RESUMEN
Gaucher disease, a prevalent lysosomal storage disease, is caused by insufficient activity of acid ß-glucosidase (GCase) and resultant glucosylceramide accumulation. Recently in Parkinson disease (PD) patients, heterozygous mutations in GCase have been associated with earlier onset and more progressive PD. To understand the pathogenic relationships between GCase variants and Parkinsonism, α-synuclein and ubiquitin distributions and levels in the brains of several mouse models containing GCase variants were evaluated by immunohistochemistry. Progressive α-synuclein and ubiquitin aggregate accumulations were observed in the cortex, hippocampus, basal ganglia, brainstem, and some cerebellar regions between 4 and 24 weeks in mice that were homozygous for GCase [D409H (9H) or V394L (4L)] variants and also had a prosaposin hypomorphic (PS-NA) transgene. In 4L/PS-NA and 9H/PS-NA mice, this was coincident with progressive neurological manifestations and brain glucosylceramide accumulation. Ultrastructural studies showed electron dense inclusion bodies in neurons and axons of 9H/PS-NA brains. α-synuclein aggregates were also observed in ventricular, brainstem, and cerebellar regions of older mice (>42-weeks) with the GCase variant (D409H/D409H) without overt neurological disease. In a chemically induced GCase deficiency, α-synuclein aggregates and glucosylceramide accumulation also occurred. These studies demonstrate a relationship between glucosylceramide accumulation and α-synuclein aggregates, and implicate glucosylceramide accumulation as risk factor for the α-synucleinopathies.
Asunto(s)
Encéfalo/patología , Enfermedad de Gaucher/patología , Ubiquitina/metabolismo , alfa-Sinucleína/metabolismo , Factores de Edad , Animales , Encéfalo/metabolismo , Modelos Animales de Enfermedad , Enfermedad de Gaucher/genética , Enfermedad de Gaucher/metabolismo , Glucosilceramidas/metabolismo , Cuerpos de Inclusión/patología , Inositol/análogos & derivados , Inositol/farmacología , Ratones , Mutación Missense , Fenotipo , Psicosina/análogos & derivados , Psicosina/metabolismo , beta-Glucosidasa/genética , beta-Glucosidasa/metabolismoRESUMEN
Rubinstein-Taybi syndrome (RTS) is a rare multiple congenital anomaly/intellectual impairment syndrome. Loss of function in CREBBP or EP300 genes has been found in about 50% of patients with RTS. Genotype-phenotype correlations were investigated in 93 patients meeting diagnostic criteria for RTS during 2 international RTS family conferences. Mutation analysis of CREBBP was performed on all 31 coding exons and exon-intron junctions; a subset of patients had FISH analysis for large deletions. A total of 64 different variations were observed in the DNA sequence, and determined to be definitive mutations in 52 patients (56%). Mutations detected included: 10 missense mutations; 36 truncating or splice-site mutations; and 6 large deletions detectable by FISH. Fourteen patients had synonymous changes of unknown significance. The majority of mutations affected the HAT domain of CREBBP or predicted termination of the protein before the HAT region. Extensive phenotypic data were collected on each patient and analyzed to determine correlations with mutation types, that is, truncating, large deletions, single amino acid substitutions, or no CREBBP mutation. All four groups displayed the characteristic facial and thumb dysmorphology. Growth retardation in height and weight was seen more frequently in patients with no CREBBP mutation; seizure disorder was more frequent in those with CREBBP mutations. Degree of mental retardation was similar in all groups, although there was a trend toward lower IQ and autistic features in patients with large deletions. Similarity in phenotype between the groups implies that the several genes involved in causing RTS likely have effects through the same pathway.
