RESUMO
Chinese hamster ovary (CHO) cells expressing recombinant human oncostatin M (rOM) were found to secrete high levels of a 28-kDa protein. Sequence analysis of the protein suggested that it was hamster tissue inhibitor of metalloproteinase-1 (TIMP-1). In this study, we show that induction of TIMP-1 mRNA and protein by CHO cells is due to rOM action in an autocrine/paracrine mode. TIMP-1 expression in rOM-producing CHO cells increased concomitantly with methotrexate-induced rOM amplification. TIMP-1 upregulation was not caused by either transfection of nonspecific DNA nor was it a direct effect of treatment of the cells with methotrexate. These results suggest that oncostatin M is a potent inducer of TIMP-1 and that its receptor-mediated expression is conserved across species.
Assuntos
Glicoproteínas/metabolismo , Metaloendopeptidases/antagonistas & inibidores , Peptídeos/farmacologia , Inibidores de Proteases/metabolismo , Animais , Células CHO , Cricetinae , Glicoproteínas/genética , Oncostatina M , RNA Mensageiro/biossíntese , Proteínas Recombinantes/farmacologia , Inibidores Teciduais de Metaloproteinases , Transfecção , Regulação para CimaRESUMO
Oncostatin M (OM) is a 28-kD glycoprotein that exhibits a panoply of biologic effects. Based on histologic observations of increased splenic megakaryocytes in nude mice implanted with an OM-secreting cell line, the thrombocytopoietic properties of OM in mice were investigated in culture and in vivo. Alone, OM did not induce megakaryocytic colony formation, but in combination with murine interleukin-3 (IL-3), OM markedly enhanced colony formation. The effects of OM on colony formation were similar to those of IL-6. OM alone augmented acetylcholinesterase in short-term marrow cultures. In normal mice, the administration of OM augmented platelet counts without increasing other circulating blood cell counts. The increment in counts exceeded that observed with IL-6. The kinetics of the OM response suggested that maximal increases in platelets occurred 3 days after the cessation of OM administration, irrespective of the duration of administration. In irradiated mice, OM administration accelerated platelet recovery and prevented the decrease in red blood cells observed in irradiated control animals. The data show that OM behaves as a megakaryocytic maturation factor in vitro and augments platelet production in vivo. Based on these animal data, OM may have potential clinical utility as a thrombocytopoietic agent.
Assuntos
Plaquetas/citologia , Hematopoese/efeitos dos fármacos , Peptídeos/administração & dosagem , Anemia/tratamento farmacológico , Animais , Sinergismo Farmacológico , Feminino , Interleucina-3/administração & dosagem , Interleucina-6/administração & dosagem , Megacariócitos/citologia , Camundongos , Camundongos Endogâmicos C3H , Oncostatina M , Contagem de Plaquetas/efeitos dos fármacos , Fatores de TempoAssuntos
Citocinas/farmacologia , Peptídeos/farmacologia , Proteínas de Fase Aguda/biossíntese , Animais , Citocinas/farmacocinética , Hematopoese/efeitos dos fármacos , Interleucina-6/biossíntese , Interleucina-6/farmacologia , Taxa de Depuração Metabólica , Camundongos , Oncostatina M , Peptídeos/farmacocinética , Contagem de Plaquetas , Proteínas Recombinantes , Proteína Amiloide A Sérica/biossíntese , Trombocitopenia/tratamento farmacológico , Distribuição TecidualRESUMO
Oncostatin M belongs to the subfamily of hematopoietin cytokines that binds a receptor complex containing gp130. To date, only the human form of oncostatin M has been identified, and its evolutionary conservation is unresolved. We have isolated a bovine gene whose open reading frame encodes a precursor protein that is 58% identical to human oncostatin M. A comparison of the bovine and human amino acid sequences predicts significant similarity, including the four-alpha-helical-bundle structure and the placement of disulfide bridges. As with the human protein, bovine oncostatin M binds specific receptors on human H2981 cells and inhibits the proliferation of human A375 tumor cells and mouse M1 leukemia cells. To identify activities regulated in vivo, we injected bovine oncostatin M fusion genes containing various tissue-specific promoters into mouse embryos. The frequencies of transgenic mice were reduced significantly, suggesting that overexpression of the bovine cytokine is detrimental to normal mouse development. In addition to deaths associated with expression in neurons and keratinized epithelia, bovine oncostatin M caused abnormalities in bone growth and spermatogenesis, stimulated fibrosis surrounding islets in the pancreas, and disrupted normal lymphoid tissue development. This work establishes the existence of a nonprimate oncostatin M gene and provides the first demonstration that this cytokine can function in a pleiotropic manner in vivo. Information regarding bovine oncostatin M may help characterize the structure and function of this cytokine in other vertebrate species.
