Expression Of Parathyroid Hormone-related Peptide Research Articles

CaSR modulates proliferation of the superficial zone cells in temporomandibular joint cartilage via the PTHrP nuclear localization sequence.

The superficial zone cells in mandibular condylar cartilage are proliferative. The present purpose was to delineate the relation of calcium-sensing receptor (CaSR) and parathyroid hormone-related peptide nuclear localization sequence (PTHrP87-139 ), and their role in the proliferation behaviors of the superficial zone cells. A gain- and loss-of-function strategy were used in an in vitro fluid flow shear stress (FFSS) model and an in vivo bilateral elevation bite model which showed mandibular condylar cartilage thickening. CaSR and PTHrP87-139 were modulated through treating the isolated superficial zone cells with activator/SiRNA and via deleting CaSR or parathyroid hormone-related peptide (PTHrP) gene in mice with the promoter gene of proteoglycan 4 (Prg4-CreERT2 ) in the tamoxifen-inducible pattern with or without additional injection of Cinacalcet, the CaSR agonist, or PTHrP87-139 peptide. FFSS stimulated CaSR and PTHrP expression, and accelerated proliferation of the Prg4-expressing superficial zone cells, in which process CaSR acted as an up-streamer of PTHrP. Proteoglycan 4 specific knockout of CaSR or PTHrP reduced the cartilage thickness, suppressed the proliferation and early differentiation of the superficial zone cells, and inhibited cartilage thickening and matrix production promoted by bilateral elevation bite. Injections of CaSR agonist Cinacalcet could not improve the phenotype caused by PTHrP mutation. Injections of PTHrP87-139 peptide rescued the cartilage from knockout of CaSR gene. CaSR modulates proliferation of the superficial zone cells in mandibular condylar cartilage through activation of PTHrP nuclear localization sequence. Our data support the therapeutic target of CaSR in promoting PTHrP production in superficial zone cartilage.

Parathyroid Hormone-Related Protein Promotes the Proliferation of Patient-Derived Glioblastoma Stem Cells via Activating cAMP/PKA Signaling Pathway.

Glioblastoma (GBM) is an aggressive primary brain tumor characterized by its heterogeneity and high recurrence and lethality rates. Glioblastoma stem cells (GSCs) play a crucial role in therapy resistance and tumor recurrence. Therefore, targeting GSCs is a key objective in developing effective treatments for GBM. The role of Parathyroid hormone-related peptide (PTHrP) in GBM and its impact on GSCs remains unclear. This study aimed to investigate the effect of PTHrP on GSCs and its potential as a therapeutic target for GBM. Using the Cancer Genome Atlas (TCGA) database, we found higher expression of PTHrP in GBM, which correlated inversely with survival. GSCs were established from three human GBM samples obtained after surgical resection. Exposure to recombinant human PTHrP protein (rPTHrP) at different concentrations significantly enhanced GSCs viability. Knockdown of PTHrP using target-specific siRNA (siPTHrP) inhibited tumorsphere formation and reduced the number of BrdU-positive cells. In an orthotopic xenograft mouse model, suppression of PTHrP expression led to significant inhibition of tumor growth. The addition of rPTHrP in the growth medium counteracted the antiproliferative effect of siPTHrP. Further investigation revealed that PTHrP increased cAMP concentration and activated the PKA signaling pathway. Treatment with forskolin, an adenylyl cyclase activator, nullified the antiproliferative effect of siPTHrP. Our findings demonstrate that PTHrP promotes the proliferation of patient-derived GSCs by activating the cAMP/PKA signaling pathway. These results uncover a novel role for PTHrP and suggest its potential as a therapeutic target for GBM treatment.

Investigation of bone invasion and underlying mechanisms of oral cancer using a cell line-derived xenograft model.

