RESUMEN
Hypertrophic scar (HS) formation is associated with the fibrosis of fibrocytes caused by excessive extracellular matrix (ECM) synthesis and deposition, the initial event of HS formation. Our high throughput screen of miRNA expression profiles identified hsa-miR31-5p, whose transcription level was most differentially in normal skin fibroblasts (NS) and HS among other miRNAs. The level of hsa-miR31-5p in HS was significantly higher than in NS. In-vitro functional experiments showed hsa-miR31-5p knockdown remarkably suppressed the proliferation of hypertrophic scar fibroblasts (HSFBs) under hypoxia, promoted cell invasion, and inhibited the expression of Collagen I and III and Fibronectin (FN), suggesting that hsa-miR31-5p knockdown effectively reduces HS formation caused by excessive ECM synthesis and deposition in HSFBs under hypoxia. Mechanism study showed that the regulation of HS formation by hsa-miR31-5p was mediated by its target gene, factor-inhibiting HIF-1 (FIH): under hypoxia, hsa-miR31-5p down-regulated FIH and thus increased the level of hypoxia inducible factor-1α (HIF-1α), which subsequently activated the HIF-1α fibrosis regulation pathway in HSFBs, and stimulated the proliferation and ECM synthesis in HSFBs, eventually resulting in fibrosis and scar formation. The data also show that knockdown of hsa-miR31-5p in HSFBs impaired the trend of increased proliferation, reduced invasion and excessive ECM synthesis and deposition caused by HIF-1a activation under hypoxia through upregulating FIH, indicating that knockdown of hsa-miR31-5p effectively inhibits the formation of HS. In conclusion, hsa-miR31 -5p plays an important role in HS formation by inhibiting FIH and regulating the HIF-1α pathway. Therefore, hsa-miR31 -5p may be a novel therapeutic target for HS.
Asunto(s)
Antagomirs/genética , Cicatriz Hipertrófica/genética , Fibroblastos/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , MicroARNs/genética , Oxigenasas de Función Mixta/genética , Proteínas Represoras/genética , Antagomirs/metabolismo , Hipoxia de la Célula , Movimiento Celular , Proliferación Celular , Cicatriz Hipertrófica/metabolismo , Cicatriz Hipertrófica/patología , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Colágeno Tipo II/genética , Colágeno Tipo II/metabolismo , Matriz Extracelular/metabolismo , Matriz Extracelular/patología , Fibroblastos/patología , Fibronectinas/genética , Fibronectinas/metabolismo , Fibrosis , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , MicroARNs/antagonistas & inhibidores , MicroARNs/metabolismo , Oxigenasas de Función Mixta/metabolismo , Cultivo Primario de Células , Proteínas Represoras/metabolismo , Transducción de SeñalRESUMEN
We have isolated and characterized the first Xenopus transmembrane Eph ligand, XLerk (Xenopus Ligand for Eph Receptor Tyrosine Kinases). While this ligand has 72% identity with the closest mammalian family member, Lerk-2, it is the cytoplasmic domain of this molecule that is the most conserved domain with 95% identity. XLerk exists as a maternally expressed mRNA, however, expression of transcripts and protein increase during gastrulation and again in the late swimming tadpole stage. In the adult, XLerk is expressed at low levels in most adult tissues with increased levels observed in the kidney, oocytes, ovary and testis. While low levels of XLerk expression are observed in the adult brain, in situ hybridization analysis demonstrates prominent expression in the developing olfactory system, retina, hindbrain, cranial ganglia, and somites. Furthermore, we have shown that XLerk transcripts are significantly elevated during mesoderm induction caused by activin and FGF, but not during noggin-induced neuralization. These results suggest a role for XLerk in the developing mesenchymal and nervous tissue.