Assuntos
Anormalidades Congênitas/genética , Peptídeos/genética , Peptídeos/fisiologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Bovinos , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Citocinas/química , Citocinas/genética , Citocinas/fisiologia , DNA/genética , Desenvolvimento Embrionário e Fetal/genética , Desenvolvimento Embrionário e Fetal/fisiologia , Expressão Gênica , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Oncostatina M , Peptídeos/química , Receptores de Citocinas/metabolismo , Receptores de Oncostatina M , Homologia de Sequência de AminoácidosRESUMO
Amphiregulin (AR) is an epidermal growth factor (EGF)-related peptide that operates exclusively through the EGF receptor and that can bind to heparin. AR also possesses nuclear localization sequences in the extended NH2-terminal region suggesting an additional intracellular site of action. AR mRNA and protein expression have been detected in primary human mammary epithelial cell strains, nontransformed human mammary epithelial cell lines, several human breast cancer cell lines, and primary human breast carcinomas. The frequency and levels of AR protein expression are generally higher in invasive breast carcinomas than in ductal carcinomas in situ or in normal, noninvolved mammary epithelium. In addition, AR can function as an autocrine and/or juxtacrine growth factor in human mammary epithelial cells that have been transformed by an activated c-Ha-ras proto-oncogene or by overexpression of c-erb B-2. AR expression is also enhanced by mammotrophic hormones such as estrogens and other growth factors such as EGF.
Assuntos
Neoplasias da Mama/genética , Glicoproteínas/genética , Substâncias de Crescimento/genética , Peptídeos e Proteínas de Sinalização Intercelular , Sequência de Aminoácidos , Anfirregulina , Neoplasias da Mama/patologia , Família de Proteínas EGF , Receptores ErbB/genética , Glicoproteínas/fisiologia , Substâncias de Crescimento/fisiologia , Humanos , Dados de Sequência Molecular , Proto-Oncogene MasRESUMO
A computer-aided homology search of the GenBank nucleotide database using the amino acid sequence of human acyl CoA-binding protein (ACBP)/diazepam-binding inhibitor (DBI)-endozepine as a probe revealed that a genomic fragment containing the gene encoding the mallard duck (Anas platyrhynchos) S-acyl fatty acid synthase thioesterase also contains sequences which encode the duck homolog of ACBP/DBI. The duck ACBP/DBI gene is positioned downstream of the thioesterase gene in a tail-to-tail orientation separated from the 3' end of the thioesterase gene by only several hundred nucleotides. Three exons were identified that have strong homology to the published cDNA sequences of human and bovine ACBP/DBI. These exons define all of the coding region except for the amino-terminal domain, which was subsequently cloned by polymerase chain reaction (PCR) amplification. The encoded amino acid sequence of the duck ACBP/DBI is 62-68% homologous to mammalian ACBP/DBI sequences. While the mammalian ACBP/DBI is expressed mainly in the liver, with smaller amounts in the brain and heart, mRNA transcripts of duck ACBP/DBI were detected only in the brain with no evidence for expression in the liver or heart. The close proximity of the genes for ACBP/DBI and S-acyl fatty acid synthase thioesterase raises the possibility of co-regulation of expression.