The cancer stroma regulates bone invasion in oral squamous cell carcinoma (OSCC). However, data on normal stroma are limited. In the present study, the effects of gingival and periodontal ligament tissue-derived stromal cells (G-SCs and P-SCs, respectively) and human dermal fibroblasts (HDFs) on bone resorption and osteoclast activation were assessed using hematoxylin and eosin and tartrate-resistant acid phosphatase staining in a cell line-derived xenograft model. The results demonstrated that G-SCs promoted bone invasion and osteoclast activation and inhibited osteoclast proliferation following crosstalk with the human OSCC HSC-3 cell line, whereas P-SCs inhibited bone resorption and promoted osteoclast proliferation in vitro but had a minimal effect on osteoclast activation both in vitro and in vivo following crosstalk with HSC-3 cells. Furthermore, the effects of G-SCs, P-SCs and HDFs on protein expression levels of matrix metalloproteinase (MMP)-9, membrane type 1 MMP (MT1-MMP), Snail, parathyroid hormone-related peptide (PTHrP) and receptor activator of NF-κB ligand (RANKL) in HSC-3 cells in OSCC bone invasion regions were assessed using immunohistochemistry. The results demonstrated that G-SCs had a more prominent effect on the expression of MMP-9, MT1-MMP, Snail, PTHrP, and RANKL, whereas P-SCs only promoted RANKL and PTHrP expression and exerted a minimal effect on MMP-9, MT1-MMP and Snail expression. The potential genes underlying the differential effects of G-SCs and P-SCs on bone invasion in OSCC were evaluated using a microarray, which indicated that cyclin-dependent kinase 1, insulin, aurora kinase A, cyclin B1 and DNA topoisomerase II alpha underlaid these differential effects. Therefore, these results demonstrated that G-SCs promoted bone invasion in OSCC by activating osteoclasts on the bone surface, whereas P-SCs exerted an inhibitory effect. These findings could indicate a potential regulatory mechanism for bone invasion in OSCC.

Involvement of Met receptor pathway in aggressive behavior of colorectal cancer cells induced by parathyroid hormone-related peptide.

Involvement of Met receptor pathway in aggressive behavior of colorectal cancer cells induced by parathyroid hormone-related peptide.

Exogenous growth hormone functionally alleviates glucocorticoid-induced longitudinal bone growth retardation in male rats by activating the Ihh/PTHrP signaling pathway

Glucocorticoid (GC)-induced longitudinal bone growth retardation is a common and severe adverse effect in pediatric patients receiving GC immunosuppressive therapy. Molecular mechanisms underlying GC-induced growth inhibition are unclear. GC withdrawal following short-term high-dose use is common, including in the immediate post-transplant period. However, whether skeleton growth recovery is sufficient or whether growth-promoting therapy is required following GC withdrawal is unknown. The aim of this study was to investigate the effect of exogenous growth hormone (GH) on growth plate impairment in GC-induced longitudinal bone growth retardation. Here, apoptotic chondrocytes in the hypertrophic layer of growth plates increased whereas Indian Hedgehog (Ihh) and Parathyroid Hormone Related Peptide (PTHrP) protein levels in the growth plate decreased following GC exposure. The hypertrophic zone of the growth plate expanded following GC withdrawal. Subcutaneously injected GH penetrated the growth plate and modified its organization in rats following GC withdrawal. Ihh and PTHrP expression in GC-induced apoptotic chondrocytes decreased in vitro. GH promoted chondrocyte proliferation by activating Ihh/PTHrP signaling. Downregulating Ihh using specific siRNAs increased chondrocyte apoptosis and inhibited PTHrP, Sox9, and type II collagen (Col2a1) protein expression. GH inhibited apoptosis of Ihh-deficient growth plate chondrocytes by upregulating PTHrP, Sox9, and Col2a1 expression. Thus, reversal of the effect of GC on growth plate impairment following its withdrawal is insufficient, and exogenous GH provides growth plate chondral protection and improved longitudinal growth following GC withdrawal by acting on the Ihh/PTHrP pathway.

Hypercalcemia and parathyroid hormone-related peptide expression in a 3 months old boy with Colon Hemangioendothelioma

Searchable abstracts of presentations at key conferences on calcified tissues ISSN 2052-1219 (online)

Involvement of Aryl Hydrocarbon Receptor in L-Kynurenine-Mediated Parathyroid Hormone-Related Peptide Expression.

