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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.

Background and Aims: A growing body of research has demonstrated that the degeneration of chondrocytes is the primary cause of osteoarthritis (OA). Parathyroid hormone-related protein (PTHrP) can alleviate the degeneration of chondrocytes via promotion of chondrocyte proliferation and inhibition of terminal differentiation, but the underlying mechanism remains unknown. This study aimed to identify the microRNAs (miRNAs) that may target PTHrP and regulate the proliferation and terminal differentiation of chondrocytes., Methods: Bioinformatic analysis was used to predict which miRNAs target PTHrP. We collected human knee cartilage specimens to acquire the primary chondrocytes, which we then used to test the expression and function of the targeted miRNAs. To explore the effects of miR-15a-5p on the putative binding sites, specific mimics or inhibitors were transfected into the chondrocytes. Furthermore, a dual-luciferase reporter gene assay and chondrocyte degeneration-related factors were used to verify the possible mechanism., Results: The expression of PTHrP was upregulated in the OA chondrocytes, whilst miR-15a-5p was downregulated in the OA chondrocytes. A negative correlation was observed between PTHrP and miR-15a-5p. The knockdown of miR-15a-5p promoted the growth of chondrocytes and inhibited calcium deposition, whilst overexpression of miR-15a-5p reversed this trend. The effect of miR-15a-5p overexpression was neutralised by PTHrP. Dual-luciferase reporter assays revealed that PTHrP can be used as a novel targeting molecule for miR-15a-5p., Conclusions: miR-15a-5p promotes the degeneration of chondrocytes by targeting PTHrP and, in addition to helping us understand the development of OA, may be a potential biomarker of OA.

During zebrafish fin regeneration, blastema cells lining the epidermis differentiate into osteoblasts and joint cells to reconstruct the segmented bony rays. We show that osteoblasts and joint cells originate from a common cell lineage, but are committed to different cell fates. Pre-osteoblasts expressing runx2a/b commit to the osteoblast lineage upon expressing sp7 , whereas the strong upregulation of hoxa13a correlates with a commitment to a joint cell type. In the distal regenerate, hoxa13a , evx1 and pthlha are sequentially upregulated at regular intervals to define the newly identified presumptive joint cells. Presumptive joint cells mature into joint-forming cells, a distinct cell cluster that maintains the expression of these factors. Analysis of evx1 null mutants reveals that evx1 is acting upstream of pthlha and downstream of or in parallel with hoxa13a Calcineurin activity, potentially through the inhibition of retinoic acid signaling, regulates evx1 , pthlha and hoxa13a expression during joint formation. Furthermore, retinoic acid treatment induces osteoblast differentiation in mature joint cells, leading to ectopic bone deposition in joint regions. Overall, our data reveal a novel regulatory pathway essential for joint formation in the regenerating fin., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

Purpose: Expression of the carboxyl PTHrP region of parathyroid hormone-related protein (PTHrP) is a positive prognostic indicator in women with lung cancer, but amino PTHrP is a negative indicator in other lung cancer patients. This project investigated whether PTHrP could be expressed as predominantly amino PTHrP or carboxyl PTHrP in individual lung carcinomas. It also assessed domain-specific effects on cancer progression and patient survival., Methods: PTHrP immunoreactivities were analyzed versus survival in a human lung cancer tissue microarray (TMA). Growth was compared in athymic mice for isogenic lung carcinoma xenografts differing in expression of amino and carboxyl PTHrP domains., Results: In the TMA, 33 of 99 patient tumors expressed only one PTHrP domain, while 54 expressed both. By Cox regression, the hazard ratio for cancer-specific mortality (95% confidence interval) was 2.6 (1.28-5.44) for amino PTHrP (P = 0.008) and 0.6 (0-2.58) for carboxyl PTHrP (P = 0.092). Xenografts of H358 lung adenocarcinoma cells that overexpressed amino PTHrP grew twice as fast as isogenic low PTHrP tumors in athymic mice, but growth of tumors expressing amino plus carboxyl PTHrP was not significantly different than growth of the control tumors. In summary, the presence of amino PTHrP signifies worse prognosis in lung cancer patients. In mouse xenografts, this effect was abrogated if carboxyl PTHrP was also present., Conclusion: Amino PTHrP and carboxyl PTHrP can vary independently in different lung carcinomas. Carboxyl PTHrP may temper the stimulatory effect of amino PTHrP on cancer progression.

A 78-year-old man was referred to our hospital with suspected liver abscess. Fever and inflammatory reaction resolved after percutaneous drainage and administration of antibiotics. However, leukocyte count was remarkably increased, and hypercalcemia was noted. The liver mass was also enlarged, as observed in the follow-up abdominal CT scans. Therefore, a percutaneous needle biopsy was performed, and the histopathological findings indicated the presence of adenocarcinoma. Additional blood examination revealed high serum levels of granulocyte colony-stimulating factor (G-CSF) and parathyroid hormone-related protein (PTHrP). Lastly, the patient was diagnosed with cholangiocarcinoma producing G-CSF and PTHrP. Chemoradiotherapy with S-1 was initiated, which was partially effective. However, the patient died 134 days after initiating the therapy. Only two cases of cholangiocarcinoma producing G-CSF and PTHrP have been reported to date. Here we reported an additional case of cholangiocarcinoma producing G-CSF and PTHrP.

A 76-year-old woman was referred to our hospital with anorexia. Computed tomography revealed a tumor lesion measuring 110mm in the liver at S4/5 with calcification and swelling of a paraaortic lymph node. The gallbladder was not visualized. Histological examination of a biopsy specimen from the liver tumor revealed squamous cell and undifferentiated carcinomas, and several tumor markers were elevated. Therefore, we diagnosed the patient with gallbladder adenosquamous cell carcinoma T3N2M0 stage III. Because the serum parathyroid hormone-related protein (PTHrP) and granulocyte-colony stimulating factor (G-CSF) levels were significantly elevated, we suspected that PTHrP and G-CSF production occurred because of adenosquamous cell carcinoma in the gallbladder. We initiated chemotherapy with S-1.

In the developing limb, differentiation of skeletal progenitors towards distinct connective tissues of the digits is correlated with the establishment of well-defined domains of Btg1 gene expression. Zones of high expression of Btg1 include the earliest digit blastemas, the condensing mesoderm at the tip of the growing digits, the peritendinous mesenchyme, and the chondrocytes around the developing interphalangeal joints. Gain- and loss-of function experiments in micromass cultures of skeletal progenitors reveal a negative influence of Btg1 in cartilage differentiation accompanied by up-regulation of Ccn1, Scleraxis and PTHrP. Previous studies have assigned a role to these factors in the aggregation of progenitors in the digit tips (Ccn1), in the differentiation of tendon blastemas (Scleraxis) and repressing hypertrophic cartilage differentiation (PTHrP). Overexpression of Btg1 up-regulates the expression of retinoic acid and thyroid hormone receptors, but, different from other systems, the influence of BTG1 in connective tissue differentiation appears to be independent of retinoic acid and thyroid hormone signaling.

