RESUMO
Duchenne muscular dystrophy (DMD) is a progressive muscle wasting disease usually resulting in death of patients by their early twenties. In contrast, mice lacking dystrophin (Dmd(mdx)), appear physically normal despite their underlying muscle pathology. Mice deficient for both dystrophin and the dystrophin-related protein, utrophin, (Dmd(mdx);Utrn-/- mice) die between 6 and 20 weeks of age suffering from severe muscle weakness with joint contractures, pronounced growth retardation and kyphosis, suggesting that dystrophin and utrophin play complementary roles. The exact cause of death in these mice was not determined. Here we show that expression of a truncated utrophin transgene solely within the skeletal muscle of these mutants prevents premature death and the development of any clinical phenotype. In the absence of full-length dystrophin and utrophin, the presence of truncated utrophin also decreases muscle fibre regeneration, relocalizes the dystrophin protein complex to the sarcolemma and re-establishes a normal expression pattern of developmental muscle proteins. These data suggest that Dmd(mdx);Utrn-/- mice succumb to a skeletal muscle defect and that their reduced lifespan is not due to cardiac or neurogenic components. The phenotypic rescue observed demonstrates that the Dmd(mdx);Utrn-/- mice are an ideal model for testing gene delivery protocols for the expression of utrophin or dystrophin in skeletal muscle. To determine the cause of death of the Dmd(mdx):Utrn-/- mice.
Assuntos
Proteínas do Citoesqueleto/genética , Distrofina/deficiência , Distrofina/genética , Expressão Gênica , Proteínas de Membrana/genética , Músculo Esquelético/metabolismo , Distrofia Muscular Animal/terapia , Transgenes , Animais , Proteínas do Citoesqueleto/deficiência , Feminino , Terapia Genética , Imuno-Histoquímica , Masculino , Proteínas de Membrana/deficiência , Camundongos , Camundongos Knockout , UtrofinaRESUMO
Duchenne and Becker muscular dystrophies are caused by mutations in the gene encoding dystrophin, a component of the subsarcolemmal cytoskeleton. Dystrophin-related proteins are identical or homologous to the cysteine-rich and C-terminal domains of dystrophin. This part of dystrophin binds to a membrane-spanning glycoprotein complex in muscle. At least five dystrophin-related proteins are encoded by the Duchenne muscular dystrophy locus. These proteins are found in many non-muscle tissues where dystrophin is not expressed and they are thought to be membrane-associated. Two other dystrophin-related proteins--utrophin and an 87 kDa postsynaptic protein--are encoded by separate loci and, like dystrophin, they are components of the neuromuscular junction.
RESUMO
Utrophin is a dystrophin-related cytoskeletal protein expressed in many tissues. It is thought to link F-actin in the internal cytoskeleton to a transmembrane protein complex similar to the dystrophin protein complex (DPC). At the adult neuromuscular junction (NMJ), utrophin is precisely colocalized with acetylcholine receptors (AChRs) and recent studies have suggested a role for utrophin in AChR cluster formation or maintenance during NMJ differentiation. We have disrupted utrophin expression by gene targeting in the mouse. Such mice have no utrophin detectable by Western blotting or immunocytochemistry. Utrophin-deficient mice are healthy and show no signs of weakness. However, their NMJs have reduced numbers of AChRs (alpha-bungarotoxin [alpha-BgTx] binding reduced to approximately 60% normal) and decreased postsynaptic folding, though only minimal electrophysiological changes. Utrophin is thus not essential for AChR clustering at the NMJ but may act as a component of the postsynaptic cytoskeleton, contributing to the development or maintenance of the postsynaptic folds. Defects of utrophin could underlie some forms of congenital myasthenic syndrome in which a reduction of postsynaptic folds is observed.
