Insulin-like Growth Factor Transport Research Articles

Integrated DNA Methylation/RNA Profiling in Middle Temporal Gyrus of Alzheimer’s Disease

Alzheimer’s disease is a neurodegenerative disorder clinically defined by gradual cognitive impairment and alteration in executive function. We conducted an epigenome-wide association study (EWAS) of a clinically and neuropathologically characterized cohort of 296 brains, including Alzheimer’s disease (AD) and non-demented controls (ND), exploring the relationship with the RNA expression from matched donors. We detected 5246 CpGs and 832 regions differentially methylated, finding overlap with previous EWAS but also new associations. CpGs previously identified in ANK1, MYOC, and RHBDF2 were differentially methylated, and one of our top hits (GPR56) was not previously detected. ANK1 was differentially methylated at the region level, along with APOE and RHBDF2. Only a small number of genes showed a correlation between DNA methylation and RNA expression statistically significant. Multiblock partial least-squares discriminant analysis showed several CpG sites and RNAs discriminating AD and ND (AUC = 0.908) and strongly correlated with each other. Furthermore, the CpG site cg25038311 was negatively correlated with the expression of 22 genes. Finally, with the functional epigenetic module analysis, we identified a protein–protein network characterized by inverse RNA/DNA methylation correlation and enriched for “Regulation of insulin-like growth factor transport”, with IGF1 as the hub gene. Our results confirm and extend the previous EWAS, providing new information about a brain region not previously explored in AD DNA methylation studies. The relationship between DNA methylation and gene expression is not significant for most of the genes in our sample, consistently with the complexities in the gene expression regulation.Graphical

Effects of Dermatan Sulfate from Marine Invertebrate Styela plicata in the Wound Healing Pathway: A Natural Resource Applied to Regenerative Therapy.

Acute and chronic dermatological injuries need rapid tissue repair due to the susceptibility to infections. To effectively promote cutaneous wound recovery, it is essential to develop safe, low-cost, and affordable regenerative tools. Therefore, we aimed to identify the biological mechanisms involved in the wound healing properties of the glycosaminoglycan dermatan sulfate (DS), obtained from ascidian Styela plicata, a marine invertebrate, which in preliminary work from our group showed no toxicity and promoted a remarkable fibroblast proliferation and migration. In this study, 2,4-DS (50 µg/mL)-treated and control groups had the relative gene expression of 84 genes participating in the healing pathway evaluated. The results showed that 57% of the genes were overexpressed during treatment, 16% were underexpressed, and 9.52% were not detected. In silico analysis of metabolic interactions exhibited overexpression of genes related to: extracellular matrix organization, hemostasis, secretion of inflammatory mediators, and regulation of insulin-like growth factor transport and uptake. Furthermore, in C57BL/6 mice subjected to experimental wounds treated with 0.25% 2,4-DS, the histological parameters demonstrated a great capacity for vascular recovery. Additionally, this study confirmed that DS is a potent inducer of wound-healing cellular pathways and a promoter of neovascularization, being a natural ally in the tissue regeneration strategy.

P-591 Cellular heterogeneity in the preovulatory follicle correlates with response to stimulation with recombinant FSH and affects somatic cell gene expression levels

