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The pre-metastatic niche (PMN) is a tumor-driven microenvironment in distant organs that can foster and support the survival and growth of disseminated tumor cells. This facilitates the establishment of secondary lesions that eventually form overt metastasis, the main cause of cancer-related death. In recent years, tumor-derived extracellular-vesicles (EVs) have emerged as potentially key drivers of the PMN. The role of the PMN in osteosarcoma metastasis is poorly understood and the potential contribution of osteosarcoma cell-derived EVs to PMN formation has not been investigated so far. Here, we characterize pulmonary PMN development using the spontaneously metastasizing 143-B xenograft osteosarcoma mouse model. We demonstrate the accumulation of CD11b + myeloid cells in the pre-metastatic lungs of tumor-bearing mice. We also establish that highly metastatic 143-B and poorly metastatic SAOS-2 osteosarcoma cell-derived EV education in naïve mice can recapitulate the recruitment of myeloid cells to the lungs. Surprisingly, despite EV-induced myeloid cell infiltration in the pre-metastatic lungs, 143-B and SAOS-2 EVs do not contribute towards the 143-B metastatic burden in the context of both spontaneous as well as experimental metastasis in severe-combined immunodeficient (SCID) mice. Taken together, OS-derived EVs alone may not be able to form a functional PMN, and may perhaps require a combination of tumor-secreted factors along with EVs to do so. Additionally, our study gives a valuable insight into the PMN complexity by providing the transcriptomic signature of the premetastatic lungs in an osteosarcoma xenograft model for the first time. In conclusion, identification of regulators of cellular and molecular changes in the pre-metastatic lungs might lead to the development of a combination therapies in the future that interrupt PMN formation and combat osteosarcoma metastasis.

Tumor-secreted extracellular vesicles (EVs) have been identified as mediators of cancer-host intercellular communication and shown to support pre-metastatic niche formation by modulating stromal cells at future metastatic sites. While osteosarcoma, the most common primary malignant bone tumor in children and adolescents, has a high propensity for pulmonary metastases, the interaction of osteosarcoma cells with resident lung cells remains poorly understood. Here, we deliver foundational in vitro evidence that osteosarcoma cell-derived EVs drive myofibroblast/cancer-associated fibroblast differentiation. Human lung fibroblasts displayed increased invasive competence, in addition to increased α-smooth muscle actin expression and fibronectin production upon EV treatment. Furthermore, we demonstrate, through the use of transforming growth factor beta receptor 1 (TGFBR1) inhibitors and CRISPR-Cas9-mediated knockouts, that TGFβ1 present in osteosarcoma cell-derived EVs is responsible for lung fibroblast differentiation. Overall, our study highlights osteosarcoma-derived EVs as novel regulators of lung fibroblast activation and provides mechanistic insight into how osteosarcoma cells can modulate distant cells to potentially support metastatic progression.

Chondrosarcoma is the second most frequent bone sarcoma. Due to the inherent chemotherapy and radiotherapy resistance and absence of known therapeutic targets, clinical management is limited to surgical resection. Consequently, patients with advanced disease face a poor prognosis. Hence, elucidating regulatory networks governing chondrosarcoma pathogenesis is vital for development of effective therapeutic strategies. Here, miRNA and mRNA next generation sequencing of different subtypes of human chondrogenic tumors in combination with in silico bioinformatics tools were performed with the aim to identify key molecular factors. We identified miR-143/145 cluster levels to inversely correlate with tumor grade. This deregulation was echoed in the miRNA plasma levels of patients and we provided the first evidence that circulating miR-145 is a potential noninvasive diagnostic biomarker and can be valuable as an indicator to improve the currently challenging diagnosis of cartilaginous bone tumors. Additionally, artificial upregulation of both miRNAs impelled a potent tumor suppressor effect in vitro and in vivo in an orthotopic xenograft mouse model. A combined in silico/sequencing approach revealed FSCN1 as a direct target of miR-143/145, and its depletion phenotypically resembled miR-143/145 upregulation in vitro. Last, FSCN1 is a malignancy-promoting factor associated with aggressive chondrosarcoma progression. Our findings underscore miR-143/145/FSCN1 as important players in chondrosarcoma and may potentially open new avenues for specific therapeutic intervention options. © 2020 American Society for Bone and Mineral Research., (© 2020 American Society for Bone and Mineral Research.)

Osteosarcoma is a highly aggressive bone cancer and the second most frequent cause of cancer-associated death in childhood and adolescence. Pulmonary metastases account for the high mortality rate in osteosarcoma patients. Therefore, novel therapeutic approaches, efficiently restraining the metastatic disease, are mandatory for a significant improvement of the currently poor patients' survival. Although initial studies with antibodies targeting insulin-like growth factor receptor (IGF-IR) showed promising potential for the treatment of patients with bone and soft tissue sarcomas, phase II clinical trials revealed variable results, which implied activation of alternative signaling pathways leading to therapy resistance. Since a cross-talk between IGF-IR and the epidermal growth factor receptor (EGFR) has been demonstrated in several cancer types, co-targeting of these two receptors was considered in the present study as a valuable therapeutic strategy to overcome single-agent treatment resistance in osteosarcoma. The effects of IGF-IR and/or EGFR targeting by intraperitoneal administration of the monospecific IGF-IR antibody R1507 or the EGFR antibody Cetuximab or the bispecific IGF-IR/EGFR antibody XGFR* on primary tumor growth and pulmonary metastasis were investigated in an intratibial human xenograft osteosarcoma mouse model. In vitro functional assays demonstrated that targeting IGF-IR and EGFR didn't affect osteosarcoma cell viability, but inhibited ligand-activated intracellular signaling and cell migratory capacity. The blocking potential of ligand-induced signaling in vitro was similar for all antibodies, but, in vivo , only XGFR* treatment significantly inhibited intratibial primary tumor growth and pulmonary metastasis. The therapeutic response to XGFR* was associated with an infiltration of innate immune system effector cells into the tumor microenvironment. Taken together, our study highlights the bispecific anti-IGF-IR/EGFR antibody XGFR* as an innovative promising effective candidate for the treatment of metastatic osteosarcoma and provides the rationale for future clinical studies., Competing Interests: None.

Osteosarcoma is an aggressive bone cancer that has a high propensity for metastasis to the lungs. Patients with metastatic disease face a very poor prognosis. Therefore, novel therapeutics, efficiently suppressing the metastatic process, are urgently needed. Integrins play a pivotal role in tumor cell adhesion, motility and metastasis. Here, we evaluated αvβ3 and αvβ5 integrin inhibition with cilengitide as a novel metastasis-suppressive therapeutic approach in osteosarcoma. Immunohistochemical analysis of αvβ3 and αvβ5 integrins expression in a tissue microarray of tumor specimens collected from osteosarcoma patients revealed that αvβ5 integrin is mainly found on tumor cells, whereas αvβ3 is predominantly expressed by stromal cells. In vitro functional assays demonstrated that cilengitide dose-dependently inhibited de novo adhesion, provoked detachment and inhibited migration of osteosarcoma cell lines. Cilengitide induced a decline in cell viability, blocked the cell cycle in the G1 phase and caused anoikis by activation of the Hippo pathway. In a xenograft orthotopic mouse model cilengitide minimally affected intratibial primary tumor growth but, importantly, suppressed pulmonary metastasis. The data demonstrate that targeting αvβ3 and αvβ5 integrins in osteosarcoma should be considered as a novel therapeutic option for patients with metastatic disease.