Parathyroid hormone-related peptide (PTHrP), produced by specific cancers such as lung cancer, profoundly influences the formation of bone metastatic lesions via the "vicious cycle" of tumor growth and bone resorption. The changes in gene expression regulated by the abnormal microenvironment components play key roles in maintaining the biological characteristics of cells, such as the organotropism of cancer metastasis. A recent study has shown that L-kynurenine (L-Kyn), one of microenvironment components, induced a substantial increase in the metastasis of lung cancer cells. What remains unclear, however, is the linkage between L-Kyn and bone metastatic lesions. In the present paper, we found that a significant upregulation of PTHrP expression was detected when 95D cells, a lung cancer cell line, were incubated with 50μM of L-Kyn. Meanwhile, L-Kyn (50/100μM) strongly strengthened aryl hydrocarbon receptor (Ahr) expression. Additionally, L-Kyn (50μM) increased the expression of the nuclear translocation of Ahr and cytochrome P450 1A1. Most importantly, the L-Kyn-induced upregulation of migration was significantly reduced when cells were co-incubated with siRNAAhr. Notably, the L-Kyn-mediated increase in PTHrP was also substantially attenuated upon siRNAAhr treatment in 95D cells. These results suggest that Ahr is involved in the L-Kyn-induced enhancement of PTHrP expression.

Parathyroid Hormone–Related Protein Contributes to Early Healing of Habu Snake Venom–Induced Glomerulonephritis in Mice

Proliferative glomerulonephritis is characterized by local inflammation and mesangial cell deterioration, followed by mesangial proliferation and glomerular healing. Parathyroid hormone-related peptide (PTHrP) is a mesangial cytokine-like growth factor implicated in mesangial proliferation and survival. No data are available about its role in glomerulonephritis. Herein, we analyzed the expression and role of PTHrP in glomerular inflammation and healing in an experimental model of glomerulonephritis induced by i.v. injection of Habu snake venom in mice. The temporal analysis showed marked renal damage in the first days after venom injection and the beginning of recovery within 7 days. Glomerular expression of PTHrP (transcript and protein) was observed in the early phase after venom injection (from day 1 to day 3), along with an inflammatory environment. The inactivation of secreted PTHrP with PTHrP-neutralizing antibody (PTH2E11; 120 μg i.p. daily) reduced the markers of local inflammation (expression of macrophage chemotactic protein-1; regulated upon activation, normal T cell expressed and secreted; cyclooxygenase 2; IL-6; and macrophage infiltration) and abolished the expression of PTHrP itself. Moreover, the glomerular cell proliferation was hampered, and the healing process was prevented on day 7 after venom injection. These results show that PTHrP has antinomic actions in glomerulonephritis, participating in both the proinflammatory condition and the healing process. Our work reveals the essential role of PTHrP in early glomerular repair in an experimental model of glomerulonephritis.

Parathyroid hormone-related protein modulates inflammation in mouse mesangial cells and blunts apoptosis by enhancing COX-2 expression.

Injury of mesangial cells (MC) is a prominent feature of glomerulonephritis. Activated MC secrete inflammatory mediators that induce cell apoptosis. Parathyroid hormone-related peptide (PTHrP) is a locally active cytokine that enhances cell survival and is upregulated by proinflammatory factors in many cell types. The aim of this study was to analyze the regulation of PTHrP expression by inflammatory cytokines and to evaluate whether PTHrP itself acts as a proinflammatory and/or survival factor on male murine MC in primary culture. Our results showed that IL-1β (10 ng/ml) and TNF-α (10 ng/ml) rapidly and transiently upregulated PTHrP expression in MC. The effects of IL-1β were both transcriptional and posttranscriptional, with stabilization of the PTHrP mRNA by human antigen R (HuR). Proteome profiler arrays showed that PTHrP itself enhanced cytokines within 2 h in cell lysates, mainly IL-17, IL-16, IL-1α, and IL-6. PTHrP also stimulated sustained expression (2-4 h) of chemokines, mainly regulated upon activation normal T cell expressed and secreted (RANTES)/C-C motif chemokine 5 (CCL5) and macrophage inflammatory protein-2 (MIP-2)/C-X-C motif chemokine 2 (CXCL2), thymus and activation-regulated chemokine (TARC)/CCL17, and interferon-inducible T cell α-chemoattractant (I-TAC)/CXCL11. Moreover, PTHrP markedly enhanced cyclooxygenase-2 (COX-2) expression and elicited its autoinduction through the activation of the NF-κB pathway. PTHrP induced MC survival via the COX-2 products, and PTHrP overexpression in MC blunted the apoptotic effects of IL-1β and TNF-α. Altogether, these findings suggest that PTHrP functions as a booster of glomerular inflammatory processes and may be a negative feedback loop preserving MC survival.