Asunto(s)
Proteína de Unión a CREB/genética , Genotipo , Mutación , Fenotipo , Síndrome de Rubinstein-Taybi/genética , Empalme Alternativo/genética , Sustitución de Aminoácidos , Trastorno Autístico/genética , Proteína de Unión a CREB/metabolismo , Estudios de Cohortes , Exones , Eliminación de Gen , Trastornos del Crecimiento/genética , Hibridación Fluorescente in Situ , Mutación Missense , Estudios Retrospectivos , Síndrome de Rubinstein-Taybi/diagnósticoRESUMEN
Recent studies have introduced prosaposin (PSAP) as a pleiotrophic growth factor for prostate cancer (PCa). We have previously reported that PSAP or one of its known active molecular derivatives, saposin C functions as an androgen-agonist and androgen-regulated gene (ARG) for androgen-sensitive (AS) PCa cell lines. Due to the potential significance of androgen receptor (AR)-expressing stroma in PCa, we evaluated a possible bi-directional paracrine regulatory interactions between DHT and PSAP in AR-positive prostate stromal (PrSt) cells. We report that saposin C in a ligand-independent manner increased AR expression, its nuclear content, and tyrosine phosphorylation. DHT treatment of PrSt cells increased PSAP expression. We also demonstrated both serum- and androgen-inducibility of a previously characterized hormone-responsive element (HRE) located in the proximal region of PSAP promoter. In addition, conditioned-media derived from PrSt cells and bone fibroblasts (i.e., MSF) differentially increased PSAP-promoter activity in androgen-independent (AI) PC-3 and AS LNCaP cells. Our data for the first time demonstrate that not only saposin C or PSAP regulates AR expression/activity, but also function as an ARG in PrSt. Ligand-independent activation of AR by PSAP or saposin C in PCa and stromal cells may contribute not only to prostate carcinogenesis at an early stage, but also in AI progression of the disease in an androgen-deprived tumor microenvironment.
Asunto(s)
Factores de Crecimiento Nervioso/química , Próstata/citología , Receptores Androgénicos/metabolismo , Saposinas/química , Saposinas/genética , Células del Estroma/metabolismo , Regulación hacia Arriba/genética , Andrógenos/farmacología , Animales , Huesos/citología , Línea Celular , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Medios de Cultivo Condicionados , Dihidrotestosterona/farmacología , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Humanos , Masculino , Ratones , Fosfotirosina/metabolismo , Neoplasias de la Próstata/metabolismo , Estructura Terciaria de Proteína , Elementos de Respuesta/genética , Saposinas/metabolismo , Suero , Células del Estroma/efectos de los fármacos , Transcripción Genética/efectos de los fármacos , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Enzyme replacement was introduced as treatment for non-neuronopathic Gaucher disease more than 15 years ago. To ensure the best use of this costly ultra-orphan agent, a systematic disease management approach has been proposed by an international panel; this includes the development, by consensus, of achievable treatment goals. Here we critically review these goals and monitoring guidelines and incorporate emerging experience of the disease in the therapeutic era, as well as contemporary clinical research. This review makes recommendations related specifically to the management of pregnancy; the appropriate use of splenectomy and bisphosphonate treatment; the relevance of biochemical markers to disease monitoring; and the use of semi-quantitative methods for assessing bone marrow infiltration. In addition, we identify key areas for development, including the requirement for a validated index of disease severity; the need to correlate widely used biomarkers with long-term disease outcomes, and the desirability of establishing agreed standards for monitoring of bone disease particularly in infants and children with Gaucher disease.
Asunto(s)
Enfermedades Óseas/diagnóstico , Difosfonatos/uso terapéutico , Enfermedad de Gaucher/terapia , Complicaciones del Embarazo/terapia , Esplenectomía , Absorciometría de Fotón , Biomarcadores , Femenino , Enfermedad de Gaucher/complicaciones , Humanos , Imagen por Resonancia Magnética , EmbarazoRESUMEN
The effect of enzyme replacement therapy (ERT) on bone crisis and bone pain was investigated in patients with Gaucher disease (GD) type 1 followed over 4 years. Data from the International Collaborative Gaucher Group Gaucher Registry were used. Only patients with bone crisis and/or bone pain data for 1 year prior to ERT, and for each of 3 years after the start of ERT, were included. Bone crises were reported in 17% of patients during the year before starting ERT. The frequencies of bone crises decreased to 5%, <1% and 3% for 1, 2, and 3 years after initiation of treatment, respectively (p < 0.0001). Bone pain followed a similar pattern of response. Bone pain was reported in 49% of patients the year before treatment and decreased to 30% in the first year, 29% in the second year, and 30% in the third year of ERT (p < 0.0001). ERT is associated with a reduction in bone crisis and bone pain in patients with GD type 1 . This study shows that significant improvements in symptoms of skeletal disease are achievable clinical outcomes and treatment goals in GD type 1.
