Parathyroid Hormone-related Protein Production Research Articles

“Superscan” Scintigraphic Version for Prostate Cancer

A rare scintigraphic phenomenon of “superscan” is considered – an abnormally high diffuse accumulation of osteotropic radiopharmaceutical by all skeletal parts in combination with the absence of kidney and tissue background imaging. This feature reflects a generalized bone system lesion of metastatic, metabolic or hematological nature. We present a clinical observation and retrospective assessment of scintigraphic data obtained during controlling androgen deprivation therapy in a patient with prostate cancer. Based on the analysis of “superscan” signs, including 99mТс-pyrophosphate hyperfixation by skull bones, as well as concomitant levels of prostatic specific antigen and parathyroid hormone (PTH), it was suggested that the identified scintigraphic phenomenon had metabolic nature, and its occurrence was possibly due to ectopic production of both PTH and parathyroid hormone-related protein, playing a well-known role in prostate cancer genesis.

Hypercalcemia of malignancy with simultaneous production of parathyroid hormone-related peptide and 1,25-Dihydroxyvitamin D by non-Hodgkin lymphoma: Case report & brief review

We report the case of a 63-year-old man with severe hypercalcemia, acute kidney injury, and concomitant diagnosis of germinal center diffuse large B-cell lymphoma (nHL). Hypercalcemia is a common finding in patients with cancer. Multiple mechanisms can lead to hypercalcemia of malignancy, and different malignancies are often associated with a specific mechanism of hypercalcemia. Parathyroid hormone-related protein (PTHrP) and excessive production of 1,25-dihydroxyvitamin D can independently (and rarely, concomitantly) lead to development of hypercalcemia in patients with a variety of malignancies. Literature review of hypercalcemia related with PTHrP and 1,25-dihydroxyvitamin D co-secretion shows that most reported cases are associated with solid tumors and responded to tumor directed therapy. Our case of nHL was associated with increased PTHrP and 1,25-dihydroxyvitamin D making this an unusual presentation.

What Lies behind Paraneoplastic Hypercalcemia Secondary to Well-Differentiated Neuroendocrine Neoplasms? A Systematic Review of the Literature.

Background: Neuroendocrine neoplasms (NEN) originate from neuroendocrine cells ubiquitously spread throughout the body. Hypercalcemia associated with cancer is the most common life-threatening metabolic disorder in patients with advanced stage cancer. Paraneoplastic hypercalcemia is more commonly associated with hematological malignancies, renal and breast carcinomas, and squamous cell carcinomas, but it has also been described in patients with well-differentiated NEN, where it often remains undiagnosed. Among its causes, systemic secretion of parathyroid hormone-related protein (PTHrP) and ectopic production of 1,25-dihydroxyvitamin D and parathyroid hormone (PTH) may be considered paraneoplastic causes of hypercalcemia. In order to clarify the diagnostic work up of paraneoplastic hypercalcemia in patients with NEN, we perform a systematic review, which is lacking in the literature. Methods: We performed a data search using MEDLINE and SCOPUS including papers from 1961 to 2021. We selected articles on paraneoplastic hypercalcemia associated with well-differentiated NEN. Results: The search led to the selection of 78 publications for a total of 114 patients. Pooled data showed that the most frequent primary tumor site associated with paraneoplastic hypercalcemia was pancreatic NEN, followed by Pheochromocytoma. In most cases, paraneoplastic hypercalcemia was caused by PTHrP production and secretion. In more than two thirds of cases, paraneoplastic hypercalcemia was present at the time of NEN diagnosis and, in metachronous cases, was related to local recurrence, distant metastasis development, or tumor progression. In most patients, a combination of therapeutic approaches was employed, and reduction of the tumor burden was essential to control the paraneoplastic syndrome. Discussion: The onset of hypercalcemia associated with cancer in patients with well-differentiated NEN represents a major clinical challenge. The complex clinical and therapeutical management of paraneoplastic hypercalcemia implies the need for a multidisciplinary approach, aimed at controlling the clinical syndrome and tumor growth.

A Review of Current Clinical Concepts in the Pathophysiology, Etiology, Diagnosis, and Management of Hypercalcemia.

