Gene expression of the IGF hormones and IGF binding proteins across time and tissues in a model reptile.

The insulin and insulin-like signaling (IIS) network regulates cellular processes including pre- and postnatal growth, cellular development, wound healing, reproduction, and longevity. Despite their importance in the physiology of vertebrates, the study of the specific functions of the top regulators of the IIS network, insulin-like growth factors (IGFs) and IGF binding proteins (IGFBPs), has been mostly limited to a few model organisms. To expand our understanding of this network, we performed quantitative gene expression of IGF hormones in liver and qualitative expression of IGFBPs across tissues and developmental stages in a model reptile, the brown anole lizard (Anolis sagrei). We found that lizards express IGF2 across all life stages (preoviposition embryos to adulthood) and at a higher level than IGF1, which is opposite to patterns seen in laboratory rodents but similar to those seen in humans and other vertebrate models. IGFBP expression was ubiquitous across tissues (brain, gonad, heart, liver, skeletal muscle, tail, and regenerating tail) in adults, apart from IGFBP5, which was variable. These findings provide an essential foundation for further developing the anole lizard as a physiological and biomedical reptile model, as well as expanding our understanding of the function of the IIS network across species.

Production of insulin-like growth factor-binding proteins during the development of hepatic fibrosis due to chronic hepatitis C. / Producción de las proteínas de unión al factor de crecimiento insulinoide durante el desarrollo de la fibrosis hepática por hepatitis C crónica.

INTRODUCTION AND AIMS: Insulin-like growth factor 1 is modulated by the insulin-like growth factor-binding proteins (IGFBPs) that are synthesized in the liver. The aim of the present study was to evaluate the concentrations of IGFBPs 1-7 in patients with chronic hepatitis C and study their association with fibrosis stage. PATIENTS AND METHODS: A prospective, cross-sectional study was conducted that included patients with chronic hepatitis C. The stages of fibrosis were determined through FibroTest and FibroScan and the patients were compared with a control group. Serum levels of IGFBPs 1-7 were quantified through multiple suspension arrays. The Kruskal-Wallis test, Mann-Whitney U test, Spearman's correlation, and ROC curves were used for the statistical analysis. RESULTS: Upon comparing the patients and controls, the highest concentrations were found in IGFBPs 1, 2, 4, and 7 (p=0.02, p=0.002, p=0.008, and p<0.001, respectively). IGFBP-3 levels had a tendency to be lower in the patients (p=0.066), whereas values were similar between patients and controls for IGFBP-5 and 6 (p=0.786 and p=0.244, respectively). Of the seven IGFBPs, IGFBP-3 concentrations were the highest. There were significant differences between fibrosis stages for IGFBP-5 and IGFBP-7. CONCLUSION: IGFBPs play a relevant role in the fibrotic process in liver damage. IGFBP-7, in particular, differentiates fibrosis stages, making it a potential serum biomarker.

Insulin-like growth factor binding protein-7: A marker of conjunctivalization in an animal model of limbal stem cell deficiency.

PURPOSE: Limbal stem cell deficiency (LSCD) is characterized by the loss of limbal epithelial stem cells, resulting in a pathological process termed 'conjunctivalization' which compromises corneal transparency, leading to blindness. Current diagnosis for LSCD is limited because reliable conjunctiva-specific biomarkers are lacking. This study sought to address this shortcoming through the serendipitous discovery of insulin-like growth factor binding protein (IGFBP)-7. METHODS: IGFBP-7 expression was determined in normal (n=83) and conjunctivalized (n=52) mouse corneas with experimentally-induced LSCD, and in cadaveric normal human corneas (n=7) and human pterygia (n=15); a disease characterized by the invasion of a conjunctivalized, fibrovascular pannus. Clinical assessments including slit-lamp microscopy, fluorescein staining and impression cytology, and biochemical, molecular and immunological assays were also conducted. RESULTS: Mass spectrometry of conditioned media from mouse limbal explant-derived cells revealed the presence of IGFBP-7. This factor was expressed in normal limbal and conjunctival epithelium and conjunctivalized corneas from mice with LSCD, and in human pterygium epithelium but not in normal mouse or human corneal epithelium. Four weeks after inducing LSCD, IGFBP-7 staining was increased by 2.9-fold in mouse corneas compared to steady-state, and by 1.6-fold in impression cytology specimens derived from the same mice. Notably, IGFBP-7 was detected approximately 2-weeks earlier than Muc5AC. CONCLUSIONS: This study provides novel insights into the specificity of IGFBP-7 for the mammalian conjunctival epithelium in health and disease. A point-of-care test for IGFBP-7 could be developed to assist clinicians in early diagnosis, and in monitoring disease progression, severity and therapeutic outcomes in patients with LSCD.

