EpCAM as multi-tumour target for near-infrared fluorescence guided surgery.

BACKGROUND: Evaluation of resection margins during cancer surgery can be challenging, often resulting in incomplete tumour removal. Fluorescence-guided surgery (FGS) aims to aid the surgeon to visualize tumours and resection margins during surgery. FGS relies on a clinically applicable imaging system in combination with a specific tumour-targeting contrast agent. In this study EpCAM (epithelial cell adhesion molecule) is evaluated as target for FGS in combination with the novel Artemis imaging system. METHODS: The NIR fluorophore IRDye800CW was conjugated to the well-established EpCAM specific monoclonal antibody 323/A3 and an isotype IgG1 as control. The anti-EpCAM/800CW conjugate was stable in serum and showed preserved binding capacity as evaluated on EpCAM positive and negative cell lines, using flow cytometry and cell-based plate assays. Four clinically relevant orthotopic tumour models, i.e. colorectal cancer, breast cancer, head and neck cancer, and peritonitis carcinomatosa, were used to evaluate the performance of the anti-EpCAM agent with the clinically validated Artemis imaging system. The Pearl Impulse small animal imaging system was used as reference. The specificity of the NIRF signal was confirmed using bioluminescence imaging and green-fluorescent protein. RESULTS: All tumour types could clearly be delineated and resected 72 h after injection of the imaging agent. Using NIRF imaging millimetre sized tumour nodules were detected that were invisible for the naked eye. Fluorescence microscopy demonstrated the distribution and tumour specificity of the anti-EpCAM agent. CONCLUSIONS: This study shows the potential of an EpCAM specific NIR-fluorescent agent in combination with a clinically validated intraoperative imaging system to visualize various tumours during surgery.

Near infrared fluorescence imaging for early detection, monitoring and improved intervention of diseases involving the joint.

Joints consist of different tissues, such as bone, cartilage and synovium, which are at risk for multiple diseases. The current imaging modalities, such as magnetic resonance imaging, Doppler ultrasound, X-ray, computed tomography and arthroscopy, lack the ability to detect disease activity before the onset of anatomical and significant irreversible damage. Optical in vivo imaging has recently been introduced as a novel imaging tool to study the joint and has the potential to image all kinds of biological processes. This tool is already exploited in (pre)clinical studies of rheumatoid arthritis, osteoarthritis and cancer. The technique uses fluorescent dyes conjugated to targeting moieties that recognize biomarkers of the disease. This review will focus on these new imaging techniques and especially where Near Infrared (NIR) fluorescence imaging has been used to visualize diseases of the joint. NIR fluorescent imaging is a promising technique which will soon complement established radiological, ultrasound and MRI imaging in the clinical management of patients with respect to early disease detection, monitoring and improved intervention.

Intraoperative fluorescence delineation of head and neck cancer with a fluorescent anti-epidermal growth factor receptor nanobody.

Intraoperative near-infrared (NIR) fluorescence imaging is a technology with high potential to provide the surgeon with real-time visualization of tumors during surgery. Our study explores the feasibility for clinical translation of an epidermal growth factor receptor (EGFR)-targeting nanobody for intraoperative imaging and resection of orthotopic tongue tumors and cervical lymph node metastases. The anti-EGFR nanobody 7D12 and the negative control nanobody R2 were conjugated to the NIR fluorophore IRDye800CW (7D12-800CW and R2-800CW). Orthotopic tongue tumors were induced in nude mice using the OSC-19-luc2-cGFP cell line. Tumor-bearing mice were injected with 25 µg 7D12-800CW, R2-800CW or 11 µg 800CW. Subsequently, other mice were injected with 50 or 75 µg of 7D12-800CW. The FLARE imaging system and the IVIS spectrum were used to identify, delineate and resect the primary tumor and cervical lymph node metastases. All tumors could be clearly identified using 7D12-800CW. A significantly higher tumor-to-background ratio (TBR) was observed in mice injected with 7D12-800CW compared to mice injected with R2-800CW and 800CW. The highest average TBR (2.00 ± 0.34 and 2.72 ± 0.17 for FLARE and IVIS spectrum, respectively) was observed 24 hr after administration of the EGFR-specific nanobody. After injection of 75 µg 7D12-800CW cervical lymph node metastases could be clearly detected. Orthotopic tongue tumors and cervical lymph node metastases in a mouse model were clearly identified intraoperatively using a recently developed fluorescent EGFR-targeting nanobody. Translation of this approach to the clinic would potentially improve the rate of radical surgical resections.

