RESUMO
Bone structure is modulated by the interaction between receptor activator of nuclear factor-κB (RANK) and RANK ligand (RANKL). Osteoprotegerin (OPG), a decoy receptor for RANKL, modifies osteoclast-mediated bone resorption directly and spares articular cartilage indirectly in rodents with immune-mediated arthritis by preventing subchondral bone destruction. The OPG/RANKL balance also seems to be critical in maintaining joint integrity in osteoarthritis, a condition featuring articular bone and cartilage damage in the absence of profound inflammation. The current study explored the role of OPG in sparing articular cartilage by evaluating joint lesions in adult C57BL/6J mice lacking osteoprotegerin (Opg (-) (/-)). At 3, 5, 7, 9, and 12 months of age, both sexes of Opg (-) (/-) mice developed severe degenerative joint disease (DJD) characterized by progressive loss of cartilage matrix and eventually articular cartilage. Lesions developed earlier and more severely in Opg (-) (/-) mice relative to age-matched, wild-type (Opg (+) (/+)), or heterozygous (Opg (+) (/-)) littermates (P ≤ .05). The femorotibial joint was affected bilaterally at 3 months, while other key weight-bearing diarthrodial joints (eg, coxofemoral, scapulohumeral, humeroradioulnar) were affected later and unilaterally. Cortical bone in subchondral plates and long bone diaphyses of Opg (-) (/-) mice but not Opg (+/+) or Opg (+) (/-) animals was osteoporotic by 3 months of age (P ≤ .05); the extent of porosity was less than the degree of DJD. Closure of the physes in long bones (P ≤ .05) and cartilage retention in the femoral primary spongiosa (P ≤ .05) affected chiefly Opg (-) (/-) mice. These data suggest that OPG plays an essential direct role in maintaining cartilage integrity in the articular surfaces and physes.
Assuntos
Artropatias/patologia , Osteoprotegerina/fisiologia , Animais , Osso e Ossos/patologia , Artropatias/fisiopatologia , Articulações/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
OBJECTIVES: Treatment with Sclerostin antibody (Scl-Ab) has shown to enhance fracture healing in rodent and non-human primate models. The current study investigated the time-dependent changes during Scl-Ab treatment in a mouse osteotomy model. METHODS: 1 day after osteotomy, C57BL mice received subcutaneous injection with vehicle or Scl-Ab at 25 mg/kg, twice/week for 2, 4, or 6 weeks. 20 mice from each group were necropsied at weeks 2, 4, and 6 for Micro-CT, histomorphmetry and mechanical testing examinations. RESULTS: The bone mineral apposition rate at fracture callus was significantly higher in the Scl-Ab treated groups at all the time points. Micro-CT analysis showed that the volumetric bone mineral density (vBMD) and bone volume over tissue volume (BV/TV) in the Scl-Ab treated groups at 4 and 6 weeks were significantly greater than that of vehicle control groups. Mechanical testing showed that the maximum load of failure at the fracture callus increased significantly by 68% at 6 weeks in the Scl-Ab treated groups. CONCLUSIONS: This study confirmed that mice treated with Scl-Ab increased bone formation from 2 weeks, bone mineral density and bone volume at 4 weeks, followed by significant increase in bone strength at the fracture site at 6 weeks. These results suggest that applying sclerostin antibody at early stage fracture healing promotes fracture healing.
Assuntos
Anticorpos/farmacologia , Consolidação da Fratura/efeitos dos fármacos , Fraturas Ósseas/tratamento farmacológico , Glicoproteínas/imunologia , Proteínas Adaptadoras de Transdução de Sinal , Animais , Fenômenos Biomecânicos , Densidade Óssea/efeitos dos fármacos , Fraturas do Fêmur/diagnóstico por imagem , Fraturas do Fêmur/tratamento farmacológico , Fraturas Ósseas/diagnóstico por imagem , Processamento de Imagem Assistida por Computador , Peptídeos e Proteínas de Sinalização Intercelular , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência , Osteotomia , Fixação de Tecidos , Tomografia Computadorizada por Raios XRESUMO
High systemic levels of osteoprotegerin (OPG) in OPG transgenic mice cause osteopetrosis with normal tooth eruption and bone elongation and inhibit the development and activity of endosteal, but not periosteal, osteoclasts. We demonstrate that both intravenous injection of recombinant OPG protein and transgenic overexpression of OPG in OPG(-/-) mice effectively rescue the osteoporotic bone phenotype observed in OPG-deficient mice. However, intravenous injection of recombinant OPG over a 4-wk period could not reverse the arterial calcification observed in OPG(-/-) mice. In contrast, transgenic OPG delivered from mid-gestation through adulthood does prevent the formation of arterial calcification in OPG(-/-) mice. Although OPG is normally expressed in arteries, OPG ligand (OPGL) and receptor activator of NF-kappaB (RANK) are not detected in the arterial walls of wild-type adult mice. Interestingly, OPGL and RANK transcripts are detected in the calcified arteries of OPG(-/-) mice. Furthermore, RANK transcript expression coincides with the presence of multinuclear osteoclast-like cells. These findings indicate that the OPG/OPGL/RANK signaling pathway may play an important role in both pathological and physiological calcification processes. Such findings may also explain the observed high clinical incidence of vascular calcification in the osteoporotic patient population.
