A radioligand binding assay for the insulin-like growth factor 2 receptor.

Insulin-like growth factors 2 and 1 (IGF2 and IGF1) and insulin are closely related hormones that are responsible for the regulation of metabolic homeostasis, development and growth of the organism. Physiological functions of insulin and IGF1 are relatively well-studied, but information about the role of IGF2 in the body is still sparse. Recent discoveries called attention to emerging functions of IGF2 in the brain, where it could be involved in processes of learning and memory consolidation. It was also proposed that these functions could be mediated by the receptor for IGF2 (IGF2R). Nevertheless, little is known about the mechanism of signal transduction through this receptor. Here we produced His-tagged domain 11 (D11), an IGF2-binding element of IGF2R; we immobilized it on the solid support through a well-defined sandwich, consisting of neutravidin, biotin and synthetic anti-His-tag antibodies. Next, we prepared specifically radiolabeled [125I]-monoiodotyrosyl-Tyr2-IGF2 and optimized a sensitive and robust competitive radioligand binding assay for determination of the nanomolar binding affinities of hormones for D11 of IGF2. The assay will be helpful for the characterization of new IGF2 mutants to study the functions of IGF2R and the development of new compounds for the treatment of neurological disorders.

From fever to immunity: A new role for IGFBP-6?

Fever is a fundamental response to infection and a hallmark of inflammatory disease, which has been conserved and shaped through millions of years of natural selection. Although fever is able to stimulate both innate and adaptive immune responses, the very nature of all the molecular thermosensors, the timing and the detailed mechanisms translating a physical trigger into a fundamental biological response are incompletely understood. Here we discuss the consequence of hyperthermic stress in dendritic cells (DCs), and how the sole physical input is sensed as an alert stimulus triggering a complex transition in a very narrow temporal window. Importantly, we review recent findings demonstrating the significant and specific changes discovered in gene expression and in the metabolic phenotype associated with hyperthermia in DCs. Furthermore, we discuss the results that support a model based on a thermally induced autocrine signalling, which rewires and sets a metabolism checkpoint linked to immune activation of dendritic cells. Importantly, in this context, we highlight the novel regulatory functions discovered for IGFBP-6 protein: induction of chemotaxis; capacity to increase oxidative burst and degranulation of neutrophils, ability to induce metabolic changes in DCs. Finally, we discuss the role of IGFBP-6 in autoimmune disease and how novel mechanistic insights could lead to exploit thermal stress-related mechanisms in the context of cancer therapy.

Cation-dependent mannose-6-phosphate receptor functions as a pattern recognition receptor in anti-bacterial immunity of Marsupenaeus japonicus.

The cation-dependent mannose-6-phosphate receptor (CD-MPR) is a member of the P-type lectin family. As a type I transmembrane glycoprotein, it functions in the delivery of newly synthesized acid hydrolases from the trans-Golgi network to endosomes for their subsequent transfer to the lysosome by binding the mannose-6-phosphate receptor-recognition moieties in the hydrolases. However, the functions of CD-MPR in immune responses are seldom reported. In the present study, we identified a CD-MPR-like molecule in Marsupenaeus japonicus and designed it as MjCD-MPR. It was significantly upregulated after challenge with Vibrio anguillarum at the mRNA and protein levels. Knockdown of MjCD-MPR resulted in a significant increase in the amount of V. anguillarum in the hemolymph of shrimp, which suggested that MjCD-MPR plays a role in shrimp antibacterial defense. The recombinant extracytoplasmic region of MjCD-MPR could bind gram-positive and gram-negative bacteria by interaction with peptidoglycan, lipopolysaccharide, and lipoteichoic acid. MjCD-MPR showed no direct bacteriostatic or bacteriocidal activity. Knockdown of MjCD-MPR decreased the expression levels of several antimicrobial peptides (Alf-C1, Alf-E1, Crustin I-2, and Crustin I-3), suggesting that MjCD-MPR promotes the expression of antimicrobial peptides in shrimp. In summary, working as a pattern recognition receptor, MjCD-MPR recognizes invading bacteria and triggers the expression of AMPs against bacterial infection in shrimp.

