RESUMEN

Extrinsic and intrinsic factors contribute to skin aging; nonetheless, they are intertwined. Moreover, intrinsic skin aging mirrors age-related declines in the entire human body's internal organs. There is evidence that skin appearance is an indicator of the general health of somebody. Earlier, it was apparent that the intrinsic factors are unalterable, but the sparkling of skin aging gene therapy on the horizon is changing this narrative. Skin aging gene therapy offers tools for skin rejuvenation and, natural beauty restoration, and therapy for diseases affecting the entire skin. However, skin aging gene therapy is an arduous and sophisticated task relying on precise interim stimulation of telomerase to extend telomeres and wend back the biological clock in the hopes to find the fountain of youth, while preserving cells innate biological features. Finding the hidden fountain of youth will be a remarkable discovery for promoting aesthetics medicine, genecosmetics, and healthy aging. Caloric restriction offers ultimate health benefits and a reproducible way to promote longevity in mammals, while delaying age-related diseases. Moreover, exercise further enhances these health benefits. This article highlights the potential of skin aging gene therapy and foretells the emerging dawn of the genecosmetics era.

Asunto(s)

Distrofia Muscular de Duchenne , Humanos , Distrofia Muscular de Duchenne/genética , Terapia Genética , Distrofina/genética , Vectores Genéticos/genética , Vectores Genéticos/uso terapéutico

Asunto(s)

Sistema Endocrino , Endocrinología

RESUMEN

Dystrophinopathies are x-linked muscular disorders which emerge from mutations in the Dystrophin gene, including Duchenne and Becker muscular dystrophy, and dilated cardiomyopathy. However, Duchenne muscular dystrophy interconnects with bone loss and osteoporosis, which are exacerbated by glucocorticoids therapy. Procedures for diagnosing dystrophinopathies include creatine kinase assay, haplotype analysis, Southern blot analysis, immunological analysis, multiplex PCR, multiplex ligation-dependent probe amplification, Sanger DNA sequencing, and next generation DNA sequencing. Pharmacological therapy for dystrophinopathies comprises glucocorticoids (prednisone, prednisolone, and deflazacort), vamorolone, and ataluren. However, angiotensin-converting enzyme (ACE) inhibitors, angiotensin receptor blockers (ARBs), and ß-blockers are the first-line to prevent dilated cardiomyopathy in dystrophinopathy patients. Duchenne muscular dystrophy gene therapy strategies involve gene transfer, exon skipping, exon reframing, and CRISPR gene editing. Eteplirsen, an antisense-oligonucleotide drug for skipping exon 51 from the Dystrophin gene, is available on the market, which may help up to 14% of Duchenne muscular dystrophy patients. There are various FDA-approved exon skipping drugs including ExonDys-51 for exon 51, VyonDys-53 and Viltolarsen for exon 53 and AmonDys-45 for exon 45 skipping. Other antisense oligonucleotide drugs in the pipeline include casimersen for exon 45, suvodirsen for exon 51, and golodirsen for exon 53 skipping. Advances in the diagnosis and therapy of dystrophinopathies offer new perspectives for their early discovery and care.

Asunto(s)

Cardiomiopatía Dilatada , Distrofia Muscular de Duchenne , Humanos , Distrofina/genética , Distrofia Muscular de Duchenne/diagnóstico , Distrofia Muscular de Duchenne/tratamiento farmacológico , Distrofia Muscular de Duchenne/genética , Antagonistas de Receptores de Angiotensina , Inhibidores de la Enzima Convertidora de Angiotensina , Oligonucleótidos Antisentido/uso terapéutico , Oligonucleótidos Antisentido/genética

RESUMEN

Alzheimer and Parkinson diseases are associated with cholinergic neuron loss and deterioration of bone mineral density. Gene therapy through either gene transfer, CRISPR gene editing, or CRISPR gene modulation holds the potential to cure Alzheimer and Parkinson diseases. The emerging role of weight-bearing exercise in the prevention of, and care for, osteoporosis, obesity, and diabetes has been previously recognized. Moreover, endurance exercise offers a viable alternative to reduce amyloid peptides deposits while increasing bone mineral density in Alzheimer and Parkinson patients. ß-amyloid peptides, α-synuclein, and tau aggregates start building up two decades before the onset of Alzheimer and Parkinson diseases. Therefore, an early intervention program for the detection of these deposits is required to prevent or delay the onset of these diseases. This article spots light on the potential of gene therapy for Alzheimer and Parkinson diseases.