Assuntos
Densidade Óssea/fisiologia , Calcinose/fisiopatologia , Glicoproteínas/metabolismo , Osteoclastos/metabolismo , Osteopetrose/metabolismo , Osteoporose/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Fosfatase Ácida/metabolismo , Animais , Aorta/patologia , Western Blotting , Células CHO , Catepsina K , Catepsinas/metabolismo , Cricetinae , Fêmur/anatomia & histologia , Fêmur/diagnóstico por imagem , Fêmur/metabolismo , Glicoproteínas/genética , Humanos , Imuno-Histoquímica , Hibridização In Situ , Isoenzimas/metabolismo , Camundongos , Camundongos Knockout , Camundongos Transgênicos , NF-kappa B/genética , NF-kappa B/metabolismo , Osteoclastos/ultraestrutura , Osteopetrose/genética , Osteoporose/genética , Osteoprotegerina , Radiografia , Receptores Citoplasmáticos e Nucleares/genética , Receptores do Fator de Necrose Tumoral , Proteínas Recombinantes de Fusão/metabolismo , Fosfatase Ácida Resistente a TartaratoRESUMO
The proinflammatory chemokine interleukin-8 (IL-8/NAP-1) has been implicated in recruiting neutrophils to sites of acute and chronic tissue inflammation. In transgenic mice, elevated serum IL-8 levels ranging from 1 to 118 ng/ml were correlated with proportional increases in circulating neutrophils and proportional decreases in L-selectin expression on the surface of blood neutrophils. No change in the expression of the beta 2-integrins Mac-1 and LFA-1 was apparent on peripheral blood neutrophils of the IL-8 transgenic mice. Additionally, L-selectin expression on bone marrow neutrophils and neutrophil precursors was normal in all transgenic lines. IL-8 transgenic mice demonstrated an accumulation of neutrophils in the microcirculation of the lung, liver and spleen. Moreover, there was no evidence of neutrophil extravasation, plasma exudation or tissue damage in any IL-8 transgenic mice. Neutrophil migration into the inflamed peritoneal cavity was severely inhibited in IL-8 transgenic mice, but not in nontransgenic littermates. The IL-8 transgenic mice should serve as useful models for studying the putative role of neutrophils in mediating tissue damage in models of inflammation, such as hepatic ischemia and reperfusion injury, cecal puncture and ligation, and glomerulonephritis.
Assuntos
Quimiotaxia de Leucócito , Interleucina-8/biossíntese , Neutrófilos/fisiologia , Animais , Antígenos de Diferenciação/biossíntese , Antígenos de Diferenciação/sangue , Apolipoproteínas E/genética , Northern Blotting , Moléculas de Adesão Celular/biossíntese , Moléculas de Adesão Celular/sangue , Ensaio de Imunoadsorção Enzimática , Humanos , Interleucina-8/sangue , Intestino Delgado/metabolismo , Íntrons , Selectina L , Fígado/metabolismo , Antígeno-1 Associado à Função Linfocitária/biossíntese , Antígeno-1 Associado à Função Linfocitária/sangue , Camundongos , Camundongos Transgênicos , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas , RNA Mensageiro/análise , RNA Mensageiro/biossíntese , Mapeamento por RestriçãoRESUMO
Alveolar type II cell proliferation occurs after many forms of lung injury and is thought to play a critical role in alveolar epithelial repair. Keratinocyte growth factor/fibroblast growth factor 7 (KGF) has been shown to promote alveolar type II cell growth in primary culture and alveolar epithelial hyperplasia in vivo. In this study, we used immunohistochemical analysis to determine the intrapulmonary distribution and cellular localization of recombinant human KGF (rhKGF) instilled into the trachea of rats. 6 h after administration, immunoreactive KGF was observed within the lung parenchyma and along alveolar epithelial cell membranes. By 18-24 h, KGF was detected intracellularly in alveolar epithelial cells and intraalveolar macrophages. Immunoreactive KGF was not demonstrable 48 h after delivery or in lung sections from PBS-treated animals. Intratracheal instillation of 5 mg/kg rhKGF stimulated a marked, time-dependent increase in the alveolar type II cell specific labeling index to a maximum level of 33 +/- 3% 48 h after rhKGF administration compared with 1.3 +/- 0.3% after PBS instillation. In addition, this increase in type II cell proliferation in vivo was documented by flow cytometric analysis of isolated type II cells which revealed a nearly fivefold increase in the proportion of cells traversing through the S and G2/M phases of the cell cycle. To test the hypothesis that KGFs effects on type II cells in vivo might affect the response to lung injury, rats were treated with rhKGF and exposed to hyperoxia. Animals that received 1 or 5 mg/kg rhKGF exhibited dramatically reduced mortality (P < 0.001, for both doses). Survival for animals treated with 0.1 mg/kg rhKGF was not significantly different from either untreated rats or animals treated with heat-denatured rhKGF. The lungs of rhKGF-treated animals that survived hyperoxia exposure had minimal hemorrhage and no exudate within the intraalveolar space. These experiments established that intratracheal administration of rhKGF stimulated alveolar type II cell proliferation in vivo and reduced hyperoxia-induced lung injury in rats. Directed delivery of KGF to the lungs may provide a therapeutic strategy to preserve or restore the alveolar epithelium during exposure to hyperoxia or other injurious agents.