Calcium is the most abundant extracellular cation in the body, and it is responsible for structural and enzymatic functions. Calcium homeostasis is regulated by 3 factors: calcitonin, vitamin D, and parathyroid hormone (PTH). Hypercalcemia is defined by a serum calcium concentration >10.5 mg/dL, and it is classified into mild, moderate, and severe, depending on calcium values. Most cases are caused by primary hyperparathyroidism and malignancies. Various mechanisms are involved in the pathophysiology of hypercalcemia, such as excessive PTH production, production of parathyroid hormone-related protein (PTHrp), bone metastasis, extrarenal activation of vitamin D, and ectopic PTH secretion. The initial approach is similar in most cases, but a definitive treatment depends on etiology, that is why etiological investigation is mandatory in all cases. The majority of patients are asymptomatic and diagnosed during routine exams; only a small percentage of patients present with severe manifestations which can affect neurological, muscular, gastrointestinal, renal, and cardiovascular systems. Clinical manifestations are related to calcium levels, with higher values leading to more pronounced symptoms. Critically ill patients should receive treatment as soon as diagnosis is made. Initial treatment involves vigorous intravenous hydration and drugs to reduce bone resorption such as bisphosphonates and, more recently, denosumab, in refractory cases; also, corticosteroids and calcitonin can be used in specific cases. This review aims to provide a clinical update on current concepts of the pathophysiology of calcium homeostasis, epidemiology, screening, clinical presentation, diagnosis, and management of hypercalcemia.

Parathyroid hormone-related protein in breast cancer bone metastasis.

Parathyroid hormone-related protein (PTHrP) was discovered as the tumor product causing the humoral hypercalcemia of malignancy. Its structural similarity to the hormone, PTH, with 8 of the first 13 amino acids identical, was sufficient to explain the sharing by PTHrP and PTH of a common receptor, PTH1R, although the remainder of the sequences are unique. PTHrP has important roles in development of several organs, including breast and bone, and functions as a paracrine factor postnatally in these and other tissues. In addition to its hormonal role in cancer, PTHrP is produced by two thirds of primary breast cancers and 90% of bone metastases from breast cancer, leading to the concept that its production in bone by breast cancer cells promotes bone resorption, thus favoring tumor establishment and expansion, and an exit from tumor dormancy in bone through downregulation of leukemia inducing factor receptor (LIFR). Cancer production of PTHrP is increased by bone-derived growth factors, with particular attention paid to TGFβ, as well as by promoter-driven transcriptional effects, such as the hedgehog signaling factor, GLI2, and microenvironment effects including changes in underlying stiffness of substrates for cells. Although interest has been focused on PTHrP-induced bone resorption in bone metastasis, a mechanistically separate, protective effect against tumor progression has been proposed. Although there is conflicting mouse data, there are clinical studies suggesting that increased production of PTHrP by breast cancers confers upon them a less invasive phenotype, an effect distinct from the bone resorption-stimulating action that favors bone metastasis.

Hypercalcaemia in a dog with lymphoma without increases in parathyroid hormone, parathyroid hormone-related protein and vitamin D metabolites concentrations

Lymphoma is one of the most common causes of hypercalcaemia in dogs. Typically, the hypercalcaemic state is driven by ectopic production of parathyroid hormone-related protein by the malignant lymphoma cells....

A Diet Rich in Fish Oil and Leucine Ameliorates Hypercalcemia in Tumour-Induced Cachectic Mice

Background: Dietary supplementation with leucine and fish oil rich in omega-3 fatty acids docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) has previously been shown to reduce cachexia-related outcomes in C26 tumour-bearing mice. To further explore associated processes and mechanisms we investigated changes in plasma Ca2+ levels, the involvement of parathyroid hormone related protein (PTHrP), and its possible interactions with cyclooxygenase 2 (COX-2). Methods: CD2F1 mice were subcutaneously inoculated with C26 adenocarcinoma cells or sham treated and divided in: (1) controls, (2) tumour-bearing controls, and (3) tumour-bearing receiving experimental diets. After 20 days, body and organ masses and total plasma Ca2+ levels were determined. Furthermore, effects of DHA, EPA and leucine on production of PTHrP were studied in cultured C26 cells. Results: The combination of leucine and fish oil reduced tumour-associated hypercalcemia. Plasma Ca2+ levels negatively correlated with carcass mass and multiple organ masses. DHA was able to reduce PTHrP production by C26 cells in vitro. Results indicate that this effect occurred independently of COX-2 inhibition. Conclusion: Our results suggest that cancer-related hypercalcemia may be ameliorated by a nutritional intervention rich in leucine and fish oil. The effect of fish oil possibly relates to a DHA-induced reduction of PTHrP excretion by the tumour.

