Hyperparathyroidism is an endocrine disorder linked to vitamin D deficiency. Reduced vitamin D receptor (VDR) activity promotes parathyroid hormone (PTH) hypersecretion by increasing heterodimerization of the type B γ-aminobutyric acid receptor 1 (GABAB1R) with the extracellular Ca2+-sensing receptor (CaSR) in parathyroid cells; however, endogenous activators of the heterodimers are unknown. We uncovered increased expression of amyloid-β peptide cleaved from the amyloid-β precursor protein (APP) in parathyroid cells of patients with hyperparathyroidism and aging mice manifesting vitamin D deficiency and the ability of exogenous amyloid-β to promote tonic PTH secretion from cultured murine or human parathyroid glands. Conversely, parathyroid cell-specific App gene deletion reduced tonic PTH secretion and lowered serum PTH concentrations in mice. The absence of the amyloid-β effect on PTH secretion in parathyroid cells lacking CaSR or GABAB1R supported direct interactions of amyloid-β with the receptor heterodimers. In situ proteomic profiling of parathyroid cells of patients with hyperparathyroidism correlated lower serum 25-hydroxyvitamin D concentrations with increased GABAB1R/CaSR heterodimer expression, β-amyloidogenesis, and phosphorylation of Tau, a downstream effector of amyloid-β. Concurrent ablation of App or the Tau-encoding Mapt gene prevented tonic PTH hypersecretion in parathyroid cell-specific Vdr knockout mice. Likewise, weekly administration of an amyloid-β-neutralizing antibody suppressed tonic PTH hypersecretion and synergized with daily administration of cinacalcet, which activates CaSR homodimers, to reduce serum PTH concentrations in aged mice. These data demonstrated amyloid-β actions in driving tonic PTH secretion by activating GABAB1R/CaSR heterodimers and the potential of targeting amyloid-β to treat hyperparathyroidism due to vitamin D deficiency.

Primary hyperparathyroidism (PHPT) is a common endocrine disorder of aging closely linked to vitamin D deficiency. Reduced vitamin D receptor activities promote parathyroid hormone (PTH) hypersecretion by increasing the heterodimerization of the type B γ-aminobutyric acid receptor 1 (GABAB1R) with the extracellular Ca2+-sensing receptor (CaSR) in parathyroid cells; however, endogenous activators of the heterodimers are unknown. Here we uncovered increased expression of the β-amyloid peptide (Aβ42) cleaved from the amyloid precursor protein in parathyroid cells from PHPT patients and aging mice, and the ability of exogenous Aβ42 to promote tonic PTH secretion from murine or human parathyroid glands ex vivo. Conversely, parathyroid-specific App gene knockout reduced tonic PTH secretion and lowered serum PTH levels in mice. The absence of an Aβ42 effect on PTH secretion in parathyroid glands lacking CaSR or GABAB1R supports direct interactions between Aβ42 and the heterodimer. In situ proteomic profiling of parathyroid glands from PHPT patients closely correlated lower serum 25-hydroxyvitamin D levels with increased GABAB1R /CaSR heterodimer expression, β-amyloidogenesis, and phosphorylation of Tau, a downstream effector of Aβ42. Concurrent ablation of App or the Tau-encoding Mapt gene prevented tonic PTH hypersecretion in parathyroid-specific Vdr-KO mice. Likewise, weekly administration of an Aβ42-neutralizing antibody suppressed tonic PTH hypersecretion and synergized with daily administration of cinacalcet, a calcimimetic that activates CaSR homodimers, to reduce serum PTH levels in aging-induced hyperparathyroidism (HPT) mice. These data demonstrated novel functions of Aβ42 in driving tonic PTH secretion by activating GABAB1R/CaSR heterodimers and suggest the potential for targeting Aβ42 in PHPT treatment.