Inhibition of Gsk3ß in cartilage induces osteoarthritic features through activation of the canonical Wnt signaling pathway.

OBJECTIVE: In the past years, the canonical Wnt/ß-catenin signaling pathway has emerged as a critical regulator of cartilage development and homeostasis. In this pathway, glycogen synthase kinase-3ß (GSK3ß) down-regulates transduction of the canonical Wnt signal by promoting degradation of ß-catenin. In this study we wanted to further investigate the role of Gsk3ß in cartilage maintenance. DESIGN: Therefore, we have treated chondrocytes ex vivo and in vivo with GIN, a selective GSK3ß inhibitor. RESULTS: In E17.5 fetal mouse metatarsals, GIN treatment resulted in loss of expression of cartilage markers and decreased chondrocyte proliferation from day 1 onward. Late (3 days) effects of GIN included cartilage matrix degradation and increased apoptosis. Prolonged (7 days) GIN treatment resulted in resorption of the metatarsal. These changes were confirmed by microarray analysis showing a decrease in expression of typical chondrocyte markers and induction of expression of proteinases involved in cartilage matrix degradation. An intra-articular injection of GIN in rat knee joints induced nuclear accumulation of ß-catenin in chondrocytes 72 h later. Three intra-articular GIN injections with a 2 days interval were associated with surface fibrillation, a decrease in glycosaminoglycan expression and chondrocyte hypocellularity 6 weeks later. CONCLUSIONS: These results suggest that, by down-regulating ß-catenin, Gsk3ß preserves the chondrocytic phenotype, and is involved in maintenance of the cartilage extracellular matrix. Short term ß-catenin up-regulation in cartilage secondary to Gsk3ß inhibition may be sufficient to induce osteoarthritis-like features in vivo.

Near-infrared fluorescence imaging in patients undergoing pancreaticoduodenectomy.

BACKGROUND: Intraoperative visualization of pancreatic tumors has the potential to improve radical resection rates. Intraoperative visualization of the common bile duct and bile duct anastomoses could be of added value. In this study, we explored the use of indocyanine green (ICG) for these applications and attempted to optimize injection timing and dose. METHODS: Eight patients undergoing a pancreaticoduodenectomy were injected intravenously with 5 or 10 mg ICG. During and after injection, the pancreas, tumor, common bile duct and surrounding organs were imaged in real time using the Mini-FLARE™ near-infrared (NIR) imaging system. RESULTS: No clear tumor-to-pancreas contrast was observed, except for incidental contrast in 1 patient. The common bile duct was clearly visualized using NIR fluorescence, within 10 min after injection, with a maximal contrast between 30 and 90 min after injection. Patency of biliary anastomoses could be visualized due to biliary excretion of ICG. CONCLUSION: No useful tumor demarcation could be visualized in pancreatic cancer patients after intravenous injection of ICG. However, the common bile duct and biliary anastomoses were clearly visualized during the observation period. Therefore, these imaging strategies could be beneficial during biliary surgery in cases where the surgical anatomy is aberrant or difficult to identify.

'In vivo' optical approaches to angiogenesis imaging.

