Ixazomib enhances parathyroid hormone-induced ß-catenin/T-cell factor signaling by dissociating ß-catenin from the parathyroid hormone receptor.

The anabolic action of PTH in bone is mostly mediated by cAMP/PKA and Wnt-independent activation of ß-catenin/T-cell factor (TCF) signaling. ß-Catenin switches the PTH receptor (PTHR) signaling from cAMP/PKA to PLC/PKC activation by binding to the PTHR. Ixazomib (Izb) was recently approved as the first orally administered proteasome inhibitor for the treatment of multiple myeloma; it acts in part by inhibition of pathological bone destruction. Proteasome inhibitors were reported to stabilize ß-catenin by the ubiquitin-proteasome pathway. However, how Izb affects PTHR activation to regulate ß-catenin/TCF signaling is poorly understood. In the present study, using CRISPR/Cas9 genome-editing technology, we show that Izb reverses ß-catenin-mediated PTHR signaling switch and enhances PTH-induced cAMP generation and cAMP response element-luciferase activity in osteoblasts. Izb increases active forms of ß-catenin and promotes ß-catenin translocation, thereby dissociating ß-catenin from the PTHR at the plasma membrane. Furthermore, Izb facilitates PTH-stimulated GSK3ß phosphorylation and ß-catenin phosphorylation. Thus Izb enhances PTH stimulation of ß-catenin/TCF signaling via cAMP-dependent activation, and this effect is due to its separating ß-catenin from the PTHR. These findings provide evidence that Izb may be used to improve the therapeutic efficacy of PTH for the treatment of osteoporosis and other resorptive bone diseases.

mTORC1 activation downregulates FGFR3 and PTH/PTHrP receptor in articular chondrocytes to initiate osteoarthritis.

OBJECTIVE: Articular chondrocyte activation, involving aberrant proliferation and prehypertrophic differentiation, is essential for osteoarthritis (OA) initiation and progression. Disruption of mechanistic target of rapamycin complex 1 (mTORC1) promotes chondrocyte autophagy and survival, and decreases the severity of experimental OA. However, the role of cartilage mTORC1 activation in OA initiation is unknown. In this study, we elucidated the specific role of mTORC1 activation in OA initiation, and identify the underlying mechanisms. METHOD: Expression of mTORC1 in articular cartilage of OA patients and OA mice was assessed by immunostaining. Cartilage-specific tuberous sclerosis complex 1 (Tsc1, mTORC1 upstream inhibitor) knockout (TSC1CKO) and inducible Tsc1 KO (TSC1CKOER) mice were generated. The functional effects of mTORC1 in OA initiation and development on its downstream targets were examined by immunostaining, western blotting and qPCR. RESULTS: Articular chondrocyte mTORC1 was activated in early-stage OA and in aged mice. TSC1CKO mice exhibited spontaneous OA, and TSC1CKOER mice (from 2 months) exhibited accelerated age-related and DMM-induced OA phenotypes, with aberrant chondrocyte proliferation and hypertrophic differentiation. This was associated with hyperactivation of mTORC1 and dramatic downregulation of FGFR3 and PPR, two receptors critical for preventing chondrocyte proliferation and differentiation. Rapamycin treatment reversed these phenotypes in KO mice. Furthermore, in vitro rescue experiments demonstrated that p73 and ERK1/2 may mediate the negative regulation of FGFR3 and PPR by mTORC1. CONCLUSION: mTORC1 activation stimulates articular chondrocyte proliferation and differentiation to initiate OA, in part by downregulating FGFR3 and PPR.

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.

Investigational parathyroid hormone receptor analogs for the treatment of osteoporosis.

