RESUMEN
Recent advances in our understanding of cellular and molecular mechanisms of rheumatoid arthritis have highlighted a critical role for interleukin-1 and tumor necrosis factor alpha. The quest for chemically amenable targets has recently led to the identification and characterization of the intracellular signaling pathways associated with these inflammatory cytokines. In particular the mitogen-activated protein kinase pathway, the nuclear factor kappaB pathway and the cross-talk between these offer several potential therapeutic opportunities for rheumatoid arthritis.
Asunto(s)
Antirreumáticos/uso terapéutico , Artritis Reumatoide/tratamiento farmacológico , Transducción de Señal/efectos de los fármacos , Animales , Antirreumáticos/farmacología , Artritis Reumatoide/fisiopatología , Citocinas/fisiología , Humanos , Proteínas Quinasas Activadas por Mitógenos/fisiología , FN-kappa B/fisiología , Transducción de Señal/fisiologíaRESUMEN
PURPOSE: There is a need to find novel oestrogen receptor (ER) ligands that antagonize oestrogen action in the reproductive tissues and would therefore have therapeutic potential in oestrogen-dependent tumours. We tested novel ER ligands in both breast and endometrial cells to profile agonism/antagonism in these oestrogen target reproductive tissues. METHODS: Novel analogues of the ER antagonist ICI 182,780 were synthesized and tested for their ability to inhibit gene expression dependent on oestrogen response elements (ERE) in human breast (MCF-7) and endometrial (Ishikawa) cell lines. This activity was correlated with inhibition of oestrogen-induced cell proliferation and ER binding. RESULTS: The sulphide analogue (compound 1) and sulphone analogue (compound 2) had no intrinsic ERE-dependent agonism in either breast cancer or endometrial cells in culture. All three compounds dose-dependently inhibited ERE-mediated oestrogen agonism. Moreover, these ER ligands inhibited oestrogen-stimulated proliferation of breast cancer and endometrial cells. ICI 182,780, compound 1 and compound 2 were all able to bind both isoforms of the ER (ER alpha and ER beta). In endometrial cells, the relative binding to ER beta correlated with the ERE-dependent antioestrogenic effect of these ligands, suggesting that in this tissue this receptor is the predominant isoform that determines antioestrogenic activity. CONCLUSIONS: The ability of these analogues of ICI 182,780 to inhibit oestrogen-stimulated transcriptional activity and cell proliferation suggests that these agents, in particular the sulphone analogue, have therapeutic potential in the treatment of breast cancer without exhibiting the unwanted oestrogenic effects in the endometrium.
Asunto(s)
Antineoplásicos/farmacología , Neoplasias de la Mama/metabolismo , Endometrio/citología , Estradiol/análogos & derivados , Estradiol/farmacología , Antagonistas de Estrógenos/farmacología , Neoplasias Hormono-Dependientes/metabolismo , Receptores de Estrógenos/efectos de los fármacos , División Celular/efectos de los fármacos , Endometrio/efectos de los fármacos , Femenino , Fulvestrant , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Células Tumorales Cultivadas/efectos de los fármacosRESUMEN
Aberrant gene expression is a fundamental cause of many disease-associated pathophysiologies. The pharmacological modulation of transcription factor activity therefore represents an attractive therapeutic approach to such disorders. With the exception of nuclear receptors, which are the direct targets of pharmaceuticals, other known classes of transcription factors are largely regulated indirectly by drugs that impact upon those signal transduction cascades that alter transcription factor phosphorylation and dephosphorylation and/or nuclear import. However, recent advances in drug discovery technologies now enable high-throughput screens that can identify molecules that act directly at the level of transcription factor complexes.