Expression of IGFBP-rP1 in ovarian and breast cancers in association with diabetes mellitus status.

INTRODUCTION: Insulin-like growth factor binding protein-related protein 1 (IGFBP-rP1) is an important component of the IGF system that regulates insulin resistance-related to tumour development. The aim of this study is to investigate the expression of IGFBP-rP1 among female cancer patients who are known or not known to have Type 2 Diabetes Mellitus (T2DM). MATERIALS AND METHODS: Using a cross-sectional design, cases of ovarian and breast cancer with clinical status of T2DM were selected over a 10-year period in Hospital Universiti Sains Malaysia. Immunohistochemical staining for IGFBP-rP1 was performed on paraffin-embedded tissues and the results were correlated with the patient's demographic and clinicopathological data. RESULTS: A total of 152 breast cancer patients were recruited into the current study with 33.5% (51/152) patients were positive T2DM. Most of the breast cancer patients with T2DM were IGFBP-rP1-negative (66.7%, 34/51). The IGFBP-rP1 expression was significantly difference between breast cancer subjects with and without T2DM (p<0.001). There was no significant association of IGFBP-rP1 expression with data on the demographic and clinicopathological profiles of patients with breast cancer. Meanwhile, positive IGFBP-rP1 expression was evident in 44 out of 108 (40.74%) ovarian cancer cases. Among these cases, 36 were T2DM. In contrast to breast cancer cases, IGFBP-rP1 was mostly expressed among ovarian cancer patients with T2DM (66.7%, 24/36, p < 0.001). However, the -positive expression was not significantly associated with any sociodemographic and clinicopathological features of ovarian cancers. CONCLUSIONS: Majority of breast cancer patients with T2DM did not express IGFBP-rP1. In contrast, majority of the ovarian cancer patients with T2DM expressed IGFBP-rP1.

IGFBP-7 inhibits the differentiation of oligodendrocyte precursor cells via regulation of Wnt/ß-Catenin signaling.

Oligodendrocytes (OLs) are glial cells that form myelin sheaths in the central nervous system. Myelin sheath plays important role in nervous system and loss of it in neurodegenerative diseases can lead to impairment of movement. Understanding the signals and factors that regulate OL differentiation can help to address novel strategies for improving myelin repair in neurodegenerative diseases. The aim of this study was to investigate the role of insulin-like growth factor-binding proteins 7 (IGFBP-7) in differentiating OL precursor cells (OPCs). It was found that oligodendrocyte precursors undergoing differentiation were accompanied by selective expression of IGFBP-7. In addition, knockdown of IGFBP-7 promoted differentiation of oligodendrocytes and increased formation of myelin in cultured cells. In contrast, excessive expression of IGFBP-7 inhibited differentiation of oligodendrocytes. Furthermore, overexpression of IGFBP-7 in oligodendrocyte precursor cells increased transcription of Wnt target genes and promoted ß-Catenin nuclear translocation. These findings suggest that IGFBP-7 negatively regulates differentiation of oligodendrocyte precursor cells via regulation of Wnt/ß-Catenin signaling.

Variation in branchial expression among insulin-like growth-factor binding proteins (igfbps) during Atlantic salmon smoltification and seawater exposure.

