Parathyroid Hormone-related Protein Research Articles

Mammary homeostasis in the mother-offspring dyad

Homeostasis during lactation is a special case in which the unit for regulation is a dyad comprising the mother and her currently nursing offspring (the mother–offspring dyad). This arrangement is not a trivial. A litter of mice can have a mass greater than the mother and nutrient demands that far exceed her. Homeostasis for milk secretion, appetite, and calcium metabolism must come under integrated control, responding seamlessly to the needs of the mother and the offspring.Serotonin (5-HT) is a primary local regulator of mammary homeostasis. 5-HT synthesis in the mammary epithelium is high during lactation and increases during milk stasis. Two important functions are attributed to the 5-HT system. Firstly, when alveolar spaces are filled with milk 5-HT inhibits milk secretion and opens tight junctions. This feedback induces early phases of involution. Secondly, 5-HT induces synthesis and secretion of parathyroid hormone-related peptide (PTHrP).

NUCLEAR PARATHYROID HORMONE‐RELATED PROTEIN IS NECESSARY FOR PITUITARY SOMATOTROPHS AND GROWTH HORMONE PRODUCTION Waleed J. Hashmi1, Noriko Kantake2, Shiu Yuan2,3, Ibiaghani Max Harry2,3, Nathan K. Hoggard1, Alex H. Fishbach2, Ramiro E. Toribio4, Thomas J. Rosol2,3, Department of Biomedical Sciences, Translation Biomedical Sciences Program, Ohio University, Athens, 45701, OH1,Department of Biomedical Sciences, Heritage College of Osteopathic Medicine, Ohio University2, Department of

Parathyroid hormone‐related protein (PTHrP) is an important regulator of bone growth, energy metabolism, and lung development. PTHrP is expressed by the pituitary endocrine cells. The pituitary gland plays an important role in homeostasis, energy metabolism, and development. The first phylogenetic identification of PTHrP in animals has shown that it originally developed as a secretory pituitary hormone in fishes. PTHrP is both a secreted and nuclear protein. A novel knock‐in mouse (PTHrP∆/∆) with a lack of the nuclear localization sequence (NLS) of PTHrP was developed. The mice fail to grow and die 4 to 5 days after birth. We hypothesized that the nuclear localization sequence (NLS) of PTHrP was necessary to many tissues for normal development and physiological processes. Histological analysis was performed to study the morphological structure of the pituitaries in these mice. Compared to the wildtype and heterozygote mice(PTHrP+/∆), the pituitary pars distalis from 1‐day‐old PTHrP∆/∆ mice was hypocellular, with a decreased density of pituitary epithelial cells throughout the gland (hypoplasia of the pituitary pars distalis). Occasional pituitary epithelial cells were apoptotic with shrunken pyknotic or fragmented nuclei, with increased cytoplasmic eosinophilia. The pituitary pars intermedia, pars nervosa, and rathke pouch epithelium was unaffected. Immunohistochemistry using GH primary antibody showed decreased GH secretion in somatotrophs which may contribute to early senescence in the mice. Immunoreactivity to GH was strongest in the pituitary epithelial cells within lateral areas of the pars distalis in PTHrP+/∆ mice. The GH immunoreactive cells in the pituitary epithelial cells of PTHrP∆/∆ mice was low compared to PTHrP+/∆ mice. The GH is expressed at low levels in the pituitaries from the PTHrP∆/∆ mice compared to PTHrP+/∆mice as verified by PCR. Understanding the mechanisms and functions of nuclear PTHrP in the pituitary provides the platform for improved medical therapies for endocrine diseases as well as novel insights into the impact of the PTHrP on processes such as growth and metabolism.