Fibroblast growth factor (FGF) signaling is a critical regulator of skeletal development. Fgf9 and Fgf18 are the only FGF ligands with identified functions in embryonic bone growth. Mice lacking Fgf9 or Fgf18 have distinct skeletal phenotypes; however, the extent of overlapping or redundant functions for these ligands and the stage-specific contributions of FGF signaling to chondrogenesis and osteogenesis are not known. To identify separate versus shared roles for FGF9 and FGF18, we generated a combined series of Fgf9 and Fgf18 null alleles. Analysis of embryos lacking alleles of Fgf9 and Fgf18 shows that both encoded ligands function redundantly to control all stages of skeletogenesis; however, they have variable potencies along the proximodistal limb axis, suggesting gradients of activity during formation of the appendicular skeleton. Congenital absence of both Fgf9 and Fgf18 results in a striking osteochondrodysplasia and revealed functions for FGF signaling in early proximal limb chondrogenesis. Additional defects were also noted in craniofacial bones, vertebrae, and ribs. Loss of alleles of Fgf9 and Fgf18 also affect the expression of genes encoding other key intrinsic skeletal regulators, including IHH, PTHLH (PTHrP), and RUNX2, revealing potential direct, indirect, and compensatory mechanisms to coordinate chondrogenesis and osteogenesis., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Background: Better methods to predict prognosis can play a supplementary role in administering individualized treatment for breast cancer patients. Altered expressions of PTHrP and TGF-β have been observed in various types of human cancers. The objective of the current study was to evaluate the association of PTHrP and TGF-β level with the clinicopathological features of the breast cancer patients., Methods: Immunohistochemistry was used to examine PTHrP and TGF-β protein expression in 497 cases of early breast cancer, and Kaplan-Meier method and COX's Proportional Hazard Model were applied to the prognostic value of PTHrP and TGF-β expression., Results: Both over-expressed TGF-β and PTHrP were correlated with the tumor in larger size, higher proportion of axillary lymph node metastasis and later clinical stage. Additionally, the tumors with a high TGF-β level developed poor differentiation, and only TGF-β expression was associated with disease-free survival (DFS) of the breast cancer patients. Followed up for a median of 48 months, it was found that only the patients with negative TGF-β expression had longer DFS (P < 0.05, log-rank test). Nevertheless, those with higher PTHrP expression tended to show a higher rate of bone metastasis (67.6 % vs. 45.8 %, P = 0.019). In ER negative subgroup, those who developed PTHrP positive expression presented poor prognosis (P < 0.05, log-rank test). The patients with both positive TGF-β and PTHrP expression were significantly associated with the high risk of metastases. As indicated by Cox's regression analysis, TGF-β expression and the high proportion of axillary lymph node metastasis served as significant independent predictors for breast cancer recurrence., Conclusions: TGF-β and PTHrP were confirmed to be involved in regulating the malignant progression in breast cancer, and PTHrP expression, to be associated with bone metastasis as a potential prognostic marker in ER negative breast cancer.

Background: An increasingly high occurrence of bone metastases in hepatocellular carcinoma (HCC) patients highlights the importance of fundamental research on HCC bone metastasis, which has been limited in its success due to the lack of a model system., Purpose: Establishment of animal and cellular models of HCC bone metastasis and discovery of HCC bone metastasis-related genes., Methods: Luciferase-transfected HCC cell lines HCCLM3, MHCC97H, and SMMC-7721 were used to inoculate nude mice intracardially. Formation of bone metastases was examined by bioluminescence imaging, SPECT, and pathology study. Metastatic cells in bone were isolated and subcultured. Differences between bone metastatic cells and their parental cells were studied by in vitro/in vivo assays., Results: Mouse model of HCC bone metastasis was successfully established. Injected tumour cells formed metastases in the skull, the spine, the hind limbs, and the sternum, causing osteolytic lesions via act of MMP-1 and recruitment of osteoclasts. Four bone metastatic cell lines were extracted from HCCLM3-inoculated mice and were demonstrated to exhibit a much stronger ability to form bone metastases as well as other phenotypes, including enhanced in vitro migration/invasion and colony formation. Moreover, the expression of PTHrP, MMP-1, and CTGF was significantly elevated in bone metastatic cells compared to parental HCC cells., Conclusion: The nude mouse model and bone metastatic cell lines together provide an effective simulation of HCC bone metastasis. This model system will become powerful tool with which to explore the mechanisms and therapies of HCC bone metastasis. Additionally, PTHrP, MMP-1, and CTGF are candidate genes related to HCC bone metastasis.

Breast cancer is the major cause of cancer death in women worldwide. The most common site of metastasis is bone. Bone metastases obstruct the normal bone remodeling process and aberrantly enhance osteoclast-mediated bone resorption, which results in osteolytic lesions. 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) is an endogenous ligand of peroxisome proliferator-activated receptor gamma (PPARγ) that has anti-inflammatory and antitumor activity at micromolar concentrations through PPARγ-dependent and/or PPARγ-independent pathways. We investigated the inhibitory activity of 15d-PGJ2 on the bone loss that is associated with breast cancer bone metastasis and estrogen deficiency caused by cancer treatment. 15d-PGJ2 dose-dependently inhibited viability, migration, invasion, and parathyroid hormone-related protein (PTHrP) production in MDA-MB-231 breast cancer cells. 15d-PGJ2 suppressed receptor activator of nuclear factor kappa-B ligand (RANKL) mRNA levels and normalized osteoprotegerin (OPG) mRNA levels in hFOB1.19 osteoblastic cells treated with culture medium from MDA-MB-231 cells or PTHrP, which decreased the RANKL/OPG ratio. 15d-PGJ2 blocked RANKL-induced osteoclastogenesis and inhibited the formation of resorption pits by decreasing the activities of cathepsin K and matrix metalloproteinases, which are secreted by mature osteoclasts. 15d-PGJ2 exerted its effects on breast cancer and bone cells via PPARγ-independent pathways. In Balb/c nu/nu mice that received an intracardiac injection of MDA-MB-231 cells, subcutaneously injected 15d-PGJ2 substantially decreased metastatic progression, cancer cell-mediated bone destruction in femora, tibiae, and mandibles, and serum PTHrP levels. 15d-PGJ2 prevented the destruction of femoral trabecular structures in estrogen-deprived ICR mice as measured by bone morphometric parameters and serum biochemical data. Therefore, 15d-PGJ2 may be beneficial for the prevention and treatment of breast cancer-associated bone diseases.

An 82-year-old man was admitted to our hospital with hemoptysis and weight loss. Chest computed tomography(CT) showed a 90 mm mass with cavity formation in the right lower lobe adjacent to chest wall. Laboratory data revealed hypercalcemia and elevation of parathyroid hormone-related protein C (PTHrP). He was diagnosed as squamous cell carcinoma of lung by transbronchial lung biopsy (TBLB) [cT3aN1M0]. Nausea and anorexia due to hypercalcemia became worse and a right middle and lower lobectomy was performed because of difficult control of symptoms by medicine and worsening of his general condition. His symptoms were improved immediately after surgery.

An 87-year-old woman was diagnosed with primary diffuse large B-cell lymphoma of the pancreas by endoscopic ultrasonography-guided fine needle aspiration. Complete remission was achieved after treatment with six courses of R-CHOP chemotherapy. However, two and a half years later, she was readmitted because of weakness during walking. At this time, laboratory tests revealed hypercalcemia associated with high plasma levels of parathyroid hormone-related protein (PTHrP), but bone lesions were not detected. Although computed tomography only revealed splenomegaly, we suspected a recurrence of her malignant lymphoma because she also had marked elevation of soluble interleukin-2 receptor and lactate dehydrogenase levels. Bone marrow examination revealed the involvement of Burkitt's lymphoma cells with malignant transformation. Immunohistochemical analysis confirmed that hypercalcemia was caused by a paraneoplastic syndrome related to PTHrP-producing B-cell lymphoma cells. Unfortunately, the patient's general condition rapidly deteriorated, and she died soon after admission. Our case is unusual because of the presentation of bone marrow relapse of malignant lymphoma.

Combined hepatocellular-cholangiocarcinoma (CHCC) is an uncommon form of primary liver cancer. A 57-year-old man was readmitted to our hospital for treatment of recurrent CHCC, 12 months after central bisegmentectomy and 4 months after limited hepatic resection. Magnetic resonance imaging (MRI) revealed multiple hepatic nodules. Laboratory data showed increased serum levels of α-fetoprotein (AFP), calcium, and parathyroid hormone-related protein (PTH-rP), to 5,571 ng/mL, 17.0 mg/dL, and 16.1 pmol/L, respectively. Palliative mass reduction surgery was indicated by the fact that the hypercalcemia was difficult to manage medically. Thus, we performed lateral segmentectomy with partial resection of segment 7 and the caudate lobe, and microwave coagulation therapy for multiple recurrent CHCC. Thereafter, the serum PTH-rP and AFP levels decreased remarkably and the hypercalcemia was controlled for the next 3 months. He died of disease progression 9 months after the last hepatic surgery. To our knowledge, this is only the second reported case of CHCC producing PTH-rP in the English-language literature.