Human sarcomas comprise a heterogeneous group of rare tumors that affect soft tissues and bone. Due to the scarcity and heterogeneity of these diseases, patient-derived cells that can be used for preclinical research are limited. In this study, we investigated whether the tissue explant technique can be used to obtain sarcoma cell lines from fresh as well as viable frozen tissue obtained from 8 out of 12 soft tissue and 9 out of 13 bone tumor entities as defined by the World Health Organization. The success rate, defined as the percent of samples that yielded sufficient numbers of outgrowing cells to be frozen, and the time to freeze were determined for a total of 734 sarcoma tissue specimens. In 552 cases (75%) enough cells were obtained to be frozen at early passage. Success rates were higher in bone tumors (82%) compared with soft tissue tumors (68%), and the mean time to freezing was lower in bone tumors (65 days) compared with soft tissue tumors (84 days). Overall, from 40% of the tissues cells could be frozen at early passage within <2 month after tissue removal. Comparable results as with fresh tissue were obtained after explant of viable frozen patient-derived material. In a selected number of bone and soft tissue sarcoma entities, conventional karyotyping and/or FISH (fluorescence in situ hybridization) analysis revealed a high amount (>60%) of abnormal cells in 41% of analyzed samples, especially in bone sarcomas (osteosarcoma and Ewing sarcoma). In conclusion, the explant technique is well suited to establish patient-derived cell lines for a large majority of bone and soft tissue sarcoma entities with adequate speed. This procedure thus opens the possibility for molecular analysis and drug testing for therapeutic decision making even during patient treatment.

Background: Osteosarcoma is a rare but highly malignant cancer of the bone. As a consequence, the number of established cell lines used for experimental in vitro and in vivo osteosarcoma research is limited and the value of these cell lines relies on their stability during culture. Here we investigated the stability in gene expression by microarray analysis and array genomic hybridization of three low metastatic cell lines and derivatives thereof with increased metastatic potential using cells of different passages., Principal Findings: The osteosarcoma cell lines showed altered gene expression during in vitro culture, and it was more pronounced in two metastatic cell lines compared to the respective parental cells. Chromosomal instability contributed in part to the altered gene expression in SAOS and LM5 cells with low and high metastatic potential. To identify metastasis-relevant genes in a background of passage-dependent altered gene expression, genes involved in "Pathways in cancer" that were consistently regulated under all passage comparisons were evaluated. Genes belonging to "Hedgehog signaling pathway" and "Wnt signaling pathway" were significantly up-regulated, and IHH, WNT10B and TCF7 were found up-regulated in all three metastatic compared to the parental cell lines., Conclusions: Considerable instability during culture in terms of gene expression and chromosomal aberrations was observed in osteosarcoma cell lines. The use of cells from different passages and a search for genes consistently regulated in early and late passages allows the analysis of metastasis-relevant genes despite the observed instability in gene expression in osteosarcoma cell lines during culture.

Osteosarcoma (OS) is the most common primary malignant bone tumour in children and adolescents. Therapy today includes surgical removal of the tumour and neoadjuvant and adjuvant chemotherapy. The 5-year survival rates for patients with localised disease are between 50 and 70%, but in patients with metastases the prognosis remains poor (∼ 20%). The aim of this study was the development of a biological relevant OS 3D microtissue model, which is suitable for drug development. Microtissues were formed by the hanging drop method with the established OS cell lines SaOS-2, HOS and MG-63, as well as with cells derived from osteoblastic and chondroblastic OS patient material. Histological characterisation of the microtissues with H/E- and Ki-67-(proliferation), as well as apoptosis staining (TUNEL) revealed the inherent histological heterogeneity of OS. Microtissues from SaOS-2 and HOS cell lines were exposed to doxorubicin, cisplatin, taurolidine, pemetrexed and taxol and the viability was assessed by the CellTiter-GLO(®) Luminescent Cell Viability Assay. The obtained IC50-values for 3D cultures were all higher (1.7 to >16,000-fold) when compared to corresponding cells grown in 2D monolayer culture, except for pemetrexed that was inactive in 2D and 3D cultures. Doxorubicin did not affect the viability of chondroblastic monolayer cultures whereas on 3D microtissues an IC50-value of 2.3 μM was obtained. The 3D microtissues reflect the tissue heterogeneity of OS and are potential suitable tools for drug development towards personalised medicine., (Copyright © 2014 Elsevier B.V. All rights reserved.)

More effective treatment of metastasizing osteosarcoma with a current mean 5-year survival rate of less than 20% requires more detailed knowledge on mechanisms and key regulatory molecules of the complex metastatic process. CXCR4, the receptor of the chemokine CXCL12, has been reported to promote tumor progression and metastasis in osteosarcoma. CXCR7 is a recently deorphanized CXCL12-scavenging receptor with so far not well-defined functions in tumor biology. The present study focused on a potential malignancy enhancing function of CXCR7 in interaction with CXCR4 in osteosarcoma, which was investigated in an intratibial osteosarcoma model in SCID mice, making use of the human 143B osteosarcoma cell line that spontaneously metastasizes to the lung and expresses endogenous CXCR4. 143B osteosarcoma cells stably expressing LacZ (143B-LacZ cells) were retrovirally transduced with a gene encoding HA-tagged CXCR7 (143B-LacZ-X7-HA cells). 143B-LacZ-X7-HA cells co-expressing CXCR7 and CXCR4 exhibited CXCL12 scavenging and enhanced adhesion to IL-1β-activated HUVEC cells compared to 143B-LacZ cells expressing CXCR4 alone. SCID mice intratibially injected with 143B-LacZ-X7-HA cells had significantly (p<0.05) smaller primary tumors, but significantly (p<0.05) higher numbers of lung metastases than mice injected with 143B-LacZ cells. Unexpectedly, 143B-LacZ-X7-HA cells, unlike 143B-LacZ cells, also metastasized with high incidence to the auriculum cordis. In conclusion, expression of the CXCL12 scavenging receptor CXCR7 in the CXCR4-expressing human 143B osteosarcoma cell line enhances its metastatic activity in intratibial primary tumors in SCID mice that predominantly metastasize to the lung and thereby closely mimic the human disease. These findings point to CXCR7 as a target, complementary to previously proposed CXCR4, for more effective metastasis-suppressive treatment in osteosarcoma.