The Role of C‐Terminus and Nuclear Localization Sequence of Parathyroid Hormone‐Related Protein (PTHrP) in Pancreatic Islet Morphology and Glucose Homeostasis

Parathyroid Hormone‐related protein (PTHrP) is an important polyhormone with multiple functions in development and cell regulation. PTHrP is expressed by pancreatic beta cells which are responsible for insulin secretion. Previous studies have focused on the ability of N‐terminal PTHrP to stimulate proliferation in beta cells. We have developed a knock‐in mouse model lacking the C‐terminal and nuclear localization sequence of PTHrP, retaining only N‐terminal PTHrP. These mice die at ~day 5, are severely stunted in growth (weigh 54% less than control mice), have chondrodystrophy, hypoinsulinemia, hypoglycemia (~40‐60mg/dL), hypotriglyceridemia, and lack body fat. Percentage stomach weight (including ingested milk) measurements in relation to body weight were consistent among all mouse genotypes indicating that inanition was not the cause of death. To characterize the pancreatic islets in these mice, islets (~10‐20) were isolated from 2‐5 day‐old‐mice using collagenase digestion and a glucose‐stimulated intracellular calcium assay was conducted at 0, 4, 8, 12, 16 and 20mM glucose. Results showed higher intracellular calcium response in knock‐in islets compared to control islets and increased insulin secretion after overnight incubation. Data from immunofluorescence also showed less glucagon staining in knock‐in islets compared to control islets. This study revealed that the C‐terminus and nuclear localization sequence of PTHrP are crucial to life, including regulation of glucose homeostasis and islet response to glucose metabolism. It also revealed that these portions of PTHrP may be important for the developmental stage of islets and differentiation into alpha or beta cells.

Abstract #1159516: A Rare Case of Hypercalcemia Mediated by PTHrP and 1,25-dihyroxyvitamin D in a Patient with Pancreatic Neuroendocrine Tumor

In patients with hypercalcemia of malignancy, the secretion of parathyroid hormone-related protein (PTHrP) is commonly seen. Rarely, neoplasms can cause hypercalcemia from multiple mechanisms, which can affect the treatment options. We present a patient with metastatic pancreatic neuroendocrine tumor who developed hypercalcemia secondary to simultaneous elevations in both PTHrP and 1,25-dihydroxyvitamin D.

Distinct Signaling of Native PTH‐related Peptide

The parathyroid hormone (PTH) type 1 receptor (PTHR) regulates a variety of key biological processes, including mineral ion and bone homeostasis, through the action of its two natural peptide ligands: PTH and PTH‐related peptide (PTHrP). Particularly, PTHrP is indispensable for the development of mammary glands, placental calcium ion transport, tooth eruption, bone formation and bone remodeling. Although mature forms of PTHR ligands consist of 84 and 141/173 amino acids for PTH and PTHrP, respectively, our current understanding on how endogenous PTHR ligands transduce signals through PTHR is largely derived from studies done with the N‐terminal fragments of both peptides, PTH1‐34 and PTHrP1‐36. While both peptides induce acute intracellular Ca2+ (iCa2+) release, they differ in the location and duration of cAMP signaling. PTH1‐34 induces sustained cAMP production from PTHR signaling complexes in endosomes, whereas PTHrP1–36 induces transient cAMP production derived from ligand–PTHR interactions at the plasma membrane. Although the transient nature of PTHrP1‐36 signaling has been regarded as a main PTHrP feature, here we demonstrate that PTHrP1‐36 cannot be considered as an analogue of the native PTHrP1‐141. We show that PTHrP1‐141 triggers sustained cAMP signaling from the plasma membrane without inducing b‐arrestin recruitment. Additionally, PTHrP1‐141 fails to stimulate iCa2+ release. We also show that the molecular basis for signaling differences between PTHrP1‐36 and native PTHrP1‐141 are caused by the stabilization of distinct PTHR conformations. These results indicate that native PTHrP1‐141 displays distinct signaling features from PTHrP1‐36 and can be considered an endogenous biased agonist for PTHR.

Abstract #1183557: Parathyroid Hormone-Related Protein Causing Hypercalcemia in a Patient with Large Cell Lung Cancer

Secretion of parathyroid hormone-related protein (PTHrP) accounts for up to 80% of cases with hypercalcemia of malignancy. It is most commonly associated with lung cancer, with a reported incidence up to 12.5% in those at advanced stages. While elevated PTHrP levels have been reported with all histologic types of lung cancer, it is rare in large cell carcinoma, with only 3 previously reported cases.

Abstract #1181995: A rare case of humoral hypercalcemia of malignancy caused by a Gallbladder Adenocarcinoma

Humoral hypercalcemia of malignancy (HHM) is a common paraneoplastic syndrome occurring secondary to oversecretion of parathyroid hormone-related peptide (PTHrP). It is usually seen in solid tumors particularly squamous cell carcinoma of the head, neck, and lungs. Gallbladder adenocarcinoma is an unusual cause of HHM. We present a rare case of hypercalcemia secondary to elevated PTHrP secretion in a patient with gallbladder adenocarcinoma.

