Upregulation of IGF-IIRα intensifies doxorubicin-induced cardiac damage.

Cardiotoxicity by doxorubicin hampers its therapeutic potential as an anticancer drug, but mechanisms leading to cardiotoxicity remain contentious. Through this study, the functional contribution of insulin-like growth factor receptor type II α (IGF-IIRα) which is a novel stress-inducible protein was explored in doxorubicin-induced cardiac stress. Employing both in vitro H9c2 cells and in vivo transgenic rat models (SD-TG [IGF-IIRα]) overexpressing IGF-IIRα specifically in heart, we found that IGF-IIRα leads to cardiac structural abnormalities and functional perturbations that were severely aggravated by doxorubicin-induced cardiac stress. Overexpression of IGF-IIRα leads to cumulative elevation of stress associated cardiac hypertrophy and apoptosis factors. There was a significant reduction of survival associated proteins p-Akt and estrogen receptor ß/α, and abnormal elevation of cardiac hypertrophy markers such as atrial natriuretic peptide, cardiac troponin-I, and apoptosis-inducing agents such as p53, Bax, and cytochrome C, respectively. IGF-IIRα also altered the expressions of AT1R, ERK1/2, and p38 proteins. Besides, IGF-IIRα also increased the reactive oxygen species production in H9c2 cells which were markedly aggravated by doxorubicin treatment. Together, we showed that IGF-IIRα is a novel stress-induced protein that perturbed cardiac homeostasis and cumulatively exacerbated the doxorubicin-induced cardiac injury that perturbed heart functions and ensuing cardiomyopathy.

Insulin-Like Growth Factor 2 Receptor Expression Is Promoted by Human Herpesvirus 8-Encoded Interleukin-6 and Contributes to Viral Latency and Productive Replication.

Human herpesvirus 8 (HHV-8) viral interleukin-6 (vIL-6) localizes largely to the endoplasmic reticulum (ER) and here associates functionally with both the gp130 signal transducer and the novel ER membrane protein vitamin K epoxide reductase complex subunit 1 variant-2 (VKORC1v2). The latter interaction contributes to the viability of latently infected primary effusion lymphoma (PEL) cells and to HHV-8 productive replication, in part via promotion of ER-associated degradation (ERAD) of nascent pro-cathepsin D (pCatD) and consequent suppression of lysosome-localized proapoptotic mature CatD. Here we report that VKORC1v2 associates with insulin-like growth factor 2 receptor (IGF2R), also known as cation-independent mannose-6-phosphate receptor, which is involved in trafficking of mannose-6-phosphate-conjugated glycoproteins to lysosomes. VKORC1v2 effected reduced IGF2R expression in a manner dependent on VKORC1v2-IGF2R interaction, while vIL-6, which could inhibit VKORC1v2-IGF2R interaction, effected increased expression of IGF2R. These effects were independent of changes in IGF2R mRNA levels, indicating likely posttranslational mechanisms. In kinetic analyses involving labeling of either newly synthesized or preexisting IGF2R, vIL-6 promoted accumulation of the former while having no detectable effect on the latter. Furthermore, vIL-6 led to decreased K48-linked ubiquitination of IGF2R and suppression of ERAD proteins effected increased IGF2R expression and loss of IGF2R regulation by vIL-6. Depletion-based experiments identified IGF2R as a promoter of PEL cell viability and virus yields from lytically reactivated cultures. Our findings identify ER-transiting nascent IGF2R as an interaction partner of VKORC1v2 and target of vIL-6 regulation and IGF2R as a positive contributor to HHV-8 biology, thereby extending understanding of the mechanisms of VKORC1v2-associated vIL-6 function.IMPORTANCE HHV-8 vIL-6 promotes productive replication in the context of reactivated lytic replication in primary effusion lymphoma (PEL) and endothelial cells and sustains latently infected PEL cell viability. Viral IL-6 is also considered to contribute significantly to HHV-8-associated pathogenesis, since vIL-6 can promote cell proliferation, cell survival, and angiogenesis that are characteristic of HHV-8-associated Kaposi's sarcoma, PEL and multicentric Castleman's disease (MCD), in addition to proinflammatory activities observed in MCD-like "Kaposi's sarcoma-associated herpesvirus-induced cytokine syndrome." We show in the present study that vIL-6 can promote productive replication and latent PEL cell viability through upregulation of the mannose-6-phosphate- and peptide hormone-interacting receptor IGF2R, which is a positive factor in HHV-8 biology via these activities. VKORC1v2-enhanced ER-associated degradation of IGF2R and vIL-6 promotion of IGF2R expression through prevention of its interaction with VKORC1v2 and consequent rescue from degradation represent newly recognized activities of VKOCR1v2 and vIL-6.

