RESUMO
Secreted WNT proteins control cell differentiation and proliferation in many tissues, and NOTUM is a secreted enzyme that modulates WNT morphogens by removing a palmitoleoylate moiety that is essential for their activity. To better understand the role this enzyme in development, the authors produced NOTUM-deficient mice by targeted insertional disruption of the Notum gene. The authors discovered a critical role for NOTUM in dentin morphogenesis suggesting that increased WNT activity can disrupt odontoblast differentiation and orientation in both incisor and molar teeth. Although molars in Notum(-/-) mice had normal-shaped crowns and normal mantle dentin, the defective crown dentin resulted in enamel prone to fracture during mastication and made teeth more susceptible to endodontal inflammation and necrosis. The dentin dysplasia and short roots contributed to tooth hypermobility and to the spread of periodontal inflammation, which often progressed to periapical abscess formation. The additional incidental finding of renal agenesis in some Notum (-/-) mice indicated that NOTUM also has a role in kidney development, with undiagnosed bilateral renal agenesis most likely responsible for the observed decreased perinatal viability of Notum(-/-) mice. The findings support a significant role for NOTUM in modulating WNT signaling pathways that have pleiotropic effects on tooth and kidney development.
Assuntos
Displasia da Dentina/enzimologia , Esterases/metabolismo , Via de Sinalização Wnt , Animais , Diferenciação Celular , Displasia da Dentina/genética , Esterases/genética , Feminino , Humanos , Incisivo/crescimento & desenvolvimento , Rim/crescimento & desenvolvimento , Masculino , Camundongos , Camundongos Knockout , Dente Molar/crescimento & desenvolvimento , Mutagênese Insercional , Odontogênese/genéticaRESUMO
Mice deficient in TMEM218 (Tmem218(-/-) ) were generated as part of an effort to identify and validate pharmaceutically tractable targets for drug development through large-scale phenotypic screening of knockout mice. Routine diagnostics, expression analysis, histopathology, and electroretinogram analyses completed on Tmem218(-/-) mice identified a previously unknown role for TMEM218 in the development and function of the kidney and eye. The major observed phenotypes in Tmem218(-/-) mice were progressive cystic kidney disease and retinal degeneration. The renal lesions were characterized by diffuse renal cyst development with tubulointerstitial nephropathy and disruption of tubular basement membranes in essentially normal-sized kidneys. The retinal lesions were characterized by slow-onset loss of photoreceptors, which resulted in reduced electroretinogram responses. These renal and retinal lesions are most similar to those associated with nephronophthisis (NPHP) and retinitis pigmentosa in humans. At least 10% of NPHP cases present with extrarenal conditions, which most often include retinal degeneration. Senior-Løken syndrome is characterized by the concurrent development of autosomal recessive NPHP and retinitis pigmentosa. Since mutations in the known NPHP genes collectively account for only about 30% of NPHP cases, it is possible that TMEM218 could be involved in the development of similar ciliopathies in humans. In reviewing all other reported mouse models of NPHP, we suggest that Tmem218(-/-) mice could provide a useful model for elucidating the pathogenesis of cilia-associated disease in both the kidney and the retina, as well as in developing and testing novel therapeutic strategies for Senior-Løken syndrome.
Assuntos
Modelos Animais de Doenças , Doenças Renais Císticas/veterinária , Amaurose Congênita de Leber/veterinária , Proteínas de Membrana/genética , Camundongos Knockout/genética , Atrofias Ópticas Hereditárias/veterinária , Degeneração Retiniana/veterinária , Animais , Ciliopatias , Eletrorretinografia/veterinária , Olho/patologia , Feminino , Rim/patologia , Doenças Renais Císticas/genética , Doenças Renais Císticas/patologia , Amaurose Congênita de Leber/patologia , Masculino , Proteínas de Membrana/fisiologia , Camundongos , Atrofias Ópticas Hereditárias/patologia , Retina/patologia , Degeneração Retiniana/genéticaRESUMO
Human deep space and planetary travel is limited by uncertainties regarding the health risks associated with exposure to galactic cosmic radiation (GCR), and in particular the high linear energy transfer (LET), heavy ion component. Here we assessed the impact of two high-LET ions 56Fe and 28Si, and low-LET X rays on genome-wide methylation patterns in human bronchial epithelial cells. We found that all three radiation types induced rapid and stable changes in DNA methylation but at distinct subsets of CpG sites affecting different chromatin compartments. The 56Fe ions induced mostly hypermethylation, and primarily affected sites in open chromatin regions including enhancers, promoters and the edges ("shores") of CpG islands. The 28Si ion-exposure had mixed effects, inducing both hyper and hypomethylation and affecting sites in more repressed heterochromatic environments, whereas X rays induced mostly hypomethylation, primarily at sites in gene bodies and intergenic regions. Significantly, the methylation status of 56Fe ion sensitive sites, but not those affected by X ray or 28Si ions, discriminated tumor from normal tissue for human lung adenocarcinomas and squamous cell carcinomas. Thus, high-LET radiation exposure leaves a lasting imprint on the epigenome, and affects sites relevant to human lung cancer. These methylation signatures may prove useful in monitoring the cumulative biological impact and associated cancer risks encountered by astronauts in deep space.
