Concurrent primary hyperparathyroidism and humoral hypercalcemia of malignancy in a patient with multiple endocrine neoplasia type 1

We report a patient with multiple endocrine neoplasia type 1 presenting with elevation of parathyroid hormone-related protein (PTHrP) from a metastatic pancreatic neuroendocrine tumor (PNET), and parathyroid hormone (PTH) from primary hyperparathyroidism, resulting in severe hypercalcemia. Parathyroid hormone-related protein production by the PNET was confirmed by immunohistochemical analysis. Hypercalcemia and elevated PTHrP improved markedly with hepatic artery chemoembolization of liver metastasis. Thus, in multiple endocrine neoplasia type 1, correct identification of the cause of hypercalcemia as PTHrP production from a PNET or PTH production from a parathyroid tumor has important therapeutic implications.     

The PTH/PTHrP receptor in Jansen's metaphyseal chondrodysplasia

JMC is a rare autosomal dominant form of short limb dwarfism characterized by asymptomatic hypercalcemia and skeletal deformities, despite low PTH and PTHrP levels. This rare disorder is likely to be caused by activating mutations in the PTH/PTHrP receptor leading to ligand-independent cAMP accumulation. The analysis of genetically altered mice which lack either PTHrP or the PTH/PTHrP receptor, as well as of transgenic mice in which the mutant receptor is targeted to the growth plate, has provided a molecular explanation for the severe skeletal abnormalities seen in JMC. In addition, the study of this rare human disorder has further elucidated the fundamental role played by the PTH/PTHrP receptor in mediating both the paracrine/autocrine actions of PTHrP in growth plate development and bone elongation, as well as the endocrine actions of PTH. The insight gained from the study of this human disease model is likely to continue to provide an important tool to define the cellular and molecular mechanisms that mediate the biological roles of the PTH, PTHrP and their receptor.     

Parathyroid hormone expression in a patient with metastatic nasopharyngeal rhabdomyosarcoma and hypercalcemia

Ectopic PTH secretion by tumor cells has been described as the cause of hypercalcemia associated with malignancy in the absence of osteolytic bone lesions. Although there have been case reports of elevated PTH and hypercalcemia in patients with rhabdomyosarcoma, to date ectopic PTH secretion by malignant cells has not been definitively shown. The possibility of PTH production by pleural-based metastatic nasopharyngeal rhabdomyosarcoma cells in a 62-yr-old Japanese male with hypercalcemia was investigated. The patient's serum PTH level was found to be elevated at 62.22 pmol/L, and pleural fluid PTH level was 47.28 pmol/L and PTHrP level was 3.7 pmol/L. RT-PCR of mRNA extracted from rhabdomyosarcoma cells in the pleural fluid was performed with the addition of PTH and PTHrP exonic primer sets yielded only a cDNA fragment of approx 150 bp consistent with the expected PTH fragment. Sequence analysis of a nested primer PCR fragment confirmed PTH mRNA sequence. We believe this patient to have had hypercalcemia secondary to ectopic PTH secretion, as we have identified the presence of PTH mRNA in tumor cells. We speculate that the overexpression of PTH in rhabdomyosarcoma cells results from molecular rearrangement of the PTH gene. The finding of a normal PTH DNA sequence of the PCR fragment suggests the likelihood of alterations in regulatory sequences.     

Lung cancer associated with hypercalcemia induced by concurrently elevated parathyroid hormone and parathyroid hormone-related protein levels

In general, many cases of malignancy-associated hypercalcemia are due to HHM. In patients with humoral hypercalcemia of malignancy (HHM), it has been reported that plasma parathyroid hormone-related protein (PTHrP) and cyclic adenosine monophosphate (cAMP) levels were elevated, while plasma PTH and active vitamin D(3) levels were suppressed. Our patient showed hypercalcemia with a concurrent increase in plasma and tumor tissue PTHrP and PTH concentrations and also high cAMP and low 1-25(OH)(2)VD(3) levels in the plasma. These data suggest that the hypercalcemia exhibited by our patient was consistent with HHM due to lung cancer and its liver metastasis. Moreover, diagnostic imaging and autopsy findings showed no appreciable lesions of the parathyroid gland. In addition, histopathologic examination of the primary and metastatic tumors revealed the existence of PTH immunohistochemically stained with anti-PTH antibodies, suggesting an ectopic-PTH-producing lung tumor. From these data, our patient was diagnosed with a rare case of lung cancer, which produced both ectopic PTH and PTHrP.     

