High endogenous expression of parathyroid hormone-related protein (PTHrP) supports osteogenic differentiation in human dental follicle cells.

Dental follicle cells (DFCs) are progenitor cells for mineralizing cells such as alveolar osteoblasts, but little is known about the mechanisms of the differentiation. Interestingly, different cell lines sometimes have different potentials to differentiate into mineralizing cells. In this study, we compared two different DFC lines, with one cell line (DFC_B) showing a high alkaline phosphatase (ALP) activity in long-term cultures with standard medium and a reliable mineralizing potential. However, the other cell line DFC_A shows low ALP activity in standard medium and almost no mineralization. Known osteogenic markers such as RUNX2 were similarly expressed in both cell lines. However, the proosteogenic signaling pathway of the bone morphogenetic protein (BMP) is induced in DFC_B, and the parathyroid hormone-related protein (PTHrP), which is involved in tooth root development, was also expressed more strongly. Previous studies have shown that the secreted PTHrP negatively regulate the transition from pre-osteoblastic progenitors to osteoblasts, but we showed that an inhibition of PTHrP gene expression reduced the ALP activity and the BMP-signaling pathway. In addition, endogenously expressed PTHrP is located in the cell nucleus. In contrast, supplementation of PTHrP or an inhibitor for the PTHrP receptor did not affect the ALP activity of DFC_B. In conclusion, our data suggest that a high endogenous expression of PTHrP in DFCs supports the induction of osteogenic differentiation via an intracrine mode.

Cancer Diagnostics via Ultrasensitive Multiplexed Detection of Parathyroid Hormone-Related Peptides with a Microfluidic Immunoarray.

Parathyroid hormone-related peptide (PTHrP) is recognized as the major causative agent of humoral hypercalcemia of malignancy (HHM). The paraneoplastic PTHrP has also been implicated in tumor progression and metastasis of many human cancers. Conventional PTHrP detection methods like immunoradiometric assay (IRMA) lack the sensitivity required to measure target peptide levels prior to the development of hypercalcemia. In general, sensitive, multiplexed peptide measurement by immunoassay represents challenges that we address in this paper. We describe here the first ultrasensitive multiplexed peptide assay to measure intact PTHrP 1-173 as well as circulating N-terminal and C-terminal peptide fragments. This versatile approach should apply to almost any collection of peptides that are long enough to present binding sites for two antibodies. To target PTHrP, we employed a microfluidic immunoarray featuring a chamber for online capture of the peptides from serum onto magnetic beads decorated with massive numbers of peptide-specific antibodies and enzyme labels. Magnetic bead-peptide conjugates were then washed and sent to a detection chamber housing an antibody-modified 8-electrode array fabricated by inkjet printing of gold nanoparticles. Limits of detection (LODs) of 150 aM (∼1000-fold lower than IRMA) in 5 µL of serum were achieved for simultaneous detection of PTHrP isoforms and peptide fragments in 30 min. Good correlation for patient samples was found with IRMA (n = 57); r(2) = 0.99 assaying PTHrP 1-86 equiv fragments. Analysis by a receiver operating characteristic (ROC) plot gave an area under the curve of 0.96, 80-83% clinical sensitivity, and 96-100% clinical specificity. Results suggest that PTHrP1-173 isoform and its short C-terminal fragments are the predominant circulating forms of PTHrP. This new ultrasensitive, multiplexed assay for PTHrP and fragments is promising for clinical diagnosis, prognosis, and therapeutic monitoring from early to advanced stage cancer patients and to examine underlying mechanisms of PTHrP overproduction.

A correlation of breast cancer and calcium levels in hair analyzed by X-ray fluorescence.