6-Shogaol Suppresses 2-Amino-1-Methyl-6-Phenylimidazo [4,5-b] Pyridine (PhIP)-Induced Human 786-O Renal Cell Carcinoma Osteoclastogenic Activity and Metastatic Potential.

2-Amino-1-methyl-6-phenylimidazo [4,5-b]pyridine (PhIP) which can be detected in processed meats and red meats, is a potential carcinogen for renal cell carcinoma (RCC). Approximately 30% of patients with metastatic RCC have bone metastases, and the prognosis of RCC with bone metastases is poor. Thus, the aim of the present study was to investigate whether PhIP induced bone metastases and to develop novel therapeutic agents. Our data revealed that PhIP pre-treatment increased the production of parathyroid hormone-related protein (PTHrP) in human 786-O renal cell carcinoma cells. Subsequently, the cultures of human osteoblasts with PhIP-stimulated condition medium of 786-O increased the expression of the macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor kappa-B ligand (RANKL), and decreased the expression of osteoprotegerin (OPG). In addition, PhIP-mediated PTHrP up-regulated as well as increased IL-8 secretion in 786-O cells, and then contributed to 786-O-mediated bone resorption. Furthermore, 6-shogaol, which is an active ingredient in ginger, showed suppressive effects on PhIP-mediated bone resorption. In summary, this is the first study to demonstrate that PhIP pre-treatment increases the stimulatory effect of human renal cell carcinoma 786-O on osteoclastogenesis activity directly by PTHrP. In addition, 6-shogaol treatment reverses PhIP-mediated bone resorption. It suggests that 6-shogaol treatment results in bone resorption activity in the RCC model in vitro.

Multiple actions of parathyroid hormone-related protein in breast cancer bone metastasis.

The sequence similarity within the amino-terminal regions of parathyroid hormone (PTH) and PTH-related protein (PTHrP) allows the two to share actions at a common site, the PTH1 receptor. A number of biological activities have been ascribed to actions of other domains within PTHrP. PTHrP production by late stage breast cancer has been shown to contribute to bone metastasis formation through promotion of osteoclast formation and bone resorption by action through PTH1 receptors. There is evidence also for a role for PTHrP early in breast cancer that is protective against tumour progression. No signalling pathway has been identified for this effect. PTHrP has also been identified as a factor promoting the emergence of breast cancer cells from dormancy in bone. In that case, PTHrP does not function through activation of PTH1 receptors, despite having very substantial effects on transcriptional activity of the breast cancer cells. This indicates actions of PTHrP that are non-canonical, that is, mediated through domains other than the amino-terminal. It is concluded that PTHrP has several distinct paracrine, autocrine, and intracrine actions in the course of breast cancer pathophysiology. Some are mediated through action at PTH1 receptors and others are controlled by other domains within PTHrP. LINKED ARTICLES: This article is part of a themed issue on The molecular pharmacology of bone and cancer-related bone diseases. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v178.9/issuetoc.

TRIENNIAL LACTATION SYMPOSIUM/BOLFA: Serotonin and the regulation of calcium transport in dairy cows.

The mammary gland regulates maternal metabolism during lactation. Numerous factors within the tissue send signals to shift nutrients to the mammary gland for milk synthesis. Serotonin is a monoamine that has been well documented to regulate several aspects of lactation among species. Maintenance of maternal calcium homeostasis during lactation is a highly evolved process that is elegantly regulated by the interaction of the mammary gland with the bone, gut, and kidney tissues. It is well documented that dietary calcium is insufficient to maintain maternal calcium concentrations during lactation, and mammals must rely on bone resorption to maintain normocalcemia. Our recent work focused on the ability of the mammary gland to function as an accessory parathyroid gland during lactation. It was demonstrated that serotonin acts to stimulate parathyroid hormone-related protein (PTHrP) in the mammary gland during lactation. The main role of mammary-derived PTHrP during mammalian lactation is to stimulate bone resorption to maintain maternal calcium homeostasis during lactation. In addition to regulating PTHrP, it was shown that serotonin appears to directly affect calcium transporters and pumps in the mammary gland. Our current working hypothesis regarding the control of calcium during lactation is as follows: serotonin directly stimulates PTHrP production in the mammary gland through interaction with the sonic hedgehog signaling pathway. Simultaneously, serotonin directly increases calcium movement into the mammary gland and, subsequently, milk. These 2 direct actions of serotonin combine to induce a transient maternal hypocalcemia required to further stimulate PTHrP production and calcium mobilization from bone. Through these 2 routes, serotonin is able to improve maternal calcium concentrations. Furthermore, we have shown that Holstein and Jersey cows appear to regulate calcium in different manners and also respond differently to serotonergic stimulation of the calcium pathway. Our data in rodents and cows indicate that serotonin and calcium are working through a unique feedback loop with PTHrP during lactation to regulate milk calcium and maternal calcium homeostasis.

