RESUMEN
Rationale: Oxaliplatin-induced peripheral neuropathy (OIPN) is a common adverse effect that causes delayed treatment and poor prognosis among colorectal cancer (CRC) patients. However, its mechanism remains elusive, and no effective treatment is available. Methods: We employed a prospective cohort study of adult patients with pathologically confirmed stage III CRC receiving adjuvant chemotherapy with an oxaliplatin-based regimen for investigating OIPN. To further validate the clinical manifestations and identify a potential therapeutic strategy, animal models, and in vitro studies on the mechanism of OIPN were applied. Results: Our work found that (1) consistent with clinical findings, OIPN was observed in animal models. Targeting the enzymatic activity of cathepsin S (CTSS) by pharmacological blockade and gene deficiency strategy alleviates the manifestations of OIPN. (2) Oxaliplatin treatment increases CTSS expression by enhancing cytosol translocation of interferon response factor 1 (IRF1), which then facilitates STIM-dependent store-operated Ca2+ entry homeostasis. (3) The cytokine array demonstrated an increase in anti-inflammatory cytokines and suppression of proinflammatory cytokines in mice treated with RJW-58. (4) Mechanistically, inhibiting CTSS facilitated olfactory receptors transcription factor 1 release from P300/CBP binding, which enhanced binding to the interleukin-10 (IL-10) promoter region, driving IL-10 downstream signaling pathway. (5) Serum CTSS expression is increased in CRC patients with oxaliplatin-induced neurotoxicity. Conclusions: We highlighted the critical role of CTSS in OIPN, which provides a therapeutic strategy for the common adverse side effects of oxaliplatin.
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Catepsinas/genética , Neuronas/metabolismo , Animales , Antineoplásicos/efectos adversos , Antineoplásicos/farmacología , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Catepsinas/antagonistas & inhibidores , Catepsinas/efectos de los fármacos , Quimioterapia Adyuvante , Estudios de Cohortes , Neoplasias Colorrectales/tratamiento farmacológico , Citocinas/metabolismo , Modelos Animales de Enfermedad , Inhibidores Enzimáticos , Femenino , Fluorouracilo/uso terapéutico , Ganglios Espinales , Humanos , Técnicas In Vitro , Leucovorina/uso terapéutico , Masculino , Ratones , Ratones Noqueados , Microglía/efectos de los fármacos , Microglía/metabolismo , Terapia Molecular Dirigida , Conducción Nerviosa , Neuronas/efectos de los fármacos , Compuestos Organoplatinos/uso terapéutico , Oxaliplatino/efectos adversos , Oxaliplatino/farmacología , Enfermedades del Sistema Nervioso Periférico/inducido químicamente , Estudios ProspectivosRESUMEN
Poligoni Multiflori Radix (PMR) is a traditional Korean medicinal herb that is known to have various pharmacological effects, including antihyperlipidemic, anticancer, and antiinflammatory effects. However, the effects of PMR on bone metabolism have not been elucidated to date. The present study aimed to investigate the in vitro and in vivo effect of PMR water extract on the regulation of osteoblast and osteoclast activity. Effects of PMR water extract on receptor activator of nuclear factorkB ligand (RANKL)induced osteoclast differentiation and survival of mouse bone marrow macrophages (BMMs) obtained from femurs were investigated by tartrateacid resistant acid phosphatase (TRAP)positive cells and XTT assay. Expression of osteoclastrelated genes was assayed by western blot analysis and reverse transcriptionquantitative polymerase chain reaction. Additionally, the effects of PMR water extract on osteoblastic proliferation and differentiation were investigated by alkaline phosphatase (ALP) activity assay, alizarin red staining, and levels of mRNA encoding known osteoblast markers. Furthermore, the effects of PMR water extract on lipopolysaccharide (LPS)induced bone loss were examined in a mouse model. PMR inhibited RANKLinduced osteoclast differentiation of BMMs in a dosedependent manner without significant cytotoxicity, and suppressed expression of the main osteoclast differentiation markers Fos protooncogene and nuclear factor of activated Tcell. In addition, PMR decreased the mRNA expression levels of NFATc1 target genes, including TRAP, osteoclastassociated receptor, ATPase H+ transporting, lysosomal 38 kDa V0 subunit d2, and Cathepsin K. These inhibitory effects were mediated by the p38 and extracellular signalregulated kinase/nuclear factorκB pathway. Simultaneously, PMR enhanced the differentiation of primary osteoblasts, and increased the mRNA expression of runtrelated transcription factor 2, ALP, osterix, and osteocalcin. Notably, PMR improved LPSinduced trabecular bone loss in mice. Collectively, the present findings demonstrated that PMR may regulate bone remodeling by reducing osteoclast differentiation and stimulating osteoblast formation. These results suggest that PMR may be used for the treatment of bone diseases, such as osteoporosis and rheumatoid arthritis.