Abstract Study question What is the difference in the composition and gene expression of pre-ovulatory follicular somatic cells between normo- and hyporesponder patients to rFSH stimulation? Summary answer Gene expression changes in the somatic cells of the preovulatory follicles of hyporesponder patients is partly attributable to distinct cell populations. What is known already Some women undergoing IVF respond suboptimally to the preceding ovarian stimulation without an indication of poor ovarian insufficiency nor advanced age. The gene expression levels in the somatic cells of their pre-ovulatory follicles have not been determined. However, the somatic cells play key roles in the transfer of gonadotrophin signals and in steroidogenesis. In the preovulatory follicle, being >20 mm in size, the somatic cells are exposed to various molecular gradients that drive their differentiation. Due to advancements in sequencing technologies the importance of somatic cell sub-populations in the etiologies of ovarian infertility can now be investigated by bioinformatic methods. Study design, size, duration Consecutive IVF patients <41 years of age were recruited at Nova Vita Clinic, Estonia, undergoing the antagonist stimulation protocol with rFSH administration. Ovarian puncture was performed for all patients after 36 hours of hCG stimulaton. rFSH dose >200 IU per one retrieved oocyte was considered as an indication for hyporesponse. Ten normo- and 9 hyporesponder patients were enrolled. Participants/materials, setting, methods Cells from the follicular fluid devoid of the cumulus oocyte complex were collected by centrifugation. RNA-seq was performed on the pooled cells of all participants. The study groups were compared by differential gene expression analysis by DESeq2. Single-cell RNA-seq was performed for >6000 individual cells of 3 normoresponder patients and cellular sub-populations were determined with Seurat package. CIBERTSORTx software was used to deconvolute the proportion of cell types from bulk RNA-seq data of all participants. Main results and the role of chance Alterations in the gene expression of ovarian somatic cells have been previously attributed to female age. We demonstrate that 407 genes are differentially expressed (FDR<0.05) between normo- and hyporesponder IVF patients after age adjustment. These genes were enriched into pathways of extracellular matrix reorganization (31 genes, FDR<0.005), post-translational protein phosphorylation (16 genes, FDR<0.006) and regulation of insulin-like growth factor transport and uptake by insulin-like growth factor binding proteins (16 genes, FDR<0.019). In addition, the sequencing of > 24 000 individual cells from 3 normoresponder patients revealed the somatic cell types of the preovulatory follicle in high resolution indicating the presence of immune cell populations (N = 4), epithelial cells, theca, cumulus cells and mural granulosa cell sub-populations (N = 7). Bioinformatic cell type deconvolution demonstrated significant differences between proportions of distinct cell sub-populations between normo- and hyporesponder patients and that several differentially expressed genes between the study groups can be attributed to a specific mural granulosa sub-population. Limitations, reasons for caution The number of study participants is small due to the high cost of the study methods. Wider implications of the findings The study demonstrates that hyporesponse to stimulation is associated with age-unrelated disturbances in gene expression of preovulatory somatic cells and that different somatic cell populations may have important underlying functions in the ovarian etiologies of infertility. Trial registration number not applicable

Predictive RNA profiles for early and very early spontaneous preterm birth

Spontaneous preterm birth remains the main driver of childhood morbidity and mortality. Because of an incomplete understanding of the molecular pathways that result in spontaneous preterm birth, accurate predictive markers and target therapeutics remain elusive. This study sought to determine if a cell-free RNA profile could reveal a molecular signature in maternal blood months before the onset of spontaneous preterm birth. Maternal samples (n=242) were obtained from a prospective cohort of individuals with a singleton pregnancy across 4 clinical sites at 12-24 weeks (nested case-control; n=46 spontaneous preterm birth <35 weeks and n=194 term controls). Plasma was processed via a next-generation sequencing pipeline for cell-free RNA using the Mirvie RNA platform. Transcripts that were differentially expressed in next-generation sequencing cases and controls were identified. Enriched pathways were identified in the Reactome database using overrepresentation analysis. Twenty five transcripts associated with an increased risk of spontaneous preterm birth were identified. A logistic regression model was developed using these transcripts to predict spontaneous preterm birth with an area under the curve=0.80 (95% confidence interval, 0.72-0.87) (sensitivity=0.76, specificity=0.72). The gene discovery and model were validated through leave-one-out cross-validation. A unique set of 39 genes was identified from cases of very early spontaneous preterm birth (<25 weeks, n=14 cases with time to delivery of 2.5±1.8 weeks); a logistic regression classifier on the basis of these genes yielded an area under the curve=0.76 (95% confidence interval, 0.63-0.87) in leave-one-out cross validation. Pathway analysis for the transcripts associated with spontaneous preterm birth revealed enrichment of genes related to collagen or the extracellular matrix in those who ultimately had a spontaneous preterm birth at <35 weeks. Enrichment for genes in insulin-like growth factor transport and amino acid metabolism pathways were associated with spontaneous preterm birth at <25 weeks. Second trimester cell-free RNA profiles in maternal blood provide a noninvasive window to future occurrence of spontaneous preterm birth. The systemic finding of changes in collagen and extracellular matrix pathways may serve to identify individuals at risk for premature cervical remodeling, with growth factor and metabolic pathways implicated more often in very early spontaneous preterm birth. The use of cell-free RNA profiles has the potential to accurately identify those at risk for spontaneous preterm birth by revealing the underlying pathophysiology, creating an opportunity for more targeted therapeutics and effective interventions.

Hepatic Steatosis Contributes to the Development of Muscle Atrophy via Inter-Organ Crosstalk.