Asunto(s)

Enfermedad de Alzheimer , Enfermedad de Parkinson , Humanos , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/terapia , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/terapia , Péptidos beta-Amiloides/genética , Edición Génica , Terapia Genética , Proteínas tau/genética

RESUMEN

Duchenne and Becker muscular dystrophies are allelic X-linked recessive neuromuscular diseases affecting both skeletal and cardiac muscles. Therefore, owing to their single X chromosome, the affected boys receive pathogenic gene mutations from their unknowing carrier mothers. Current pharmacological drugs are palliative that address the symptoms of the disease rather than the genetic cause imbedded in the Dystrophin gene DNA sequence. Therefore, alternative therapies like gene drugs that could address the genetic cause of the disease at its root are crucial, which include gene transfer/implantation, exon skipping, and gene editing. Presently, it is possible through genetic reprogramming to engineer AAV vectors to deliver certain therapeutic cargos specifically to muscle or other organs regardless of their serotype. Similarly, it is possible to direct the biogenesis of exosomes to carry gene editing constituents or certain therapeutic cargos to specific tissue or cell type like brain and muscle. While autologous exosomes are immunologically inert, it is possible to camouflage AAV capsids, and lipid nanoparticles to evade the immune system recognition. In this review, we highlight current opportunities for Duchenne muscular dystrophy gene therapy, which has been known thus far as an incurable genetic disease. This article is a part of Gene Therapy of Rare Genetic Diseases thematic issue.

RESUMEN

Juvenile idiopathic arthritis (JIA) is the most common pediatric rheumatological condition. Although it has been proposed that JIA has an autoimmune component, the autoantigens are still unknown. Using biochemical and proteomic approaches, we identified the molecular chaperone transthyretin (TTR) as an antigenic target for B and T cell immune responses. TTR was eluted from IgG complexes and affinity purified from 3 JIA patients, and a statistically significant increase in TTR autoantibodies was observed in a group of 43 JIA patients. Three cryptic, HLA-DR1-restricted TTR peptides, which induced CD4+ T cell expansion and IFN-γ and TNF-α production in 3 out of 17 analyzed patients, were also identified. Misfolding, aggregation and oxidation of TTR, as observed in the synovial fluid of all JIA patients, enhanced its immunogenicity in HLA-DR1 transgenic mice. Our data point to TTR as an autoantigen potentially involved in the pathogenesis of JIA and to oxidation and aggregation as a mechanism facilitating TTR autoimmunity.

RESUMEN

PURPOSE: Recent advances in the treatment of ACL ruptures employ platelet-rich plasma combined with collagen to modulate growth factor release from platelets to stimulate healing. Among the most notable of these growth factors is VEGF, which is a potent mitogen and stimulator of vascular growth and healing. However, the effect of such a growth factor on healing depends on the cellular ability to bind with its receptor. The purpose of this study was to test (1) whether the strength of a tissue-engineered ACL repair is associated with VEGF receptors' mRNA expression of ACL cells and (2) whether age influences this association. METHOD: Nineteen female Yucatan pigs underwent enhanced ACL repair. Biomechanical testing was performed after 15 weeks of healing. Messenger RNA of VEGF receptors 1 and 2 in ACL fibroblasts was assessed by RT-PCR. The ACL structural properties were regressed on receptor expression levels in a multivariate model including serum levels of VEGF, age, and weight as potential confounders. RESULT: While maximum load and linear stiffness were independent of VEGF receptor expression, VEGF receptor 1 was associated with displacement (positively) and yield load (negatively). In a multivariate model of VEGF receptor expression and biomechanics, age was associated with maximum load and yield load. CONCLUSION: These findings suggest that high VEGF receptor expression, even more so at higher age, results in a more compliant scar, which in turn may lead to greater knee laxity and a compromised clinical result.