Asunto(s)
Enfermedades Óseas/tratamiento farmacológico , Enfermedad de Gaucher/tratamiento farmacológico , Glucosilceramidasa/uso terapéutico , Adolescente , Adulto , Enfermedades Óseas/etiología , Huesos/efectos de los fármacos , Huesos/patología , Niño , Femenino , Estudios de Seguimiento , Enfermedad de Gaucher/complicaciones , Glucosilceramidasa/deficiencia , Humanos , Masculino , Dolor/tratamiento farmacológico , Sistema de Registros , Resultado del TratamientoRESUMEN
Androgen-regulated genes (ARG) are implicated in normal and neoplastic growth of the prostate. Recently, we reported genomic amplification and/or overexpression of a previously known neurotrophic factor, prosaposin, in androgen-independent (AI) or metastatic prostate cancer (PCa) cells and tissues. Prosaposin and/or its known active molecular derivatives (e.g., saposin C) function as a pluripotent growth factor with diverse biological activities that favor malignant phenotypes in PCa cells. In addition, prosaposin or saposin C upregulates androgen receptor (AR) and AR-target genes (i.e., prostate-specific antigen, Probasin) expression and activity in LNCaP cells. Here, we examined prosaposin as an ARG. We report that DHT treatment of LNCaP cells increases prosaposin expression. In addition, we demonstrate androgen-responsiveness of prosaposin promoter and AR occupancy to a hormone-responsive element located in the proximal region of the prosaposin promoter. Our data for the first time identify prosaposin as an ARG. This observation, together with the pleiotropic growth factor activity of prosaposin, might suggest a role for this molecule in AR-dependent progression of prostate cancer at its early or late AI-state.
Asunto(s)
Andrógenos/farmacología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Saposinas/genética , Western Blotting , Línea Celular Tumoral , Dihidrotestosterona/farmacología , Humanos , Factor I del Crecimiento Similar a la Insulina/farmacología , Interleucina-6/farmacología , Luciferasas/genética , Luciferasas/metabolismo , Masculino , Plásmidos/genética , Regiones Promotoras Genéticas/genética , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Unión Proteica/efectos de los fármacos , Receptores Androgénicos/genética , Receptores Androgénicos/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Saposinas/metabolismo , Transducción de Señal/efectos de los fármacos , TransfecciónRESUMEN
We investigated 10 unrelated Chinese patients with type 2 Gaucher disease and performed ex vivo expression for the novel mutations to characterize their functional defects. These patients were diagnosed by enzymatic assays and clinicopathologic features over the past five years in a national centre in China. Genomic DNA was sequenced by a two-stage PCR approach for mutations in the functional GBA gene. Novel mutations were expressed with baculovirus-transfected Sf21 cells. Six novel mutations were found (in traditional nomenclature): P122L, Y363C, N382K, L383R, L385P, and M416V. Review of reported mutations indicated clustering of type 2 mutations in three regions of the GBA gene. Expression of novel mutations revealed that the enzyme defect could arise from one of two mechanisms: loss of catalytic activity (Y363C and M416V) or enzyme instability (P122L and N382K).
Asunto(s)
Pueblo Asiatico/genética , Enfermedad de Gaucher/enzimología , Enfermedad de Gaucher/genética , Glucosilceramidasa/genética , Glucosilceramidasa/metabolismo , Mutación/genética , Edad de Inicio , Catálisis , China , Análisis Mutacional de ADN , Estabilidad de Enzimas/genética , Enfermedad de Gaucher/clasificación , Glucosilceramidasa/química , Humanos , LactanteRESUMEN
Gaucher disease is an autosomal recessive trait and the most common lysosomal storage disease. The pathogenesis evolves from the diminished activity of the lysosomal hydrolase, acid beta-glucosidase and the resultant accumulation of glucosylceramide within lysosomes. The pathogenic mechanisms are poorly understood. During the past 2 decades, progress has been made in understanding the biochemical basis and molecular biology of the disease, but more fundamental knowledge is required to relate these advances to the cell and whole body phenotypes. Despite this lack of understanding, enzyme replacement therapy has proved a successful and effective management for Gaucher disease. However, basic details of this therapeutic efficacy require elucidation. Here, we review the current state of the molecular pathogenesis and provide our perspective of some major issues for continued advances in this prototype lysosomal storage disease.