Asunto(s)
Regulación de la Expresión Génica/efectos de los fármacos , Factores de Transcripción/efectos de los fármacos , Animales , Regulación de la Expresión Génica/genética , Humanos , Factores de Transcripción/química , Factores de Transcripción/genéticaRESUMEN
OBJECTIVE: To examine the expression pattern of human cartilage glycoprotein 39 (HC gp-39) mRNA in human cartilage and bone. DESIGN: In-situ hybridization analysis was used to examine the expression pattern of human cartilage glycoprotein 39 (HC gp-39) mRNA in adult human osteoarthritic articular cartilage from various stages of disease, as well as in human osteophytic tissue and in human fetal bone. RESULTS: In cartilage from patients with mild osteoarthritic cartilage degeneration, HC gp-39 was expressed at moderate to high levels only in chondrocytes of the superficial zone. In advanced OA cartilage, cloning chondrocytes of the superficial zone expressed high levels of HC gp-39 and chondrocytes of the mid- and deep zones were also positive. HC gp-39 was undetectable in the chondrocytes of normal articular cartilage. In osteophytic tissue, the expression of HC gp-39 mRNA was intense in flattened, end-stage osteoblasts and in primary osteocytes in both endochondral and intramembranous bone formation. Proliferating osteoblasts expressed low to moderate levels. Notably, mature osteocytes were negative for HC gp-39 expression. Chondrocytes in the secondary ossification center of developing fetal cartilage demonstrated high expression while growth plate and mineralized cartilage chondrocytes had lower expression. Osteoblasts at sites of endochondral and intramembranous bone formation were positive for expression of HC gp-39. CONCLUSIONS: The stage-specific expression of HC gp-39 in fetal development and adult remodelling bone and cartilage provides evidence for a specific functional or structural role for HC gp-39 in bone and cartilage tissue. HC gp-39 is expressed in diseased human osteoarthritic cartilage and osteophyte, but not in non-diseased tissue, and its distribution within the tissue changes as disease progresses. OA is characterized not only by cartilage degeneration, but by increased subchondral bone formation and osteophytosis. The results from this study indicate that the increased HC gp-39 expression in OA serum and synovial fluid may reflect not only cartilage degeneration but increased osteogenesis.
Asunto(s)
Remodelación Ósea/fisiología , Cartílago Articular/embriología , Cartílago Articular/metabolismo , Glicoproteínas/metabolismo , Osteoartritis/metabolismo , Adipoquinas , Adulto , Huesos/embriología , Proteína 1 Similar a Quitinasa-3 , Condrocitos/metabolismo , Expresión Génica , Humanos , Hibridación in Situ , Lectinas , Osteoblastos/metabolismo , Osteoclastos/metabolismo , ARN Mensajero/metabolismoRESUMEN
Bipotential cells in human trabecular bone explant cultures that express osteoblast characteristics are able to undergo adipogenesis in the presence of 3-isobutyl-1-methylxanthine plus dexamethasone (Nuttall et al. [1998] J Bone Miner Res 13:371-382). The initial studies of these bipotential cells in explant cultures have been extended to examine differential gene expression during osteoblast/adipocyte transdifferentiation. Using differential display, we have identified a gene expressed in trabecular bone explant cultures that is downregulated as these cells differentiate from an osteoblast to an adipocyte phenotype. Homology searching identified this gene as the human urea transporter HUT11. The expression and downregulation of HUT11 have been observed in multiple patient bone explant cultures. The size of the bone explant-derived HUT11 mRNA is approximately 4.4 kb, which is identical to the largest splice variant reported. In this article, we report the cloning and sequencing of this gene from primary human osteoblasts. In addition, we report tissue distribution for the bone explant-derived form of HUT11 mRNA and show a reciprocal relationship between the expression of HUT11 and the nuclear hormone receptor peroxisome proliferator-activated receptor gamma 2, which is a marker of adipocyte differentiation. Because the control of osteoblast/adipocyte transdifferentiation is unknown, selective downregulation of HUT11 during adipogenesis suggests that HUT11 expression may be a marker of the switch from an osteoblast to an adipocyte phenotype. Understanding the role of HUT11 in osteoblasts may provide insights into the mechanism controlling osteoblast and adipocyte differentiation.
