Upregulation Of IGF-1 Research Articles

Abstract 2810: Loss of microRNA-122 in mice causes hepatic inflammation and hepatocarcinogenesis

Abstract Background: miR-122 is the most abundant liver-specific microRNA that modulates hepatic lipid metabolism, hepatitis C viral replication and is downregulated in hepatocellular carcinoma (HCC) of both rodent and human origin. However, it is not clear whether loss of miR-122 directly promotes or induces hepatocarcinogenesis. To address this question we have generated liver specific (LKO) and systemic (KO) miR-122 knockout mice and have studied the effect of the miR-122 deficiency on the liver function, hepatocyte proliferation and hepatocarcinogenesis with or without administration of a chemical carcinogen, diethylnitrosamine (DEN). Materials and methods: LKO and KO mice were generated by crossing miR-122 conditional KO (floxed) mice to Albumin-Cre and E2A-Cre mice, respectively. Southern blot/PCR and Northern blot/RT-PCR analysis were performed to confirm deletion of the gene and depletion of miR122 in the mutants compared to the controls (floxed). Phenotypic changes in the mutants were identified by histopathological and biochemical analysis of the liver and serum, respectively. Microarray analysis of hepatic RNA was performed to identify molecular pathways altered in LKO mice. Identify novel miR-122 targets among the upregulated genes in LKO mice by RT-PCR, luciferase assay, immunoblot and immunohistochemical analysis. Development of HCC in LKO, KO and control mice with or without DEN injection was monitored. Results and conclusions: Both LKO and KO mice were born alive and fertile but developed inflammation, steatosis and fibrosis with age. Hepatic miR-122 level was depleted by ∼83% and 98% on postnatal day 0 in LKO and KO mice, respectively and by 99% at adult stage in both mice. Serology showed reduced total, HDL- and LDL-associated cholesterol. Increased CK19- and A6-postitve cells as well as elevated serum alkaline phosphatase indicate abnormality in the biliary system in knockout mice. These mice developed hepatic inflammation in early adult life, which progressed to bridging inflammation and fibrosis with age. T cells and macrophages predominantly constituted the inflammatory cells in the mutant mice. Upregulation of Ccng1, Ccnd1 and Igf2 correlated with increased proliferation (as determined by Ki-67 positivity) and dedifferentiation (increased expression of Afp, Igf2 and H19) of miR-122 deficient hepatocytes. Eight months after DEN injection, LKO mice exhibited pronounced liver damage, higher incidence of HCC compared to the control mice. After one year, both LKO and KO mice developed spontaneous liver cancers, including HCC and cholangiocarcinoma, and exhibited liver damage as demonstrated by increased serum ALT, ALP, GGT and bilirubin levels. Taken together, these observations suggest that miR-122 deficiency causes inflammation that leads to hepatocarcinogenesis. Data will be presented on the underlying molecular mechanism of the development of liver damage and cancer in the knockout mice. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2810. doi:10.1158/1538-7445.AM2011-2810

Phosphoproteomic Analysis of Signaling Pathways in Head and Neck Squamous Cell Carcinoma Patient Samples

Molecular targeted therapy represents a promising new strategy for treating cancers because many small-molecule inhibitors targeting protein kinases have recently become available. Reverse-phase protein microarrays (RPPAs) are a useful platform for identifying dysregulated signaling pathways in tumors and can provide insight into patient-specific differences. In the present study, RPPAs were used to examine 60 protein end points (predominantly phosphoproteins) in matched tumor and nonmalignant biopsy specimens from 23 patients with head and neck squamous cell carcinoma to characterize the cancer phosphoproteome. RPPA identified 18 of 60 analytes globally elevated in tumors versus healthy tissue and 17 of 60 analytes that were decreased. The most significantly elevated analytes in tumor were checkpoint kinase (Chk) 1 serine 345 (S345), Chk 2 S33/35, eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) S65, protein kinase C (PKC) ζ/ι threonine 410/412 (T410/T412), LKB1 S334, inhibitor of kappaB alpha (IκB-α) S32, eukaryotic translation initiation factor 4E (eIF4E) S209, Smad2 S465/67, insulin receptor substrate 1 (IRS-1) S612, mitogen-activated ERK kinase 1/2 (MEK1/2) S217/221, and total PKC ι. To our knowledge, this is the first report of elevated PKC ι in head and neck squamous cell carcinoma that may have significance because PKC ι is an oncogene in several other tumor types, including lung cancer. The feasibility of using RPPA for developing theranostic tests to guide personalized therapy is discussed in the context of these data.