Asunto(s)

Reconstrucción del Ligamento Cruzado Anterior/métodos , Fibroblastos/metabolismo , Traumatismos de la Rodilla/cirugía , ARN Mensajero/metabolismo , Receptor 1 de Factores de Crecimiento Endotelial Vascular/metabolismo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo , Cicatrización de Heridas/fisiología , Factores de Edad , Animales , Ligamento Cruzado Anterior/metabolismo , Ligamento Cruzado Anterior/cirugía , Lesiones del Ligamento Cruzado Anterior , Reconstrucción del Ligamento Cruzado Anterior/instrumentación , Fenómenos Biomecánicos , Modelos Animales de Enfermedad , Femenino , Análisis Multivariante , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Porcinos , Ingeniería de Tejidos , Andamios del Tejido , Receptor 1 de Factores de Crecimiento Endotelial Vascular/sangre , Receptor 1 de Factores de Crecimiento Endotelial Vascular/genética , Receptor 2 de Factores de Crecimiento Endotelial Vascular/sangre , Receptor 2 de Factores de Crecimiento Endotelial Vascular/genética , Soporte de Peso

RESUMEN

AIMS: Inadequate capillary growth in pressure-overload hypertrophy impairs myocardial perfusion and substrate delivery, contributing to progression to failure. Capillary growth is tightly regulated by angiogenesis growth factors like vascular endothelial growth factor (VEGF) and endogenous inhibitors such as the splice variant of VEGF receptor-1, sVEGFR-1. We hypothesized that inadequate expression of VEGF and up-regulation of VEGFR-1 and its soluble splice variant, sVEGFR-1, restrict capillary growth in pressure-overload hypertrophy. METHODS AND RESULTS: Neonatal New Zealand White rabbits underwent aortic banding. mRNA (qRT-PCR) and protein levels (immunoblotting) were determined in hypertrophied and control myocardium (7/group) for total VEGF, VEGFR-1, sVEGFR-1, VEGFR-2, and phospho-VEGFR-1 and -R-2. Free VEGF was determined by enzyme-linked immunoassay (ELISA) in hypertrophied myocardium, controls, and hypertrophied hearts following inhibition of sVEGFR-1 with placental growth factor (PlGF). VEGFR-1 and sVEGFR-1 mRNA (seven-fold up-regulation, P = 0.001) and protein levels were significantly up-regulated in hypertrophied hearts vs. controls (VEGFR-1: 44 ± 8 vs. 23 ± 1, P = 0.031; sVEGFR-1: 71 ± 13 vs. 31 ± 3, P = 0.016). There was no change in VEGF and VEGFR-2 mRNA or protein levels in hypertrophied compared with controls hearts. A significant decline in free, unbound VEGF was found in hypertrophied myocardium which was reversed following inhibition of sVEGFR-1 with PlGF, which was accompanied by phosphorylation of VEGFR-1 and VEGFR-2. CONCLUSION: Up-regulation of the soluble VEGFR-1 in pressure-loaded myocardium prevents capillary growth by trapping VEGF. Inhibition of sVEGFR-1 released sufficient VEGF to induce angiogenesis and preserved contractile function. These data suggest sVEGFR-1 as possible therapeutic targets to prevent heart failure.

Asunto(s)

Capilares/metabolismo , Cardiomegalia/metabolismo , Miocardio/metabolismo , Neovascularización Fisiológica , Receptor 1 de Factores de Crecimiento Endotelial Vascular/metabolismo , Envejecimiento , Animales , Animales Recién Nacidos , Aorta/cirugía , Western Blotting , Capilares/efectos de los fármacos , Capilares/fisiopatología , Cardiomegalia/diagnóstico por imagen , Cardiomegalia/genética , Cardiomegalia/fisiopatología , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática , Ligadura , Neovascularización Fisiológica/efectos de los fármacos , Fosforilación , Factor de Crecimiento Placentario , Proteínas Gestacionales/administración & dosificación , ARN Mensajero/metabolismo , Conejos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Ultrasonografía , Regulación hacia Arriba , Factor A de Crecimiento Endotelial Vascular/metabolismo , Receptor 1 de Factores de Crecimiento Endotelial Vascular/genética , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo

RESUMEN

Bone matrix mineralisation plays a critical role in the determination of the overall biomechanical competence of bone. However, the molecular mechanisms of bone matrix mineralisation have not been fully elucidated. We used a proteomic approach to identify proteins and genes that may play a role in osteoblast matrix mineralisation. Proteomic differential display revealed a protein band that appeared only in mineralising mouse 7F2 osteoblasts. In-gel protein digestion and mass spectrometry proteomic analysis of this protein band identified 16 proteins. Furthermore, their corresponding transcripts were upregulated. This identification of proteins that may be associated with bone matrix mineralisation presents important new information toward a better understanding of the precise mechanisms of this process.