Asunto(s)
Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Mesodermo/fisiología , Neuronas/fisiología , Proteínas de Xenopus , Xenopus laevis/embriología , Xenopus laevis/genética , Activinas , Factores de Edad , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión , Clonación Molecular , Secuencia Conservada , Citoplasma/metabolismo , ADN Complementario , Embrión no Mamífero/fisiología , Inducción Embrionaria/genética , Efrina-B1 , Evolución Molecular , Femenino , Regulación del Desarrollo de la Expresión Génica , Hibridación in Situ , Inhibinas/metabolismo , Inhibinas/farmacología , Masculino , Mamíferos/genética , Mesodermo/efectos de los fármacos , Datos de Secuencia Molecular , Proteínas/genética , Proteínas Tirosina Quinasas Receptoras/metabolismo , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Distribución Tisular , Xenopus laevis/crecimiento & desarrolloRESUMEN
The host range of retroviral oncogenes is naturally limited by the host range of the retroviral vector. The question of whether the transforming host range of retroviral oncogenes is also restricted by the host species has not been directly addressed. Here we have tested in avian and murine host species the transforming host range of two retroviral onc genes, myc of avian carcinoma viruses MH2 and MC29 and mht/raf of avian carcinoma virus MH2 and murine sarcoma virus MSV 3611. Virus vector-mediated host restriction was bypassed by recombining viral oncogenes with retroviral vectors that can readily infect the host to be tested. It was found that, despite high expression, transforming function of retroviral myc genes is restricted to avian cells, and that of retroviral mht/raf genes is restricted to murine cells. Since retroviral oncogenes encode the same proteins as certain cellular genes, termed protooncogenes, our data must also be relevant to the oncogene hypothesis of cancer. According to this hypothesis, cancer is caused by mutation of protooncogenes. Because protooncogenes are conserved in evolution and are presumed to have conserved functions, the oncogene hypothesis assumes no host range restriction of transforming function. For example, mutated human proto-myc is postulated to cause Burkitt lymphoma, because avian retroviruses with myc genes cause cancer in birds. But there is no evidence that known mutated protooncogenes can transform human cells. The findings reported here indicate that host range restriction appears to be one of the reasons (in addition to insufficient transcriptional activation) why known, mutated protooncogenes lack transforming function in human cells.
Asunto(s)
Transformación Celular Neoplásica , Genes myc , Oncogenes , Proteínas Oncogénicas de Retroviridae/genética , Retroviridae/genética , Animales , Evolución Biológica , Aves , Línea Celular , Células Cultivadas , Mapeo Cromosómico , Embrión de Mamíferos , Embrión no Mamífero , Vectores Genéticos , Humanos , Proteínas Oncogénicas v-raf , Proto-Oncogenes , Ratas , Ratas Endogámicas F344 , Virus del Sarcoma Murino/genética , Especificidad de la Especie , TransfecciónRESUMEN
The product of protooncogene c-mos, pp39mos, is expressed and functions during oocyte maturation. We have previously found that pp39mos is complexed with and phosphorylates tubulin. In addition, part of pp39mos is localized on mitotic spindle and spindle pole regions in c-mosxe-transformed NIH/3T3 cells. Here, we further characterized the interaction between pp39mos and tubulin. We show that mos product synthesized in vitro appears in a 500 kD complex and can oligomerize with tubulin in vitro under tubulin polymerization conditions. Moreover, conditions which favor microtubule depolymerization facilitate pp39mos extraction from c-mosxe-transformed NIH/3T3 cells. We also show by immunofluorescence and immunoelectron microscopy that pp39mos is localized on microtubules. Thus, in vitro and in vivo the mos product is associated with tubulin and microtubules, respectively. Therefore, the mos product may be involved in the modification of microtubules and formation of the spindle.
Asunto(s)
Microtúbulos/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Tubulina (Proteína)/metabolismo , Animales , Línea Celular , Técnicas In Vitro , Ratones , Microscopía Electrónica , Oocitos/metabolismo , Unión Proteica , Proteínas Proto-Oncogénicas c-mosRESUMEN
The mos proto-oncogene product, pp39mos, is a protein kinase and has been equated with cytostatic factor (CSF), an activity in unfertilized eggs that is thought to be responsible for the arrest of meiosis at metaphase II. The biochemical properties and potential substrates of pp39mos were examined in unfertilized eggs and in transformed cells in order to study how the protein functions both as CSF and in transformation. The pp39mos protein associated with polymers under conditions that favor tubulin oligomerization and was present in an approximately 500-kilodalton "core" complex under conditions that favor depolymerization. beta-Tubulin was preferentially coprecipitated in pp39mos immunoprecipitates and was the major phosphorylated product in a pp39mos-dependent immune complex kinase assay. Immunofluorescence analysis of NIH 3T3 cells transformed with Xenopus c-mos showed that pp39mos colocalizes with tubulin in the spindle during metaphase and in the midbody and asters during telophase. Disruption of microtubules with nocodazole affected tubulin and pp39mos organization in the same way. It therefore appears that pp39mos is a tubulin-associated protein kinase and may thus participate in the modification of microtubules and contribute to the formation of the spindle. This activity expressed during interphase in somatic cells may be responsible for the transforming activity of pp39mos.