BACKGROUND: In preparation for migration from freshwater to marine habitats, Atlantic salmon (Salmo salar L.) undergo smoltification, a transformation that includes the acquisition of hyposmoregulatory capacity. The growth hormone (Gh)/insulin-like growth-factor (Igf) axis promotes the development of branchial ionoregulatory functions that underlie ion secretion. Igfs interact with a suite of Igf binding proteins (Igfbps) that modulate hormone activity. In Atlantic salmon smolts, igfbp4,-5a,-5b1,-5b2,-6b1 and-6b2 transcripts are highly expressed in gill. We measured mRNA levels of branchial and hepatic igfbps during smoltification (March, April, and May), desmoltification (July) and following seawater (SW) exposure in March and May. We also characterized parallel changes in a broad suite of osmoregulatory (branchial Na+/K+-ATPase (Nka) activity, Na + /K + /2Cl - cotransporter 1 (nkcc1) and cystic fibrosis transmembrane regulator 1 (cftr1) transcription) and endocrine (plasma Gh and Igf1) parameters. RESULTS: Indicative of smoltification, we observed increased branchial Nka activity, nkcc1 and cftr1 transcription in May. Branchial igfbp6b1 and -6b2 expression increased coincidentally with smoltification. Following a SW challenge in March, igfbp6b1 showed increased expression while igfbp6b2 exhibited diminished expression. igfbp5a,-5b1 and-5b2 mRNA levels did not change during smolting, but each had lower levels following a SW exposure in March. CONCLUSIONS: Salmonids express an especially large suite of igfbps. Our data suggest that dynamic expression of particular igfbps accompanies smoltification and SW challenges; thus, transcriptional control of igfbps may provide a mechanism for the local modulation of Igf activity in salmon gill.

Inhibition of Cervical Cancer by Promoting IGFBP7 Expression Using Ellagic Acid from Pomegranate Peel.

BACKGROUND The aim of this study was to explore the mechanism by which cervical cancer is inhibited by promoting IGFBP7 expression using ellagic acid from pomegranate peel extract. MATERIAL AND METHODS HeLa cells were divided into 6 groups: control group (NC), blank control group (BL), and IGFBP7 overexpression group (IGFBP7), and 2.5 uM, 5. 0 uM, and 10.0 uM ellagic acid-treated groups. The cell proliferation ability was detected and the degree of invasion in the 6 groups was measured by Transwell assay. The expression levels of IGFBP7 and AKT/mTOR in the 6 groups of cells were detected by RT-PCR technique. RESULTS Compared with NC and BL groups, The IGFBP7 gene expressions of the IGFPB7 and ellagic acid-treated groups were significantly increased (P<0.05). There was a dose-effect dependence in the ellagic acid-treated groups. The invasion ability of the IGFBP7 group and ellagic acid-treated groups was significantly lower than that of NC and BL groups in HeLa cells (P<0.05). The apoptosis rate of the IGFBP7 group and ellagic acid-treated groups was significantly higher than that of the NC and BL groups in HeLa cells (P<0.05). AKT and mTOR mRNA and protein expressions of the IGFBP7 group and ellagic acid-treated groups were significantly lower than that of the NC and BL groups (P<0.05). There was a dose-effect dependence in the ellagic acid-treated groups. CONCLUSIONS The ellagic acid in pomegranate peel extract can inhibit the AKT/mTOR signaling pathway by enhancing the expression level of IGFBP7, which can inhibit the HeLa cells in cervical cancer.

Molecular characterization and expression patterns of insulin-like growth factor-binding protein genes in postnatal Nanjiang brown goats.