BRAF-V600E expression correlates with ameloblastoma aggressiveness.

The aim of this study was to investigate whether the expression of BRAF-V600E determines an aggressive clinical and molecular presentation of ameloblastoma. Ninety-three cases of solid ameloblastomas were arranged in a 1.0-mm tissue microarray (TMA) block. Immunohistochemistry against a large panel of cytokeratins (CK), epidermal growth factor receptor (EGFR), parathyroid hormone-related peptide (PTHrP), syndecan-1, Ki67, p53 and BRAF-V600E were performed. Clinicopathological parameters, including sex, age, tumour size, tumour duration, tumour location, treatment, recurrences, radiographic pattern, vestibular/lingual and basal cortical plates disruption and follow-up data, were obtained from patients' medical records. Immunoexpression of BRAF-V600E was investigated in 73 cases that remained available in TMA sections. Our results indicated that 46.6% (34 cases) demonstrated cytoplasm positivity (six weak and 28 strong positivity). BRAF-V600E expression was associated significantly with the expression of CK8 (P = 0.00077), CK16 (P = 0.05), PTHrP (P = 0.0082) and p53 (P = 0.0087). Additionally, a significant association was seen with the presence of recurrences (P = 0.0008), multilocular radiographic appearance (P = 0.044) and disruption of basal bone cortical (P = 0.05). Univariate analysis showed that BRAF-positive cases (P = 0.001), EGFR-negative/weak positive cases (P = 0.03) and multilocular tumours (P = 0.04) had a significantly lower disease-free survival rate, but these parameters were not considered independent prognostic factors in the multivariate analysis (P > 0.05). Our findings suggest an association of BRAF-V600E with parameters of a more aggressive behaviour of ameloblastoma, supporting the future use of BRAF inhibitors for targeted therapy of this neoplasm.

Effects of corticosterone on the metabolic activity of cultured chicken chondrocytes.

BackgroundCorticosterone is one of the most crucial glucocorticoids (GCs) in poultry. Our previous study shows that corticosterone can retard the longitudinal growth of bones by depressing the proliferation and differentiation of chondrocytes in broilers. The present study was designed to investigate whether corticosterone affect the development of chondrocytes and the synthesis of collagen in vitro. The chondrocytes were isolated from proximal tibial growth plates of 6-week-old broiler chickens and cultured with different doses of corticosterone for 48 h. Then the cell viability, alkaline phosphatase (ALP) activity and the expression of parathyroid hormone-related peptide (PTHrP) and type X collagen (Col X) were detected.ResultsAt 10−9-10−6 M concentration, corticosterone significantly inhibited the viability and differentiation of chondrocytes, as indicated by decreases in ALP and type X collagen expression. Conversely, there was completely opposite effect at 10−10 M. In addition, the expression of PTHrP was significantly downregulated at 10−6 M and 10−8 M, and was upregulated at 10−10 M.ConclusionsThe results suggested that corticosterone regulated chicken chondrocytes performance depending on its concentration with high concentrations inhibiting the viability and differentiation of chondrocytes and light concentrations promoting them, and these roles of corticosterone may be in part mediated through PTHrP.Electronic supplementary materialThe online version of this article (doi:10.1186/s12917-015-0398-5) contains supplementary material, which is available to authorized users.

Phosphate interacts with PTHrP to regulate endochondral bone formation.