PTH-related peptide (PTHrP) is a widely distributed cytokine, which shares the cognate receptor PTHR1 with PTH. Originally identified as a causal factor of humoral hypercalcemia of malignancy twenty years ago, PTHrP is now recognized as a critical physiological regulator of various biological processes, including bone and cartilage metabolism.

Parathyroid hormone-related protein (PTHrP) can be secreted from cells and interact with its receptor, the Type 1 PTH/PTHrP Receptor (PTHR1) in an autocrine, paracrine or endocrine fashion. PTHrP can also remain inside cells and be transported into the nucleus, where its functions are unclear, although recent experiments suggest that it may broadly regulate cell survival and senescence. Disruption of either the PTHrP or PTHR1 gene results in many abnormalities including a failure of embryonic mammary gland development in mice and in humans. In order to examine the potential functions of nuclear PTHrP in the breast, we examined mammary gland development in PTHrP (1-84) knock-in mice, which express a mutant form of PTHrP that lacks the C-terminus and nuclear localization signals and which can be secreted but cannot enter the nucleus. Interestingly, we found that PTHrP (1-84) knock-in mice had defects in mammary mesenchyme differentiation and mammary duct outgrowth that were nearly identical to those previously described in PTHrP-/- and PTHR1-/- mice. However, the mammary buds in PTHrP (1-84) knock-in mice had severe reductions in mutant PTHrP mRNA levels, suggesting that the developmental defects were due to insufficient production of PTHrP by mammary epithelial cells and not loss of PTHrP nuclear function. Examination of the effects of nuclear PTHrP in the mammary gland in vivo will require the development of alternative animal models.

Breast cancer frequently spreads to bone. The interaction between bone metastases and microenvironment, referred as the "vicious cycle", increases both tumor burden and bone destruction. Therefore, inhibition at any point in this "vicious cycle" can reduce malignant osteolytic lesions in patients with advanced breast cancer. In this study, we evaluated whether tetrahydrofurofuran-type lignans derived from Magnoliae Flos, commonly used in traditional Asian medicine to treat inflammatory diseases, could block breast cancer-mediated bone loss. Aschatin, fargesin, lirioresinol B dimethyl ether, and magnolin at noncytotoxic concentrations suppressed mRNA expression and secretion of osteolytic factor PTHrP in MDA-MB-231 metastatic human breast cancer cells. Fargesin inhibited TGF-β-stimulated cell viability, migration, and invasion and decreased TGF-β-induced PTHrP production in MDA-MB-231 cells. In addition, these lignans reduced RANKL/OPG ratio in PTHrP-treated hFOB1.19 human osteoblastic cells and inhibited RANKL-mediated osteoclast differentiation in mouse bone marrow macrophages. Aschatin, fargesin, lirioresinol B dimethyl ether, and magnolin substantially reduced bone-resorbing activity of osteoclasts by inhibiting MMP-9 and cathepsin K activities. Furthermore, orally administered fargesin inhibited tumor growth and cancer-mediated bone destruction in mice with MDA-MB-231 cells injected into calvarial tissues. Aschatin, fargesin, lirioresinol B dimethyl ether, and magnolin blocked initiation and progression of the "vicious cycle" between breast cancer metastases and bone microenvironment by inhibiting PTHrP production in breast cancer cells and osteoclastic bone resorption. Therefore, these tetrahydrofurofuran-type lignans have the potential to serve as beneficial agents to prevent and treat cancer-induced bone destruction in breast cancer patients.

Susceptibly to the induction of rat tongue cancer (TC) by oral 4-nitroquinoline 1-oxide (4NQO) exposure is a polygenic trait. Among several quantitative trait loci identified by crosses between TC-susceptible Dark Agouti (DA) rats and TC-resistant Wistar-Furth (WF) rats, we focused on tongue cancer susceptibility locus (Tcas3) of chromosome 4. We examined tongue carcinogenesis in the reciprocal congenic strains DA.WF-Tcas3 and WF.DA-Tcas3 and in their parental strains. The Tcas3DA allele, and not the Tcas3WF allele, significantly favored tumor latency, incidence and TC number/size. In genomic DNA of TCs induced in (DA x WF) F1 rats, the resistant Tcas3WF allele was frequently and selectively lost, particularly in larger tumors. Thus, we searched the possible candidate genes in the Tcas3 region using microarray analysis of TCs in F1 rats and revealed significant upregulation of 2 cancer-related genes, parathyroid hormone-like hormone (Pthlh) and Kras2. The relevance of the WF allele of Pthlh as a cancer modifier was indicated by 3 single nucleotide polymorphisms specific to this strain. In contrast, no consistent strain-specific variations were found in Kras2. Moreover, the plasma Ca2+ level was consistently higher in DA rats when compared to the level in WF rats bearing TCs; moreover, the Pthlh-mRNA expression level was >30-fold higher in TCs when compared to this level in the normal tongue mucosa. Immunostaining experiments showed strong PTHrP protein expression in TCs of DA rats, and the signal was intensified in larger TCs. Kras2 was also upregulated in TCs, but to a lesser degree than PTHrP. Thus, Pthlh is a promising candidate modifier gene in the development and progression of rat TCs.

To promote their survival and progression in the skeleton, osteotropic malignancies of breast, lung, and prostate produce parathyroid hormone-related protein (PTHrP), which induces hypercalcemia. PTHrP serum elevations have also been described in multiple myeloma (MM), although their role is not well defined. When we investigated MM cells from patients and cell lines, we found that PTHrP and its receptor (PTH-R1) are highly expressed, and that PTHrP is secreted both as a full-length molecule and as small subunits. Among these subunits, the mid-region, including the nuclear localization sequence (NLS), exerted a proliferative effect because it was accumulated in nuclei of MM cells surviving in starvation conditions. This was confirmed by increased transcription of several genes enrolled in proliferation and apoptosis control. PTHrP was also found to stimulate PTH-R1 in MM cells. PTH-R1's selective activation by the full-length PTHrP molecule or the NH2 -terminal fragment resulted in a significant increase of intracellular Ca(2+) influx, cyclic adenosine monophosphate (cAMP) content, and expression of receptor activator of NF-κB ligand (RANKL) and monocyte chemoattractant protein-1 (MCP-1). Our data definitely clarify the role of PTHrP in MM. The PTHrP peptide is functionally secreted by malignant plasma cells and contributes to MM tumor biology and progression, both by intracrine maintenance of cell proliferation in stress conditions and by autocrine or paracrine stimulation of PTH-R1, which in turn reinforces the production of osteoclastogenic factors. © 2014 American Society for Bone and Mineral Research., (© 2014 American Society for Bone and Mineral Research.)

To investigate the correlation between parathyroid hormone-related protein (PTHrP), erythropoietin (EPO), and vascular endothelial growth factor (VEGF) expression in clear cell renal cell carcinoma (ccRCC). Immunohistochemical studies on PTHrP, EPO and VEGF were performed in 249 patients with ccRCC. Serum calcium level and haematocrit were analyzed. The expression of the factors and clinicopathological parameters were studied statistically for possible correlations. The incidence for hypercalcaemia and polycythaemia were 15.3% and 2.0% respectively. Expression of PTHrP, EPO, and VEGF were respectively related to advanced stage (P < 0.0001 respectively). PTHrP was not related to tumour grade. Expressions of EPO and VEGF were correlated to tumour grade significantly. All factors were expressed higher in hypercalcaemic patients. PTHrP, EPO, and VEGF were positively correlated with each other in non-hypercalcaemic patients yet not in hypercalcaemic ones. PTHrP and EPO are related to VEGF expression and to the progression of ccRCC. This finding offers us new insight on the behaviour of ccRCC and offers possible targets in RCC treatment.