Assuntos
Proteínas do Citoesqueleto/deficiência , Proteínas do Citoesqueleto/genética , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Junção Neuromuscular/fisiopatologia , Sinapses/patologia , Animais , Western Blotting , Feminino , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Camundongos Endogâmicos ICR , Camundongos Knockout , Músculo Esquelético/química , Músculo Esquelético/imunologia , Junção Neuromuscular/química , Junção Neuromuscular/metabolismo , Fenótipo , Receptores Colinérgicos/metabolismo , Transmissão Sináptica , UtrofinaRESUMO
During the past year significant progress has been made in understanding how dystrophin deficiency leads to muscle cell necrosis in Duchenne muscular dystrophy and Becker muscular dystrophy. Dystrophin interacts with a glycoprotein complex spanning the muscle sarcolemma, effectively linking the actin cytoskeleton to the extracellular matrix. The carboxyl terminus of dystrophin is required for glycoprotein binding. Interestingly, at least three mRNAs transcribed from the distal end of the DMD gene in tissues other than muscle have been shown to encode this domain. Deficiency of a second component of the dystrophin-associated glycoprotein complex has been shown to occur in another muscle-wasting disorder, severe childhood autosomal recessive muscular dystrophy. Sequence analysis of the entire cDNA for the autosomal dystrophin-related protein utrophin has shown that dystrophin and utrophin are closely related. Furthermore, both of these proteins have been shown to bind to the same or a similar glycoprotein complex in muscle.
Assuntos
Distrofina/genética , Proteínas de Membrana , Distrofias Musculares/genética , Animais , Proteínas do Citoesqueleto/genética , Distrofina/metabolismo , Humanos , UtrofinaRESUMO
Utrophin is a close homolog of dystrophin, the protein whose mutations cause Duchenne muscular dystrophy (DMD). Utrophin is present at low levels in normal and dystrophic muscle, whereas dystrophin is largely absent in DMD. In such cases, the replacement of dystrophin using a utrophin gene transfer strategy could be more advantageous because utrophin would not be a neoantigen. To establish if adenovirus (AV)-mediated utrophin gene transfer is a possible option for the treatment of DMD, an AV vector expressing a shortened version of utrophin (AdCMV-Utr) was constructed. The effect of utrophin overexpression was investigated following intramuscular injection of this AV into mdx mice, the mouse model of DMD. When the tibialis anterior (TA) muscles of 3- to 5-day-old animals were injected with 5 microl of AdCMV-Utr (7.0 x 10(11) virus/ml), an average of 32% of fibers were transduced and the transduction level remained stable for at least 60 days. The presence of utrophin restored the normal histochemical pattern of the dystrophin-associated protein complex at the cell surface and resulted in a reduction in the number of centrally nucleated fibers. The transduced fibers were largely impermeable to the tracer dye Evans blue, suggesting that utrophin protects the surface membrane from breakage. In vitro measurements of the force decline in response to high-stress eccentric contractions demonstrated that the muscles overexpressing utrophin were more resistant to mechanical stress-induced injury. Taken together, these data indicate that AV-mediated utrophin gene transfer can correct various aspects of the dystrophic phenotype. However, a progressive reduction in the number of transduced fibers was observed when the TA muscles of 30- to 45-day-old mice were injected with 25 microl of AdCMV-Utr. This reduction coincides with a humoral response to the AV and transgene, which consists of a hybrid mouse-human cDNA.
Assuntos
Adenoviridae , Proteínas do Citoesqueleto/uso terapêutico , Técnicas de Transferência de Genes , Vetores Genéticos , Proteínas de Membrana/uso terapêutico , Distrofias Musculares/terapia , Animais , Animais Recém-Nascidos , Proteínas do Citoesqueleto/genética , Expressão Gênica , Humanos , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos mdx , Músculos/patologia , Distrofias Musculares/patologia , Fenótipo , UtrofinaRESUMO
Utrophin is an autosomally-encoded homologue of dystrophin, the protein product of the Duchenne muscular dystrophy (DMD) gene. Although, utrophin is very similar in sequence to dystrophin and possesses many of the protein-binding properties ascribed to dystrophin, both proteins are expressed in an apparently reciprocal manner and may be coordinately regulated. In normal skeletal muscle, utrophin is found at the neuromuscular junction (NMJ) whereas dystrophin predominates at the sarcolemma. However, during development, and in some myopathies including DMD, utrophin is also found at the sarcolemma. This re-distribution is often associated with a significant increase in the levels of utrophin. At the NMJ utrophin co-localizes with the acetylcholine receptors (AChR) and may play a role in the stabilization of the synaptic cytoskeleton. Because utrophin and dystrophin are so similar, utrophin may be able to replace dystrophin in dystrophin deficient muscle. This review compares the structure and function of utrophin to dystrophin and discusses the rationale behind the use of utrophin as a potential therapeutic agent.