Asunto(s)
Diferenciación Celular/efectos de los fármacos , Medicina Tradicional Coreana , Osteoblastos/citología , Osteoclastos/citología , Extractos Vegetales/farmacología , Animales , Biomarcadores/metabolismo , Células de la Médula Ósea/citología , Células de la Médula Ósea/efectos de los fármacos , Células de la Médula Ósea/metabolismo , Resorción Ósea/patología , Catepsinas/genética , Catepsinas/metabolismo , Cromatografía Líquida de Alta Presión , Modelos Animales de Enfermedad , Lipopolisacáridos , Sistema de Señalización de MAP Quinasas , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Masculino , Ratones Endogámicos ICR , FN-kappa B/metabolismo , Factores de Transcripción NFATC/metabolismo , Osteoblastos/efectos de los fármacos , Osteoblastos/metabolismo , Osteoclastos/efectos de los fármacos , Osteoclastos/metabolismo , Proteínas Proto-Oncogénicas c-fos/metabolismo , Ligando RANK/farmacología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Transducción de Señal , Proteínas Smad/metabolismo , Fosfatasa Ácida Tartratorresistente/genética , Fosfatasa Ácida Tartratorresistente/metabolismo , ATPasas de Translocación de Protón Vacuolares/genética , ATPasas de Translocación de Protón Vacuolares/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
BACKGROUND: Cytoreductive surgery and hyperthermic intraperitoneal chemotherapy (CRS-HIPEC) is a treatment option for peritoneal surface malignancies. The ability to detect microscopic foci of peritoneal metastasis intraoperatively may ensure the completeness of cytoreduction. In this study, we evaluated the suitability of a hand-held cathepsin-based fluorescent imaging system for intraoperative detection of appendiceal and colorectal peritoneal metastasis. METHODS: Peritoneal tumors and normal peritoneal tissues were collected from patients with appendiceal and colorectal peritoneal metastasis. Expression of different cathepsins (CTS-B, -D, -F, -G, -K, -L, -O, and -S) was determined by quantitative RT-PCR and immunohistochemistry. The hand-held cathepsin-based fluorescent imaging system was used to detect peritoneal xenografts derived from human colon cancer cells (HT29, LoVo and HCT116) in nu/nu mice. RESULTS: While the expression levels of CTS-B, -D, -L, and -S could be higher in peritoneal tumors than normal peritoneum with a median (range) of 6.1 (2.9-25.8), 2.0 (1.0-15.8), 1.4 (0.8-7.0), and 2.1 (1.6-13.9) folds by quantitative RT-PCR, respectively, CTS-B was consistently the major contributor of the overall cathepsin expression in appendiceal and colonic peritoneal tumors, including adenocarcinomas and low-grade appendiceal mucinous neoplasms. Using peritoneal xenograft mouse models, small barely visible colonic peritoneal tumors (<2.5 mm in maximum diameter) could be detected by the hand-held cathepsin-based fluorescent imaging system. CONCLUSIONS: Because cathepsin expression is higher in peritoneal tumors than underlying peritoneum, the hand-held cathepsin-based fluorescent imaging system could be useful for intraoperative detection of microscopic peritoneal metastasis during CRS-HIPEC and clinical trial is warranted.
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Neoplasias del Apéndice/patología , Catepsinas/análisis , Neoplasias Colorrectales/patología , Procedimientos Quirúrgicos de Citorreducción , Hipertermia Inducida , Imagen Óptica/instrumentación , Neoplasias Peritoneales/diagnóstico por imagen , Neoplasias Peritoneales/terapia , Adulto , Anciano , Animales , Catepsina B/análisis , Catepsinas/genética , Femenino , Fluorescencia , Células HCT116 , Células HT29 , Humanos , Inmunohistoquímica , Masculino , Ratones , Persona de Mediana Edad , Trasplante de Neoplasias , Imagen Óptica/métodos , Neoplasias Peritoneales/química , Neoplasias Peritoneales/secundario , Periodo Preoperatorio , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Provision of AA has shown success in attenuating proteolytic activity in monogastrics suffering from metabolic acidosis. However, it is unknown whether AA supplementation can provide any beneficial effects to ruminants with nutritionally induced metabolic acidosis. The objective of the current study was to examine the effects of glutamine infusion on various protein degradation components across several tissues in sheep with induced metabolic acidosis. Sheep were assigned to a randomized complete block design with 2 x 2 factorial arrangement of treatments (n = 6 sheep/treatment) consisting of a control or acidosis diet, and receiving a saline or L-glutamine infusion. Sheep were fed diets for 10 d and slaughtered on d 11. Liver, kidney, and muscle samples were collected at slaughter and examined for relative messenger RNA (mRNA) expression of ubiquitin, C8, E2, cathepsin L, cathepsin B, caspase-3, and m-calpain, as well as protein expression of ubiquitin. Relative mRNA expression of C8 (P = 0.02), E2 (P = 0.06), and ubiquitin (P = 0.07) was less in kidney in acidotic vs. control sheep. Additionally, mRNA expression of m-calpain in kidney was greater (P = 0.01) as a result of glutamine infusion. There were no significant alterations (P > 0.10) in mRNA of any component as a result of acidosis in the liver or muscle. This study demonstrates the inability of metabolic acidosis to increase expression of the ubiquitin-mediated proteolytic pathway in skeletal muscle; however, downregulation of renal mRNA expression of these components is apparent during the induction of metabolic acidosis.