In recent years, molecular imaging gained significant importance in biomedical research. Optical imaging developed into a modality which enables the visualization and quantification of all kinds of cellular processes and cancerous cell growth in small animals. Novel gene reporter mice and cell lines and the development of targeted and cleavable fluorescent "smart" probes form a powerful imaging toolbox. The development of systems collecting tomographic bioluminescence and fluorescence data enabled even more spatial accuracy and more quantitative measurements. Here we describe various bioluminescent and fluorescent gene reporter models and probes that can be used to specifically image and quantify neovascularization or the angiogenic process itself.

Sclerostin: current knowledge and future perspectives.

In recent years study of rare human bone disorders has led to the identification of important signaling pathways that regulate bone formation. Such diseases include the bone sclerosing dysplasias sclerosteosis and van Buchem disease, which are due to deficiency of sclerostin, a protein secreted by osteocytes that inhibits bone formation by osteoblasts. The restricted expression pattern of sclerostin in the skeleton and the exclusive bone phenotype of good quality of patients with sclerosteosis and van Buchem disease provide the basis for the design of therapeutics that stimulate bone formation. We review here current knowledge of the regulation of the expression and formation of sclerostin, its mechanism of action, and its potential as a bone-building treatment for patients with osteoporosis.

Variations in surgical procedures for hind limb ischaemia mouse models result in differences in collateral formation.

OBJECTIVE: To identify the optimal mouse model for hind limb ischaemia, which offers a therapeutic window that is large enough to detect improvements of blood flow recovery, for example, using cell therapies. MATERIALS AND METHODS: Different surgical approaches were performed: single coagulation of femoral and iliac artery, total excision of femoral artery and double coagulation of femoral and iliac artery. Blood flow restoration was analysed with laser Doppler perfusion imaging (LDPI). Immuno-histochemical stainings, angiography and micro-computed tomography (CT) scans were performed for visualisation of collaterals in the mouse. RESULTS: Significant differences in flow restoration were observed depending on the surgical procedure. After single coagulation, blood flow already restored 100% in 7 days, in contrast to a significant delayed flow restoration after double coagulation (54% after 28 days, P<0.001). After total excision, blood flow was 100% recovered within 28 days. Compared with total excision, double coagulation displayed more pronounced corkscrew phenotype of the vessels typical for collateral arteries on angiographs. CONCLUSION: The extent of the arterial injury is associated with different patterns of perfusion restoration. The double coagulation mouse model is, in our hands, the best model for studying new therapeutic approaches as it offers a therapeutic window in which improvements can be monitored efficiently.

Synergistic effect of bisphosphonate and docetaxel on the growth of bone metastasis in an animal model of established metastatic bone disease.

Bisphosphonates decrease bone resorption and reduce significantly the rate of skeletal complications in patients with metastatic bone disease. Bisphosphonates have also been shown to exhibit antitumor activity in vitro but in vivo results have been equivocal. In the present study, we investigated the effects of bisphosphonate treatment alone or in combination with the cytostatic docetaxel on the growth of breast cancer cells in bone. Tumor gowth was studied in an athymic nude mice model inoculated with MDA-231-B/luc+ breast cancer cells. Two days after the inoculation, mice were treated with risedronate, zolendronate or docetaxel alone or with a combination of risedronate and docetaxel. Bone destruction and tumor growth were evaluated radiographically, histologically and by whole-body bioiluminescent reporter imaging (BLI). Five week treatment with high doses risedronate or zoledronate (37.5-150 microg/kg, 5 times/week), fully protected the bones from osteolysis, but did not affect tumour growth. Docetaxel (2, 4, and 8 mg/kg, 2 times/week) inhibited tumour growth dose-dependently and after 5 weeks treatment with the highest dose, there was no detectable tumour in bone. The combination of a dose of docetaxel (4 mg/kg) that demonstrated only a minimal effect on tumour growth, with risedronate (150 microg/kg), protected bone integrity and nearly completely inhibited the growth of the cancer cells. Risedronate and docetaxel act synergistically to protect bone and decrease tumour burden in an animal model of established bone metastases from breast cancer cells.