Assuntos
Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/fisiologia , Proteínas de Membrana , Animais , Embrião de Mamíferos/metabolismo , Desenvolvimento Embrionário e Fetal , Genes , Humanos , Músculo Esquelético/metabolismo , Junção Neuromuscular/fisiologia , Músculos Papilares/metabolismo , Relação Estrutura-Atividade , Distribuição Tecidual , UtrofinaRESUMO
Utrophin is a large cytoskeletal protein which shows high homology to dystrophin. In contrast to the sarcolemmal distribution of dystrophin, utrophin accumulates at the postsynaptic membrane of the neuromuscular junction. Because of its localization within this compartment of muscle fibers, expression of utrophin may be significantly influenced by the presence of the motor nerve. We tested this hypothesis by denervating muscles of mdx mouse and monitoring levels of utrophin and its mRNA by immunofluorescence, immunoblotting and RT-PCR. A significant increase in the number of utrophin positive fibers was observed by immunofluorescence 3 to 21 days after sectioning of the sciatic nerve. Quantitative analyses of utrophin and its transcripts in hindlimb muscles denervated for two weeks showed only a moderate increase in the levels of both utrophin (approximately 2-fold) and its transcript (approximately 60 to 90%). The present data suggest that although utrophin is a component of the postsynaptic membrane, its neural regulation is distinct from that of the acetylcholine receptor.
Assuntos
Proteínas do Citoesqueleto/genética , Expressão Gênica , Proteínas de Membrana , Denervação Muscular , Músculos/metabolismo , Distrofias Musculares/metabolismo , RNA Mensageiro/metabolismo , Animais , Sequência de Bases , Imunofluorescência , Immunoblotting , Camundongos , Camundongos Endogâmicos mdx , Dados de Sequência Molecular , Músculos/inervação , Reação em Cadeia da Polimerase , DNA Polimerase Dirigida por RNA , Receptores Colinérgicos/metabolismo , Nervo Isquiático/fisiologia , UtrofinaRESUMO
This paper reviews the evidence that utrophin, the autosomally encoded protein related to dystrophin, may be capable of performing the same cellular functions as dystrophin. If this is the case, it may be possible to modify the regulation of utrophin expression as an alternative route to dystrophin gene therapy for sufferers of DMD and/or BMD.
Assuntos
Proteínas do Citoesqueleto/biossíntese , Distrofina/biossíntese , Proteínas de Membrana , Distrofias Musculares/genética , Distrofias Musculares/terapia , Adulto , Animais , Proteínas do Citoesqueleto/genética , Distrofina/genética , Desenvolvimento Embrionário e Fetal , Regulação da Expressão Gênica , Terapia Genética , Idade Gestacional , Humanos , Camundongos , Camundongos Mutantes Neurológicos , Desenvolvimento Muscular , Músculos/embriologia , Músculos/metabolismo , Distrofias Musculares/metabolismo , Distrofia Muscular Animal/metabolismo , UtrofinaRESUMO
The analysis of dystrophin gene expression has led to the identification of multiple transcripts and varying isoforms. The data indicate that transcription of the dystrophin gene occurs from several promoters, which involves developmental and tissue-dependent regulation. These discoveries have complicated the interpretation of immunolocalization studies, although there is a strong correlation between the amount and size of dystrophin and the severity of the clinical phenotype. The importance of using protein-specific antibodies for dystrophin analysis has been underscored by the identification of a protein, designated utrophin, which exhibits significant sequence homology with dystrophin. This review addresses the recent studies of dystrophin and utrophin expression in an attempt to illustrate the transcriptional diversity of these large genes and the localization of their protein products within various tissues.
Assuntos
Distrofina/biossíntese , Expressão Gênica , Proteínas do Tecido Nervoso/biossíntese , Sequência de Aminoácidos , Animais , Sequência de Bases , Distrofina/genética , Regulação da Expressão Gênica , Humanos , Dados de Sequência Molecular , Músculos/metabolismo , Distrofias Musculares/genética , Distrofias Musculares/metabolismo , Proteínas do Tecido Nervoso/genética , Regiões Promotoras Genéticas , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico , Transcrição GênicaRESUMO
Duchenne muscular dystrophy (DMD) is an inherited, severe muscle wasting disease caused by the loss of the cytoskeletal protein, dystrophin. Patients usually die in their late teens or early twenties of cardiac or respiratory failure. We have previously demonstrated that the dystrophin related protein, utrophin is able to compensate for the loss of dystrophin in the mdx mouse, the mouse model of the disease. Expression of a utrophin transgene under the control of an HSA promoter results in localization of utrophin to the sarcolemma and prevents the muscle pathology. Here we show that the over-expression of full-length utrophin in a broad range of tissues is not detrimental in the mdx mouse. These findings have important implications for the feasibility of the up-regulation of utrophin in therapy for DMD since they suggest that tissue specific up-regulation may not be necessary.