Asunto(s)
Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Tubulina (Proteína)/metabolismo , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales , Línea Celular , Transformación Celular Neoplásica/metabolismo , Sustancias Macromoleculares , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Fosfoproteínas/metabolismo , Pruebas de Precipitina , Unión Proteica , Proteínas Proto-Oncogénicas c-mosAsunto(s)
Transformación Celular Neoplásica/genética , Neoplasias/genética , Oncogenes , Recombinación Genética , Animales , Transformación Celular Viral , Regulación Neoplásica de la Expresión Génica , Humanos , Modelos Biológicos , Regiones Promotoras Genéticas , Proto-Oncogenes , Retroviridae/genética , Retroviridae/fisiología , Transducción GenéticaRESUMEN
Lymphomas of certain strains of chickens infected by retroviruses frequently contain recombinant transforming genes in which the promoter of the cellular proto-myc gene is replaced by that of a defective rather than an intact retrovirus. Here we ask whether the resulting hybrid genes are sufficient for tumorigenic transformation like viral myc genes. Further, we ask whether retroviruses must be defective in order to mutate proto-myc to a transforming gene or whether the defectiveness plays a transformation-independent function in tumorigenesis. For this purpose the defective provirus of proviral-proto-myc recombinants from lymphomas were repaired, or intact proviruses were recombined with proto-myc genes in vitro, and then compared to recombinant proto-myc genes with defective proviruses for transforming function in quail embryo fibroblasts. It was found that a single copy of a provirus-proto-myc recombinant gene with an intact provirus is sufficient to transform a quail embryo cell in vitro. Moreover, our analyses showed that multiple internal retroviral deletions [corrected] eliminate or inhibit provirus expression. The effect of these deletions [corrected] was detectable only because the inactive proviruses were linked to the selectable, transforming proto-myc gene marker. It is consistent with our results that proviral defectiveness of recombinant proto-myc genes is necessary in vivo for the clonal growth of a transformed cell into a tumor to escape antiviral immunity. The large discrepancy between the probabilities of provirus insertion and tumorigenesis is suggested to reflect the low probabilities of spontaneous deletion of the provirus and of rare, strain-specific defects of tumor-resistance genes of the host.
Asunto(s)
Virus del Sarcoma Aviar/genética , Transformación Celular Neoplásica , Virus Defectuosos/genética , Proto-Oncogenes , Animales , Inversión Cromosómica , ADN Viral/genética , ADN Viral/aislamiento & purificación , Embrión no Mamífero , Exones , Intrones , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-myc , Provirus/genética , Codorniz , Recombinación Genética , Mapeo RestrictivoRESUMEN
Retroviral onc genes are as yet the only proven cancer genes. They are generated by rare illegitimate recombinations between retroviruses and cellular genes, termed proto-onc genes. The claims that these proto-onc genes cause virus-free cancers upon "activation" by mechanisms that do not alter their germline structure are challenged. Instead, it is proposed that retroviral onc genes and cellular cancer genes are generated de novo by illegitimate recombinations that alter the germline structure of normal genes.
Asunto(s)
Oncogenes , Recombinación Genética , Retroviridae/genética , Animales , Regulación de la Expresión Génica , Humanos , Mutación , Proto-OncogenesRESUMEN
To define conditions under which the chicken protooncogene p-myc is converted to a viral and possibly to a cellular transforming gene, we assayed transforming function of hybrid genes put together from cloned retroviral and p-myc elements and of p-myc genes isolated from spontaneous viral lymphomas. Transforming function was measured in quail embryo cells transfected with cloned myc genes. We found that only myc genes with a promoter of a retroviral long terminal repeat (LTR) located between the native p-myc promoter and the second p-myc exon have transforming function. Transforming efficiencies decreased with increasing lengths of unspliced sequences between the LTR and p-myc exon 2. p-myc DNAs with LTRs downstream of the coding region or upstream but in the opposite transcriptional orientation failed to transform embryo cells. Likewise, only those retroviral-p-myc combinations from chicken B-cell lymphomas with a LTR positioned as promoter upstream of p-myc exon 2 had transforming function. We conclude that substitution of a retroviral LTR for the promoter and for as yet poorly defined, untranscribed regulatory elements of p-myc is sufficient to convert chicken p-myc to a transforming gene. However, retroviral LTRs can only convert p-myc genes to embryo-cell-transforming genes from a limited number of positions, and not as position-independent enhancers. Further, we deduce that there are two classes of viral chicken B-cell lymphomas, those with and those without embryo-cell-transforming p-myc genes.