Osteosarcoma (OS) is the most common primary bone malignancy in children and adolescents. More than 30% of patients develop lung metastasis, which is the leading cause of mortality. Recently, the extracellular matrix protein Cyr61 has been recognized as a malignancy promoting protein in OS mouse model with prognostic potential in human OS. In this study, we aimed at the identification of novel Cyr61-interacting proteins. Here we report that Cyr61 associates with Caprin-1, and confocal microscopy showed that stable ectopic expression of Caprin-1 leads to the formation of stress granules containing Caprin-1 and Cyr61, confers resistance to cisplatin-induced apoptosis, and resulted in constitutive phosphorylation of Akt and ERK1/2. Importantly, ectopic expression of Caprin-1 dramatically enhanced primary tumor growth, remarkably increased lung metastatic load in a SCID intratibial OS mouse model, and decreased significantly (p<0.0018) the survival of the mice. Although Caprin-1 expression, evaluated with a tissue microarray including samples from 59 OS patients, failed to be an independent predictor for the patients' outcome in this limited cohort of patients, increased Caprin-1 expression indicated a tendency to shortened overall survival, and more strikingly, Cyr61/Caprin-1 co-expression was associated with worse survival than that observed for patients with tumors expressing either Cyr61 or Caprin-1 alone or none of these proteins. The findings imply that Caprin-1 may have a metastasis promoting role in OS and show that through resistance to apoptosis and via the activation of Akt and ERK1/2 pathways, Caprin-1 is significantly involved in the development of OS metastasis., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Background: Osteosarcoma is the most common malignant bone tumor in children and young adults. Since the introduction of chemotherapy, the 5-year survival rate of patients with non-metastatic osteosarcoma is ~70%. The main problems in osteosarcoma therapy are the occurrence of metastases, severe side-effects and chemoresistance. Antiproliferative and apoptotic effects of quercetin were shown in several types of cancers, including breast cancer and lung carcinoma., Materials and Methods: The present study investigates the cytotoxic potential of quercetin, a dietary flavonoid, in a highly metastasizing human osteosarcoma cell line, 143B., Results: We found that quercetin induces growth inhibition, G2/M phase arrest, and apoptosis in the 143B osteosarcoma cell line. We also observed impaired adhesion and migratory potential after the addition of quercetin., Conclusion: Since quercetin has already been shown to have low side effects in a clinical phase I trial in advanced cancer patients, this compound may have considerable potential for osteosarcoma treatment.

Formation of metastases in the lungs is the major cause of death in patients suffering from osteosarcoma (OS). Metastases at presentation and poor response to preoperative chemotherapy are strong predictors for poor patient outcome. The elucidation of molecular markers that promote metastasis formation and/or chemoresistance is therefore of importance. CD44 is a plasma membrane glycoprotein that binds to the extracellular matrix component hyaluronan (HA) and has been shown to be involved in metastasis formation in a variety of other tumors. Here we investigated the role of CD44 expression on OS tumor formation and metastasis. High CD44 expression, evaluated with a tissue microarray including samples from 53 OS patients and stained with a pan-CD44 antibody (Hermes3), showed a tendency (p < 0.08) to shortened overall survival. However, nonresponders and patients with lung metastases and high CD44 expression had significantly poorer prognosis than patients with low CD44 expression. Overexpression of the standard CD44 isoform (CD44s) and its HA-binding defective mutant R41A in osteoblastic SaOS-2 cells resulted in HA-independent higher migration rates and increased chemoresistance, partially dependent on HA. In an orthotopic mouse model of OS, overexpression of CD44s in SaOS-2 cells resulted in an HA-dependent increased primary tumor formation and increased numbers of micrometastases and macrometastases in the lungs. In conclusion, although CD44 failed to be an independent predictor for patient outcome in this limited cohort of OS patients, increased CD44 expression was associated with even worse survival in patients with chemoresistance and with lung metastases. CD44-associated chemoresistance was also observed in vitro, and increased formation of lung metastases was found in vivo in SCID mice., (Copyright © 2013 American Society for Bone and Mineral Research.)

Proteolytic degradation of the extracellular matrix (ECM) is an important process during tumor invasion. Matrix Metalloproteinase 1 (MMP-1) is one of the proteases that degrade collagen type I, a major component of bone ECM. In the present study, the biological relevance of MMP-1 in osteosarcoma (OS) tumor growth and metastasis was investigated in vitro and in vivo. Human OS cells in primary culture expressed MMP-1 encoding mRNA at considerably higher levels than normal human bone cells. In addition, MMP-1 mRNA and protein expression in the highly metastatic human osteosarcoma 143-B cell line was remarkably higher than in the non-metastatic parental HOS cell line. Stable shRNA-mediated downregulation of MMP-1 in 143-B cells impaired adhesion to collagen I and anchorage-independent growth, reflected by a reduced ability to grow in soft agar. Upon intratibial injection into SCID mice, 143-B cells with shRNA-downregulated MMP-1 expression formed smaller primary tumors and significantly lower numbers of lung micro- and macrometastases than control cells. Conversely, HOS cells stably overexpressing MMP-1 showed an enhanced adhesion capability to collagen I and accelerated anchorage-independent growth compared to empty vector-transduced control cells. Furthermore, and most importantly, individual MMP-1 overexpression in HOS cells enabled the formation of osteolytic primary tumors and lung metastasis while the HOS control cells did not develop any tumors or metastases after intratibial injection. The findings of the present study reveal an important role of MMP-1 in OS primary tumor and metastasis formation to the lung, the major organ of OS metastasis., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Metastasis is the major cause of death of osteosarcoma patients and its diagnosis remains difficult. In preclinical studies, however, forced expression of reporter genes in osteosarcoma cells has remarkably improved the detection of micrometastases and, consequently, the quality of the studies. We recently showed that Dunn cells equipped with a lacZ reporter gene disseminated from subcutaneous primary tumors as frequently as their highly metastatic subline LM8, but only LM8 cells grew to macrometastases. In the present time-course study, tail-vein-injected Dunn and LM8 cells settled within 24 h at the same frequency in the lung, liver, and kidney of mice. Furthermore, Dunn cells also grew to macrometastases, but, compared to LM8, with a delay of two weeks in lung and one week in liver and kidney tissue, consistent with prolonged survival of the mice. Dunn- and LM8-cell-derived ovary and spine metastases occurred less frequently. In vitro, Dunn cells showed less invasiveness and stronger contact inhibition and intercellular adhesion than LM8 cells and several cancer- and dormancy-related genes were differentially expressed. In conclusion, Dunn cells, compared to LM8, have a similar capability but a longer latency to form macrometastases and provide an interesting new experimental system to study tumor cell dormancy.

Osteosarcoma (OS) is the most frequent primary malignant bone cancer in children and adolescents with a high propensity for lung metastasis. Therefore, it is of great importance to identify molecular markers leading to increased metastatic potential in order to devise more effective therapeutic strategies that suppress metastasis, the major cause of death in OS. CD44, the principal receptor for the extracellular matrix component hyaluronan (HA), is frequently found overexpressed in tumor cells and has been implicated in metastatic spread in various cancer types. Here, we investigated the effects of stable shRNA-mediated silencing of CD44 gene products on in vitro and in vivo metastatic properties of the highly metastatic human 143-B OS cell line. In vitro, CD44 knockdown resulted in a 73% decrease in the adhesion to HA, a 57% decrease in the migration rate in a trans-filter migration assay, and a 28% decrease in the cells' capacity for anchorage-independent growth in soft agar compared to the control cells, implicating that CD44 expression contributes to the metastatic activity of 143-B cells. However, making use of an orthotopic xenograft OS mouse model, we demonstrated that reduced CD44 expression facilitated primary tumor growth and formation of pulmonary metastases. The enhanced malignant phenotype was associated with decreased adhesion to HA and reduced expression of the tumor suppressor merlin in vivo. In conclusion, our study identified CD44 as a metastasis suppressor in this particular experimental OS model.