Parathyroid hormone-related peptide as an endogenous inducer of parietal endoderm differentiation.

Parathyroid hormone related peptide (PTHrP), first identified in tumors from patients with the syndrome of "Humoral Hypercalcemia of Malignancy," can replace parathyroid hormone (PTH) in activating the PTH-receptor in responsive cells. Although PTHrP expression is widespread in various adult and fetal tissues, its normal biological function is as yet unknown. We have examined the possible role of PTHrP and the PTH/PTHrP-receptor in early mouse embryo development. Using F9 embryonal carcinoma (EC) cells and ES-5 embryonic stem (ES) cells as in vitro models, we demonstrate that during the differentiation of these cells towards primitive and parietal endoderm-like phenotypes, PTH/PTHrP-receptor mRNA is induced. This phenomenon is correlated with the appearance of functional adenylate cyclase coupled PTH/PTHrP-receptors. These receptors are the mouse homologues of the recently cloned rat bone and opossum kidney PTH/PTHrP-receptors. Addition of exogenous PTH or PTHrP to RA-treated EC or ES cells is an efficient replacement for dBcAMP in inducing full parietal endoderm differentiation. Endogenous PTHrP is detectable at very low levels in undifferentiated EC and ES cells, and is upregulated in their primitive and parietal endoderm-like derivatives as assessed by immunofluorescence. Using confocal laser scanning microscopy on preimplantation mouse embryos, PTHrP is detected from the late morula stage onwards in developing trophectoderm cells, but not in inner cell mass cells. In blastocyst stages PTHrP is in addition found in the first endoderm derivatives of the inner cell mass. Together these results indicate that the PTH/PTHrP-receptor signalling system serves as a para- or autocrine mechanism for parietal endoderm differentiation in the early mouse embryo, thus constituting the earliest hormone receptor system involved in embryogenesis defined to date.

Novel mutations in the parathyroid hormone (PTH)/PTH-related peptide receptor type 1 causing Blomstrand osteochondrodysplasia types I and II.

CONTEXT: The PTH/PTHrP receptor type 1 (PTHR1) has a key role in endochondral ossification, which is emphasized by diseases resulting from mutations in the PTHR1 gene. Among these diseases is Blomstrand osteochondrodysplasia (BOCD). OBJECTIVE: BOCD can be divided into two types, depending on the severity of the skeletal abnormalities. The molecular basis for this heterogenic presentation is unknown. DESIGN AND PATIENTS: We performed mutation analysis in two families with type I and in three families with the less severe form of BOCD type II. RESULTS: In one of the type I BOCD cases, a homozygous nonsense mutation (R104X) was found, resulting in a truncated PTHR1. In the second type I BOCD case, no mutation was found. A homozygous nucleotide change (intron M4+27C>T) was demonstrated in one of the type II BOCD cases creating a novel splice site. In dermal fibroblasts of the patient, this novel splice site was preferentially used, resulting in an aberrant transcript. The wild-type transcript remained, however, present, albeit at low levels. In the other two families with type II BOCD, a previously identified homozygous missense mutation (P132L) was found. Functional analysis demonstrated that the P132L mutant had low residual activity. CONCLUSIONS: In combination with data presented in literature, we conclude that type I BOCD is caused by a complete inactivation of the PTHR1, whereas low levels of residual activity due to a near complete inactivation of the PTHR1 result in the relatively milder presentation of type II BOCD.

Inducible production of nitric oxide in osteoblast-like cells and in fetal mouse bone explants is associated with suppression of osteoclastic bone resorption.