Abstract #1174395: Calcium Balance in Rhabdomyolysis and Acute Kidney Injury. A biphasic disease

Calcium homeostasis is closely regulated mainly by the parathyroid hormone (PTH) and to a lesser degree by the activated form of vitamin D. Primary hyperparathyroidism and underlying malignancy are the two most common causes of hypercalcemia. Few cases of rebound hypercalcemia have been reported in the recovery period of rhabdomyolysis-induced acute kidney injury (AKI) despite the relatively high incidence from up to one third of cases. Here we report a case of severe and symptomatic hypercalcemia in the recovery period of rhabdomyolysis and AKI with low PTH, PTH-related peptide (PTHrP) and 1,25-dihydroxycholecalciferol levels, and normal 25-hydroxycholecalciferol.

Metabolic Bone Disease of Prematurity.

Metabolic Bone Disease of Prematurity.

Abstract #1182064: Recurrent Hypercalcemia Following Calcium Sulfate Bead Placement

Stimulan® (Smith & Nephew) beads are antibiotic-loaded calcium sulfate beads (CSB) that are used to prevent and treat joint infections intraoperatively. They are used to prevent systemic toxicity of oral and intravenous (IV) antibiotics and can also be used as bone void fillers. We present a case of recurrent transient hypercalcemia due to calcium sulfate beads leading to prolonged hospitalization and IV bisphosphonate treatment. A 65-year-old female with chronic kidney disease (stage 3b) and no prior calcium disorder presented to her orthopedic surgeon with severe osteoarthritis in her left hip after having failed multiple non-surgical interventions. She subsequently underwent left total hip arthroplasty (THA) that was complicated by a prosthetic joint infection requiring THA revision for which she received 10 cc of CSB in the hip joint during the 1st stage revision surgery. On post-operative day 3, her calcium level rapidly increased from 8.3 to 12.6 mg/dL. She was treated with intravenous fluids for dehydration with resolution of hypercalcemia and discharged home. One month later, she was admitted for revision of left THA with insertion of CSB. On post-operative day 3, her calcium increased from 9.3 to 13.8 mg/dL requiring fluids and calcitonin with suspicion that CSB may be the etiology. Two months later, she was admitted with polymicrobial drug resistant infection of the left hip. She underwent another left THA revision with insertion of 20 cc of CSB beads instead of the 10 cc kits previously used. Once again, on post-operative day 3, her calcium levels increased from 8.2 to 14.5 mg/dL. At this time, she was treated with intravenous fluids, calcitonin and 60mg IV pamidronate. Hypercalcemia workup did not show another etiology for hypercalcemia with a suppressed PTH level and normal PTH-related peptide, 25-hydroxy and 1,25-dihydroxy vitamin D levels. Kidney function was at baseline during each admission. The patient endorsed fatigue and constipation. As highlighted above, each episode of hypercalcemia peaked on post-operative day three and resolved within one week. When 20 cc of CSB were used, the peak calcium was more severe necessitating IV bisphosphonate therapy. CSB were not used in subsequent surgical revisions resulting in normal calcium levels. While CSB-induced hypercalcemia has been reported, this case highlights the importance of recognizing this entity and being aware that susceptible patients may be more likely to have recurrence with re-use. Pre- and post-CSB calcium levels should be measured in patients who receive CSB and they should be used at the minimum effective dose. Treatment of CSB-induced hypercalcemia may be considered when hypercalcemia is severe.

Expression Profile of Myoepithelial Cells in DCIS: Do They Change From Protective Angels to Wicked Witches?