Photodynamic therapy (PDT) is a minimally invasive therapeutic modality approved for palliative and curative treatment of some forms of local cancers, precancerous lesions and nononcological disorders. As a prerequisite for future studies in animal models aiming at an intraoperative application of PDT in osteosarcoma (OS), in the present study, we investigated the uptake and the dark- and photo-toxicity of the photosensitizer mTHPC in the metastatic human OS cell line 143B, which, intratibially injected into SCID mice, reproduces spontaneous, aggressive lung metastasis, the main cause of death in OS patients. The uptake of mTHPC by 143B cells was time- and dose-dependent. mTHPC accumulated to higher levels in the 143B than in the parental low-metastatic HOS cell line. A significant decrease in viability of 143B cells, reflecting mTHPC dark-toxicity, occurred upon incubation in the dark at mTHPC concentrations ≥2.5 μg mL(-1). In phototoxicity experiments with illumination by 652 nm laser light (2.5-10 J cm(-2)), the half-maximal lethal doses of mTHPC ranged from 0.012 to 0.047 μg mL(-1). This treatment activated caspase-3, -7 and -9 and Z-VAD-FMK-inhibitable PARP cleavage, indicating caspase-dependent apoptosis. In conclusion, PDT with mTHPC is effective in the metastatic 143B human osteosarcoma cell line in vitro., (© 2012 Wiley Periodicals, Inc. Photochemistry and Photobiology © 2012 The American Society of Photobiology.)

Background: Metastasizing osteosarcoma has a mean 5-year survival rate of only 20% to 30%. Therefore, novel chemotherapeutics for more effective treatment of this disease are required., Methods: The antineoplastic activity of honokiol, which was demonstrated previously in numerous malignancies, was studied in vivo in C3H mice subcutaneously injected with syngeneic β-galactosidase bacterial gene (lacZ)-expressing LM8 osteosarcoma (LM8-lacZ) cells. In vitro cytotoxic effects of honokiol were investigated in 8 human and 2 murine osteosarcoma cell lines with different in vivo metastatic potential., Results: Seven days after subcutaneous flank injection of LM8-lacZ cells, daily intraperitoneal treatment of mice with 150 mg/kg honokiol reduced the number of micrometastases in the lung by 41% and reduced the number of macrometastases in the lung and liver by 69% and 80%, respectively, compared with control. Primary tumor growth was not inhibited. In osteosarcoma cell lines, honokiol inhibited the metabolic activity with a half-maximal concentration (IC(50) ) between 8.0 μg/mL and 16 μg/mL. Cyclosporin A partially reversed the inhibition of metabolic activity in LM8-lacZ cells. Cell proliferation and wound healing migration of LM8-lacZ cells were inhibited by honokiol with an IC(50) between 5.0 μg/mL and 10 μg/mL. Higher concentrations caused rapid cell death, which was distinct from necrosis, apoptosis, or autophagy but was associated with swelling of the endoplasmic reticulum, cytoplasmic vacuolation, and morphologically altered mitochondria., Conclusions: Honokiol exhibited prominent antimetastatic activity in experimental osteosarcoma and caused rapid cell death in vitro that was unrelated to necrosis, apoptosis, or autophagy. The authors concluded that honokiol has considerable potential for the treatment of metastasizing osteosarcoma., (Copyright © 2011 American Cancer Society.)

Osteosarcoma (OS) is a rare bone neoplasm that affects mainly adolescents. It is associated with poor prognosis in case of metastases formation. The search for metastasis predicting markers is therefore imperative to optimize treatment strategies for patients at risk and important for the search of new drugs for the treatment of this devastating disease. Here, we have analyzed by microarray the differential gene expression in four human and two mouse OS cell line systems consisting of parental cell lines with low metastatic potential and derivatives thereof with increased metastatic potential. Using two osteoblastic cell line systems, the most common OS phenotype, we have identified forty-eight common genes that are differentially expressed in metastatic cell lines compared to parental cells. The identified subset of metastasis relevant genes in osteoblastic OS overlapped only minimally with differentially expressed genes in the other four preosteoblast or nonosteoblastic cell line systems. The results imply an OS phenotype specific expression pattern of metastasis regulating proteins and form a basis for further investigation of gene expression profiles in patients' samples combined with survival analysis with the aim to optimize treatment strategies to develop new drugs and to consequently improve the survival of patients with the most common form of osteoblastic OS.

Osteosarcoma is the most frequent primary malignant bone tumor in children and adolescents with a high propensity for lung metastasis, the major cause of disease-related death. Reliable outcome-predictive markers and targets for osteosarcoma metastasis-suppressing drugs are urgently needed for more effective treatment of metastasizing osteosarcoma, which has a current mean 5-year survival rate of approximately 20%. This study investigated the prognostic value and the biological relevance of the extracellular matrix-associated growth factor Cyr61 of the CCN family of secreted proteins in osteosarcoma and metastasis. The prognostic value of Cyr61 was assessed with Kaplan-Meier analyses based on Cyr61 immunostaining of a tissue microarray of osteosarcoma biopsies collected from 60 patients with local or metastatic disease. Effects of Cyr61 overexpression on intratibial tumor growth and lung metastasis of the low metastatic human SaOS-2 osteosarcoma cell line were examined in severe combined immunodeficiency (SCID) mice. Cyr61-provoked signaling was studied in vitro in nonmanipulated SaOS-2 cells. Cyr61 immunostaining of osteosarcoma tissue cores correlated significantly (p = 0.02) with poor patient survival. Mice intratibially injected with Cyr61-overexpressing SaOS-2 cells showed faster tumor growth and an increase in number and outgrowth of lung metastases and consequently significantly (p = 0.0018) shorter survival than mice injected with control SaOS-2 cells. Cyr61-evoked PI-3K/Akt/GSK3β signaling in SaOS-2 cells resulted in a subcellular redistribution of the cell cycle inhibitor p21(Cip1/WAF1). Cyr61 has considerable potential as a novel marker for poor prognosis in osteosarcoma and is an attractive target for primary tumor- and metastases-suppressing drugs., (Copyright © 2012 American Society for Bone and Mineral Research.)