Assuntos
Proteínas de Transporte/genética , Tioléster Hidrolases/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Northern Blotting , Bovinos , Clonagem Molecular , DNA , Inibidor da Ligação a Diazepam , Patos , Humanos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Homologia de Sequência de AminoácidosRESUMO
Recently, gp130, the signal transducer for interleukin 6 (IL-6), leukemia inhibitory factor (LIF), and ciliary neurotrophice factor (CNTF), was identified as the low-affinity receptor for oncostatin M (OM). However, it is not yet clear if OM binding to gp130 requires accessory factor(s) and if gp130 alone can mediate OM signalling. Here we report that: (a) expressing murine gp130 in BAF-B03 cells (BAF-m130) resulted in the appearance of a single class of low-affinity OM binding sites; (b) chemical cross-linking studies with 125I-OM identified a 180 kDa labelled complex on BAF-m130 cells; (c) OM cross-linking to the H2981 cell line which expresses both low- and high-affinity OM receptor, identified a 180 kDa and an additional 280 kDa species; (d) 125I-OM was specifically cross-linked to soluble recombinant gp130 (sgp130-Rg) in solution; and (e) the cellular proliferation of BAF-m130 was unaffected by OM treatment. These data indicate that gp130 can act as the low-affinity receptor for OM, however, gp130-OM interactions alone are unable to elicit cellular proliferation. This suggests that an additional factor(s) are required to interact with the OM/gp130 complex to form the high-affinity functional receptor. We propose that the 280 kDa species detected on H2981 cells is likely a complex of OM, gp130, and the putative beta chain of the functional OM high-affinity receptor. Recently, OM has been shown to be the major growth factor for Kaposi's sarcoma derived cells.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Antígenos CD , Inibidores do Crescimento/metabolismo , Interleucina-6/fisiologia , Glicoproteínas de Membrana/metabolismo , Peptídeos/metabolismo , Peptídeos/farmacologia , Receptores de Citocinas/metabolismo , Transdução de Sinais , Animais , Linhagem Celular , Chlorocebus aethiops , Receptor gp130 de Citocina , Citocinas/metabolismo , DNA/biossíntese , Replicação do DNA/efeitos dos fármacos , Humanos , Cinética , Glicoproteínas de Membrana/isolamento & purificação , Camundongos , Peso Molecular , Oncostatina M , Peptídeos/isolamento & purificação , Receptores de Citocinas/isolamento & purificação , Receptores de Oncostatina M , Proteínas Recombinantes de Fusão/metabolismo , Sarcoma de Kaposi/patologia , Timidina/metabolismo , Transfecção , Células Tumorais CultivadasRESUMO
Interleukin 6 (IL-6) is a major growth factor for tumor plasma cells involved in human multiple myeloma (MM). In particular, human myeloma cell lines (HMCL), whose growth is completely dependent on addition of exogenous IL-6, can be obtained reproducibly from every patient with terminal disease. Four cytokines, ciliary neurotropic factor (CNTF), IL-11, leukemia inhibitory factor (LIF), and oncostatin M (OM), use the same transducer chain (signal transducer gp130) as IL-6 and share numerous biological activities with this IL. We found that these four cytokines stimulated proliferation and supported the long-term growth of two out of four IL-6-dependent HMCL obtained in our laboratory. Half-maximal proliferation was obtained with cytokine concentrations ranging from 0.4 to 1.2 ng/ml for IL-11, LIF, and OM. CNTF worked at high concentrations only (90 ng/ml), but addition of soluble CNTF receptor increased sensitivity to CNTF 30-fold. The growth-promoting effect of these four cytokines was abrogated by anti-gp130 antibodies, contrary to results for anti-IL-6 receptor or anti-IL-6 antibodies. No detectable changes in the morphology and phenotype were found when myeloma cells were cultured with one of these four cytokines instead of IL-6. Concordant with their IL-6-dependent growth, the four HMCL expressed membrane IL-6R and gp130 detected by FACS analysis. LIF-binding chain gene (LIFR) was expressed only in the two HMCL responsive to LIF and OM.
Assuntos
Antígenos CD , Substâncias de Crescimento/fisiologia , Interleucina-6/fisiologia , Glicoproteínas de Membrana/fisiologia , Mieloma Múltiplo/patologia , Transdução de Sinais , Fator Neurotrófico Ciliar , Receptor gp130 de Citocina , Inibidores do Crescimento/genética , Inibidores do Crescimento/fisiologia , Humanos , Interleucina-11/fisiologia , Fator Inibidor de Leucemia , Linfocinas/genética , Linfocinas/fisiologia , Mieloma Múltiplo/imunologia , Mieloma Múltiplo/metabolismo , Proteínas do Tecido Nervoso/fisiologia , Oncostatina M , Peptídeos/fisiologia , Receptor do Fator Neutrófico Ciliar , Receptores de Fatores de Crescimento/fisiologia , Células Tumorais CultivadasRESUMO
Amphiregulin (AR), a member of the epidermal growth factor (EGF) family, was found to be as potent as EGF in stimulating the anchorage-dependent growth (ADG) of immortalized, nontransformed human mammary epithelial MCF-10A cells. MCF-10A cells transformed by either an activated human c-Ha-ras protooncogene (MCF-10A ras) or by overexpression of a nonactivated rat c-neu gene (MCF-10A neu) exhibited a 35% reduction in the response to AR in ADG when compared to MCF-10A cells, but AR was still as potent as EGF in these transformants. Exogenous AR exhibited only 15-20% of the activity of EGF in stimulating the anchorage-independent growth, a response that is normally dependent upon exogenous EGF, of the oncogene-transformed MCF-10A cells. MCF-10A cells express low levels of a 1.4-kb AR mRNA transcript, while MCF-10A ras and MCF-10A neu cells display a 15- to 30-fold increase in the levels of AR mRNA and endogenous AR protein as determined by Western blot analysis. Exogenous EGF was found to induced both the AR mRNA and protein in the MCF-10A parental and transformed cells. A 20-mer phosphorothioate antisense deoxyoligonucleotide complementary to the 5' sequence of AR mRNA was able to significantly reduce the levels of endogenous AR protein and to inhibit the EGF-stimulated ADG and anchorage-independent growth of MCF-10A ras and MCF-10A neu cells. These data suggest that AR may function as an EGF-dependent autocrine growth factor in mammary epithelial cells that have been transformed by either a point-mutated c-Ha-ras or c-neu.