Asunto(s)
Acidosis/inducido químicamente , Regulación de la Expresión Génica/efectos de los fármacos , Glutamina/farmacología , Ovinos/fisiología , Equilibrio Ácido-Base/efectos de los fármacos , Aminoácidos/sangre , Alimentación Animal , Fenómenos Fisiológicos Nutricionales de los Animales , Animales , Calpaína/genética , Calpaína/metabolismo , Caspasa 3/genética , Caspasa 3/metabolismo , Catepsina B/genética , Catepsina B/metabolismo , Catepsina L , Catepsinas/genética , Catepsinas/metabolismo , Cisteína Endopeptidasas/genética , Cisteína Endopeptidasas/metabolismo , Dieta/veterinaria , Glutamina/administración & dosificación , Enfermedades de las Ovejas/inducido químicamente , Ubiquitina/genética , Ubiquitina/metabolismoRESUMEN
This study aims to test the feasibility of introducing functional chemical groups into biogenic silica spicules by examining the effect of supplementing a silican coupler [3-(trimethoxysilyl)propyl]urea (3-TMOSPU) as silica source in the cultures of archaeocytes-dominant-cell-population (ADCP) primmorphs and explants of the marine sponge Hymeniacidon perleve. Analysis by Fourier Transform Infrared Spectroscopy (FT-IR) confirmed that the organic group in 3-TMOSPU was introduced into silica spicules. By comparing ADCP-primmorph cultures when supplemented with Na2SiO3, 3-TMOSPU supplementation showed no notable effect on the primmorphs development and cell locomotion behaviors. A decline in silicatein expression quantified by real-time RT-PCR was, however, observed during spiculogenesis. The decline was slower for the 3-TMOSPU group whereas significantly fewer spicules were formed. When sponge papillae explants were cultured, 3-TMOSPU supplementation had no negative effect on sponge growth but inhibited the growth biofouling of the diatom Nitzschia closterium. By monitoring the detectable Si concentration, it seemed that 3-TMOSPU was converted by the sponge and its conversion was related to spiculogenesis. Analysis of spicule dimensional changes indicated that the inhibition of spiculogenesis by 3-TMOSPU supplementation was less in ADCP-primmorphs culture due to lower 3-TMOSPU/detectable Si ratio in the media.
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Catepsinas/metabolismo , Poríferos/metabolismo , Silicio/metabolismo , Animales , Biomasa , Catepsinas/genética , Técnicas de Cultivo de Célula/métodos , Diferenciación Celular , Poríferos/citología , Poríferos/crecimiento & desarrollo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Silicio/administración & dosificación , Silicio/química , Espectroscopía Infrarroja por Transformada de FourierRESUMEN
It has been reported that Cordyceps sinensis, a traditional Chinese medicine, has various pharmacological effects. The aim of this study was to clarify the effect of water extract of Cordyceps sinensis (WECS) on osteoclast differentiation in vitro. In mouse bone marrow cells and monocyte/macrophage cell line RAW264.7, WECS dose-dependently inhibited the receptor activator of nuclear factor kappa B (NF-kappaB) ligand (RANKL)-induced osteoclast differentiation by tartrate-resistant acid phosphatase (TRAP) staining. In fact, cytotoxic effect was not observed in the RAW264.7 cells treated with WECS. Moreover, the mRNA expression of osteoclast related genes (calcitonin receptor, cathepsin K, matrix metalloprotease 9 and nuclear factor of activated T cells c1) was also inhibited by WECS. Investigation of inhibitory mechanism by using electrophoretic mobility shift assay (EMSA) and Western blot analysis revealed that WECS inhibited the activation of NF-kappaB through the prevention of IkappaBalpha phosphorylation. In conclusion, the present results demonstrate for the first time that WECS is a potent inhibitor of the RANKL-induced osteoclast differentiation through a mechanism involving the NF-kappaB pathway.
Asunto(s)
Diferenciación Celular/efectos de los fármacos , Cordyceps/química , Osteoclastos/efectos de los fármacos , Ligando RANK/farmacología , Animales , Células de la Médula Ósea/efectos de los fármacos , Catepsina K , Catepsinas/genética , Células Cultivadas , Expresión Génica/efectos de los fármacos , Macrófagos/efectos de los fármacos , Masculino , Metaloproteinasa 9 de la Matriz/genética , Ratones , Ratones Endogámicos C57BL , Monocitos/efectos de los fármacos , FN-kappa B/antagonistas & inhibidores , Factores de Transcripción NFATC/genética , Receptores de Calcitonina/genéticaRESUMEN
OBJECTIVE: Obesity is associated with reduced insulin sensitivity and extensive reorganization of adipose tissue. As polyunsaturated fatty acids (PUFA) appear to inhibit diabetes development, we investigated PUFA effects on markers of matrix remodeling in white adipose tissue. METHODS AND PROCEDURE: Male obese diabetic (db/db) mice were treated with either a low-fat standard diet (LF), or high-fat diets rich in saturated and monounsaturated fatty acids (HF/S), n-6 PUFA (HF/6) or the latter including marine n-3 PUFA (HF/3). White adipose tissue was analyzed for gene expression, fatty acid composition and by immunofluorescence. RESULTS: HF/S treatment increased adipose tissue expression of a number of genes involved in matrix degradation including matrix metalloproteinase (MMP)-12, -14 and cathepsin K, L and S compared with LF. MMP-12 gene was expressed in macrophages and adipocytes, and MMP-12 protein colocalized with both cell types. In addition, mean adipocyte area increased by 1.6-fold in HF/S-treated mice. Genes essential for collagen production, such as procollagen I, III, VI, tenascin C and biglycan were upregulated in HF/S-treated animals as well. N-3 PUFA supplementation resulted in enrichment of these fatty acids in adipose tissue. Moreover, n-3 PUFA inhibited the HF/S-induced upregulation of genes involved in matrix degradation and production I restored mean adipocyte area and prevented MMP-12 expression in macrophages and adipocytes. CONCLUSION: N-3 PUFA prevent high-fat diet-induced matrix remodeling and adipocyte enlargement in adipose tissue of obese diabetic mice. Such changes could contribute to diabetes prevention by n-3 PUFA in obese patients.