Twist on a classic: vitamin D and hypercalcaemia of malignancy

Malignancy is the most common cause of hypercalcaemia in the inpatient setting. Most cases are caused by tumour production of parathyroid hormone-related protein and osseous metastases. In less than 1%...

Trousseau's syndrome caused by bladder cancer producing granulocyte colony-stimulating factor and parathyroid hormone-related protein: A case report.

The present study reports a rare case of bladder cancer that caused Trousseau's syndrome, a malignancy-associated hypercoagulative state leading to venous or arterial thrombosis. A 38-year-old Japanese female patient experienced a sudden onset of visual field defects and lower limb weakness. Cerebral magnetic resonance imaging revealed multi-regional hypointense areas compatible with acute cerebral infarction. Despite the repeated administration of anticoagulant treatment with heparin, the condition could not be adequately controlled and thromboembolic events occasionally recurred. Several tumor markers, including carbohydrate antigen 19-9, cancer antigen 125, carcinoembryonic antigen, cytokeratin 19 fragment and squamous cell carcinoma antigen levels, were elevated. Consequently, computed tomography scans were performed, which revealed a massive bladder tumor with multiple bone and lymph node metastases. The patient also exhibited other paraneoplastic disorders, including leukocytosis due to granulocyte colony-stimulating factor (G-CSF) production, and hypercalcemia due to parathyroid hormone-related protein (PTHrP) production. Transurethral resection of the bladder tumor was performed, and the tumor was pathologically confirmed as urothelial cell carcinoma. Immunohistochemical testing revealed positive staining for G-CSF and PTHrP. Despite undergoing gemcitabine/cisplatin-based systemic chemotherapy, the disease developed rapidly and the patient succumbed to the disease within 3 months of initial symptoms. The present case indicates that occult visceral malignancy should be considered in patients with unexplained thromboembolism.

Bone metabolism during pregnancy

During pregnancy, mineral concentrations, of calcium and phosphorus in particular, are maintained at a high level in fetal blood so that the developing skeleton may accrete adequate mineral content. The placenta actively transports minerals for this purpose. Maternal intestinal absorption increases in order to meet the fetal demand for calcium, which is only partly dependent on calcitriol. Mineral regulation is essentially dependent on parathyroid hormone (PTH) and PTH-related protein (PTHrP). The calcium-sensing receptor (CaSR) regulates PTH and PTHrP production. If calcium intake is insufficient, the maternal skeleton will undergo resorption due to PTHrP. After birth, a switch from fetal to neonatal homeostasis occurs through increase in PTH and calcitriol, and developmental adaptation of the kidneys and intestines with bone turnover contributing additional mineral to the circulation. Calcium absorption becomes progressively active and dependent on calcitriol. The postnatal skeleton can transiently present with osteoposis but adequate mineral diet usually allows full restoration. Cases of primary osteoporosis must be identified. Loss of trabecular mineral content occurs during lactation in order to provide calcium to the newborn. This programmed bone loss is dependent on a “brain-breast-bone” circuit. The physiological bone resorption during reproduction does not normally cause fractures or persistent osteoporosis. Women who experience fracture are likely to have other causes of bone loss.

Induction of chronic pancreatitis by pancreatic duct ligation activates BMP2, apelin, and PTHrP expression in mice.

Chronic pancreatitis (CP) is a devastating disease with no treatments. Experimental models have been developed to reproduce the parenchyma and inflammatory responses typical of human CP. For the present study, one objective was to assess and compare the effects of pancreatic duct ligation (PDL) to those of repetitive cerulein (Cer)-induced CP in mice on pancreatic production of bone morphogenetic protein-2 (BMP2), apelin, and parathyroid hormone-related protein (PTHrP). A second objective was to determine the extent of cross talk among pancreatic BMP2, apelin, and PTHrP signaling systems. We focused on BMP2, apelin, and PTHrP since these factors regulate the inflammation-fibrosis cascade during pancreatitis. Findings showed that PDL- and Cer-induced CP resulted in significant elevations in expression and peptide/protein levels of pancreatic BMP2, apelin, and PTHrP. In vivo mouse and in vitro pancreatic cell culture experiments demonstrated that BMP2 stimulated pancreatic apelin expression whereas apelin expression was inhibited by PTHrP exposure. Apelin or BMP2 exposure inhibited PTHrP expression, and PTHrP stimulated upregulation of gremlin, an endogenous inhibitor of BMP2 activity. Transforming growth factor-β (TGF-β) stimulated PTHrP expression. Together, findings demonstrated that PDL- and Cer-induced CP resulted in increased production of the pancreatic BMP2, apelin, and PTHrP signaling systems and that significant cross talk occurred among pancreatic BMP2, apelin, and PTHrP. These results together with previous findings imply that these factors interact via a pancreatic network to regulate the inflammation-fibrosis cascade during CP. More importantly, this network communicated with TGF-β, a key effector of pancreatic pathophysiology. This novel network may be amenable to pharmacologic manipulations during CP in humans.