Nitric oxide (NO) has been suggested to be involved in the regulation of osteoclast activity. Since osteoblasts, through the release of various factors, are the main regulators of osteoclastic resorption, first we have investigated whether osteoblast-like cells and fetal mouse long bone explants are able to produce NO. Second, we have assessed the effect of NO on osteoclastic resorption in whole bone cultures. In this study we show that primary rat osteoblast-like cells as well as the clonal rat osteoblast-like cell line UMR-106, stimulated with IFN-gamma together with TNF-alpha and LPS, produce NO, measured as nitrite production. IL-1 alpha enhanced while TGF-beta 2 inhibited TNF-alpha + IFN-gamma + LPS-stimulated NO production in UMR-106 cells dose dependently. Both the cytokines, however, had no effect when given alone. The competitive inhibitor of NO production, NG-monomethyl-arginine (L-NMMA), and cycloheximide abolished the increase in nitrite production induced by TNF-alpha + IFN-gamma + LPS, while hydrocortisone had no effect, as previously reported for chondrocytes. Calciotropic hormones had either no effect [1,25(OH)2D3] or had a small inhibitory effect (parathyroid hormone) on stimulated NO production. Furthermore, we found that in cultured fetal mouse long bone explants the combination of TNF-alpha + IFN-gamma + LPS as well as the NO donor sodium nitroprusside could inhibit osteoclastic resorption, measured as 45Ca release. The inhibition of resorption was prevented by concurrent administration of L-NMMA. Histological evaluation revealed that the TNF-alpha + IFN-gamma + LPS-induced inhibition of 45Ca release was associated with a decrease in the number of tartrate-resistant acid phosphatase-positive osteoclasts. We propose that the NO production by osteogenic cells (osteoblasts and chondrocytes) may represent an important regulatory mechanism of osteoclastic activity especially under pathological conditions characterized by release of bone-resorbing inflammatory cytokines.

Preclinical uPAR-targeted multimodal imaging of locoregional oral cancer.

OBJECTIVES: Establishing adequate resection margins and lymphatic mapping are crucial for the prognosis of oral cancer patients. Novel targeted imaging modalities are needed, enabling pre- and intraoperative detection of tumour cells, in combination with improved post-surgical examination by the pathologist. The urokinase-receptor (uPAR) is overexpressed in head and neck cancer, where it is associated with tumour progression and metastasis. MATERIAL AND METHODS: To determine suitability of uPAR for molecular imaging of oral cancer surgery, human head and neck tumours were sectioned and stained for uPAR to evaluate the expression pattern compared to normal mucosa. Furthermore, metastatic oral squamous carcinoma cell line OSC-19 was used for targeting uPAR in in vivo mouse models. Using anti-uPAR antibody ATN-658, equipped with a multimodal label, the in vivo specificity was investigated and the optimal dose and time-window were evaluated. RESULTS: All human oral cancer tissues expressed uPAR in epithelial and stromal cells. Hybrid ATN-658 clearly visualized tongue tumours in mice using either NIRF or SPECT imaging. Mean fluorescent TBRs over time were 4.3±0.7 with the specific tracer versus 1.7±0.1 with a control antibody. A significant difference in TBRs could be seen between 1nmol (150µg) and 0.34nmol (50µg) dose groups (n=4, p<0.05). Co-expression between BLI, GFP and the NIR fluorescent signals were seen in the tongue tumour, whereas human cytokeratin staining confirmed presence of malignant cells in the positive cervical lymph nodes. CONCLUSION: This study shows the applicability of an uPAR specific multimodal tracer in an oral cancer model, combining SPECT with intraoperative guidance.

Accelerated atherosclerosis and calcification in vein grafts: a study in APOE*3 Leiden transgenic mice.