Severe growth retardation and early lethality in mice lacking the nuclear localization sequence and C-terminus of PTH-related protein

Parathyroid hormone (PTH) plays a central role in the regulation of serum calcium and phosphorus homeostasis, while parathyroid hormone-related protein (PTHrP) has important developmental roles. Both peptides signal through the same G protein-coupled receptor, the PTH/PTHrP or PTH type 1 receptor (PTH1R). PTHrP, normally a secreted protein, also contains a nuclear localization signal (NLS) that in vitro imparts functionality to the protein at the level of the nucleus. We investigated this functionality in vivo by introducing a premature termination codon in Pthrp in ES cells and generating mice that express PTHrP (1–84), a truncated form of the protein that is missing the NLS and the C-terminal region of the protein but can still signal through its cell surface receptor. Mice homozygous for the knock-in mutation (Pthrp KI) displayed retarded growth, early senescence, and malnutrition leading postnatally to their rapid demise. Decreased cellular proliferative capacity and increased apoptosis in multiple tissues including bone and bone marrow cells were associated with altered expression and subcellular distribution of the senescence-associated tumor suppressor proteins p16^INK4a and p21 and the oncogenes Cyclin D, pRb, and Bmi-1. These findings provide in vivo experimental proof that substantiates the biologic relevance of the NLS and C-terminal portion of PTHrP, a polypeptide ligand that signals mainly via a cell surface G protein-coupled receptor.     

高糖诱导NRK-52E细胞甲状旁腺素相关肽及其受体1表达及氯沙坦干预研究

为探讨甲状旁腺素相关肽(PTHrP)及其受体1(PTH1R)在高糖诱导大鼠肾小管导管上皮细胞株NRK-52E表达及氯沙坦对其表达干预作用.应用RT-PCR及Western印迹检测空白组(0 mmol/L),正常葡萄糖组(5 mmol/L)、中高浓度葡萄糖组(16.7 mmol/L)及高浓度葡萄糖组(25 mmol/L)中PTHrP及PTHIR在NRK-52E细胞表达.然后10 μoml/L氯沙坦及25 mmol/L葡萄糖分别干预72 h,应用Western印迹检测PTHrP及PTM1R表达情况.结果发现高浓度葡萄糖能上调PTHrP/PTH1R蛋白(PTHrP:0.63±0.12,0.68±0.06,1.02±0.11和1.04±0.08;PTH1R:0.88±0.05,0.87±0.10,1.05±0.11和1.12±0.09)及mRNA表达(PTHrP:0.66±0.08,0.84±0.13,1.57±0.15和1.73±0.21;PTH1R:0.26±0.08,0.28±0.07,2.35±0.10和2.47±0.05).氯沙坦能显著下调高浓度葡萄糖状态下PTHrP及PTH1R表达(PTHrP 0.74±0.15,PTH1R 0.98±0.06,均P<0.01).提示氯沙坦可以抑制高浓度葡萄糖诱导PTHrP/PTH1R的表达水平,从而可能影响肾小管导管上皮转分化.     

Circulating PTH and PTHrP levels before and after treatment of tumor induced hypercalcemia with Pamidronate Disodium (APD).

The effect of lowering ionized calcium on circulating parathyroid hormone (PTH) and parathyroid hormone-related protein (PTHrP) was assessed in twenty patients with hypercalcemia of malignancy following treatment with Pamidronate Disodium. Ionized calcium levels fell rapidly in all treated patients. PTH concentrations were initially suppressed below normal in 18 patients, but rose from 0.48 +/- 0.42 pmol/L to 3.63 +/- 3.13 pmol/L (p less than 0.01) after treatment, reaching higher than normal values in some patients even in the presence of persistent hypercalcemia. PTHrP concentrations did not change significantly after treatment. These findings are consistent with an increased sensitivity of parathyroid tissue to changes in ionized calcium following prolonged exposure to hypercalcemia. Regulation of tumor secretion of PTHrP by calcium was not apparent within the range of calcium concentrations in this study.     

