RESUMO
AIM: To explore the value of radiomics for predicting the expression of programmed death ligand 1 (PD-L1) in non-small-cell lung cancer (NSCLC) based on multiparameter spectral computed tomography (CT) images. MATERIALS AND METHODS: A total of 220 patients with NSCLC were enrolled retrospectively and divided into the training (n=176) and testing (n=44) cohorts. The radiomics features were extracted from the conventional CT images, mono-energy 40 keV images, iodine density (ID) maps, Z-effective maps, and electron density maps. The logistic regression (LR) and support vector machine (SVM) algorithms were employed to build models based on radiomics signatures. The prediction abilities were qualified by the area under the curve (AUC) obtained from the receiver operating characteristic (ROC) curve. Internal validation was performed on the independent testing dataset. RESULTS: The combined model for PD-L1 ≥1%, which consisted of the radiomics score (rad-score; p<0.0001), white blood cell (WBC; p=0.027) counts, and air bronchogram (p=0.003), reached the highest performance with the AUCs of 0.873 and 0.917 in the training and testing dataset, respectively, which was better than the radiomics model with the AUCs of 0.842 and 0.886. The combined model for PD-L1 ≥50%, which consisted of rad-score (p<0.0001) and WBC counts (p=0.027), achieved the highest performance in the training and testing dataset with AUCs of 0.932 and 0.903, respectively, which was better than the radiomics model with AUCs of 0.920 and 0.892, respectively. CONCLUSION: The radiomics model based on the multiparameter images of spectral CT can predict the expression level of PD-L1 in NSCLC. The combined model can obtain higher prediction efficiency and serves as a promising method for immunotherapy selection.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Humanos , Antígeno B7-H1 , Carcinoma Pulmonar de Células não Pequenas/diagnóstico por imagem , Radiômica , Estudos Retrospectivos , Neoplasias Pulmonares/diagnóstico por imagem , Tomografia Computadorizada por Raios XRESUMO
BACKGROUND: With the growing evidence that other tissues, apart from adipose, could have strong relevance to obesity, it is necessary to comprehensively understand the relationship between obesity and other tissues, and to point out the most relevant tissues. METHODS: There were 549 participants with 20 different tissue types involved in this study. We firstly employed both Spearman's correlation test and WGCNA (weighted correlation network analysis) to identify body mass index (BMI)-related genes. Subsequently, we performed enrichment analyses with obesity genes and pathways to see the different regulation patterns among tissues. In addition, we compared obesity genes identified by genome-wide association studies (GWAS) with BMI-related genes to find the overlapping proportion in each tissue. Finally, we integrated preceding results to identify six strong obesity relevant tissues and indicate three categories to represent different obesity relevant tissues. RESULTS: Statistical analyses revealed diverse BMI-related genes and tissue-specific enrichment patterns among tissues. Comparison between BMI-related genes and GWAS findings showed tissue-specific expression changes of GWAS genes. Ultimately, six tissues that showed predominant performance in enrichment analyses and significantly embraced GWAS genes were referred to as strong obesity relevant tissues, including adipose, esophagus, nerve, pancreas, pituitary and skin. We also proposed three categories to represent different obesity relevant tissues. CONCLUSIONS: We performed the first study to investigate the BMI-related gene expression changes across 20 tissues at the same time. With valid data analyses and comparison with GWAS findings, our study provides a holistic view of how different tissues correlate with obesity, and proposes target tissues for obesity pathogenesis investigation.