Asunto(s)
Enfermedad de Gaucher/genética , Animales , Citocinas/biosíntesis , Enfermedad de Gaucher/diagnóstico , Enfermedad de Gaucher/enzimología , Enfermedad de Gaucher/etiología , Regulación de la Expresión Génica , Genotipo , Glucosilceramidasa/genética , Glucosilceramidasa/metabolismo , Humanos , Ratones , Modelos Biológicos , Mutación , FenotipoRESUMEN
The hydrolysis of glucosylceramide (GC) to ceramide and glucose requires the action of the lysosomal enzyme, acid beta-glucosidase (GCase), encoded by gba in the mouse. Gaucher disease, an autosomal recessive disorder, results from the inherited deficiency of this enzyme. Although enzyme activity is present in all mammalian tissues, the patterns of mRNA expression have not been explored. In situ hybridization analyses of mouse embryonic, newborn, and adult tissues were conducted to evaluate the spectrum of gba mRNA expression. Signals were present in all tissues and cell types. Distinct patterns of differential expression were identified in specific tissues and cell types, and at defined developmental stages. Differential expression was first observed around E14 in the intestinal tract, kidneys, skeletal system, and skin. At E18, moderate intensity signals were in adipocytes of brown fat and pancreatic cells. Differential expression remained in skin, bone, and the GI tract postnatally. In the postnatal and adult animals increasing expression was observed throughout the CNS, esophageal epithelium, intestinal villi, pancreas, and thymus and lymph node capsular cells. These tissue-, cell-, and developmental stage-specific variations of the gba mRNA level indicate major developmentally regulated changes in the expression pattern of gba in the late gestational period and postnatally.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Glucosilceramidasa/genética , Animales , Animales Recién Nacidos , Northern Blotting , Enfermedad de Gaucher/fisiopatología , Hibridación in Situ , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C3H , Sondas ARN , ARN Mensajero/biosíntesis , Distribución TisularRESUMEN
Lysosomal acid lipase (LAL) is the critical enzyme for the hydrolysis of the triglycerides (TG) and cholesteryl esters (CE) delivered to lysosomes. Its deficiency produces two human phenotypes, Wolman disease (WD) and cholesteryl ester storage disease (CESD). A targeted disruption of the LAL locus produced a null (lal( -/-)) mouse model that mimics human WD/CESD. The potential for enzyme therapy was tested using mannose terminated human LAL expressed in Pichia pastoris (phLAL), purified, and administered by tail vein injections to lal( -/-) mice. Mannose receptor (MR)-dependent uptake and lysosomal targeting of phLAL were evidenced ex vivo using competitive assays with MR-positive J774E cells, a murine monocyte/macrophage line, immunofluorescence and western blots. Following (bolus) IV injection, phLAL was detected in Kupffer cells, lung macrophages and intestinal macrophages in lal( -/-) mice. Two-month-old lal( -/-) mice received phLAL (1.5 U/dose) or saline injections once every 3 days for 30 days (10 doses). The treated lal( -/-) mice showed nearly complete resolution of hepatic yellow coloration; hepatic weight decreased by approximately 36% compared to PBS-treated lal( -/-) mice. Histologic analyses of numerous tissues from phLAL-treated mice showed reductions in macrophage lipid storage. TG and cholesterol levels decreased by approximately 50% in liver, 69% in spleen and 50% in small intestine. These studies provide feasibility for LAL enzyme therapy in human WD and CESD.