15-deoxy-δ12,14-prostaglandin j2 inhibits osteolytic breast cancer bone metastasis and estrogen deficiency-induced bone loss.

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.

The effects of static pressure on chondrogenic and osteogenic differentiation in condylar chondrocytes from temporomandibular joint

ObjectiveThe goal of the study was to investigate the production of collagen, type II, alpha 1 (COL2A1), SOX9, alkaline phosphatase (ALP), runt-related transcription factor 2 (Runx2), Indian hedgehog (Ihh), and periarticular cell-derived parathyroid hormone-related protein (PTHrP) in mandibular condylar chondrocytes under static pressure stimuli. MethodsMandibular condylar chondrocytes separated from rabbit temporomandibular joint (TMJ) were treated with a static pressure of 100kPa for 0, 1, 2, 3, and 4h by an in-house-designed pressure chamber. A CCK-8 kit was used to analyze the cell viability. The production of COL2A1, SOX9, ALP, Runx2, Ihh, and PTHrP was detected by Western blot or real-time polymerase chain reaction (PCR). Changes in cell morphology were observed by scanning electron microscopy. ResultsCompared with the control group (0h), the cytoplasmic processes of treated chondrocytes obviously increased and elongated, and the cell viability of pressurized chondrocytes were 91.13% (1h), 103.41% (2h), 103.47% (3h), and 104.94% (4h), respectively. The exposure of condylar chondrocytes to a static pressure of 100kPa for 3–4h resulted in a significant increase in COL2A1, SOX9, ALP, and Runx2. After a static pressure loading of 100kPa, the activation of Ihh and PTHrP was also observed. ConclusionsMandibular condylar chondrocytes adapt to alterations of the microenvironment. Ihh and PTHrP are sensitive to static pressure. Our findings suggest that static pressure accelerated the chondrogenic and osteogenic differentiation of condylar chondrocytes, which may influence the pathological progress of temporomandibular diseases.

Effects of simvastatin on human breast cancer osteolytic bone metastasis in a nude mice model

Objective To observe the effect of simvastatin on bone metastasis of breast cancer in nude mice model. Methods Sixty mice were divided into three groups randomly with 20 in each group. Mice were inoculated with MDA-MB-231 cells into the left cardiac ventricle. After 7 days, mice were treated with either simvastatin, saline, or nothing twice per week for 19 days. The area of osteolytic metastases was subsequently measured in long bones of all mice using an image analysis system. After sacrifice, parathyroid hormone-related protein (PTHrP) concentrations in bone marrow from all mice were determined using a two-site immunoradiometric assay. Osteoclast number expressed per millimeter of tumor/bone interface was assessed using an OsteoMeasure System. Measured data were compared with analysis of variance, and P<0.05 for the difference was statistically significant. Results The area of osteolytic lesions was significantly lower in mice treated with simvastatin compared with mice receiving saline and no treatment (0.51±0.18 mm2vs 2.13±1.24 mm2vs 2.29±1.22 mm2;F=15.600, P=0.002; F=15.673, P=0.001). In addition, treatment with simvastatin decreased the concentrations of PTHrP in bone marrow plasma (0.98±0.20 pmol/L vs 2.11±0.31 pmol/L vs 1.99±0.29 pmol/L; F=61.469, P<0.001; F=58.274, P<0.001) and the osteoclast numbers per millimeter of tumor/bone interface (4.00±1.73 vs 11.40±4.93 vs 10.91±3.87; F=17.820, P=0.001; F=17.184, P=0.002) compared with controls. Conclusion Simvastatin may reduce the production of PTHrP of breast cancer cells, which suppresses the development of destructive bone lesions as well as the growth of breast cancer cells in bone. Key words: Breast neoplasms; Neoplasm metastasis; Simvastatin; Parathyroid hormone

Wnt signaling induces gene expression of factors associated with bone destruction in lung and breast cancer.