More effective treatment of patients with metastasizing osteosarcoma (OS) with a mean 5-year survival rate of <20% requires more detailed knowledge on the complex mechanisms of metastasis for the design of new drugs, which selectively target metastasizing cells. Moreover, novel diagnostic imaging technology for early detection of metastases is needed. Mouse models, which reproduce human metastasizing OS and allow visualization of single metastatic cells are instrumental for preclinical testing of new pharmaceuticals and diagnostic instruments. Here, the low metastatic Dunn cell line and its highly metastatic LM8 subline, both equipped with a constitutively expressed lacZ gene, were used to improve the well-established OS models in syngeneic C3H mice to achieve ex vivo visualization of single metastatic cells in affected organs by X-gal staining. These models, combined with a technique for in situ high quality lung tissue-maintaining perfusion revealed, as a novel finding, single metastasizing Dunn cells in lung and liver. Importantly, constitutive lacZ gene expression did not affect in vitro and in vivo tumorigenic and metastatic properties of Dunn and LM8 cells. Thus, these improved Dunn and LM8 OS mouse models will in the future serve as a benchmark for the development of new metastasis-targeting drugs and metastasis-imaging technology., (Copyright © 2011 Orthopaedic Research Society.)

Various micro-devices have been used to assess single cell mechanical properties. Here, we designed and implemented a novel, mechanically actuated, two dimensional cell culture system that enables a measure of cell stiffness based on quantitative functional imaging of cell-substrate interaction. Based on parametric finite element design analysis, we fabricated a soft (5 kPa) polydimethylsiloxane (PDMS) cell substrate coated with collagen-I and fluorescent micro-beads, thus providing a favorable terrain for cell adhesion and for substrate deformation quantification, respectively. We employed a real-time tracking system that analyzes high magnification images of living cells under stretch, and compensates for gross substrate motions by dynamic adjustment of the microscope stage. Digital image correlation (DIC) was used to quantify substrate deformation beneath and surrounding the cell, leading to an estimate of cell stiffness based upon the ability of the cell to resist the applied substrate deformation. Sensitivity of the system was tested using chemical treatments to both "soften" and "stiffen" the cell cytoskeleton with either 0.5 μg/ml Cytochalasin-D or 3% Glutaraldehyde, respectively. Results indicate that untreated osteosarcoma cells (SAOS-2) exhibit a 1.5 ± 0.7% difference in strain from an applied target substrate strain of 8%. Compared to untreated cells, those treated with Cyochalasin-D passively followed the substrate (0.5 ± 0.5%, p < 0.001), whereas Glutaraldehyde enhanced cellular stiffness and the ability to resist the substrate deformation (2.9 ± 1.6%, p < 0.001). Nano-indentation testing showed differences in cell stiffness based on culture treatment, consistent with DIC findings. Our results indicate that mechanics and image analysis approaches do hold promise as a method to quantitatively assess tensile cell constitutive properties.

Curcumin (diferuloylmethane), one of the main components of the Indian spice turmeric, is known to possess potent anti-inflammatory and anti-oxidant properties. In addition, curcumin has also been shown to have in vitro and in vivo efficacy against a variety of malignancies. In the current study we examined the cytotoxic effect of curcumin on seven osteosarcoma (OS) cell lines with varying degrees of in vivo metastatic potential. Curcumin inhibited the growth of all OS cell lines tested with half-maximal inhibitory concentration values ranging from 14.4 to 24.6 microM. Growth inhibition was associated with a dose dependent increase in the number of apoptotic cells and accumulation of cells in the G(2)/M phase of the cell cycle. Curcumin treatment also resulted in cleavage of caspase-3 and poly adenosine diphosphate-ribose polymerase. Moreover, curcumin treatment was associated with an increase in cellular levels of the apoptotic B-cell leukemia/lymphoma 2 (Bcl-2)-associated X protein and a decrease in cellular content of the anti-apoptotic protein Bcl-2. In addition, curcumin treatment also inhibited the migration of OS cell lines. These data indicate that the potent cytotoxic activity of curcumin on OS cell lines is mediated by induction of apoptotic processes. Thus, curcumin has potential to be a novel OS chemotherapeutic agent.

Glaucoma, frequently associated with high IOP (intra-ocular pressure), is a leading cause of blindness, characterized by a loss of retinal ganglion cells and the corresponding optic nerve fibres. In the present study, acutely and transiently elevated IOP, characteristic of acute angle-closure glaucoma in humans, was observed in CLR (calcitonin receptor-like receptor) transgenic mice between 1 and 3 months of age. Expression of CLR under the control of a smooth muscle alpha-actin promoter in these mice augmented signalling of the smooth-muscle-relaxing peptide adrenomedullin in the pupillary sphincter muscle and resulted in pupillary palsy. Elevated IOP was prevented in CLR transgenic mice when mated with hemizygote adrenomedullin-deficient mice with up to 50% lower plasma and organ adrenomedullin concentrations. This indicates that endogenous adrenomedullin of iris ciliary body origin causes pupillary palsy and angle closure in CLR transgenic mice overexpressing adrenomedullin receptors in the pupillary sphincter muscle. In human eyes, immunoreactive adrenomedullin has also been detected in the ciliary body. Furthermore, the CLR and RAMP2 (receptor-activity-modifying protein 2), constituting adrenomedullin receptor heterodimers, were identified in the human pupillary sphincter muscle. Thus, in humans, defective regulation of adrenomedullin action in the pupillary sphincter muscle, provoked in the present study in CLR transgenic mice, may cause acute and chronic atony and, thereby, contribute to the development of angle-closure glaucoma. The CLR transgenic mice used in the present study provide a model for acute angle-closure glaucoma.

Osteosarcoma is the most frequent malignant bone tumor with a poor survival rate for patients with metastasis. Previous studies have shown that beside other proteases, distinct sets of cathepsins are involved in the process of metastasis of different tumors. In this study we investigated the expression of cathepsin proteases in human osteosarcoma metastasis. First, the mRNA expression of 14 human cathepsins was studied in SAOS-2 osteosarcoma cells and the highly metastatic LM5 and LM7 sublines by reverse transcriptase (RT)-polymerase chain reaction (PCR). The expression of cathepsin D, K, and L mRNA was found upregulated and that of cathepsin F, H, and V downregulated in the highly metastatic LM5 and LM7 cells. A subgroup of the cathepsin proteases was further studied at the protein level by Western blot analysis of cell extracts. The expression of cathepsin B and H was decreased and that of cathepsin D, K, and L was increased in the highly metastatic cell lines as compared to the SAOS-2 cell line. Diagnostic relevance of cathepsin K expression in osteosarcoma was revealed upon correlation of survival and metastasis with immunohistochemical cathepsin K staining of biopsies collected from 92 patients prior to chemotherapy. Patients with metastatic high-grade osteosarcoma and low cathepsin K expression at diagnosis had a better prognosis than those with high expression. Thus, it appears that cathepsin K expression is of predictive prognostic value for patients with high-grade tumors and metastasis at diagnosis., ((c) 2007 Wiley-Liss, Inc.)