Placental expression of pituitary hormones is an ancestral feature of therian mammals.

BackgroundThe placenta is essential for supplying nutrients and gases to the developing mammalian young before birth. While all mammals have a functional placenta, only in therian mammals (marsupials and eutherians) does the placenta closely appose or invade the uterine endometrium. The eutherian placenta secretes hormones that are structurally and functionally similar to pituitary growth hormone (GH), prolactin (PRL) and luteinizing hormone (LH). Marsupial and eutherian mammals diverged from a common ancestor approximately 125 to 148 million years ago and developed distinct reproductive strategies. As in eutherians, marsupials rely on a short-lived but functional placenta for embryogenesis.ResultsWe characterized pituitary GH, GH-R, IGF-2, PRL and LHβ in a macropodid marsupial, the tammar wallaby, Macropus eugenii. These genes were expressed in the tammar placenta during the last third of gestation when most fetal growth occurs and active organogenesis is initiated. The mRNA of key growth genes GH, GH-R, IGF-2 and PRL were expressed during late pregnancy. We found significant up-regulation of GH, GH-R and IGF-2 after the start of the rapid growth phase of organogenesis which suggests that the placental growth hormones regulate the rapid phase of fetal growth.ConclusionsThis is the first demonstration of the existence of pituitary hormones in the marsupial placenta. Placental expression of these pituitary hormones has clearly been conserved in marsupials as in eutherian mammals, suggesting an ancestral origin of the evolution of placental expression and a critical function of these hormones in growth and development of all therian mammals.

Wt1 ablation and Igf2 upregulation in mice result in Wilms tumors with elevated ERK1/2 phosphorylation

Wilms tumor (WT) is a genetically heterogeneous childhood kidney tumor. Several genetic alterations have been identified in WT patients, including inactivating mutations in WT1 and loss of heterozygosity or loss of imprinting at 11p15, which results in biallelic expression of IGF2. However, the mechanisms by which one or a combination of genetic alterations results in tumorigenesis has remained challenging to determine, given the lack of a mouse model of WT. Here, we engineered mice to sustain mosaic, somatic ablation of Wt1 and constitutional Igf2 upregulation, mimicking a subset of human tumors. Mice with this combination of genetic alterations developed tumors at an early age. Mechanistically, Wt1 ablation blocked mesenchyme differentiation, and increased Igf2 expression upregulated ERK1/2 phosphorylation. Importantly, a subset of human tumors similarly displayed upregulation of ERK1/2 phosphorylation, which suggests ERK signaling might contribute to WT development. Thus, we have generated a biologically relevant mouse model of WT and defined one combination of driver alterations for WT. This mouse model will provide a powerful tool to study the biology of WT initiation and progression and to investigate therapeutic strategies for cancers with IGF pathway dysregulation.

Insulin-like Growth Factor 2 Expression Modulates Taxol Resistance and Is a Candidate Biomarker for Reduced Disease-Free Survival in Ovarian Cancer