Assuntos
Fatores de Crescimento de Fibroblastos , Substâncias de Crescimento/farmacologia , Oxigênio/toxicidade , Alvéolos Pulmonares/efeitos dos fármacos , Animais , Divisão Celular/efeitos dos fármacos , Fator 10 de Crescimento de Fibroblastos , Fator 7 de Crescimento de Fibroblastos , Substâncias de Crescimento/administração & dosagem , Humanos , Masculino , Alvéolos Pulmonares/citologia , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes/farmacologia , Superóxido Dismutase/metabolismo , Traqueia/efeitos dos fármacosRESUMO
Retroviral gene transfer to liver without prior injury has not yet been accomplished. We hypothesized that recombinant human keratinocyte growth factor would stimulate proliferation of hepatocytes and allow for efficient in vivo gene transfer with high titer murine Moloney retroviral vectors. This report shows that 48 h after intravenous injection of keratinocyte growth factor, hepatocyte proliferation increased approximately 40-fold compared to non-stimulated livers. When keratinocyte growth factor treatment was followed by intravenous injection of high titer (1 x 10(8) colony forming units/ml) retrovirus coding for the Escherichia Coli beta-galactosidase gene, there was a 600-fold increase in beta-galactosidase expression, with 2% of hepatocytes transduced. Thus, by exploiting the mitogenic properties of keratinocyte growth factor, retrovirus-mediated gene transfer to liver may be accomplished in vivo without the use of partial hepatectomy or pretreatment with other toxins to induce hepatocyte cell division.
Assuntos
Divisão Celular/efeitos dos fármacos , Fatores de Crescimento de Fibroblastos , Técnicas de Transferência de Genes , Substâncias de Crescimento/farmacologia , Animais , Células Cultivadas , Primers do DNA/química , Primers do DNA/genética , Escherichia coli/enzimologia , Escherichia coli/genética , Fator 10 de Crescimento de Fibroblastos , Fator 7 de Crescimento de Fibroblastos , Expressão Gênica/genética , Vetores Genéticos/genética , Vetores Genéticos/metabolismo , Imuno-Histoquímica , Óperon Lac/genética , Fígado/metabolismo , Camundongos , Reação em Cadeia da Polimerase , Retroviridae/metabolismo , Transdução Genética/genética , beta-Galactosidase/genética , beta-Galactosidase/metabolismoRESUMO
To identify DNA sequence elements from the human apolipoprotein B (apoB) gene required for high-level, correct tissue-specific expression in transgenic mice, we made several constructs that included one or more of the key regulatory elements that were previously characterized with cultured liver-derived and intestine-derived cell lines. Our data show that the apoB promoter alone (-898 to +121) is not sufficient to direct transcription in transgenic mice. An enhancer located in the second intron is absolutely required to specify transcription by the homologous apoB promoter in the livers of transgenic mice; this enhancer does not direct transcription in the small intestines. Thus, the elements controlling transcriptional activation of the apoB gene in the liver and the intestine in vivo are distinct and separable. Analysis of the DNase I hypersensitivity of the integrated human transgenes in various lines of expressing and nonexpressing mice suggests that the formation of DH4, a strong hypersensitive site in intron 2, may be a prerequisite for hepatic expression of the apoB gene. Nuclear matrix association regions (MARs) of the apoB gene may play a role in transgene expression. Constructs including MAR sequences displayed higher levels of expression than those lacking them. However, these MARs did not completely insulate the associated transgenes from position effects.