Assuntos
Radiação Cósmica/efeitos adversos , Metilação de DNA/efeitos da radiação , Epigenômica , Neoplasias Pulmonares/genética , Astronautas , Brônquios/patologia , Brônquios/efeitos da radiação , Metilação de DNA/genética , Células Epiteliais/efeitos da radiação , Humanos , Transferência Linear de Energia , Neoplasias Pulmonares/etiologia , Neoplasias Pulmonares/patologia , Voo Espacial , Raios XRESUMO
A second generation genetically-engineered cell-based drug delivery system, referred to as apoptotic-induced drug delivery (AIDD), was developed using endothelial cells (ECs) that undergo apoptosis upon binding of vascular endothelial growth factor (VEGF) to a Flk-1:Fas fusion protein (FF). This new AIDD was redesigned using mouse brain derived ECs, bEnd3 cells, and an adenovirus vector in order to enhance and control the expression of FF. The FF was tagged with a HA epitope (FFHA) and designed to be coexpressed with green fluorescence protein (GFP) by the regulation of cytomegalovirus promoters in the adenovirus vector. bEnd3 cells showed favorable coexpression of FFHA and GFP consistent with the multiplicity of infection of the adenovirus. Immunofluorescence analysis demonstrated that FFHA was localized at the plasma membrane, whereas GFP was predominantly located in the cytoplasm of ECs. Cell death was induced by VEGF, but not by platelet derived growth factor or fibroblast growth factor in a dose-dependent manner (range 2-20 ng/ml), and revealed caspase-dependent apoptotic profiles. The FFHA expressing bEnd3 cells underwent apoptosis when cocultured with a glioma cell (SF188V+) line able to overexpress VEGF. The combined data indicated that the FFHA adenovirus system can induce apoptotic signaling in ECs in response to VEGF, and thus, is an instrumental modification to the development of AIDD.
Assuntos
Apoptose/efeitos dos fármacos , Encéfalo/citologia , Sistemas de Liberação de Medicamentos/métodos , Fator A de Crescimento do Endotélio Vascular/farmacologia , Adenoviridae/genética , Animais , Apoptose/genética , Sequência de Bases , Células Cultivadas , Neoplasias do Sistema Nervoso Central/tratamento farmacológico , Neoplasias do Sistema Nervoso Central/patologia , Técnicas de Cocultura , Células Endoteliais/virologia , Epitopos/genética , Epitopos/metabolismo , Vetores Genéticos/genética , Glioma/tratamento farmacológico , Glioma/patologia , Humanos , Camundongos , Dados de Sequência Molecular , Receptores do Fator de Necrose Tumoral/genética , Receptores do Fator de Necrose Tumoral/metabolismo , Receptores de Fatores de Crescimento do Endotélio Vascular/genética , Receptores de Fatores de Crescimento do Endotélio Vascular/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Receptor fasRESUMO
Expression of insulin-like growth factor I (IGF-I) mRNA by some tumor cell lines of neuroectodermal origin has been described. To further explore the significance of IGF-I mRNA expression in these tumors, a more extensive analysis was performed. Most (9 of 10) neuroectodermal tumor cell lines with a t(11;22) translocation (primitive neuroectodermal tumor [PNET], Ewing's sarcoma, esthesioneuroblastoma) expressed IGF-I mRNA, whereas 0 of 15 cell lines without the translocation (PNET, neuroblastoma) expressed IGF-I. Furthermore, inasmuch as all neuroblastoma (12 of 12) cell lines examined expressed IGF-II RNA, the pattern of IGF expression could distinguish between these closely related tumors. CHP-100, a PNET cell line with the t(11;22) translocation, was shown to secrete both IGF-I protein and an IGF binding protein, IGFBP-2. This cell line also expressed the type I IGF receptor mRNA, and blockade of this receptor by a monoclonal antibody (alpha IR3) inhibited serum-free growth. These data demonstrate that IGF-I expression is a property of neuroectodermal tumors with a t(11;22) translocation and that interruption of an IGF-I autocrine loop inhibits the growth of these tumor cells.