This study was undertaken to examine the role of the insulin-like growth factor (IGF) signaling pathway in the response of ovarian cancer cells to Taxol and to evaluate the significance of this pathway in human epithelial ovarian tumors. The effect of Taxol treatment on AKT activation in A2780 ovarian carcinoma cells was evaluated using antibodies specific for phospho-AKT. To study the drug-resistant phenotype, we developed a Taxol-resistant cell line, HEY-T30, derived from HEY ovarian carcinoma cells. IGF2 expression was measured by real-time PCR. A type 1 IGF receptor (IGF1R) inhibitor, NVP-AEW541, and IGF2 small interfering RNA were used to evaluate the effect of IGF pathway inhibition on proliferation and Taxol sensitivity. IGF2 protein expression was evaluated by immunohistochemistry in 115 epithelial ovarian tumors and analyzed in relation to clinical/pathologic factors using the chi(2) or Fisher's exact tests. The influence of IGF2 expression on survival was studied with Cox regression. Taxol-induced AKT phosphorylation required IGF1R tyrosine kinase activity and was associated with upregulation of IGF2. Resistant cells had higher IGF2 expression compared with sensitive cells, and IGF pathway inhibition restored sensitivity to Taxol. High IGF2 tumor expression correlated with advanced stage (P < 0.001) and tumor grade (P < 0.01) and reduced disease-free survival (P < 0.05). IGF2 modulates Taxol resistance, and tumor IGF2 expression is a candidate prognostic biomarker in epithelial ovarian tumors. IGF pathway inhibition sensitizes drug-resistant ovarian carcinoma cells to Taxol. Such novel findings suggest that IGF2 represents a therapeutic target in ovarian cancer, particularly in the setting of Taxol resistance.

Upregulation of IGF2 is associated with an acquired resistance for cis-diamminedichloroplatinum in human head and neck squamous cell carcinoma

Ten head and neck squamous cell carcinoma (HNSCC) cell lines that had acquired cis-diamminedichloroplatinum (CDDP) resistance were successfully established by means of in vitro culture with CDDP. Flow cytometry analysis showed the significantly decreased induction of apoptosis after the CDDP treatment in the acquired CDDP-resistant sublines. Among these, RPMI2650CR showed a 9.38-fold increased IC50 in comparison with the parental cell line, RPMI2650. The identification of the resistance-related gene clusters was conducted between RPMI2650CR and RPMI2650 using two distinct microarray platforms. The expressional profiles were quite similar, suggesting that a limited number of genes regulate the acquisition of CDDP resistance. IGF2 was found to be one of the candidates for acquired CDDP-resistance. The introduction of IGF2 into RPMI2650 caused CDDP resistance along with suppression of CDDP-dependent apoptosis. On the other hand, siRNA-mediated knockdown of IGF2 caused reduction of acquired CDDP-resistance in RPMI2650CR. These results demonstrate that expression of IGF2 plays an important and critical role in CDDP resistance in a HNSCC cell line RPMI2650. These findings suggest the possibility for developing a new strategy for treating patients with HNSCC, particularly those with acquired CDDP resistance, by combining CDDP with inhibition of the IGF2 pathway.

Epigenomic alterations and gene expression profiles in respiratory epithelia exposed to cigarette smoke condensate

Limited information is available regarding epigenomic events mediating initiation and progression of tobacco-induced lung cancers. In this study, we established an in vitro system to examine epigenomic effects of cigarette smoke in respiratory epithelia. Normal human small airway epithelial cells and cdk-4/hTERT-immortalized human bronchial epithelial cells (HBEC) were cultured in normal media with or without cigarette smoke condensate (CSC) for up to 9 months under potentially relevant exposure conditions. Western blot analysis showed that CSC mediated dose- and time-dependent diminution of H4K16Ac and H4K20Me3, while increasing relative levels of H3K27Me3; these histone alterations coincided with decreased DNA methyltransferase 1 (DNMT1) and increased DNMT3b expression. Pyrosequencing and quantitative RT-PCR experiments revealed time-dependent hypomethylation of D4Z4, NBL2, and LINE-1 repetitive DNA sequences; up-regulation of H19, IGF2, MAGE-A1, and MAGE-A3; activation of Wnt signaling; and hypermethylation of tumor suppressor genes such as RASSF1A and RAR-beta, which are frequently silenced in human lung cancers. Array-based DNA methylation profiling identified additional novel DNA methylation targets in soft-agar clones derived from CSC-exposed HBEC; a CSC gene expression signature was also identified in these cells. Progressive genomic hypomethylation and locoregional DNA hypermethylation induced by CSC coincided with a dramatic increase in soft-agar clonogenicity. Collectively, these data indicate that cigarette smoke induces 'cancer-associated' epigenomic alterations in cultured respiratory epithelia. This in vitro model may prove useful for delineating early epigenetic mechanisms regulating gene expression during pulmonary carcinogenesis.