Antibodies that neutralize cellular uptake of elosulfase alfa are not associated with reduced efficacy or pharmacodynamic effect in individuals with Morquio A syndrome.

Many enzyme replacement therapies (ERTs) for lysosomal storage disorders use the cell-surface cation-independent mannose-6 phosphate receptor (CI-M6PR) to deliver ERTs to the lysosome. However, neutralizing antibodies (NAb) may interfere with this process. We previously reported that most individuals with Morquio A who received elosulfase alfa in the phase 3 MOR-004 trial tested positive for NAbs capable of interfering with binding to CI-M6PR ectodomain in an ELISA-based assay. However, no correlation was detected between NAb occurrence and clinical efficacy or pharmacodynamics. To quantify and better characterize the impact of NAbs, we developed a functional cell-based flow cytometry assay with a titer step that detects antibodies capable of interfering with elosulfase alfa uptake. Serum samples collected during the MOR-004 trial were tested and titers were determined. Consistent with earlier findings on NAb positivity, no correlations were observed between NAb titers and the clinical outcomes of elosulfase alfa-treated individuals with Morquio A.

Targeted therapy of osteosarcoma with radiolabeled monoclonal antibody to an insulin-like growth factor-2 receptor (IGF2R).

INTRODUCTION: Osteosarcoma overall survival has plateaued around 70%, without meaningful improvements in over 30years. Outcomes for patients with overt metastatic disease at presentation or who relapse are dismal. In this study we investigated a novel osteosarcoma therapy utilizing radioimmunotherapy (RIT) targeted to IGF2R, which is widely expressed in OS. METHODS: Binding efficiency of the Rhenium-188(188Re)-labeled IGF2R-specific monoclonal antibody (mAb) to IGF2R on OS17 OS cells was assessed with Scatchard plot analysis. Biodistribution studies were performed in heterotopic murine osteosarcoma xenografts. Tumor growth was compared over a 24-day period post-treatment between mice randomized to receive 188Re-labeled IGF2R-specific murine mAb MEM-238 (188Re-MEM-238) or one of three controls: 188Re-labeled isotype control mAb, unlabeled MEM-238, or no treatment. RESULTS: Results demonstrate that the radioimmunoconjugate had a high binding constant to IGF2R. Both 188Re-MEM-238 and the isotype control had similar initial distribution in normal tissue. After 48h 188Re-MEM-238 exhibited a 1.8 fold selective uptake within tumor compared to the isotype control (p=0.057). Over 24days, the tumor growth ratio was suppressed in animals treated with RIT compared to unlabeled and untreated controls (p=0.005) as demonstrated by a 38% reduction of IGF2R expressing osteosarcoma cells in the RIT group (p=0.002). CONCLUSIONS: In conclusion, given the lack of new effective therapies in osteosarcoma, additional investigation into this target is warranted. ADVANCES IN KNOWLEDGE: High expression of IGF2R on osteosarcoma tumors, paired with the specificity and in vivo anti-cancer activity of 188Re-labeled IGF2R-specific mAb suggests that IGF2R may represent a novel therapeutic target in the treatment of osteosarcoma. IMPLICATIONS FOR PATIENT CARE: This targeted approach offers the benefits of being independent of a specific pathway, a resistance mechanism, and/or an inherent biologic tumor trait and therefore is relevant to all OS tumors that express IGF2R.

The late endosomal transporter CD222 directs the spatial distribution and activity of Lck.

The spatial and temporal organization of T cell signaling molecules is increasingly accepted as a crucial step in controlling T cell activation. CD222, also known as the cation-independent mannose 6-phosphate/insulin-like growth factor 2 receptor, is the central component of endosomal transport pathways. In this study, we show that CD222 is a key regulator of the early T cell signaling cascade. Knockdown of CD222 hampers the effective progression of TCR-induced signaling and subsequent effector functions, which can be rescued via reconstitution of CD222 expression. We decipher that Lck is retained in the cytosol of CD222-deficient cells, which obstructs the recruitment of Lck to CD45 at the cell surface, resulting in an abundant inhibitory phosphorylation signature on Lck at the steady state. Hence, CD222 specifically controls the balance between active and inactive Lck in resting T cells, which guarantees operative T cell effector functions.