Vein grafts fail due to development of intimal hyperplasia and accelerated atherosclerosis. Many murine genetic models in which genes are overexpressed, deleted, or mutated have been introduced recently. Therefore, mouse models are very well suited to dissect the relative contribution of different genes in the development of accelerated atherosclerosis. In the present study, we evaluated whether accelerated atherosclerosis in human vein grafts could be mimicked in hypercholesterolemic APOE*3 Leiden transgenic mice. Venous bypass grafting was performed in the carotid artery in APOE*3 Leiden mice fed either a standard chow diet or a high cholesterol-rich diet for 4 weeks. At several time points (0 hour to 28 days), mice were euthanized and the morphology of the vein grafts was analyzed. In normocholesterolemic mice, vein graft thickening up to 10-fold original thickness, predominantly consisting of alpha-smooth muscle cell actin-positive cells, was observed after 28 days. In hypercholesterolemic mice, accelerated atherosclerosis with accumulation of lipid-loaded foam cells was observed within 7 days after surgery. This accelerated atherosclerosis progressed in time and resulted in significant increase in vein graft thickening up to 50 times original thickness with foam cell-rich lesions and calcification within 28 days after surgery. The atherosclerotic lesions observed in these murine grafts show high morphological resemblance with the atherosclerotic lesions observed in human vein grafts. This accelerated, diet-dependent induction of atherosclerotic-like lesions in murine vein grafts provides a valuable tool in evaluating the mechanisms of accelerated atherosclerosis and therapeutic interventions of vein graft disease.

Modulation of the production of a parathyroid hormone-like protein in human squamous carcinoma cell lines by interaction with fibroblasts.

Normal human keratinocytes as well as human squamous cell carcinomas produce a parathyroid hormone-like protein (PLP). However, PLP production by these cells is not a constant phenomenon. Since nothing is known about factors which regulate the production of PLP, in vitro studies were performed with normal keratinocytes and squamous carcinoma cell lines in order to establish conditions under which PLP production may vary. PLP was measured as cyclic AMP production in parathyroid hormone target cells (osteoblasts) which could be inhibited by a parathyroid hormone antagonist. The presence of PLP was confirmed using a radioimmunoassay specific for PLP. Results from the bioassay correlated very well with the data obtained by radioimmunoassay for PLP. The results confirm that human squamous carcinoma cells and normal keratinocytes produce PLP. PLP production appeared to be very sensitive to modulation of coculture of squamous carcinoma cells with fibroblasts. The effect of fibroblasts was not mediated by an effect on squamous carcinoma cell viability. Murine transformed fibroblasts (3T3 cells) as well as human normal foreskin fibroblasts were equally effective in inducing PLP production in these cells. The fibroblastic factor was apparently present in a soluble form in the coculture system which prevented direct cell-cell contact but allowed communication through the medium. Nevertheless, conditioned medium from 3T3 cells failed to induce PLP production by squamous carcinoma cells. This suggests a more complicated interaction between the two cell types than a one way message from fibroblasts to keratinocytes. Production of PLP by a number of squamous carcinoma cell lines was variable and not evidently correlated with the ability of these carcinoma cells to differentiate. Production of parathyroid hormone-like protein not only is the expression of a disturbed metabolism of a specific cell type but also reflects the cell-cell interaction in tumor tissue.

Bone sialoprotein peptides are potent inhibitors of breast cancer cell adhesion to bone.

Bone and bone marrow are important sites of metastasis formation in breast cancer. Extracellular matrix proteins with attachment properties are generally believed to play a key role in tumorigenesis and metastasis formation. We have investigated whether mammary carcinoma cells (MDA-MB-231) can recognize constructs of the fairly bone-specific human bone sialoprotein, which encompass the RGD sequence (EPRGD-NYR). Exogenously added bone sialoprotein peptides with this amino acid sequence in their backbone structure, but not the more common fibronectin-derived GRGDS peptide, strongly inhibited breast cancer cell adhesion to extracellular bone matrix at micromolar concentrations. Most cyclic derivatives with the EPRGDNYR sequence were more effective inhibitors of tumor cell adhesion to bone than their linear equivalents. Furthermore, changes in the RGD-tripeptide of the backbone structure of the constructs, removal of the NYR flanking sequence, or a different tertiary cyclic structure significantly decreased their inhibitory potencies. In addition, the RGE-analogue EPRGENYR was capable of inhibiting breast cancer cell adhesion to bone, albeit to a lesser extent. We conclude therefore, that the inhibitory potency of the bone sialoprotein-derived peptides on breast cancer cell adhesion to bone is not solely due to a properly positioned RGD-motif alone but is also determined by its flanking regions, together with the tertiary structure of the EPRGDNYR peptide. Synthetic cyclic constructs with the EPRGDNYR sequence may, therefore, be potentially useful as antiadhesive agents for cancer cells to bone in vivo.