Asunto(s)
Adipocitos/metabolismo , Proteínas Portadoras/genética , Diferenciación Celular/genética , Regulación de la Expresión Génica/genética , Glicoproteínas de Membrana/genética , Proteínas de Transporte de Membrana , Osteoblastos/metabolismo , 1-Metil-3-Isobutilxantina/farmacología , Secuencia de Bases , Biomarcadores , Proteínas Portadoras/metabolismo , Células Cultivadas , Clonación Molecular , Dexametasona/farmacología , Regulación hacia Abajo/genética , Histocitoquímica , Humanos , Glicoproteínas de Membrana/metabolismo , Datos de Secuencia Molecular , ARN Mensajero/metabolismo , Receptores Citoplasmáticos y Nucleares/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Transcripción/genética , Transportadores de UreaRESUMEN
Herpesvirus entry mediator (HVEM), a member of the tumor necrosis factor (TNF) receptor family, mediates herpesvirus entry into cells during infection. Upon overexpression, HVEM activates NF-kappaB and AP-1 through a TNF receptor-associated factor (TRAF)-mediated mechanism. Using an HVEM-Fc fusion protein, we screened soluble forms of novel TNF-related proteins derived from an expressed sequence tag data base. One of these, which we designated HVEM-L, specifically bound to HVEM-Fc with an affinity of 44 nM. This association was confirmed with soluble and membrane forms of both receptor and ligand. HVEM-L mRNA is expressed in spleen, lymph nodes, macrophages, and T cells and encodes a 240-amino acid protein. A soluble, secreted form of the protein stimulates proliferation of T lymphocytes during allogeneic responses, inhibits HT-29 cell growth, and weakly stimulates NF-kappaB-dependent transcription.
Asunto(s)
Antineoplásicos/metabolismo , Sustancias de Crecimiento/metabolismo , Proteínas de la Membrana/metabolismo , Receptores del Factor de Necrosis Tumoral/metabolismo , Receptores Virales/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Secuencia de Aminoácidos , Antineoplásicos/farmacología , Proteínas Bacterianas/metabolismo , Regulación de la Expresión Génica , Inhibidores de Crecimiento/metabolismo , Inhibidores de Crecimiento/farmacología , Sustancias de Crecimiento/farmacología , Células HT29/efectos de los fármacos , Humanos , Ligandos , Prueba de Cultivo Mixto de Linfocitos , Proteínas de la Membrana/genética , Proteínas de la Membrana/farmacología , Datos de Secuencia Molecular , FN-kappa B/metabolismo , Unión Proteica , Miembro 14 de Receptores del Factor de Necrosis Tumoral , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/farmacología , Homología de Secuencia de Aminoácido , Linfocitos T/efectos de los fármacos , Distribución Tisular , Factor de Transcripción AP-1/metabolismo , Miembro 14 de la Superfamilia de Ligandos de Factores de Necrosis Tumoral , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
TRAIL is a tumor necrosis factor-related ligand that induces apoptosis upon binding to its death domain-containing receptors, DR4 and DR5. Two additional TRAIL receptors, TRID/DcR1 and DcR2, lack functional death domains and function as decoy receptors for TRAIL. We have identified a fifth TRAIL receptor, namely osteoprotegerin (OPG), a secreted tumor necrosis factor receptor homologue that inhibits osteoclastogenesis and increases bone density in vivo. OPG-Fc binds TRAIL with an affinity of 3.0 nM, which is slightly weaker than the interaction of TRID-Fc or DR5-Fc with TRAIL. OPG inhibits TRAIL-induced apoptosis of Jurkat cells. Conversely, TRAIL blocks the anti-osteoclastogenic activity of OPG. These data suggest potential cross-regulatory mechanisms by OPG and TRAIL.