Individuals with hepatic steatosis often display several metabolic abnormalities including insulin resistance and muscle atrophy. Previously, we found that hepatic steatosis results in an altered hepatokine secretion profile, thereby inducing skeletal muscle insulin resistance via inter-organ crosstalk. In this study, we aimed to investigate whether the altered secretion profile in the state of hepatic steatosis also induces skeletal muscle atrophy via effects on muscle protein turnover. To investigate this, eight-week-old male C57BL/6J mice were fed a chow (4.5% fat) or a high-fat diet (HFD; 45% fat) for 12 weeks to induce hepatic steatosis, after which the livers were excised and cut into ~200-µm slices. Slices were cultured to collect secretion products (conditioned medium; CM). Differentiated L6-GLUT4myc myotubes were incubated with chow or HFD CM to measure glucose uptake. Differentiated C2C12 myotubes were incubated with chow or HFD CM to measure protein synthesis and breakdown, and gene expression via RNA sequencing. Furthermore, proteomics analysis was performed in chow and HFD CM. It was found that HFD CM caused insulin resistance in L6-GLUT4myc myotubes compared with chow CM, as indicated by a blunted insulin-stimulated increase in glucose uptake. Furthermore, protein breakdown was increased in C2C12 cells incubated with HFD CM, while there was no effect on protein synthesis. RNA profiling of C2C12 cells indicated that 197 genes were differentially expressed after incubation with HFD CM, compared with chow CM, and pathway analysis showed that pathways related to anatomical structure and function were enriched. Proteomics analysis of the CM showed that 32 proteins were differentially expressed in HFD CM compared with chow CM. Pathway enrichment analysis indicated that these proteins had important functions with respect to insulin-like growth factor transport and uptake, and affect post-translational processes, including protein folding, protein secretion and protein phosphorylation. In conclusion, the results of this study support the hypothesis that secretion products from the liver contribute to the development of muscle atrophy in individuals with hepatic steatosis.

Post-translational protein deimination signatures in sea lamprey (Petromyzon marinus) plasma and plasma-extracellular vesicles

Lampreys are a jawless vertebrate species belonging to an ancient vertebrate lineage that diverged from a common ancestor with humans ~500 million years ago. The sea lamprey (Petromyzon marinus) has a filter feeding ammocoete larval stage that metamorphoses into a parasitic adult, feeding both on teleost and elasmobranch fish. Lampreys are a valuable comparative model species for vertebrate immunity and physiology due to their unique phylogenetic position, unusual adaptive immune system, and physiological adaptions such as tolerance to salinity changes and urea.Peptidylarginine deiminases (PADs) are a phylogenetically conserved enzyme family which catalyses post-translational deimination/citrullination in target proteins, enabling proteins to gain new functions (moonlighting). The identification of deiminated protein targets in species across phylogeny may provide novel insights into post-translational regulation of physiological and pathophysiological processes. Extracellular vesicles (EVs) are membrane vesicles released from cells that carry cargos of small molecules and proteins for cellular communication, involved in both normal and pathological processes. The current study identified deimination signatures in proteins of both total plasma and plasma-EVs in sea lamprey and furthermore reports the first characterisation of plasma-EVs in lamprey. EVs were poly-dispersed in the size range of 40–500 nm, similar to what is observed in other taxa, positive for CD63 and Flotillin-1. Plasma-EV morphology was confirmed by transmission electron microscopy. Assessment of deimination/citrullination signatures in lamprey plasma and plasma-EVs, revealed 72 deimination target proteins involved in immunity, metabolism and gene regulation in whole plasma, and 37 target proteins in EVs, whereof 24 were shared targets. Furthermore, the presence of deiminated histone H3, indicative of gene-regulatory mechanisms and also a marker of neutrophil extracellular trap formation (NETosis), was confirmed in lamprey plasma. Functional protein network analysis revealed some differences in KEGG and GO pathways of deiminated proteins in whole plasma compared with plasma-EVs. For example, while common STRING network clusters in plasma and plasma-EVs included Peptide chain elongation, Viral mRNA translation, Glycolysis and gluconeogenesis, STRING network clusters specific for EVs only included: Cellular response to heat stress, Muscle protein and striated muscle thin filament, Nucleosome, Protein processing in endoplasmic reticulum, Nucleosome and histone deacetylase complex. STRING network clusters specific for plasma were: Adipokinetic hormone receptor activity, Fibrinogen alpha/beta chain family, peptidase S1A, Glutathione synthesis and recycling-arginine, Fructose 1,6-bisphosphate metabolic process, Carbon metabolism and lactate dehydrogenase activity, Post-translational protein phosphorylation, Regulation of insulin-like growth factor transport and clotting cascade. Overall, for the EV citrullinome, five STRING network clusters, 10 KEGG pathways, 15 molecular GO pathways and 29 Reactome pathways were identified, compared with nine STRING network clusters, six KEGG pathways, two Molecular GO pathways and one Reactome pathway specific for whole plasma; while further pathways were shared. The reported findings indicate that major pathways relevant for immunity and metabolism are targets of deimination in lamprey plasma and plasma-EVs, with some differences, and may help elucidating roles for the conserved PAD enzyme family in regulation of immune and metabolic function throughout phylogeny.