Phosphate and parathyroid hormone related peptide (PTHrP) are required for normal growth plate maturation. Hypophosphatemia impairs hypertrophic chondrocyte apoptosis leading to rachitic expansion of the growth plate; however, the effect of phosphate restriction on chondrocyte differentiation during endochondral bone formation has not been examined. Investigations were, therefore, undertaken to address whether phosphate restriction alters the maturation of embryonic d15.5 murine metatarsal elements. Metatarsals cultured in low phosphate media exhibited impaired chondrocyte differentiation, analogous to that seen with PTHrP-treatment of metatarsals cultured in control media. Because phosphate restriction acutely increases PTHrP expression in cultured metatarsals, studies were undertaken to determine if this increase in PTHrP plays a pathogenic role in the impaired chondrocyte differentiation observed under low phosphate conditions. In contrast to what was observed with wild-type metatarsal elements, phosphate restriction did not impair the differentiation of metatarsals isolated from PTHrP heterozygous or PTHrP knockout mice. In vivo studies in postnatal mice demonstrated that PTHrP haploinsufficiency also prevents the impaired hypertrophic chondrocyte apoptosis observed with phosphate restriction. To determine how signaling through the PTH/PTHrP receptor antagonizes the pro-apoptotic effects of phosphate, investigations were performed in primary murine hypertrophic chondrocytes. Receptor activation impaired phosphate-induced Erk1/2 phosphorylation specifically in the mitochondrial fraction and decreased levels of mitochondrial Bad, while increasing cytosolic phospho-Bad. Thus, these data demonstrate that phosphate restriction attenuates chondrocyte differentiation as well as impairing hypertrophic chondrocyte apoptosis and implicate a functional role for the PTH/PTHrP signaling pathway in the abnormalities in chondrocyte differentiation and hypertrophic chondrocyte apoptosis observed under phosphate restricted conditions.

Regulation of chondrocyte differentiation by IRE1α depends on its enzymatic activity

Bone morphogenetic protein 2(BMP2) is known to activate unfolded protein response (UPR) signal molecules in chondrogenesis. Inositol-requiring enzyme-1α (IRE1α),as one of three unfolded protein sensors in UPR signaling pathways, can be activated during ER stress. However, the influence on IRE1α in chondrocyte differentiation has not yet been elucidated. Here we present evidence demonstrating that overexpression of IRE1α inhibits chondrocyte differentiation, as revealed by reduced expression of collagen II (ColII), Sox9, collagen X (ColX), matrix metalloproteinase 13 (MMP-13), Indian hedgehog (IHH), Runx2 and enhanced expression of parathyroid hormone-related peptide (PTHrP). Furthermore, IRE1α-mediated inhibition of chondrogenesis depends on its enzymatic activity, since its point mutant lacking enzymatic activity completely loses this activity. The RNase and Kinase domains of IRE1α C-terminal are necessary for its full enzymatic activity and inhibition of chondrocyte differentiation. Mechanism studies demonstrate that granulin–epithelin precursor(GEP), a growth factor known to stimulate chondrogenesis, induced IRE1α expression in chondrogenesis. The expression of IRE1α is depended on GEP signaling, and IRE1α expression is hardly detectable in GEP−/− embryos. In addition, IRE1α inhibits GEP-mediated chondrocyte differentiation as a negative regulator. Altered expression of IRE1α in chondrocyte hypertrophy was accompanied by altered levels of IHH and PTHrP. Collectively, IRE1α may be a novel regulator of chondrocyte differentiation by 1) inhibition GEP-mediated chondrocyte differentiation as a negative regulator; 2) promoting IHH/PTHrP signaling.

Parathyroid hormone-related peptide (PTHrP): prokaryotic expression, purification, and preparation of a polyclonal antibody.