Assuntos
Cromossomos Humanos Par 11 , Cromossomos Humanos Par 22 , Fator de Crescimento Insulin-Like I/fisiologia , Neuroblastoma/fisiopatologia , Tumores Neuroectodérmicos Primitivos Periféricos/fisiopatologia , Receptores de Superfície Celular/fisiologia , Divisão Celular , Amplificação de Genes , Regulação Neoplásica da Expressão Gênica , Humanos , Fator de Crescimento Insulin-Like I/genética , Proteínas Proto-Oncogênicas c-myc/genética , RNA Mensageiro/genética , RNA Neoplásico/genética , Receptores de Superfície Celular/genética , Receptores de Somatomedina , Sarcoma/genética , Translocação GenéticaRESUMO
The rate of transcription of the hepatic phosphoenolpyruvate carboxykinase (PEPCK) and insulin-like growth factor-binding protein 1 (IGFBP-1) genes is stimulated by glucocorticoids and inhibited by insulin. In both cases, the effect of insulin is dominant, since it suppresses both basal and glucocorticoid-stimulated PEPCK or IGFBP-1 gene transcription. Analyses of both promoters by transfection of PEPCK or IGFBP-1-chloramphenicol acetyltransferase fusion genes into rat hepatoma cells has led to the identification of insulin response sequences (IRSs) in both genes. The core IRS, T(G/A)TTTTG, is the same in both genes, but the PEPCK promoter has a single copy of this element whereas the IGFBP-1 promoter has two copies arranged as an inverted palindrome. The IGFBP-1 IRS and PEPCK IRS both bind the alpha and beta forms of hepatic nuclear factor 3 (HNF-3), although the latter does so with a sixfold-lower relative affinity. Both the PEPCK and the IGFBP-1 IRSs also function as accessory factor binding sites required for the full induction of gene transcription by glucocorticoids. A combination of transient transfection and DNA binding studies suggests that HNF-3 is the accessory factor that supports glucocorticoid-induced gene transcription. In both genes, the HNF-3 binding site overlaps the IRS core motif(s). A model in which insulin is postulated to mediate its negative effect on glucocorticoid-induced PEPCK and IGFBP-1 gene transcription indirectly by inhibiting HNF-3 action is proposed.
Assuntos
Proteínas de Transporte/biossíntese , Proteínas de Ligação a DNA/metabolismo , Dexametasona/farmacologia , Regulação da Expressão Gênica , Insulina/farmacologia , Fígado/metabolismo , Proteínas Nucleares/metabolismo , Fosfoenolpiruvato Carboxiquinase (GTP)/biossíntese , Fatores de Transcrição/metabolismo , Animais , Sequência de Bases , Sítios de Ligação , Carcinoma Hepatocelular , Linhagem Celular , DNA/química , DNA/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Fator 3-alfa Nuclear de Hepatócito , Fator 3-beta Nuclear de Hepatócito , Fator 3-gama Nuclear de Hepatócito , Humanos , Proteína 1 de Ligação a Fator de Crescimento Semelhante à Insulina , Cinética , Neoplasias Hepáticas , Neoplasias Hepáticas Experimentais , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Oligodesoxirribonucleotídeos , Regiões Promotoras Genéticas , Ratos , Receptores de Glucocorticoides/biossíntese , Receptores de Glucocorticoides/fisiologia , Proteínas Recombinantes de Fusão/biossíntese , Sequências Reguladoras de Ácido Nucleico , Transcrição Gênica , Transfecção , Células Tumorais CultivadasRESUMO
A high-throughput, retrovirus-mediated mutagenesis method based on gene trapping in embryonic stem cells was used to identify a novel mouse gene. The human ortholog encodes a transmembrane protein containing five extracellular immunoglobulin-like domains that is structurally related to human NEPHRIN, a protein associated with congenital nephrotic syndrome. Northern analysis revealed wide expression in humans and mice, with highest expression in kidney. Based on similarity to NEPHRIN and abundant expression in kidney, this protein was designated NEPH1 and embryonic stem cells containing the retroviral insertion in the Neph1 locus were used to generate mutant mice. Analysis of kidney RNA from Neph1(-/-) mice showed that the retroviral insertion disrupted expression of Neph1 transcripts. Neph1(-/-) pups were represented at the expected normal Mendelian ratios at 1 to 3 days of age but at only 10% of the expected frequency at 10 to 12 days after birth, suggesting an early postnatal lethality. The Neph1(-/-) animals that survived beyond the first week of life were sickly and small but without edema, and all died between 3 and 8 weeks of age. Proteinuria ranging from 300 to 2,000 mg/dl was present in all Neph1(-/-) mice. Electron microscopy demonstrated NEPH1 expression in glomerular podocytes and revealed effacement of podocyte foot processes in Neph1(-/-) mice. These findings suggest that NEPH1, like NEPHRIN, may play an important role in maintaining the structure of the filtration barrier that prevents proteins from freely entering the glomerular urinary space.