Selective induction of apoptosis in MCF7 cancer-cell by targeted liposomes functionalised with mannose-6-phosphate.

Liposomes are versatile platforms to carry anticancer drugs in targeted drug delivery; they can be surface modified by different strategies and, when coupled with targeting ligands, are able to increase cellular internalisation and organelle-specific drug delivery. An interesting strategy of antitumoral therapy could involve the use of lysosomotropic ligand-targeted liposomes loaded with molecules, which can induce lysosomal membrane permeabilization (LMP), leakage of cathepsins into the cytoplasm and subsequent apoptosis. We have previously demonstrated the ability of liposomes functionalised with a mannose-6-phosphate to reach lysosomes; in this research we compare the behaviour of M6P-modified and non-functionalised liposomes in MCF7 tumour cell and in HDF normal cells. With this aim, we first demonstrated by Western blotting the overexpression of mannose-6-phosphate/insulin-like growth factor (M6P/IGF-II) receptor in MCF7. Then, we prepared calcein-loaded liposomes and we revealed the increased uptake of M6P-functionalised liposomes in MCF7 cells respect to HDF cells by flow cytometry analysis. Finally, we loaded functionalised and not functionalised liposomes with N-hexanoyl-d-erythro-sphingosine (C6Cer), able to initiate LMP-induced apoptosis; after having studied the stability of both vesicles in the presence of serum by Dynamic Light Scattering and Spectrophotometric turbidity measurements, we showed that ceramide-loaded M6P-liposomes significantly increased apoptosis in MCF7 with respect to HDF cells.

Cloning, Expression, and Purification of the Glycosylated Transmembrane Protein, Cation-Dependent Mannose 6-Phosphate Receptor, from Sf9 Cells Using the Baculovirus System.

The cation-dependent mannose 6-phosphate receptor (CD-MPR) is a single-pass type I membrane protein. This protein functions to transport lysosomal enzymes displaying phosphomannosyl residues from the Golgi complex and the cell surface to the lysosome. This glycosylated protein contains three disulfide bridges in its 159-residue extracytoplasmic domain. One of the problems with studying eukaryotic membrane proteins is isolating sufficient quantities. Structural studies typically require several hundred milligrams of highly purified protein. Here we present a method to isolate milligram quantities of CD-MPR/Asn81 suitable for structural studies.

Vitrification, not cryoprotectant exposure, alters the expression of developmentally important genes in in vitro produced porcine blastocysts.

The vitrification of embryos is common practice in advanced livestock breeding programs and in human fertility clinics. Recent studies have revealed that vitrification results in aberrant expression of a number of stress related genes. However, few studies have examined the effect that vitrification has on developmentally important genes, and none have been conducted in porcine embryos. The aim of this study was to determine the effects that different vitrification procedures and cryoprotectant combinations have on the expression of imprinted genes in in vitro produced (IVP) porcine blastocysts. The transcript levels of insulin-like growth factor 2 (IGF2) were lower in all groups of vitrified blastocysts compared to that in non-vitrified control blastocysts (P < 0.05). Expression levels of IGF2 and IGF2 receptor (IGF2R) in blastocysts that had been exposed to cryoprotectants without being vitrified were similar to that in non-vitrified control blastocysts (P > 0.05). Furthermore, blastocysts vitrified using ethylene glycol and propanediol combined, and those vitrified in a closed device, had IGF2R transcript levels similar to that in non-vitrified control blastocysts (P > 0.05). In conclusion, vitrification, but not exposure to cryoprotectants, caused aberrant expression of the imprinted genes IGF2 and IGF2R. Vitrification protocols that incorporated propanediol or a closed device were found to be least disruptive of gene expression in IVP porcine blastocysts.