Asunto(s)

Calcificación Fisiológica/fisiología , Proteínas de la Matriz Extracelular/metabolismo , Osteoblastos/citología , Proteómica/métodos , Animales , Western Blotting , Células Cultivadas , Electroforesis en Gel Bidimensional , Proteínas de la Matriz Extracelular/genética , Expresión Génica , Ratones , Osteoblastos/metabolismo , ARN Mensajero/metabolismo

RESUMEN

LOX, the principal enzyme involved in crosslinking of collagen, was the first of several lysyl oxidase isotypes to be characterized. Its active form was believed to be exclusively extracellular. Active LOX was later reported to be present in cell nuclei; its function there is unknown. LOX expression opposes the effect of mutationally activated Ras, which is present in about 30% of human cancers. The mechanism of LOX in countering the action of Ras is also unknown. In the present work, assessment of nuclear protein for possible effects of lysyl oxidase activity led to the discovery that proliferating cells dramatically increase their nuclear protein content when exposed to BAPN (beta-aminopropionitrile), a highly specific lysyl oxidase inhibitor that reportedly blocks LOX inhibition of Ras-induced oocyte maturation. In three cell types (PC12 cells, A7r5 smooth muscle cells, and NIH 3T3 fibroblasts), BAPN caused a 1.8-, 1.7-, and 2.1-fold increase in total nuclear protein per cell, respectively, affecting all major components in both nuclear matrix and chromatin fractions. Since nuclear size is correlated with proliferative status, enzyme activity restricting nuclear growth may be involved in the lysyl oxidase tumor suppressive effect. Evidence is also presented for the presence of apparent lysyl oxidase isotype(s) containing a highly conserved LOX active site sequence in the nuclei of PC12 cells, which do not manufacture extracellular lysyl oxidase substrates. Results reported here support the hypothesis that nuclear lysyl oxidase regulates nuclear growth, and thereby modulates cell proliferation.

Asunto(s)

Núcleo Celular/enzimología , Proliferación Celular , Proteína-Lisina 6-Oxidasa/antagonistas & inhibidores , Proteína-Lisina 6-Oxidasa/metabolismo , Transporte Activo de Núcleo Celular/efectos de los fármacos , Aminopropionitrilo/farmacología , Animales , Dominio Catalítico , Bovinos , Ciclo Celular , ADN/metabolismo , Inhibidores Enzimáticos/farmacología , Proteínas de la Matriz Extracelular/metabolismo , Humanos , Ratones , Células 3T3 NIH , Células PC12 , Ratas

RESUMEN

Tissue engineering approaches that harness the stimulatory power of platelet-rich plasma have produced encouraging results in anterior cruciate ligament (ACL) repair. However, a number of recent studies have demonstrated age-dependent differences in cellular responses to such an approach. Identifying the reasons for these differences would allow counteracting them and consequently improve outcomes. In this study we hypothesized that these age-related effects are caused by differences in the expression of the receptors for growth factors released from platelet-rich plasma (PRP). Porcine ACL fibroblasts from a predetermined number of animals of different ages were obtained, and mRNA levels of the receptors of platelet-derived growth factor (PDGF), transforming growth factor beta (TGF-beta), fibroblast growth factor (FGF), and vascular endothelial growth factor (VEGF) were determined. Expression levels were compared across age groups (young and adolescent) and regressed on age in days. While no significant difference was seen across groups, the regression analysis showed decreases in receptor expression with increasing age. These differences were statistically significant for TGF-beta receptor 1, FGF receptor, and VEGF receptor 2; and borderline significant for TGF-beta receptor 3 and PDGF receptor. The only receptor that was not associated with age was VEGF receptor 1, a regulator of VEGF receptor 2. These findings suggest that the decrease in growth factor receptor expression as a likely reason for reduced PRP action with increasing age.

Asunto(s)

Ligamento Cruzado Anterior/citología , Receptores de Factores de Crecimiento de Fibroblastos/biosíntesis , Receptores del Factor de Crecimiento Derivado de Plaquetas/biosíntesis , Receptores de Factores de Crecimiento Transformadores beta/biosíntesis , Receptores de Factores de Crecimiento Endotelial Vascular/biosíntesis , Envejecimiento , Animales , Ligamento Cruzado Anterior/metabolismo , Femenino , Plasma Rico en Plaquetas/metabolismo , Porcinos

RESUMEN

Osteopontin is a noncollagenous, phosphorylated extracellular glycoprotein, expressed in mineralized and nonmineralized tissues, organs and body fluids. The protein contains an RGD tripeptide cell-binding motif, and is subjected to a variety of posttranslational modifications that play important roles in its multiple biological functions, such as bone remodeling and inhibition of pathological calcification. In this study, we have expressed bovine osteopontin in a prokaryotic system and identified the seven amino acid residues phosphorylated in vitro by CKII.