Assuntos
Rim/anormalidades , Proteínas de Membrana/fisiologia , Proteínas/fisiologia , Proteinúria/etiologia , Sequência de Aminoácidos , Animais , Sequência de Bases , DNA Complementar , Perfilação da Expressão Gênica , Humanos , Rim/metabolismo , Rim/patologia , Rim/ultraestrutura , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Proteínas/genéticaRESUMO
Sequencing of the human genome has yielded thousands of potential drug targets. The difficulty now is in determining which targets have real therapeutic value and should be the focus of a drug discovery effort. The available evidence suggests that knockout technology can be used prospectively to identify targets that are amenable to drug development for the treatment of a variety of diseases. This review compares the knockout phenotypes of 21 potential obesity targets with the effects of therapeutics designed for those targets on rodents and, when data were available, on humans. The phenotypes of obesity target knockouts model the effects seen when therapeutics designed for those obesity targets are delivered to rodents; of the 21 obesity targets reviewed, 16 showed a correspondence between knockout phenotype and drug effect in mice and/or rats. This suggests that, at least in terms of evaluating obesity targets, it is rare for compensatory developmental changes caused by the gene knockout to prevent detection of the relevant phenotype. In the majority of cases, the knockout phenotypes also modelled the effects seen when the relevant therapeutics were delivered to humans. Thus, it seems rational to use mouse knockout technology prospectively to identify genes that regulate body fat in vivo, and then to develop anti-obesity therapeutics by targeting the human protein products of these genes. Ultimately, the value of using this approach to identify novel targets for human anti-obesity therapies will be judged by future studies examining the anti-obesity effect, in humans, of the therapeutics that result from this approach.
Assuntos
Fármacos Antiobesidade/uso terapêutico , Obesidade/tratamento farmacológico , Animais , Fármacos Antiobesidade/farmacologia , Enzimas/efeitos dos fármacos , Humanos , Ligantes , Camundongos , Camundongos Knockout , Fenótipo , Ratos , Receptores Citoplasmáticos e Nucleares/efeitos dos fármacos , Receptores Acoplados a Proteínas G/efeitos dos fármacosRESUMO
BACKGROUND: The insulin-like growth factors (IGFs) play an important role in normal growth and development. Evidence suggests they may also regulate the growth of several cancer cell types. This regulation is mediated by interactions between the receptors and ligands. There is now ample evidence to suggest that these interactions are also influenced by extracellular IGF-binding proteins (IGFBPs). Six different IGFBPs have been cloned. Some species may act to inhibit the mitogenic effects of the IGFs. Since breast cancer cells are responsive to the IGFs, it is possible that regulated expression of the IGFBPs affects tumor growth. Furthermore, inhibitory binding proteins could be used as neutralizers of IGF action. PURPOSE: We conducted this study to fully characterize the expression and hormonal regulation of IGF-binding protein expression in human MCF-7 breast cancer cells and to test the ability of purified IGFBP-1 to inhibit IGF-I action. METHODS: We used ribonuclease protection assays and Western ligand blotting to examine IGFBP expression in MCF-7 cells. The effect of IGF-I, IGFBP-1, and 17 beta-estradiol on serum-free cell growth was also studied. RESULTS: MCF-7 cells expressed IGFBP-2, IGFBP-4, and IGFBP-5 RNA and protein. These cells are dependent on estrogen for growth. In short-term culture, IGF-I can substitute for estrogen. Concomitant addition of IGF-I and estrogen enhanced stimulation above the level achieved by either factor alone. Estrogen also increased IGFBP production, making it unlikely that the IGFBPs induced by estrogen in MCF-7 cells could function as major inhibitors of IGF action. In contrast, exogenous addition of IGFBP-1 could block IGF-I-induced mitogenesis; this effect was reversible by excess IGF-I. CONCLUSIONS: The studies suggest that cancer cell growth may be regulated by endogenous IGFBP expression. Furthermore, the exogenous addition of the IGFBP-1 blocked IGF-I action and potentially could be used as a pharmacologic inhibitor of IGF action.
Assuntos
Neoplasias da Mama/metabolismo , Proteínas de Transporte/biossíntese , Proteínas de Transporte/farmacologia , Estradiol/farmacologia , Fator de Crescimento Insulin-Like I/farmacologia , RNA Mensageiro/biossíntese , Western Blotting , Neoplasias da Mama/patologia , Proteínas de Transporte/análise , Divisão Celular/efeitos dos fármacos , Sinergismo Farmacológico , Humanos , Proteína 1 de Ligação a Fator de Crescimento Semelhante à Insulina , Proteína 2 de Ligação a Fator de Crescimento Semelhante à Insulina , Proteína 4 de Ligação a Fator de Crescimento Semelhante à Insulina , Proteína 5 de Ligação a Fator de Crescimento Semelhante à Insulina , Fator de Crescimento Insulin-Like I/antagonistas & inibidores , Fator de Crescimento Insulin-Like I/metabolismo , Células Tumorais CultivadasRESUMO
Hydride attack at a ferric heme-NO to give an Fe-HNO intermediate is a key step in the global N-cycle. We demonstrate differential reactivity when six- and five-coordinate ferric heme-NO models react with hydride. Although Fe-HNO formation is thermodynamically favored from this reaction, Fe-H formation is kinetically favored for the 5C case.