Parathyroid hormone-related peptide (PTHrP) plays important roles in promoting cancer occurrence and in the development of bone metastases. To increase our knowledge of the biological functions of PTHrP, the prokaryotic expression vector pET-PTHrP was successfully constructed and the His-PTHrP fusion protein was expressed in Escherichia coli. Anti-PTHrP polyclonal antibody was then prepared from rabbits. Finally, the goat tissue expression profile of PTHrP was analyzed by Western blot with the anti-PTHrP polyclonal antibody. The results showed that the expression of PTHrP in goat mammary glands was significantly higher than that in other organs. This indicates that PTHrP may play important roles in the goat mammary gland. The antibody prepared will be a useful tool for detecting PTHrP and will be valuable in future studies investigating the role of PTHrP in calcium metabolism in the goat model.

Hu/elav RNA-binding protein HuR regulates parathyroid hormone related peptide expression in human lung adenocarcinoma cells.

In 54 stage I and II human lung adenocarcinomas, HuR and PTHrP levels were positively correlated and the PTHrP-HuR status of the tumor was an independent prognostic marker of the clinical outcomes of patients. The possibility that HuR could upregulate PTHrP expression in lung adenocarcinoma was investigated by immunohistochemical, Western blot and RT-PCR analyses in HCC44 and DV90 human lung adenocarcinoma cell lines. In both cell lines, knockdown of HuR by specific siRNAs reduced PTHrP mRNAs and both cellular and secreted protein. Moreover, it inhibited cell growth and induced cell apoptosis, as revealed by the increase of caspase-3 activity. These effects were partially rescued by the addition of exogenous PTHrP (1-34). Analysis by actinomycin D assay revealed that in both cell lines HuR silencing produced a decrease of PTHrP mRNA half-life by about 70%. These findings add PTHrP to the list of lung cancer-associated genes, whose mRNA is stabilized by HuR.

Characterization of neuroblastoma bone invasion/metastasis in established bone metastatic model of SY5Y and KCNR cell lines

To determine the mechanism of neuroblastoma (NB) bone invasion/metastasis, it is necessary to investigate the bone invasion/metastasis-related factors in the bone invasion/metastasis process. Some evidence has suggested that various proteins were involved in bone osteolytic response. The invasion/metastasis property and gene expression of NB, however, are still unknown. Single-cell suspensions of SY5Y and KCNR cells were injected directly into the femur of nude mice. Radiological and histological analyses, immunohistochemistry analyses, and western blot assay were performed to characterize bone metastasis mechanism in these bone metastasis models. SY5Y and KCNR NB cells result in osteolytic responses in bone metastasis model. Osteoprotegerin (OPG), receptor activator of NF-kappaB ligand (RANKL), parathyroid hormone-related peptide (PTHrP), endothelin 1 (ET-1), and CXCR4 were examined and compared among in vitro, in vivo, and normal bone, respectively. PTHrP, OPG, RANKL, and ET-1 except CXCR4 in SY5Y and KCNR NB cells xenografts were strikingly upregulated compared with normal bone and NB cells. However, significantly stronger expression of PTHrP and RANKL was presented than ET-1 and OPG; furthermore, the ratios of expression of PTHrP, RANKL to OPG, and ET-1 were also markedly increased in vivo versus in vitro. Our study provided evidence that NB cell may enhance bone invasion through PTHrP, OPG, RANKL, and ET-1, especially PTHrP and RANKL which may display stronger effects. CXCR4 appeared not participating in bone invasion, but in tumor growth, and homing to bone. Targeting PTHrP, OPG, ET-1, and RANKL may provide a new insight and method for patient therapy by inhibiting NB bone metastasis and invasiveness.

Hypercalcemia in Disseminated Coccidioidomycosis: Expression of Parathyroid Hormone-Related Peptide Is Characteristic of Granulomatous Inflammation