Assuntos
Proteínas do Citoesqueleto/biossíntese , Proteínas de Membrana/biossíntese , Distrofia Muscular Animal/metabolismo , Envelhecimento , Animais , Western Blotting , Peso Corporal , Creatinina/urina , Proteínas do Citoesqueleto/genética , Modelos Animais de Doenças , Progressão da Doença , Estudos de Viabilidade , Expressão Gênica , Terapia Genética , Homozigoto , Imuno-Histoquímica , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos mdx , Camundongos Transgênicos , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Distrofia Muscular Animal/genética , Distrofia Muscular Animal/patologia , Distrofia Muscular Animal/terapia , Especificidade de Órgãos , Regiões Promotoras Genéticas , Distribuição Tecidual/genética , Transgenes , Regulação para Cima/genética , UtrofinaRESUMO
31P NMR spectroscopy was used to study the energy metabolism of dystrophin-deficient skeletal muscle of mdx mice, an animal model of Duchenne muscular dystrophy, in which expression of a truncated form of utrophin has been obtained through transgenesis technology. Measurements of ATP, phosphocreatine (PCr), inorganic phosphates (Pi) and intracellular pH (pHi) were made at rest, during a fatigue protocol and during the subsequent recovery. Mechanical fatigue of transgenic muscles was similar to normal muscle, while mdx muscle showed larger force loss. At rest, muscles of all groups had similar values for [ATP], [PCr], [Pi] and pHi. During fatigue, [PCr] decreases mirrored [Pi] increases and were similar in all groups. The major difference between mdx muscles and the group of normal and trc-utrophin muscles concerned the values and evolution of pHi. The mdx muscles showed a more severe intracellular acidosis during exercise and a slower and incomplete post-exercise recovery of normal pHi. In contrast, in trc-utrophin muscles, the kinetics and amplitude of pHi changes were remarkably close to normal behaviour. We conclude that the impaired proton washout which is present in mdx muscles, is corrected to a great extent by the expression of trc-utrophin.
Assuntos
Proteínas do Citoesqueleto/química , Proteínas do Citoesqueleto/metabolismo , Hidrogênio/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Atividade Motora/fisiologia , Distrofia Muscular Animal/fisiopatologia , Animais , Concentração de Íons de Hidrogênio , Espectroscopia de Ressonância Magnética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos mdx/metabolismo , Camundongos Endogâmicos mdx/fisiologia , Camundongos Transgênicos , Distrofia Muscular Animal/diagnóstico , Distrofia Muscular Animal/metabolismo , Fósforo , UtrofinaRESUMO
Reversal of the A-ring amide link in 1,2-dibenzamidobenzene 1 (fXa K(ass) = 0.81 x 10(6) L/mol) led to a series of human factor Xa (hfXa) inhibitors based on N(2)-aroylanthranilamide 4. Expansion of the SAR around 4 showed that only small planar substituents could be accommodated in the A-ring for binding to the S1 site of hfXa. Bulky groups such as 4-isopropyl, 4-tert-butyl, and 4-dimethylamino were favored in the B-ring to interact with the S4 site of hfXa. The central (C) ring containing a 5-methanesulfonamido group yielded greater activity than carbamoyl groups. Combining the beneficial features from the B- and C-ring SAR, compound 55 represents the most potent hfXa inhibitor in the N(2)-aroylanthranilamide 4 series with hfXa K(ass) = 58 x 10(6) L/mol (K(i) = 11.5 nM).