Asunto(s)
Tejido Adiposo Blanco/fisiopatología , Diabetes Mellitus Experimental/fisiopatología , Grasas de la Dieta/administración & dosificación , Obesidad/fisiopatología , Adipocitos/fisiología , Tejido Adiposo Blanco/metabolismo , Animales , Biomarcadores/análisis , Catepsinas/genética , Tamaño de la Célula , Colagenasas/genética , Diabetes Mellitus Experimental/complicaciones , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Tipo 2/complicaciones , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/fisiopatología , Ácidos Grasos/administración & dosificación , Ácidos Grasos/análisis , Ácidos Grasos Omega-3/administración & dosificación , Regulación de la Expresión Génica/fisiología , Gónadas/metabolismo , Gónadas/fisiopatología , Hígado/metabolismo , Masculino , Metaloproteinasa 12 de la Matriz/análisis , Ratones , Ratones Endogámicos C3H , Obesidad/complicaciones , Obesidad/genética , Inhibidores Tisulares de Metaloproteinasas/genética , Triglicéridos/análisisRESUMEN
Simon extracts are vitamin K(1)-rich food materials extracted from the leaves of the Simon sweet potato. Although vitamin K is known to stimulate bone formation, we postulated that Simon extracts also contain unknown biological compounds having the ability to regulate bone resorption. Here we prepared the vitamin K-free fraction from the Simon extracts and investigated the ability of this fraction on the differentiation of osteoclasts. A remarkable inhibitory effect of osteoclastogenesis was observed when osteoclast precursors were treated with this fraction in rat bone marrow culture systems as well as in a pure differentiation system using murine osteoclast precursor cell line. The vitamin K-free Simon extracts markedly suppressed severe bone destruction mediated by abundant osteoclasts associated with adjuvant-induced arthritis in rats. High performance liquid chromatography (HPLC) analysis revealed that the vitamin K-free Simon extracts contained three types of low molecular weight inhibitors for osteoclastogenesis; caffeic acid, chlorogenic acids and isochlorogenic acids. Among these substances, caffeic acid showed the most powerful inhibitory effects on osteoclastogenesis. Caffeic acid significantly suppressed expression of NFATc1, a key transcription factor for the induction of osteoclastogenesis. Our current study enlightened a high utility of the Simon extracts and their chemical components as effective regulators for bone resorption accompanied with inflammation and metabolic bone diseases.
Asunto(s)
Artritis Experimental/tratamiento farmacológico , Ácidos Cafeicos/farmacología , Osteoclastos/efectos de los fármacos , Animales , Artritis Experimental/genética , Artritis Experimental/patología , Secuencia de Bases , Resorción Ósea/tratamiento farmacológico , Resorción Ósea/genética , Resorción Ósea/patología , Catepsina K , Catepsinas/genética , Células Cultivadas , Cartilla de ADN/genética , Gliceraldehído-3-Fosfato Deshidrogenasas/genética , Técnicas In Vitro , Ipomoea batatas/química , Masculino , Osteoclastos/patología , Fitoterapia , Extractos Vegetales/química , Extractos Vegetales/farmacología , Ratas , Ratas Endogámicas Lew , Ratas Sprague-Dawley , Receptor Activador del Factor Nuclear kappa-B/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Vitamina K/aislamiento & purificaciónRESUMEN
OBJECTIVE: To establish an effective assay to access the effects of natural products on cathepsin K for screening antiosteoporosis drugs. METHODS: To obtain the purified cathepsin K, we cloned the target fragment from the mRNA of human osteosacoma cell line MG63 and demonstrated its correctness through DNA sequencing. Cathepsin K was expressed in a high amount in E. coli after IPTG induction, and was purified to near homogenetity through resolution and column purification. The specificity of the protein was shown by Western blotting experiment. The biological activity of the components in the fermentation broth was assayed by their inhibitory effects on cathepsin K and its analog papain. RESULTS: With the inhibition of papain activity as a screen index, the fermentation samples of one thousand strains of fungi were tested and 9 strains among them showed strong inhibitory effects. The crude products of the fermentation broth were tested for their specific inhibitory effects on the purified human cathepsin K, the product of fungi 2358 shows the highest specificity against cathepsin K. CONCLUSIONS: The compounds isolated from fungi 2358 show the highest biological activity and are worth further structure elucidation and function characterization.