Parathyroid hormone-related protein (PTHrP) is a protein member of the parathyroid hormone family that regulates the dynamic balance between blood and bone calcium during lactation. However, the mechanism of its regulation is not very clear. In order to establish a framework for further functional studies of the PTHrP gene in goat mammary gland epithelial cells during the lactation period, PTHrP cDNA was isolated from Xinong Saanen dairy goats. Its coding sequence is 534 bp in size. We also designed a short hairpin RNA (shRNA) to efficiently inhibit PTHrP expression and constructed recombinant adenoviruses carrying a template encoding this shRNA (AD-PTHrP-322) using the Block-iT shRNA interference system. Finally, the inhibition of PTHrP expression by the recombinant adenoviruses was confirmed by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) and Western blotting. qRT-PCR results showed that the expression of PTHrP mRNA in mammary epithelial cells was downregulated by 29.2, 68.1, and 82.6% 24, 48, and 72 h after the cells were infected with AD-PTHrP-322, respectively. Western blotting also showed that the expression of PTHrP was reduced in a time-dependent manner. These results suggest that AD-PTHrP-322 significantly inhibits the expression of PTHrP.

Objectives: To investigate the correlation between the percentage of metastatic tumor present in lymph nodes resected from patients with squamous cell carcinoma of the head and neck (HNSCC) and level of expression of 3 marker genes: pemphigus vulgaris antigen (PVA), parathyroid hormone-related peptide (PTHrP), and tumor-associated calcium signal transducer 1 (TACSTD1). In addition, we investigated whether the level of expression of these 3 markers was associated with clinical outcomes for patients with HNSCC., Study Design: Retrospective analysis of previously harvested patient samples., Setting: The University of Pittsburgh., Subjects and Methods: A total of 448 lymph nodes from 92 patients with HNSCC were evaluated for expression of the gene markers PVA, PTHrP, and TACSTD1 using real-time polymerase chain reaction. Confirmation of metastasis was determined by histologic examination. The expression level of these markers versus tumor percentage was analyzed., Results: All 3 markers were studied independently and were associated with tumor percentage in metastatic lymph nodes. PVA had the strongest correlation, followed by PTHrP and then TACSTD1. PVA levels had a trend toward association with clinical outcome, specifically time to death caused by cancer, but this was confounded by tumor stage., Conclusion: All 3 tumor gene markers were associated with percentage of tumor cells in metastatic lymph nodes. PVA had the strongest correlation. PVA may add prognostic utility beyond pathologic staging, but this requires analysis of a larger cohort. Prospective studies of tumor volume in metastatic nodes should determine a lower limit threshold of molecular marker detection.

Background: Bone is a common site of metastasis for lung cancer, and is associated with significant morbidity and a dismal prognosis. MicroRNAs (miRNAs) are increasingly implicated in regulating the progression of malignancies., Methods: The efficacy of miR-33a or anti-miR-33a plasmid was assessed by Real-time PCR. Luciferase assays were using One-Glo Luciferase Assay System. Measurement of secreted factors was determined by ELISA kit., Results: We have found that miR-33a, which is downregulated in lung cancer cells, directly targets PTHrP (parathyroid hormone-related protein), a potent stimulator of osteoclastic bone resorption, leading to decreased osteolytic bone metastasis. We also found that miR-33a levels are inversely correlated with PTHrP expression between human normal bronchial cell line and lung cancer cell lines. The reintroduction of miR-33a reduces the stimulatory effect of A549 on the production of osteoclastogenesis activator RANKL (receptor activator of nuclear factor kappa-B ligand) and M-CSF (macrophage colony-stimulating factor) on osteoblasts, while the expression of PTHrP is decreased in A549 cells. miR-33a overexpression also reduces the inhibitory activity of A549 on the production of OPG (osteoprotegerin), an osteoclastogenesis inhibitor. In addition, miR-33a-mediated PTHrP downregulation results in decreased IL-8 secretion in A549, which contributes to decreased lung cancer-mediated osteoclast differentiation and bone resorption., Conclusions: These findings have led us to conclude that miR-33a may be a potent tumor suppressor, which inhibits direct and indirect osteoclastogenesis through repression of PTHrP., General Significance: miR-33a may even predict a poor prognosis for lung cancer patients., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Translocations are chromosomal rearrangements that are frequently associated with a variety of disease states and developmental disorders. We identified 2 families with brachydactyly type E (BDE) resulting from different translocations affecting chromosome 12p. Both translocations caused downregulation of the parathyroid hormone-like hormone (PTHLH) gene by disrupting the cis-regulatory landscape. Using chromosome conformation capturing, we identified a regulator on chromosome 12q that interacts in cis with PTHLH over a 24.4-megabase distance and in trans with the sex-determining region Y-box 9 (SOX9) gene on chromosome 17q. The element also harbored a long noncoding RNA (lncRNA). Silencing of the lncRNA, PTHLH, or SOX9 revealed a feedback mechanism involving an expression-dependent network in humans. In the BDE patients, the human lncRNA was upregulated by the disrupted chromosomal association. Moreover, the lncRNA occupancy at the PTHLH locus was reduced. Our results document what we believe to be a novel in cis- and in trans-acting DNA and lncRNA regulatory feedback element that is reciprocally regulated by coding genes. Furthermore, our findings provide a systematic and combinatorial view of how enhancers encoding lncRNAs may affect gene expression in normal development.

MicroRNAs (miRNAs) as modulators of gene expression have been described to display both tumor-promoting and tumor-suppressive functions. Although their role has been studied in different tumor types, little is known about how they regulate nuclear factor κB (NF-κB) signaling in breast cancer. Here, we performed an unbiased whole genome miRNA (miRome) screen to identify novel modulators of NF-κB pathway in breast cancer. The screen identified 13 miRNA families whose members induced consistent effects on NF-κB activity. Among those, the miR-520/373 family inhibited NF-κB signaling through direct targeting of RELA and thus strongly reduced expression and secretion of the pro-inflammatory cytokines interleukin (IL)-6 and IL-8. With a combination of in vitro and in vivo approaches, we propose a metastasis-suppressive role of miR-520/373 family. miR-520c and miR-373 abrogated both in vitro cell invasion and in vivo intravasation of highly invasive MDA-MB-231 cells. However, knockdown of RELA did not affect their metastatic ability. mRNA profiling of MDA-MB-231 cells on overexpression of miR-520/373 members revealed a strong downregulation of transforming growth factor-β (TGF-β) signaling. Mechanistically, the metastasis-suppressive role of miR-520/373 can be attributed to direct suppression of TGFBR2, as the silencing of TGFBR2 phenocopied the effects of miR-520/373 overexpression on suppression of Smad-dependent expression of the metastasis-promoting genes parathyroid hormone-related protein, plasminogen activator inhibitor-1 and angiopoietin-like 4 as well as tumor cell invasion, in vitro and in vivo. A negative correlation between miR-520c and TGFBR2 expression was observed in estrogen receptor negative (ER(-)) breast cancer patients but not in the ER positive (ER(+)) subtype. Remarkably, decreased expression of miR-520c correlated with lymph node metastasis specifically in ER(-) tumors. Taken together, our findings reveal that miR-520/373 family has a tumor-suppressive role in ER(-) breast cancer by acting as a link between the NF-κB and TGF-β pathways and may thus contribute to the interplay of tumor progression, metastasis and inflammation.