Expression cloning of a common receptor for parathyroid hormone and parathyroid hormone-related peptide from rat osteoblast-like cells: a single receptor stimulates intracellular accumulation of both cAMP and inositol trisphosphates and increases intracellular free calcium.

Parathyroid hormone (PTH), a major regulator of mineral ion metabolism, and PTH-related peptide (PTHrP), which causes hypercalcemia in some cancer patients, stimulate multiple signals (cAMP, inositol phosphates, and calcium) probably by activating common receptors in bone and kidney. Using expression cloning, we have isolated a cDNA clone encoding rat bone PTH/PTHrP receptor from rat osteosarcoma (ROS 17/2.8) cells. The rat bone PTH/PTHrP receptor is 78% identical to the opossum kidney receptor; this identity indicates striking conservation of this receptor across distant mammalian species. Additionally, the rat bone PTH/PTHrP receptor has significant homology to the secretin and calcitonin receptors but not to any other G protein-linked receptor. When expressed in COS cells, a single cDNA clone, expressing either rat bone or opossum kidney PTH/PTHrP receptor, mediates PTH and PTHrP stimulation of both adenylate cyclase and phospholipase C. These properties could explain the diversity of PTH action without the need to postulate other receptor subtypes.     

PTHrP and insulin levels following oral glucose and calcium administration

BACKGROUND: Parathyroid hormone-related peptide (PTHrP), identified as a tumor product, is responsible for humoral malignant hypercalcemia. Unlike PTH, PTHrP is found in almost every body tissue including pancreatic alpha, beta, delta, and pp cells, where it is processed into multiple secretory forms, co-packaged with insulin, and secreted in a regulated fashion in response to insulin secretagogues. Ionized calcium is a stimulator for the release of several peptide hormones. METHODS: In the present study, we examined the effect of an oral calcium (1 g elemental calcium) and glucose (75 g) load on insulin and PTHrP release in 16 healthy volunteers and of an oral calcium load in 16 non-insulin-dependent diabetes mellitus (NIDDM) patients. Serum calcium, glucose, insulin, and PTHrP levels were determined at 0, 5, 10, 15, 30, and 60 min. RESULTS: Our results indicate that, at each time point, type 2 diabetic patients exhibited greater basal values of PTHrP than controls (200.3+/-110.5 pg/ml vs. 82.0+/-22.3 pg/ml, respectively, p<0.0001). The PTHrP level was consistently higher in response to the glucose load than the calcium load at each time point observed (p<0.0001). NIDDM patients exhibited greater basal serum PTHrP levels than the control group. CONCLUSION: PTHrP was proven for the first time to be released from beta cells in parallel to insulin and in response to glucose stimulation.     

Humoral hypercalcemia of malignancy caused by parathyroid hormone-related peptide-secreting neuroendocrine tumors: Report of six cases

We report the clinical characteristics and management of six patients with metastatic gastroentero-pancreatic neuroendocrine tumor (NET) presenting with severe hypercalcemia due to elevation of parathyroid hormone-related protein (PTHrP). All patients had histological confirmation of NET, five well-differentiated and one poorly differentiated. In 5 patients, hypercalcemia developed after years after the initial diagnosis of NET. One patient presented with concomitant elevation of PTHrP and intact parathyroid hormone (PTH) in the setting of multiple endocrine neoplasia 1 (MEN1). In all the other cases, PTH levels were low or undetectable. Management of malignant hypercalcemia due to PTHrP-producing NET is challenging, and optimal therapy depends on the extent of metastatic disease and the grade of malignancy. Aggressive tumor cytoreduction in addition to the systemic treatment modalities is frequently used to control disease progression and endocrine symptoms. To our knowledge, this is the largest series to date of hypercalcemia mediated by PTHrP-secreting NET.     