Asunto(s)
Apoptosis/fisiología , Glicoproteínas/fisiología , Glicoproteínas de Membrana/fisiología , Receptores Citoplasmáticos y Nucleares , Receptores del Factor de Necrosis Tumoral/fisiología , Factor de Necrosis Tumoral alfa/fisiología , Animales , Proteínas Reguladoras de la Apoptosis , Unión Competitiva , Proteínas Ligadas a GPI , Humanos , Fragmentos Fc de Inmunoglobulinas/genética , Inmunohistoquímica , Células Jurkat , Ligandos , Ratones , Microscopía Fluorescente , Oligopéptidos , Osteoclastos/citología , Osteoprotegerina , Péptidos/inmunología , Unión Proteica/fisiología , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF , Receptores del Factor de Necrosis Tumoral/clasificación , Receptores del Factor de Necrosis Tumoral/metabolismo , Miembro 10c de Receptores del Factor de Necrosis Tumoral , Miembro 25 de Receptores de Factores de Necrosis Tumoral , Proteínas Recombinantes de Fusión/metabolismo , Ligando Inductor de Apoptosis Relacionado con TNF , Receptores Señuelo del Factor de Necrosis TumoralRESUMEN
Human cartilage glycoprotein 39 (HC gp-39) has been described as a major secreted product of cultured articular chondrocytes, synovial fibroblasts, and the osteosarcoma line MG63. However, its expression in these cells types has not been directly linked to corresponding cell types in vivo. In this report, expression of HC gp-39 is demonstrated from peripheral blood-derived macrophages in association with their differentiation from monocytes to macrophages. Consistent with macrophage specificity, HC gp-39 expression is also induced upon selective stimulation of the pluripotent promyelocytic leukemia cell line HL-60 toward the monocyte/macrophage lineage with vitamin D3 or phorbol 12-myristate 13-acetate (PMA), while treatments stimulating granulocyte and eosinophilic pathways do not induce expression. Furthermore, HC gp-39 expression levels correlate with the degree of morphological differentiation induced by PMA and vitamin D3 treatments. PMA-induced mRNA expression occurs by 36 h and is a secondary transcriptional response since its synthesis is inhibited by cycloheximide. Apparently, HC gp-39 expression is tied to later events in the differentiation of monocytes into macrophages. The in vivo significance of these results is validated by the in situ detection of HC gp-39 mRNA in inflammatory macrophages associated with rheumatoid synovium. Thus, macrophages appear to be an important source of HC gp-39, which has been shown to be present at elevated levels in the blood and synovium of rheumatoid arthritis patients. The implications of this extend well beyond the previously restricted observations in cell types associated with the joint and suggest a potential involvement of macrophage-derived HC gp-39 in other aspects of inflammation, tissue remodeling, and host defense.
Asunto(s)
Artritis Reumatoide/metabolismo , Cartílago Articular/metabolismo , Glicoproteínas/biosíntesis , Macrófagos/metabolismo , Monocitos/metabolismo , Adipoquinas , Artritis Reumatoide/patología , Cartílago Articular/patología , Diferenciación Celular/efectos de los fármacos , Línea Celular , Células Cultivadas , Proteína 1 Similar a Quitinasa-3 , Colecalciferol/farmacología , Cicloheximida/farmacología , Células HL-60/metabolismo , Humanos , Inflamación , Células Jurkat/metabolismo , Cinética , Lectinas , Macrófagos/citología , Macrófagos/patología , Monocitos/citología , Reacción en Cadena de la Polimerasa , ARN Mensajero/biosíntesis , Proteínas Recombinantes/biosíntesis , Valores de Referencia , Membrana Sinovial/metabolismo , Membrana Sinovial/patología , Acetato de Tetradecanoilforbol/farmacología , Transcripción Genética/efectos de los fármacosRESUMEN
A human term placental cDNA library was screened at low stringency with a human prolactin cDNA probe. One of the cDNAs isolated hybridizes to a 1.8 kb mRNA present in all four tissues of the placenta as well as to every nucleated tissue and cell line tested. The sequence of the full-length cDNA was determined. An extended open reading frame predicted an encoded protein product of 20.5 kDa. This was directly confirmed by the in vitro translation of a synthetic mRNA transcript. Based upon the characteristic placement of cysteine (C) and histidine (H) residues in the predicted protein structure, this molecule contains four putative zinc fingers. The first and third fingers are of the C4 class while the second and fourth are of the C2HC class. Based upon sequence similarities between the first two and last two zinc fingers and sequence similarities to a related rodent protein, cysteine-rich intestinal protein (CRIP), these four finger domains appear to have evolved by duplication of a preexisting two finger unit. Southern blot analyses indicate that this human cysteine-rich protein (hCRP) gene has been highly conserved over the span of evolution from yeast to man. The characteristics of this protein suggest that it serves a fundamental role in cellular function.