Parathyroid hormone related protein (PTHrP) has important biological functions in calcium metabolism. The aim of this study was to silence the expression of PTHrP by RNA interference and recombinant adenovirus, and to provide a material to investigate the relative functions of PTHrP in goat mammary gland epithelial cell. The Block-iT shRNA interference system was used in this experiment. We designed and synthesized two pairs of complementary single-strand DNA oligonucleotides (shRNA-322/357) targeting two different sites of PTHrP mRNA. Then the oligonucleotides were inserted into shuttle vector pENTR/CMV-GFP/U6. After detection of the interference efficiency by Western blotting, we chose pENTR/CMV-GFP/U6-322 and adenovirus backbone vector pAD/PL-DEST to produce recombinant vector pAD/PL-DEST/CMV-GFP/U6-322. The first generation recombinant adenovirus particles (AD-PTHrP-322) were produced and further amplified by transfecting HEK-293 cells. The titer of the recombinant adenovirus reached 2.0 x 1(9) PFU/mL determined by TCID50 assays. The result of real-time quantitative PCR indicated that mRNA expression levels of gene were reduced 29.2%, 68.1% and 82.6% (P < 0.05), respectively, when goat mammary gland epithelial cells were infected with AD-PTHrP-322 after 24, 48 and 72 h, in which PTHrP. Western blotting also showed that the expression of PTHrP was reduced by infecting the cells with AD-PTHrP-322. AD-PTHrP-322 has been proved with significant interference effect on expression of PTHrP.

Aims/hypothesis: A major focus in the treatment of diabetes is to identify factors that stimulate endogenous beta cell growth while preserving function. The first 36 amino acids of parathyroid hormone-related protein (PTHrP) are sufficient to enhance proliferation and function in rodent and human beta cells in vitro. This study examined whether acute and systemic administration of the amino-terminal PTHrP(1-36) peptide can achieve similar effects in rodent beta cells in vivo., Methods: Adult male mice were injected with 40, 80 or 160 μg of PTHrP(1-36) per kg body weight or with vehicle for 25 days. Glucose and beta cell homeostasis, as well as expression of differentiation markers and cell cycle genes were analysed., Results: All three doses of PTHrP(1-36) significantly enhanced beta cell proliferation in vivo at day 25, with 160 μg/kg PTHrP(1-36) increasing proliferation as early as day 5. Importantly, the two higher doses of PTHrP(1-36) caused a significant 30% expansion of beta cell mass, with a short-term improvement in glucose tolerance. PTHrP(1-36) did not cause hypercalcaemia, or change islet number, beta cell size, beta cell death or expression of differentiation markers. Analysis of islet G1/S cell cycle proteins revealed that chronic overabundance of PTHrP(1-139) in the beta cell significantly increased the cell cycle activator cyclin D2 and decreased levels of cyclin-dependent kinase 4 inhibitor (p16( Ink4a ) [Ink4a also known as Cdkn2a]), but acute treatment with PTHrP(1-36) did not., Conclusions/interpretation: Acute and systemic administration of PTHrP(1-36) increases rodent beta cell proliferation and mass without negatively affecting function or survival. These findings highlight the future potential therapeutic effectiveness of this peptide under diabetes-related pathophysiological conditions.

Background: Graves' ophthalmopathy (GO) and lymphedema share some pathogenetic mechanisms, such as edema, inflammation, and adipogenesis. The aim of this study was to examine similarities and differences between chronic GO and chronic lymphedema., Methods: Intraorbital adipose tissue was collected from patients with active (n = 10) or chronic GO (n = 10) and thyroid-healthy controls (n = 10). Arm subcutaneous adipose tissue was obtained from patients with chronic arm lymphedema (n = 10), where the unaffected arm served as a control. Gene expression was studied using microarray and real-time polymerase chain reaction., Results: The following genes were significantly upregulated (p < 0.05) in lymphedema but not in GO and have functions in wound healing, fibrosis, fat metabolism, inflammation, differentiation, development, adhesion, and the cytoskeleton: ATP-binding cassette, sub-family G (WHITE), member 1 (ABCG1), actin, alpha 2, smooth muscle, aorta (ACTA2), secreted frizzled-related protein 2 (SFRP2), tenascin C (TNC), pentraxin-related gene, rapidly induced by IL-1 beta (PTX3), and carboxypeptidase X (M14 family), member 1 (CPMX1). In chronic GO, but not in lymphedema, adipocyte-related immediate early genes known to be overexpressed in patients with active GO were upregulated but at a lower level than previously shown for the active phase. Genes of the Wnt pathway, such as secreted frizzled-related protein 1, 2, and 3, were up- and downregulated in both chronic GO and lymphedema. Parathyroid hormone-like hormone (PTHLH) was downregulated (p = 0.01) and apolipoprotein L domain containing 1 (APOLD1) was upregulated (p = 0.05) in both active and chronic GO., Conclusions: There are more differences than similarities between chronic ophthalmopathy and chronic lymphedema, but both conditions exhibit less inflammation and adipogenesis compared to the active phases. In lymphedema, fibrosis dominates. PTHLH, which can inhibit adipogenesis, is downregulated both in active and chronic ophthalmopathy, indicating the possibility of an increased risk of adipogenesis.

The calcium-sensing receptor (CaR) is responsible for the regulation of extracellular calcium (Ca(2+) (o)) homeostasis. CaR activation has been shown to increase proliferation in several cancer cell lines; however, its presence or function has never been documented in lung cancer. We report that Ca(2+) (o)-activated CaR results in MAPK-mediated stimulation of parathyroid hormone-related protein (PTHrP) production in human lung squamous cell carcinoma (SCC) lines and humoral hypercalcemia of malignancy (HHM) in vivo. Furthermore, a single nucleotide polymorphism in CaR identified from a hypercalcemia-inducing lung SCC reduced the receptor's activation threshold leading to increased PTHrP expression and secretion. Increasing the expression of either wild-type CaR or a CaR variant with a single nucleotide polymorphism in the cytoplasmic domain was both necessary and sufficient for lung SCC to induce HHM. Because lung cancer patients who frequently develop HHM and PTHrP expression in lung cancer has been only partially explained, the significance of our findings indicates that CaR variants may provide a positive feedback between PTHrP and calcium and result in the syndrome of HHM.

Elevated parathyroid hormone-related protein (PTHrP) is responsible for humoral hypercalcemia of malignancy (HHM), which is of clinical significance in treatment of terminal patients with malignancies. Steroid hormones were known to cause suppression of PTHrP expression. However, detailed studies linking multiple steroid hormones to PTHrP expression are lacking. Here we studied PTHrP expression in response to steroid hormones in four cell lines with excessive PTHrP production. Our study established that steroid hormones negatively regulate PTHrP expression. Vitamin D receptor, estrogen receptor α, glucocorticoid receptor, and progesterone receptor, were required for repression of PTHrP expression by the cognate ligands. A notable exception was the androgen receptor, which was dispensable for suppression of PTHrP expression in androgen-treated cells. We propose a pathway(s) involving nuclear receptors to suppress PTHrP expression., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Background: Gastrectomy may disturb the body's mineral homeostasis, with osteopenia and osteoporosis being among the late outcomes. Parathyroid hormone-like hormone (PTHLH) was detected in rat gastric enterochromaffin-like (ECL) cells in 2005, and some researchers suggested that it was the hypothetical hormone gastrocalcin that is believed to lead to osteoporosis., Aims: Our objective was to learn whether PTHLH is expressed in human gastric ECL cells and to form a basic understanding about the relationship between PTHLH and gastrin., Methods: We collected normal human gastric mucosa specimens and serum samples from 28 patients., Results: RT-PCR and immunohistochemical analysis demonstrated weak expression of PTHLH in ECL cells at the RNA and protein levels. A low level of PTHLH expression was also found in the serum. Serum gastrin did have a significant positive correlation with the relative ratio of PTHLH mRNA to β-actin levels in gastric mucosa (rs=0.569, p=0.002)., Conclusions: This indicates that PTHLH has a low signal expression in human gastric ECL cells and that serum gastrin levels correlate with PTHLH RNA levels in gastric mucosa. Further work is needed to evaluate the functional role of PTHLH in ECL cells and to determine whether PTHLH is gastrocalcin.