Pancreatic neuroendocrine tumor with extensive vascularisation and parathyroid hormone-related protein (PTHrP)--associated hypercalcemia of malignancy.

We report the case of a 34 year old male presenting with symptomatic hypercalcemia due to excessive PTHrP secretion from a pancreatic neuroendocrine carcinoma with extensive hypervascularization and without any evidence for metastatic disease. In the early phase of the disease conventional chemotherapy with streptozocin and doxorubicin was able to control functional activity as well as tumor growth. However, after 2 years tumor escape was indicated by severe therapy-resistant hypercalcemia. Therapeutic options were reduced due to the excessive tumor vascularization and the patient died from his disease after a short period of intensified therapy. The role of PTHrP in hypercalcemia of malignancy (HHM) and its association with neuroendocrine pancreatic tumors as well as possible therapeutic options are reviewed.     

Jansen's metaphyseal chondrodysplasia A disorder due to a PTH/PTHrP receptor gene mutation.

Jansen's metaphyseal chondrodysplasia (JMC) is a rare genetic disorder that is characterized by short-limbed dwarfism and severe, agonist-independent hypercalcemia. An activating PTH/PTHrP receptor mutation that results in constitutive cAMP accumulation was recently identified in the genomic DNA of a patient with this disorder. These findings provide a plausible explanation for the abnormal regulation of growth-plate chondrocytes and mineral ion homeostasis in JMC, and may have significant implications for understanding the broader biological role of PTHrP and its receptor.     

Expressions of parathyroid hormone‐related protein (PTHrP) and PTH/PTHrP‐receptor (PTH/PTHrP‐R) in gastrointestinal stromal tumours (GISTs), leiomyomas and schwannomas

Background: Gastrointestinal stromal tumours (GISTs) are rare, c‐kit and CD34 positive, and different from other mesenchymal tumours of the gastrointestinal tract (e.g. leiomyomas and schwannomas). The purpose of this study was to investigate the roles of parathyroid hormone‐related protein (PTHrP) and parathyroid hormone/parathyroid hormone‐related protein‐receptor (PTH/PTHrP‐R) in the growth and differentiation of GISTs. Methods: Nineteen GISTs, six leiomyomas and five schwannomas were examined in this study. Results: All of the GISTs and leiomyomas, and four of the schwannomas (80.0%) were positive for PTHrP. Immunohistochemical staining revealed that all of the leiomyomas, 90% of the GISTs and 80% of the schwannomas expressed PTH/PTHrP‐R. Furthermore, both PTHrP and PTH/PTHrP‐R were expressed in the cytoplasm of identical cells in all of these tumours. Conclusion: Our results suggest that both PTHrP and PTH/PTHrP‐R play an important role in the growth and differentiation of GISTs, leiomyomas and schwannomas.     

Ischemic injury activates PTHrP and PTH1R expression in human ventricular cardiomyocytes

The PTHrP/PTH1R signalling system induces calciotropic and myorelaxant effects on the vascular system and plays critical roles in the development of the cardiovascular system. In animal models, PTHrP exerts important effects on heart rate and contractility, particularly under ischemic conditions, while, in human hearts, the expression of PTHrP by cardiomyocytes remains to be defined in both normal and ischemic conditions. The present study has been conducted on 101 autoptical cases and confirmed on nine explanted hearts in order to analyze the expression of the PTHrP/PTH1R system by ventricular myocardium in respect to morphological aspects of the myocardial ischemic damage, myofiber hypertrophy and disarray, coronarosclerosis, age and sex. Immunohistochemistry showed positive cytoplasmic immunostaining for both PTHrP and PTH1R in ventricular cardiomyocytes. The expression levels of the PTHrP/PTH1R system resulted significantly increased (P = 0.0008 and P < 0.0001, respectively) in association with the myocardial ischemic damage and the presence of cardiomyocyte hypertrophy (P = 0.02 and P = 0.009 respectively). Conversely, increased expression levels of PTHrP alone were observed in myofiber disarray (P = 0.04), whereas PTH1R was augmented in coronarosclerosis (P = 0.004) and age (P = 0.001). Taken together, these results demonstrate that human ventricular cardiomyocytes express PTHrP and PTH1R and suggest that the activation of the PTHrP/PTH1R system could represent an aspect of the embryonic gene program typically reactivated by the myocardium when subjected to ischemia and/or hypertrophy. P. Di Nardo and A. Capelli contributed equally to this work. Returned for 1. Revision: 5 May 2008 1. Revision received: 31 October 2008     