Asunto(s)
Enfermedad de Acumulación de Colesterol Éster/tratamiento farmacológico , Lectinas Tipo C , Lipasa/uso terapéutico , Lectinas de Unión a Manosa , Enfermedad de Wolman/tratamiento farmacológico , Animales , Anticuerpos/inmunología , Células Cultivadas , Enfermedad de Acumulación de Colesterol Éster/sangre , Enfermedad de Acumulación de Colesterol Éster/enzimología , Enfermedad de Acumulación de Colesterol Éster/patología , Modelos Animales de Enfermedad , Sistemas de Liberación de Medicamentos , Humanos , Técnicas para Inmunoenzimas , Intestinos/patología , Lipasa/deficiencia , Lipasa/genética , Lipasa/inmunología , Lípidos/sangre , Hígado/patología , Lisosomas/metabolismo , Macrófagos/metabolismo , Masculino , Receptor de Manosa , Ratones , Fenotipo , Pichia , Receptores de Superficie Celular/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/uso terapéutico , Bazo/patología , Enfermedad de Wolman/sangre , Enfermedad de Wolman/enzimología , Enfermedad de Wolman/patologíaAsunto(s)
Antineoplásicos/farmacología , Ceramidas/biosíntesis , Fenretinida/farmacología , Glicoproteínas/biosíntesis , Integrinas/metabolismo , Precursores de Proteínas/biosíntesis , Adhesión Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica , Modelos Biológicos , Estrés Oxidativo , Saposinas , Transducción de Señal , Células Tumorales Cultivadas , Regulación hacia ArribaRESUMEN
Saposins are small, heat-stable glycoprotein activators of lysosomal glycosphingolipid hydrolases that derive from a single precursor, prosaposin, by proteolytic cleavage. Three of these saposins (B, C, and D) share common structural features including a lack of tryptophan, a single glycosylation sequence, the presence of three conserved disulfide bonds, and a common multiamphipathic helical bundle motif. Saposin A contains an additional glycosylation site and a single tryptophan. The oligosaccharides on saposins are not required for in vitro activation functions. Saposins A and C were produced in Escherichia coli to contain single tryptophans at various locations to serve as intrinsic fluorescence reporters, i.e. as topological probes, for interaction with phospholipid membranes. Maximum emission shifts, aqueous and solid quenching, and resonance energy transfer were quantified by fluorescence spectroscopy. Amphipathic helices at the amino- and carboxyl termini of saposins A and C were shown to insert into the lipid bilayer to about five carbon bond lengths. In comparison, the middle region of saposins A or C were either embedded in the bilayer or solvent-exposed, respectively. Conformational changes of saposin C induced by phosphatidylserine interaction suggested the reorientation of functional helical domains. Differential interaction models are proposed for the membrane-bound saposins A and C. By site-directed mutagenesis of saposin A and C, their membrane topological structures were correlated with their activation effects on acid beta-glucosidase. These findings show that proper orientation of the middle segment of saposin C to the outside of the membrane surface is critical for its specific and multivalent interaction with acid beta-glucosidase. Such membrane interactions and orientations of the saposins determine the proximity of their activation and/or binding sites to lysosomal hydrolases or lipoid substrates.
Asunto(s)
Glicoproteínas/metabolismo , Secuencia de Aminoácidos , Membrana Celular/metabolismo , Transferencia de Energía , Fluorescencia , Glicoproteínas/química , Humanos , Membrana Dobles de Lípidos , Datos de Secuencia Molecular , Fosfolípidos/metabolismo , Conformación Proteica , Proteolípidos/química , Proteolípidos/metabolismo , Surfactantes Pulmonares/química , Surfactantes Pulmonares/metabolismo , SaposinasRESUMEN
The alpha1,6 fucosyltransferase (alpha1,6 FucT) catalyzes the transfer of a fucose from GDP-fucose to the innermost GlcNAc residue of N-linked glycans via an alpha1,6 linkage. alpha1,6 FucT was overexpressed in transgenic mice under the control of a combined cytomegalovirus and chicken beta-actin promoter. Histologically numerous small vacuoles, in which lipid droplets had accumulated, were observed in hepatocytes and proximal renal tubular cells. Electron microscopic studies showed that the lipid droplets were membrane-bound and apparently localized within the lysosomes. Cholesterol esters and triglycerides were significantly increased in liver and kidney of the transgenic mice. Liver lysosomal acid lipase (LAL) activity was significantly lower in the transgenic mice compared to the wild mice, whereas LAL protein level, which was detected immunochemically, was increased, indicating that the specific activity of LAL was much lower in the transgenic mice. In all of the transgenic and nontransgenic mice examined, the activity of liver LAL was negatively correlated with the level of alpha1,6 FucT activity. As evidenced by lectin and immunoblot analysis, LAL was found to be more fucosylated in the transgenic mice, suggesting that the aberrant fucosylation of LAL causes an accumulation of inactive LAL in the lysosomes. Such an accumulation of inactive LAL could be a likely cause for a steatosis in the lysosomes of the liver and kidney in the case of the alpha1,6 FucT transgenic mice.