Insulin-like growth factor-2 enhances functions of antigen (Ag)-specific regulatory B cells.

Regulatory B cells (Bregs) are important in immune regulation. The factors that regulate Breg functions are less clear. Insulin-like growth factor 2 (IGF2) is capable of inducing hematopoietic stem cell differentiation. This study aimed to investigate the role of IGF2 in the development of Bregs and the enhancement of their function. In this study, the expression of IGF1 receptor (IGF1R) and IGF2R in ovalbumin (OVA)-specific B cells (OVAsBCs) was assessed by real time RT-PCR and Western blotting. The release of interleukin (IL)-10 from OVAsBCs and OVAsBC proliferation were assessed by enzyme-linked immunoassay and proliferation assay. The role of IGF2 in enhancing the function of OVAsBCs was tested with an intestinal allergic inflammation mouse model. The results showed that OVAsBCs expressed high levels of IGF2R. Exposure to both IGF2 and a specific antigen (Ag), OVA, markedly enhanced the expression of IL-10 in OVAsBCs as well as enhanced the IL-10(+) OVAsBC proliferation. The concurrent exposure to IGF2 and specific Ag markedly induced the IL-10 promoter DNA demethylation via activating the STAT5 pathway. IGF2 also enhanced both the OVAsBC proliferation in vivo and the effect of Ag-specific immunotherapy on inhibiting allergic inflammation in the intestine. We conclude that OVAsBCs express high levels of IGF2R and that IGF2 increases the expression of IL-10 in OVAsBCs and enhances OVAsBC proliferation and the inhibitory effect on allergic inflammation.

Development of IGF signaling antibody arrays for the identification of hepatocellular carcinoma biomarkers.

PURPOSE: Our objective was to develop a system to simultaneously and quantitatively measure the expression levels of the insulin-like growth factor (IGF) family proteins in numerous samples and to apply this approach to profile the IGF family proteins levels in cancer and adjacent tissues from patients with hepatocellular carcinoma (HCC). EXPERIMENTAL DESIGN: Antibodies against ten IGF family proteins (IGF-1, IGF-1R, IGF-2, IGF-2R, IGFBP-1, IGFBP-2, IGFBP-3, IGFBP-4, IGFBP-6, and Insulin) were immobilized on the surface of a glass slide in an array format to create an IGF signaling antibody array. Tissue lysates prepared from patient's liver cancer tissues and adjacent tissues were then applied to the arrays. The proteins captured by antibodies on the arrays were then incubated with a cocktail of biotinylated detection antibodies and visualized with a fluorescence detection system. By comparison with standard protein amount, the exact protein concentrations in the samples can be determined. The expression levels of the ten IGF family proteins in 25 pairs of HCC and adjacent tissues were quantitatively measured using this novel antibody array technology. The differential expression levels between cancer tissues and adjacent tissues were statistically analyzed. RESULTS: A novel IGF signaling antibody array was developed which allows the researcher to simultaneously detect ten proteins involved in IGF signal pathway with high sensitivity and specificity. Using this approach, we found that the levels of IGF-2R and IGFBP-2 in HCC tissues were higher than those in adjacent tissues. CONCLUSION: Our IGF signaling antibody array which can detect the expression of ten IGF family members with high sensitivity and specificity will undoubtedly prove a powerful tool for drug and biomarker discovery.

Selective plasma pharmacokinetics and brain uptake in the mouse of enzyme fusion proteins derived from species-specific receptor-targeted antibodies.

Enzymes may be re-engineered for brain drug targeting as an IgG-enzyme fusion protein, where the IgG is a monoclonal antibody (MAb) against an endogenous blood-brain barrier (BBB) receptor transporter, such as the insulin receptor or transferrin receptor (TfR). Iduronate 2-sulfatase (IDS) is fused to the heavy chain of a genetically engineered MAb against the human insulin receptor (HIR). Neither the HIRMAb alone, nor the HIRMAb-IDS fusion protein, is delivered across the BBB in the mouse, owing to lack of cross-reactivity of the HIRMAb with the insulin receptor in the mouse. The uptake of the HIRMAb-IDS fusion protein in peripheral organs exceeds that of the HIRMAb, which is attributed to uptake mediated via the mannose-6 phosphate receptor in non-brain organs. In contrast to the lack of BBB transport of the HIRMAb-IDS fusion protein, there is high BBB penetration in the mouse of an IDS fusion protein and a chimeric MAb against the mouse TfR. The comparison of the brain distribution of two different IgG-IDS fusion proteins, with different reactivity for an endogenous BBB receptor, illustrates the difference in brain targeting of a biopharmaceutical caused by the targeting properties of the IgG domain of the fusion protein.