ZBED6, a Novel Transcription Factor Derived from a Domesticated DNA Transposon Regulates IGF2 Expression and Muscle Growth

Author SummaryThe molecular identification of genes and mutations affecting complex traits and disorders has proven to be very challenging in humans as well as in model organisms. These so-called quantitative traits arise from interactions between two or more genes and their environment, and can be mapped to their underlying genes via closely linked stretches of DNA called quantitative trait loci (QTL). Previously, we identified a single nucleotide substitution in a noncoding region of the insulin-like growth factor 2 gene (IGF2) in pigs that is underlying a major QTL affecting muscle growth, heart size, and fat deposition. The mutation disrupts interaction with an unknown nuclear protein acting as a repressor of IGF2 transcription. In the present study, we have isolated a zinc finger protein of unknown function and show that it regulates the expression of IGF2. The protein, which we named ZBED6, is encoded by a domesticated DNA transposon that was inserted into the genome prior to the radiation of placental mammals. ZBED6 is exclusive to placental mammals and highly conserved among species. Our functional characterization of ZBED6 shows that it has a broad tissue distribution and may affect the expression of thousands of other genes, besides IGF2, that control fundamental biological processes. We postulate that ZBED6 is an important transcription factor affecting development, cell proliferation, and growth in placental mammals.

척수타박손상 후 트레드밀운동이 근 위축지연과 기능적 회복에 미치는 효과

본 연구는 흰쥐의 흉추 9번을 타박 손상시키고 1주간의 휴식 후 스트레스를 유발하지 않는 저강도 트레드밀운동을 7주동안 수행하여, 가자미근 위축지연 변화를 western blotting과 H&E 염색 그리고 면역형광염색법으로 관찰하였으며, BBB-검사를 이용하여 기능적 회복을 관찰하였다. 형태학적 변화의 결과 척수손상 후 트레드밀 운동에 의해 하지부위 가자미 근과 근섬유의 횡단면 크기가 증가하였으며, 근섬유 세포에서 IGF-I 단백질 발현이 증가하였다. 또한 생화학적인 결과 척수손상 후 트레드밀 운동에 의해 하지부위 골격근 IGF-I 단백질 발현이 증가하였으며, 행동학적 검사를 통해 트레드밀 운동그룹이 비 운동그룹보다 기능적 회복이 개선된 것을 확인하였다. 이러한 결과는 근 비대에 직접적으로 관여하는 IGF-I 단백질 발현량이 운동에 의해 증가하여 가자미근크기 증가와 같은 근 위축 지연을 확인하였으며, 이러한 변화를 통해 기능적 회복에 긍정적인 결과를 보여주었다. 따라서 운동은 척수손상 환자에게 효과적인 기능재활 치료방법이 될 수 있으며, 장애를 최소화하여 자발적인 일상생활을 도모할 수 있을 것이라 생각된다. Physical activity and exercise can promote sensorimotor recovery from central nerve injury. It has been suggested that the functional recovery promoted by exercise training after spinal cord injury might be associated with insulin-like growth factor-I in the inflicted muscle. To investigate morphological and biochemical change of the soleus muscle after spinal cord injury, all tissues were used for H&E, immunofluorescence staining and Western blot. Also, BBB-test was used to evaluate behavioral improvement after spinal cord contusion. Thirty male Sprague-Dawley rats (<TEX>$230{\pm}10\;g$</TEX>; 7week in age) were assigned equally to three different groups; Normal (n=10), SCI (n=10), SCI+TMT (n=10). Every rat in SCI and SCI+TMT groups underwent laminectomy at T9 level and then contusion on the exposed spinal cord site in anesthetized condition. After one week-recovery from contusion, every rat in the SCI+TMT group exercised on a motorized treadmill for 30min/d, 5d/wk for 7wks. TMT followed by injury increased IGF-I induction levels in the soleus muscle and inhibited muscle atrophy. Behavioral scales for 4 and 8 weeks after spinal cord injury were improved in the SCI+TMT group compared to the SCI group. These results suggest that treadmill exercise after spinal cord injury might promote functional recovery along with muscle regrowth through the up-regulation of IGF-1 in muscle tissue.