The mechanism of transition of ductal carcinoma in situ (DCIS) to invasive cancer is elusive but recently changes in the myoepithelial cells (MECs) have been implicated. The aim of this study is to investigate the changes in gene profile of MECs in DCIS that could compromise their tumor suppressor function leading to promotion of tumor progression. Immuno-laser capture microdissection (LCM) was used to isolate MECs from normal and DCIS breast tissues followed by whole genome expression profiling using Affymetrix HGU-133 plus2.0 arrays. The data were analyzed using Bioconductor packages then validated by using real-time quantitative polymerase chain reaction and immunohistochemistry. Ingenuity Pathways software analysis showed clustering of most of the altered genes in cancer and cell death networks, with the Wnt/B-catenin pathway as the top canonical pathway. Validation revealed a 71.4% correlation rate with the array results. Most dramatic was upregulation of Fibronectin 1 ( FN1 ) in DCIS-associated MECs. Immunohistochemistry analysis for FN1 on normal and DCIS tissues confirmed a strong correlation between FN1 protein expression by MECs and DCIS ( P <0.0001) and between high expression level and presence of invasion ( P =0.006) in DCIS. Other validated alterations in MEC expression profile included upregulation of Nephronectin and downregulation of parathyroid hormone like hormone ( PTHLH ), fibroblast growth factor receptor 2 ( FGFR2 ), ADAMTS5 , TGFBR3 , and CAV1 . In vitro experiments revealed downregulation of PTHLH in DCIS-modified MECs versus normal lines when cultured on Fibronectin matrix. This is the first study to use this in vivo technique to investigate molecular changes in MECs in DCIS. This study adds more evidences to the molecular deviations in MECs toward tumor progression in DCIS through upregulation of the tumor-promoting molecules that may lead to novel predictive and therapeutic targets.

Actions of Parathyroid Hormone Ligand Analogues in Humanized PTH1R Knockin Mice.

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.

Parathyroid Hormone-Related Peptide and Its Analog, Abaloparatide, Attenuate Lethal Myocardial Ischemia-Reperfusion Injury.

Parathyroid hormone-related peptide (PTHrP) is well-known to play a role in bone formation, and abaloparatide, an analog of PTHrP(1-34), is approved for the treatment of osteoporosis in post-menopausal women. PTHrP has also been reported to have cardiovascular effects, with recent data demonstrating that exogenously administered PTHrP can limit the death of isolated cardiomyocytes subjected to oxidative stress via upregulation of classic ‘survival kinase’ signaling. Our aim in the current study was to extend this concept and, employing both in vitro and in vivo models, establish whether PTHrP(1-36) and abaloparatide are cardioprotective in the setting of lethal myocardial ischemia-reperfusion injury. We report that preischemic administration of PTHrP(1-36) and abaloparatide attenuated cell death in HL-1 cardiomyocytes subjected to simulated ischemia-reperfusion, an effect that was accompanied by the augmented expression of phospho-ERK and improved preservation of phospho-Akt, and blocked by co-administration of the MEK-ERK inhibitor PD98059. Moreover, using the translationally relevant swine model of acute coronary artery occlusion-reperfusion, we make the novel observation that myocardial infarct size was significantly reduced in pigs pretreated with PTHrP(1-36) when compared with placebo-controls (13.1 ± 3.3% versus 42.0 ± 6.6% of the area of at-risk myocardium, respectively; p < 0.01). Taken together, these data provide the first evidence in support of the concept that pretreatment with PTHrP(1-36) and abaloparatide renders cardiomyocytes resistant to lethal myocardial ischemia-reperfusion injury.

Functional Properties of Two Distinct PTH1R Mutants Associated With Either Skeletal Defects or Pseudohypoparathyroidism.