Assuntos
Genes ras , Glicoproteínas/farmacologia , Substâncias de Crescimento/farmacologia , Peptídeos e Proteínas de Sinalização Intercelular , Proteínas Oncogênicas Virais/genética , Anfirregulina , Divisão Celular/efeitos dos fármacos , Linhagem Celular Transformada , Transformação Celular Neoplásica , Família de Proteínas EGF , Fator de Crescimento Epidérmico/farmacologia , Epitélio/metabolismo , Glicoproteínas/análise , Glicoproteínas/genética , Substâncias de Crescimento/análise , Substâncias de Crescimento/genética , Humanos , Oligonucleotídeos Antissenso/farmacologia , RNA Mensageiro/análise , Receptor ErbB-2 , Tionucleotídeos/farmacologiaRESUMO
Propagation of the undifferentiated pluripotential phenotype of embryonic stem (ES) cells is dependent on the cytokine differentiation inhibiting activity/leukemia inhibitory factor (DIA/LIF). The DIA/LIF receptor complex is a heterodimer of DIA/LIF receptor (DIA/LIF-R) and gp130. The latter is also a component of the interleukin-6 (IL-6) receptor complex. We report that a combination of IL-6 and soluble IL-6 receptor (sIL-6R), which can induce homodimerisation of gp130 and activation of signalling processes, sustains self-renewal of pluripotential ES cells. Our findings indicate that the IL-6/sIL-6R complex acts on ES cells through gp130 alone, bypassing DIA/LIF-R, and therefore implicate gp130 as the key component in the signalling pathway responsible for stem cell renewal.
Assuntos
Antígenos CD , Inibidores do Crescimento/fisiologia , Interleucina-6 , Linfocinas/fisiologia , Glicoproteínas de Membrana/fisiologia , Receptores de Citocinas/fisiologia , Receptores de Interleucina/fisiologia , Transdução de Sinais , Células-Tronco/citologia , Sequência de Aminoácidos , Animais , Blastocisto/citologia , Diferenciação Celular , Células Cultivadas , Quimera , Receptor gp130 de Citocina , Feminino , Regulação da Expressão Gênica , Inibidores do Crescimento/farmacologia , Fator Inibidor de Leucemia , Subunidade alfa de Receptor de Fator Inibidor de Leucemia , Linfocinas/farmacologia , Masculino , Glicoproteínas de Membrana/química , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Fenótipo , Receptores de Citocinas/química , Receptores de Interleucina/química , Receptores de Interleucina-6 , Receptores de OSM-LIF , Células-Tronco/efeitos dos fármacos , Células-Tronco/fisiologiaRESUMO
Oncostatin M (OM) is a member of the cytokine family that includes leukaemia inhibitory factor (LIF), granulocyte colony stimulating factor (G-CSF), and interleukin 6 (IL-6). We previously reported the characterization of a monoclonal antibody (MAb) to OM, termed OM2, which neutralizes its functional activity. To gain information about the epitope detected by this MAb, we utilized a sandwich enzyme-linked immunoassay (EIA) to examine OM2 binding on a series of mutant recombinant OM molecules generated by site-directed mutagenesis, encompassing amino acid insertions, deletions, or alterations throughout the molecule. Carboxy-terminal deletions of a putative amphiphilic alpha helix past residue 185 abrogated binding of the OM2 MAb; alteration of a hydrophobic residue in the helix to a neutral one also prevented antibody binding. Analysis of mutants in which cysteines involved in intrachain disulfide binding were changed to serines revealed that one of two disulphide bonds was essential for OM2 binding. Two mutant molecules containing deletions in the amino-terminal one-fourth of the molecule were not bound by OM2, while a third mutant OM molecule with a C-terminal proximal internal deletion outside of the amphiphilic alpha helix was bound. The binding pattern of MAb OM2 to the OM mutant molecules correlated well with their functional activities. The data suggest that residues in both the C-terminal alpha helix and N-terminal one-fourth of the molecule are involved in neutralizing antibody binding and functional activity.