Time variations of elemental concentrations and their abnormalities due to breast cancer have been observed along single hair strands by X-ray fluorescence excited by synchrotron radiation. The renal-controlled elements Ca, Sr, S, K, Cl, Br and P have upper and lower levels associated with gating and closing of ion channels in the hair-making cells. The Ca lower level is normal. In cases of Ca deficiency, with a decrease from the normal, store-operated Ca channel gating occurs so as to keep the hair Ca at the normal, and paradoxically high Ca levels near or at the upper level are produced by PTH-operated channel gating of the cells. Chronic Ca deficiency shows a temporal pattern along the hair consisting of a long-term duration of the upper [Ca] level, 10-month long decay to the lower level and abrupt increase to the upper level. The observation for hair from breast-cancer patients also shows the upper Ca level for the time period well before detection, and suggests that cancer is always generated at the long-lasting [Ca] upper level and the hair [Ca] decreases gradually toward the lower level with the cancer growth. This decay of [Ca] is accompanied by those of [Sr] and [K]. Their different decay forms can be explained by parathyroid hormone related peptide (PTHrP) in serum secreted from the cancer having 150 times longer dwell time on the PTH receptors than that of PTH. Patient hair has a memory for the entire cancer process from the state before cancer generation, and the pattern can be distinguished from concentration variation due to the chronic Ca deficiency without cancer, leading to a criterion for cancer detection by the ratio of [Sr]/[Ca]. The hair analysis is useful for early detection of cancer.

Effects of PTHrP on chondrocytes of sika deer antler.

Parathyroid-hormone-related peptide (PTHrP) is an important regulator of chondrocyte differentiation in growth plates but little is known about its role in deer antler cartilage. The aim of the present study was to use the deer antler as a model to determine the possible role of PTHrP in regulating chondrocyte differentiation of deer antler. PTHrP and its receptor PTH1R mRNA were highly expressed in the perichondrium and cartilage of sika deer antler, as shown by in situ hybridization. Chondrocytes of deer antler were identified by toluidine blue staining of glycosaminoglycan and immunocytochemical staining of type II collagen (Col II). Treatment with PTHrP (1-34) reduced the expression of prehypertrophic chondrocyte marker Col IX and hypertrophic chondrocyte marker Col X. In order to confirm the mechanism of action of PTHrP, we initially examined the expression of cyclin D1, Bcl-2 and runt-related transcription factor 2 (Runx2) in sika deer antler by in situ hybridization and found that cyclin D1, Runx2 and Bcl-2 mRNA were also expressed in antler chondrocytes. Exogenous PTHrP induced the expression of cyclin D1 and Bcl-2 mRNA by various signalling pathways, whereas it inhibited Runx2 expression through PKA, p38MAPK, MEK and PI3K signalling pathways. Thus, PTHrP might promote the proliferation of antler chondrocytes and prevent their differentiation; it might furthermore influence the growth and development of sika deer antler.

Localization of parathyroid hormone-related protein in the preimplantation mouse embryo is associated with events of blastocyst hatching.

PURPOSE: To determine the pattern of expression of parathyroid hormone-related protein (PTHrP) and its receptor, parathyroid hormone receptor 1 (PTHR1), in mouse embryos in different stages of preimplantation development. METHODS: Embryos were cultured from the pronuclear zygote stage and harvested as 2-cell, 4-cell and 8-cell embryos, morulae and blastocysts. RT-PCR was carried out on mRNAs of these and of trophoblast outgrowths for detection of PTHrP and PTHR1. Whole mounted embryos intact or stripped of zonae pellucidae were immunofluorescently stained for PTHrP and PTH receptor and observed with confocal microscopy. RESULTS: PTHrP mRNA was present in the pronuclear zygote, not present in 2-cell, 4-cell and uncompacted 8-cell embryos, present in the 8-cell compacting embryo, and not detected in 16-cell morulae or blastocysts. The mRNA was present in trophoblasts growing on fibronectin beds. mRNA for PTHR1 was detected in the pronuclear zygote, then undetected until the compacted 8-cell stage and thereafter. PTH receptor protein was observed in 2-cell embryos, morulae and in the inner cell mass and trophectoderm of blastocysts. PTHrP was observed dispersed in the cytoplasm of 2-cell, 4-cell and uncompacted 8-cell embryos, and in distinct foci near the nuclei of morulae. In blastocysts, PTHrP appeared on the apical surface of only trophoblast cells which had extruded from the zona pellucida. Fully hatched blastocysts expressed the protein on the apical side of all trophoblasts. When morulae were prematurely stripped of their zonae, PTHrP was observed on the embryos' outer surface. CONCLUSIONS: PTHrP protein is expressed throughout early embryo development, and its receptor PTHR1 is expressed from the morula stage. Embryo hatching is associated with translocation of PTHrP to the apical plasma membrane of trophoblasts. PTHrP may thus have autocrine effects on the developing blastocyst.