Molecular inflammation and adipose tissue matrix remodeling precede physiological adaptations to pregnancy.

Changes in adipose tissue metabolism are central to adaptation of whole body energy homeostasis to pregnancy. To gain insight into the molecular mechanisms supporting tissue remodeling, we have characterized the longitudinal changes of the adipose transcriptome in human pregnancy. Healthy nonobese women recruited pregravid were followed in early (8-12 wk) and in late (36-38 wk) pregnancy. Adipose tissue biopsies were obtained in the fasting state from the gluteal depot. The adipose transcriptome was examined via whole genome DNA microarray. Expression of immune-related genes and extracellular matrix components was measured using real-time RT-PCR. Adipose mass, adipocyte size, and cell number increased in late pregnancy compared with pregravid measurements (P < 0.001) but remained unchanged in early pregnancy. The adipose transcriptome evolved during pregnancy with 10-15% of genes being differently expressed compared with pregravid. Functional gene cluster analysis revealed that the early molecular changes affected immune responses, angiogenesis, matrix remodeling, and lipid biosynthesis. Increased expression of macrophage markers (CD68, CD14, and the mannose-6 phosphate receptor) emphasized the recruitment of the immune network in both early and late pregnancy. The TLR4/NF-κB signaling pathway was enhanced specifically in relation to inflammatory adipokines and chemokines genes. We conclude that early recruitment of metabolic and immune molecular networks precedes the appearance of pregnancy-related physiological changes in adipose tissue. This biphasic pattern suggests that physiological inflammation is an early step preceding the development of insulin resistance, which peaks in late pregnancy.

Mannose-6-phosphate/insulin-like growth factor-II receptor in human melanoma cells: effect of ligands and antibodies on the receptor expression.

BACKGROUND: The M6P/IGF-II receptor belongs to the IGF system which plays a crucial role in tumorigenicity. While the role of the IGF-I receptor in signal transduction is well documented, previous experiments failed to uncover a clear signalling function for the M6P/IGF-II receptor. However, more recent studies have shown the capability of M6P/IGF-II receptor to initiate transmembrane signalling. MATERIALS AND METHODS: Human melanoma cells were used to detect the cell surface expression of the M6P/IGF-II receptor and its modulation by different effectors and monoclonal anti-receptor antibodies. RESULTS: M6P (5 mM) caused an increase of the luminescent receptor signal of about 50% . Pre-incubation of cells with Act-D (5 microg/mL) or CHI (10 microg/mL) following M6P stimulation in the presence of the inhibitors caused a reduction of receptor cell surface expression of 27% or 31%, respectively. The monoclonal antibody (mAb) 2G11 was able to mimic the M6P effect on the receptor up-regulation but the mAb MEM-238 did not. The synergistic effect detected with the combination of M6P and the mAb 2G11 and the failure of 2G11 to compete with the M6P action suggests that both effectors have different binding sites on the receptor. Unlike 2G11 the mAb MEM-238 prevented the M6P effect on receptor up-regulation confirming partially overlapping binding epitopes of both effectors. Brefeldin A was shown to have an inhibiting effect on the vesicular transport of the receptor protein to the plasma membrane and forskolin had an activating effect on the receptor exocytosis with the following enhanced integration into the plasma membrane. CONCLUSION: Up-regulation of the tumour suppressor M6P/IGF-II receptor might represent an approach for anticancer therapy. In addition, results support recent data on the receptor's capability of signal transduction.

Characterization of the mannose 6-phosphate receptor (Mr 300 kDa) protein dependent pathway of lysosomal enzyme targeting in Biomphalaria glabrata mollusc cells.