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Anticorpos Monoclonais/metabolismo , Citocinas/metabolismo , Peptídeos/metabolismo , Animais , Anticorpos , Biotina , Linhagem Celular , Chlorocebus aethiops , Técnicas Imunoenzimáticas , Rim , Cinética , Mutagênese Sítio-Dirigida , Testes de Neutralização , Oncostatina M , Peptídeos/imunologia , Coelhos/imunologia , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Deleção de Sequência , TransfecçãoRESUMO
Oncostatin M and LIF are related members of a cytokine family that also includes IL-6, CNTF and G-CSF. These proteins exhibit overlapping biological properties and with the exception of G-CSF, they all appear to utilize gp130 as a signaling component of their high affinity receptor complexes. Recently it has been demonstrated that monomeric, membrane bound gp130 can directly bind OM. To further investigate the binding properties of gp130 we generated a soluble form of gp130, sgp130-Rg, to investigate potential gp130 interactions with OM and other members of this cytokine family. Using chemical crosslinking techniques we demonstrate that OM and LIF but not CNTF or IL-6 directly interact with sgp130-Rg. Since OM signaling can be prevented by binding gp130 with anti gp130 mAbs we also investigated the potential of sgp130-Rg to prevent the biological activities of both LIF and OM. Here we demonstrate that sgp130-Rg can bind LIF and OM preventing their biological activities on the TF-1 erythroleukemia cell line. This property suggests that sgp130-Rg may have therapeutic value in the specific prevention of LIF or OM mediated pathologies.
Assuntos
Antígenos CD , Inibidores do Crescimento/metabolismo , Interleucina-3/metabolismo , Interleucina-6/metabolismo , Linfocinas/metabolismo , Glicoproteínas de Membrana/metabolismo , Peptídeos/metabolismo , Receptores de Interleucina/fisiologia , Transdução de Sinais , Animais , Proteínas de Transporte/metabolismo , Linhagem Celular , Fator Neurotrófico Ciliar , Reagentes de Ligações Cruzadas , Receptor gp130 de Citocina , Citocinas , Inibidores do Crescimento/antagonistas & inibidores , Radioisótopos do Iodo , Fator Inibidor de Leucemia , Linfocinas/antagonistas & inibidores , Camundongos , Fatores de Crescimento Neural/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Oncostatina M , Peptídeos/antagonistas & inibidores , Receptores de Interleucina-6 , Transfecção/métodosRESUMO
We recently reported the molecular cloning of HER4/p180erbB4, a new member of the epidermal growth factor receptor family, as well as its activation by a partially purified ligand (Plowman, G. D., Culouscou, J.-M., Whitney, G. S., Green, J. M., Carlton, G. W., Foy, L., Neubauer, M. G., and Shoyab, M. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 1746-1750). In this report we describe the purification to homogeneity of a 45-kDa protein (p45) that induces the differentiation of MDA-MB-453 human breast cancer cells and stimulates the tyrosine phosphorylation of p180erbB4, the HER4-encoded protein. Hydrophobic interaction, ion-exchange, heparin, and size exclusion chromatographies were used to purify this p180erbB4 activator to homogeneity. N-terminal amino acid sequencing suggests that p45 is related to heregulin, a recently reported ligand for p185erbB2. Binding and cross-linking experiments demonstrated that p45 specifically binds to cells expressing recombinant p180erbB4 and not cells expressing recombinant p185erbB2.