Asunto(s)

Remodelación Ósea/fisiología , Huesos/metabolismo , Calcinosis/prevención & control , Quinasa de la Caseína II/metabolismo , Osteopontina/metabolismo , Procesamiento Proteico-Postraduccional , Animales , Calcificación Fisiológica/fisiología , Quinasa de la Caseína II/química , Bovinos , Escherichia coli/genética , Escherichia coli/metabolismo , Matriz Extracelular/metabolismo , Osteogénesis/fisiología , Fosforilación , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Tripsina/metabolismo

RESUMEN

Vascular endothelial growth factor (VEGF) gene gives rise to several distinct isoforms of VEGF. Those isoforms differ in biochemical and biological properties, and it has been reported that their expression patterns are tissue and age specific as well. We investigated the expression levels of VEGF isoforms (VEGF121, VEGF165, VEGF183, VEGF189) and its receptors (VEGFR-1, flt-1 and VEGFR-2, flk-1/KDR) in the anterior cruciate ligament (ACL) of 2- to 3-week-, 2-month-, and 18-month-old New Zealand White rabbits using Sybr green Real-Time RT-PCR. VEGF isoforms and both receptors were expressed in the ACL at all investigated ages. VEGF121 was found to be the most abundant isoform at the ages under investigation, followed by VEGF165, VEGF189 and VEGF183. All isoforms showed decreased expression levels with age, however the larger membrane bound isoforms, VEGF183 and VEGF189, showed the most striking age-associated decrease in expression level. VEGFR-1 expression levels increased with age, while the expression level of VEGFR-2 expression was highest at 2-3 weeks and was significantly lower at 2 and 18 months of age. Distinct age-associated differences in the expression level of VEGF isoforms as well as their receptors suggest differential physiological functions during development, maturation and ageing of the ACL.

Asunto(s)

Ligamento Cruzado Anterior/metabolismo , Receptores de Factores de Crecimiento Endotelial Vascular/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Factores de Edad , Animales , Benzotiazoles , Cartilla de ADN , Diaminas , Compuestos Orgánicos , Isoformas de Proteínas/metabolismo , Quinolinas , Conejos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa

RESUMEN

Bone sialoprotein is an extracellular noncollagenous acidic protein that plays a role in bone mineralization and remodeling. Its expression is restricted to mineralized tissues and is subjected to variety of posttranslational modifications including phosphorylation and glycosylation. We have expressed the full-length and half domains of bovine bone sialoprotein in a prokaryotic system and identified the phosphorylation sites of casein kinase II. The N-terminal automated solid-phase sequencing defined four phosphorylated peptides: residues 28-38 (LEDS(P)EENGVFK), 51-86 (FYPELKRFAVQSSS(P)DS(P)S(P)EENGNGDS(P)S(P)EEEEEEEETS(P)), 151-165 (EDES(P)DEEEEEEEEEE), and 295-305 (GRGYDS(P)YDGQD). Nine phosphoserines were identified within the four peptides. Seven of them were in the N-terminus (S31, S64, S66, S67, S75, S76, and S86) and two were in the C-terminus (S154 and S300) of the protein.

Asunto(s)

Quinasa de la Caseína II/química , Quinasa de la Caseína II/metabolismo , Sialoglicoproteínas/química , Sialoglicoproteínas/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Escherichia coli/genética , Escherichia coli/metabolismo , Sialoproteína de Unión a Integrina , Datos de Secuencia Molecular , Fosforilación , Unión Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Sialoglicoproteínas/genética

RESUMEN

We investigated the effects of short- (8- and 24-h) and long-term (3 weeks) exposure to systemic normobaric hypoxia (13%) on the gene expression level of structural proteins and growth factors in knee joint cartilage of rabbits. Collagen type Ia2, II, and Va1, TGF-beta1, and b-FGF were upregulated after short-term hypoxia in both menisci, but not in articular cartilage. In contrast, long-term hypoxia downregulated gene expression level of collagens, aggrecan, and growth factors in articular cartilage and meniscal fibrocartilage. Interestingly, gene expression levels of non-collagenous proteins biglycan, decorin, and versican were not affected by short-term or by long-term hypoxia in knee joint cartilage. The present study suggests that changes in oxygen level differentially affect gene expression levels of growth factors, collagens, and non-collagenous proteins in normal knee joint cartilage in rabbits.