Malignancy-related Hypercalcemia Caused by Metameric Cutaneous Metastasis of Parathyroid Hormone-related Protein-producing Bladder Carcinoma with Squamous Cell Differentiation: An Autopsy Case of Cobb Syndrome.

A 74-year-old woman was admitted with hypercalcemia and prolonged disturbance of consciousness. The left buttock to the anterior aspect of the left thigh was swollen and erythematous, with a collection of 1.0-cm large, firm, elastic nodules distributed in a zosteriform pattern in the L1-L4 region. Based on autopsy findings, a very rare case of Cobb syndrome was diagnosed due to a spinal vascular malformation at the Th12-L4 level and L5 vertebral hemangioma. Cobb syndrome-associated cutaneous metastasis extending along the same metamere was complicated by immunohistochemically proven parathyroid hormone-related protein-producing advanced bladder carcinoma in this case.

Evidence for in vivo nicotine-induced alveolar interstitial fibroblast-to-myofibroblast transdifferentiation.

Nicotine exposure alters normal homeostatic pulmonary epithelial-mesenchymal paracrine signaling pathways, resulting in alveolar interstitial fibroblast (AIF)-to-myofibroblast (MYF) transdifferentiation. Though this has been described under in vitro conditions, it is not known if the same phenomenon also takes place in vivo. A well-established rodent model of lung damage following perinatal nicotine exposure was used. By probing for the well-established markers of fibroblast differentiation (parathyroid hormone-related protein [PTHrP], peroxisome proliferator-activated receptor gamma [PPARgamma], adipocyte differentiation-related protein, alpha-smooth muscle actin, and fibronectin) at the mRNA, protein, and tissue levels, the authors provide the first in vivo evidence for nicotine-induced AIF-to-MYF transdifferentiation. In addition, these data also provide the first evidence for nicotine-induced up-regulation of Wnt signaling, accompanying the down-regulation of PTHrP/PPARgamma signaling in vivo following nicotine exposure during pregnancy. These data provide an integrated mechanism for in utero nicotine-induced lung damage and how it could permanently alter the "developmental program" of the developing lung by disrupting critically important epithelial-mesenchymal interactions. More importantly, these data are likely to provide specific interventions to augment the pulmonary mesenchymal lipogenic pathway to ameliorate nicotine-induced in utero lung injury.

Modification of the analysis of parathyroid hormone-related protein in milk and concentrations of this protein in commercial milk and milk products in Japan.