Regulation of PTH/PTH-related protein receptor expression by endogenous PTH-related protein in the rat osteosarcoma cell line ROS 17/2.8

We have utilized clonal lines of the rat osteoblastic cell line ROS 17/2.8 stably transfected with full-length parathyroid hormone-related protein (PTHrP) cDNA in a sense or an antisense orientation to examine the effects of alteration in the production of endogenous PTHrP on expression of the PTH/PTHrP receptor. In the stably transfected clonal cell lines, changes in PTH/PTHrP receptor expression were evaluated by Northern blot analysis, whole-cell ligand binding of ¹²⁵I-[Tyr³⁶] PTHrP (1–36), and exogenous PTHrP (1–34)-stimulated cyclic adenosine monophosphate (cAMP) accumulation. Compared to control (vector-transfected) cells, PTHP-overproducing (sense-transfected) cells exhibited a marked decrease in the expression of PTH/PTHrP receptor mRNA and PTHrP ligand binding, as well as a corresponding decrease in the PTHrP (1–34)-stimulated cAMP response. By contrast, the antisense-transfected cells showed a marked increase in expression of PTH/PTHrP receptor mRNA and PTHrP (1–34) ligand binding, but a significant increase in the PTHrP (1–34)-stimulated cAMP response was not detected. Using antisense-transfected ROS cells, PTH/PTHrP receptor mRNA expression and ¹²⁵I-[Tyr³⁶] PTHrP (1–36) binding were downregulated by treatment for 24 h with exogenous PTHrP (1–36), forskolin, or dibutyryl cAMP. The findings extend those of earlier studies showing receptor downregulation by exogenous PTH by indicating that endogenous PTHrP, as well as circulating PTH, may help regulate receptor production; and suggesting that even very low concentrations of the peptide may influence receptor production.     

Elevation of circulating plasma cytokines in cancer patients with high plasma parathyroid hormone-related protein levels

Parathyroid hormone (PTH) and PTH-related protein/peptide (PTHrP) bind to the same PTH/PTHrP receptor and stimulate osteoblasts to secrete pro-inflammatory cytokines like interleukin (IL)-6. In patients with primary hyperparathyroidism, elevation of plasma levels of tumor necrosis factor (TNF)-alpha and IL-6 was also described. We, therefore, postulated that PTHrP secreted from cancer cells stimulates the secretion of cytokines and causes increases in their blood levels. Blood concentrations of several cytokines (TNF-alpha, IL-1beta, IL-5, IL-6, IL-8, IL-11 and IL-12) in cancer-bearing patients with or without elevation of blood PTHrP were measured by ELISA. The patients with high plasma PTHrP levels (n=29, intact PTHrP: 8.5 +/- 1.4 pmol/l, normal: <1.1) had higher serum type 1 collagen C-telopeptide (ICTP). Twenty of the patients were hypercalcemic. Plasma concentrations of TNF-alpha, IL-6 and IL-8 were significantly increased in patients with high PTHrP, in either the presence or absence of hypercalcemia. The concentrations of TNF-alpha and IL-6 were also significantly correlated with those of PTHrP. Our observations indicate that high plasma levels of PTHrP in cancer-bearing patients contribute not only to the development of hypercalcemia, but also to the development of the syndrome caused by an excess of pro-inflammatory cytokines.     