Assuntos
Anticoagulantes/síntese química , Benzamidas/síntese química , Inibidores Enzimáticos/síntese química , Inibidores do Fator Xa , Sulfonamidas/síntese química , Anticoagulantes/química , Anticoagulantes/metabolismo , Benzamidas/química , Benzamidas/metabolismo , Sítios de Ligação , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Fator Xa/química , Fator Xa/metabolismo , Humanos , Modelos Moleculares , Relação Estrutura-Atividade , Sulfonamidas/química , Sulfonamidas/metabolismoRESUMO
High-throughput screening of a combinatorial library of diamidophenols yielded lead compounds with the ability to inhibit human factor Xa (fXa) at micromolar concentrations (e.g. compound 4, fXa apparent K(ass) = 0.64 x 10(6) L/mol). SAR studies in this novel structural series of fXa inhibitors showed that the phenolic hydroxyl group was not essential for activity. The best activity was found in substituted 1,2-dibenzamidobenzenes in which the phenyl group of one benzoyl group (A-ring) was substituted in the 4-position with relatively small lipophilic or polarizable groups such as methoxy, vinyl, or chloro and the phenyl group of the other benzoyl group (B-ring) was substituted in the 4-position with larger lipophilic groups such as tert-butyl or dimethylamino. The central phenyl ring (C-ring) tolerated a wide variety of substituents, but methoxy, methanesulfonamido, hydroxyl, and carboxyl substitution produced slightly higher levels of activity than other substituents when present in combination with favorable B-ring substitution. Methylation of the amide nitrogen atoms was found to greatly decrease activity. Compound 12 is the highest affinity fXa inhibitor in this group of compounds, having fXa apparent K(ass) = 25.5 x 10(6) L/mol, about 40x more active than the original lead. This lead series does not show potent inhibition of human thrombin. A model for the binding of these ligands to the fXa active site is proposed. The model is consistent with the observed SAR and can serve to guide future SAR studies.
Assuntos
Anticoagulantes/síntese química , Inibidores Enzimáticos/síntese química , Inibidores do Fator Xa , Fenilenodiaminas/síntese química , Sulfonamidas/síntese química , Trombina/antagonistas & inibidores , Anticoagulantes/química , Anticoagulantes/metabolismo , Sítios de Ligação , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Fator Xa/química , Fator Xa/metabolismo , Humanos , Modelos Moleculares , Fenilenodiaminas/química , Fenilenodiaminas/metabolismo , Fenilenodiaminas/farmacologia , Inibidores de Serina Proteinase/síntese química , Inibidores de Serina Proteinase/química , Inibidores de Serina Proteinase/metabolismo , Relação Estrutura-Atividade , Sulfonamidas/química , Sulfonamidas/metabolismo , Sulfonamidas/farmacologia , Trombina/metabolismoRESUMO
To enhance the potency of 1,2-dibenzamidobenzene-derived inhibitors of factor Xa (fXa), an amidine substituent was incorporated on one of the benzoyl side chains to interact with Asp189 in the S1 specificity pocket. Lead molecule 1 was docked into the active site of fXa to facilitate inhibitor design. Subsequently, iterative SAR studies and molecular modeling led to a 1000-fold increase in fXa affinity and a refined model of the new inhibitors in the fXa active site. Strong support for the computational model was achieved through the acquisition of an X-ray crystal structure using thrombin as a surrogate protein. The amidines in this series show high levels of selectivity for the inhibition of fXa relative to other trypsin-like serine proteases. Furthermore, the fXa affinity of compounds in this series (K(ass) = 50-500 x 10(6) L/mol) translates effectively into both anticoagulant activity in vitro and antithrombotic activity in vivo.