RESUMO
Myelodysplastic syndrome (MDS) is characterized by ineffective hematopoiesis with resultant cytopenias. Increased apoptosis and aberrantly functioning progenitors are thought to contribute to this phenotype. As is the case for other malignancies, overcoming apoptosis is believed to be important in progression toward acute myeloid leukemia (AML). Using the NUP98-HOXD13 (NHD13) transgenic mouse model of MDS, we previously reported that overexpression of the anti-apoptotic protein BCL2, blocked apoptosis and improved cytopenias, paradoxically, delaying leukemic progression. To further understand this surprising result, we examined the role of p53 and its pro-apoptotic effectors, PUMA and NOXA in NHD13 mice. The absence of p53 or PUMA but not NOXA reduced apoptosis and expanded the numbers of MDS-repopulating cells. Despite a similar effect on apoptosis and cell numbers, the absence of p53 and PUMA had diametrically opposed effects on progression to AML: absence of p53 accelerated leukemic progression, while absence of PUMA significantly delayed progression. This may be explained in part by differences in cellular responses to DNA damage. The absence of p53 led to higher levels of γ-H2AX (indicative of persistent DNA lesions) while PUMA-deficient NHD13 progenitors resolved DNA lesions in a manner comparable to wild-type cells. These results suggest that targeting PUMA may improve the cytopenias of MDS without a detrimental effect on leukemic progression thus warranting further investigation.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Apoptose , Síndromes Mielodisplásicas/patologia , Proteínas Supressoras de Tumor/metabolismo , Animais , Proteínas Reguladoras de Apoptose/genética , Transplante de Medula Óssea , Dano ao DNA , Modelos Animais de Doenças , Progressão da Doença , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Humanos , Estimativa de Kaplan-Meier , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Síndromes Mielodisplásicas/metabolismo , Síndromes Mielodisplásicas/mortalidade , Proteínas de Fusão Oncogênica/genética , Fenótipo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteína Supressora de Tumor p53/deficiência , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Proteínas Supressoras de Tumor/genéticaRESUMO
Recent genome searches suggest a putative linkage of many loci to susceptibility to type I diabetes. The chromosome 2q31-35 region is reported to be linked to susceptibility to type I diabetes and is thought to contain several diabetes susceptibility loci. These candidate genes include the HOXD gene cluster, BETA2, CTLA4, CD28, IGFBP2, and IGFBP5. Association studies in populations and families are required to confirm and/or identify the actual susceptibility loci. We hereby report several previously unknown DNA polymorphisms for HOXD8, BETA2, and IGFBP5, which we have used along with previously known polymorphisms of HOXD8 and CTLA4 to test whether these candidate loci are the susceptibility genes on chromosome 2q31-35. Using a case-control design with a subsequent family-association approach to confirm associations, we find no evidence that these candidate genes are associated with susceptibility to type I diabetes.
Assuntos
Cromossomos Humanos Par 2/genética , Proteínas de Ligação a DNA/genética , Diabetes Mellitus Tipo 1/genética , Proteínas de Homeodomínio/genética , Proteína 5 de Ligação a Fator de Crescimento Semelhante à Insulina/genética , Transativadores/genética , Fatores de Transcrição/genética , Alelos , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Antígenos CD28/genética , Estudos de Casos e Controles , Mapeamento Cromossômico , Primers do DNA/química , Diabetes Mellitus Tipo 1/imunologia , Família , Frequência do Gene , Ligação Genética , Antígenos HLA-DR/imunologia , Humanos , Proteína 2 de Ligação a Fator de Crescimento Semelhante à Insulina/genética , Repetições de Microssatélites/genética , Família Multigênica , Reação em Cadeia da Polimerase , Polimorfismo Conformacional de Fita SimplesRESUMO
Because overexpression of the glucose-6-phosphatase catalytic subunit (G-6-Pase) in both type 1 and type 2 diabetes may contribute to the characteristic increased rate of hepatic glucose production, we have investigated whether the insulin response unit (IRU) identified in the mouse G-6-Pase promoter is conserved in the human promoter. A series of human G-6-Pase-chloramphenicol acetyltransferase (CAT) fusion genes was transiently transfected into human HepG2 hepatoma cells, and the effect of insulin on basal CAT expression was analyzed. The results suggest that the IRU identified in the mouse promoter is conserved in the human promoter, but that an upstream multimerized insulin response sequence (IRS) motif that is only found in the human promoter appears to be functionally inactive. The G-6-Pase IRU comprises two distinct promoter regions, designated A and B. Region B contains an IRS, whereas region A acts as an accessory element to enhance the effect of insulin, mediated through region B, on basal G-6-Pase gene transcription. We have previously shown that the accessory factor binding region A is hepatocyte nuclear factor-1, and we show here that the forkhead protein FKHR is a candidate for the insulin-responsive transcription factor binding region B.
