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.

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.

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.

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.

Tail-specific antibodies that block return of 46,000 M(r) mannose 6-phosphate receptor to the trans-Golgi network.

Recycling of 46,000 M(r) mannose 6-phosphate receptor (MPR 46) was investigated by microinjection of Fab fragments against small epitopes within the cytoplasmic domain of the receptor. Fab fragments against the peptide 43-47 (Ala-Tyr-Arg-Gly-Val) efficiently blocked return of MPR 46 to the TGN. Antibody-induced redistribution resulted in accumulation of MPR 46 within an endosomal compartment, from which it recycled to the plasma membrane. Rab5 and rab7, markers for early and late endosomes, respectively, were not detectable in the compartment of redistributed MPR 46, suggesting that it represents a specialized endosomal subcompartment. The bulk of redistributed MPR 46 did not colocalize with endocytosed fluid-phase marker, suggesting that it accumulates at a site where MPR 46 has been segregated from endocytosed material, which is destined for transport to lysosomes. Peptide 43-47 contains a tyrosine (residue 44) which has been shown earlier to be part of an internalization signal for MPR 46 (Johnson, K. F., W. Chan, and S. Kornfeld. 1990. Proc. Natl. Acad. Sci. USA. 87:10010-10014). The role of tyrosine residue 44 as part of a putative multifunctional sorting signal is discussed.

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.

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.

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.

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.

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.

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.

Stimulation of human extravillous trophoblast migration by IGF-II is mediated by IGF type 2 receptor involving inhibitory G protein(s) and phosphorylation of MAPK.

We have earlier shown that migration and invasiveness of first trimester human extravillous trophoblast cells are stimulated by IGF-II, independently of IGF type 1 receptor and that migration stimulation is the primary reason for increased extravillous trophoblast cell invasiveness induced by IGF-II. In the present study we examined the functional role of IGF type II receptor in IGF-II stimulation of extravillous trophoblast cell migration and the underlying signal transduction pathways including the participation of inhibitory G protein(s) and MAPK. The migratory ability of a well characterized in vitro propagated human first trimester extravillous trophoblast cell line expressing the phenotype of extravillous trophoblast cells in situ was quantitated with a Transwell migration assay under different experimental conditions. We found that the extravillous trophoblast cells expressed an abundance of IGF type 2 receptor as detected by immunostaining and Western blots, and recombinant human IGF-II promoted their migration in a dose- and time-dependent manner. Both polyclonal and monoclonal IGF type 2 receptor-blocking antibodies blocked migration-stimulating effects of IGF-II. Two synthetic IGF-II analogs ([Leu27]IGF-II, which can bind to IGF type 2 receptor and IGF-binding proteins, but not IGF type 1 receptor, and [QAYL-Leu27]IGF-II, which can bind to IGFR-II, but neither IGFR-I nor IGF-binding proteins) both stimulated extravillous trophoblast cell migration to levels higher than those induced by wild-type IGF-II. These results reveal that IGF-II action was mediated by IGF type 2 receptor, independently of IGF type 1 receptor and IGF-binding proteins. Treatment of extravillous trophoblast cell membrane preparations with IGF-II decreased adenylyl cyclase activity in a concentration-dependant manner, indicating the participation of inhibitory G proteins in IGF-II action. This was substantiated further with the findings that increasing intracellular cAMP using forskolin or (Bu)2cAMP inhibited basal extravillous trophoblast cell migration and blocked IGF-II stimulation of migration. IGF-II treatment rapidly stimulated phosphorylation of MAPK (ERK-1 and -2), which was blocked by pretreatment of extravillous trophoblast cells with the MAPK kinase (MEK) inhibitor PD98059. Treatment with this inhibitor also blocked extravillous trophoblast cell migration in the presence or absence of IGF-II. These results, taken together, reveal that IGF-II stimulates extravillous trophoblast cell migration by signaling through IGF type 2 receptor, involving inhibitory G proteins and activating the MAPK pathway.

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.

Insulin-like growth factor II promotes in vitro cholinergic development of mouse septal neurons: comparison with the effects of insulin-like growth factor I.

We investigated the effect of insulin-like growth factors II and I (IGFII and IGFI) on septal primary cultures from mouse embryonic day 15 brains. The addition of IGFII to septal cultures enhanced total choline acetyltransferase (ChAT) activity in a dose-dependent manner. Maximal stimulation of ChAT activity was observed at 10 ng/ml IGFII. The effect of IGFII on ChAT activity was completely blocked by anti-IGFII/M-6-P receptor antibodies, whereas the antisera alone had no effect on the enzyme activity. Double-labeled immunohistochemical studies revealed that most ChAT-positive neurons expressed IGFII/M-6-P receptor immunoreactivity. These results indicate that the trophic effect of IGFII results from the direct action of this molecule through the IGFII/M-6-P receptor in septal cholinergic neurons. IGFI also stimulated ChAT activity, but with less potency than IGFII. Antibodies against the IGFII/M-6-P receptor inhibited approximately 50% of the IGFI response, suggesting that the effect of IGFI is mediated in part by the IGFII/M-6-P receptor. Thus, it appears that IGFII and IGFI are potent trophic factors for central cholinergic neurons and could potentially play a significant role in the differentiation, maintenance and regeneration of these neurons.

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.

The insulin-like growth factor-II/mannose-6-phosphate receptor is present in fetal bovine tissues throughout gestation.

The insulin-like growth factor-II (IGF-II) and the IGF-II/mannose-6-phosphate (M6P) receptor are thought to play an important role in fetal growth and development. We have studied the expression of the IGF-II/M6P receptor in fetal bovine tissues from 5 through 36 weeks' gestation. Tissues from bovine fetuses were extracted in buffer containing 2% Triton-X-100 and 2% sodium dodecyl sulfate (SDS). Aliquots of the protein extracts were analyzed by SDS polyacrylamide gel electrophoresis and the protein bands were transferred onto nitrocellulose. Immunoblotting was performed with anti-bovine IGF-II/M6P receptor antiserum. In a subset of experiments, ligand blotting was carried out with radiolabeled IGF-II and subsequent autoradiography. IGF-II/M6P receptors were expressed in all tissues examined, with the highest amount of receptor being present in fetal lung and liver. Low amounts of receptors were measured in fetal brain. The amount of receptor was developmentally regulated throughout fetal life. The developmental regulation of receptor expression varied among the different tissues. In conclusion, the IGF-II/M6P receptor is present in all fetal bovine tissues examined. The presence of the IGF-II/M6P receptor seems to be developmentally regulated during bovine fetal life. We hypothesize that this receptor exerts important biologic effects during fetal growth and tissue and organ development.

[Molecular therapy of breast carcinoma in the advanced phase]. / Terapia molecolare del carcinoma della mammella in fase avanzata.

Conventional chemotherapy regimens for the treatment of breast cancer have limited efficacy and are associated with significant toxicity, highlighting the need for novel targeted therapies. Increased expression and activation of receptor tyrosine kinases frequently occurs in human breast carcinomas and, therefore, several clinical trials are currently evaluating therapies targeting these receptors. Therapeutic strategies include blockade of individual receptors with monoclonal antibodies (e.g., trastuzumab) and inhibition of tyrosine kinase function (e.g., gefitinib). Trastuzumab is the first agent that has been approved for patients with human epidermal growth factor receptor 2 (HER2)-overexpressing breast cancer. Other growth-factor targeted drugs are in clinical development such as STI-571, farnesyl-transferase inhibitors and antibodies directed at the insulin-like growth factor.