Inhibition of HSF2 SUMOylation via MEL18 upregulates IGF-IIR and leads to hypertension-induced cardiac hypertrophy.

Cardiac hypertrophy is a major characteristic of early-stage hypertension-related heart failure. We have found that the insulin-like growth factor receptor II (IGF-IIR) signaling was critical for hypertensive angiotensin II-induced cardiomyocyte hypertrophy and apoptosis. Moreover, this IGF-IIR signaling was elegantly modulated by the heat shock transcription factors (HSFs) during heart failure. However, the detailed mechanism by which HSFs regulates IGF-IIR during hypertension-induced cardiac hypertrophy remains elusive. In this study, we found that heat shock transcription factor 2 (HSF2) activated IGF-IIR to induce cardiac hypertrophy for hypertension-induced heart failure. The transcriptional activity of HSF2 appeared to be primarily mediated by SUMOylation via conjugation with small ubiquitin-like modifier-1 (SUMO-1). The SUMOylation of HSF2 was severely attenuated by MEL18 (also known as polycomb group ring finger 2 or PCGF2) in the heart of spontaneously hypertensive rats (SHR). Inhibition of HSF2 SUMOylation severely induced cardiac hypertrophy via IGF-IIR-mediated signaling in hypertensive rats. Angiotensin II receptor type I blocker (ARB) treatment in spontaneously hypertensive rats restored HSF2 SUMOylation and alleviated the cardiac defects. Thus, our study uncovered a novel MEL18-SUMO-1-HSF2-IGF-IIR pathway in the heart that profoundly influences cardiac hypertrophy for hypertension-induced heart failure.

microRNA-352 regulates collateral vessel growth induced by elevated fluid shear stress in the rat hind limb.

Although collateral vessel growth is distinctly enhanced by elevated fluid shear stress (FSS), the underlying regulatory mechanism of this process remains incompletely understood. Recent studies have shown that microRNAs (miRNAs) play a pivotal role in vascular development, homeostasis and a variety of diseases. Therefore, this study was designed to identify miRNAs involved in elevated FSS-induced collateral vessel growth in rat hind limbs. A side-to-side arteriovenous (AV) shunt was created between the distal stump of one of the bilaterally occluded femoral arteries and the accompanying vein. The miRNA array profile showed 94 differentially expressed miRNAs in FSS-stressed collaterals including miRNA-352 which was down-regulated. Infusion of antagomir-352 increased the number and proliferation of collateral vessels and promoted collateral flow restoration in a model of rat hind limb ligation. In cell culture studies, the miR-352 inhibitor increased endothelial proliferation, migration and tube formation. In addition, antagomir-352 up-regulated the expression of insulin-like growth factor II receptor (IGF2R), which may play a part in the complex pathway leading to arterial growth. We conclude that enhanced collateral vessel growth is controlled by miRNAs, among which miR-352 is a novel candidate that negatively regulates arteriogenesis, meriting additional studies to unravel the pathways leading to improved collateral circulation.

The role of insulin-like growth factor I-II receptor on development of pleomorphic adenoma.

Pleomorphic adenoma is a slow-growing salivary gland tumor frequently arising from the parotid gland. In this study, we investigated the role of the insulin-like growth factor I-II receptor (IGFI-IIR) levels on the development of parotid gland pleomorphic adenomas. The study included 20 males and 20 females who had superficial parotidectomy with a histopathological diagnosis of pleomorphic adenoma in Firat University Otorhinolaryngology Clinic between 2000 and 2011. The ages of the patients ranged between 20 and 50 years. The control tissues were obtained unilaterally from the parotid glands of five female and five male cadavers during autopsy, and consisted of 0.5 × 0.5 cm sized normal parotid gland tissues. The expression of IGFI-IIR were measured in both tumor and tumor-free normal parotid tissue in the study group while only the normal parotid tissues were studied in the cadavers. Primary polyclonal antibodies against IGFI-IIR were used with "Streptavidin-Biotin Complex" method for immunohistochemical staining of both the study and the control groups' tissue sections. In this study, the IGFI-IIR levels were found significantly higher in the pleomorphic adenoma tissue (p < 0.05). In addition, IGFI-IIR expression was greater in normal parotid tissues of the study group when compared to the normal parotid tissues of the cadavers. However, the difference was not statistically significant (p > 0.017). Greater expression for IGFI-IIR in pleomorphic adenoma when compared to normal parotid tissues of the patients and the cadavers suggests that IGFI-II may be important factors in the development of pleomorphic adenoma.