ABSTRACTConsistent with a vital role of parathyroid hormone (PTH) receptor type 1 (PTH1R) in skeletal development, homozygous loss‐of‐function PTH1R mutations in humans results in neonatal lethality (Blomstrand chondrodysplasia), whereas such heterozygous mutations cause a primary failure of tooth eruption (PFE). Despite a key role of PTH1R in calcium and phosphate homeostasis, blood mineral ion levels are not altered in such cases of PFE. Recently, two nonlethal homozygous PTH1R mutations were identified in two unrelated families in which affected members exhibit either dental and skeletal abnormalities (PTH1R‐V204E) or hypocalcemia and hyperphosphatemia (PTH1R‐R186H). Arg186 and Val204 map to the first transmembrane helix of the PTH1R, and thus to a critical region of this class B G protein‐coupled receptor. We used cell‐based assays and PTH and PTH‐related protein (PTHrP) ligand analogs to assess the impact of the R186H and V204E mutations on PTH1R function in vitro. In transiently transfected HEK293 cells, PTH1R‐R186H mediated cyclic adenosine monophosphate (cAMP) responses to PTH(1‐34) and PTHrP(1‐36) that were of comparable potency to those observed on wild‐type PTH1R (PTH1R‐WT) (half maximal effective concentrations [EC50s] = 0.4nM to 1.2nM), whereas the response‐maxima were significantly reduced for the PTH1R‐V204E mutant (maximum effect [Emax] = 81%–77% of PTH1R‐WT, p ≤ 0.004). Antibody binding to an extracellular hemagglutinin (HA) tag was comparable for PTH1R‐R186H and PTH1R‐WT, but was significantly reduced for PTH1R‐V204E (maximum binding level [Bmax] = 44% ± 11% of PTH1R‐WT, p = 0.002). The potency of cAMP signaling induced by a PTH(1‐11) analog was reduced by ninefold and threefold, respectively, for PTH1R‐R186H and PTH1R‐V204E, relative to PTH1R‐WT, and a PTH(1‐15) radioligand analog that bound adequately to PTH1R‐WT exhibited little or no specific binding to either mutant receptor. The data support a general decrease in PTH1R surface expression and/or function as a mechanism for PFE and a selective impairment in PTH ligand affinity as a potential PTH1R‐mutation‐based mechanism for pseudohypoparathyroidism. © 2022 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Parathyroid Hormone-Related Protein Inhibition Blocks Triple-Negative Breast Cancer Expansion in Bone Through Epithelial to Mesenchymal Transition Reversal.

ABSTRACTParathyroid hormone‐related protein (PTHrP) plays a major role in skeletal metastasis but its action mechanism has not been fully defined. We previously demonstrated the crucial importance of PTHrP in promoting mammary tumor initiation, growth, and metastasis in a mouse model with a mammary epithelium‐targeted Pthlh gene ablation. We demonstrate here a novel mechanism for bone invasion involving PTHrP induction of epithelial to mesenchymal transition (EMT) and cancer stem cells (CSCs) regulation. Clustered regularly interspaced short palindromic repeats (CRISPR)‐mediated Pthlh gene ablation was used to study EMT markers, phenotype, and invasiveness in two triple‐negative breast cancer (TNBC) cell types (established MDA‐MB‐231 and patient‐derived PT‐TNBC cells). In vitro, Pthlh ablation in TNBC cells reduced EMT markers, mammosphere‐forming ability, and CD44high/CD24low cells ratio. In vivo, cells were injected intratibially into athymic nude mice, and therapeutic treatment with our anti‐PTHrP blocking antibody was started 2 weeks after skeletal tumors were established. In vivo, compared to control, lytic bone lesion from Pthlh ‐ablated cells decreased significantly over 2 weeks by 27% for MDA‐MB‐231 and by 75% for PT‐TNBC‐injected mice (p < 0.001). Micro‐CT (μCT) analyses also showed that antibody therapy reduced bone lytic volume loss by 52% and 48% for non‐ablated MDA‐MB‐231 and PT‐TNBC, respectively (p < 0.05). Antibody therapy reduced skeletal tumor burden by 45% and 87% for non‐ablated MDA‐MB‐231 and PT‐TNBC, respectively (p < 0.002) and caused a significant decrease of CSC/EMT markers ALDH1, vimentin, and Slug, and an increase in E‐cadherin in bone lesions. We conclude that PTHrP is a targetable EMT molecular driver and suggest that its pharmacological blockade can provide a potential therapeutic approach against established TNBC‐derived skeletal lesions. © 2021 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Uncarboxylated osteocalcin promotes proliferation and metastasis of MDA-MB-231 cells through TGF-\u03b2/SMAD3 signaling pathway