Parathyroid hormone-related protein (PTHrP) is an important regulator of bone destruction in bone metastatic tumors. Transforming growth factor-beta (TGF-β) stimulates PTHrP production in part through the transcription factor Gli2, which is regulated independent of the Hedgehog signaling pathway in osteolytic cancer cells. However, inhibition of TGF-β in vivo does not fully inhibit tumor growth in bone or tumor-induced bone destruction, suggesting other pathways are involved. While Wnt signaling regulates Gli2 in development, the role of Wnt signaling in bone metastasis is unknown. Therefore, we investigated whether Wnt signaling regulates Gli2 expression in tumor cells that induce bone destruction. We report here that Wnt activation by β-catenin/T cell factor 4 (TCF4) over-expression or lithium chloride (LiCl) treatment increased Gli2 and PTHrP expression in osteolytic cancer cells. This was mediated through the TCF and Smad binding sites within the Gli2 promoter as determined by promoter mutation studies, suggesting cross-talk between TGF-β and Wnt signaling. Culture of tumor cells on substrates with bone-like rigidity increased Gli2 and PTHrP production, enhanced autocrine Wnt activity and led to an increase in the TCF/Wnt signaling reporter (TOPFlash), enriched β-catenin nuclear accumulation, and elevated Wnt-related genes by PCR-array. Stromal cells serve as an additional paracrine source of Wnt ligands and enhanced Gli2 and PTHrP mRNA levels in MDA-MB-231 and RWGT2 cells in vitro and promoted tumor-induced bone destruction in vivo in a β-catenin/Wnt3a-dependent mechanism. These data indicate that a combination of matrix rigidity and stromal-secreted factors stimulate Gli2 and PTHrP through Wnt signaling in osteolytic breast cancer cells, and there is significant cross-talk between the Wnt and TGF-β signaling pathways. This suggests that the Wnt signaling pathway may be a potential therapeutic target for inhibiting tumor cell response to the bone microenvironment and at the very least should be considered in clinical regimens targeting TGF-β signaling.

The paracrine feedback loop between vitamin D₃ (1,25(OH)₂D₃) and PTHrP in prehypertrophic chondrocytes.

The endocrine feedback loop between vitamin D3 (1,25(OH)2D3) and parathyroid hormone (PTH) plays a central role in skeletal development. PTH-related protein (PTHrP) shares homology and its receptor (PTHR1) with PTH. The aim of this study was to investigate whether there is a functional paracrine feedback loop between 1,25(OH)2D3 and PTHrP in the growth plate, in parallel with the endocrine feedback loop between 1,25(OH)2D3 and PTH. This was investigated in ATDC5 cells treated with 10−8 M 1,25(OH)2D3 or PTHrP, Col2-pd2EGFP transgenic mice, and primary Col2-pd2EGFP growth plate chondrocytes isolated by FACS, using RT-qPCR, Western blot, PTHrP ELISA, chromatin immunoprecipitation (ChIP) assay, silencing of the 1,25(OH)2D3 receptor (VDR), immunofluorescent staining, immunohistochemistry, and histomorphometric analysis of the growth plate. The ChIP assay confirmed functional binding of the VDR to the PTHrP promoter, but not to the PTHR1 promoter. Treatment with 1,25(OH)2D3 decreased PTHrP protein production, an effect which was prevented by silencing of the VDR. Treatment with PTHrP significantly induced VDR production, but did not affect 1α- and 24-hydroxylase expression. Hypertrophic differentiation was inhibited by PTHrP and 1,25(OH)2D3 treatment. Taken together, these findings indicate that there is a functional paracrine feedback loop between 1,25(OH)2D3 and PTHrP in the growth plate. 1,25(OH)2D3 decreases PTHrP production, while PTHrP increases chondrocyte sensitivity to 1,25(OH)2D3 by increasing VDR production. In light of the role of 1,25(OH)2D3 and PTHrP in modulating chondrocyte differentiation, 1,25(OH)2D3 in addition to PTHrP could potentially be used to prevent undesirable hypertrophic chondrocyte differentiation during cartilage repair or regeneration.

Deletion of the nuclear localization sequences and C-terminus of PTHrP impairs embryonic mammary development but also inhibits PTHrP production.

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.