Background: Metastasis is the leading cause of death in patients with osteosarcoma (OS). High alkaline phosphatase (ALP) activity and resistance to chemotherapy are independent predictors of poor clinical outcome of osteosarcoma. Here, the osteoblastic phenotype, cell and nuclear morphology, cell adhesion and drug resistance of the SAOS-2 cell line and two in vivo selected highly metastatic derivatives, LM5 and LM7, were compared., Results: ALP activity and deposition of mineralized extracellular matrix were the same in the parental SAOS-2 and the LM5 and LM7 cells, but parathyroid hormone (PTH)-stimulated cAMP accumulation was lost in the LM7 cells. The LM5 and LM7 cells were smaller than the parental SAOS-2 cells, and 10% of the LM7 cells had distorted nuclei. The adhesion of LM5 and LM7 cells was decreased when compared to SAOS-2 cells. The cytotoxic responses of the SAOS-2, LM5 and LM7 cells to Cisplatin, Doxorubicin and Etoposide were indistinguishable., Conclusion: The increased metastatic potential of LM5 and LM7 as compared to SAOS-2 cells is not associated with a substantial change of the osteoblastic phenotype or of the cytotoxic response to current chemotherapeutic drugs. The decrease in cell size and altered cell adhesion, reflecting cytoskeletal rearrangement, together with increased nuclear instability and partial dedifferentiation, as revealed by the loss of PTH responsiveness in LM7 cells, may account for the higher metastatic potential of the LM5 and LM7 sublines as compared to the parental SAOS-2 cells.

Taurolidine, the active agent of Taurolin, is a broad spectrum anti-biotic that has been used for over 15 years for the treatment of severe surgical infections. Recently, taurolidine has been shown to possess anti-neoplastic properties in vitro and in vivo against a variety of cancers including ovarian, colon and prostate. In this study we assessed the cytotoxic activity of taurolidine against human osteosarcoma (OS) cell lines and normal human bone cells. Treatment with taurolidine inhibited the growth of all ten osteosarcoma cell lines tested and taurolidine was equally potent against cell lines with and without distinct genetic defects (i.e. p53, Rb). Moreover, taurolidine-induced growth inhibition was found to be associated with a dose dependent increase in the number of apoptotic cells and apoptosis was shown to be caspase-dependent. Taurolidine treatment was also found to inhibit adhesion of OS cell lines. Compared to OS cell lines, normal bone cells in primary culture were found to be less sensitive to the cytotoxic and anti-adhesive effects of taurolidine. These data indicate that taurolidine possesses potent anti-neoplastic activity against osteosarcoma cell lines and may have potential as a novel OS chemotherapeutic agent.

A high propensity to aggregate into intractable deposits is a common problem limiting the production and use of many peptides and proteins in a wide range of biotechnological and pharmaceutical applications. Many therapeutic polypeptides are frequently abandoned at an early stage in their development because of problems with stability and aggregation. It has been shown recently that parameters describing the physicochemical properties of polypeptides can be used as predictors of protein aggregation. Here we demonstrate that these and similar tools can be applied to the rational redesign of bioactive molecules with a significantly reduced aggregation propensity without loss of physiological activity. This strategy has been exemplified by designing variants of the hormone calcitonin that show a significantly reduced aggregation propensity, yet maintain, or even increase, their potency when compared to the current therapeutic forms. The results suggest that this approach could be used successfully to enhance the solubility and efficacy of a wide range of other peptide and protein therapeutics.

The calcitonin receptor-like receptor (CLR) requires the associated receptor activity-modifying protein (RAMP)1 to reveal a calcitonin gene-related peptide (CGRP) receptor. Here, the subdomain of the CLR that associates with RAMP1 has been identified in chimeras between the CLR and the parathyroid hormone (PTH) receptor 1 (PTHR). The PTHR alone does not interact with RAMP1. RAMP1 requires the CLR for its transport to the cell surface. Thus, receptor-dependent RAMP1 delivery to the plasma membrane and coimmunoprecipitation from the cell surface were used as measures for receptor/RAMP1 interaction. Several chimeric CLR-PTHR included the N-terminal amino acids 23-60 of the CLR transported RAMP1 to the surface of COS-7 cells much like the intact CLR. Moreover, RAMP1 coimmunoprecipitated with these receptors from the cell surface. A CLR deletion mutant, consisting of the N-terminal extracellular domain, the first transmembrane domain, and the C-terminal intracellular region, revealed the same results. Cyclic AMP was stimulated by CGRP in CLR/RAMP1 expressing cells (58 +/- 19-fold, EC(50) = 0.12 +/- 0.03 nM) and by PTH-related protein in cells expressing the PTHR (50 +/- 10-fold, EC(50) = 0.25 +/- 0.03 nM) or a PTHR with the N-terminal amino acids 23-60 of the CLR (23 +/- 5-fold, EC(50) > 1000 nM). Other chimeric CLR-PTHR were inactive. In conclusion, structural elements in the extreme N-terminus of the CLR between amino acids 23-60 are required and sufficient for CLR/RAMP1 cotransport to the plasma membrane and heterodimerization.

The hormone calcitonin (CT) of thyroid C-cell origin, the neuropeptides alpha- and beta-calcitonin gene-related peptide (CGRP), the widely expressed hormone and tissue factor adrenomedullin (AM), and amylin (AMY) that is co-produced with insulin in pancreatic beta-cells, are structurally related peptides. They have in common six or seven amino acid ring structures, linked by disulfide bridges between cysteine residues, and amidated carboxyl termini that are both required for biological activity. The actions of the peptides in vivo have traditionally been studied after intravenous and intracerebroventricular administration. As a result, CT lowers serum calcium and reduces pain perception. alpha- and beta CGRP and AM are highly potent vasodilatory peptides. AMY inhibits food intake through its action in the area postrema of the brain. Physiological actions of the peptides summarized in the present review have been defined through gene knockout and overexpression strategies.

The calcitonin-like receptor (CLR) associated with receptor-activity-modifying proteins (RAMP) 1 or -2 recognizes calcitonin gene-related peptide (CGRP) and adrenomedullin (AM), respectively. The amino acid sequence CNRTWDGWLCW corresponding to residues 64-74 in the extracellular N-terminus of the CLR is conserved. The Asp(69) (D(69)) is present in all family B1 G-protein-coupled receptors. Here the D(69) of a V5-tagged mouse CLR has been mutated to Ala (A), Glu (E), and Asn (N). The function of the intact and the mutant CLR was investigated in COS-7 cells coexpressing myc-tagged mouse RAMP1 or -2. In CLR/RAMP1 and -2 expressing cells CGRP and AM stimulated cAMP formation with an EC(50) of 0.17 and 0.50 nM, respectively. The expression of the D69A, D69E, and D69N mutants at the cell surface was comparable to that of the intact CLR. cAMP stimulation by CGRP and AM was abolished in the D69A mutant. With the D69E mutant the EC(50) of CGRP and AM were 1000-fold higher than those with the intact CLR. With the D69N mutant the EC(50) of CGRP was 0.48 nM and that of AM 0.44 nM, but the maximal cAMP formation was reduced to 24% and to 12% of cells with the intact CLR. Co-immunoprecipitation of RAMP1 with the CLR, indicating complex formation, was reduced with the D69A, D69N, and D69E mutants. RAMP2 co-precipitated with the mutant receptors indistinguishable from the intact CLR. In conclusion, mutation of D69 to N, E or A in the CLR did not affect its expression at the cell surface, but impaired or abolished the CGRP and AM receptor function in the presence of RAMP1 and -2, respectively.