Mutation of phenylalanine-34 of parathyroid hormone disrupts NHERF1 regulation of PTH type I receptor signaling

Internalization of the PTH type I receptor (PTH1R) is regulated in a cell- and ligand-specific manner. We previously demonstrated that the sodium/proton exchanger regulatory factor type 1 (NHERF1; EBP50) is pivotal in determining the range of peptides that internalize the PTH1R. Antagonist PTH fragments can internalize the PTH1R in some kidney and bone cell models. PTH(7-34), which binds to, but does not activate, the PTH1R, internalizes the PTH1R in kidney distal tubule (DT) cells, where NHERF1 is not expressed. The effect of antagonist PTHrP peptides has not, to this point, been assessed. PTH1R internalization was measured by real-time confocal fluorescence microscopy of DT cells stably expressing 10⁵ EGFP-tagged PTH1R/cell (1). PTHrP(7-34) internalized the PTH1R in a manner indistinguishable from PTH(7-34). Introduction of NHERF1 into DT cells, however, blocked PTH(7-34)—, but not PTHrP(7-34)—, induced PTH1R internalization. To delineate the sequences within PTHrP that determine whether PTH1R internalization is affected by NHERF1, chimeric PTH/PTHrP fragments were tested for their ability to induce PTH1R internalization. PTH(7-21)/PTHrP (22-34), PTH(7-32)/PTHrP(33-34), and PTH(7-33)/PTHrP(34) at 1 μM each internalized the PTH1R 50–70% in a NHERF1-independent manner. When the C terminus of PTHrP was replaced with homologous amino acids from PTH, NHERF1 inhibited PTH1R internalization. It was determined that simply mutating F34 to A in PTH induced PTH1R internalization in a NHERF1-independent manner. None of the chimeric peptides activated the PTH1R but all effectively competed for 1 nM PTH(1-34) in cyclic AMP assays. In addition, all chimeric peptides competed for radiolabeled PTH(1-34) in binding assays in DT cells. PTH(1-34) and PTHrP(7-34), but not PTH(7-34), efficiently recruited β-arrestin1 to plasma membrane PTH1Rs. We, therefore, conclude that PTH(1-34) and PTHrP(7-34) induce a conformational change in the PTH1R that promotes arrestin binding and dissociates NHERF1 from PTH1R internalization.     

The Role of PTHrP in Skeletal Metastases and Hypercalcemia of Malignancy

Since its discovery as the principal mediator of humoral hypercalcemia of malignancy, parathyroid hormone-related protein (PTHrP) has emerged as a key player in skeletal complications associated with solid tumor metastasis to bone. In addition to functioning as an endocrine factor, this pleiotropic peptide mediates its actions locally on tumor and stromal cells in paracrine, autocrine, and intracrine fashion when cancer metastasizes to the bone compartment. Multiple splice variants and newly described PTHrP fragments confer diverse functions to PTHrP that extend beyond binding to its common receptor with parathyroid hormone. Here, we summarize the causal role of PTHrP in humoral hypercalcemia of malignancy and its local involvement in the progression of osteolytic and osteoblastic cancer bone metastases. Clinical and preclinical findings describing how PTHrP regulates tumor and stromal cell interactions are summarized, with emphasis on emerging evidence of PTHrP’s role in tumorigenesis and cancer cell proliferation. Finally, we examine therapeutic opportunities and limitations to targeting PTHrP directly and indirectly in cancer patients.     

甲状旁腺素相关肽对骨髓基质细胞中RANKL及OPG基因的表达影响

目的 研究甲状旁腺素相关肽(PTHrP)对骨髓基质细胞RANKL／OPG基因表达的影响。方法 分离小鼠骨髓细胞后，45ng／ml的PTHrP刺激，第6天时观察破骨细胞生成情况。90ng／mlPTHrP刺激贴壁鼠骨髓基质细胞3d和6d，实时荧光PCR检测破骨细胞分化因子RANKL及骨保护素OPG基因的表达情况。结果在PTHrP的刺激下，大量破骨细胞生成。骨髓基质细胞在PTHrP的刺激下，RANKI基因表达升高，OPG基因表达降低。结论 PTHrP刺激破骨细胞生成是通过上调RANKI基因表达，下调OPG基因的表达实现的。