Assuntos
Amidinas/síntese química , Anticoagulantes/síntese química , Inibidores Enzimáticos/síntese química , Inibidores do Fator Xa , Fibrinolíticos/síntese química , Amidinas/química , Amidinas/metabolismo , Amidinas/farmacologia , Animais , Anticoagulantes/química , Anticoagulantes/metabolismo , Anticoagulantes/farmacologia , Sítios de Ligação , Cristalografia por Raios X , Cães , Desenho de Fármacos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Fator Xa/química , Fator Xa/metabolismo , Fibrinolíticos/química , Fibrinolíticos/metabolismo , Fibrinolíticos/farmacologia , Humanos , Técnicas In Vitro , Masculino , Modelos Moleculares , Tempo de Protrombina , Coelhos , Ratos , Ratos Sprague-Dawley , Relação Estrutura-Atividade , Trombina/química , Trombina/metabolismo , Trombose/tratamento farmacológicoAssuntos
Distrofina/química , Distrofina/metabolismo , Proteínas de Membrana , Sequência de Aminoácidos , Animais , Proteínas do Citoesqueleto/química , Proteínas do Citoesqueleto/metabolismo , Distrofina/genética , Humanos , Dados de Sequência Molecular , Distrofias Musculares/genética , Distrofias Musculares/metabolismo , Mutação , Ligação Proteica , Conformação Proteica , Estrutura Terciária de Proteína , UtrofinaAssuntos
Anticoagulantes/farmacologia , Benzamidas/farmacologia , Derivados de Benzeno/farmacologia , Inibidores do Fator Xa , Fibrinolíticos/farmacologia , Animais , Anticoagulantes/química , Anticoagulantes/uso terapêutico , Benzamidas/química , Benzamidas/uso terapêutico , Derivados de Benzeno/química , Derivados de Benzeno/uso terapêutico , Modelos Animais de Doenças , Fator Xa/metabolismo , Fibrinolíticos/química , Fibrinolíticos/uso terapêutico , Humanos , Piperidinas/química , Coelhos , Relação Estrutura-Atividade , Trombose/prevenção & controleAssuntos
Proteínas do Citoesqueleto , Distrofina , Proteínas de Membrana , Distrofias Musculares/genética , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Distrofina/química , Distrofina/genética , Distrofina/metabolismo , Feminino , Terapia Genética , Humanos , Masculino , Distrofias Musculares/terapia , UtrofinaRESUMO
The effect of inhibition of protein synthesis on interferon gamma (IFN-gamma) mRNA induction has been examined in human peripheral blood leukocytes and growing T lymphoblasts. Inhibition of protein synthesis by cycloheximide or puromycin when lymphocytes were stimulated with mitogen alone had little effect on the steady-state levels of IFN-gamma mRNA induced. Activation of the IFN-gamma gene appears to occur independently of synthesis of protein factors. Synergistic induction of IFN-gamma by mitogen plus the phorbol ester mezerein was at least in part accounted for by increased levels of IFN-gamma mRNA in both fresh lymphocytes and growing lymphoblasts. Inhibition of protein synthesis abolished the synergistic effect on mRNA leading to levels similar to those observed in cells treated with mitogen alone. Synergistic induction is dependent upon synthesis of protein factors either within the cells or produced as soluble mediators; these factors are not simply lymphokines since addition of exogenous factors to the cultures did not reverse the block on synergy by the inhibition. These data suggest that protein factors though they may be important in exerting qualitative control on the level of production of IFN-gamma have no role in the initial activation of the gene.
Assuntos
Proteínas Sanguíneas/biossíntese , Diterpenos , Interferon gama/biossíntese , Mitógenos/farmacologia , Linfócitos T/metabolismo , Proteínas Sanguíneas/fisiologia , Meios de Cultura/farmacologia , Cicloeximida/farmacologia , Sinergismo Farmacológico , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Indutores de Interferon/farmacologia , Interferon gama/genética , Ativação Linfocitária/efeitos dos fármacos , RNA Mensageiro/biossíntese , Terpenos/farmacologia , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
The promoter region of a keratin 6 (K6) gene was used to regulate expression of the early region of human papillomavirus type 1 (HPV-1e) in transgenic mice. In one line of mice the K6-HPV1e transgene was transcribed in several regions of the skin, the predominant transcript being a 1.1 kb RNA including the E4 open reading frame, and E1-E4 protein was detected in the upper suprabasal layers of the skin in paws and tail. A 1.7 kb RNA corresponding to the E6/E7 transcript was also prominent in tails of homozygous transgenic animals. In young homozygous transgenic mice the epidermis of the tail showed dysplasia and hyperplasia of the suprabasal layers with both hyperkeratosis and focal parakeratosis in the stratum corneum. A similar though milder phenotype was also observed sporadically in hemizygous transgenics. Analysis of the pattern of mouse keratins present in the affected tail skin showed strong up-regulation of the endogenous keratins 6 and 16 throughout the basal and suprabasal layers, suggesting a positive feedback mechanism for the strong transgene activation. Expression of the major differentiation-specific keratins 1 and 10 was repressed. The pattern of E1-E4 expression and the perturbation of normal epithelial differentiation parallel many of the characteristics of HPV-1 warts or verrucae, suggesting that HPV transgenic mice could be useful for analysis of the interactions of HPV gene products with cellular regulatory pathways within an otherwise normal epithelium.