Assuntos
Apolipoproteínas B/biossíntese , Apolipoproteínas B/genética , DNA/genética , Elementos Facilitadores Genéticos , Regulação da Expressão Gênica , Íntrons , Fígado/metabolismo , Transcrição Gênica , Animais , Sequência de Bases , Southern Blotting , Linhagem Celular , DNA/análise , Desoxirribonuclease I , Feminino , Humanos , Fígado/citologia , Masculino , Camundongos , Camundongos Transgênicos , Especificidade de Órgãos , Proteínas Recombinantes/análise , Proteínas Recombinantes/biossíntese , Mapeamento por Restrição , Transfecção , Células Tumorais Cultivadas , beta-Galactosidase/análise , beta-Galactosidase/biossínteseRESUMO
The fibroblast growth factors (FGFs) play key roles in controlling tissue growth, morphogenesis, and repair in animals. We have cloned a novel member of the FGF family, designated FGF-18, that is expressed primarily in the lungs and kidneys and at lower levels in the heart, testes, spleen, skeletal muscle, and brain. Sequence comparison indicates that FGF-18 is highly conserved between humans and mice and is most homologous to FGF-8 among the FGF family members. FGF-18 has a typical signal sequence and was glycosylated and secreted when it was transfected into 293-EBNA cells. Recombinant murine FGF-18 protein (rMuFGF-18) stimulated proliferation in the fibroblast cell line NIH 3T3 in vitro in a heparan sulfate-dependent manner. To examine its biological activity in vivo, rMuFGF-18 was injected into normal mice and ectopically overexpressed in transgenic mice by using a liver-specific promoter. Injection of rMuFGF-18 induced proliferation in a wide variety of tissues, including tissues of both epithelial and mesenchymal origin. The two tissues which appeared to be the primary targets of FGF-18 were the liver and small intestine, both of which exhibited histologic evidence of proliferation and showed significant gains in organ weight following 7 (sometimes 3) days of FGF-18 treatment. Transgenic mice that overexpressed FGF-18 in the liver also exhibited an increase in liver weight and hepatocellular proliferation. These results suggest that FGF-18 is a pleiotropic growth factor that stimulates proliferation in a number of tissues, most notably the liver and small intestine.
Assuntos
Fatores de Crescimento de Fibroblastos/metabolismo , Intestino Delgado/citologia , Fígado/citologia , Células 3T3 , Sequência de Aminoácidos , Animais , Sequência de Bases , Divisão Celular , Linhagem Celular Transformada , Células Cultivadas , Clonagem Molecular , DNA Complementar , Escherichia coli , Fatores de Crescimento de Fibroblastos/genética , Expressão Gênica , Humanos , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Tamanho do Órgão , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Homologia de Sequência de Aminoácidos , Distribuição TecidualRESUMO
Expression of human keratinocyte growth factor (KGF/FGF-7) was directed to hepatocytes during the later period of mouse gestation using a human apolipoprotein E (ApoE) gene promoter and its associated liver-specific enhancer. Human KGF was detectable in liver extracts and serum prepared from e17.5-e19.5 embryos, concomitant with the appearance of morphological abnormalities in several organs which express KGF receptor. The most striking phenotypic aberration in the ApoE-hKGF transgenic embryos was marked hyperplasia and cystic dilation of the cortical and medullary kidney collecting duct system, a phenotype resembling infantile polycystic kidney disease in humans. Transgenic embryos had enlarged livers, with prominent biliary epithelial hyperplasia, and also exhibited enhanced bronchiolar epithelial and type II pneumocyte proliferation. There was variable hyperplasia of intestinal epithelia, and urothelium of the urinary bladder and ureters. When compared to age-matched littermate controls, marked epidermal papillomatous acanthosis and hyperkeratosis in the skin, with a notable decrease in the number of developing hair follicles was seen in transgenic embryos. The pancreas exhibited significant ductal hyperplasia, with an increase in the number of ductal epithelial cells staining positive for insulin expression. High systemic levels of KGF during the latter stages of embryogenesis causes abnormalities in epithelial growth and differentiation within multiple organ systems and results in perinatal lethality. Correct temporal and spatial expression of KGF during the latter stages of organ development is likely to play a critical role in mesenchymal-epithelial signaling required for normal embryonic growth and development.