Asunto(s)
Bioensayo , Catepsinas/antagonistas & inhibidores , Evaluación Preclínica de Medicamentos , Osteoporosis/tratamiento farmacológico , Papaína/antagonistas & inhibidores , Western Blotting , Catepsina K , Catepsinas/genética , Catepsinas/aislamiento & purificación , Línea Celular , Clonación Molecular , ADN Complementario , Escherichia coli/enzimología , Escherichia coli/genética , Expresión Génica , Humanos , Análisis de Secuencia de ADN , Homología de Secuencia de AminoácidoRESUMEN
In scrapie-infected cells, the abnormal isoform of the prion protein, PrP(Sc), accumulates in endosomes/lysosomes. In this study, the involvement of two lysosomal proteases, cathepsin B and L, in cellular processing of PrP(Sc) was analyzed in immortalized neuronal gonadotropin-releasing hormone cells (GT1-1) infected with scrapie. Treatment with inhibitors of either cathepsin B or L resulted in accumulation of PrP(Sc). Such an increased accumulation also occurred when the activities of both cathepsins were inhibited using RNA interference. We conclude that cathepsin B and L are involved in the degradation of PrP(Sc) in scrapie-infected GT1-1 cells and that they can compensate for each other's functions. This study shows that specific proteases, abundantly present in neurons, have the capacity to degrade PrP(Sc).
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Catepsina B/metabolismo , Catepsinas/metabolismo , Neuronas/metabolismo , Priones/metabolismo , Animales , Western Blotting/métodos , Catepsina B/antagonistas & inhibidores , Catepsina B/genética , Catepsina L , Catepsinas/antagonistas & inhibidores , Catepsinas/genética , Línea Celular , Cisteína Endopeptidasas , Hormona Liberadora de Gonadotropina/metabolismo , Hipotálamo , Ratones , Neuronas/virología , Proteínas PrPSc/metabolismo , ARN Mensajero/biosíntesis , ARN Interferente Pequeño/metabolismo , ARN Interferente Pequeño/farmacología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Transfección/métodosRESUMEN
In some sponges peculiar proteins called silicateins catalyze silica polymerization in ordered structures, and their study is of high interest for possible biotechnological applications in the nanostructure industry. In this work we describe the isolation and the molecular characterization of silicatein from spicules of Petrosia ficiformis, a common Mediterranean sponge, and the development of a cellular model (primmorphs) suitable for in vitro studies of silicatein gene regulation. The spicule of P. ficiformis contains an axial filament composed of 2 insoluble proteins, of 30 and 23 kDa. The 23-kDa protein was characterized, and the full-length cDNA was cloned. The putative amino acid sequence has high homology with previously described silicateins from other sponge species and also is very similar to cathepsins, a cystein protease family. Finally, P. ficiformis primmorphs express the silicatein gene, suggesting that they should be a good model for biosilicification studies.
Asunto(s)
Catepsinas/genética , Modelos Animales , Filogenia , Poríferos/genética , Silicio/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Catepsinas/metabolismo , Clonación Molecular , Análisis por Conglomerados , Cartilla de ADN , ADN Complementario/genética , Italia , Datos de Secuencia Molecular , Técnicas de Amplificación de Ácido Nucleico , Poríferos/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Alineación de Secuencia , Análisis de Secuencia de ADNRESUMEN
OBJECTIVE: To identify genes regulated in human cholesteatoma compared with normal skin tissue using complementary DNA arrays. STUDY DESIGN: In vitro analysis. METHODS: Eight cholesteatoma and retroauricular skin samples were obtained from the same patients during surgery. Upregulated and downregulated genes were highlighted using complementary DNA arrays for screening. Reverse transcriptase-polymerase chain reaction and immunohistochemical staining were performed to confirm the results of the complementary DNA array. RESULTS: Twelve genes were found to be induced or upregulated in cholesteatoma compared with skin samples. These included genes involved in cell proliferation and differentiation (eg, calgranulin A, calgranulin B, psoriasin, thymosin beta-10) and cell invasion (eg, cathepsin C, cathepsin D, cathepsin H). Analyses by means of reverse transcription-polymerase chain reaction showed enhanced expression of several genes including calgranulin A, calgranulin B, psoriasin, thymosin beta-10, cathepsin C, cathepsin D, and cathepsin H in cholesteatoma, supporting the findings from the gene array. In addition, it was verified by immunohistochemical analysis that the expressions of Calgranulin A, Calgranulin B, and Cathepsin D were mainly located in cholesteatoma epithelium. CONCLUSION: The observed alteration in gene expression may play a role in various mechanisms of pathogenesis in cholesteatoma.