Differential alternative splicing between hepatocellular carcinoma with normal and elevated serum alpha-fetoprotein

BackgroundSerum alpha-fetoprotein (AFP) is the approved serum marker for hepatocellular carcinoma (HCC) screening. However, not all HCC patients show high (≥ 20 ng/mL) serum AFP, and the molecular mechanisms of HCCs with normal (< 20 ng/mL) serum AFP remain to be elucidated. Therefore, we aimed to identify biological features of HCCs with normal serum AFP by investigating differential alternative splicing (AS) between HCCs with normal and high serum AFP.MethodsWe performed a genome-wide survey of AS events in 249 HCCs with normal (n = 131) and high (n = 118) serum AFP levels using RNA-sequencing data obtained from The Cancer Genome Atlas.ResultsIn group comparisons of RNA-seq profiles from HCCs with normal and high serum AFP levels, 161 differential AS events (125 genes; ΔPSI > 0.05, FDR < 0.05) were identified to be alternatively spliced between the two groups. Those genes were enriched in cell migration or proliferation terms such as “the cell migration and growth-cone collapse” and “regulation of insulin-like growth factor (IGF) transport and uptake by IGF binding proteins”. Most of all, two AS genes (FN1 and FAM20A) directly interact with AFP; these relate to the regulation of IGF transport and post-translational protein phosphorylation. Interestingly, 42 genes and 27 genes were associated with gender and vascular invasion (VI), respectively, but only eighteen genes were significant in survival analysis. We especially highlight that FN1 exhibited increased differential expression of AS events (ΔPSI > 0.05), in which exons 25 and 33 were more frequently skipped in HCCs with normal (low) serum AFP compared to those with high serum AFP. Moreover, these events were gender and VI dependent.ConclusionWe found that AS may influence the regulation of transcriptional differences inherent in the occurrence of HCC maintaining normal rather than elevated serum AFP levels.

TNFRSF12A and a new prognostic model identified from methylation combined with expression profiles to predict overall survival in hepatocellular carcinoma.

BackgroundIt has been proved that DNA methylation, as an epigenetic regulatory mode, plays a crucial role in the initiation, progression and invasion of hepatocellular carcinoma (HCC). However, there still are some pathways and factors that regulates the carcinogenesis of HCC remains unclear.MethodsThe original datasets comparing DNA methylation, clinical information and transcriptome profiling between HCC and normal controls were downloaded from The Cancer Genome Atlas (TCGA) database. R software was used to screen for methylation-differential genes (MDGs) and methylation-driven genes. Gene-functional enrichment analysis, ConsensusPathDB pathway analysis, protein-protein interaction (PPI) network construction and survival analysis were performed; methylation-specific polymerase chain reaction (MSP) and real-time quantitative polymerase chain reaction (RT-qPCR) were used for validation.ResultsOne hundred and sixty-seven MDGs and 285 methylation-driven genes were identified. Function and pathway enrichment analysis revealed that they are associated with sequence-specific DNA binding, nuclear nucleosome, regulation of insulin-like growth factor transport, etc. An eight-gene (HIST1H1D, RP11-476B1.1, OR2AK2, TNFRSF12A, CTD-2313N18.8, AC133644.2, RP11-467L13.4 and LINC00989) prognostic model was identified from the MDGs; its methylation degree can strongly predict the overall survival of HCC. Among them, TNFRSF12A being the only one belongs to both MDGs and methylation-driver genes, shows a significant independent correlation with the prognosis of HCC. That was validated in further details.ConclusionsOur research has identified a registry of novel genes and pathways that’s important for regulating the carcinogenesis of HCC. In addition, we identified a strong molecular model for prognostic prediction. These findings will not only provide guidance for clinical individualized treatment, but also to set us targets for further research on the molecular mechanism of HCC.