Parathyroid hormone-related protein (PTHrP), which causes hypercalcemia associated with malignant tumors, is known to be present in milk. Gene expression of PTHrP in the mammary gland increases markedly during parturition and with the onset of lactation. Even when circulating PTHrP levels are extremely low or below the detection limit, milk PTHrP levels are remarkably high. Parathyroid hormone-related protein derived from the mammary gland is assumed to play a role in maintaining the maternal calcium homeostasis and calcium transport from blood to milk. In previous studies that determined the PTHrP concentrations in milk, the pretreatments and diluent composition were not standardized. Here, we investigated the effect of various pretreatment procedures and diluent constitutions and the consequent PTHrP concentrations in commercial milk and milk products in Japan. Significant differences were found in PTHrP concentrations in raw milk samples subjected to different combinations of pretreatments (mixing, centrifugation, acidification, and heating) and diluents (0pM standard solution of PTHrP, plasma treated with protease inhibitors, and original diluent). We measured the PTHrP concentrations in normal liquid milk, processed milk, milk drinks, formulated milk powders, and skim milk powder by using the appropriate combination of pretreatment (acidification) and diluent (plasma treated with protease inhibitors). The PTHrP concentration in normal liquid milk, processed milk, and skim milk powder was as high as that in raw milk (>5nM), whereas that in milk drinks differed considerably. The PTHrP concentration in infant formulas (<2nM) was lower than that in the other milk products. These results indicate that a certain amount of PTHrP is ingested when milk and milk products are consumed.

Humoral Hypercalcemia in a Patient with Cholangiocellular Carcinoma - Effective Therapy with Denosumab.

BACKGROUND Hypercalcemia in cholangiocellular carcinoma is a highly uncommon event, mainly reported in Asian patients. In the absence of bone metastases, humoral hypercalcemia of malignancy (HHM) can be assumed. This is mostly the consequence of an elevated parathormone-related peptide (PTHrP) level. The standard therapeutic options in HHM are sometimes limited by the underlying disease or concomitant diseases. CASE REPORT We report the case of a 65-year-old Caucasian male. A syncope due to a hypercalcemia of 4.16 mmol/L (normal range, 2.19-2.54 mmol/L) was the initial symptom that eventually led to the diagnosis of cholangiocellular carcinoma. He had no metastatic bone disease; HHM was suspected. PTHrP was moderately elevated. Since there were contraindications for the standard therapeutic options, a therapy with 120 mg denosumab was initiated and proved effective, safe, and restored the patient's quality of life for 11 months. CONCLUSIONS The moderate elevation of parathyroid hormone-related peptide (PTHrP) in this case is addressed in context with the recent insights of a substantial underestimation of this parameter by many commercial assays which can explain our observation. Denosumab, a human monoclonal antibody which acts as a RANKL-inhibitor (receptor activator of nuclear factor kappaB ligand) was recently suggested as a therapeutic alternative. In this case, the therapy of the hypercalcemia with denosumab due to contraindications for other therapies led to an effective and long-standing remission of hypercalcemia. Its effectivity should be studied in larger case samples.

Wnt/beta-catenin signaling regulates cranial base development and growth.

Wnt proteins and beta-catenin signaling regulate major processes during embryonic development, and we hypothesized that they regulate cranial base synchondrosis development and growth. To address this issue, we analyzed cartilage-specific beta-catenin-deficient mice. Mutant synchondroses lacked typical growth plate zones, and endochondral ossification was delayed. In reciprocal transgenic experiments, cartilage overexpression of a constitutive active Lef1, a transcriptional mediator of Wnt/beta-catenin signaling, caused precocious chondrocyte hypertrophy and intermingling of immature and mature chondrocytes. The developmental changes seen in beta-catenin-deficient synchondroses were accompanied by marked reductions in Ihh and PTHrP as well as sFRP-1, an endogenous Wnt signaling antagonist and a potential Ihh signaling target. Thus, Wnt/beta-catenin signaling is essential for cranial base development and synchondrosis growth plate function. This pathway promotes chondrocyte maturation and ossification events, and may exert this important role by dampening the effects of Ihh-PTHrP together with sFRP-1.

Clinical tumor growth and comparison with proliferation markers in non-functioning (inactive) pituitary adenomas.