BackgroundTriple-negative breast cancer (TNBC) is the most severe type of breast cancer owing to its high heterogeneity, aggressiveness and lack of treatment. Studies have reported that uncarboxylated osteocalcin (GluOC) promotes the development of prostate and other cancers. Studies have also found elevated levels of serum osteocalcin in breast cancer patients with bone metastasis, and serum osteocalcin can be a marker of bone metastasis. However, whether GluOC promotes the development of TNBC and the related mechanisms need to be further clarified.ResultsOur results revealed that GluOC is associated with the proliferation and metastasis of MDA-MB-231 cells. GluOC increased the viability and proliferation of MDA-MB-231 cells. In addition, GluOC enhanced the metastatic ability of MDA-MB-231 cells by promoting the expression of matrix metalloproteinase-2 (MMP2), matrix metalloproteinase-13 (MMP13), and vascular endothelial growth factor (VEGF) and inducing epithelial-mesenchymal transition (EMT). We also found that GluOC upregulated the expression of interleukin-8 (IL-8) and parathyroid hormone-related protein (PTHrP) genes in MDA-MB-231 breast cancer cells. Moreover, the promoting effect of GluOC was reversed in MDA-MB-231 breast cancer cells treated with specific inhibitor of SMAD3 (SIS3), a SMAD3 phosphorylation inhibitor.ConclusionOur research proved for the first time that GluOC facilitates the proliferation and metastasis of MDA-MB-231 cells by accelerating the transforming growth factor-β (TGF-β)/SMAD3 signaling pathway. Moreover, GluOC also promotes the gene expression of IL-8 and PTHrP. Both IL-8 and PTHrP can act as osteolytic factors in breast cancer cells. This study indicates that GluOC may be a useful target for preventing TNBC bone metastasis.

Polygonum cuspidatum Extract (Pc-Ex) Containing Emodin Suppresses Lung Cancer-Induced Cachexia by Suppressing TCF4/TWIST1 Complex-Induced PTHrP Expression.

Cachexia, which is characterised by the wasting of fat and skeletal muscles, is the most common risk factor for increased mortality rates among patients with advanced lung cancer. PTHLH (parathyroid hormone-like hormone) is reported to be involved in the pathogenesis of cancer cachexia. However, the molecular mechanisms underlying the regulation of PTHLH expression and the inhibitors of PTHLH have not yet been identified. The PTHLH mRNA levels were measured using quantitative real-time polymerase chain reaction, while the PTHrP (parathyroid hormone-related protein) expression levels were measured using Western blotting and enzyme-linked immunosorbent assay. The interaction between TCF4 (Transcription Factor 4) and TWIST1 and the binding of the TCF4–TWIST1 complex to the PTHLH promoter were analysed using co-immunoprecipitation and chromatin immunoprecipitation. The results of the mammalian two-hybrid luciferase assay revealed that emodin inhibited TCF4–TWIST1 interaction. The effects of Polygonum cuspidatum extract (Pc-Ex), which contains emodin, on cachexia were investigated in vivo using A549 tumour-bearing mice. Ectopic expression of TCF4 upregulated PTHLH expression. Conversely, TCF4 knockdown downregulated PTHLH expression in lung cancer cells. The expression of PTHLH was upregulated in cells ectopically co-expressing TCF4 and TWIST1 when compared with that in cells expressing TCF4 or TWIST1 alone. Emodin inhibited the interaction between TCF4 and TWIST1 and consequently suppressed the TCF4/TWIST1 complex-induced upregulated mRNA and protein levels of PTHLH and PTHrP. Meanwhile, emodin-containing Pc-Ex significantly alleviated skeletal muscle atrophy and downregulated fat browning-related genes in A549 tumour-bearing mice. Emodin-containing Pc-Ex exerted therapeutic effects on lung cancer-associated cachexia by inhibiting TCF4/TWIST1 complex-induced PTHrP expression.

Inhibition of epidermal growth factor receptor suppresses parathyroid hormone-related protein expression in tumours and ameliorates cancer-associated cachexia.