Assuntos
Neoplasias da Mama/patologia , Diferenciação Celular/efeitos dos fármacos , Receptores ErbB/metabolismo , Proteínas Tirosina Quinases/metabolismo , Proteínas/farmacologia , Sequência de Aminoácidos , Animais , Células CHO , Carcinoma Hepatocelular/metabolismo , Cromatografia , Cricetinae , Receptores ErbB/efeitos dos fármacos , Glicoproteínas/química , Glicoproteínas/metabolismo , Glicoproteínas/farmacologia , Humanos , Neoplasias Hepáticas/metabolismo , Dados de Sequência Molecular , Peso Molecular , Neurregulinas , Fosforilação , Fosfotirosina , Proteínas Tirosina Quinases/efeitos dos fármacos , Proteínas/química , Proteínas/metabolismo , Receptor ErbB-4 , Receptores de Superfície Celular/metabolismo , Proteínas Recombinantes/metabolismo , Células Tumorais Cultivadas , Tirosina/análogos & derivados , Tirosina/metabolismoRESUMO
Oncostatin M (OM), a 28 kilodalton glycoprotein cytokine, is structurally and functionally related to interleukin 6 and leukemia-inhibitory factor. We reported previously that OM strongly up-regulated low density lipoprotein (LDL) receptors in human liver cells by a tyrosine kinase-mediated mechanism. Now, we demonstrate that the transcription factor Egr-1 is induced by OM. The induction of Egr-1 was time and concentration dependent; maximal inductions of 10-fold occurred by 30 min at concentrations of 10-25 ng/ml and higher. This concentration dependency was identical to those for OM-mediated tyrosine phosphorylation and LDL receptor up-regulation. The Egr-1, tyrosine kinase, and LDL receptor responses were inhibited at similar concentrations of genistein, suggesting that induction of Egr-1 and up-regulation of LDL receptors depended on activation of tyrosine kinase by OM. In contrast, depletion of protein kinase C by preincubation with 4 beta-phorbol 12-myristate 13 alpha-acetate did not affect OM-mediated induction of Egr-1 or up-regulation of LDL receptors, indicating that protein kinase C is not required for the OM action. Other similar cytokines were investigated, and, of these, only interleukin 1 could increase both Egr-1 and LDL receptor activity. The correlation among tyrosine kinase phosphorylation, Egr-1 induction, and LDL receptor regulation suggests that Egr-1 may be a nuclear signal transducer utilized by OM to induce transcription of the LDL receptor gene. In support of this possibility is the discovery of an Egr-1 consensus sequence (GAGGGGGCG) at approximately 330 base pairs upstream from the transcription initiation site of the LDL receptor promoter region.
Assuntos
Carcinoma Hepatocelular/metabolismo , Citocinas/farmacologia , Proteínas de Ligação a DNA/biossíntese , Proteínas Imediatamente Precoces , Neoplasias Hepáticas/metabolismo , Peptídeos/farmacologia , Receptores de LDL/efeitos dos fármacos , Fatores de Transcrição/biossíntese , Sequência de Bases , Proteína 1 de Resposta de Crescimento Precoce , Humanos , Dados de Sequência Molecular , Oncostatina M , Células Tumorais Cultivadas , Regulação para Cima/efeitos dos fármacos , Dedos de ZincoRESUMO
Oncostatin M (OM) is structurally and functionally related to a subclass of hematopoietic cytokines including leukemia-inhibitory factor (LIF), ciliary neurotrophic factor (CNTF), granulocyte colony-stimulating factor (G-CSF), and interleukin-6 (IL-6). Using human endothelial cells (HEC) as a model for cytokine regulation of hematopoietic growth factor expression, we tested OM as an inducer of colony-stimulating activity. Colony-forming cell assays supplemented with culture supernatants from OM-treated HEC contained a threefold increase in colony-forming unit granulocyte-macrophage colonies. Specific immunoassay (enzyme-linked immunosorbent assay) of culture supernatants indicated that OM treatment of HEC resulted in a dose- and time-dependent increase in the accumulation of G-CSF and granulocyte-macrophage CSF (GM-CSF) (> 28-fold). The ED50 for OM induction of G-CSF and GM-CSF protein expression was 17 and 7 pmol/L, respectively. Increased protein expression was associated with a similar increase in steady-state expression of G-CSF and GM-CSF mRNA. Furthermore, a period of 12 to 24 hours elapsed before there were measurable increases in CSF expression, suggesting that OM may stimulate CSF production through a mechanism requiring the synthesis or activation of a secondary mediating factor or pathway. These findings provide the first evidence that OM may regulate myelopoiesis by inducing the cellular expression of hematopoietic growth factors.