INTRODUCTION: Intermittent parathyroid hormone (PTH) administration, acting through multiple signaling pathways, exerts an osteoanabolic effect on the skeleton that surpasses the effect of other antiosteoporotic agents. However, its efficacy is limited by the coupling effect and relatively common adverse events. Thus, the development of more sophisticated PTH receptor analogs seems imperative. AREAS COVERED: In this review, the authors summarize the role of PTH signaling pathway in bone remodeling. The authors also summarize investigational analogs targeting this pathway, which may be potential treatments for osteoporosis. EXPERT OPINION: ß-arrestins are multifunctional cytoplasmic molecules that are decisive for regulating intracellular PTH signaling. Recently, in preclinical studies, arrestin analogs have achieved the anabolic bone effect of PTH without an accompanying increase in bone resorption. However, it is not yet known whether these analogs have adverse effects and there are no clinical data for their efficacy to date. On the other hand, several molecules derived either from PTH and PTH-related protein (PTHrP) molecules have been developed. Alternative routes of PTH 1 - 34 delivery (oral, transdermal), the PTH analog ostabolin and the N-terminal PTHrP analogs PTHrP 1 - 36 and abaloparatide, have recently been or are currently being tested in clinical trials and are more likely to become available for use in the near future.

Effects of nicotine on PTHrP and PTHrP receptor expression in rat coronary endothelial cells.

AIMS: The study was aimed to investigate whether nicotine affects endothelial expression of PTHrP and PTHrP receptor, a peptide system involved in endothelial protection against apoptosis. METHODS: Isolated and cultured rat coronary endothelial cells were used. Immunoblot techniques were used to study activation of mitogen-activated protein (MAP) kinases and to quantify PTHrP and PTHrP receptor expression. Real-time RT-PCR was used to quantify PTHrP, PTHrP-receptor, bcl-2, and bax mRNA expression. The rate of apoptosis was determined by HOE33258 staining and confirmed by quantification of the bcl-2-to-bax ratio. In vitro data were compared to hearts from rats exposed to cigarette smoking. RESULTS: Nicotine induced PTHrP protein expression at nanomolar levels and small increases of PTHrP release (≈8%). Antagonists directed against the α7 subunit of cholinergic receptors, the most prominent isoform, attenuated nicotine-dependent increases of PTHrP expression. This effect of nicotine was p38 MAPK dependent. Nicotine at micromolar concentrations reduced PTHrP receptor expression. In vitro and in vivo we found a correlation between PTHrP receptor expression and bcl-2 expression. CONCLUSION: Nicotine induces PTHrP expression in endothelial cells but excessive concentrations of nicotine reduce PTHrP receptor expression thereby attenuating any protective effects of PTHrP against apoptosis.

Peptide-peptoid hybrids based on (1-11)-parathyroid hormone analogs.

A series of peptide-peptoid hybrids, containing N-substituted glycines, were synthesized based on the H-Aib-Val-Aib-Glu-Ile-Gln-Leu-Nle-His-Gln-Har-NH(2) (Har = Homoarginine) as the parent parathyroid hormone (1-11) analog. The compounds were pharmacologically characterized in their agonistic activity at the parathyroid hormone 1 receptor.

Upregulation of PTH receptor mRNA expression by dexamethasone in UMR-106 osteoblast-like cells.

OBJECTIVES: Glucocorticoids influence receptor interactions of the parathyroid hormone (PTH) that are crucial for osteoblast function. As mechanisms linking receptor mRNA with glucocorticoids are incompletely understood, we investigated regulation of PTH receptor (PTH1R) mRNA expression in rat osteoblast-like UMR-106 cells by using dexamethasone (Dex), a synthetic glucocorticoid. MATERIALS AND METHODS: UMR-106 cells were exposed to 10(-8) to 10(-5) M Dex, while some cells were also exposed to a transcriptional inhibitor (DRB) for 24 h with or without Dex. PTH-stimulated cyclicAMP activities were measured by an enzyme-linked immunosorbent assay. PTH1R mRNA was determined by Northern analysis. Transcriptional activities were measured as heretogeneous nuclear PTH1R RNA and also as luciferase activity in constructs, including the PTH1R gene promoter. RESULTS: Dexamethasone dose-dependently increased PTH-stimulated adenylyl cyclase activity at 72 h. Dex markedly increased PTH1R mRNA accumulation, but did not change transcriptional activity. PTH1R mRNA stability was significantly increased by Dex in transcriptionally arrested cells. CONCLUSION: In osteoblast-like cells, Dex induced upregulation of PTH1R mRNA followed by increased functional PTH receptor expression. This was caused by posttranscriptional mechanisms increasing mRNA stability.