Bacterial expression of the phosphodiester-binding site of the cation-independent mannose 6-phosphate receptor for crystallographic and NMR studies.

The cation-independent mannose 6-phosphate receptor (CI-MPR) is a multifunctional protein that interacts with diverse ligands and plays central roles in autophagy, development, and tumor suppression. By delivering newly synthesized phosphomannosyl-containing acid hydrolases from the Golgi to endosomal compartments, CI-MPR is an essential component in the generation of lysosomes that are critical for the maintenance of cellular homeostasis. The ability of CI-MPR to interact with ∼60 different acid hydrolases is facilitated by its large extracellular region, with four out of its 15 domains binding phosphomannosyl residues. Although the glycan specificity of CI-MPR has been elucidated, the molecular basis of carbohydrate binding has not been determined for two out of these four carbohydrate recognition domains (CRD). Here we report expression of CI-MPR's CRD located in domain 5 that preferentially binds phosphodiester-containing glycans. Domain 5 of CI-MPR was expressed in Escherichia coli BL21 (DE3) cells as a fusion protein containing an N-terminal histidine tag and the small ubiquitin-like modifier (SUMO) protein. The His6-SUMO-CRD construct was recovered from inclusion bodies, refolded in buffer to facilitate disulfide bond formation, and subjected to Ni-NTA affinity chromatography and size exclusion chromatography. Surface plasmon resonance analyses demonstrated that the purified protein was active and bound phosphorylated glycans. Characterization by NMR spectroscopy revealed high quality (1)H-(15)N HSQC spectra. Additionally, crystallization conditions were identified and a crystallographic data set of the CRD was collected to 1.8Šresolution. Together, these studies demonstrate the feasibility of producing CI-MPR's CRD suitable for three-dimensional structure determination by NMR spectroscopic and X-ray crystallographic approaches.

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.

Autophagy induced by conventional chemotherapy mediates tumor cell sensitivity to immunotherapy.

Autophagy attenuates the efficacy of conventional chemotherapy but its effects on immunotherapy have been little studied. Here, we report that chemotherapy renders tumor cells more susceptible to lysis by CTL in vivo. Moreover, bystander tumor cells that did not express antigen were killed by CTL. This effect was mediated by transient but dramatic upregulation of the mannose-6-phosphate receptor (MPR) on the tumor cell surface. Antitumor effects of combined treatment related to the kinetics of MPR upregulation and abrogation of this event abolished the combined effect of immunotherapy and chemotherapy. MPR accumulation on the tumor cell surface during chemotherapy was observed in different mouse tumor models and in patients with multiple myeloma. Notably, this effect was the result of redistribution of the receptor caused by chemotherapy-inducible autophagy. Together, our findings reveal one molecular mechanism through which the antitumor effects of conventional cancer chemotherapy and immunotherapy are realized.

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.

An in situ hybridization study of the insulin-like growth factor system in developing condylar cartilage of the fetal mouse mandible.