Assuntos
Obesidade/genética , Obesidade/metabolismo , Especificidade de Órgãos/genética , Transdução de Sinais/genética , Transcriptoma/genética , Índice de Massa Corporal , Estudo de Associação Genômica Ampla , HumanosRESUMO
This study was conducted to evaluate the influence of back-fat thickness (BF), at mating of sows, on the maternal and newborn circulating lipids, expression of placental fatty acids (FA) transporters and lipid accumulation in placenta. Full-term placentas were obtained by vaginal delivery from BFI (9-14 mm; n = 37), BFII (15-19 mm; n = 43) and BFIII (20-27 mm; n = 38) sows according to BF at mating, and frozen placental sections were analysed for fat accumulation. Blood samples were collected from the sows of day 105 pregnancy and from cord blood at delivery. mRNA and protein expression levels were evaluated with real-time RT-PCR and Western blotting. Our results demonstrated that BFII females had significantly increased litter weight and placental efficiency, decreased maternal triglyceride (TG) and non-esterified fatty acids (NEFA) levels, decreased maternal IL-6, TNFα and leptin levels compared to BFIII females (p < .05). BFIII sows were associated with significantly decreased newborn TG levels, increased newborn glucose, IL-6 and TNFα levels compared to BFI or BFII sows (p < .05). BFI and BFII females had significantly decreased placental TG, NEFA and cholesterol (CHOL) contents compared to BFIII females (p < .05). Moreover, decreased CD36, FATP1, FABP4, and FABP1 mRNA and protein and FATP4 protein expression, and increased LPL activity were also observed in BFIII group compared with BFII group (p < .05). PPARγ mRNA and protein and lipogenic genes such as SREBP-1c, ACSL1, ACCα, FAS and SCD mRNA expression were downregulated or upregulated, respectively, in the placentas of BFIII sows compared to BFI or BFII sows (p < .05). Overall, this study demonstrated that there is no advantage, in terms of litter live size, litter weight and placental FA transport and metabolism, in performing the mating of sows with BF>19 mm.
Assuntos
Proteínas de Transporte de Ácido Graxo/metabolismo , Metabolismo dos Lipídeos/fisiologia , Obesidade/veterinária , Placenta/metabolismo , Doenças dos Suínos/metabolismo , Animais , Proteínas de Transporte de Ácido Graxo/genética , Ácidos Graxos/metabolismo , Feminino , Obesidade/metabolismo , Gravidez , SuínosRESUMO
OBJECTIVES: With ENCODE epigenomic data and results from published genome-wide association studies (GWASs), we aimed to find regulatory signatures of obesity genes and discover novel susceptibility genes. METHODS: Obesity genes were obtained from public GWAS databases and their promoters were annotated based on the regulatory element information. Significantly enriched or depleted epigenomic elements in the promoters of obesity genes were evaluated and all human genes were then prioritized according to the existence of the selected elements to predict new candidate genes. Top-ranked genes were subsequently applied to validate their associations with obesity-related traits in three independent in-house GWAS samples. RESULTS: We identified RAD21 and EZH2 as over-represented, and STAT2 (signal transducer and activator of transcription 2) and IRF3 (interferon regulatory transcription factor 3) as depleted transcription factors. Histone modification of H3K9me3 and chromatin state segmentation of 'poised promoter' and 'repressed' were over-represented. All genes were prioritized and we selected the top five genes for validation at the population level. Combining results from the three GWAS samples, rs7522101 in ESRRG (estrogen-related receptor-γ) remained significantly associated with body mass index after multiple testing corrections (P=7.25 × 10(-5)). It was also associated with ß-cell function (P=1.99 × 10(-3)) and fasting glucose level (P<0.05) in the meta-analyses of glucose and insulin-related traits consortium (MAGIC) data set.Cnoclusions:In summary, we identified epigenomic characteristics for obesity genes and suggested ESRRG as a novel obesity-susceptibility gene.
Assuntos