NO-1886 suppresses diet-induced insulin resistance and cholesterol accumulation through STAT5-dependent upregulation of IGF1 and CYP7A1

Insulin resistance and dyslipidemia are both considered to be risk factors for metabolic syndrome. Low levels of IGF1 are associated with insulin resistance. Elevation of low-density lipoprotein cholesterol (LDL-C) concomitant with depression of high-density lipoprotein cholesterol (HDL-C) increase the risk of obesity and type 2 diabetes mellitus (T2DM). Liver secretes IGF1 and catabolizes cholesterol regulated by the rate-limiting enzyme of bile acid synthesis from cholesterol 7alpha-hydroxylase (CYP7A1). NO-1886, a chemically synthesized lipoprotein lipase activator, suppresses diet-induced insulin resistance with the improvement of HDL-C. The goal of the present study is to evaluate whether NO-1886 upregulates IGF1 and CYP7A1 to benefit glucose and cholesterol metabolism. By using human hepatoma cell lines (HepG2 cells) as an in vitro model, we found that NO-1886 promoted IGF1 secretion and CYP7A1 expression through the activation of signal transducer and activator of transcription 5 (STAT5). Pretreatment of cells with AG 490, the inhibitor of STAT pathway, completely abolished NO-1886-induced IGF1 secretion and CYP7A1 expression. Studies performed in Chinese Bama minipigs pointed out an augmentation of plasma IGF1 elicited by a single dose administration of NO-1886. Long-term supplementation with NO-1886 recovered hyperinsulinemia and low plasma levels of IGF1 suppressed LDL-C and facilitated reverse cholesterol transport by decreasing hepatic cholesterol accumulation through increasing CYP7A1 expression in high-fat/high-sucrose/high-cholesterol diet minipigs. These findings indicate that NO-1886 upregulates IGF1 secretion and CYP7A1 expression to improve insulin resistance and hepatic cholesterol accumulation, which may represent an alternative therapeutic avenue of NO-1886 for T2DM and metabolic syndrome.

Increased regeneration in ARI-treated diabetic nerve is associated with upregulation of IGF-1 and NGF receptors

Increased regeneration in ARI-treated diabetic nerve is associated with upregulation of IGF-1 and NGF receptors

Introduction: Uveal melanoma

Introduction: Uveal melanoma

Gene Expression Changes in Dorsal Root Ganglion of Rat Experimental Lumber Disc Herniation Models

Comprehensive overviews of gene expression changes in dorsal root ganglion (DRG) were obtained using microarrays in 2 rat models of experimental lumbar disc herniation (LDH). To clarify the mechanisms of painful radiculopathy caused by LDH from the viewpoint of gene expression changes in DRG of 2 rat models of LDH. Mechanical compression and chemical irritation are considered to be the 2 major causative factors of radiculopathy associated with LDH. Several basic studies have revealed histologic and functional changes in the nerve root and DRG induced by mechanical compression or application of nucleus pulposus. However, the effects of the 2 major factors have not been investigated in detail. METHODS.: The effects of mechanical and chemical factors were assessed in 2 models and in sham-operated rats. The mechanical compression model had a stainless steel rod inserted through a drill hole in the L5 lamina; the nucleus pulposus model had autologous nucleus pulposus placed in the drill hole, and only a drill hole was made in the L5 lamina of sham-operated rats. Samples from the left L5 DRG were harvested from the models with mechanical allodynia and from sham rats and analyzed using microarrays at 3 and 7 days after surgery. The gene expression profiles differed in the 2 models at 7 days, but were similar at 3 days after surgery. Expression of the growth factor gene, insulin-like growth factor 1, and of the cyclinD1, cell division cycle 2 homolog A, and cyclinA2 genes related to the cell cycle was significantly upregulated in the DRG of the mechanical compression group at 7 days after surgery. Mechanical and chemical factors caused altered gene expression in the DRG at 7 days after surgery, suggesting that the mechanisms of nerve injury induced by these factors differ. The upregulation of IGF-1 might be a key factor in painful radiculopathy induced by mechanical factors.