Insulin-like growth factor-binding proteins (IGFBPs) play a key role in modulating insulin-like growth factors (IGFs), and are considered candidate genes for growth traits in livestock. In this study, we identified the complete coding sequences of IGFBP-1 to -6 in the Nanjiang brown goat, and assessed gene tissue expression patterns by quantitative polymerase chain reaction. Expression of mRNA for the six gene targets was detectable in liver, heart, and longissimus dorsi (LD) muscle. Expression levels of IGFBP-1, -2 and -5 mRNA were higher in liver than in heart and LD muscle (P < 0.01), while IGFBP-6 expression was highest in LD muscle, and IGFBP-3 and -4 were predominantly expressed in LD muscle and liver. Higher IGFBP-2, -3, -4, and -6 mRNA levels were observed in LD, compared to triceps brachii muscle (P < 0.01). Additionally, the target genes had different temporal expression profiles during postnatal development. Histological assessment of muscle sections revealed a constant increase in muscle fiber diameter with aging. These results suggest that IGFBPs may be important for liver and skeletal muscle development, and may contribute to the biological function of these tissues in goats.

Mammalian cell transient expression, non-affinity purification, and characterization of human recombinant IGFBP7, an IGF-1 targeting therapeutic protein.

Targeted inhibiting insulin-like growth factor 1 is an effective approach for cancer therapy. Insulin-like growth factor binding protein 7 (IGFBP7) is considered as a potential therapeutic protein. However, producing high quality of such non-IgG proteins in mammalian cells is still a challenge in biopharmaceutical development. Here, we report a rapid production process by using transient gene transfection in HEK 293E cells. A set of constructs combining several expression promoters, leader sequences, and 5' un-translated regions were generated and optimized, from which the best vector with expression level at ~50mg/L was selected for production at 2L cell culture scale. Comparison study in downstream purification methods led to development of a scalable, non-affinity chromatography strategy through Super Q, Fast Flow Q, and Heparin columns. The product was characterized in purity (99%), isoelectric point, molecule weight, glycosylation, and stability by using SEC-HPLC, SDS-PAGE, isoelectric focusing and mass spectrometry. The highly purified product shows IGF-1 binding activity and inhibits IGF-1-induced cell proliferation. This process not only provides a remarkable high expression at ~50mg/L and pure glycosylated mammalian rhIGFBP7, also highlights that transient gene expression technology is practical to be used for production and early development of recombinant non-IgG therapeutic proteins.

Low levels of IGFBP7 expression in high-grade serous ovarian carcinoma is associated with patient outcome.

BACKGROUND: Insulin-like growth factor binding protein 7 (IGFBP7) has been suggested to act as a tumour suppressor gene in various human cancers, yet its role in epithelial ovarian cancer (EOC) has not yet been investigated. We previously observed that IGFBP7 was one of several genes found significantly upregulated in an EOC cell line model rendered non-tumourigenic as consequence of genetic manipulation. The aim of the present study was to investigate the role of IGFBP7 in high-grade serous ovarian carcinomas (HGSC), the most common type of EOC. METHODS: We analysed IGFBP7 gene expression in 11 normal ovarian surface epithelial cells (NOSE), 79 high-grade serous ovarian carcinomas (HGSC), and seven EOC cell lines using a custom gene expression array platform. IGFBP7 mRNA expression profiles were also extracted from publicly available databases. Protein expression was assessed by immunohistochemistry of 175 HGSC and 10 normal fallopian tube samples using tissue microarray and related to disease outcome. We used EOC cells to investigate possible mechanisms of gene inactivation and describe various in vitro growth effects of exposing EOC cell lines to human recombinant IGFBP7 protein and conditioned media. RESULTS: All HGSCs exhibited IGFBP7 expression levels that were significantly (p = 0.001) lower than the mean of the expression value of NOSE samples and that of a whole ovary sample. IGFBP7 gene and protein expression were lower in tumourigenic EOC cell lines relative to a non-tumourigenic EOC cell line. None of the EOC cell lines harboured a somatic mutation in IGFBP7, although loss of heterozygosity (LOH) of the IGFBP7 locus and epigenetic methylation silencing of the IGFBP7 promoter was observed in two of the cell lines exhibiting loss of gene/protein expression. In vitro functional assays revealed an alteration of the EOC cell migration capacity. Protein expression analysis of HGSC samples revealed that the large majority of tumour cores (72.6%) showed low or absence of IGFBP7 staining and revealed a significant correlation between IGFBP7 protein expression and a prolonged overall survival (p = 0.044). CONCLUSION: The low levels of IGFPB7 in HGSC relative to normal tissues, and association with survival are consistent with a purported role in tumour suppressor pathways.