Assuntos
Anormalidades Congênitas/etiologia , Fatores de Crescimento de Fibroblastos , Substâncias de Crescimento/fisiologia , Fígado/embriologia , Fígado/fisiologia , Doenças Renais Policísticas/etiologia , Animais , Apolipoproteínas E/genética , Diferenciação Celular/fisiologia , Divisão Celular/fisiologia , Anormalidades Congênitas/genética , Células Epiteliais , Epitélio/crescimento & desenvolvimento , Fator 10 de Crescimento de Fibroblastos , Fator 7 de Crescimento de Fibroblastos , Expressão Gênica , Substâncias de Crescimento/biossíntese , Substâncias de Crescimento/genética , Humanos , Imuno-Histoquímica , Fígado/metabolismo , Pulmão/anormalidades , Pulmão/citologia , Camundongos , Camundongos Endogâmicos , Camundongos Transgênicos , Doenças Renais Policísticas/genética , Doenças Renais Policísticas/patologia , Regiões Promotoras Genéticas/fisiologia , RNA Mensageiro/metabolismo , TransgenesRESUMO
The levels of cholesterol and dolichyl phosphate in the liver of an abortus with severe mevalonic aciduria were found to be approximately 60% of the mean of 5 age matched controls, while the level of squalene was within the normal range. Thus, despite a level of mevalonate kinase reported to be less than 1% of normal (Hoffmann, H. et al. (1986) N. Engl. J. Med. 314, 1610-1614), the liver was able to synthesize normal or near normal levels of isoprenoid lipids.
Assuntos
Fosfatos de Dolicol/metabolismo , Erros Inatos do Metabolismo/metabolismo , Ácido Mevalônico/urina , Fosfatos de Poli-Isoprenil/metabolismo , Colesterol/sangue , Feminino , Doenças Fetais/metabolismo , Humanos , Hidroximetilglutaril-CoA Redutases/metabolismo , Fígado/metabolismo , Masculino , Gravidez , Esqualeno/metabolismoRESUMO
The mechanisms by which thyroid hormones increase hepatic 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase mRNA levels were investigated in hypophysectomized rats. Feeding these rats a diet supplemented with 0.5% desiccated porcine thyroid powder resulted in a 5-fold increase in the rate of transcription of the HMG-CoA reductase gene as measured by in vitro "run-on" transcription assays in isolated rat liver nuclei. Time courses of change in reductase mRNA, showing the kinetics of approach to new steady-state levels, indicate that reductase mRNA is also 4-6-fold more stable in thyroid hormone-treated animals than in non-treated animals. Reductase mRNA decayed with a half-life of 2.5 h when mevinolin, a potent inhibitor of HMG-CoA reductase, and colestipol, a bile acid sequesterant, were removed from the diet of hypophysectomized rats. When these drugs were removed from the diet of thyroid hormone-treated hypophysectomized rats, reductase mRNA decayed with a half-life of 15 h. Treating rats with only mevinolin and colestipol increased reductase mRNA levels without stabilizing the mRNA. Administration of cycloheximide to thyroid hormone treated rats rapidly decreased HMG-CoA reductase mRNA levels by destabilizing reductase mRNA and decreasing reductase gene transcription. Cycloheximide treatment had no effect on beta-actin gene transcription or steady state levels of beta-actin mRNA. These results suggest that a short-lived protein(s) may mediate the transcriptional and post-transcriptional effects of thyroid hormones on HMG-CoA reductase mRNA levels.
Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , Hidroximetilglutaril-CoA Redutases/genética , Fígado/enzimologia , Hormônios Tireóideos/farmacologia , Transcrição Gênica/efeitos dos fármacos , Actinas/genética , Animais , Colestipol/administração & dosagem , Colestipol/farmacologia , Cicloeximida/farmacologia , Dieta , Meia-Vida , Hipofisectomia , Cinética , Lovastatina/administração & dosagem , Lovastatina/farmacologia , Masculino , RNA Mensageiro/metabolismo , Ratos , Ratos EndogâmicosRESUMO
When normal or hypophysectomized rats maintained on a diet containing mevinolin and colestipol were switched to a normal chow diet, HMG-CoA reductase mRNA fell rapidly with a half-life of 3 hrs. After a 90 min lag period, reductase activity and immunoreactive protein fell in parallel with the mRNA in normal rats. In hypophysectomized rats, reductase activity and protein required 10 and 18 hrs, respectively to fall to 50% of their original levels. Administration of thyroid hormones to hypophysectomized rats resulted in a 3 to 4 fold stabilization of reductase mRNA suggesting that the increased mRNA levels are due in part to a posttranscriptional regulatory effect of thyroid hormones.