The receptors for the neuropeptide calcitonin (CT) gene-related peptide (CGRP) and the multifunctional peptide hormone adrenomedullin (AM) are calcitonin-like receptor (CLR)/receptor-activity-modifying protein (RAMP) 1 and CLR/RAMP2 heterodimers, respectively. Here, the amino acid sequence TRNKIMT, corresponding to the residues 14-20 of the N terminus of the mouse (m) CLR, was found to be required for a functional mCLR/RAMP2 AM receptor. The deletion of amino acids 14-20 (Delta14-20) or their substitution by alanine (14-20A) did not affect the heterodimerization of the mCLR with mRAMP1 or mRAMP2, and the levels of expression at the surface of transiently transfected COS-7 cells were not altered. In mRAMP1/mCLR- or mRAMP1/mCLR-(Delta14-20)-expressing cells CGRP stimulated cAMP formation with EC(50) values of 0.12 +/- 0.01 and 1.5 +/- 0.4 nm, respectively. In mRAMP2/mCLR-expressing cells the EC(50) of AM was 0.8 +/- 0.2 nm. However, in cells expressing mRAMP2/mCLR-(Delta14-20) up to 10(-6) m AM failed to stimulate cAMP production. In mRAMP2/mCLR-(14-20A) expressing cells the cAMP response to AM was minimally restored, and the EC(50) was >100 nm. In conclusion, the deletion of the amino acid sequence TRNKIMT of the extreme N terminus of the mCLR maintained CGRP receptor function of mRAMP1/receptor heterodimers, but AM no longer activated the mutant mCLR-(Delta14-20) in the presence of mRAMP2. The TRNKIMT sequence is required for normal mCLR/mRAMP2 association, and as a consequence, high affinity AM binding signaling the activation of adenylyl cyclase.

Purpose: The objective of this study was to evaluate key motif requirements of human calcitonin (hCT)-derived peptides for the permeation through the plasma membrane of MDCK monolayers, as epithelial model., Methods: Truncated and sequence-modified fluorescent-labeled hCT-derived peptides were synthesized through Fmoc chemistry. Peptide uptake by confluent MDCK was observed by confocal laser scanning microscopy. The cytotoxic effect of the peptides on cellular integrity was followed by LDH release. For direct comparison we covered the cellular uptake of established cell penetrating peptides, Tat(47-57) and penetratin(43-58)., Results: Truncated sequences of hCT, from hCT(9-32) to hCT(18-32), penetrated the plasma membrane and demonstrated a sectoral, punctuated cytoplasmic distribution. The uptake process appeared to be temperature-, time- and concentration-dependent. Amino acid modifications of hCT(18-32) indicated that both the proline in position 23 and the positive charge of lysine in position 18 are crucial for peptide uptake. The reverse sequence hCT(32-18) did not penetrate the membrane, indicating the importance of sequence orientation. Tat(47-57) and penetratin(43-58) showed a similar punctuated cytoplasmic distribution in MDCK and HeLa cell lines. No relevant toxicity was observed., Conclusions: Selected hCT-derived peptides have cell penetrating properties. The uptake mechanism seems to involve an endocytic pathway.

The receptor-activity-modifying protein (RAMP) 1 is a single-transmembrane-domain protein associated with the calcitonin-like receptor (CLR) to reveal a calcitonin gene-related peptide (CGRP) receptor. The extracellular region of RAMP1 contains six conserved cysteines. Here, Cys(27) in myc-tagged human (h) RAMP1 was deleted (hRAMP1Delta1), and Cys(40), Cys(57), Cys(72), Cys(82) and Cys(104) were each replaced by Ala. In COS-7 cells expressing hCLR/myc-hRAMP1Delta1 or -C82A, cell surface expression, [(125)I]halphaCGRP binding and cAMP formation in response to halphaCGRP were similar to those of hCLR/myc-hRAMP1. Cell surface expression of myc-hRAMP1-C72A was reduced to 24+/-7% of myc-hRAMP1, and that of -C40A, -C57A and -C104A was below 10%. [(125)I]halphaCGRP binding of hCLR/myc-hRAMP1-C72A was 13+/-3% of hCLR/myc-hRAMP1 and it was undetectable in hCLR/myc-hRAMP1-C40A-, -C57A- and -C104A-expressing cells. Maximal cAMP stimulation by halphaCGRP in hCLR/myc-hRAMP1-C40A- and -C72A-expressing cells was 14+/-1% and 33+/-2% of that of the hCLR/myc-hRAMP1 with comparable EC(50). But cAMP stimulation was abolished in cells expressing hCLR/myc-hRAMP1-C57A and -C104A. In conclusion, CGRP receptor function was not affected by the deletion of Cys(27) or the substitution of Cys(82) by Ala in hRAMP1, but it was impaired by the substitution of Cys(40), Cys(57), Cys(72) and Cys(104) by Ala. These four cysteines are required for the transport of hRAMP1 together with the CLR to the cell surface.

Receptors for calcitonin gene-related peptide (CGRP) and adrenomedullin (AM) are heterodimeric complexes of the calcitonin-like receptor (CLR) together with associated receptor-activity-modifying proteins (RAMP)1, -2 or -3. The RAMP define the specificity of the CLR for CGRP or AM. Here, mouse (m)CLR/mRAMP1, -2 and -3 were expressed in COS-7 cells that lack detectable CGRP and AM receptors. myc epitope-tagged non-glycosylated mRAMP1 required V5-tagged mCLR for its translocation to the cell surface. The glycosylated myc-mRAMP2 and -3, on the other hand, were expressed at the cell surface in the absence of co-transfected mCLR. Selective binding of [125I]h alpha CGRP to mCLR/mRAMP1 expressing cells was inhibited by rat (r)alpha CGRP(1-37) and the CGRP antagonist r alpha CGRP(8-37) with IC(50) of 7.0+/-1.6 nM and 1.0+/-0.1 nM (mean+/-SEM). rAM(1-50) and the AM antagonist rAM(20-50) inhibited [125I]h alpha CGRP binding at over 36-fold higher concentrations than r alpha CGRP. In mCLR/mRAMP2 expressing cells, selective [125I]rAM binding was inhibited by rAM(1-50) and -(20-50) with IC(50) of 8.9+/-2.6 nM and 34+/-9 nM. r alpha CGRP(1-37) and -(8-37) displaced the binding at over 25-fold higher concentrations. mCLR/mRAMP3 expressing cells recognized both [125I]h alpha CGRP and -rAM. The IC(50) of rAM and r alpha CGRP(8-37) ranged between 5.8 and 7.0 nM, and those of r alpha CGRP and rAM(20-50) were only 4- to 8-fold higher. r alpha CGRP and rAM stimulated and r alpha CGRP(8-37) and rAM(20-50) antagonized mCLR/mRAMP1, -2 and -3 mediated cAMP formation with relative potencies that reflected the observed CGRP and AM selectivity of the three receptor types. In conclusion, mCLR/mRAMP1 and -2 are CGRP- and AM-selective receptors, respectively, whereas mCLR/mRAMP3 is an AM/CGRP receptor.