Insulin like growth factor binding protein 7 (IGFBP7) expression is linked to poor prognosis but may protect from bone disease in multiple myeloma.

BACKGROUND: Insulin like growth factor binding protein 7 (IGFBP7) is a secreted protein binding insulin like growth factor 1 (IGF-1), insulin, vascular endothelial growth factor A (VEGFA), and activin A. It antagonizes bone morphogenetic proteins and is involved in the tumour propagation of solid as well as haematological malignancies. Its role in multiple myeloma (MM) is not defined so far. We therefore aim here to investigate its prognostic and pathophysiological role in MM. METHODS: The clinical significance of IGFBP7 gene expression was investigated by gene expression profiling in two independent cohorts (n = 948) of newly-diagnosed MM patients. Methylation of the IGFBP7 promoter was analysed by pyrosequencing and treatment of MM cell lines with 5-aza-2-deoxycytidine. The impact of IGFBP7 on MM cells was studied by CCK-8 assay, BrdU assay and flow cytometry, respectively. IGFBP7 expression in bone marrow stromal cells (BMSCs) was studied by quantitative RT-PCR. For osteoblast development, immortalized and primary human BMSCs were cultured in osteogenic differentiation medium for 7-14 days in the presence of recombinant human IGFBP7 and/or activin A. RESULTS: Median IGFBP7 expression is significantly lower in CD138-purified plasma cells from individuals with MGUS and MM, compared to normal bone marrow plasma cells. IGFBP7 gene expression in MM cells is regulated by methylation, shown by pyrosequencing and exposure to demethylating agents (5-aza-2-deoxycytidine). High expression of IGFBP7 in MM cells is associated with adverse survival in two independent cohorts of 247 and 701 newly-diagnosed MM patients treated with high-dose therapy and autologous stem cell transplantation. IGFBP7 is associated with prognostically adverse chromosomal aberrations (t(4;14) and gain of 1q21), MMSET expression, and higher myeloma cell proliferation. In vitro, IGFBP7 overcomes activin A induced osteoblast suppression and promotes osteogenesis. MM cells downregulate IGFBP7 in stromal cells, possibly contributing to the osteoblast suppression found in MM. Conversely, higher IGFBP7 expression is associated with a lower probability of myeloma bone disease. CONCLUSIONS: Our data indicate that IGFBP7 expression is a marker for a specific methylation pattern in myeloma, linked to translocation t(4;14) associated MMSET expression, showing clinical features of adverse prognosis with absence of myeloma bone disease.

IGFBP-rP1 suppresses epithelial-mesenchymal transition and metastasis in colorectal cancer.

Epithelial-mesenchymal transition (EMT) was initially recognized during organogenesis and has recently been reported to be involved in promoting cancer invasion and metastasis. Cooperation of transforming growth factor-ß (TGF-ß) and other signaling pathways, such as Ras and Wnt, is essential to inducing EMT, but the molecular mechanisms remain to be fully determined. Here, we reported that insulin-like growth factor binding protein-related protein 1 (IGFBP-rP1), a potential tumor suppressor, controls EMT in colorectal cancer progression. We revealed the inhibitory role of IGFBP-rP1 through analyses of clinical colorectal cancer samples and various EMT and metastasis models in vitro and in vivo. Moreover, we demonstrated that IGFBP-rP1 suppresses EMT and tumor metastasis by repressing TGF-ß-mediated EMT through the Smad signaling cascade. These data establish that IGFBP-rP1 functions as a suppressor of EMT and metastasis in colorectal cancer.