Identification of potential crucial genes in atrial fibrillation: a bioinformatic analysis

BackgroundAtrial fibrillation (AF) is at least partially heritable, affecting 2–3% of the population in Europe and the USA. However, a substantial proportion of heritability is still lacking. In the present study, we aim to identify potential crucial genes associated with AF through bioinformatic analyses of public datasets.MethodsMicroarray data sets of GSE115574, GSE31821, GSE79768, GSE41177 and GSE14975 from the Gene Expression Omnibus (GEO) database were retrieved. After merging all microarray data and adjusting batch effect, differentially expressed genes (DEGs) were identified. Functional enrichment analyses based on Gene Ontology (GO) resource, Kyoto Encyclopedia of Genes and Genomes (KEGG) resource, Gene Set Enrichment Analysis (GSEA), Reactome Pathway Database and Disease Ontology (DO) were carried out. Protein-protein interaction (PPI) network was constructed using the STRING database. Combined with aforementioned significant bioinformatics information, potential crucial genes were subsequently selected. The comparative toxicogenomics database (CTD) was used to explore the interaction between potential crucial genes and AF.ResultWe identified 27 of DEGs with gene expression fold change (FC) ≥ 1.5 or ≤ 2/3 (|log2 FC| ≥ 0.58) and 5 with FC ≥ 2 or ≤ 0.5 (|log2 FC| ≥ 1) of AF patients compared with sinus rhythm controls. The most significantly enriched pathway was regulation of insulin-like growth factor transport and uptake by insulin-like growth factor binding proteins. IGFBP2, C1orf105, FHL2, CHGB, ATP1B4, IGFBP3, SLC26A9, CXCR4 and HTR2B were considered the potential crucial genes. CTD showed CXCR4, IGFBP2, IGFBP3 and FHL2 had higher scores with AF.ConclusionsThe 9 potential crucial genes, especially CXCR4, IGFBP2, IGFBP3 and FHL2, may be associated with risk of AF. Our study provided new insights of AF into genetics, molecular pathogenesis and new therapeutic targets.

Role of Tumor and Stroma-Derived IGF/IGFBPs in Pancreatic Cancer.

Pancreatic cancer (PC) is the utmost stroma-rich cancer, which is accompanied by fibrotic reactions that stimulate interactions between tumor cells and stroma to promote tumor progression. Considerable research evidence denotes that insulin-like growth factor (IGF)/IGF binding proteins (IGFBP) signaling axis facilitate tumor growth, metastasis, drug resistance, and thereby facilitate PC into an advanced stage. The six members of IGFBPs were initially considered as passive carriers of free IGFs; however, current evidence revealed their functions beyond the endocrine role in IGF transport. Though numerous efforts have been made in blocking IGF/IGFBPs, the targeted therapies remain unsuccessful due to the complexity of tumor-stromal interactions in the pancreas. In this review, we explore the emerging evidence of the various roles of the tumor as well as stroma derived IGF/IGFBPs and highlight as a novel therapeutic target against PC progression.

Amyloid-beta peptide neurotoxicity in human neuronal cells is associated with modulation of insulin-like growth factor transport, lysosomal machinery and extracellular matrix receptor interactions.

Extracellular deposits of the amyloid-beta peptide (Aβ) are known as the main pathological hallmark of Alzheimer’s disease. In Alzheimer’s disease, neurons are injured and die throughout the brain, a process in which Aβ neurotoxicity is considered to play an important role. However, the molecular mechanisms underlying Aβ toxicity that lead to neurodegeneration are not clearly established. Here we have elucidated the molecular pathways and networks which are impacted by Aβ in neurons using SH-SY5Y human neuroblastoma cells as a model. These cells were treated with Aβ1–42 peptides to study changes in biochemical networks using tandem mass tag labeled quantitative proteomic technique followed by computational analysis of the data. The molecular impacts of Aβ on cells were evident in a time- and dose-dependent manner, albeit the duration of treatment induced greater differential changes in cellular proteome compared to the effects of concentration. Aβ induced early changes in proteins associated with lysosomes, collagen chain trimerization and extracellular matrix receptor interaction, complement and coagulation cascade, oxidative stress induced senescence, ribosome biogenesis, regulation of insulin-like growth factor transport and uptake by insulin-like growth factor-binding protein. These novel findings provide molecular insights on the effects of Aβ on neurons, with implications for better understanding the impacts of Aβ on early neurodegeneration in Alzheimer’s disease pathology.