Asunto(s)
Transformación Celular Neoplásica , Elementos de Facilitación Genéticos , Regiones Promotoras Genéticas , Retroviridae/genética , Animales , Transformación Celular Viral , Embrión de Pollo , Enzimas de Restricción del ADN/metabolismo , Electroforesis en Gel de Poliacrilamida , Linfoma/genética , Linfoma/microbiología , Proto-Oncogenes , Codorniz , Secuencias Repetitivas de Ácidos Nucleicos , Transcripción GenéticaRESUMEN
The transforming (onc) genes of retroviruses contain specific sequences, derived from as yet poorly defined, normal cellular genes, termed proto-onc genes. Proto-onc genes must be defined to explain their docility compared to the oncogenicity of the viral derivatives. Here we set out to determine the borders of the chicken proto-fps gene from which the onc genes of avian Fujinami (FSV) and PRC sarcoma viruses (PRCSV) are derived. These onc genes are hybrids of an element from the gag gene of retroviruses (delta gag) linked to a 2.8-kb domain from proto-fps. To identify the 5' border of proto-fps we have sequenced 1.5 kb beyond the 5' border of overlap with viral fps utilizing a proto-fps clone derived previously. A possible promoter was identified that maps 736 nucleotides from this border. The 736 nucleotides contain two possible exons with 121 codons, and short regions of homology with the delta gag termini of FSV and PRCII. A translation stop codon and an adjacent polyadenylation signal were identified just prior to the 3' border of overlap with viral fps within a 1.15-kb sequence of a newly isolated proto-fps clone. Comparing four exons within this 1.15 kb proto-fps sequence with known fps equivalents of FSV and PRCSV, we have detected strain-specific, but no common point mutations in each viral genome. A 3.3-kb polyadenylated proto-fps mRNA was detected in chicken liver RNA by gel electrophoresis and hybridization with proto-fps DNA. We conclude that the coding capacity of proto-fps is just over 3 kb, consistent with the size of the putative proto-fps protein of 98 kDa and hence slightly larger than that of viral fps. Thus proto-fps and the viral delta gag-fps genes each contain distinct 5' regulatory and coding sequences and share the 3' terminal fps domains. It is suggested that this difference, rather than scattered point mutations, is responsible for the oncogenic function of the viral genes and the unknown cellular function of proto-fps.
Asunto(s)
Virus del Sarcoma Aviar/genética , Secuencia de Bases , Pollos/genética , Genes Virales , Oncogenes , Proto-Oncogenes , Animales , Codón , Mutación , Regiones Promotoras Genéticas , Biosíntesis de Proteínas , Recombinación Genética , Transcripción GenéticaRESUMEN
Avian carcinoma virus MH2 contains two potential transforming genes, delta gag-mht and delta gag-myc. Thus, MH2 may be a model for two-gene carcinogenesis in which transformation depends on two synergistic genes. Most other directly oncogenic viruses contain single, autonomous transforming (onc) genes and are models for single-gene carcinogenesis. To determine which role each potential onc gene of MH2 plays in oncogenesis, we have prepared deletion and frameshift mutants of each of the two MH2 genes by in vitro mutagenesis of cloned proviral DNA and have tested transforming function and virus production in cultured primary quail cells. We have found that mht deletion mutants and wild-type virus transform primary cells and that myc deletion and frameshift mutants do not. The morphologies of cells transformed by the mht deletion mutants and by wild-type MH2 are similar yet vary considerably. Nevertheless, typical mutant transformed cells can often be distinguished from cells transformed by wild-type MH2. We conclude that the delta gag-myc gene transforms primary cells by itself, without the second potential onc gene. This myc-related gene is the smallest that has direct transforming function. delta gag-mht is without detectable transforming function but may affect transformation by delta gag-myc. Thus, MH2 behaves like a virus with a single onc gene, although it expresses two potential onc genes, and it appears not to be a model for two-gene carcinogenesis. Further work is necessary to determine whether the delta gag-mht gene possibly enhances oncogenic function of delta gag-myc or has independent oncogenic function in animals.