Asunto(s)

Cartílago Articular/metabolismo , Colágeno/genética , Proteínas de la Matriz Extracelular/genética , Regulación de la Expresión Génica , Sustancias de Crecimiento/genética , Hipoxia/genética , Articulación de la Rodilla/metabolismo , Proteoglicanos/genética , Agrecanos , Animales , Secuencia de Bases , Cartilla de ADN , Lectinas Tipo C , ARN Mensajero/genética , ARN Mensajero/metabolismo , Conejos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa

RESUMEN

Vascular endothelial growth factor (VEGF) gene gives rise to several distinct isoforms of VEGF, which differ in their expression patterns as well as their biochemical and biological properties. We examined the expression levels of VEGF isoforms and their receptors in the medial and lateral meniscus of rabbits under normal physiologic conditions as well their expression levels after 8 and 24 h of systemic normobaric hypoxia (13%). VEGF121 is the most abundant VEGF isoform in the medial and lateral meniscus, followed by VEGF165, VEGF189, and VEGF183. While the soluble VEGF121 and VEGF165 are only upregulated at 8 h of hypoxia, the membrane-bound VEGF183 and VEGF189 are further increased at 24 h. VEGFR-2 is expressed at a much higher level than VEGFR-1 under normal conditions, and both receptors are upregulated under hypoxia. Differential expression levels under normoxia as well as a differential response to hypoxia may indicate different functions of VEGF isoforms in the meniscus.

Asunto(s)

Hipoxia , Articulación de la Rodilla/metabolismo , Isoformas de Proteínas/química , Factor A de Crecimiento Endotelial Vascular/biosíntesis , Receptor 1 de Factores de Crecimiento Endotelial Vascular/biosíntesis , Receptor 2 de Factores de Crecimiento Endotelial Vascular/biosíntesis , Animales , Cartílago/metabolismo , Cartilla de ADN/química , ARN/metabolismo , ARN Mensajero/metabolismo , ADN Polimerasa Dirigida por ARN/metabolismo , Conejos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Tiempo , Factor A de Crecimiento Endotelial Vascular/química , Receptor 1 de Factores de Crecimiento Endotelial Vascular/química , Receptor 2 de Factores de Crecimiento Endotelial Vascular/química

RESUMEN

HB13 human myoblasts express physiological and biochemical markers associated with myoblast differentiation in non-human cell culture model systems. During differentiation, HB13 myoblasts also demonstrate fusion-related increases in cathepsin B activity and protein levels. These increases are associated with an increase in levels of cathepsin B mRNA suggesting the involvement of transcriptional regulatory mechanisms. To examine these mechanisms human myoblasts were transfected with cathepsin B nested deletion promoter constructs within the 1.8 kb 5' promoter 1 region of the human catB gene. Transfected myoblasts that were maintained under differentiating conditions demonstrated higher promoter activity than those maintained in proliferating conditions. The highest activity was obtained with pSCB2-3 (-1279/+56 bp), a construct containing two putative upstream E-box elements. Co-transfection experiments demonstrated that MyoD and myogenin transactivate cathepsin B promoter activity. Electrophoretic mobility shift assays of nuclear extracts incubated with an oligonucleotide containing two upstream E-box elements found within the cathepsin B promoter demonstrated two band shifts. The band shifts were abolished using an oligonucleotide with mutations in both E-box elements. Moreover, the shifted bands were super-shifted and abolished when incubated with anti-myogenin and anti-MyoD, respectively. Collectively, these data support myogenic transcription factor-mediated activation of cathepsin B expression during myogenesis.

Asunto(s)

Catepsina B/biosíntesis , Elementos E-Box/genética , Proteína MioD/genética , Mioblastos/metabolismo , Factores de Transcripción/genética , Northern Blotting , Western Blotting , Catepsina B/genética , Diferenciación Celular , Células Cultivadas , Creatina Quinasa/metabolismo , ADN Complementario/biosíntesis , ADN Complementario/genética , Ensayo de Cambio de Movilidad Electroforética , Regulación Enzimológica de la Expresión Génica/genética , Humanos , Miogenina/biosíntesis , Miogenina/genética , Cadenas Pesadas de Miosina/metabolismo , Proteínas Nucleares/metabolismo , Sondas de Oligonucleótidos , Fenotipo , Unión Proteica , Biosíntesis de Proteínas , ARN Mensajero/biosíntesis , Transfección