Assuntos
Colestipol/farmacologia , Hidroximetilglutaril-CoA Redutases/genética , Hipofisectomia , Fígado/enzimologia , Naftalenos/farmacologia , Poliaminas/farmacologia , RNA Mensageiro/metabolismo , Hormônios Tireóideos/farmacologia , Animais , Cinética , Fígado/efeitos dos fármacos , Lovastatina , Masculino , RNA Mensageiro/genética , Ratos , Ratos Endogâmicos , Suínos , Glândula Tireoide/fisiologia , Extratos de Tecidos/farmacologiaRESUMO
Administration of dexamethasone to hypophysectomized rats treated with thyroid hormones blocked the increase in hepatic 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase mRNA and enzyme activity which occurred in response to thyroid hormone treatment. The rate of transcription of the rat liver HMG-CoA reductase gene measured by "run-on" assays in isolated nuclei was not diminished by dexamethasone. The half-life of HMG-CoA reductase mRNA was decreased from 12-15 to 2-3 h by dexamethasone treatment of hypophysectomized rats fed thyroid powder. Adrenalectomy caused the half-life of HMG-CoA reductase mRNA to increase from 3 to 10 h, suggesting that endogenous glucocorticoids also regulate reductase mRNA stability. Reductase mRNA levels were increased only 5-fold in thyroidectomized rats fed thyroid powder compared to a 20- to 40-fold increase in similarly treated hypophysectomized rats. In thyroidectomized rats, reductase mRNA had a half-life of only 1.5 h. Thyroid hormone treatment increased this to 4.5 h, significantly less than that of similarly treated hypophysectomized rats. Hydrocortisone, like dexamethasone, lowered reductase mRNA levels, but the biologically inactive analogue epihydrocortisone did not affect reductase mRNA or activity. These results suggest that glucocorticoids decrease the abundance of HMG-CoA reductase mRNA by stimulating its degradation.
Assuntos
Adrenalectomia , Dexametasona/farmacologia , Hidroximetilglutaril-CoA Redutases/genética , Hipofisectomia , Fígado/enzimologia , Glândulas Paratireoides/fisiologia , Processamento Pós-Transcricional do RNA/efeitos dos fármacos , RNA Mensageiro/genética , Animais , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Hidrocortisona/farmacologia , Fígado/efeitos dos fármacos , Masculino , Microssomos Hepáticos/enzimologia , RNA Mensageiro/efeitos dos fármacos , Ratos , Ratos Endogâmicos , Extratos de Tecidos/farmacologia , Transcrição Gênica/efeitos dos fármacosRESUMO
The human apolipoprotein (apo) E and apoC-I genes are located 5 kilobases apart in the same transcriptional orientation on chromosome 19, and they are expressed at high levels in the liver with lower levels of expression in selected other tissues. Analysis of a series of overlapping human apoE and apoC-I genomic fragments in transgenic mice revealed that the expression of these transgenes in the liver requires a common cis-acting regulatory domain. This hepatic control region (HCR) was localized to a 764-base pair region that is located about 18 kilobases downstream of the apoE promoter and about 9 kilobases downstream of the apoC-I promoter. All the transgenic animals that had been prepared with a construct that contained this region had relatively high levels of transgene expression in the liver, whereas constructs that lacked this region showed no expression in the liver. In situ hybridization studies showed that the HCR directed apoE and apoC-I transgene expression in hepatocytes. When the HCR from the apoE/C-I gene locus was ligated proximal to a human apoA-IV gene fragment, which is not normally expressed in the liver, the resulting apoA-IV/HCR fusion construct was expressed at high levels in the liver, indicating that the HCR could direct high level liver expression of a heterologous promoter/gene construct. Expression of the apoE transgene in the liver and kidney, and perhaps other tissues, required the presence of a nonspecific proximal enhancer element in the apolipoprotein E gene promoter, located between 161 and 141 bp relative to the transcription initiation site. However, the proximal apoE gene promoter, including this enhancer element, contained no sequences capable of directing hepatocyte expression in the absence of the HCR. Thus, the far-downstream HCR appears to contain all of the sequences necessary for determining high level liver-specific gene expression.
Assuntos
Apolipoproteínas C/genética , Apolipoproteínas E/genética , Cromossomos Humanos Par 19 , Regulação da Expressão Gênica , Fígado/metabolismo , Animais , Elementos Antissenso (Genética) , Apolipoproteína C-I , Apolipoproteínas C/biossíntese , Apolipoproteínas E/biossíntese , Elementos Facilitadores Genéticos , Humanos , Hibridização In Situ , Rim/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Especificidade de Órgãos , Regiões Promotoras Genéticas , RNA/genética , RNA/isolamento & purificação , Sondas RNA , RNA Mensageiro/análise , RNA Mensageiro/genética , Mapeamento por RestriçãoRESUMO
To investigate the mechanisms controlling tissue-specific expression of the human apolipoprotein (apo) E/C-I gene locus, human apoE and apoC-I gene constructs containing various lengths of the 5'-flanking or 3'-flanking region were used to create transgenic mice. Several essential tissue-specific regulatory elements were identified in the region between the apoE and the apoC-I genes, as well as in a distal domain found downstream of the apoC-I gene. Most notably, transcription of both the apoE and apoC-I genes in the liver, their major site of expression, required downstream regulatory elements, possibly located within a common regulatory domain more than 2 kilobases 3' of the apoC-I gene (about 14 kilobases downstream of the apoE gene promoter). In the region between the apoE and apoC-I genes, a single strong positive element directed apoE and apoC-I gene expression in the skin. The intergenic region also contained elements that stimulated apoE gene expression in the brain and silenced apoE gene expression only in the kidney. These results demonstrate that multiple independent regulatory elements control expression of the human apoE/C-I gene locus in various tissues. Transgenic mice expressing human apoC-I in the liver exhibited plasma triglyceride levels that were 2-3-fold higher than those in control mice, an effect not found when transgenic human apoE was produced. This result suggests that apoC-I may modulate the metabolism of triglyceride-rich lipoproteins.