Production of functional human insulin-like growth factor binding proteins (IGFBPs) using recombinant expression in HEK293 cells.

Insulin-like growth factor binding proteins (IGFBPs) display many functions in humans including regulation of the insulin-like growth factor (IGF) signaling pathway. The various roles of human IGFBPs make them attractive protein candidates in drug discovery. Structural and functional knowledge on human proteins with therapeutic relevance is needed to design and process the next generation of protein therapeutics. In order to conduct structural and functional investigations large quantities of recombinant proteins are needed. However, finding a suitable recombinant production system for proteins such as full-length human IGFBPs, still remains a challenge. Here we present a mammalian HEK293 expression method suitable for over-expression of secretory full-length human IGFBP-1 to -7. Protein purification of full-length human IGFBP-1, -2, -3 and -5 was conducted using a two-step chromatography procedure and the final protein yields were between 1 and 12mg protein per liter culture media. The recombinant IGFBPs contained PTMs and exhibited high-affinity interactions with their natural ligands IGF-1 and IGF-2.

Identification of coexistence of DNA methylation and H3K27me3 specifically in cancer cells as a promising target for epigenetic therapy.

Alterations of epigenetic modifications are promising targets for cancer therapy, and several epigenetic drugs are now being clinically utilized. At the same time, individual epigenetic modifications have physiological functions in normal cells, and cancer cell specificity is considered difficult to achieve using a drug against a single epigenetic modification. To overcome this limitation, a combination of epigenetic modifications specifically or preferentially present in cancer cells is a candidate target. In this study, we aimed to demonstrate (i) the presence of a cancer cell-specific combination of epigenetic modifications by focusing on DNA methylation and trimethylation of histone H3 lysine 27 (H3K27me3) and (ii) the therapeutic efficacy of a combination of DNA demethylation and EZH2 inhibition. Analyses of DNA methylation and H3K27me3 in human colon, breast and prostate cancer cell lines revealed that 24.7±4.1% of DNA methylated genes had both DNA methylation and H3K27me3 (dual modification) in cancer cells, while it was 11.8±7.1% in normal cells. Combined treatment with a DNA demethylating agent, 5-aza-2'-deoxycytidine (5-aza-dC) and an EZH2 inhibitor, GSK126, induced marked re-expression of genes with the dual modification, including known tumor-suppressor genes such as IGFBP7 and SFRP1, and showed an additive inhibitory effect on growth of cancer cells in vitro. Finally, an in vivo combined treatment with 5-aza-dC and GSK126 inhibited growth of xenograft tumors more efficiently than a single treatment with 5-aza-dC. These results showed that the dual modification exists specifically in cancer cells and is a promising target for cancer cell-specific epigenetic therapy.

IGFBP7, a novel tumor stroma marker, with growth-promoting effects in colon cancer through a paracrine tumor-stroma interaction.

The activated tumor stroma participates in many processes that control tumorigenesis, including tumor cell growth, invasion and metastasis. Cancer-associated fibroblasts (CAFs) represent the major cellular component of the stroma and are the main source for connective tissue components of the extracellular matrix and various classes of proteolytic enzymes. The signaling pathways involved in the interactions between tumor and stromal cells and the molecular characteristics that distinguish normal 'resting' fibroblasts from cancer-associated or '-activated' fibroblasts remain poorly defined. Recent studies emphasized the prognostic and therapeutic significance of CAF-related molecular signatures and a number of those genes have been shown to serve as putative therapeutic targets. We have used immuno-laser capture microdissection and whole-genome Affymetrix GeneChip analysis to obtain transcriptional signatures from the activated tumor stroma of colon carcinomas that were compared with normal resting colonic fibroblasts. Several members of the Wnt-signaling pathway and gene sets related to hypoxia, epithelial-to-mesenchymal transition (EMT) and transforming growth factor-ß (TGFß) pathway activation were induced in CAFs. The putative TGFß-target IGFBP7 was identified as a tumor stroma marker of epithelial cancers and as a tumor antigen in mesenchyme-derived sarcomas. We show here that in contrast to its tumor-suppressor function in epithelial cells, IGFPB7 can promote anchorage-independent growth in malignant mesenchymal cells and in epithelial cells with an EMT phenotype when IGFBP7 is expressed by the tumor cells themselves and can induce colony formation in colon cancer cells co-cultured with IGFBP7-expressing CAFs by a paracrine tumor-stroma interaction.