Breast cancer frequently metastasizes to bone, in which tumor cells receive signals from the bone marrow microenvironment. One relevant factor is TGF-β, which upregulates expression of the Hedgehog (Hh) signaling molecule, Gli2, which in turn increases secretion of important osteolytic factors such as parathyroid hormone-related protein (PTHrP). PTHrP inhibition can prevent tumor-induced bone destruction, whereas Gli2 overexpression in tumor cells can promote osteolysis. In this study, we tested the hypothesis that Hh inhibition in bone metastatic breast cancer would decrease PTHrP expression and therefore osteolytic bone destruction. However, when mice engrafted with human MDA-MB-231 breast cancer cells were treated with the Hh receptor antagonist cyclopamine, we observed no effect on tumor burden or bone destruction. In vitro analyses revealed that osteolytic tumor cells lack expression of the Hh receptor, Smoothened, suggesting an Hh-independent mechanism of Gli2 regulation. Blocking Gli signaling in metastatic breast cancer cells with a Gli2-repressor gene (Gli2-rep) reduced endogenous and TGF-β-stimulated PTHrP mRNA expression, but did not alter tumor cell proliferation. Furthermore, mice inoculated with Gli2-Rep-expressing cells exhibited a decrease in osteolysis, suggesting that Gli2 inhibition may block TGF-β propagation of a vicious osteolytic cycle in this MDA-MB-231 model of bone metastasis. Accordingly, in the absence of TGF-β signaling, Gli2 expression was downregulated in cells, whereas enforced overexpression of Gli2 restored PTHrP activity. Taken together, our findings suggest that Gli2 is required for TGF-β to stimulate PTHrP expression and that blocking Hh-independent Gli2 activity will inhibit tumor-induced bone destruction.

Cortactin, an F-actin binding protein, stabilizes F-actin networks and promotes actin polymerization by activating the Arp2/3 complex. Overexpression of cortactin has been reported in several human cancers. Cortactin stimulates cell migration, invasion, and experimental metastasis. However, the underlying mechanism is not still understood. In the present study, we therefore evaluated the possibility that cortactin could be appropriate as a molecular target for cancer gene therapy. In 70 primary oral squamous cell carcinomas and 10 normal oral mucosal specimens, cortactin expression was evaluated by immunological analyses, and the correlations of the overexpression of cortactin with clinicopathologic factors were evaluated. Overexpression of cortactin was detected in 32 of 70 oral squamous cell carcinomas; significantly more frequently than in normal oral mucosa. Cortactin overexpression was more frequent in higher grade cancers according to T classification, N classifications, and invasive pattern. Moreover, RNAi-mediated decrease in cortactin expression reduced invasion. Downregulation of cortactin expression increased the expression levels of E-cadherin, β-catenin, and EpCAM. The siRNA of cortactin also reduced PTHrP expression via EGF signaling. These results consistently indicate that the overexpression of cortactin is strongly associated with an aggressive phenotype of oral squamous cell carcinoma. In conclusion, we propose that cortactin could be a potential molecular target of gene therapy by RNAi targeting in oral squamous cell carcinoma.

In the advanced stages of prostate cancer, tumor cells can evolve to become androgen-independent and resistant to injury-induced apoptosis. Tumor cell expression of parathyroid hormone-related protein (PTHrP) may contribute to the apoptosis phenotype. Expression of the adenovirus E1A oncogene repressed PTHrP promoter and mRNA expression in human PC-3 prostate cancer cells and increased the caspase 3 activation and sensitivity of these cells to apoptosis triggered by tumor necrosis factor alpha. These results suggest that strategies aimed at modulating PTHrP expression may increase the efficacy of innate immune effector mechanisms and proapoptotic, therapeutics in prostate cancer.

Background: Breast cancer (BC) cells often metastasize to bone where they express large amounts of parathyroid hormone-related protein (PTHrP). In this study, we investigated the possibility that PTHrP may have roles in breast cancer bone metastasis independently of, or in addition to, its roles in osteoclastic function., Materials and Methods: A mouse model of bone metastasis was prepared by inoculating mice with suspensions of the human BC cell line MDA-MB-231 tumor cells via the left cardiac ventricle. Matrix metalloproteinase-13 (MMP-13) expression in the bone microenvironment was examined by Western blot and Real-time RT-PCR (RT-PCR) analysis, as well as by confocal microscopy., Results: The invading MDA-MB-231 cells contained conspicuous amounts of both PTHrP and MMP-13, an important matrix-degrading enzyme; and treatment of the cells in culture with exogenous PTHrP markedly stimulated MMP13 gene expression. Analysis of signaling mechanisms showed that PTHrP treatment led to rapid increases in the levels of phosphorylated protein kinase C (PKCα) and extracellular signal-regulated kinase (ERK1/2). Pharmacologic inhibition of ERK1/2 and PKC as well as of PKA activities counteracted the PTHrP-dependent stimulation of MMP13 expression. Indeed, pharmacologic activation of PKA or PKC was sufficient for stimulation of MMP13 expression., Conclusion: Consistent with these findings, the inhibition of PKC prevented PTHrP-induced activation of ERK1/2, whereas 12-O-tetradecanoylphorbol-13-acetate (TPA), a stimulator of PKC, up-regulated the PTHrP-induced activation of ERK1/2. Taken together, our data indicate that the MDA-MB-231 breast cancer cells may carry out bone destruction and favor their own metastatic behavior by producing MMP-13. Given that the cells expressed PTHrP and that this factor stimulated MMP-13 expression, metastatic bone destruction may result from a PTHrP autocrine loop involving a PKC-ERK1/2 signaling pathway.

Parathyroid hormone-related protein (PTHrP) is expressed by human colon cancer tissue and cell lines. Rac1 GTPase enhances colon cancer cell migration and invasion. Here we report a positive correlation between PTHrP expression and Rac1 activity in LoVo (human colon cancer) cells. The positive effects of PTHrP on Rac1 activity and on cell migration and invasion are mediated via the guanine nucleotide exchange factor Tiam1. Knockdown of integrin α6β4, which is upregulated by PTHrP, negates the PTHrP-mediated increase in Rac1 activation. Integrin α6β4 signals synergistically with growth factor receptors to activate the phosphatidylinositol 3-kinase (PI3-K) pathway. Chemical inhibition of PI3-K negates the PTHrP-mediated effects on Tiam1 and Rac1 activity. Tumors from PTHrP-overexpressing LoVo cells also show increased expression of Tiam1. Taken together, these observations provide evidence of a link between PTHrP and Rac1 activity through integrin α6β4, resulting in enhanced cell migration and invasion. Targeting PTHrP production in colon cancer may thus prove therapeutically beneficial., (Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.)

We experienced a case of parathyroid hormone-related peptide (PTHrP)-producing pheochromocytoma, which was found in a 12-year-old boy with hypercalcemia. The leading symptom was abdominal pain, and severe hypertension and tachycardia were noticed at the initial visit. His medical and familial histories were unremarkable. Laboratory examinations showed hypercalcemia (3.3 mmol/L of serum-calcium). Computed tomography showed a heterogeneous mass measuring 5.0 cm in the right adrenal gland, which had abnormal uptake with 123-I metaiodobenzylguanidine scintigraphy. Serum/urine catecholamines were highly elevated, and serum PTHrP also increased (1.4 pmol/L). The patient underwent laparoscopic right adrenalectomy. The tumor was histologically diagnosed as typical pheochromocytoma and the expression of PTHrP was confirmed with immunohistochemistry. The serum PTHrP level was normalized after surgery. He was free of disease postoperatively for 12 months. There has been no described pediatric patient with PTHrP-producing pheochromocytoma. We showed evidence that the present tumor is a complex neoplasm involving various neuroendocrine activities with the dual-lineage differentiation.