The objective of this study was to investigate the involvement of the insulin-like growth factor (IGF) system in the developing mandibular condylar cartilage and temporomandibular joint (TMJ). Fetal mice at embryonic day (E) 13.0-18.5 were used for in situ hybridization studies using [35S]-labeled RNA probes for IGF-I, IGF-II, IGF-I receptor (-IR), and IGF binding proteins (-BPs). At E13.0, IGF-I and IGF-II mRNA were expressed in the mesenchyme around the mandibular bone, but IGF-IR mRNA was not expressed within the bone. At E14.0, IGF-I and IGF-II mRNA were expressed in the outer layer of the condylar anlage, and IGF-IR mRNA was first detected within the condylar anlage, suggesting that the presence of IGF-IR mRNA in an IGF-rich environment triggers the initial formation of the condylar cartilage. IGFBP-4 mRNA was expressed in the anlagen of the articular disc and lower joint cavity from E15.0 to 18.5. When the upper joint cavity was formed at E18.5, IGFBP-4 mRNA expression was reduced in the fibrous mesenchymal tissue facing the upper joint cavity. Enhanced IGFBP-2 mRNA expression was first recognized in the anlagen of both the articular disc and lower joint cavity at E16.0 and continued expression in these tissues as well as in the fibrous mesenchymal tissue facing the upper joint cavity was observed at E18.5. IGFBP-5 mRNA was continuously expressed in the outer layer of the perichondrium/fibrous cell layer in the developing mandibular condyle. These findings suggest that the IGF system is involved in the formation of the condylar cartilage as well as in the TMJ.

The glycan-binding properties of the cation-independent mannose 6-phosphate receptor are evolutionary conserved in vertebrates.

The 300-kDa cation-independent mannose 6-phosphate receptor (CI-MPR) plays an essential role in the biogenesis of lysosomes by delivering newly synthesized lysosomal enzymes from the trans Golgi network to the endosomal system. The CI-MPR is expressed in most eukaryotes, with Saccharomyces cerevisiae and Caenorhabditis elegans being notable exceptions. Although the repertoire of glycans recognized by the bovine receptor has been studied extensively, little is known concerning the ligand-binding properties of the CI-MPR from non-mammalian species. To assess the evolutionary conservation of the CI-MPR, surface plasmon resonance analyses using lysosomal enzymes with defined N-glycans were carried out to probe the glycan-binding specificity of the Danio rerio CI-MPR. The results demonstrate that the D. rerio CI-MPR harbors three glycan-binding sites that, like the bovine CI-MPR, map to domains 3, 5 and 9 of its 15-domain-containing extracytoplasmic region. Analyses on a phosphorylated glycan microarray further demonstrated the unique binding properties of each of the three sites and showed that, similar to the bovine CI-MPR, only domain 5 of the D. rerio CI-MPR is capable of recognizing Man-P-GlcNAc-containing glycans.

Expression of insulin-like growth factor (IGF) family members in porcine pregnancy.

Prenatal mortality is a prime concern for commercial swine industry in North America. Fetal losses occur throughout gestation but cluster in early (~day20) and mid (~day50) pregnancy. Adequate vascularization of the attachment site has emerged as a key factor contributing to fetal success. Since Insulin-Like Growth Factor (IGF) family members regulate angiogenesis in addition to promoting fetal development and growth, we hypothesized that conceptus success is governed by members of the IGF family. Using quantitative real time PCR, we analyzed expression of IGF family members (IGF-I, IGF-II, IGF-I Receptor (IGF-IR), IGF-IIR and their binding proteins, IGFBPs) in matched maternal and fetal tissues of healthy and arresting conceptuses at gestation days (gd) 20 and 50. IGF-II transcripts were 100 fold increased in both maternal and fetal tissues compared to IGF-I, but receptor transcripts were found in similar abundance irrespective of health status and gestation point. IGFBP3 was the most abundantly transcribed of the binding proteins. Using immunohistochemistry we confirmed the expression of IGF family members in maternal luminal and glandular epithelial cells, the endothelium of blood vessels and some scattered stromal cells. Our results suggest that IGF-I and II and their receptors are differentially expressed at the maternal and fetal components of the attachment site.

Growth hormone affects gene expression and proliferation in human prostate cancer cells.