BACKGROUND: As the development of clinically silent pituitary adenomas is not yet fully understood, the radiologically measured growth of inactive pituitary adenomas should be compared with adenoma classification and immunostainings for proliferation markers. MATERIAL AND METHODS: In 32 patients with non-functioning adenomas (NFA) from 45 operations with retrospectively available preoperative series of magnetic resonance imaging (MRI) we measured the largest growing diameter (LGD) in mm/ year. The adenomas were immunostained for Ki-67 (MiB-1), PCNA, p53 protein, IGF- and PTH-related protein. The positive nuclei for MiB-1, PCNA, and p53 protein were counted and their labelling indices (LI) were calculated. The clinical measurements were compared with these data and were statistically analysed (Spearman test, Whitney-U-test). The growth rate per year was available in 28 cases. We chosed three grades of LGD: less than 1.5 mm in diameter in 9 patients (32%), between 1.5 and 3.0 mm in 11 patients (39%) and more than 3.0 mm in 8 patients (29%). RESULTS: MiB-1 positive nuclei were found in 42% of adenomas, PCNA positive nuclei in 58% and p53 positive nuclei in 16%. IGF 1 was immuno-stained in 84% of adenomas. The mean LI for MiB-1 was 0.12 in adenomas growing less than 1.5 mm and 0.34 in adenomas growing more than 1.5 mm per year. For non-invasive adenomas, the MiB-1 LI was 0.03, for invasive adenomas it was 0.126 and for strongly invasive adenomas 0.212. The MiB-1 LI was lower in null cell adenomas than in FSH/LH adenomas. All these data for MiB-1 showed no statistically significant differences (p<0.05). PCNA LI in adenomas growing less than 1.5 mm per year was 0.51 in contrast to LI of 1.12 for those growing more than 1.5 mm. In non-invasive adenomas the PCNA LI was 0.796, in invasive adenomas 0.655 and in diffuse strongly invasive ones 1.011. Null cell adenomas had a lower PCNA LI than FSH/LH cell adenomas. CONCLUSIONS: Statistically significant differences were measured for the growth rate und the PCNA expression. P53 was immunostained in invasive adenomas only. There were no correlations to the clinical growth rate, but p53 expression correlated significantly to numbers of MiB-1 positive nuclei and PCNA positive nuclei. IGF-I expression was found to correlate inversely with age of patients. We recommend the use of PCNA if correlations to progression of tumor growth are wanted.

Parathyroid hormone related protein concentration in human serum and CSF correlates with age.

BACKGROUND: Parathyroid Hormone-Related Protein (PTHrP) is involved in intracellular calcium (Ca) regulation, and has been demonstrated to participate in regulation of Ca in brain cells, activation of neurons, and modulation of pain. However, there are conflicting reports regarding the presence of PTHrP in CSF. DESIGN AND METHODS: PTHrP and Ca were quantified in paired CSF and serum samples using mass spectrometry-based methods. Associations between PTHrP and Ca concentrations with age, sex and concentrations of nine CSF diagnostic markers in a set of 140 paired serum and CSF patient samples were evaluated. RESULTS: The observed median PTHrP concentration in CSF was 51 times higher than in serum; the median concentration of Ca in CSF was 1.8 times lower than in serum. We observed positive correlation between concentrations of PTHrP in CSF and serum (p=0.013). Distribution of PTHrP concentrations in serum was associated with age (p=0.0068) and the concentrations were higher in women. In samples with serum calcium concentrations within the reference intervals (n=118), central 95% distribution of concentrations for Ca-CSF, PTHrP-serum and PTHrP-CSF were 5.4 (4.5-6.1) mg/dL, 1.2 (0.5-2.5) pmol/L, 62 (22-125) pmol/L, respectively. CONCLUSIONS: Our data demonstrate that PTHrP is a normal constituent of human CSF with median concentrations 51 fold higher than in serum. Elevated serum PTHrP concentrations were positively correlated with age and significantly higher in women. Our data suggest that CSF could be a significant source of circulating PTHrP.

In situ monitoring of PTHLH secretion in neuroblastoma cells cultured onto nanoporous membranes.