Combinatorial prospects of nano-targeted chemoimmunotherapy.

Despite the significant increase in our knowledge on cancer initiation and progression, and the development of novel cancer treatments, overall patient survival rates have thus far only marginally improved. However, it can be expected that lasting tumor control will be attainable for an increasing number of cancer patients in the foreseeable future, which is likely to be achieved by combining cancer chemotherapy with anticancer immunotherapy. A plethora of new cancer chemotherapy reagents are expected to become accessible to the clinic in the coming years which can then be used for efficient tumor debulking and aid in antigen exposure to the immune system. Durable remission and the eradication of micrometastases are likely to be achieved with specialized monoclonal antibodies and therapeutic cancer vaccines that modulate the immune system to overcome immunosuppression and kill distant cancer cells. Moreover, the method of drug delivery to tumors, stromal and immune cells is expected to shift largely from conventional 'free' drug molecules to encapsulated in targeted nano-vehicles, therapeutics often referred to or considered part of "nanomedicine". Several biocompatible nano-vehicles, such as metal-nanoparticles, biodegradable-nanoparticles, liposomes or dendrimers are potential candidates for targeted drug delivery but may also serve additional purposes. A dexterous combination of nanomedicine, cancer immunotherapy and chemotherapeutic engineering are likely to become the basis for new hope in the form of targeted cancer therapies that could attack tumors early in their development. One can envision nano-vehicles that would selectively deliver effective doses of chemotherapeutic agents to cancer cells while leaving healthy cells untouched. Furthermore, given that after chemotherapeutic treatment there often remains a limited number of chemo-resistant tumor cells, which go on to drive tumor progression, nano-vehicles could also be engineered to provoke an appropriate immune response to destroy these cells. Here, we discuss the potential of the combinatorial role of cancer chemotherapy, cancer immunotherapy and the prospective of nanotechnology for the targeted delivery of chemoimmunotherapeutic agents.

Parathyroid hormone-related peptide (PTHrP) induces parietal endoderm formation exclusively via the type I PTH/PTHrP receptor.

A number of studies suggest a role for PTHrP and the classical PTH/PTHrP receptor (type I) in one of the first differentiation processes in mouse embryogenesis, i.e. the formation of parietal endoderm (PE). We previously reported that although in type I receptor (-/-) embryos PE formation seemed normal, the embryos were smaller from at least day 9.5 p.c. and 60% had died before day 12.5 p.c. Here we show that the observed growth defect commences even earlier, at day 8.5 p.c. Using two novel antibodies, we show that the expression of the type I receptor protein at this stage is confined to extraembryonic endoderm only. In addition, we show that large amounts of PTHrP protein are present in the adjacent trophoblast giant cells, suggesting a paracrine interaction of PTHrP and the type I PTH/PTHrP receptor in PE formation. The involvement in PE differentiation of other recently described receptors for PTHrP would explain a possible redundancy for the type I receptor in PE formation. However, deletion of the type I PTH/PTHrP receptor in ES cells by homologous recombination completely prevents PTHrP-induced PE differentiation. Based upon these observations, we propose that PTHrP and the type I PTH/PTHrP receptor, although not required for the initial formation of PE, are required for its proper differentiation and/or functioning.

Expression of the parathyroid hormone-related peptide gene in retinoic acid-induced differentiation: involvement of ETS and Sp1.