Assuntos
Endotélio Vascular/metabolismo , Fator Estimulador de Colônias de Granulócitos/metabolismo , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Hematopoese/efeitos dos fármacos , Peptídeos/farmacologia , Células da Medula Óssea , Células Cultivadas , Ensaio de Unidades Formadoras de Colônias , Expressão Gênica , Fator Estimulador de Colônias de Granulócitos/genética , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Humanos , Técnicas In Vitro , Oncostatina M , RNA Mensageiro/genética , Fatores de TempoRESUMO
Tissue inhibitor of metalloproteinases (TIMP-1) is a potent inhibitor of activated matrix metalloproteinases (MMP) such as collagenase, stromelysin, and gelatinase, and thus helps to control extracellular matrix metabolism and deposition by connective tissue cells. Since various cytokines and growth factors can modify the production of MMP and TIMP-1, we explored the action of oncostatin M (OM), a unique lymphocyte- and monocyte-derived cytokine, on expression of these proteins. We examined the regulation of TIMP-1 expression in cultured human fibroblasts by cytokines including OM, IL-6, leukemia inhibitory factor (LIF), and IL-1 alpha. When used at levels of 5 to 50 ng/ml, OM, IL-6, LIF, and IL-1 alpha elevated the TIMP-1 expression at the RNA level in fibroblasts of lung or synovial origin. Interestingly, OM stimulation resulted in highest levels of TIMP-1 RNA and protein synthesis. However, unlike IL-1 alpha, the cytokines OM, IL-6, and LIF did not induce MMP or PGE2 release. OM also enhanced TIMP-1 mRNA levels in the H2981 lung carcinoma and HepG2 hepatoma cell lines. The results suggest that OM as well as IL-6 and LIF, other cytokines acting through similar receptor pathways, may act to inhibit net MMP activity by specifically up-regulating TIMP-1 expression. The selective induction of TIMP-1 by OM may be influential in altering matrix destruction in chronic inflammation and tumor metastasis.
Assuntos
Citocinas/farmacologia , Glicoproteínas/biossíntese , Metaloendopeptidases/antagonistas & inibidores , Peptídeos/farmacologia , Células Cultivadas , Dinoprostona/metabolismo , Fibroblastos/metabolismo , Glicoproteínas/genética , Humanos , Interleucina-6/metabolismo , Neoplasias/metabolismo , Oncostatina M , RNA Mensageiro/análise , Inibidores Teciduais de MetaloproteinasesRESUMO
Epithelin 1 and 2 are cysteine-rich proteins that act as growth modulators of epithelial cells. In this report, we have characterized the epithelins receptors of MDA-MB-468 human breast carcinoma cells using both binding and cross-linking techniques. Equilibrium binding studies of iodinated epithelin 1 indicated that two classes of binding sites are expressed at the surface of breast carcinoma cells. The high affinity sites had a dissociation constant of approximately 2 x 10(-10) M, with approximately 290 receptors/cell. The low affinity sites had a dissociation constant of approximately 10(-8) M, with approximately 32,000 receptors/cell. Binding of iodinated epithelin 1 was specifically inhibited by unlabeled epithelin 1, 2, or 3, but not by other growth factors tested. We also performed competition binding studies of 125I-epithelin 1 to cell surface receptors in the presence of unlabeled epithelin 1, 2, or 3. Binding results analyzed by the method of Scatchard suggested that all three epithelins interact with a same receptor. A 140-145-kDa epithelin 1-binding protein complex was identified by chemical cross-linking of 125I-epithelin 1 to breast cancer cells. Formation of such a complex was prevented by coincubation of 125I-epithelin 1 with an excess of unlabeled epithelin 1, 2, or 3.
Assuntos
Inibidores do Crescimento/metabolismo , Inibidores do Crescimento/farmacologia , Substâncias de Crescimento/metabolismo , Substâncias de Crescimento/farmacologia , Peptídeos e Proteínas de Sinalização Intercelular , Receptores de Superfície Celular/metabolismo , Sítios de Ligação , Ligação Competitiva , Neoplasias da Mama , Divisão Celular/efeitos dos fármacos , Citocinas/farmacologia , Feminino , Granulinas , Humanos , Cinética , Receptores de Superfície Celular/isolamento & purificação , Células Tumorais CultivadasRESUMO
This report describes the isolation and recombinant expression of a cDNA clone encoding HER4, the fourth member of the human epidermal growth factor receptor (EGFR) family. The HER4/erbB4 gene encodes a 180-kDa transmembrane tyrosine kinase (HER4/p180erbB4) whose extracellular domain is most similar to the orphan receptor HER3/p160erbB3, whereas its cytoplasmic kinase domain exhibits 79% and 77% identity with EGFR and HER2/p185erbB2, respectively. HER4 is most predominantly expressed in several breast carcinoma cell lines, and in normal skeletal muscle, heart, pituitary, brain, and cerebellum. In addition, we describe the partial purification of a heparin-binding HER4-stimulatory factor from HepG2 cells. This protein was found to specifically stimulate the intrinsic tyrosine kinase activity of HER4/p180erbB4 while having no direct effect on the phosphorylation of EGFR, HER2, or HER3. Furthermore, this heparin-binding protein induces phenotypic differentiation, and tyrosine phosphorylation, of a human mammary tumor cell line that overexpresses both HER4 and HER2. These findings suggest that this ligand-receptor interaction may play a role in the growth and differentiation of some normal and transformed cells.