In this work, we propose for the first time the use of anodic aluminum oxide (AAO) nanoporous membranes for in situ monitoring of parathyroid hormone-like hormone (PTHLH) secretion in cultured human cells. The biosensing system is based on the nanochannels blockage upon immunocomplex formation, which is electrically monitored through the voltammetric oxidation of Prussian blue nanoparticles (PBNPs). Models evaluated include a neuroblastoma cell line (SK-N-AS) and immortalized keratinocytes (HaCaT) as a control of high PTHLH production. The effect of total number of seeded cells and incubation time on the secreted PTHLH levels is assessed, finding that secreted PTHLH levels range from approximately 60 to 400 ng/mL. Moreover, our methodology is also applied to analyse PTHLH production following PTHLH gene knockdown upon transient cell transfection with a specific silencing RNA (siRNA). Given that inhibition of PTHLH secretion reduces cell proliferation, survival and invasiveness in a number of tumors, our system provides a powerful tool for the preclinical evaluation of therapies that regulate PTHLH production. This nanoporous membrane - based sensing technology might be useful to monitor the active secretion of other proteins as well, thus contributing to characterize their regulation and function.

The axolotl limb: a model for bone development, regeneration and fracture healing.

Among vertebrates, urodele amphibians (e.g., axolotls) have the unique ability to perfectly regenerate complex body parts after amputation. The limb has been the most widely studied due to the presence of three defined axes and its ease of manipulation. Hence, the limb has been chosen as a model to study the process of skeletogenesis during axolotl development, regeneration and to analyze this animal's ability to heal bone fractures. Extensive studies have allowed researchers to gain some knowledge of the mechanisms controlling growth and pattern formation in regenerating and developing limbs, offering an insight into how vertebrates are able to regenerate tissues. In this study, we report the cloning and characterization of two axolotl genes; Cbfa-1, a transcription factor that controls the remodeling of cartilage into bone and PTHrP, known for its involvement in the differentiation and maturation of chondrocytes. Whole-mount in situ hybridization and immunohistochemistry results show that Cbfa-1, PTHrP and type II collagen are expressed during limb development and regeneration. These genes are expressed during specific stages of limb development and regeneration which are consistent with the appearance of skeletal elements. The expression pattern for Cbfa-1 in late limb development was similar to the expression pattern found in the late stages of limb regeneration (i.e. re-development phase) and it did not overlap with the expression of type II collagen. It has been reported that the molecular mechanisms involved in the re-development phase of limb regeneration are a recapitulation of those used in developing limbs; therefore the detection of Cbfa-1 expression during regeneration supports this assertion. Conversely, PTHrP expression pattern was different during limb development and regeneration, by its intensity and by the localization of the signal. Finally, despite its unsurpassed abilities to regenerate, we tested whether the axolotl was able to regenerate non-union bone fractures. We show that while the axolotl is able to heal a non-stabilized union fracture, like other vertebrates, it is incapable of healing a bone gap of critical dimension. These results suggest that the axolotl does not use the regeneration process to repair bone fractures.

Bladder cancer outcome and subtype classification by gene expression.

Models of bladder tumor progression have suggested that genetic alterations may determine both phenotype and clinical course. We have applied expression microarray analysis to a divergent set of bladder tumors to further elucidate the course of disease progression and to classify tumors into more homogeneous and clinically relevant subgroups. cDNA microarrays containing 10,368 human gene elements were used to characterize the global gene expression patterns in 80 bladder tumors, 9 bladder cancer cell lines, and 3 normal bladder samples. Robust statistical approaches accounting for the multiple testing problem were used to identify differentially expressed genes. Unsupervised hierarchical clustering successfully separated the samples into two subgroups containing superficial (pT(a) and pT(1)) versus muscle-invasive (pT(2)-pT(4)) tumors. Supervised classification had a 90.5% success rate separating superficial from muscle-invasive tumors based on a limited subset of genes. Tumors could also be classified into transitional versus squamous subtypes (89% success rate) and good versus bad prognosis (78% success rate). The performance of our stage classifiers was confirmed in silico using data from an independent tumor set. Validation of differential expression was done using immunohistochemistry on tissue microarrays for cathepsin E, cyclin A2, and parathyroid hormone-related protein. Genes driving the separation between tumor subsets may prove to be important biomarkers for bladder cancer development and progression and eventually candidates for therapeutic targeting.