720-P: Exercise Training Causes Vast Changes in the Circulating Factor Profile of Human Subjects

Exercise training induces adaptations to the body that improve metabolic health; however, the molecular mechanisms mediating these beneficial adaptations to training are not well characterized. We hypothesized that exercise training causes adaptations to multiple tissues (e.g., muscle, adipose) that lead to altered secretion of factors from these tissues into the circulation, and that these circulating factors may contribute to the effects of exercise training on metabolic health. To identify novel exercise training-stimulated circulating factors, healthy subjects (n=8, 3 males and 5 females; age=27.0 ± 2.2 years; BMI=21.9 ± 0.9 kg/m2; V?O2peak=28.4 ± 1.2 ml/kg/minute) underwent 10-weeks of aerobic exercise training. Training increased V?O2peak by 17% (p=&amp;lt;0.003). Fasting blood samples were collected pre- and post-training, and 1,310 circulating factors were measured in plasma using aptamer-based proteomics. Remarkably, exercise training significantly altered 293 (22%) of all circulating factors. 164 proteins were increased and 129 proteins were decreased; the majority of which have not been previously identified as exercise training-induced factors. Change in V?O2peak most significantly correlated with baseline levels of Mesothelin (p&amp;lt;0.001, R=0.53), a cell-surface protein that may regulate Insulin-like Growth Factor transport. Change in V?O2peak was also highly correlated with Protein Kinase C Zeta (p=0.005, R=0.3), a kinase involved in AKT3-PI3K signaling and adipocyte glucose transport. Pathway analysis revealed downregulation of the lysosomal pathway, and upregulation of pathways including FC Epsilon Ri signaling, Mtor signaling and WNT signaling, suggesting that exercise training adaptations involve multiple processes, including cellular growth, proliferation and protein degradation. These data define proteomic adaptations to exercise training, and identify biological processes that may mediate the beneficial effects of exercise training in humans. Disclosure R. Middelbeek: None. J.J. Richard: None. L.A. Rowland: None. P. Nigro: None. H. Pan: None. J. Dreyfuss: None. L.J. Goodyear: None. Funding National Institutes of Health (R01DK099511 to L.J.G.), (K23DK114550 to R.M.); Joslin Diabetes Center (5P30DK36836); Daiichi Sankyo (to L.J.G.); Boston Area Diabetes Endocrinology Research Center (to R.M.)

Differential proteomic comparison of breast cancer secretome using a quantitative paired analysis workflow

BackgroundWorldwide, breast cancer is the main cause of cancer mortality in women. Most cases originate in mammary ductal cells that produce the nipple aspirate fluid (NAF). In cancer patients, this secretome contains proteins associated with the tumor microenvironment. NAF studies are challenging because of inter-individual variability. We introduced a paired-proteomic shotgun strategy that relies on NAF analysis from both breasts of patients with unilateral breast cancer and extended PatternLab for Proteomics software to take advantage of this setup.MethodsThe software is based on a peptide-centric approach and uses the binomial distribution to attribute a probability for each peptide as being linked to the disease; these probabilities are propagated to a final protein p-value according to the Stouffer’s Z-score method.ResultsA total of 1227 proteins were identified and quantified, of which 87 were differentially abundant, being mainly involved in glycolysis (Warburg effect) and immune system activation (activated stroma). Additionally, in the estrogen receptor-positive subgroup, proteins related to the regulation of insulin-like growth factor transport and platelet degranulation displayed higher abundance, confirming the presence of a proliferative microenvironment.ConclusionsWe debuted a differential bioinformatics workflow for the proteomic analysis of NAF, validating this secretome as a treasure-trove for studying a paired-organ cancer type.

IGF binding proteins in cancer: mechanistic and clinical insights.

The six members of the family of insulin-like growth factor (IGF) binding proteins (IGFBPs) were originally characterized as passive reservoirs of circulating IGFs, but they are now understood to have many actions beyond their endocrine role in IGF transport. IGFBPs also function in the pericellular and intracellular compartments to regulate cell growth and survival - they interact with many proteins, in addition to their canonical ligands IGF-I and IGF-II. Intranuclear roles of IGFBPs in transcriptional regulation, induction of apoptosis and DNA damage repair point to their intimate involvement in tumour development, progression and resistance to treatment. Tissue or circulating IGFBPs might also be useful as prognostic biomarkers.