Mammalian mannose 6-phosphate receptors (MPR 300 and 46) are involved in the targeting of newly synthesized lysosomal enzymes and only MPR 300 also participates in the endocytosis of various exogenous ligands. The present study describes for the first time the MPR 300 dependent pathway of lysosomal enzyme sorting in the Biomphalaria glabrata embryonic (Bge) cells. Lysosomal enzymes (arylsulfatase A, beta-hexosaminidase and alpha-fucosidase) were identified by their enzymatic activities and by immunoprecipitation with specific antisera. Exposure of Bge cells to unio MPR 300 antiserum resulted in a dramatic loss of MPR 300 protein with a shortened half life of approximately 20 min as compared to control cells exposed to preimmune serum in which the half life of MPR 300 was of approximately 13 h. Loss of receptor proteins resulted in a significant misrouting of newly synthesized lysosomal enzymes and their secretion in cell culture medium as demonstrated by immunoprecipitation. The ability of Bge cells to uptake and internalize labeled arylsulfatase A, beta-hexosaminidase and alpha-fucosidase enzymes contained in cell secretion products also indicated the role of B. glabrata MPR 300 (CIMPR) protein in internalization and targeting of lysosomal enzymes. M6P dependent binding of lysosomal enzymes to MPR 300 was shown by confocal microscopy and coimmunoprecipitation experiments.

The homeostatic properties of the mannose receptor in health and disease

El estudio de la especificidad y distribución anatómica del Receptor de la Manosa (MR) y la caracterización fenotípica de ratones deficientes en el MR han permitido discernir la función de este receptor en condiciones fisiológicas normales y patológicas. En esta revisión se considera la biología del MR en tres contextos diferentes: A nivel molecular como miembro de la familia de receptores de manosa; como receptor de macrófagos y células dendríticas; y como un marcador de vasos linfáticos. Resultados recientes en estas tres áreas resaltan las propiedades únicas de este receptor y la necesidad de mantenerla mente abierta a la hora de establecer su función fisiológica (AU)

Research into the binding properties and anatomical distribution of the Mannose Receptor (MR), and the phenotypic characterization of MR deficient animals have provided highly valuable information regarding the role of the MR in health and disease. In this review, the biology of MR is considered in three different contexts: At the molecular level as a member of the mannose receptor family of proteins; as a macrophage and dendritic cell receptor; and as marker of lymphatic endothelia. Novel observations in these three areas highlight the unique properties of MR and the need to keep an open mind when assessing its physiological role (AU)

Single transmembrane domain insulin-like growth factor-II/mannose-6-phosphate receptor regulates central cholinergic function by activating a G-protein-sensitive, protein kinase C-dependent pathway.

The insulin-like growth factor-II/mannose-6-phosphate (IGF-II/M6P) receptor is a single-pass transmembrane glycoprotein that plays an important role in the intracellular trafficking of lysosomal enzymes and endocytosis-mediated degradation of IGF-II. However, its role in signal transduction after IGF-II binding remains unclear. In the present study, we report that IGF-II/M6P receptor in the rat brain is coupled to a G-protein and that its activation by Leu27IGF-II, an analog that binds rather selectively to the IGF-II/M6P receptor, potentiates endogenous acetylcholine release from the rat hippocampal formation. This effect is mediated by a pertussis toxin (PTX)-sensitive GTP-binding protein and is dependent on protein kinase Calpha (PKCalpha)-induced phosphorylation of downstream substrates, myristoylated alanine-rich C kinase substrate, and growth associated protein-43. Additionally, treatment with Leu27IGF-II causes a reduction in whole-cell currents and depolarization of cholinergic basal forebrain neurons. This effect, which is blocked by an antibody against the IGF-II/M6P receptor, is also sensitive to PTX and is mediated via activation of a PKC-dependent pathway. These results together revealed for the first time that the single transmembrane domain IGF-II/M6P receptor expressed in the brain is G-protein coupled and is involved in the regulation of central cholinergic function via the activation of specific intracellular signaling cascades.

The discovery and mechanism of action of novel tumor-selective and apoptosis-inducing 3,5-diaryl-1,2,4-oxadiazole series using a chemical genetics approach.