Characterisation of ligand binding to the parathyroid hormone/parathyroid hormone-related peptide receptor in MCF7 breast cancer cells and SaOS-2 osteosarcoma cells.

Parathyroid hormone-related peptide (PTHrP) and parathyroid hormone (PTH)/PTHrP-receptor, PTH/PTHrP-R, are frequently expressed in mammary carcinomas as well as in bone cells. In this study we compared the ligand binding characteristics of the PTH/PTHrP-R in SaOS-2 human osteosarcoma cells with those in MCF7 breast cancer cells. We used both Scatchard analysis of saturation kinetics for iodinated ligand and the level of expressed receptor protein by visualising the single radio-labelled receptor-ligand complex from isolated membrane preparations from the two cell lines. In MCF7 cells, ligand binding, (receptor number) was increased by prior exposure of the cultured cells to epidermal growth factor (EGF), estradiol (E2), or dexamethasone (DEX), and decreased following calcitriol (1,25 DHCC). In contrast in the SaOS-2 cells, PTH/PTHrP-R number was increased by exposure to E2 and 1,25DHCC and decreased by DEX while EGF had no effect. These data were confirmed when the PTH/PTHrP-R was cross linked with (125)I-PTHrP-1-34(Tyr), and extended by visualising the intensity of the isolated radiolabelled receptor complex by autoradiography following SDS-PAGE at several time points during the treatment.

The parathyroid hormone-related protein system and diabetic nephropathy outcome in streptozotocin-induced diabetes.

The pathophysiology of the diabetic kidney (e.g., hypertrophy, increase urinary albumin excretion (UAE) is still ill-defined. Parathyroid hormone-related protein (PTHrP) is overexpressed in several nephropathies, but its role remains unclear. We evaluated the effect of high glucose on PTHrP and the PTH1 receptor (PTH1R) protein (by Western blot and immunohistochemistry) in the kidney of mice ith streptozotocin-induced diabetes, and in several mouse renal cells in vitro. Diabetic mice showed a significantly increased renal expression of PTHrP and PTH1R proteins with 2-8 weeks from the onset of diabetes. These animals exhibited an intense immunostaining for both proteins in the renal tubules and glomeruli. Using transgenic mice overexpressing PTHrP targeted to the renal proximal tubule, we found a significant increase in the renal hypertrophy index and in UAE in these diabetic mice relative to their control littermates. Moreover, logistic regression analysis showed a significant association between both PTHrP and PTH1R protein levels and UAE in all diabetic mice throughout the study. High-glucose (25 mm) medium was found to increase PTHrP and PTH1R in tubuloepithelial cells, mesangial cells and podocytes in vitro. Moreover, this increase in PTHrP (but not that of PTH1R) was inhibited by the AT1 receptor antagonist losartan. Collectively, these results indicate that the renal PTHrP/PTH1R system is upregulated in streptozotozin-induced diabetes in mice, and appears to adversely affect the outcome of diabetic renal disease. Our findings also suggest that angiotensin II might have a role in the PTHrP upregulation in this condition.

Direct action of parathyroid hormone-related peptide to enhance corticosterone production stimulated by adrenocorticotropic hormone in adrenocortical cells of hens.

The presence of receptor for chicken parathyroid hormone-related peptide (cPTHrP) in the cortical cells of the adrenal gland of the hen was demonstrated by radioligand binding assays. When the cortical cells were incubated in vitro with chicken adrenocorticotropic hormone (cACTH) in the presence of cPTHrP, greater production of corticosterone was observed than when incubated with cACTH alone. The results suggest that PTHrP may act directly on the adrenocortical cells via its receptor binding and increase the response to ACTH for corticosterone secretion in the hen.