Assuntos
Receptores ErbB/genética , Receptores ErbB/fisiologia , Proteínas Tirosina Quinases/genética , Receptores de Superfície Celular/genética , Sequência de Bases , Células , Clonagem Molecular , Receptores ErbB/metabolismo , Expressão Gênica , Ligantes , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos/química , Proteínas Tirosina Quinases/metabolismo , RNA Mensageiro/genética , Receptor ErbB-4 , Receptores de Superfície Celular/fisiologia , Alinhamento de Sequência , Transdução de Sinais , Distribuição TecidualRESUMO
The COOH-terminal half of the amphiregulin (AR) molecule has sequence homology to epidermal growth factor (EGF). The ability of AR to elicit in vivo phosphorylation of the EGF receptor (EGFR) and p185erbB2 was studied in four human epithelial cell lines which expressed either or both of the receptor tyrosine kinases. AR induced the phosphorylation of the EGFR and p185erbB2, and phosphoamino acid analysis revealed enhanced phosphorylation of tyrosine residues in both receptor proteins. A monoclonal antibody (mAb) which binds to the extracellular domain of the EGFR blocked the phosphorylation of the EGFR and p185erbB2 as well as AR-induced mitogenesis indicating that the EGFR mediated these responses. In MDA-MB-453 cells which lack EGFRs, AR did not induce phosphorylation of p185erbB2, did not affect proliferation, and had no detectable effect on the phosphorylation of cellular proteins isolated using an anti-phosphotyrosine mAb. Qualitatively, in vivo phosphorylations induced by AR and EGF were found to be indistinguishable as demonstrated by analysis of cellular 32P-labeled proteins isolated with the anti-phosphotyrosine mAb. Moreover, in the presence of the anti-EGFR mAb, AR had no effect on the proliferation of cells. These results provide strong evidence that the EGFR is the sole cell surface mediator of the action of AR in human epithelial cells.
Assuntos
Receptores ErbB/metabolismo , Glicoproteínas/fisiologia , Substâncias de Crescimento/fisiologia , Peptídeos e Proteínas de Sinalização Intercelular , Proteínas Proto-Oncogênicas/metabolismo , Tirosina/análogos & derivados , Anfirregulina , Anticorpos Monoclonais , Divisão Celular , Células Cultivadas , Família de Proteínas EGF , Epitélio/metabolismo , Humanos , Técnicas In Vitro , Fosforilação , Fosfotirosina , Proteínas Tirosina Quinases/metabolismo , Receptor ErbB-2 , Tirosina/metabolismoRESUMO
The growth regulatory protein oncostatin M was initially discovered in macrophage-conditioned medium. We investigated the effects of oncostatin M on cultured rabbit aorta smooth muscle cells (SMCs) and found that the peptide stimulated an increase in the incorporation of [3H]thymidine into DNA. The magnitude of the stimulation was dependent on oncostatin M concentration and SMC confluency. In subconfluent cultures, 1-2 nM stimulated 4- to 5-fold increases in DNA synthesis after 20 hr. Other structurally related cytokines (granulocyte colony-stimulating factor, leukemia inhibitory factor, interleukin 6, ciliary neurotrophic factor) did not affect SMC DNA synthesis. After 5 or 8 days, oncostatin M caused a doubling in SMC number and also induced a transformed phenotype. The combination of oncostatin M and platelet-derived growth factor for 8 days resulted in a 4-fold increase in cell number, approximately the same increase in cell number as induced by the addition of 10% fetal calf serum. Further investigation suggested that the mitogenic effect of oncostatin M was in part due to tyrosine kinase activation. Within 1-2 min, the factor increased phosphotyrosine levels of several SMC proteins. In addition, detectable increases in diacylglycerol levels occurred within 2-5 min, reached 50% above control by 30 min, and remained elevated through 45 min of incubation with oncostatin M. SMC inositol phosphate levels were also elevated within 2 min and then returned to near control values by 20 min. Within 30 min, oncostatin M induced expression of the immediate-early gene EGR-1. These data indicate that oncostatin M may be an important, naturally occurring mitogen for vascular SMCs.