Hypercalcemia secondary to neoplastic disease is a frequent entity caused in the majority of cases by ectopic secretions of PTHrP. Despite this there are certain tumours, such as uterine carcinomas, in which this type of paraneoplastic manifestation has been described very little. We present a case of humoral hypercalcemia in a mixed endometrial carcinoma.

Serratia marcescens is an ubiquitous, saprophytic gram-negative bacillus that is associated with infections such as bacteremia, pneumonia and osteomyelitis. However, it has not been known to form granulomas. A 72-year-old man with a history of tricuspidal insufficiency, mitral insufficiency and ureterolithiasis presented with lumbago on the left side. He was admitted to our hospital, where abscess formation in the subcapsular space and perirenal fat space of the left kidney, and left renal calculi were identified by computed tomography of the abdomen. As infection and/or a tumor were suspected, nephrectomy was performed. The histopathological findings in the resected kidney indicated severe infiltration by inflammatory cells with lymphoid follicles in the interstitium, and the proliferation of mesangial cells and matrix in glomerulus. Furthermore, giant cell granulomas were observed in the soft tissue around the kidney. As an aerobic culture of the abscess from the granulomas only produced Serratia marcescens, these granulomas were diagnosed as Serratia marcescens granulomas. In addition, expressions of PTHrP and PTH/PTHrP-receptor were observed in the giant cells in Serratia granuloma, which suggested that PTHrP might be involved in giant cell formation in Serratia granuloma by autocrine and/or paracrine mechanisms.

Purpose: Despite remarkable progress in resuscitation and intensive care, the morbidity and mortality rates in congenital diaphragmatic hernia (CDH) remain high due to severe pulmonary hypoplasia. The pathogenesis of pulmonary hypoplasia associated with CDH is still not clearly understood. Pulmonary parathyroid hormone-related protein (PTHrP) is expressed in the type II epithelial cells and stimulates surfactant production by a paracrine feedback loop regulated by PTHrP receptor (PTHrP-R), which is expressed in the mesenchyme, during terminal airway differentiation. It has been reported that PTHrP knockout and PTHrP-R null mice both exhibit pulmonary hypoplasia, disrupting alveolar maturation before birth. We designed this study to test the hypothesis that gene expression of PTHrP and PTHrP-R is downregulated in the late stages of lung morphogenesis in the nitrofen-induced hypoplastic lung., Methods: Pregnant rats were exposed to either olive oil or nitrofen on day 9 of gestation (D9). Fetal lungs were harvested on D15, 18, and 21 and divided into three groups: control, nitrofen without CDH [CDH(-)], and nitrofen with CDH [CDH(+)] (n = 8 at each time point for each group, respectively). Total mRNA was extracted from fetal lungs and mRNA expression of PTHrP and PTHrP-R was analyzed by real-time RT-PCR and the significant differences between the groups were accepted at P < 0.05 by statistical analysis. Immunohistochemical studies were also performed to evaluate PTHrP and PTHrP-R protein expression at each time point., Results: Pulmonary mRNA expression of PTHrP-R was significantly decreased in both nitrofen groups [CDH(-) and CDH(+)] compared to controls at D18 and 21. The mRNA level of PTHrP was significantly decreased at D21 in both nitrofen groups compared to controls. Immunoreactivity of PTHrP and PTHrP-R at D18 and 21 was diminished in the distal epithelium and in the mesenchyme, respectively, in the nitrofen-induced hypoplastic lung compared to control lungs. There were no significant differences in both gene/protein expression of PTHrP and PTHrP-R on D15., Conclusion: Downregulation of PTHrP and PTHrP-R gene expression during late lung morphogenesis may cause pulmonary hypoplasia in the nitrofen CDH model, disrupting alveolar maturation and surfactant production by interfering with mesenchymal-epithelial interactions.

Objective: Little is known about the natural progression of the disease process of temporomandibular joint (TMJ) osteoarthritis (OA), which affects approximately 1% of the US population. The goal of this study was to examine the early microarchitectural and molecular changes in the condylar cartilage and subchondral bone in biglycan/fibromodulin (Bgn/Fmod) double-deficient mice, which develop TMJ-OA at 6 months., Methods: TMJs from 3-month-old (n=44) and 9-month-old (n=52) wild-type (WT n=46) and Bgn/Fmod (n=50) double-deficient mice were evaluated. Micro-CT analysis of the subchondral bone (n=24), transmission electron microscopy for condylar cartilage fibril diameters (n=26), and real-time PCR analysis for gene expression for bone and cartilage maturation markers (n=45) was performed., Results: A statistically significant increase in collagen fibril diameter of the condylar cartilage and a decrease in expression of Parathyroid related protein in the mandibular condylar head were observed in the 3-month Bgn/Fmod double-deficient mice compared to WT controls. The 9-month Bgn/Fmod double-deficient mouse demonstrated an increase in bone volume and total volume in subchondral bone, and an increase in the expression of Collagen Type X and Aggrecan in the mandibular condylar head compared to the WT controls., Conclusion: We found that changes in the microarchitecture of the condylar cartilage preceded changes in the subchondral bone during OA in the TMJ in Bgn/Fmod double-deficient mice.

Parathyroid hormone-related protein (PTHrP) is required for mammary gland development and promotes the growth of breast cancer metastases within bone. However, there are conflicting reports of the prognostic significance of its expression in primary breast cancers. To study the role of PTHrP in early breast cancer, the effect of conditional deletion of PTHrP was examined in the context of neu-induced mammary tumorigenesis. Loss of PTHrP resulted in a higher tumor incidence. Transcriptional profiling of the tumors revealed that PTHrP influenced genes relevant to heterotypic cell signaling, including regulators of monocyte recruitment. Immunohistochemical analysis of human breast cancers revealed that PTHrP expression was associated with both HER-2/neu expression and macrophage infiltration in preinvasive ductal carcinoma in situ. The gene expression signature associated with loss of PTHrP expression in vivo correlated with poorer outcome in human breast cancer. Together, these data indicate that loss of PTHrP accelerates mammary tumorigenesis possibly by a non-cell-autonomous tumor suppressor pathway.

A 64-year-old woman had normal serum calcium and plasma parathyroid hormone levels, despite an extremely high plasma parathyroid hormone-related protein (PTHrP) level. She underwent medical screening at our hospital and several neck tumors were detected by ultrasonography. After surgical resection of these tumors, her plasma PTHrP level was normalized. Histological examination showed that the resected tumors were thyroid follicular adenomas, while immunohistochemistry revealed positive staining with a monoclonal antibody for PTHrP. This is a rare case of thyroid follicular adenoma producing PTHrP in a patient with a normal serum calcium level despite elevation of plasma PTHrP.

Objectives: Parathyroid hormone-related protein (PTHrP), the main factor responsible for malignant hypercalcemia, is produced by a wide range of normal and malignant tissues. Prior studies in the rabbit model demonstrated that partial bladder outlet obstruction results in calcium-dysregulation characterized by a marked increase in free calcium within the smooth muscle compartment and the stimulation of calcium-activated enzymes, such as calpain and phospholipase A(2)., Methods: Twenty-four male New Zealand white rabbits were divided into four groups of six each. Following 4 weeks of obstruction, one group of animals was killed, while outlet obstruction was reversed in two additional groups of animals, which were killed 4 and 8 weeks after relieving the obstruction. A group with six sham-operated rabbits served as controls. The expression and localization of PTHrP were detected in muscle and mucosa by immunohistochemistry, using a PTHrP-specific antibody., Results: In the sham-operated group, rabbit bladders showed a low expression of PTHrP in both the mucosa and muscle layers. PTHrP in the 4-week obstructed bladder group, in muscle and mucosa, were 266% and 134% higher than the sham group, respectively. Strong PTHrP immunostaining persisted in the 4-week reversal groups, but it returned to the sham level after 8 weeks of reversal in the muscle layer. As mentioned about the mucosa layer, the PTHrP level returned to control levels more rapidly after 4 weeks of reversal and continued after 8 weeks of reversal., Conclusion: This study showed that PTHrP is increased after partial bladder outlet obstruction and decreased after relieving the obstruction.