Assuntos
Apolipoproteínas C/genética , Apolipoproteínas E/genética , Regulação da Expressão Gênica , Animais , Apolipoproteína C-I , Colesterol/sangue , Genes , Humanos , Fígado/fisiologia , Camundongos , Camundongos Transgênicos , RNA Mensageiro/genética , Sequências Reguladoras de Ácido Nucleico , Fenômenos Fisiológicos da Pele , Triglicerídeos/sangueRESUMO
Acid aspiration is a serious complication of anesthesia and other forms of unconsciousness that can result in the adult respiratory distress syndrome (ARDS), which continues to have a very high mortality despite our current therapeutic interventions. This type of injury damages the alveolar epithelium, principally alveolar type I cells, and requires proliferation of alveolar type II cells to restore gas exchange units. Since keratinocyte growth factor (KGF) has been shown to be a potent mitogen for alveolar type II cells, we evaluated whether intrabronchial administration of KGF would minimize lung injury due to the unilateral instillation of 0.1 N hydrochloric acid (HCl). Rats were pretreated or post-treated by intrabronchial instillation of KGF (5 mg/kg) into the left lung before HCl instillation. All rats receiving KGF at 48 or 72 h before HCl instillation survived for the 7-day observation period, whereas the mortality rate for those receiving HCl alone or saline followed by HCl was 31% and 33%, respectively. Pretreatment with KGF at 72 h but not at 24 or 48 h considerably ameliorated morphologic damage produced by HCl. Inflammatory cells in bronchoalveolar lavage were markedly decreased 3 and 7 days after HCl instillation by the 72-h KGF pretreatment. Pretreatment with KGF at 72 h also attenuated the reduction of total lung capacity, decreased the alpha 1(I) procollagen mRNA levels, and diminished hydroxyproline accumulation due to HCl instillation. Saline pretreatment at 72 h had no significant effect on the HCl injury and subsequent physiologic abnormalities. Our attempts to improve outcome with post-treatment instillation of KGF were unsuccessful. We conclude that KGF pretreatment reduces lung injury due to acid instillation and can prevent subsequent pulmonary fibrosis.
Assuntos
Fatores de Crescimento de Fibroblastos , Substâncias de Crescimento/administração & dosagem , Ácido Clorídrico/toxicidade , Pulmão/efeitos dos fármacos , Administração por Inalação , Animais , Lavagem Broncoalveolar , Contagem de Células/efeitos dos fármacos , Fator 10 de Crescimento de Fibroblastos , Fator 7 de Crescimento de Fibroblastos , Pulmão/patologia , Ratos , Ratos Endogâmicos F344RESUMO
Keratinocyte growth factor (KGF) caused cystic dilation of mouse fetal lung explants in vitro, markedly increasing the luminal volume of lung buds and disrupting branching morphogenesis. Effects of KGF were dose dependent, were detected within 4 h of treatment, and were blocked by cycloheximide but not by actinomycin D, indicating that de novo protein synthesis mediated the response. Effects of KGF were inhibited by bumetanide, an inhibitor of the Na(+)-K(+)-Cl- cotransporter, and ouabain, an inhibitor of the Na(+)-K(+)-ATPase. KGF stimulated fluid secretion equally in lung buds from cystic fibrosis transmembrane conductance regulators (CFTR) -/- and wild-type embryos, indicating that the effects were mediated by CFTR-independent Cl- transport. Microelectrode studies demonstrated that, whereas KGF did not acutely alter the transepithelial potential difference (PD) across the respiratory epithelium, the PD decreased while luminal volume increased during chronic exposure. KGF inhibited expression of alpha-subunit of epithelial Na+ channel (alpha-ENaC) mRNA, suggesting that KGF may inhibit Na+ absorption, which may contribute to KGF-induced fluid accumulation. KGF-induced fluid accumulation is driven by CFTR-independent Cl- transport and associated with decreased expression of alpha-ENaC.