Assuntos
Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Glucose-6-Fosfatase/genética , Insulina/farmacologia , Regiões Promotoras Genéticas , Elementos de Resposta , Fatores de Transcrição/genética , Animais , Sequência de Bases , Domínio Catalítico , Cloranfenicol O-Acetiltransferase/genética , Sequência Conservada , Glucose-6-Fosfatase/química , Humanos , Camundongos , Dados de Sequência Molecular , Proteínas Recombinantes de Fusão/efeitos dos fármacosRESUMO
We have characterized the structure of the human chromosomal gene for the 25 kilodalton insulin-like growth factor binding protein (BP-25) as a first step toward understanding both the factors which regulate BP-25 transcription and also the evolution of the insulin-like growth factor binding proteins. The BP-25 gene is present in the human genome as a single copy which spans 5.2 kilobases and contains four exons. Primer extension localizes the mRNA cap site 165 base pairs (bp) upstream of the ATG translational start codon. Preliminary analysis of the putative promoter region for BP-25 demonstrates characteristics consistent with those of many eukaryotic promoters; these include a consensus TATA box beginning 28 bp 5' to the cap site and a consensus CCAAT promoter element beginning 72 bp upstream from the cap site.
Assuntos
Proteínas de Transporte/genética , Genes , Somatomedinas/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Cromossomos Humanos , Clonagem Molecular , Genes Homeobox , Humanos , Proteínas de Ligação a Fator de Crescimento Semelhante a Insulina , Dados de Sequência Molecular , Peso Molecular , Regiões Promotoras Genéticas , Capuzes de RNA/genética , Mapeamento por RestriçãoRESUMO
The primary structure of an insulin-like growth factor (IGF) binding protein produced by human HEP G2 hepatoma cells has been deduced from the cDNA sequence. The 234 amino acid protein has a predicted molecular mass of 25,274 and contains a single, distinctive cysteine-rich region. The N-terminal sequence of this protein is quite similar to the limited sequence data available for a rat IGF binding protein produced by BRL-3A cells and suggests a common ancestral origin. In contrast, the HEP G2 IGF binding protein sequence bears no similarity to the N-terminal 15 amino acids of a 53 kilodalton binding protein purified from human plasma. Comparison of full-length protein sequences for the IGF-I and IGF-II receptors with that of the HEP G2 IGF binding protein also fails to demonstrate any significant similarities among these three proteins, and suggests that each contains a unique binding domain for the IGF peptides.
Assuntos
Proteínas de Transporte , Receptor de Insulina , Somatomedinas , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação , Proteínas de Transporte/genética , Proteínas de Transporte/isolamento & purificação , Mapeamento Cromossômico , Clonagem Molecular , Enzimas de Restrição do DNA , DNA de Neoplasias/genética , Humanos , Proteínas de Ligação a Fator de Crescimento Semelhante a Insulina , Dados de Sequência Molecular , Receptores de Somatomedina , Somatomedinas/genética , Somatomedinas/isolamento & purificação , Células Tumorais CultivadasRESUMO
Insulin-like growth factor-I (IGF-I) initiates its diverse biological effects by binding to type I IGF receptors on cells. In addition, IGF-I associates with distinct proteins that can modulate its actions. One of these IGF-binding proteins, IGFBP-3, is the major circulating form in adults and is produced by many cells in culture. We investigated the effect of purified bovine IGFBP-3 on IGF-I binding and IGF-I stimulation of amino acid uptake and DNA synthesis in cultured bovine fibroblasts, a cell culture system highly suitable for these types of studies. Incubation of cells with IGF-I resulted in time- and dose-dependent decreases in [125I]IGF-I binding and IGF-I stimulated [3H]aminoisobutyric acid uptake and [3H]thymidine incorporation. Preincubation with 4 nM IGF-I resulted in a 50-60% decrease in IGF-I receptor binding, accompanied by marked decreases in IGF-I-stimulated [3H]aminoisobutyric acid uptake (50-60%) and [3H]thymidine incorporation (80-90%). Preincubation with the IGF-I analog [QAYL]IGF-I (4 nM) or with 100 nM insulin, growth factors that bind and activate type I IGF receptor signalling but have little or no affinity for IGFBP-3, had effects comparable to IGF-I, decreasing both IGF-I binding and action 50-95%. The addition of IGFBP-3 during the preincubation period with IGF-I blocked the decrease in receptor availability and prevented the cells from becoming desensitized. IGFBP-3 did not prevent the [QAYL]IGF-I- or insulin-induced receptor loss and cellular resistance to IGF-I. These data indicate that IGFBP-3 can prevent IGF-I-induced receptor down-regulation, a process that renders cells refractory to further stimulation by IGF-I. Thus, cell-derived IGFBP-3 may function in a buffering capacity to restrict IGF-I and target cell interaction, thereby modulating the biological response to changes in local IGF-I levels.