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Colesteatoma del Oído Medio/genética , ADN Complementario/genética , Expresión Génica/genética , Actinas/genética , Anticuerpos/inmunología , Proteínas de Unión al Calcio/genética , Calgranulina A/genética , Calgranulina B/genética , Catepsina C/genética , Catepsina D/genética , Catepsina H , Catepsinas/genética , Diferenciación Celular , Movimiento Celular , Colesteatoma del Oído Medio/inmunología , Colesteatoma del Oído Medio/patología , Cisteína Endopeptidasas/genética , Cartilla de ADN/genética , Regulación hacia Abajo , Humanos , Inmunohistoquímica , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteína A7 de Unión a Calcio de la Familia S100 , Proteínas S100 , Timosina/genética , Regulación hacia ArribaRESUMEN
To study gene expression in the endometrium at different stages of the menstrual cycle, differential mRNA display and reverse Northern analysis were performed on uterine tissues from cynomolgus monkeys. Eutopic endometrial RNA was prepared from uteri of animals that were either ovariectomized and supplemented with hormones, or were not ovariectomized but were subjected to surgically induced endometriosis. A number of genes were identified whose levels fluctuated between the proliferative and secretory phases of the cycle. Expression of four genes thus identified was further examined by in situ hybridization to normal human endometrial biopsies. Iodothyronine deiodinase was uniformly expressed at all stages of the human cycle that were studied: proliferative, secretory, and menstrual. Fibulin 1, osteopontin, and cathepsin H exhibited complex temporal and spatial regulation. Fibulin 1 was expressed in glandular epithelia during the menstrual phase, but expression switched to the stroma during the secretory phase. During the menstrual phase, osteopontin was expressed at high levels in glandular epithelia and in isolated stromal cells that may be of immune origin. Secretory phase expression of osteopontin was confined to a sub-population of epithelial cells. Cathepsin H was expressed in proliferative and menstrual phase endometrium, but expression disappeared in the secretory phase. Messenger RNA for fibulin 1, osteopontin, and iodothyronine deiodinase was detected in an endometriosis sample. Our data support functional roles for fibulin 1, osteopontin, cathepsin H and thyroid hormone in endometrium.
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Endometrio/fisiología , Fase Folicular , Regulación de la Expresión Génica , Fase Luteínica , Animales , Proteínas de Unión al Calcio/genética , Catepsina H , Catepsinas/genética , Cisteína Endopeptidasas/genética , Femenino , Expresión Génica , Perfilación de la Expresión Génica , Hibridación in Situ , Yoduro Peroxidasa/genética , Macaca fascicularis , Osteopontina , Sialoglicoproteínas/genética , Útero/fisiología , Yodotironina Deyodinasa Tipo IIRESUMEN
Cathepsin E is an intracellular aspartic proteinase that is considered to have a number of physiological roles including antigen processing. Quantitation of procathepsin E mRNA by LightCyclertrade mark technology indicated that the gene was transcribed in lung but not in kidney of both human and mouse origin. In contrast, the transcript was present in mouse spleen and alveolar macrophages but not in the counterpart tissue/cells from humans. Regulation of human and mouse procathepsin E gene expression was shown not to be influenced by the extent of CpG methylation but depended on the recognition of potential binding motifs in each promoter region by transcription factors such as GATA1, PU1 and YY1, as revealed by functional analysis using a series of promoter/luciferase reporter gene fusion constructs. Thus the extent to which the procathepsin E gene is expressed in a particular cell type may depend on the balance between the effects produced by positive-acting, cell-specific transcription factors such as GATA1 and PU1 and the negative influence of the ubiquitous YY1 factor. In this way, the relative abundance and influence of general and cell-specific transcription factors can govern the production of cathepsin E and thereby account for the sporadic cell and tissue distribution of this enzyme in different species.
Asunto(s)
Catepsinas/genética , Precursores Enzimáticos/genética , Péptidos , Animales , Antibacterianos , Secuencia de Bases , Catepsina E , Línea Celular , ADN/química , ADN/genética , ADN/metabolismo , Metilación de ADN , Cartilla de ADN/genética , Proteínas de Unión al ADN/metabolismo , Factores de Unión al ADN Específico de las Células Eritroides , Factor de Transcripción GATA1 , Regulación Enzimológica de la Expresión Génica , Humanos , Ratones , Datos de Secuencia Molecular , Oxazoles , Reacción en Cadena de la Polimerasa , Proteínas Proto-Oncogénicas/metabolismo , Distribución Tisular , Transactivadores/metabolismo , Factores de Transcripción/metabolismo , Transfección , Factor de Transcripción YY1RESUMEN
The major skeletal elements in the (Porifera) sponges, are spicules formed from inorganic material. The spicules in the Demospongiae class are composed of hydrated, amorphous silica. Recently an enzyme, silicatein, which polymerizes alkoxide substrates to silica was described from the sponge Tethya aurantia. In the present study the cDNA encoding silicatein was isolated from the sponge Suberites domuncula. The deduced polypeptide comprises 331 amino acids and has a calculated size of Mr 36 306. This cDNA was used as a probe to study the potential role of silicate on the expression of the silicatein gene. For these studies, primmorphs, a special form of aggregates composed of proliferating cells, have been used. It was found that after increasing the concentration of soluble silicate in the seawater medium from around 1 microM to approximately 60 microM, this gene is strongly upregulated. Without additional silicate only a very weak expression could be measured. Because silica as well as collagen are required for the formation of spicules, the expression of the gene encoding collagen was measured in parallel. It was also found that the level of transcripts for collagen strongly increases in the presence of 60 microM soluble silicate. In addition, it is demonstrated that the expression of collagen is also upregulated in those primmorphs which were treated with recombinant myotrophin obtained from the same sponge. Myotrophin, however, had no effect on the expression of silicatein. From these data we conclude that silicate influences the expression of the enzyme silicatein and also the expression of collagen, (via the mediator myotrophin).