Endoplasmic Reticulum Stress Induction of Insulin-like Growth Factor-binding Protein-1 Involves ATF4

Endoplasmic reticulum (ER) stress is sensed by cells in different physiopathological conditions in which there is an accumulation of unfolded proteins in the ER. A coordinated adaptive program called the unfolded protein response is triggered and includes translation inhibition, transcriptional activation of a set of genes encoding mostly intracellular proteins, and ultimately apoptosis. Here we show that insulin-like growth factor (IGF)-binding protein-1 (IGFBP-1), a secreted protein that modulates IGF bioavailability and has other IGF-independent effects, is potently induced during ER stress in human hepatocytes. Various ER stress-inducing agents were able to increase IGFBP-1 mRNA levels, as well as cellular and secreted IGFBP-1 protein up to 20-fold. A distal regulatory region of the human IGFBP-1 gene (-6682/-6384) containing an activating transcription factor 4 (ATF4) composite site was required for promoter activation upon ER stress. Mutation of the ATF4 composite site led to the loss of IGFBP-1 regulation. Electrophoretic mobility shift assay revealed an ER stress-inducible complex that was displaced by an ATF4 antibody. Knockdown of ATF4 expression using two specific small interfering RNAs impaired up-regulation of IGFBP-1 mRNA, which highlights the relevance of ATF4 in endogenous IGFBP-1 gene induction. In addition to intracellular proteins involved in secretory and metabolic pathways, we conclude that ER stress induces the synthesis of secreted proteins. Increased secretion of IGFBP-1 during hepatic ER stress may thus constitute a signal to modulate cell growth and metabolism and induce a systemic adaptive response.

Als2-deficient mice exhibit disturbances in endosome trafficking associated with motor behavioral abnormalities.

ALS2 is an autosomal recessive form of spastic paraparesis (motor neuron disease) with juvenile onset and slow progression caused by loss of function of alsin, an activator of Rac1 and Rab5 small GTPases. To establish an animal model of ALS2 and derive insights into the pathogenesis of this illness, we have generated alsin-null mice. Cytosol from brains of Als2(-/-) mice shows marked diminution of Rab5-dependent endosome fusion activity. Furthermore, primary neurons from Als2(-/-) mice show a disturbance in endosomal transport of insulin-like growth factor 1 (IGF1) and BDNF receptors, whereas neuronal viability and endocytosis of transferrin and dextran seem unaltered. There is a significant decrease in the size of cortical motor neurons, and Als2(-/-) mice are mildly hypoactive. Altered trophic receptor trafficking in neurons of Als2(-/-) mice may underlie the histopathological and behavioral changes observed and the pathogenesis of ALS2.

Roles of insulin-like growth factor (IGF) binding proteins in regulating IGF actions

The insulin-like growth factor (IGF) system is an evolutionarily conserved signaling pathway that is composed of two IGF ligands, two IGF receptors, and six IGF binding proteins. Studies in a variety of species suggest that the IGF signaling system plays a fundamental role in regulating embryonic growth and differentiation as well as in maintaining homeostasis in the adults. In extracellular fluids, IGFs are present in a complex with an IGF-binding protein (IGFBP). These IGFBPs are traditionally thought to function as carrier proteins and regulate circulating IGF turnover, transport, and distribution. Locally expressed IGFBPs can also inhibit and/or potentiate IGF activities. Recent studies have shown that some IGFBPs, in particular IGFBP-3 and -5, possess intrinsic biological activities and can act through IGF-independent mechanisms. In this article, we provide a brief overview of our current understanding of the IGF signaling system with particular reference to IGFBPs.

The role of the acid-labile subunit in regulating insulin-like growth factor transport across human umbilical vein endothelial cell monolayers.

We have established a human umbilical vein endothelial cell (HUVEC) monolayer system to study the role of complex formation with IGF binding protein (IGFBP) and acid-labile subunit (ALS) on the transendothelial transport of IGF. Incubation with recombinant human IGFBP-3 alone did not retard (125)I-IGF-I or -II transport, but addition of ALS caused marked inhibition. Transport of (125)I-des(1-3)IGF-I was more rapid than (125)I-IGF-I, suggesting the presence of some endogenous IGFBPs, although these were undetectable by affinity labeling of cells or medium. In the presence of ALS, recombinant human IGFBP-5 also retarded IGF transport, although significantly less than IGFBP-3, despite their similar ternary complex formation. In contrast, IGFBP-3 mutated in its ALS-binding domain was not inhibitory. To study IGF transport by pregnancy-proteolyzed IGFBP-3, we prepared [Tyr(31)]monoiodoIGF-I, the only iodoIGF-I form that reacts normally with proteolyzed IGFBP-3. In the presence of ALS, IGFBP-3 isolated by immunoaffinity chromatography from second-trimester pregnancy serum significantly retarded IGF transport, but to a lower extent than IGFBP-3 isolated from normal serum, despite normal ALS binding. This study demonstrates the key role of ALS in regulating transendothelial IGF transport, but indicates that other factors are also involved. Our data suggest that pregnancy-proteolyzed IGFBP-3, despite forming normal ternary complexes, is less effective than intact IGFBP-3 in retarding IGF egress from the circulation.