We previously showed that growth hormone (GH) receptors (GHR) are expressed in the most commonly studied human prostate cancer (PCa) cell lines and that GHR isoforms undergo differential, cell-type-specific hormonal regulation. We now report that human GH (hGH) can stimulate/modulate insulin-like growth factor (IGF) and ß-oestradiol (E(2) ) receptor (ER(ß) ) gene expressions in these cells and interact with IGF-I and E(2) to stimulate androgen-dependent LNCaP cell proliferation. We observed a cell type-dependent, differential regulation of IGF axis gene expression by GH: IGF-I was stimulated in the androgen-dependent LNCaP cells; IGF-II was stimulated in androgen-insensitive (AI) PC3 cells; the IGF-I cognate receptor, IGF-IR, was stimulated in LNCaP cells, but inhibited in PC3 cells; IGF-IIR was stimulated in both LNCaP and PC3 cells. GH also stimulated ER(ß) gene expression in LNCaP and PC3 cells, but had little or no effect on any of those genes in AI DU145 cells. The potent androgen analogue, mibolerone, also stimulated IGF-I, IGF-IR and ER(ß) , but reduced IGF-IIR mRNAs in LNCaP cells. Furthermore, triiodothyronine (T(3) ) and E(2) also stimulated the expression of those four genes in LNCaP cells, but co-administration of GH had almost no effect. Finally, we also studied the effects of GH, IGF-I and E(2) , alone or in combination, on LNCaP cell proliferation. Importantly, we demonstrated, for the first time, that although GH and IGF-I alone had no effect on LNCaP cell proliferation, concomitant administration for 96 h revealed a permissive role of GH on IGF-I-induced proliferation. GH also appeared to exert a synergistic effect on E(2) -stimulated LNCaP cell proliferation. Taken together, these findings indicate that GH via GHRs, most likely in concert with gonadal steroids, T(3) , IGF system axis and probably other hormones and growth factors, potentially plays an important role in the mechanisms underlying tumour cell growth in PCa.

Insulinlike growth factor receptor type 1 and type 2 are downregulated in the nitrofen-induced hypoplastic lung.

PURPOSE: In congenital diaphragmatic hernia (CDH), high mortality rates are attributed to severe pulmonary hypoplasia. The insulinlike growth factor receptor type 1 (IGF-1R) and type 2 (IGF-2R) play a critical role in the alveologenesis during lung development. The IGF-1R null mutation mice die after birth because of respiratory failure. The IGF-2R knockout mice showed retarded lungs with poorly formed alveoli. We hypothesized that IGF-1R and IGF-2R gene expression levels are downregulated in the nitrofen-induced CDH model. METHODS: Pregnant rats were exposed to either olive oil or 100 mg of nitrofen on day 9.5 (D9.5) of gestation. Fetuses were harvested on D18 and D21 and divided into control and nitrofen groups. Relative messenger RNA (mRNA) levels of IGF-1R and IGF-2R were determined using real time reverse transcription polymerase chain reaction. Immunohistochemistry was performed to determine protein expression. RESULTS: Relative levels of IGF-1R mRNA were significantly decreased in the nitrofen group (2.91 +/- 0.81) on D21 compared to controls (5.29 +/- 2.59) (P < .05). Expression levels of IGF-2R mRNA on D21 were also significantly decreased in nitrofen group (1.76 +/- 0.49) compared to controls (3.59 +/- 2.45) (P < .05). Immunohistochemistry performed on D21 showed decreased IGF-1R and also IGF-2R expression in nitrofen group. CONCLUSION: Downregulation of IGF-1R and IGF-2R gene expression may interfere with normal alveologenesis causing pulmonary hypoplasia in the nitrofen-induced CDH model.

An in vitro ES cell imprinting model shows that imprinted expression of the Igf2r gene arises from an allele-specific expression bias.