BackgroundLung cancer is the primary cause of cancer deaths worldwide. Activation of epidermal growth factor receptor (EGFR) leads to lung cancer progression and poor prognosis while involuntary weight loss remains a major problem. Tumour‐derived parathyroid hormone‐related protein (PTHrP) emerged as a potential mediator of cachexia. Here, we investigated the modulatory role of EGFR signalling in PTHrP (encoded by Pthlh) gene expression and the impact of this relationship on cancer cachexia.MethodsGlobal gene expression profiles of Lewis lung carcinoma (LLC) cells were analysed. Pthlh mRNA levels were measured by qRT‐PCR in LLC cells treated with EGFR ligands and tyrosine kinase inhibitors (TKIs). LLC tumour‐bearing mice received EGFR TKI erlotinib for 7 days via intraperitoneal injection or oral gavage. Tumour Pthlh mRNA, weight of fat/muscle tissue, and grip strength were assessed. RNA‐seq data from The Cancer Genome Atlas and gene expression analysis tools were used to characterize expression profiles of PTHLH and EGFR along with correlation analysis of PTHLH with EGFR and transforming growth factor alpha (TGFA) in human lung cancer and head and neck squamous carcinoma (HNSC). Survival of lung squamous cell carcinoma (LUSC) and lung adenocarcinoma (LUAD) patients with EGFR gene alterations was analysed in regard to PTHLH expression.ResultsExpression of EGFR ligands, EGFR itself, and PTHrP co‐clusters in LLC cells. Activation of EGFR signalling with its ligands significantly increases (3.8‐fold, P < 0.0005) while EGFR TKIs significantly decrease (90%, P < 0.0005) Pthlh mRNA levels in LLC cells. Pthlh mRNA drops 65–75% (P < 0.0005) in tumours upon treatment of LLC tumour‐bearing mice with erlotinib while their muscle mass and grip strength increase (9.2% P < 0.05, 23% P < 0.005, respectively) compared with tumour‐bearing control mice. PTHLH is overexpressed in tumours of LUSC (45.8‐fold, P < 0.05) and HNSC (17.5‐fold, P < 0.05) compared with normal tissue. PTHLH expression correlates with EGFR and its ligand TGFA in both cancers (LUSC: n = 745, R = 0.32, P < 0.0001 and R = 0.51, P < 0.0001; HNSC: n = 545, R = 0.34, P < 0.001 and R = 0.50, P < 0.001, respectively). High PTHLH mRNA associates with poor overall survival in LUAD patients with activating EGFR mutations (n = 40, log‐rank test, P = 0.0451).ConclusionsEpidermal growth factor receptor signalling regulates expression of cachexia mediator PTHrP. EGFR inhibition reduces PTHrP expression in LLC tumours and ameliorates cachexia in LLC tumour‐bearing mice.

87 Refractory Parathyroid Hormone-Related Peptide–Mediated Hypercalcemia of Pregnancy Treated With Cinacalcet

87 Refractory Parathyroid Hormone-Related Peptide–Mediated Hypercalcemia of Pregnancy Treated With Cinacalcet

Protein kinase A is activated during bone morphogenetic protein 2-induced osteogenic differentiation of dental follicle stem cells via endogenous parathyroid hormone-related protein

ObjectiveThe aim of this study was to investigate the mechanisms of how protein kinase A (PKA) is activated during bone morphogenetic protein 2 (BMP2)-induced osteogenic differentiation in dental follicle stem cells. DesignHuman dental follicle stem cells were cultured and treated with a BMP2-containing osteogenic differentiation medium or differentiation medium without BMP2. Specific siRNAs and substances/proteins were used to modulate pathways. PKA activity and activity of alkaline phosphatase were determined. Expression of targets was measured by Western Blots and reverse transcription-quantitative polymerase chain reaction, while protein interactions were investigated by immunoprecipitation. Immunofluorescence staining was used for subcellular target localization. ResultsPKA activity is stimulated after osteogenic induction by BMP2. Differentiation medium without BMP2 strongly induces BMP2 gene expression, which correlates with downstream target expression. Elevation of cAMP levels does not affect alkaline phosphatase activity and PKA does not directly interact with Smad 4. However, PKA activation requires expression of parathyroid hormone-related protein (PTHrP), which is stimulated after BMP2-induced differentiation. Furthermore, neither supplementation with PTHrP nor with the receptor antagonist parathyroid hormone (7−34) affects PKA activity. Thus, endogenous PTHrP expression is required for PKA activation and immunofluorescence staining shows that PTHrP is mainly located in the nucleus of dental follicle stem cells. Beyond, knockdown of PKA stimulates the BMP2 signaling pathway and down-stream expression of PTHrP. ConclusionsBMP2-induced osteogenic differentiation activates PKA in dental follicle stem cells via endogenous expression of PTHrP. Additionally, PKA inhibits BMP2 signaling and expression of PTHrP in a negative feedback loop.