides, including drugs for osteoporosis and hypoparathyroidism. The PTHR1 engages peptide agonists via a two-step mechanism. Initial contact involves the extracellular domain (ECD), which has been thought to contribute primarily to receptor-peptide binding, and then the N terminus of the peptide engages the receptor transmembrane domain (TMD), which is thought to control the message conveyed to intracellular partners. This mechanism has been suggested to apply to other Class B GPCRs as well. Here, we show that modification of a PTHR1 agonist at ECD-contact sites can alter the signaling profile, an outcome that is not accommodated by the current two-step binding model. Our data support a modified two-step binding model in which agonist orientation on the ECD surface can influence the geometry of agonist-TMD engagement. This expanded binding model offers a mechanism by which altering ECD-contact residues can affect signaling profile. Our discoveries provide a rationale for exploring agonist modifications distal from the TMD-contact region in future efforts to optimize therapeutic performance of peptide hormone analogs.
Assuntos
Receptor Tipo 1 de Hormônio Paratireóideo , Transdução de Sinais , Receptor Tipo 1 de Hormônio Paratireóideo/metabolismo , Ligação Proteica , Domínios Proteicos , Peptídeos/metabolismoRESUMO
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.
Assuntos
Proteína Relacionada ao Hormônio Paratireóideo , Hormônio Paratireóideo , Receptor Tipo 1 de Hormônio Paratireóideo , Sequência de Aminoácidos , Animais , Ligantes , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Hormônio Paratireóideo/genética , Hormônio Paratireóideo/metabolismo , Proteína Relacionada ao Hormônio Paratireóideo/genética , Proteína Relacionada ao Hormônio Paratireóideo/metabolismo , Ratos , Receptor Tipo 1 de Hormônio Paratireóideo/genética , Receptor Tipo 1 de Hormônio Paratireóideo/metabolismo , Receptores de Hormônios Paratireóideos/genética , Receptores de Hormônios Paratireóideos/metabolismo , Transdução de SinaisRESUMO
Osteoblast lysyl oxidase (LOX) is a strongly up-regulated mRNA and protein by the glucose-dependent insulinotropic polypeptide (GIP). LOX is critically required for collagen maturation, and was shown to be dramatically down-regulated in a mouse model of type 1 diabetes, consistent with known low collagen cross-linking and poor bone quality in diabetic bone disease in humans and in mouse models. GIP is a gastric hormone released by the gut upon consumption of nutrients, which then stimulates insulin release from ß-cells in the pancreas. GIP is directly anabolic to osteoblasts and to bone, while gut-derived dopamine attenuates effects of GIP on osteoblast anabolic pathways, including LOX expression. GIP-stimulation of LOX expression was shown to be dependent on increased cAMP levels and protein kinase A activity, consistent with the fact that GIP receptors are G protein coupled receptors. Downstream signaling events resulting in increased LOX expression remain, however, unexplored. Here we provide evidence for ß-catenin mediation of signaling from GIP to increase LOX expression. Moreover, we have identified a TCF/LEF element in the Lox promoter that is required for GIP-upregulation of LOX. These findings will be of importance in designing potential therapeutic approaches to address deficient LOX production in diabetic bone disease by pointing to the importance of exploring strategies to stimulate ß-catenin signaling in osteoblasts under diabetic conditions as potential therapeutic strategies.
RESUMO
LA-PTH is a long-acting parathyroid hormone (PTH) peptide analogue in preclinical development for hypoparathyroidism (HP). Like native PTH, LA-PTH contains a methionine at position 8 (Met8) that is predicted to be critical for function. We assessed the impact of Met oxidation on the functional properties of LA-PTH and control PTH ligands. Oxidation of PTH(1-34) resulted in marked (~20-fold) reductions in binding affinity on the PTH receptor-1 (PTHR1) in cell membranes, similarly diminished potency for 3',5'-cyclic AMP signaling in osteoblastic cell lines (SaOS-2 and UMR106), and impaired efficacy for raising blood calcium in mice. Surprisingly, oxidation of LA-PTH resulted in little or no change in these functional responses. The signaling potency of oxidized-LA-PTH was, however, reduced approximately 40-fold compared to LA-PTH in cells expressing a PTHR1 construct that lacks the N-terminal extracellular domain (ECD). Molecular modeling revealed that while Met8 of both LA-PTH and PTH(1-34) is situated within the orthosteric ligand-binding pocket of the receptor's transmembrane domain bundle (TMD), the Met8 sidechain position is shifted for the 2 ligands so that on Met8 oxidation of PTH(1-34), steric clashes occur that are not seen with oxidized LA-PTH. The findings suggest that LA-PTH and PTH(1-34) engage the receptor differently in the Met8-interaction environment of the TMD bundle, and that this interaction environment can be allosterically influenced by the ECD component of the ligand-receptor complex. The findings should be useful for the future development of novel PTH-based peptide therapeutics for diseases of bone and mineral ion metabolism.
Assuntos
Hipoparatireoidismo/tratamento farmacológico , Hormônio Paratireóideo/análogos & derivados , Receptor Tipo 1 de Hormônio Paratireóideo/agonistas , Animais , Cálcio/sangue , Linhagem Celular Tumoral , Avaliação Pré-Clínica de Medicamentos , Feminino , Células HEK293 , Humanos , Metionina/metabolismo , Camundongos , Modelos Moleculares , Norleucina , Oxirredução , Hormônio Paratireóideo/metabolismo , Hormônio Paratireóideo/farmacologia , Hormônio Paratireóideo/uso terapêutico , Ratos , Receptor Tipo 1 de Hormônio Paratireóideo/metabolismoRESUMO
Diabetic bone disease is a complication of type I and type II diabetes, both of which are increasing in the United States and elsewhere. Increased hip and foot fracture rates do not correlate well with changes in bone mineral density (BMD), whereas studies support the importance of collagen structure to bone strength. Extracellular lysyl oxidase (LOX) catalyzes the oxidative deamination of hydroxylysine and lysine residues in collagens resulting in aldehydes that subsequently form critically important biosynthetic crosslinks that stabilize functional collagens. Although LOX-dependent biosynthetic crosslinks in bone collagen are deficient in diabetic bone, the expression and regulation of bone LOXs in diabetes have not been comprehensively studied. Here, we found that LOX is profoundly downregulated in bone in diabetes. Moreover, we have identified a novel metabolic regulatory relationship that is dysregulated in diabetes using mouse models. Data indicate that the incretin (gastric hormone) known as glucose-dependent insulinotropic polypeptide (GIP) that is anabolic to osteoblasts strongly upregulates LOX, and that this regulation is disrupted in the streptozotocin-induced model of diabetes in mice. In vivo and in vitro studies support that diabetes results in elevated circulating peripheral dopamine, likely also derived from the gut, and is responsible for blocking GIP signaling and LOX levels in osteoblasts. Moreover, peripheral administration of the dopamine D2 receptor antagonist amisulpride to diabetic mice restored trabecular bone structure to near normal and partially reversed downregulation of LOX. Taken together our data identifies a novel metabolic relationship between the gut-derived hormone GIP and bone-derived LOX, and points to the importance of LOX dysregulation in the pathology of diabetic bone disease. © 2019 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of the American Society for Bone and Mineral Research.