Asunto(s)
Fucosiltransferasas/metabolismo , Riñón/enzimología , Lipasa/metabolismo , Hepatopatías/enzimología , Hígado/enzimología , Lisosomas/enzimología , Animales , Humanos , Lípidos/sangre , Ratones , Ratones TransgénicosRESUMEN
Lysosomal acid lipase (LAL) is essential for the hydrolysis of triglycerides (TG) and cholesteryl esters (CE) in lysosomes. A mouse model created by gene targeting produces no LAL mRNA, protein, or enzyme activity. The lal-/- mice appear normal at birth, survive into adulthood, and are fertile. Massive storage of TG and CE is observed in adult liver, adrenal glands, and small intestine. The age-dependent tissue and gross progression in this mouse model are detailed here. Although lal-/- mice can be bred to give homozygous litters, they die at ages of 7 to 8 months. The lal-/- mice develop enlargement of a single mesenteric lymph node that is full of stored lipids. At 6;-8 months of age, the lal-/- mice have completely absent inguinal, interscapular, and retroperitoneal white adipose tissue. In addition, brown adipose tissue is progressively lost. The plasma free fatty acid levels are significantly higher in lal-/- mice than age-matched lal+/+ mice, and plasma insulin levels were more elevated upon glucose challenge. Energy intake was also higher in lal-/- male mice, although age-matched body weights were not significantly altered from age-matched lal+/+ mice. Early in the disease course, hepatocytes are the main storage cell in the liver; by 3;-8 months, the lipid-stored Kupffer cells progressively fill the liver. The involvement of macrophages throughout the body of lal-/- mice provide evidence for a critical nonappreciated role of LAL in cellular cholesterol and fatty acid metabolism, adipocyte differentiation, and fat mobilization.
Asunto(s)
Adipocitos/citología , Tejido Adiposo Pardo/metabolismo , Tejido Adiposo/metabolismo , Hepatomegalia/patología , Lipasa/metabolismo , Esplenomegalia/patología , Adipocitos/metabolismo , Envejecimiento , Animales , Glucemia/análisis , Diferenciación Celular , Enfermedad de Acumulación de Colesterol Éster/genética , Enfermedad de Acumulación de Colesterol Éster/metabolismo , Enfermedad de Acumulación de Colesterol Éster/patología , Ésteres del Colesterol/metabolismo , Hepatomegalia/metabolismo , Humanos , Resistencia a la Insulina , Mucosa Intestinal/metabolismo , Intestinos/patología , Lipasa/deficiencia , Lipasa/genética , Lipoproteínas/sangre , Hígado/metabolismo , Hígado/patología , Ganglios Linfáticos/patología , Lisosomas/enzimología , Lisosomas/metabolismo , Lisosomas/ultraestructura , Masculino , Ratones , Ratones Noqueados , Bazo/metabolismo , Bazo/patología , Esplenomegalia/metabolismo , Triglicéridos/metabolismo , Enfermedad de Wolman/genética , Enfermedad de Wolman/metabolismo , Enfermedad de Wolman/patologíaRESUMEN
Wolman disease is a lethal lysosomal storage disease due to deficiency of lysosomal acid lipase (LAL). Wolman disease is characterized by pronounced hepatic involvement while neurological symptoms are uncommon, making Wolman disease an attractive candidate for liver-directed gene therapy. This study was performed to test the effects of gene replacement in fibroblasts lacking LAL, using a recombinant adenovirus encoding the human LAL cDNA (AdhLAL). Human fibroblasts from a Wolman disease patient were infected with AdhLAL and showed a dose-dependent increase in LAL protein and activity up to 5-fold above levels in control fibroblasts. Furthermore, 72 hr after infection with AdhLAL there was a dose-dependent correction of the severe lipid storage phenotype of Wolman disease fibroblasts. Electron microscopy confirmed significant correction of the lysosomal lipid storage in AdhLAL-infected Wolman disease fibroblasts at the ultrastructural level. Intravenous injection of AdhLAL into wild-type mice resulted in a 13.5-fold increase in hepatic LAL activity, and overexpression of LAL was not associated with toxic side effects. These data demonstrate high-level lysosomal expression of recombinant LAL in vitro and in vivo and show the feasibility of gene therapeutic strategies for the treatment of Wolman disease.