Differentiation of P19 embryonal carcinoma (EC) and embryonal stem (ES)-5 cells with retinoic acid (RA) induces expression of PTH-related peptide (PTHrP) mRNA. In this study we have characterized a region between nucleotide (nt) -88 and -58 relative to the transcription start site in the murine PTHrP gene that was involved in this expression. Sequence analysis identified two partially overlapping binding sites for the Ets family of transcription factors and an inverted Sp1-binding site. Two major specific bands were detected in a bandshift assay using an oligonucleotide spanning nt -88 and -58 as a probe and nuclear extracts from both undifferentiated and RA-differentiated P19 EC cells. The lower complex consisted of Ets-binding proteins as demonstrated by competition with consensus Ets-binding sites, while the upper complex contained Sp1-binding activity as demonstrated by competition with consensus Sp1-binding sites. The observed bandshift patterns using nuclear extracts of undifferentiated or RA-differentiated P19 cells were indistinguishable, suggesting that the differentiation-mediated expression was not caused by the induction of expression of new transcription factors. Mutations in either of the Ets-binding sites or the Sp1-binding site completely abolished RA-induced expression of PTHrP promoter reporter constructs, indicating that the RA effect was dependent on the simultaneous action of both Ets- and Sp1-like activities. Furthermore, these mutations also abolished promoter activity in cells that constitutively expressed PTHrP mRNA, suggesting a central role for the Ets and Sp1 families of transcription factors in the expression regulation of the mouse PTHrP gene.

Identification of a retinoic acid-inducible element in the murine PTH/PTHrP (parathyroid hormone/parathyroid hormone-related peptide) receptor gene.

We have shown previously that the PTH/PTHrP (PTH-related peptide) receptor mRNA becomes expressed very early in murine embryogenesis, i.e. during the formation of extraembryonic endoderm. Retinoic Acid (RA) is a potent inducer of extraembryonic endoderm formation and PTH/PTHrP-receptor expression in embryonal carcinoma (EC) and embryonal stem (ES) cells. Using the P19 EC cell line, we have characterized promoter elements of the murine PTH/PTHrP-receptor gene that are involved in this RA-induced expression. The data show that RA-induced expression of the PTH/ PTHrP-receptor gene is mediated by the downstream P2 promoter. Analysis of promoter reporter constructs in transiently transfected P19 cells treated with RA identified an enhancer region between nucleotides -2714 and -2702 upstream of the P2 transcription start site that is involved in the RA effect. This region matches a consensus hormone response element consisting of a direct repeat with an interspacing of 1 bp (R-DR1). The R-DR1 efficiently binds retinoic acid receptor-alpha (RARalpha)-retinoid X receptor-alpha (RXRalpha) and chicken ovalbumin upstream promoter (COUP)-transcription factor I (TFI)-RXRalpha heterodimers and RXRalpha and COUP-TFI homodimers in a bandshift assay using extracts of transiently transfected COS-7 cells. RA differentiation of P19 EC cells strongly increases protein binding to the R-DR1 in a band-shift assay. This is caused by increased expression of RXR (alpha, beta, or gamma) and by the induction of expression of RARbeta and COUP TFI/TFII, which bind to the R-DR1 as shown by supershifting antibodies. The presence of RXR (alpha, beta, or gamma) in the complexes binding to the R-DR1 suggests that RXR homodimers are involved in RA-induced expression of the PTH/PTHrP-receptor gene. The importance of the R-DR1 for RA-induced expression of PTH/ PTHrP-receptor was shown by an inactivating mutation of the R-DR1, which severely impairs RA-induced expression of PTH/PTHrP-receptor promoter reporter constructs. Since this mutation does not completely abolish RA-induced expression of PTH/PTHrP-receptor promoter reporter constructs, sequences other than the R-DR1 might also be involved in the RA effect. Finally, we show that the RA-responsive promoter region is also able to induce expression of a reporter gene in extraembryonic endoderm of 7.5 day-old transgenic mouse embryos.