Asunto(s)
Catepsinas/biosíntesis , Colágeno/biosíntesis , Regulación de la Expresión Génica , Sustancias de Crecimiento/metabolismo , Péptidos y Proteínas de Señalización Intercelular , Poríferos/química , Silicatos/metabolismo , Secuencia de Aminoácidos , Aminoácidos/química , Animales , Secuencia de Bases , Northern Blotting , Catepsinas/genética , Clonación Molecular , Colágeno/genética , ADN Complementario/metabolismo , Biblioteca de Genes , Germanio/farmacología , Datos de Secuencia Molecular , Filogenia , Reacción en Cadena de la Polimerasa , Homología de Secuencia de Aminoácido , Transcripción Genética , Regulación hacia ArribaRESUMEN
Previous studies have shown that lysine residues on the surface of cathepsins and other lysosomal proteins are a shared component of the recognition structure involved in mannose phosphorylation. In this study, the involvement of specific lysine residues in mannose phosphorylation of cathepsin D was explored by site-directed mutagenesis. Mutation of two lysine residues in the mature portion of the protein, Lys-203 and Lys-293, cooperated to inhibit mannose phosphorylation by 70%. Other positively charged residues could not substitute for lysine at these positions, and comparison of thermal denaturation curves for the wild type and mutant proteins indicated that the inhibition could not be explained by alterations in protein folding. Structural comparisons of the two lysine residues with those required for phosphorylation of cathepsin L, using models generated from recently acquired crystal structures, revealed several relevant similarities. On both molecules, the lysine residues were positioned approximately 34 A apart (34.06 A for cathepsin D and 33.80 A for cathepsin L). When the lysine pairs were superimposed, N-linked glycosylation sites on the two proteins were found to be oriented so that oligosaccharides extending out from the sites could share a common region of space. Further similarities in the local environments of the critical lysines were also observed. These results provide details for a common lysosomal targeting structure based on a specific arrangement of lysine residues with respect to each other and to glycosylation sites on the surface of lysosomal proteins.
Asunto(s)
Catepsina D/metabolismo , Catepsinas/metabolismo , Endopeptidasas , Lisina/metabolismo , Lisosomas/enzimología , Manosa/metabolismo , Animales , Secuencia de Bases , Células COS , Catepsina D/química , Catepsina D/genética , Catepsina L , Catepsinas/química , Catepsinas/genética , Cisteína Endopeptidasas , ADN Complementario , Humanos , Lisina/química , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Fosforilación , Conformación ProteicaRESUMEN
Earth's biota produces vast quantities of polymerized silica at ambient temperatures and pressures by mechanisms that are not understood. Silica spicules constitute 75% of the dry weight of the sponge Tethya aurantia, making this organism uniquely tractable for analyses of the proteins intimately associated with the biosilica. Each spicule contains a central protein filament, shown by x-ray diffraction to exhibit a highly regular, repeating structure. The protein filaments can be dissociated to yield three similar subunits, named silicatein alpha, beta, and gamma. The molecular weights and amino acid compositions of the three silicateins are similar, suggesting that they are members of a single protein family. The cDNA sequence of silicatein alpha, the most abundant of these subunits, reveals that this protein is highly similar to members of the cathepsin L and papain family of proteases. The cysteine at the active site in the proteases is replaced by serine in silicatein alpha, although the six cysteines that form disulfide bridges in the proteases are conserved. Silicatein alpha also contains unique tandem arrays of multiple hydroxyls. These structural features may help explain the mechanism of biosilicification and the recently discovered activity of the silicateins in promoting the condensation of silica and organically modified siloxane polymers (silicones) from the corresponding silicon alkoxides. They suggest the possibility of a dynamic role of the silicateins in silicification of the sponge spicule and offer the prospect of a new synthetic route to silica and siloxane polymers at low temperature and pressure and neutral pH.