Predisposição Genética para Doença/genética , Estudo de Associação Genômica Ampla , Obesidade/genética , Regiões Promotoras Genéticas/genética , Receptores de Estrogênio/genética , Epigenômica , Humanos , MicroRNAs , Fenótipo , Receptores de Estrogênio/metabolismo , Fatores de TranscriçãoRESUMO
OBJECTIVE: To explore the proliferation property of vascular smooth muscle cells (VSMCs) in the stable CMKLR1 gene knock-down mouse VSMCs line and explore related mechanism. METHODS: The short hairpin RNA sequence targeting to knockdown the coding regions of mouse CMKLR1 mRNA was synthesized and subsequently employed to construct recombinant lentivirus vector.Mouse VSMCs were cultured and infected with the recombinant lentivirus (knockdown VSMCs). mRNA and protein CMKLR1 expression in Knockdown VSMCs was measured by real-time PCR and Western blot and compared with those in normal VSMCs (vehicle VSMCs) and lentivirus control VSMCs (control VSMCs). The proliferation of normal, knockdown and control VSMCs was induced by platelet-derived growth factor-BB (PDGF VSMCs) and measured by cell number counting and BrdU.The phosphorylated c-Jun N-terminal kinase (p-JNK) protein was investigated by Western blot. RESULTS: The relative level of CMKLR1 mRNA in knockdown VSMCs (0.23±0.04) was significantly downregulated compared with which in vehicle VSMCs (1.05±0.05) as well as control VSMCs (0.99±0.04) (P<0.01). The relative level of CMKLR1 protein in knockdown VSMCs (0.29±0.04) was also significantly decreased, compared with which in vehicle VSMCs (1.06±0.04) as well as control VSMCs (0.95±0.02) (P<0.01). The VSMCs number ((50.33±1.20)×10(3)/cm(2)) and BrdU A450 nm value (1.80±0.05) in PDGF VSMCs were significantly increased in vehicle VSMCs ((42.02±1.53)×10(3)/cm(2,) 1.55±0.04) (both P<0.05). Compared with those in vehicle VSMCs, the VSMCs number ((23.33±2.03)×10(3)/cm(2)) and BrdU A450 nm value (1.32±0.02) in knockdown VSMCs were significantly decreased.The proliferation property between PDGF VSMCs and control VSMCs was similar(P>0.05). Compared with the relative level of p-JNK protein (1.03±0.03) in vehicle VSMCs, the p-JNK protein level was significantly increased in PDGF VSMCs (1.36±0.02, P<0.05) and significantly downregulated in knockdown VSMCs (0.79±0.05, P<0.05). CONCLUSION: Knockdowning CMKLR1 gene can reduce the proliferation of mouse vascular smooth muscle cells, which was related with the down-regulation of p-JNK expression.
Assuntos
Miócitos de Músculo Liso/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Animais , Becaplermina , Proliferação de Células , Células Cultivadas , Regulação para Baixo , Técnicas de Silenciamento de Genes , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Camundongos , Músculo Liso Vascular/citologia , Fosforilação , Proteínas Proto-Oncogênicas c-sis/farmacologia , RNA Mensageiro/metabolismo , Receptores de QuimiocinasRESUMO
In this study, we investigated the relationship between serum glutamic acid decarboxylase (GAD) autoantibody (Ab) levels and single nucleotide polymorphisms (SNPs) of the glutamic acid de-carboxylase 2 (GAD2) 5'-untranslated region and the susceptibility to type 2 diabetes in the Han population. The distributions of patients with SNPs in the GAD2 5'-untranslated region (rs2236418, rs185649317, and rs8190590) and type 2 diabetes and that of the healthy group were genotyped and analyzed using Sequenom MassArray SNP genotyp-ing. GAD-Ab levels were also detected. The frequency distributions of the AA, AG, and GG genotypes in the polymorphic site rs2236418 in the diabetes GAD-Ab-positive group were 45.9, 42.8, and 11.4%, respectively, whereas those in the control group were 36.6, 43.7, and 19.8%, respectively. The difference between the 2 groups was statis-tically significant (P < 0.05). Unlike the GG genotype, the AA and AA + AG genotypes increased the risk of GAD-Ab (odds ratios (95% confidence intervals) = 2.623 (1.351-4.937) and 2.152 (1.375-4.202), respectively). The associations of the 3 SNPs of the GAD2 gene 5'-un-translated region polymorphisms with susceptibility to type 2 diabe-tes in the Chongqing Han population were significant. The SNP of rs2236418 in the Chongqing Han population of diabetic patients with serum GAD-Ab levels was significantly correlated with the SNPs rs185649317 and rs8190590.