Upregulation of IGF-2 and IGF-1 receptor expression in oral cancer cell lines

The insulin-like growth factors (IGFs) are a family of mitogenic proteins involved in the regulation of cell growth and differentiation. The presence and role of the IGF system in oral mucosal epithelium is not clear but could influence our understanding of the pathogenesis of oral cancer. We characterised the expression and function of IGF-1, IGF-2 and IGF receptor in human oral squamous carcinoma cell lines and normal oral epithelial cells as well as normal oral and squamous cell carcinoma tissues. Using reverse transcription followed by PCR, IGF-1 mRNA was only detected in normal cells, whereas IGF-2 and IGF-1R mRNA transcripts were highly expressed in tumour cell lines and tissues. Similar observations were seen by Western blot analysis and immunohistochemistry. Exogenous IGF-2, but not IGF-1, caused significant increases in DNA synthesis in the cell lines. IGF-2 also increased cell proliferation which was significantly attenuated in the presence of an IGF-2 neutralizing antibody or one which blocked IGF-1R. Taken together, these studies suggest that autocrine production of IGF-2, together with over-expression of IGF-1R, may be important components controlling the proliferation of oral carcinoma cells.

Transcriptome analysis of the response to chronic constant hypoxia in zebrafish hearts

Insufficient blood supply during acute infarction and chronic ischemia leads to tissue hypoxia which can significantly alter gene expression patterns in the heart. In contrast to most mammals, some teleost fishes are able to adapt to extremely low oxygen levels. We describe here that chronic constant hypoxia (CCH) leads to a smaller ventricular outflow tract, reduced lacunae within the central ventricular cavity and around the trabeculae and an increase in the number of cardiac myocyte nuclei per area in the hearts of two teleost species, zebrafish (Danio rerio) and cichlids (Haplochromis piceatus). In order to identify the molecular basis for the adaptations to CCH, we profiled the gene expression changes in the hearts of adult zebrafish. We have analyzed over 15,000 different transcripts and found 376 differentially regulated genes, of which 260 genes showed increased and 116 genes decreased expression levels. Two notch receptors (notch-2 and notch-3) as well as regulatory genes linked to cell proliferation were transcriptionally upregulated in hypoxic hearts. We observed a simultaneous increase in expression of IGF-2 and IGFbp1 and upregulation of several genes important for the protection against reactive oxygen species (ROS). We have identified here many novel genes involved in the response to CCH in the heart, which may have potential clinical implications in the future.Electronic supplementary materialThe online version of this article (doi:10.1007/s00360-007-0201-4) contains supplementary material, which is available to authorized users.

Novel Roles of Local Insulin-Like Growth Factor-1 Activation in Gastric Ulcer Healing: Promotes Actin Polymerization, Cell Proliferation, Re-Epithelialization, and Induces Cyclooxygenase-2 in a Phosphatidylinositol 3-Kinase-Dependent Manner