Transport and binding of insulin-like growth factor I through articular cartilage

This study focused on the role of insulin-like growth factor (IGF) binding proteins (IGFBPs) in cartilage on the transport and binding of IGF-I within the tissue. We have developed experimental and theoretical modeling techniques to quantify and contrast the roles of diffusion, binding, fluid convection, and electrical migration on the transport of IGF-I within cartilage tissue. Bovine articular cartilage disks were equilibrated in buffer containing 125I-IGF-I and graded levels of unlabeled IGF-I. Equilibrium binding, as measured by the uptake ratio of 125I-IGF-I in the tissue (free plus bound) to the concentration of labeled species in the buffer, was found to be consistent with a first-order reversible binding model involving one dominant family of binding sites within the matrix. Western ligand blots revealed a major IGF binding doublet around 23 kDa, which has been previously shown to coincide with IGFBP-6. Diffusive transport of 125I-IGF-I through cartilage was measured and found to be consistent with a diffusion-limited reaction theoretical model incorporating first-order reversible binding. Addition of excess amounts of unlabeled IGF-I during steady state transport of 125I-IGF-I resulted in release of bound 125I-IGF-I from the tissue, as predicted by the diffusion–reaction model. In contrast, addition of the low-affinity Des(1-3)IGF-I analog did not result in release of bound 125I-IGF-I. Application of electric current was used to augment transport of IGF-I through cartilage via electroosmosis and electrophoresis. Taken together, our results suggest that a single dominant substrate family, the high-affinity IGFBPs, is responsible for much of the observed binding of IGF-I within cartilage. The data suggest that intratissue fluid flow, such as that induced by mechanical loading of cartilage in vivo may be expected to enhance IGF transport by an order of magnitude and that this increment may help to counterbalance the restrictions encountered by the immobilization of IGFs by the binding proteins.

Alpha 2-Macroglobulin: a new component in the insulin-like growth factor/insulin-like growth factor binding protein-1 axis.

Insulin-like growth factors (IGFs) are crucial for many aspects of development, growth, and metabolism yet control of their activity by IGF-binding proteins (IGFBPs) remains controversial. The effect of IGFBP-1 depends on its phosphorylation status; phosphorylated IGFBP-1 inhibits IGF actions whereas the nonphosphorylated isoform is stimulatory. In order to understand this phenomenon, we purified phosphorylated IGFBP-1 from normal human plasma by immunoaffinity chromatography. Unexpectedly, the resulting preparation enhanced IGF-stimulated 3T3-L1 fibroblast proliferation, due to the presence of a co-purified protein of approximately 700 kDa. Matrix-assisted laser desorption ionization-mass spectrometry and Western immunoblotting analysis identified this co-purified protein as alpha(2)-macroglobulin (alpha(2)M). Anti-alpha(2)M antibodies co-immunoprecipitated IGFBP-1 from human plasma and from (125)I-IGFBP-1.alpha(2)M complexes formed in vitro. The (125)I-IGFBP-1/alpha(2)M association could be inhibited with excess unlabeled IGFBP-1. Surface plasmon resonance analysis indicated that alpha(2)M preferentially associates with the phosphorylated isoform of IGFBP-1 and that when complexed to alpha(2)M, IGFBP-1 can still bind IGF-I. These findings have functional significance since alpha(2)M protects IGFBP-1 from proteolysis and abrogates the inhibitory effect of phosphorylated IGFBP-1 on IGF-I stimulated 3T3-L1 cell proliferation. We conclude that alpha(2)M is a binding protein of IGFBP-1 which modifies IGF-I/IGFBP-1 actions resulting in enhanced IGF effects. In line with its role in regulating the clearance and activity of other growth factors, we predict that alpha(2)M has a novel and important role in controlling the transport and biological activity of IGFs.