IGFBP7 induces apoptosis of acute myeloid leukemia cells and synergizes with chemotherapy in suppression of leukemia cell survival.

Despite high remission rates after chemotherapy, only 30-40% of acute myeloid leukemia (AML) patients survive 5 years after diagnosis. This extremely poor prognosis of AML is mainly caused by treatment failure due to chemotherapy resistance. Chemotherapy resistance can be caused by various features including activation of alternative signaling pathways, evasion of cell death or activation of receptor tyrosine kinases such as the insulin growth factor-1 receptor (IGF-1R). Here we have studied the role of the insulin-like growth factor-binding protein-7 (IGFBP7), a tumor suppressor and part of the IGF-1R axis, in AML. We report that IGFBP7 sensitizes AML cells to chemotherapy-induced cell death. Moreover, overexpression of IGFBP7 as well as addition of recombinant human IGFBP7 is able to reduce the survival of AML cells by the induction of a G2 cell cycle arrest and apoptosis. This effect is mainly independent from IGF-1R activation, activated Akt and activated Erk. Importantly, AML patients with high IGFBP7 expression have a better outcome than patients with low IGFBP7 expression, indicating a positive role for IGFBP7 in treatment and outcome of AML. Together, this suggests that the combination of IGFBP7 and chemotherapy might potentially overcome conventional AML drug resistance and thus might improve AML patient survival.

IGFBP7 is associated with poor prognosis in oesophageal adenocarcinoma and is regulated by promoter DNA methylation.

BACKGROUND: We examined whether silencing of IGFBP7 was associated with survival in patients with oesophageal adenocarcinoma. METHODS: Protein expression of IGFBP7 was determined using immunohistochemistry in a tissue microarray representing tumours from 65 patients with oesophageal adenocarcinoma who had not had neoadjuvant therapy. DNA methylation of the IGFBP7 promoter was determined with the melt curve analysis in cell lines and patient tissues. RESULTS: Expression of IGFBP7 was observed in the oesophageal adenocarcinoma of 34 out of 65 (52%) patients and was associated with significantly reduced median (11 vs 92 months) and 5-year survival (25% vs 52%). Multivariate analysis identified expression as an independent prognostic indicator for survival (hazard ratio=3.24, 95% confidence interval=1.58-6.67, P-value=0.0014). Hypermethylation of IGFBP7 was associated with silencing of gene expression in cell lines and patient tissues (P-value=0.0225). Methylation was observed in the squamous mucosa of 2 out of 15 (13%) patients with Barrett's oesophagus and 3 out of 17 (18%) with oesophageal adenocarcinoma. Methylation was observed in 14 out of 18 (78%) of biopsies of Barrett's mucosa and 23 out of 34 (68%) patients with oesophageal adenocarcinoma. CONCLUSION: Reduced IGFBP7 protein expression was associated with longer survival in patients with oesophageal adenocarcinoma. Methylation of the IGFBP7 promoter was associated with silencing of gene expression and was frequent in Barrett's oesophagus and oesophageal adenocarcinoma.

Expression of insulin-like growth factor binding proteins during mouse cochlear development.