Assuntos
Autoanticorpos/sangue , Diabetes Mellitus Tipo 2/enzimologia , Diabetes Mellitus Tipo 2/imunologia , Glutamato Descarboxilase/genética , Idoso , Autoanticorpos/genética , Autoanticorpos/imunologia , Estudos de Casos e Controles , Diabetes Mellitus Tipo 2/sangue , Diabetes Mellitus Tipo 2/genética , Feminino , Frequência do Gene , Predisposição Genética para Doença , Glutamato Descarboxilase/sangue , Glutamato Descarboxilase/imunologia , Humanos , Masculino , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo ÚnicoRESUMO
OBJECTIVE: Diabetic nephropathy (DN) is a major diabetic micro-vascular complication, and podocyte apoptosis induced by high glucose (HG) is a typical early feature of DN. Studies have shown that microRNAs (miRNAs) play a crucial role in the pathogenesis of DN. The purpose of the current study was to explore the role and molecular mechanism of miR-770-5p in podocyte apoptosis in DN. PATIENTS AND MATERIALS AND METHODS: In vitro podocyte model of DN was conducted by treatment conditionally immortalized mouse podocytes with HG (30 mM D-glucose). The level of miR-770-5p in podocytes was detected using quantitative real-time PCR (qRT-PCR), and protein levels were measured using Western blot assay in our current study. The relationship between miR-770-5p and TP53 regulated inhibitor of apoptosis 1 (TRIAP1) was revealed by TargetScan and dual luciferase reporter assay. Cell proliferation ability and cell apoptosis were determined by using cell counting kit-8 (CCK-8) assay and flow cytometer (FCM), respectively. RESULTS: We found that miR-770-5p was significantly upregulated in podocytes under HG condition. TRIAP1 was a target gene of miR-770-5p and it was down-regulated in podocytes by HG treatment. Further analysis indicated that HG induced cell proliferation ability reduction, cell apoptosis enhancement and apoptotic peptidase activating factor 1(APAF1)/Caspase9 pathway exaltation in podocytes were prevented by miR-770-5p down-regulation. More importantly, the results showed that all the effects of miR-770-5p inhibitor on HG induced podocytes were eliminated by TRIAP1 silencing. S.-Z. Zhang, X.-J. Qiu, S.-S. Dong, L.-N. Zhou, Y. Zhu, M.-D. Wang, L.-W. Jin We showed that miR-770-5p was upregulated in the in vitro model of DN, and it might promote the development of DN through regulating podocyte apoptosis by targeting TRIAP1.
Assuntos
Apoptose , Nefropatias Diabéticas/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , MicroRNAs/metabolismo , Modelos Biológicos , Podócitos/metabolismo , Animais , Linhagem Celular , Nefropatias Diabéticas/patologia , Glucose/farmacologia , Humanos , Camundongos , Podócitos/patologia , Ligação ProteicaRESUMO
Sudden death syndrome (SDS) is one of the most serious diseases of fast-growing broilers. The incidence of SDS may result from a decrease in ventricular function. The purpose of this study was to explore the mechanism of sexual difference in the sensitivity of broilers to SDS by measuring their ventricular vulnerability, serum enzyme activities, and serum electrolyte levels. Results were as follows. 1) Ventricular fibrillation thresholds induced by injection of KCl and by electrical stimulus of male broilers were both significantly lower than those of female broilers (P < 0.05), suggesting that the ventricular vulnerability of male broilers was higher than that of female broilers. 2) Serum lactate dehydrogenase and creatine kinase activities of male broilers were significantly higher than those of female broilers (P < 0.01), but there was not a significant difference in serum aspartate aminotransferase activity between male and female broilers. 3) No significant difference was observed in serum electrolyte levels of potassium, sodium, and chloride between males and females. From these results, we concluded that there is a significant difference between males and females in their ventricular vulnerability and serum enzyme activities, which may result in a higher sensitivity of male broilers to injury of the myocardium by stress and may further result in a sexual difference in sensitivity to SDS.
Assuntos
Galinhas/metabolismo , Morte Súbita/veterinária , Eletrólitos/sangue , Doenças das Aves Domésticas/metabolismo , Caracteres Sexuais , Fibrilação Ventricular/veterinária , Animais , Aspartato Aminotransferases/sangue , Galinhas/sangue , Galinhas/crescimento & desenvolvimento , Creatina Quinase/sangue , Feminino , L-Lactato Desidrogenase/sangue , Masculino , Cloreto de Potássio/toxicidade , Doenças das Aves Domésticas/sangue , Fibrilação Ventricular/induzido quimicamenteRESUMO
We aimed to summarize the results of genetic association studies for obesity and provide a comprehensive annotation of all susceptibility single nucleotide polymorphisms (SNPs). A total of 72 studies were summarized, resulting in 90,361 susceptibility SNPs (738 index SNPs and 89,623 linkage disequilibrium SNPs). Over 90% of the susceptibility SNPs are located in non-coding regions, and it is challenging to understand their functional significance. Therefore, we annotated these SNPs by using various functional databases. We identified 24,623 functional SNPs, including 4 nonsense SNPs, 479 missense SNPs, 399 untranslated region SNPs which might affect microRNA binding, 262 promoter and 5,492 enhancer SNPs which might affect transcription factor binding, 7 splicing sites, 76 SNPs which might affect gene methylation levels, 1,839 SNPs under natural selection and 17,351 SNPs which might modify histone binding. Expression quantitative trait loci analyses for functional SNPs identified 98 target genes, including 69 protein coding genes, 27 long non-coding RNAs and 3 processed transcripts. The percentage of protein coding genes that could be correlated with obesity-related pathways directly or through gene-gene interaction is 75.36 (52/69). Our results may serve as an encyclopaedia of obesity susceptibility SNPs and offer guide for functional experiments.