Genomic imprinting is an epigenetic process that results in parental-specific gene expression. Advances in understanding the mechanism that regulates imprinted gene expression in mammals have largely depended on generating targeted manipulations in embryonic stem (ES) cells that are analysed in vivo in mice. However, genomic imprinting consists of distinct developmental steps, some of which occur in post-implantation embryos, indicating that they could be studied in vitro in ES cells. The mouse Igf2r gene shows imprinted expression only in post-implantation stages, when repression of the paternal allele has been shown to require cis-expression of the Airn non-coding (nc) RNA and to correlate with gain of DNA methylation and repressive histone modifications. Here we follow the gain of imprinted expression of Igf2r during in vitro ES cell differentiation and show that it coincides with the onset of paternal-specific expression of the Airn ncRNA. Notably, although Airn ncRNA expression leads, as predicted, to gain of repressive epigenetic marks on the paternal Igf2r promoter, we unexpectedly find that the paternal Igf2r promoter is expressed at similar low levels throughout ES cell differentiation. Our results further show that the maternal and paternal Igf2r promoters are expressed equally in undifferentiated ES cells, but during differentiation expression of the maternal Igf2r promoter increases up to 10-fold, while expression from the paternal Igf2r promoter remains constant. This indicates, contrary to expectation, that the Airn ncRNA induces imprinted Igf2r expression not by silencing the paternal Igf2r promoter, but by generating an expression bias between the two parental alleles.

Expression of insulin-like growth factor system components in colorectal tissue and its relation with serum IGF levels.

CONTEXT: The insulin-like growth factor (IGF)-system has been implicated in colorectal tumor carcinogenesis. Although both tumor expression levels and serum concentrations of IGF-system components are related to colorectal cancer risk, it is unknown whether IGF levels in tissue and serum are correlated. OBJECTIVE: The objective of this study was to determine expression levels of various IGF-system components in different locations of the colorectum, and to investigate whether normal tissue IGF expression levels are correlated with serum IGF-I and IGF-II concentrations. DESIGN: Biopsies from macroscopically normal mucosa at four locations in the colorectum (ascending, transverse, sigmoid colon, and rectum) and a fasting serum sample were obtained from 48 asymptomatic patients at increased risk of colorectal cancer. Expression levels of IGF-I, IGF-II, IGF-IR, IGF-IIR, and IGFBP-3 messenger RNA (mRNA) in tissue were quantitatively evaluated using real-time RT-PCR. Expression of IGF-IR protein in the ascending colon and rectum tissue specimens was assessed semi-quantitatively by immunohistochemistry. Serum IGF-I and IGF-II concentrations were determined using immunometric assays. RESULTS: With the exception of IGF-IIR, mRNA levels of all the IGF-system components investigated, as well as IGF-IR protein expression, were significantly higher in the rectum compared with the ascending colon (p

Histamine suppresses fibulin-5 and insulin-like growth factor-II receptor expression in melanoma.

We previously showed that transgenic enhancement of histamine production in B16-F10 melanomas strongly supports tumor growth in C57BL/6 mice. In the present study, gene expression profiles of transgenic mouse melanomas, secreting different amounts of histamine, were compared by whole genome microarrays. Array results were validated by real-time PCR, and genes showing histamine-affected behavior were further analyzed by immunohistochemistry. Regulation of histamine-coupled genes was investigated by checking the presence and functional integrity of all four known histamine receptors in experimental melanomas and by administering histamine H1 receptor (H1R) and H2 receptor (H2R) antagonists to tumor-bearing mice. Finally, an attempt was made to integrate histamine-affected genes in known gene regulatory circuits by in silico pathway analysis. Our results show that histamine enhances melanoma growth via H1R rather than through H2R. We show that H1R activation suppresses RNA-level expression of the tumor suppressor insulin-like growth factor II receptor (IGF-IIR) and the antiangiogenic matrix protein fibulin-5 (FBLN5), decreases their intracellular protein levels, and also reduces their availability in the plasma membrane and extracellular matrix, respectively. Pathway analysis suggests that because plasma membrane-bound IGF-IIR is required to activate matrix-bound, latent transforming growth factor-beta1, a factor suggested to sustain FBLN5 expression, the data can be integrated in a known antineoplastic regulatory pathway that is suppressed by H1R. On the other hand, we show that engagement of H2R also reduces intracellular protein pools of IGF-IIR and FBLN5, but being a downstream acting posttranslational effect with minimal consequences on exported IGF-IIR and FBLN5 protein levels, H2R is rather irrelevant compared with H1R in melanoma.