A regulatory pathway involving retinoic acid and calcineurin demarcates and maintains joint cells and osteoblasts in regenerating fin.

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.

A combined series of Fgf9 and Fgf18 mutant alleles identifies unique and redundant roles in skeletal development.

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.

Intrahepatic cholangiocarcinoma producing granulocyte colony-stimulating factor and parathyroid hormone-related protein.

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.

The tumor suppressor BTG1 is expressed in the developing digits and regulates skeletogenic differentiation of limb mesodermal progenitors in high density cultures.

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.

A case of adenosquamous cell carcinoma of the gallbladder with markedly elevated PTHrP and G-CSF levels.

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.

Co-expression of parathyroid hormone related protein and TGF-beta in breast cancer predicts poor survival outcome.

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-ß have been observed in various types of human cancers. The objective of the current study was to evaluate the association of PTHrP and TGF-ß level with the clinicopathological features of the breast cancer patients. METHODS: Immunohistochemistry was used to examine PTHrP and TGF-ß 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-ß expression. RESULTS: Both over-expressed TGF-ß 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-ß level developed poor differentiation, and only TGF-ß 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-ß 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-ß and PTHrP expression were significantly associated with the high risk of metastases. As indicated by Cox's regression analysis, TGF-ß expression and the high proportion of axillary lymph node metastasis served as significant independent predictors for breast cancer recurrence. CONCLUSIONS: TGF-ß 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.

[Parathyroid Hormone-related Peptide (PTHrP) Producing Lung Cancer Presenting Hypercalcemia; Report of a Case].

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.

MicroRNA-33a functions as a bone metastasis suppressor in lung cancer by targeting parathyroid hormone related protein.

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.

Tetrahydrofurofuran-type lignans inhibit breast cancer-mediated bone destruction by blocking the vicious cycle between cancer cells, osteoblasts and osteoclasts.

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-ß-stimulated cell viability, migration, and invasion and decreased TGF-ß-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.

Combined hepatocellular-cholangiocarcinoma producing parathyroid hormone-related protein: report of a case.

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.

[Role for PTHrP in bone and cartilage metabolism].

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.

[A case of hypercalcemia associated with parathyroid hormone-related protein produced by the recurrence of B-cell lymphoma of the pancreas].

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.

Paraneoplastic hormones: parathyroid hormone-related protein (PTHrP) and erythropoietin (EPO) are related to vascular endothelial growth factor (VEGF) expression in clear cell renal cell carcinoma.

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.

Construction of recombinant adenoviruses carrying the optimal shRNA template against goat PTHrP and successful suppression of PTHrP expression in mammary epithelial cells.

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.

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.

Systemic and acute administration of parathyroid hormone-related peptide(1-36) stimulates endogenous beta cell proliferation while preserving function in adult mice.

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.

Negative regulation of parathyroid hormone-related protein expression by steroid hormones.

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.

The expression of PTHLH in human gastric mucosa enterochromaffin-like cells.

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 ß-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.

A case of Serratia granuloma in the soft tissue around the left kidney: a role of PTHrP in the formation of Serratia granuloma.

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.

Parathyroid hormone-related peptide-producing non-familial pheochromocytoma in a child.

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.