Asunto(s)
Cisteína/análisis , Proteínas de Unión al ADN/genética , Metaloproteínas/genética , Hormonas Placentarias/genética , Proteínas Gestacionales/genética , Zinc , Secuencia de Aminoácidos , Secuencia de Bases , Evolución Biológica , Southern Blotting , ADN/genética , Femenino , Humanos , Datos de Secuencia Molecular , Placenta , Proteínas Gestacionales/biosíntesis , Proteínas Gestacionales/aislamiento & purificación , Prolactina/genética , ARN Mensajero/análisis , Homología de Secuencia de Ácido NucleicoRESUMEN
We have sought direct evidence for the in vivo expression of the human growth hormone-variant (hGH-V) gene by screening a placental cDNA library with a hGH-V-specific oligonucleotide. Nine independent hGH-V cDNA clones were isolated and analyzed, and three distinct species were detected. Five of these hGH-V cDNAs represent mRNAs spliced and processed in a pattern analogous to that of the highly homologous human growth hormone and human chorionic somatomammotropin gene transcripts. Each of the remaining four hGH-V cDNAs contains an additional segment of 253 nucleotides corresponding in position and sequence to the fourth intron of the hGH-V gene. In addition, one of the mRNAs in this second group uses an alternative downstream polyadenylation site. The alternatively spliced hGH-V mRNA, which we refer to as hGH-V2 mRNA, constitutes approximately 30% of the hGH-V transcripts both in the human term placenta and in a stable mouse fibroblast line expressing the transfected hGH-V gene. The placental expression of the hGH-V gene is specific to villous tissue. The hGH-V2 mRNA is predicted to encode a protein which substitutes the 65 carboxyl-terminal amino acids of hGH-V with a new 104-residue carboxyl terminus resulting in significant divergence in their relative physical properties. The alternative splicing of the hGH-V transcripts to hGH-V and hGH-V2 mRNAs expands the potential complexity of the hGH-V gene's role in normal placental function.
Asunto(s)
Genes , Variación Genética , Hormona del Crecimiento/genética , Placenta/metabolismo , ARN Mensajero/genética , Transcripción Genética , Secuencia de Aminoácidos , Secuencia de Bases , Gonadotropina Coriónica/genética , Clonación Molecular , ADN/genética , Femenino , Humanos , Datos de Secuencia Molecular , Hibridación de Ácido Nucleico , Embarazo , Conformación ProteicaRESUMEN
Thyroidectomy for giant goitres in Northern Nigeria is associated with a high incidence of postoperative asphyxia. Tracheostomy may be a life saving procedure in these circumstances, but delay may prove fatal when its need arises insidiously. It is therefore better established prophylactically in patients who are more than likely to develop asphyxia, as in the case of preoperative complications followed by prolonged surgery. During the dry, dust-laden and desiccating Harmattan season of Northern Nigeria, however, tracheostomy poses life-threatening dangers and should be established only in patients who need it for survival. Postoperative asphyxia can be minimised by adopting certain operative techniques which reduce the risks of postoperative haematoma and laryngeal oedema. Establishment of a thyroidectomy team for surgery and for postoperative management improves results.