We report two cases of squamous cell carcinoma of upper urinary tract with hypercalcemia. Case 1; a 54 year old female with primary squamous cell carcinoma (SCC) of right ureter showed marked hypercalcemia and leukocytosis. High levels of serum parathyroid hormone-related peptide (PTHrP) and granulocyte colony stimulating factor (G-CSF) were detected. Although chemotherapy of cisplatin and 5-fluorouracil with radiotherapy was effective, thereafter recurrence was occurred in renal pelvis, and the patient died 17 months after the initiation of therapy. Case 2; a 54 year old male of primary SCC of right renal pelvis with local lymphadenopathy and anterior mediastinal metastases showed marked hypercalcemia. High levels of PTHrP were detected. Although the patient was administered UFT with palliative radiotherapy to the anterior mediastinum, he died 2 months after the initiation of therapy. To our knowledge, the case 1 is the third case that of the high levels of serum PTHrP and G-CSF simultaneously in squamous cell carcinoma of upper urinary tract.

Parathyroid hormone-related protein (PTHrP) is an autocrine/paracrine factor produced by breast cancer cells that is speculated to play a major role in permitting breast cancer cells to grow into the bone microenvironment by stimulating the bone resorption axis. It has been previously shown that EGFR signaling induces the production of PTHrP in several primary and transformed epithelial cell types. Therefore, we investigated the relationship between EGFR and PTHrP gene expression in human breast cancer cells. Of a panel of 7 breast epithelial and cancer cell lines, the osteolytic, EGFR- positive lines (MDA-MB-231 and NS2T2A1) exhibited higher levels of PTHrP transcript expression. Amphiregulin mRNA levels in all lines were approximately 2 orders of magnitude higher than those of TGFalpha or HB-EGF. In the EGFR bearing lines, the receptor was phosphorylated at tyrosine 992 under basal conditions, and the addition of 100 nM amphiregulin did not lead to the phosphorylation of other tyrosine residues typically phosphorylated by the prototypical ligand EGF. Treatment of the EGFR positive lines with the EGFR inhibitor PD153035 and amphiregulin-neutralizing antibodies reduced PTHrP mRNA levels by 50-70%. Stable EGFR expression in the MCF7 line failed to increase basal PTHrP mRNA levels; however, treatment of this cell line with exogenous EGF or amphiregulin increased PTHrP transcription 3-fold. Transient transfection analysis suggests that the MAPK pathway and ETS transcription factors mediate EGFR coupling to PTHrP gene expression. Taken together, it appears that autocrine stimulation of EGFR signaling by amphiregulin is coupled to PTHrP gene expression via EGFR Tyr992 and MAPK, and that this pathway may contribute to PTHrP expression by breast tumor cells.

Background: Adult T-cell leukemia/lymphoma (ATLL) is initiated by infection with human T-lymphotropic virus type-1 (HTLV-1); however, additional host factors are also required for T-cell transformation and development of ATLL. The HTLV-1 Tax protein plays an important role in the transformation of T-cells although the exact mechanisms remain unclear. Parathyroid hormone-related protein (PTHrP) plays an important role in the pathogenesis of humoral hypercalcemia of malignancy (HHM) that occurs in the majority of ATLL patients. However, PTHrP is also up-regulated in HTLV-1-carriers and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) patients without hypercalcemia, indicating that PTHrP is expressed before transformation of T-cells. The expression of PTHrP and the PTH/PTHrP receptor during immortalization or transformation of lymphocytes by HTLV-1 has not been investigated., Results: We report that PTHrP was up-regulated during immortalization of lymphocytes from peripheral blood mononuclear cells by HTLV-1 infection in long-term co-culture assays. There was preferential utilization of the PTHrP-P2 promoter in the immortalized cells compared to the HTLV-1-transformed MT-2 cells. PTHrP expression did not correlate temporally with expression of HTLV-1 tax. HTLV-1 infection up-regulated the PTHrP receptor (PTH1R) in lymphocytes indicating a potential autocrine role for PTHrP. Furthermore, co-transfection of HTLV-1 expression plasmids and PTHrP P2/P3-promoter luciferase reporter plasmids demonstrated that HTLV-1 up-regulated PTHrP expression only mildly, indicating that other cellular factors and/or events are required for the very high PTHrP expression observed in ATLL cells. We also report that macrophage inflammatory protein-1alpha (MIP-1alpha), a cellular gene known to play an important role in the pathogenesis of HHM in ATLL patients, was highly expressed during early HTLV-1 infection indicating that, unlike PTHrP, its expression was enhanced due to activation of lymphocytes by HTLV-1 infection., Conclusion: These data demonstrate that PTHrP and its receptor are up-regulated specifically during immortalization of T-lymphocytes by HTLV-1 infection and may facilitate the transformation process.

Parathyroid hormone-related peptide (PTHrP) is released under ischaemic conditions and it improves contractile function of stunned myocardium. The actions of PTHrP are mediated primarily by the type 1 parathyroid hormone receptor (PTH.1R), while PTHrP and PTH.1R expression levels are increased in ventricular hypertrophy associated with experimental hyperthyroidism. Since chronic administration of thyroxine (T4) improves postischaemic recovery in isolated heart models subjected to ischaemia-reperfusion stress, we tested the hypothesis that experimentally induced hyperthyroidism is associated with elevated expression of PTHrP and PTH.1R in rat myocardium. Hyperthyroid and control male Wistar rats were subjected to ischaemia-reperfusion stress using the Langendorff technique, and the PTHrP and PTH.1R expression was assessed by relative quantitative reverse transcriptase-polymerase chain reaction, Western blot analysis and immunohistochemistry. In the Langendorff model, the recovery of left ventricular developed pressure at the end of the stablization period and 45 min into the reperfusion period was used to assess the cardioprotective actions of T4 administration. Our data show that hyperthyroid animals had increased tolerance to the ischaemia-reperfusion stress and that this was associated with an increase of PTHrP and PTH.1R expression levels compared with those of control animals. In the control animals, the expression of PTHrP was increased 45 min into the reperfusion phase, while the PTH.1R expression pattern was significantly and gradually decreased throughout the ischaemia and reperfusion phases. In the hyperthyroid animals, the PTHrP and PTH.1R expression pattern was significantly higher throughout the ischaemia and reperfusion phases compared with that of control hearts. Our data suggest that increasing levels of PTHrP and PTH.1R expression can mediate, at least in part, the T4 administration-induced cardioprotection in rat ventricular myocardium.

Introduction: Parathyroid-hormone-related protein (PTHrP) is considered as an autocrine/paracrine regulator of growth and/or differentiation in normal and malignant tissues. We determined the distribution and density of the expression of PTHrP in the rabbit bladder during growth in response to partial outlet obstruction and its relation with the smooth muscle/collagen ratio., Materials and Methods: A total of 30 male New Zealand White rabbits were studied. After 7, 14, 28 or 56 days of obstruction, 6 rabbits per group were sacrificed and the bladder extracted. Six sham-operated rabbits served as controls. The expression and localization of PTHrP or collagen type III were detected by immunohistochemistry using specific antibodies., Results: The levels of PTHrP were progressively increased by 14 days of obstruction, whereas they decreased after 14 days, although the PTHrP positivity was higher than in sham controls by 28 and 56 days of obstruction. PTHrP staining was related to the smooth muscle/collagen ratio, both showing a peak in rabbits obstructed for 14 days., Conclusions: These data indicate that PTHrP may play an important regulatory role in the cellular and vascular response to partial outlet obstruction., ((c) 2008 S. Karger AG, Basel.)