Abstract Disclosure: C. Tu: None. Z. Cheng: None. K.A. Pena: None. S. Savransky: None. T. Glinin: None. N. Szeto: None. J.A. Sosa: Research Investigator; Self; Institutional research funding and Data Monitoring Committee of the Medullary Thyroid Cancer Consortium Registry, supported by, AstraZeneca, Novo Nordisk, Eli Lilly & Company, Exelixis, Inc.. J. Vilardaga: None. J. Koh: None. W. Chang: None. Primary hyperparathyroidism (PHPT) is a common endocrinopathy characterized by elevated parathyroid hormone (PTH) secretion. Low serum 25-hydroxyvitamin D (25OHD) levels are more prevalent in PHPT patients than in the general population, however, the mechanistic basis for this association is unclear. Previous studies (Nat Metab 2:243) demonstrated that increased heterodimerization and co-activation (by GABA and Ca2+, respectively) of the type B γ-aminobutyric acid receptor 1 (GABAB1R) and the extracellular Ca2+-sensing receptor (CaSR) promote tonic PTH secretion from the parathyroid glands (PTGs) isolated from PHPT patients and HPT mice. In searching for additional ligands of the GABAB1R/CaSR heterodimer, we found upregulation of a putative GABAB1R ligand, the amyloid precursor protein (APP), and one of its derivatives, β-amyloid (Aβ1-42) in the parathyroid adenoma of PHPT patients verse age-matched normal PTGs (p<0.005) by in situ proteomic profiling. Those tumors also showed increased levels of the proteolytic enzymes, β-secretase and γ-secretase, that make Aβ1-42 and enhanced phosphorylation of the microtubule-associated protein TAU, a downstream effector of Aβ1-42-induced signaling in the degenerative neurons of dementia patients. Adding exogenous Aβ1-42 (0.3 to 1000 nM) in cultures concentration-dependently stimulated tonic PTH secretion by up to 1.6-fold in murine (p<0.001 vs control) and 1.9-fold in human PTGs (p=0.03 vs control) without shifting the Ca2+-set point. This stimulatory effect was absent in murine PTGs lacking either CaSR or GABAB1R. Furthermore, parathyroid cell (PTC)-specific knockout (KO) of the App (PTCAppΔflox/Δflox) gene to remove Aβ1-42 or the Gabbr1 (PTCGabbr1Δflox/Δflox) gene to disrupt GABAB1R/CaSR heterodimer similarly reduced tonic PTH secretion in PTG cultures and produced hypoparathyroidism in vivo, supporting a role for Aβ1-42 as a ligand in stimulating PTH secretion via GABAB1R/CaSR heterodimer. The proteomic profiles revealed a significant inverse correlation between the pre-operative 25OHD levels of PHPT patients and increased Tau phosphorylation in their PTG tumors (r2=0.225, p<0.0001), suggesting a role for Tau signaling in stimulating PTH secretion in 25OHD deficiency. In support of this idea, inhibition of Tau phosphorylation by a staurosporine analogue (K252a) blocked the ability of Aβ1-42 to stimulate PTH secretion in vitro, and the increased tonic PTH hypersecretion seen in mice with PTC-targeted Vdr gene KO (PTCVdrΔflox/Δflox), mimicking vitamin D deficiency, was reversed by a concurrent ablation of App (PTCVdrΔflox/Δflox;AppΔflox/Δflox) or the TAU-encoding Mapt (PTCVdrΔflox/Δflox;Mapt-/-) gene. Collectively, we demonstrate novel roles of Aβ1-42/p-Tau signaling in sustaining tonic PTH secretion in physiological states and in promoting PTH hypersecretion due to 25OHD deficiency. Presentation: 6/3/2024