Chondrocyte-specific knockout of the G protein G(s)alpha leads to epiphyseal and growth plate abnormalities and ectopic chondrocyte formation.

UNLABELLED: G(s)alpha is a ubiquitously expressed G protein alpha-subunit that couples receptors to adenylyl cyclase. Mice with chondrocyte-specific ablation of the G(s)alpha gene had severe epiphyseal and growth plate abnormalities and ectopic cartilage formation within the metaphyseal region of the tibia. These results show that G(s)alpha negatively regulates chondrocyte differentiation and is the critical signaling mediator of the PTH/PTH-rP receptor in growth plate chondrocytes. INTRODUCTION: G(s)alpha is a ubiquitously expressed G protein alpha-subunit that mediates signaling through G protein-coupled receptors to activate the cAMP/protein kinase A signaling pathway. Although studies suggest an important role for G(s)alpha in regulating growth plate development, direct in vivo results examining this role are lacking. MATERIALS AND METHODS: The G(s)alpha gene was ablated in murine cartilage by mating mice with loxP sites surrounding the G(s)alpha promoter and first exon with collagen 2a1 promoter-Cre recombinase transgenic mice. Skeletal tissues were studied by gross and microscopic pathology, and gene expression was determined by in situ hybridization. RESULTS AND CONCLUSIONS: Mice with complete chondrocyte-specific G(s)alpha deficiency (homozygotes) died within minutes after birth and had severe epiphyseal and growth plate defects with shortening of the proliferative zone and accelerated hypertrophic differentiation of growth plate chondrocytes, a phenotype similar to that of PTH/PTH-related peptide (PTHrP) receptor knockout mice. Indian hedgehog and PTH/PTHrP receptor expression in prehypertrophic chondrocytes was unaffected in mutant mice. PTHrP expression in periarticular cartilage was increased in the mutant mice, probably because of the closer proximity of Ihh-secreting chondrocytes to the periarticular zone. In addition, these mice developed ectopic cartilage at the anterior side of the metaphyseal region in the tibia. Mice with partial G(s)alpha deficiency (heterozygotes) exhibited no phenotype. These results show that G(s)alpha negatively regulates chondrocyte differentiation and is the critical signaling mediator of the PTH/PTHrP receptor in epiphyseal and growth plate chondrocytes.

Hypercalcemia and pancreatic endocrine neoplasia with elevated PTH-rP: report of two new cases and subject review.

Humoral hypercalcemia of malignancy is widely associated with tumor production of parathyroid hormone related protein (PTH-rP). This peptide functions in endocrine, autocrine and paracrine mechanisms in a manner similar to PTH; increasing renal uptake of calcium, decreasing retention of phosphorous, and stimulating adenylate cyclase and phospholipase C. Although PTH-rP production has been well documented in neoplasms of the exocrine pancreas, we present here two cases of endocrine pancreatic neoplasms elaborating PTH-rP. We then review the literature of previous cases and delve into the pathophysiology of this peptide.

Properties of bisphosphonates in the 13762 rat mammary carcinoma model of tumor-induced bone resorption.