The precise role of insulin-like growth factor (IGF)-1 in gastric ulcer healing is unknown. In experimental rat gastric ulcers, we examined expression of IGF-1 mRNA and protein by reverse transcriptase-polymerase chain reaction or enzyme-linked immunosorbent assay and immunostaining, respectively. In cultured rat gastric epithelial RGM1 cells, we examined effects of exogenous IGF-1 on cell migration, re-epithelialization, and proliferation-essential components of ulcer healing. We also examined whether IGF-1 induces cyclooxygenase (COX)-2 expression and determined the role of phosphatidylinositol 3-kinase and mitogen-activated protein kinase signaling pathways in mediating IGF-1 actions. Gastric ulceration triggered an approximately threefold increase in IGF-1 expression in epithelial cells of the ulcer margins (P < 0.001 versus control), especially in cells re-epithelizing granulation tissue and in mucosa in proximity to the ulcer margin. Treatment of RGM1 cells with IGF-1 caused a dramatic increase in actin polymerization, an eightfold increase in cell migration (P < 0.001), a 195% increase in cell proliferation (P < 0.05), and a sixfold increase in COX-2 expression (P < 0.01). Inhibitor of phosphatidylinositol 3-kinase abolished IGF-1-induced RGM1 cell migration and proliferation, actin polymerization, and COX-2 expression. The up-regulation of IGF-1 in gastric ulcer margin accelerates gastric ulcer healing by promoting cell re-epithelization, proliferation, and COX-2 expression via the phosphatidylinositol 3-kinase pathway.

Effect of temperature on gene expression of the Gh/Igf system during embryonic development in rainbow trout (Oncorhynchus mykiss).

In fish, the GH/IGF system installs very early during development suggesting that this system could promote embryonic growth and development. In contrast to mammals, the embryonic growth rate of poikilotherms depends considerably on the incubation temperature. Therefore, the aim of this study was to determine if variations of embryo growth in response to temperature could be associated with modifications in the gene expression of the GH/IGF system. In this study, using whole mount in situ hybridisation, we demonstrated that embryo incubation temperature (4, 8, and 12 degrees C) did not change the timing of GH-1 and GH-2 mRNA expression in somatotroph cells (stage 24). Similarly, at hatching (stage 30), we did not observe an obvious difference in GH protein and GH-1 and GH-2 transcript amounts in relation to the incubation temperature. Furthermore, from stage 22 to 25, the highest temperature led to a specific up-regulation of IGF-2 (2-fold between 4 and 12 degrees C), and both IGF-RIa and IGFRIb mRNA (1.5-fold between 4 and 12 degrees C), while no difference was observed for IGF-1 mRNA. Conversely, at hatching, the highest temperature specifically down-regulated IGF-2 (3-fold between 4 and 12 degrees C) and both IGF receptor mRNAs (2 fold between 4 and 12 degrees C) present in the head, while no difference was observed in the trunk. Our results demonstrated that different incubation temperatures during trout embryonic development did not change the stage of somatotroph cell appearance. Before hatching, IGF-2 and both IGF receptors, but not IGF-1 mRNA, were specifically up-regulated by high temperatures and could be related to the enhancement of embryonic growth rate.

Focal overexpression of insulin-like growth factor 2 by hepatocytes and cholangiocytes in viral liver cirrhosis

Insulin-like growth factor (IGF)-2 is overexpressed in hepatocellular carcinoma and accompanying dysplastic lesions. IGF-2 signalling is mediated through IGF-1 receptor (IGF-1R), while mannose 6-phosphate/insulin-like growth factor-2 receptor (M6P/IGF-2R) controls pericellular levels of free IGF-2. We studied, by in situ hybridisation and immunohistology, 18 liver specimens with cirrhosis of different aetiology without neoplastic or dysplastic lesions. Immunohistology was also performed for insulin receptor IGF-1R and IGF-binding proteins 3 and 4. High focal levels of IGF-2 RNA were found in some hepatocytes of all livers with HBV- or HCV-induced cirrhosis (n=10), but in only one of the cirrhoses with nonviral aetiology (n=8). IGF-2 was overexpressed in biliary duct epithelial cells in one case. Compared with noncirrhotic liver, all cirrhotic specimens showed reduced hepatocellular expression of M6P/IGF-2R protein, which contrasted with enhanced expression in perisinusoidal cells. Immunostaining for the other antigens did not reveal significant differences. Upregulation of IGF-2 in some hepatocytes may lead to high focal IGF-2 levels sufficient to saturate local IGF-2 binding capacities, and may result in an increased susceptibility to cellular dedifferentiation and, ultimately, liver cancer. Downregulation of hepatocellular M6P/IGF-2R and upregulation of IGF-2 seem to be early events in hepatocarcinogenesis prior to the appearance of morphologically distinct dysplastic lesions. Elevated focal IGF-2 transcript levels may therefore indicate an increased risk for hepatocellular and cholangiocellular carcinomas.