Asunto(s)
Fibroblastos/metabolismo , Técnicas de Transferencia de Gen , Lipasa/metabolismo , Lisosomas/enzimología , Enfermedad de Wolman/enzimología , Enfermedad de Wolman/terapia , Adenoviridae/genética , Animales , Western Blotting , Células COS , Colesterol/metabolismo , ADN Complementario/metabolismo , Relación Dosis-Respuesta a Droga , Femenino , Fibroblastos/enzimología , Fibroblastos/ultraestructura , Terapia Genética , Humanos , Hígado/metabolismo , Ratones , Ratones Endogámicos C57BL , Microscopía Electrónica , Fenotipo , Factores de Tiempo , Triglicéridos/metabolismo , Enfermedad de Wolman/genéticaAsunto(s)
Glucosilceramidasa/genética , Glucosilceramidasa/metabolismo , Glicoproteínas/genética , Glicoproteínas/metabolismo , Enfermedad de Gaucher/enzimología , Enfermedad de Gaucher/genética , Glucosilceramidasa/química , Glicoproteínas/química , Glicoproteínas/fisiología , Humanos , Cinética , Unión Proteica , Biosíntesis de Proteínas , SaposinasRESUMEN
Prosaposin has a central role in intracellular glycosphingolipid catabolism and also has extracellular functions. This locus is regulated temporally and spatially. The highest mRNA expression occurs in the central nervous system (CNS) and reproductive system. In vitro, the CNS-expressed proteins Sp4 and RORalpha bind to Sp1 and RORE sites within a 310-bp fragment directly upstream of the transcription start site. These transcription factors exhibit negative cooperativity in vitro for prosaposin expression. Mice deficient in RORalpha and Sp4 (Staggerer [Sg(-/-)] and Sp4 knockout [Sp4 KO], respectively) containing selected prosaposin promoter deletion transgenes were used in comparative expression studies to evaluate this negative cooperativity in vivo. Constructs containing the RORE or Sp1/U cluster alone were independently stimulatory. Deletion of the Sp1/U site led to a decrease in reporter activity only in the cerebellum of Sg(-/-) mice. The deletion of RORE and Sp1/U sites did alter the increase of reporter activity in the brain and eye, but not in the spinal cord, of Sg(-/-) mice. These results indicate that Sp4 and RORalpha play minor and major roles, respectively, in regional expression of the prosaposin locus in the brain, whereas expression in the spinal cord is independent of RORalpha.
Asunto(s)
Regulación de la Expresión Génica , Glicoproteínas/genética , Receptores Citoplasmáticos y Nucleares/fisiología , Transactivadores/fisiología , Factores de Transcripción/fisiología , Animales , Línea Celular , Cartilla de ADN/química , Glicoproteínas/metabolismo , Luciferasas/metabolismo , Ratones , Ratones Noqueados , Ratones Transgénicos , Mutagénesis Sitio-Dirigida , Miembro 1 del Grupo F de la Subfamilia 1 de Receptores Nucleares , Reacción en Cadena de la Polimerasa , Regiones Promotoras Genéticas , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , ARN Mensajero/metabolismo , Saposinas , Factor de Transcripción Sp4RESUMEN
The expression of prosaposin is temporally and spatially regulated at the transcriptional and post-translational levels. In vitro, the mouse prosaposin promoter contains functional RORE [retinoic acid-receptor-related orphan receptor alpha subunit (RORalpha)-binding element], Sp1 and U (unknown) sites within 310 bp directly 5' to the transcription start site and additional elements within 2400 bp 5' to the transcription start site. To elucidate promoter regions important to tissue-preferential expression in vivo, transgenic mice were created with 5'-flanking deletions of the prosaposin gene fused to a luciferase reporter. Nearly exclusive expression was observed in cerebrum, cerebellum and eyes of adult transgenic mice containing constructs with 234-310 bp of 5'-flanking DNA. This central nervous system (CNS) expression was due to the presence of RORE and overlapping Sp1 sites in this region. Internal deletion of RORE and the Sp1 cluster from the longer constructs with 2400 bp of 5'-flanking DNA significantly diminished expression in the CNS. The appearance of substantial visceral tissue (e.g. liver, spleen, lung, kidney, thymus and heart) expression was obtained with transgenic mice bearing constructs with 742-2400 bp of 5'-flanking DNA. The cellular localization of luciferase reporter-gene expression from these constructs corresponded closely with that for prosaposin. These results define important CNS and visceral regulatory regions in the promoter in vivo and may be sufficient to account for the majority of prosaposin's tissue-preferential expression.