Hypercalcemia is an uncommon complication of disseminated granulomatous infections. The pathogenesis of hypercalcemia associated with infection is not clear. We investigated a case of disseminated coccidioidomycosis with hypercalcemia. We used a sensitive radioimmunoassay to measure serum parathyroid hormone-related peptide (PTHrP) and a mouse monoclonal antibody to PTHrP to immunostain biopsies. We found elevated serum levels of PTHrP while the patient was hypercalcemic that became undetectable when serum calcium normalized. We also found that the inflammatory cells and some surrounding tissues in skin biopsies expressed PTHrP. PTHrP was expressed by all biopsied lesions of patients with coccidioidomycosis that we examined, whether localized to the lung or disseminated, but no other cases were hypercalcemic. PTHrP was also expressed by the 3 mycobacterial granulomas we examined, and in a lymph node from a patient with sarcoidosis. The expression of PTHrP is a property of infectious granulomas regardless of etiology or the tissue involved, suggesting that PTHrP expression is part of the normal granulomatous immune response. Hypercalcemia may result if there is disseminated infection and multiple granulomas. We propose that excess production of PTHrP is the cause of hypercalcemia in granulomatous infections.

Gene and protein expression of cartilage canal and osteochondral junction chondrocytes and full-thickness cartilage in early equine osteochondrosis

The objective of this study was to investigate the expression of several regulatory factors associated with cartilage maturation in horses with early osteochondrosis (OC) compared to normal controls. The hypothesis was that expression levels of Indian hedgehog (Ihh), parathyroid hormone-related peptide (PTH-rP), vascular endothelial growth factor (VEGF), platelet-derived growth factor-A (PDGF-A), and matrix metalloproteinase-13 and -3 (MMP-13, -3) would be increased in OC. Articular cartilage and osteochondral samples were collected from the femoropatellar joints from seven OC and eight normal young (1-6 months) horses after euthanasia and snap frozen or suspended in 4% paraformaldehyde. Laser capture microdissection was used to capture cells surrounding cartilage canals and the osteochondral junction. Total RNA was isolated from whole cartilage and laser-captured cells. Equine-specific Ihh, PTH-rP, VEGF, PDGF-A, MMP-13, and MMP-3 mRNA expression levels were evaluated by real-time (RT)-PCR. Spatial tissue protein expression was determined by immunohistochemistry. In laser-captured samples, there was significantly increased MMP-13 and PDGF-A gene expression in chondrocytes adjacent to cartilage canals and increased PDGF-A gene expression in osteochondral junction chondrocytes of OC-affected foals. In full-thickness cartilage samples, there was significantly increased Ihh, MMP-3, and MMP-13 gene expression in OC samples, while PTH-rP protein expression was significantly higher along the osteochondral junction. The results suggest that pathways involving cartilage maturation and ossification are altered in early OC and may be associated with disease pathogenesis.

Macrophages induce neuroendocrine differentiation of prostate cancer cells via BMP6‐IL6 Loop

Frequently associated with hormone refractory prostate cancer are neuroendocrine cells. Because these cells do not express androgen receptors and are castration-resistant, further understanding the mechanism of neuroendocrine differentiation (NED) of prostate cancer cells may yield novel intervention methods in hormone refractory prostate cancer. In this regard, the present study investigated the effect of macrophages on prostate cancer NED. THP-1 and LNCaP or RAW264.7 and TRAMP-C2 cell line co-cultures were used to investigate NED-macrophage interactions. Also interleukin-6 (IL-6) knockout mice and macrophage-depleted mice were used to test NED in vivo. We found that co-culturing with THP-1 human monocytic cell line and RAW 264.7 murine macrophage cell line led to the NED of LNCaP and TRAMP-C2 prostate cancer cells, respectively. Specifically, the conditioned media of activated macrophages stimulated the expression of parathyroid hormone-related peptide (PTHrP), a marker of NED, in both LNCaP and TRAMP-C2 cells. Mechanistically, bone morphogenetic protein-6 (BMP-6) derived from prostate cancer cells increased the expression of IL-6 in macrophages. Subsequently, IL-6 induced the NED of prostate cancer cells. When this feedback loop was disrupted with neutralizing antibodies to either BMP-6 or IL-6, NED was no longer observed. In human prostate cancer tissues, neuroendocrine cells frequently co-localized with macrophages and BMP-6. In mice, the removal of IL-6 or macrophages blocked the BMP-6-induced NED of prostate cancer cells. Therefore, we propose that BMP-6 secreted by prostate cancer cells induces IL-6 expression in macrophages; IL-6, in turn, stimulates the NED of prostate cancer cells.

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.