Bone metastasis from primary tumors is a clinically important complication of neoplastic progression. The role of parathyroid hormone-related protein (PTHrP) and transforming growth factor (TGF)-beta1 in this process has been clearly established. The current study describes an in vivo model of 13762 rat mammary carcinoma tumor cell-induced osteolysis in which PTHrP and TGF-beta1 expression is observed. Exposure of in vitro-cultured 13762 cells to doxorubicin, cis-platinum, carboplatin, methotrexate, 5-fluorouracil, paclitaxel, alendronate, risedronate, or pamidronate for 72 h resulted in varying effects on cell proliferation (IC(50) values of 0.005, 0.4, 1.9, >40, 17.9, 0.003, >40, >40, and 33.6 micro M, respectively). Tumor cells were implanted into the intramedullary space of the proximal tibia of rats, and the time course of tumor progression was evaluated using radiographic and microcomputed tomography scanning techniques. Trabecular bone mineral density, cortical bone mineral density, and whole bone mineral density were measured (in mg/cm(3)). In untreated animals, radiographic evidence of osteolysis was evident 7 days after implantation. Trabecular bone mineral density and whole bone mineral density were significantly decreased by 21 days after implantation (48% and 26%, respectively). Bisphosphonates showed broad protective activity against tumor-driven osteolysis, Immunohistochemical evaluation of s.c. and intratibially implanted cells demonstrated the expression of PTHrP and TGF-beta1. The results of this study demonstrate the ability of 13762 rat mammary carcinoma cells to elicit a measurable osteolysis and that bisphosphonates inhibit the tumor-induced bone resorption in this model.

ATP dependent Ca2+ transport across basal membrane of human syncytiotrophoblast in pregnancies complicated by intrauterine growth restriction or diabetes.

Neonates born after pregnancies complicated by diabetes or intrauterine growth restriction (IUGR) have increased incidence of hypocalcaemia. Furthermore, IUGR is associated with reduced bone mineralization in infancy and osteoporosis in adult life. We tested the hypothesis that placental calcium transport is altered in these pregnancy complications. Transport of calcium into syncytiotrophoblast basal plasma membrane (BM) vesicles was studied by rapid filtration and protein expression of Ca(2+) ATPase by Western blot. In IUGR Ca(2+) ATPase activity was increased by 48 per cent (n=13; P< 0.05) whereas protein expression was 15 per cent lower (n=13; P< 0.05) than in controls (n=16). Basal membrane ATP dependent calcium transport was unaltered in gestational diabetes (GDM) but increased by 54 per cent in insulin dependent diabetes (IDDM) compared to controls (P< 0.05; n =14). Diabetes did not affect Ca(2+) ATPase expression in BM. We have previously shown that the mid-molecular fragment of parathyroid hormone related peptide (PTHrP midmolecule) stimulates BM Ca(2+) ATPase in vitro. PTHrP midmolecule concentrations in umbilical cord plasma were measured using radioimmunoassay. The concentrations in umbilical cord plasma were increased in IUGR, but unaltered in diabetes. In conclusion, placental calcium pump is activated in IUGR and IDDM, which may be secondary to increased foetal calcium demand. We speculate that PTHrP midmolecule may be one mechanism for activating BM Ca(2+) ATPase in IUGR.

Gene expression profiling of mouse condylar cartilage during mastication by means of laser microdissection and cDNA array.

Little is known about the mechanisms of mandibular condylar growth. In this study, gene expression in the mandibular condylar cartilage of young post-natal mice was monitored by means of a cDNA microarray, real-time PCR, and laser microdissection before and after the initiation of mastication (newborn, 7 days, 21 days, initiation of mastication, and 35 days). Insulin-like growth factor-1 (IGF-I), transforming-growth-factor-beta-2 (TGFbeta2), and aggrecan mRNAs were clearly expressed at 21 days, while the expression of osteopontin mRNAs was most clear at 35 days. Parathyroid-hormone-related protein (PTHrP), Indian-hedgehog (Ihh), and insulin-like growth factor-2 (IGF-2) mRNAs were clearly expressed during lactation (newborn and 7 days). Heat-shock-protein 84 (HSP-84) and heat-shock-protein 86 (HSP-86) were clearly expressed at 35 days. These results revealed that gene expression changed during mandibular condylar cartilage growth, and that, interestingly, these changes coincided with the initiation of mastication.