Asunto(s)
Catepsinas/genética , Endopeptidasas , Poríferos/genética , Secuencia de Aminoácidos , Animales , Catepsina L , Clonación Molecular , Cisteína Endopeptidasas/genética , ADN Complementario/genética , ADN Complementario/aislamiento & purificación , Datos de Secuencia Molecular , Alineación de SecuenciaRESUMEN
We have shown previously that cathepsin K, a recently identified member of the papain superfamily of cysteine proteases, is expressed selectively in osteoclasts and is the predominant cysteine protease in these cells. Based upon its abundant cell type-selective expression, potent endoprotease activity at low pH and cellular localization at the bone interface, cathepsin K has been proposed to play a specialized role in osteoclast-mediated bone resorption. In this study, we evaluated a series of peptide aldehydes and demonstrated that they are potent cathepsin K inhibitors. These compounds inhibited osteoclast-mediated bone resorption in fetal rat long bone (FRLB) organ cultures in vitro in a concentration-dependent manner. Selected compounds were also shown to inhibit bone resorption in a human osteoclast-mediated assay in vitro. Chz-Leu-Leu-Leu-H (in vitro enzyme inhibition Ki,app = 1.4 nM) inhibited parathyroid hormone (PTH)-stimulated resorption in the FRLB assay with an IC-50 of 20 nM and inhibited resorption by isolated human osteoclasts cultured on bovine cortical bone slices with an IC-50 of 100 nM. In the adjuvant-arthritic (AA) rat model, in situ hybridization studies demonstrated high levels of cathepsin K expression in osteoclasts at sites of extensive bone loss in the distal tibia. Cbz-Leu-Leu-Leu-H (30 mg/kg, intraperitoneally) significantly reduced this bone loss, as well as the associated hind paw edema. In the thyroparathyriodectomized rat model, Cbz-Leu-Leu-Leu-H inhibited the increase in blood ionized calcium induced by a 6 h infusion of PTH. These data indicate that inhibitors of cathepsin K are effective at reducing osteoclast-mediated bone resorption and may have therapeutic potential in diseases of excessive bone resorption such as rheumatoid arthritis or osteoporosis.
Asunto(s)
Aldehídos/farmacología , Resorción Ósea , Catepsinas/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Oligopéptidos/farmacología , Animales , Artritis Experimental/metabolismo , Calcio/sangre , Catepsina K , Catepsinas/genética , Bovinos , Femenino , Humanos , Hormona Paratiroidea/farmacología , Paratiroidectomía , Embarazo , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes/antagonistas & inhibidores , Tiroidectomía , Células Tumorales CultivadasRESUMEN
Acute promyelocytic leukemia (APML) is characterized by abnormal myeloid development, resulting an accumulation of leukemic promyelocytes that are often highly sensitive to retinoic acid. A balanced t(15;17) (q22;q21) reciprocal chromosomal translocation is found in approximately 90% of APML patients; this translocation fuses the PML gene on chromosome 15 to the retinoic acid receptor alpha (RAR alpha) gene on chromosome 17, creating two novel fusion genes, PML-RAR alpha and RAR alpha-PML. The PML-RAR alpha fusion gene product, which is expressed in virtually all patients with t(15;17), is thought to play a direct role in the pathogenesis of APML. To determine whether PML-RAR alpha is sufficient to cause APML in an animal model, we used the promyelocyte-specific targeting sequences of the human cathepsin G (hCG) gene to direct the expression of a PML-RAR alpha cDNA to the early myeloid cells of transgenic mice. Mice expressing the hCG-PML-RAR alpha transgene were found to have altered myeloid development that was characterized by increased percentages of immature and mature myeloid cells in the peripheral blood, bone marrow, and spleen. In addition, approximately 30% of transgene-expressing mice eventually developed acute myeloid leukemia after a long latent period. The splenic promyelocytes of mice with both the nonleukemic and leukemic phenotypes responded to all-trans retinoic acid (ATRA) treatment, which caused apoptosis of myeloid precursors. Although low-level expression of the hCG-PML-RAR alpha transgene is not sufficient to directly cause acute myeloid leukemia in mice, its expression alters myeloid development, resulting in an accumulation of myeloid precursors that may be susceptible to cooperative transforming events.
Asunto(s)
Catepsinas/genética , Regulación Neoplásica de la Expresión Génica , Hematopoyesis/genética , Leucemia Promielocítica Aguda/genética , Ratones Transgénicos , Proteínas de Neoplasias/genética , Proteínas de Fusión Oncogénica/genética , Animales , Catepsina G , ADN Complementario/genética , ADN Complementario/aislamiento & purificación , Técnicas de Transferencia de Gen , Humanos , Leucemia Promielocítica Aguda/patología , Ratones , Serina EndopeptidasasRESUMEN
Two cathepsin L-related cysteine proteinase molecular clones were isolated from cDNA libraries of mRNA from the eyestalk nervous system and the stomach of Nephrops norvegicus and sequenced. The cDNA from nervous system was first obtained by screening an eyestalk cDNA library with an oligonucleotide whose sequence derived from the amino acid sequence of a peptide isolated previously and subsequently with the 5' end of the longest cDNA probe thus obtained. Several clones were isolated and analysed: one of these clones contains the complete cDNA (NCP1:AC = X80989). It encodes a preproenzyme of 324 amino acid residues, and a putative mature enzyme of 217 residues. Only one variant (with one amino acid change) was identified. The screening of the stomach cDNA library was carried out with a cDNA probe corresponding to the 5' terminal region of the nervous system cysteine proteinase cDNA previously obtained. A near full-length cDNA (NCP2:AC = X80990) was isolated. The sequence for mature stomach cathepsin L-related cysteine proteinase is 215 residues long. Nervous system cathepsin L-like NCP1 is very similar to the American lobster cysteine proteinase LCP1 (81% identity) and the stomach cathepsin L-related NCP2 is very similar to cysteine protease LCP3 (82% identity). Moreover, comparison of the two Nephrops norvegicus cathepsin L-related cysteine proteinases revealed only 68% identity.