Assuntos
Proteínas de Transporte/farmacologia , Regulação para Baixo/efeitos dos fármacos , Fibroblastos/metabolismo , Fator de Crescimento Insulin-Like I/farmacologia , Receptores de Superfície Celular/metabolismo , Ácidos Aminoisobutíricos/metabolismo , Animais , Ligação Competitiva , Bovinos , Células Cultivadas , Reagentes de Ligações Cruzadas , Fibroblastos/efeitos dos fármacos , Proteínas de Ligação a Fator de Crescimento Semelhante a Insulina , Fator de Crescimento Insulin-Like I/metabolismo , Peso Molecular , Receptores de SomatomedinaRESUMO
Insulin-like growth factor binding protein-3 (IGFBP-3) purified from bovine serum shares 19 of 25 amino-terminal amino acid residues with IGFBP-3 purified from human, rat, and porcine sources. A newly characterized bovine fibroblast model was used to investigate the biological effects of purified bovine IGFBP-3 (bIGFBP-3). Coincubation of insulin-like growth factor I (IGF-I) with increasing concentrations of bIGFBP-3 produced a dose-dependent inhibition of IGF-I-stimulated [3H]aminoisobutyric acid (AIB) uptake in cultured bovine fibroblasts. Inhibition was complete at equimolar concentrations of IGF-I and bIGFBP-3. Inhibition of IGF-I-stimulated [3H]AIB uptake paralleled the ability of bIGFBP-3 to prevent [125I]IGF-I cell surface binding. In contrast, preincubation with bIGFBP-3 resulted in a dose-dependent enhancement of IGF-I-stimulated [3H]AIB uptake; a 32-86% increase in IGF-I bioactivity was seen after a 24 h preexposure to 10 nM bIGFBP-3, and a 2- to 6-fold potentiation was seen after a 72 h preincubation. Preincubation with bIGFBP-3 increased both the sensitivity and maximal responsiveness of the cells to IGF-I. The potentiating effects of bIGFBP-3 were associated with increased [125I]IGF-I binding to cultured bovine fibroblasts. Affinity cross-linking experiments indicated that the increase in IGF-I binding was due to increased membrane-associated bIGFBP-3 rather than to a bIGFBP-3-induced increase in type I IGF receptors. bIGFBP-3 had no effect on insulin stimulation of [3H]AIB uptake under either experimental condition. These data suggest that soluble bIGFBP-3 inhibits IGF-I action by sequestering and preventing IGF-I receptor binding, whereas surface-associated bIGFBP-3 enhances the growth-promoting effects of IGF-I in bovine fibroblasts. We propose that IGFBP-3 serves a dual function in modulating IGF action in vivo.
Assuntos
Proteínas de Transporte/metabolismo , Sequência de Aminoácidos , Ácidos Aminoisobutíricos/metabolismo , Animais , Proteínas de Transporte/genética , Proteínas de Transporte/isolamento & purificação , Bovinos , Células Cultivadas , Cromatografia Líquida de Alta Pressão , Replicação do DNA/efeitos dos fármacos , Fibroblastos/metabolismo , Humanos , Proteínas de Ligação a Fator de Crescimento Semelhante a Insulina , Fator de Crescimento Insulin-Like I/metabolismo , Fator de Crescimento Insulin-Like I/farmacologia , Dados de Sequência Molecular , Peso Molecular , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Homologia de Sequência do Ácido Nucleico , Pele/metabolismoRESUMO
The insulin-like growth factors (IGFs) exist primarily bound to cell surface receptors or complexed to specific binding proteins (IGFBPs). The IGFBPs modulate the bioavailability of the IGFs and may enhance or inhibit IGF actions. Several distinct forms of IGFBPs have been described on the basis of size, immunological determinants, and distribution in biological fluids; the IGFBPs may differ as well in their biological function. Sheep thyroid cells produce IGFBPs under hormonal regulation. Cells grown in basal medium or with six-hormone (6H) medium supplements (transferrin, glycyl-histidyl-lysine, hydrocortisone, somatostatin, insulin, and TSH) release nonglycosylated BPs that migrate at 24, 27, 29, and 32 kDa on Western ligand blot. Cells cultured with the thyroid mitogens epidermal growth factor and phorbol ester release additional glycosylated IGFBPs of 40-44 kDa. Immunoprecipitation experiments indicate that 29- and 32-kDa IGFBPs are antigenically related to IGFBP-2, and the 40- to 44-kDa proteins are related to IGFBP-3. Using specific cDNA probes IGFBP-1, -2, and -3, we examined the regulation of IGFBP mRNA levels in sheep thyroid cultures. The rat IGFBP-2 cDNA probe hybridized to an approximately 1.6-kilobase mRNA species in cells under all culture conditions. However, IGFBP-3 mRNA was detectable only in epidermal growth factor- or phorbol ester-treated cells and appeared within 4 h, preceding the release of IGFBP-3 protein into the medium. The 6H additives, which stimulate differentiated function in thyroid cells, inhibited the mRNA levels of both IGFBP-2 and IGFBP-3. IGFBP-1 mRNA was not detectable. The distinct regulation of these IGFBPs suggest that they may play different biological roles in modulating thyroid physiology.