Abstract Disclosure: C. Tu: None. Z. Cheng: None. K.A. Pena: None. N. Szeto: None. J.A. Sosa: None. J. Vilardaga: None. J. Koh: None. W. Chang: None. Understanding the mechanisms driving parathyroid hormone (PTH) hypersecretion in primary hyperparathyroidism (PHPT) is essential for better management of this common endocrinopathy. Prior studies showed that reduced Ca2+-sensing receptor (CaSR) expression and the subsequent increases in heterodimerization of the CaSR with the type B γ-aminobutyric acid receptor 1 (GABAB1R) are causally linked to PTH hypersecretion in PHPT mouse models (Nat Metab 2:243-255). We further showed that expression of the putative GABAB1R ligands, amyloid precursor protein (APP) and its proteolytic product, β-amyloid (Aβ1-42), is significantly upregulated in adenomas of PHPT patients associated with vitamin D insufficiency/deficiency when compared to normal donor controls. The current study aims to delineate the actions of Aβ1-42 in promoting tonic PTH secretion and its interactions with vitamin D receptor (VDR) signaling in parathyroid glands (PTGs) in basal and PHPT states. We show that Aβ1-42 (200 nM) stimulated tonic PTH secretion in cultures of normal human PTGs without shifting the calcium/PTH secretion setpoint (Ca2+-setpoint). In contrast, conditional knockout (KO) of the App gene in the parathyroid cell (PTC) of PTCAppΔflox/Δflox mice significantly reduced serum PTH levels (KO: 64±13 pg/ml vs Control: 109±12 pg/ml; p<0.05, n=8) despite hypocalcemia, indicating hypoparathyroidism. In PTGs cultured from the PTCAppΔflox/Δflox mice, supplementation of Aβ1-42 dose-dependently (EC50=5.6 nM, p<0.001) increased PTH secretion without altering the Ca2+-setpoint. However, the stimulatory effects of Aβ1-42 on tonic PTH secretion were completely abrogated in the PTGs with concurrent deletions of App, Casr and Gabbr1 genes, supporting a direct action of Aβ1-42 on CaSR and/or GABAB1R. The latter notion is further supported by the ability of Aβ1-42 to stimulate cAMP production in cells co-expressing CaSR and GABAB1R. PTC-specific deletion of the Vdr gene in the PTCVdrΔflox/Δflox mice led to elevated serum PTH levels in vivo and increased tonic PTH secretion with unaffected Ca2+-setpoint in PTGs in vitro. Concurrent ablation of the App gene completely normalized serum PTH levels in the PTCVdrΔflox/Δflox mice and prevented PTH hypersecretion in their PTGs in culture. These findings support a critical role of the APP-derived Aβ1-42 in mediating tonic PTH secretion in the normal physiological state and suggest a new mechanism driving PTH hypersecretion in PHPT due to vitamin D deficiency. Presentation: Saturday, June 17, 2023

The parathyroid hormone (PTH) type 1 receptor (PTHR) is a class B G protein–coupled receptor (GPCR) that regulates mineral ion, vitamin D, and bone homeostasis. Activation of the PTHR by PTH induces both transient cell surface and sustained endosomal cAMP production. To address whether the spatial (location) or temporal (duration) dimension of PTHR-induced cAMP encodes distinct biological outcomes, we engineered a biased PTHR ligand (PTH7d) that elicits cAMP production at the plasma membrane but not at endosomes. PTH7d stabilized a unique active PTHR conformation that mediated sustained cAMP signaling at the plasma membrane due to impaired β-arrestin coupling to the receptor. Experiments in cells and mice revealed that sustained cAMP production by cell surface PTHR failed to mimic the pharmacological effects of sustained endosomal cAMP production on the abundance of the rate-limiting hydroxylase catalyzing the formation of active vitamin D, as well as increases in circulating active vitamin D and Ca2+ and in bone formation in mice. Thus, similar amounts of cAMP generated by PTHR for similar lengths of time in different cellular locations, plasma membrane and endosomes, mediate distinct physiological responses. These results unveil subcellular signaling location as a means to achieve specificity in PTHR-mediated biological outcomes and raise the prospect of rational drug design based upon spatiotemporal manipulation of GPCR signaling.

Structural defects in primary cilia have robust effects in diverse tissues and systems. However, how disorders of ciliary length lead to functional outcomes are unknown. We examined the functional role of a ciliary length control mechanism of FBW7-mediated destruction of NDE1, in mesenchymal stem cell (MSC) differentiation. We show that FBW7 functions as a master regulator of both negative (NDE1) and positive (TALPID3) regulators of ciliogenesis, with an overall positive net effect on primary cilia formation, MSC differentiation to osteoblasts, and bone architecture. Deletion of Fbxw7 suppresses ciliation, Hedgehog activity, and differentiation, which are partially rescued in Fbxw7/Nde1 -null cells. We also show that NDE1, despite suppressing ciliogenesis, promotes MSC differentiation by increasing the activity of the Hedgehog pathway by direct binding and enhancing GLI2 activity in a cilia-independent manner. We propose that FBW7 controls a protein-protein interaction network coupling ciliary structure and function, which is essential for stem cell differentiation. Petsouki et al. dissect the importance of FBW7-mediated regulation of NDE1 and TALPID3 in mesenchymal stem cells (MSCs). They find that by modulating the abundance of negative (NDE1) and positive (TALPID3) cilia regulators, FBW7 contributes to both the assembly and signaling functions of primary cilia that are necessary for osteoblast differentiation.