Effects of Exogenous Growth Hormone(GH)-Treatment on Carbon Tetrachloride(CCl4)-Induced Acute Liver Injury in GH-Deficient Mini Rats.

The effects of exogenous growth hormone (GH)-treatment on the development and regeneration of CCl4-induced acute hepatic injury were examined in GH-deficient Mini rats. Alanin aminotransferase (ALT) and asparate aminotransferase (AST) activities increased significantly at 24 and 48 hours (h) after CCl4-administration in both GH-treated and non treated groups, and they tended to be suppressed by GH-treatment. The expression level of insulin-like growth factor (IGF)-1 mRNA was higher in GH (+) CCl4 (+) group than in GH (-) CCl4 (+) group, especially at 72 h, and that of hepatocyte growth factor (HGF) mRNA was also higher in some points in the former group. Histologically, degenerative changes of hepatocytes observed at 24 and 48 h was less severe in GH (+) CCl4 (+) group than in GH (-) CCl4 (+) group. At 72 h, depositions of reticular fibers and extracellular matrix materials were less severe and the number of desmin- or α-SMA-positive cells was larger in GH (-) CCl4 (+) group than in GH (+) CCl4 (+) group. Proliferating cell nuclear antigen (PCNA)-labeling index of centrilobular hepatocytes increased at 48 h in both GH (+) CCl4 (+) and GH (-) CCl4 (+) groups, and it returned to the control level at 72 h in the former group while that in the latter group showed still higher level at 72 h. In this study, it was shown that CCl4-induced acute liver injury in Mini rats can be restored by exogenous GH-treatment, and upregulation of IGF-1 and HGF mRNAs may be involved in this phenomenon.

Expression of Insulin-like Growth Factor-binding Protein 5 in Neuroblastoma Cells Is Regulated at the Transcriptional Level by c-Myb and B-Myb via Direct and Indirect Mechanisms

Neuroblastoma (NB), a malignant childhood tumor deriving from the embryonic neural crest, is sensitive to the growth-stimulating effects of insulin-like growth factors (IGFs). Aggressive cases of this disease often acquire autocrine loops of IGF production, but the mechanisms through which the different components of the IGF axis are regulated in tumor cells remain unclear. Upon conditional expression of c-Myb in a NB cell line, we detected up-regulation of IGF1, IGF1 receptor, and insulin-like growth factor-binding protein 5 (IGFBP-5) expression. Analysis of the IGFBP-5 promoter revealed two potential Myb binding sites at position -59 to -54 (M1) and -429 to -424 (M2) from the transcription start site; both sites were bound by c-Myb and B-Myb in vitro and in vivo. Reporter assays carried out using the proximal region of the human IGFBP-5 promoter demonstrated that c-Myb and B-Myb enhanced transcription. However, site-directed mutagenesis and deletion of the Myb binding sites coupled with reporter assays revealed that M2 but not M1 was important for Myb-dependent transactivation of the IGFBP-5 promoter. The double mutant M1/M2 was still transactivated by c-Myb, suggesting the existence of Myb binding-independent mechanisms of IGFBP-5 promoter regulation. A constitutively active AKT transactivated the IGFBP-5 promoter, whereas the phosphatidylinositol 3-kinase inhibitor LY294002 suppressed it. Moreover, the kinase dead dominant negative K179M AKT mutant was able to inhibit transcription from the M2 and M1/M2 IGFBP-5 mutant promoters. Deletion analysis of the IGFBP-5 promoter revealed that the AKT-responsive region lies between nucleotides -334 and -83. Together, these data suggest that the Myb binding-independent transactivation of the IGFBP-5 promoter was due to the activation of the phosphatidylinositol 3-kinase/AKT pathway likely mediated by IGF1 receptor-dependent signals. Finally, IGFBP-5 was able to modulate proliferation of NB cells in a manner dependent on its concentration and on the presence of IGFs.