Assuntos
Proteínas de Transporte/genética , Regulação da Expressão Gênica , RNA Mensageiro/metabolismo , Glândula Tireoide/metabolismo , Actinas/genética , Animais , Disponibilidade Biológica , Western Blotting , Proteínas de Transporte/química , Células Cultivadas , Meios de Cultura , Sondas de DNA , Fator de Crescimento Epidérmico/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Técnicas de Imunoadsorção , Proteínas de Ligação a Fator de Crescimento Semelhante a Insulina , Peso Molecular , Hibridização de Ácido Nucleico , Ovinos , Especificidade da Espécie , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
During extrauterine life, insulin-like growth factors (IGFs) circulate in a ternary serum complex with one IGF-binding protein-3 (IGFBP-3) or IGFBP-5 protein and with a single acid-labile subunit (ALS). GH increases levels of this ternary complex; in mice, this effect is achieved in part by the ability of GH to stimulate mouse ALS (mALS) transcription through an interferon-gamma-activated sequence-like element (GLE) in the mALS promoter. To begin studying how GH regulates human ALS (hALS) gene expression, we cloned the hALS gene and found that it spans approximately 3.3 kb of DNA at chromosomal region 16p13.3. The hALS gene has two exons separated by a 1235-bp intron, which is found at the identical site in rat and mouse ALS genes. Sequence analysis reveals that the hALS 5'-flanking sequence is homologous to the mALS promoter, and that the GH-responsive GLE in the mALS promoter is conserved in both sequence and location in the hALS gene. The region spanning from -755 to -4 bp 5' to the hALS ATG translation start codon directs expression of a luciferase reporter gene in primary rat hepatocytes, and GH increases reporter expression in the presence of the native, but not a mutant, GLE in the hALS promoter. These data suggest that GH stimulates hALS and mALS gene expression by a similar mechanism, which involves at least in part a conserved GLE in the ALS promoter.
Assuntos
Proteínas de Transporte/genética , Cromossomos Humanos Par 16 , Glicoproteínas/genética , Hormônio do Crescimento/fisiologia , Hormônio do Crescimento Humano/fisiologia , Regiões Promotoras Genéticas , Animais , Sequência de Bases , Proteínas de Transporte/metabolismo , Mapeamento Cromossômico , Clonagem Molecular , Éxons , Glicoproteínas/metabolismo , Humanos , Íntrons , Camundongos , Dados de Sequência Molecular , Ratos , Mapeamento por Restrição , Alinhamento de Sequência , Homologia de Sequência do Ácido Nucleico , Somatomedinas/metabolismoRESUMO
Cell-association and processing of insulin-like growth factor binding protein-3 (IGFBP-3) by cultured bovine fibroblasts results in markedly enhanced type I IGF receptor signaling at a step distal to ligand binding. The purpose of the present study was to determine the intracellular mediators of IGFBP-3's potentiating effect. Preincubation of cultured bovine fibroblasts with 50 nM IGFBP-3 had no effect alone, but enhanced by 3- to 4-fold IGF-I-stimulated 3H-aminoisobutryric acid (AIB) uptake. IGFBP-3-induced potentiation was specifically prevented if an inhibitor of phosphatidylinositol 3 (PI3)-kinase activation (LY294002), but not an inhibitor of mitogen-activated protein kinase activation (PD98059), was present during the preincubation period. IGFBP-3 did not directly activate the downstream effector of PI3-kinase, protein kinase B (PKB)/Akt. However, the sensitivity of PKB/Akt to activation by IGF-I was increased by 2- to 4-fold with IGFBP-3 pretreatment. This increased sensitivity was accompanied by altered mobility of PKB/Akt on SDS-polyacrylamide gels, suggestive of a diminished phosphorylation state. Consistent with this, okadaic acid, a potent serine/threonine phosphatase inhibitor, was able to block the potentiation effect of IGFBP-3 and prevent the altered mobility of the PKB/Akt molecule in response to IGFBP-3 treatment. PKB/Akt immunoprecipitated from IGFBP-3-pretreated cells was no longer recognized by an antibody specific for phosphorylated threonine followed by proline. These data indicate that IGFBP-3 modulates type I IGF receptor signaling through an effect on PI-3-kinase pathway substrates and suggest a novel mechanism of dephosphorylation whereby PKB/Akt is transformed into a more sensitive substrate of type I IGF receptor signaling.