BACKGROUND: Insulin-like growth factor (IGF) signaling plays important roles in growth and cellular differentiation in the cochlear sensory epithelium. However, the roles of IGF binding proteins (IGFBPs), a family of IGF modulators, remain to be elucidated in this system. To begin to examine the role of IGFBPs, we used reverse transcription polymerase chain reaction (RT-PCR) and in situ hybridization to determine the temporal and spatial patterns of expression for Igfbps within the developing mouse cochlea. RESULTS: RT-PCR analysis indicates that Igfpb2-5 are expressed in the cochlea between embryonic day (E) 13.5 and postnatal day (P) 0. In addition, the expression of each Igfbp significantly increased between E13.5 and P0. In situ hybridization indicates that Igfbp2, 3, 4, and 5 have distinct and complementary expression patterns in the developing cochlea. Moreover, expression patterns of Igfbp3 and 5 demonstrate contrasting gradients along the basal-to-apical axis of the cochlea. CONCLUSIONS: Igfbp2-5 are expressed in distinct and complementary patterns during cochlear development. These data suggest that IGFBPs may act to precisely regulate activation of IGF signaling in the developing cochlea in a cell type-specific manner, contributing to cellular patterning and differentiation in the cochlea.

Expression of specific IGFBPs is associated with those of the proliferating and differentiating markers in regenerating rat plantaris muscle.

To examine the relationship between specific insulin-like growth factor (IGF)-binding proteins (IGFBPs) and myogenesis during muscle regeneration in vivo, we measured mRNA expression of IGFBPs and myogenic markers in rat plantaris muscle after bupivacaine administration. IGF-I Ea, MGF, IGFBPs and myogenic marker mRNAs were analyzed 12, 24, 48 and 72 h after bupivacaine injection. IGFBP-1, -2, -3 and -4 proteins were immunostained after the treatment. MGF, IGF-I Ea and IGFBP-4 mRNAs started to increase 12 or 24 h after bupivacaine injection and increased further after that. IGFBP-1, -2, -3 and -4 proteins were strongly stained in the immature muscle fiber nuclei and the extracellular matrix after bupivacaine injection. PCNA, MyoD, IGFBP-2 and IGFBP-3 mRNAs increased at 12 or 24 h and did not show further increases after that. Myogenin, p21, IGFBP-1 and IGFBP-5 mRNAs sharply increased after 72 h. These results suggest that specific IGFBPs are individually expressed and differently associated with the expression of myogenic markers in regenerating muscles.

Human macrophage ATP7A is localized in the trans-Golgi apparatus, controls intracellular copper levels, and mediates macrophage responses to dermal wounds.

The copper transporter ATP7A has attracted significant attention since the discovery of its gene mutation leading to human Menkes disease. We previously reported that ATP7A is highly expressed in the human vasculature and identified a novel vascular function of ATP7A in modulation of the expression and activity of extracellular superoxide dismutase. We recently identified that ATP7A expression in THP-1 cells (a monocyte/macrophage model cell line) plays a role in the oxidation of low density lipoproteins, indicating that it is necessary to further investigate its expression and function in monocytes/macrophages. In the current study, we demonstrated the protein and mRNA expression of ATP7A in human peripheral blood mononuclear cell (PBMC)-derived macrophages and alveolar macrophages. ATP7A was strongly co-localized with the trans-Golgi apparatus in PBMC-derived macrophages. Intracellular copper, detected by synchrotron X-ray fluorescence microscopy, was found to be distributed to the nucleus and cytoplasm in human THP-1 cells. To confirm the role of endogenous ATP7A in macrophage copper homeostasis, we performed inductively coupled plasma mass spectrometry in murine peritoneal macrophages, which showed markedly increased intracellular copper levels in macrophages isolated from ATP7A-deficient mice versus control mice. Moreover, the role of ATP7A in regulating macrophage responses to dermal wounds was studied by introduction of control and ATP7A-downregulated THP-1 cells into dermal wounds of nude mice. Infiltration of THP-1 cells into the wounded area (detected by expression of human macrophage markers MAC2 and CD68) was reduced in response to downregulation of ATP7A, hinting decreased macrophage accumulation subsequent to dermal wounds. In summary, alongside our previous studies, these findings indicate that human macrophage ATP7A is localized in the trans-Golgi apparatus, regulates intracellular copper levels, and mediates macrophage responses to a dermal wound.