Assuntos
Cloretos/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Fatores de Crescimento de Fibroblastos , Substâncias de Crescimento/farmacologia , Pulmão/metabolismo , Animais , Líquido da Lavagem Broncoalveolar , Bumetanida/farmacologia , Proteínas de Transporte/metabolismo , Técnicas de Cultura , Feminino , Fator 10 de Crescimento de Fibroblastos , Fator 7 de Crescimento de Fibroblastos , Pulmão/embriologia , Camundongos , Ouabaína/farmacologia , Gravidez , Simportadores de Cloreto de Sódio-Potássio , ATPase Trocadora de Sódio-Potássio/metabolismoRESUMO
Transgenic mouse lines were produced that expressed low levels of a receptor-binding-defective variant of human apolipoprotein (apo) E, apoE(Arg112, Cys142). In transgenic mice, the human apoE was produced only by the kidney, whereas endogenous mouse apoE was produced mainly by the liver. The plasma concentration of the transgenic protein was about half that of endogenous apoE. The expression of transgenic apoE did not affect total plasma cholesterol and triglyceride levels, but the distribution of the human variant differed from that of endogenous apoE in the intermediate size and density range, where the transgenic protein accumulated selectively. Immunoblots of agarose gels of lipoprotein fractions showed that the transgenic protein occurred primarily on large alpha-migrating particles (HDL1). This phenomenon was not observed in transgenic mice expressing normal human apoE-3, which distributed like endogenous apoE, suggesting that the defective apoE variant perturbed HDL1 metabolism. In mice fed a high-fat, high-cholesterol diet, the transgenic apoE associated primarily with the apoB-containing lipoproteins. A significantly higher increase in very low density lipoprotein cholesterol was observed in fat-fed transgenics compared to fat-fed nontransgenic mice, suggesting a metabolic perturbation of apoB-containing lipoproteins. Thus, the receptor-binding-defective variant, apoE(Arg112, Cys142), expressed at low levels by the kidney, alters lipoprotein metabolism in transgenic mice, presumably by interfering with apoE-mediated removal of the lipoproteins from circulation.
Assuntos
Apolipoproteínas E/genética , Lipoproteínas/sangue , Mutação , Animais , Apolipoproteínas E/metabolismo , Southern Blotting , Western Blotting , Colesterol/sangue , Expressão Gênica , Humanos , Immunoblotting , Lipoproteínas HDL/sangue , Camundongos , Camundongos Endogâmicos ICR , Camundongos Transgênicos , Tamanho da Partícula , Triglicerídeos/sangueRESUMO
Human apolipoprotein (apo) E gene constructs with 30 or 5 kilobases of 5'-flanking and 1.5 kilobases of 3'-flanking regions were used to create transgenic mice. High levels of human apoE mRNA were present in the transgenic kidney, but none was detected in the liver, which is normally the major source of apoE. When a construct with 5 kilobases of 5'- and 23 kilobases of 3'-flanking regions was used, only trace levels of human apoE mRNA were detected in the kidney, whereas high levels were found in the liver. These results indicated that regulatory elements downstream of the human apoE gene interacted with the transcription initiation complex to stimulate gene expression in the liver while suppressing expression in the kidney. In each case, human apoE was secreted into the plasma. The source of human apoE in the transgenic kidney was the epithelial cells lining the proximal tubule and Bowman's capsule.
Assuntos
Apolipoproteínas E/genética , Expressão Gênica , Rim/metabolismo , Animais , Clonagem Molecular , DNA Recombinante , Humanos , Técnicas Imunoenzimáticas , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos ICR , Camundongos Transgênicos , Hibridização de Ácido Nucleico , Sondas RNA , RNA Mensageiro/análise , Sequências Reguladoras de Ácido Nucleico , Pele/análise , Testículo/análise , Distribuição Tecidual , Transcrição GênicaRESUMO
Intratracheal instillation of bleomycin produces pulmonary fibrosis in rats. Alveolar type II cell proliferation is thought to minimize the fibrotic response after lung injury. Because keratinocyte growth factor (KGF) stimulates type II cell proliferation in the rat, we designed experiments to evaluate whether intratracheal KGF before or after intratracheal bleomycin would prevent pulmonary fibrosis. Intratracheal bleomycin without KGF resulted in moderate to severe lung injury and subsequent fibrosis. Conversely, intratracheal KGF pretreatment at 48 or 72 hr before bleomycin resulted in minimal to no visible lung injury. Rats pretreated with phosphate buffered saline before bleomycin had significantly more neutrophils and protein in bronchoalveolar lavage fluid at 4 and 6 days and higher hydroxyproline levels after bleomycin as compared to KGF-pretreated rats. Pretreatment with KGF at 48 hr protected against bleomycin-induced alterations in pulmonary physiology and increased surfactant protein C-positive (SP-C)-positive cells and SP-A, SP-B, SP-C, and SP-D mRNA levels after bleomycin instillation when compared to saline pretreated rats on day 1 or day 7. KGF posttreatment protocols did not prevent bleomycin lung injury and fibrosis. We conclude that KGF pretreatment attenuates bleomycin lung injury and increases type II cell proliferation and surfactant protein gene expression after bleomycin instillation in the rat.