Co-expression of an initially orphan calcitonin receptor-like (CL)1 receptor with individual receptor-activity-modifying proteins (RAMP)1, -2 and -3 results in CL receptor/RAMP1, -2 and -3 proteins at the cell surface. The RAMP define the selectivity of the CL receptor for the vasodilatory peptides adrenomedullin (AM) and calcitonin gene-related peptide (CGRP). The selectivity for AM and CGRP agonists and antagonists of human, rat, porcine and bovine CL receptors, co-expressed with RAMP2 and -3, has been studied in different cell types. This revealed CL receptor/RAMP2 and CL receptor/RAMP3 as AM1 and AM2 receptor subtypes, respectively. The AM1 receptor crossreacts with CGRP at high and the AM2 receptor at lower concentrations. Here the pharmacological properties of the cloned AM receptors are compared to those revealed in tissues and cell lines. According to nomenclature recommendation of the IUPHAR (International Union of Pharmacology) subcommittee XXXII, the former CRLR is now the CL receptor (1).

The calcitonin (CT)-like (CL) receptor is a CT gene-related peptide (CGRP) receptor or an adrenomedullin (AM) receptor when co-expressed with receptor-activity-modifying proteins (RAMP) 1 or 2, respectively. The CL receptor shows 57% overall sequence identity with the CT receptor, but the homology is much lower in the extreme N-terminus. An N-terminal deletion mutant of the human (h) CL receptor (Delta18-hCL) and a chimeric receptor consisting of the N-terminal amino acids of the porcine (p) CT receptor fused to the Delta18-hCL receptor (pCT-hCL) were therefore analyzed. The Delta18-hCL receptor function was abolished when co-expressed with RAMP1 or -2. The pCT-hCL receptor was a fully functional CGRP receptor when co-expressed with RAMP1, but the RAMP2-dependent AM receptor function was impaired. Limited sequence similarities in the N-terminus of the pCT and the hCL receptors rescue CGRP but not AM receptor binding and signalling.

The presence of adrenomedullin (AM) and of an AM receptor were investigated in highly enriched primary cultures of Schwann cells and perineural fibroblasts of newborn and adult rats. AM was released into the conditioned medium of adult perineural fibroblasts (1749+/-629 pgeq/10(5) cells per 24 h). mRNA encoding AM was also predominantly expressed in adult perineural fibroblasts. mRNA encoding the calcitonin receptor-like receptor (CRLR) and the receptor-activity-modifying proteins (RAMP) 1, -2 and -3 were demonstrated in all the primary cells, but the levels of RAMP1 mRNA relative to 18s rRNA were 10-fold lower than those of CRLR and RAMP2 and -3 encoding mRNA. The results are consistent with the expression of CRLR/RAMP2 and CRLR/RAMP3 heterodimeric AM receptors in all the primary cells examined. AM stimulated cAMP accumulation in newborn (EC(50) 0.62+/-0.29 nM) and adult (EC(50) 0.45+/-0.03 nM) rat Schwann cells and in newborn (EC(50) 0.79+/-0.50 nM) and adult (EC(50) 1.06+/-0.72 nM) rat perineural fibroblasts. The EC(50) of calcitonin gene-related peptide stimulated cAMP production was 93- to 100-fold higher than those of AM in the four types of primary cells studied. The co-expression of AM and its receptor in perineural fibroblasts and the expression of an AM receptor in Schwann cells suggest autocrine and/or paracrine modes of action of AM in peripheral nerves.

Three receptor-activity-modifying proteins (RAMP) define specific interactions between calcitonin (CT) gene-related peptide (CGRP), adrenomedullin (AM) and amylin, and a CT receptor or a CT receptor-like receptor (CRLR). Both form heterodimeric RAMP/receptor complexes at the cell surface. This association represents a novel principle of G protein-coupled receptor function. RAMP1 is transported to the cell surface together with the CRLR or the CT receptor. Here, we have investigated the functional relevance of the short C-terminal intracellular tail QSKRTEGIV and of the single transmembrane domain of human (h) RAMP1 for their interactions with the hCRLR to constitute a CGRP receptor. To this end, hRAMP1 has been sequentially truncated from the C-terminus, and [(125)I]h alpha CGRP/hRAMP1/hCRLR association at the cell surface and cAMP accumulation in response to h alpha CGRP have been examined. With the C-terminal truncation of hRAMP1 by four amino acids wild-type hRAMP1 function was maintained, and the hCRLR was required for the transport of hRAMP1 to the cell surface. Further truncation of hRAMP1 through removal of the remaining five intracellular amino acids revealed CRLR-independent cell surface delivery but otherwise normal hRAMP1 activity. Sequential shortening of the hRAMP1 transmembrane domain resulted in progressively impaired association with the hCRLR and, as a consequence, abolished CGRP receptor function. In conclusion, the intracellular QSKRT sequence adjacent to the transmembrane domain of hRAMP1 provides a signal for intracellular retention. The sequence is unrelated to consensus endoplasmic reticulum retention/retrieval motives and overridden by the presence of the hCRLR. The entire single transmembrane domain of hRAMP1 together with one hydrophilic amino acid residue at its C-terminus is required for the formation of a fully functional CGRP/hRAMP1/hCRLR receptor complex.

The calcitonin family of peptides comprises calcitonin, amylin, two calcitonin gene-related peptides (CGRPs), and adrenomedullin. The first calcitonin receptor was cloned in 1991. Its pharmacology is complicated by the existence of several splice variants. The receptors for the other members the family are made up of subunits. The calcitonin-like receptor (CL receptor) requires a single transmembrane domain protein, termed receptor activity modifying protein, RAMP1, to function as a CGRP receptor. RAMP2 and -3 enable the same CL receptor to behave as an adrenomedullin receptor. Although the calcitonin receptor does not require RAMP to bind and respond to calcitonin, it can associate with the RAMPs, resulting in a series of receptors that typically have high affinity for amylin and varied affinity for CGRP. This review aims to reconcile what is observed when the receptors are reconstituted in vitro with the properties they show in native cells and tissues. Experimental conditions must be rigorously controlled because different degrees of protein expression may markedly modify pharmacology in such a complex situation. Recommendations, which follow International Union of Pharmacology guidelines, are made for the nomenclature of these multimeric receptors.

Adrenomedullin (AM), alpha- and beta-calcitonin gene-related peptide (CGRP), calcitonin (CT), and amylin are homologous polypeptides with overlapping biological actions such as vasodilatation and inhibition of bone resorption. They are brought about through receptors that include the CT receptor (CTR) and an initially orphan CT receptor-like receptor (CRLR) in association with receptor-activity-modifying proteins (RAMP)1, -2, and -3. Co-expression of CRLR with RAMP1 or -2 revealed CGRP or AM receptors, respectively. The CTR interacts with CT and does not require a known RAMP for functional expression. The same CTR is a CGRP/amylin or an amylin receptor upon co-expression with RAMP1 or -3, respectively. Interactions between CRLR and RAMP are thought to be required for their delivery to the cell surface. There, heterodimeric complexes between CRLR or CTR and the corresponding RAMP reveal high-affinity receptors for AM, CGRP, and amylin. Here we review the current knowledge on interactions of G protein-coupled receptors with defined associated proteins., (Copyright 2002 Wiley-Liss, Inc.)