A novel series of 3,5-diaryl-oxadiazoles was identified as apoptosis-inducing agents through our cell and chemical genetics-based screening assay for compounds that induce apoptosis using a chemical genetics approach. Several analogues from this series including MX-74420 and MX-126374 were further characterized. MX-126374, a lead compound from this series, was shown to induce apoptosis and inhibit cell growth selectively in tumor cells. To elucidate the mechanism(s) by which this class of compounds alters the signal transduction pathway that ultimately leads to apoptosis, expression profiling using the Affymetrix Gene Chip array technology was done along with other molecular and biochemical analyses. Interestingly, we have identified several key genes (cyclin D1, transforming growth factor-beta1, p21, and insulin-like growth factor-BP3) that are altered in the presence of this compound, leading to characterization of the pathway for activation of apoptosis. MX-126374 also showed significant inhibition of tumor growth as a single agent and in combination with paclitaxel in murine tumor models. Using photoaffinity labeling, tail-interacting protein 47, an insulin-like growth factor-II receptor binding protein, was identified as the molecular target. Further studies indicated that down-regulation of tail-interacting protein 47 in cancer cells by small interfering RNA shows a similar pathway profile as compound treatment. These data suggest that 3,5-diaryl-oxadiazoles may be a new class of anticancer drugs that are tumor-selective and further support the discovery of novel drugs and drug targets using chemical genetic approaches.

HCG increases trophoblast migration in vitro via the insulin-like growth factor-II/mannose-6 phosphate receptor.

We have previously shown that both HCG and insulin-like growth factor-II (IGF-II) stimulate trophoblastic invasion. Furthermore, the invasion-promoting function of IGF-II resulted from IGF-II mannose 6-phosphate receptor (IGF-II/M6PR) activation. Since HCG and IGF-II did not have an additive effect on cell migration of extravillous trophoblast (EVT) cell line, HTR-8 SVneo, we hypothesized that HCG actions are mediated via alterations in the expression and/or function of IGF-II axis. HCG treatment (50-50,000 mU/ml) of the HTR-8/SVneo cells did not alter the expression of either insulin-like growth factor-I or IGF-II mRNA or peptide synthesis, but caused (i) an increase in the (125)I-IGF-II binding to EVT cells, and (ii) an increase in the externalization rate of the IGF-II binding sites without affecting their internalization. This effect was due to the increase in the number of IGF-II binding sites in the plasma membrane without any change in the IGF-II binding affinity. Although HCG did not influence the abundance of IGF-II/M6PR mRNA or protein, anti-IGF-II/M6PR antibody decreased HCG-induced migration of EVT, supporting the hypothesis that HCG might stimulate EVT migration by increasing IGF-II binding to the plasma membrane and subsequently by increasing the IGF-II effect probably mediated via the IGF-II/M6PR.

The insulin-like growth factor system in advanced breast cancer.

Despite improvements in therapy, the prognosis for advanced breast cancer is poor and a search for new treatment targets and key regulators of tumour growth is warranted. Extensive data are available on the importance of the insulin-like growth factor (IGF) system in growth regulation of breast cancer cell lines in vitro, indicating that the IGF-I receptor (IGF-IR), IGF-I (and IGF-II) function as survival factors, while IGF binding protein (IGFBP)-3 may act as a growth inhibitor. There is a tight link between the growth regulatory pathways of IGFs and oestrogens in oestrogen-receptor(OR)-positive breast cancer cells. In vivo studies indicate a role of IGF-I and IGF-IR in breast cancer development. However, the importance of the IGF system in metastatic and highly aggressive breast tumours in vivo is not clear, and therapeutic strategies designed to interrupt IGF signalling have not yet proved to be an effective treatment modality in patients with metastatic breast cancer.

An immuno-affinity method for the purification of mannose 6-phosphate receptor proteins.

In a recent study, we have developed an ELISA method to quantify the mannose 6-phosphate receptor (MPR) proteins [J. Biochem. Biophys. Methods 52 (2002) 111]. In the present study, we have used the goat MPR 300 antibody and peptide specific antibodies to human MPR 46 to develop simple and efficient immuno-affinity matrices, which can be used to purify the MPR proteins from goat liver in a single step. The identity of the immuno-affinity purified receptors is confirmed by their molecular masses as well as by their immunoreactivity.