Assuntos
Predisposição Genética para Doença/genética , Obesidade/genética , Estudo de Associação Genômica Ampla , Humanos , Desequilíbrio de Ligação , Polimorfismo de Nucleotídeo Único , Locos de Características QuantitativasAssuntos
Carcinoma Hepatocelular/virologia , Hepatite B/complicações , Neoplasias Hepáticas Experimentais/virologia , Transativadores/genética , Animais , Apoptose , Linhagem Celular Tumoral , Proliferação de Células , DNA Viral/análise , Células Hep G2 , Hepatite B/virologia , Humanos , Camundongos , Camundongos Nus , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas Virais Reguladoras e Acessórias , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Overlapping cDNA fragments encoding avian cathepsin B were cloned from an osteoclast cDNA library and sequenced. The primary structure of the prepro enzyme deduced from this sequence has 340 amino acids. The mature portion of the enzyme is 80% identical with murine cathepsin B; regions found in other papain superfamily enzymes are conserved. In osteoclasts and cultured macrophages, which produce large quantities of cathepsin B, mRNAs of 1.8 and 2.4 kb are produced in approximately equal quantities, while cells producing smaller quantities of the enzyme produce predominantly the 2.4 kb form. This variation in mRNAs suggests transcriptional differences related to production of large quantities of the enzyme.
Assuntos
Catepsina B/genética , DNA Complementar/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Catepsina B/análise , Galinhas , Clonagem Molecular , DNA Complementar/química , Dados de Sequência Molecular , Osteoclastos/enzimologia , RNA Mensageiro/isolamento & purificaçãoRESUMO
Mast cells accumulate in hyperparathyroid bone, but the reason is not clear. We compared the distribution of mast cells and related growth factors in normal and hyperparathyroid bone. Mast cell formation was strongly affected by proximity to bone-forming surfaces of hyperparathyroid bone. Hyperparathyroidism greatly increased the production by active, bone-synthesizing osteoblasts of stem cell factor (SCF) but not of IL-3. Osteoblast SCF was distributed to the basolateral cell membranes, and its cDNA sequence (GenBank AF119835) is homologous to the murine membrane-bound SCF. Quiescent osteoblasts did not produce detectable SCF. Synthetic osteoblasts in normal bone were SCF positive, but comprised a much smaller population of cells, in keeping with the slow turnover of normal bone. Major SCF isoforms on immunoblot analysis of osteoblast-fraction proteins from high-turnover bone had M(r)s of about 48 and 40 kDa. Similar SCF isoforms were produced by MG63 osteoblast-derived cells and were identified by several anti-SCF antibodies. SCF is expressed in several mesenchymal cell types in a complementary fashion with cells bearing its receptor. SCF potently facilitates differentiation of mast cells, so the increase in paratrabecular mast cells in hyperparathyroid bone is probably driven by osteoblastic SCF. However, since mast cells are not normal components of bone, osteoblastic SCF probably regulates other cells, with mast cell differentiation occurring as a side effect greatly increased osteoblastic activity.
Assuntos
Osso e Ossos/metabolismo , Hiperparatireoidismo/metabolismo , Mastócitos/patologia , Osteoblastos/metabolismo , Fator de Células-Tronco/biossíntese , Osso e Ossos/citologia , Diferenciação Celular , Feminino , Humanos , Hiperparatireoidismo/patologia , Immunoblotting , Imuno-Histoquímica , Interleucina-3/metabolismo , Pessoa de Meia-Idade , Isoformas de Proteínas/metabolismo , Células Tumorais CultivadasRESUMO
A normal phase liquid chromatographic method was developed for the determination of berberine-type alkaloids in rhizoma Coptidis and traditional Chinese medicine--Angong Niuhuang Wan. Experimental evidences indicate that a normal phase system of a silica column (3.9 x 250 mm) as stationary phase with an eluent of ethyl acetate-formic acid-ethanol (15:3:2) as mobile phase can separate excellently (R5 greater than 1.5) four berberine-type alkaloids in the samples tested. According to the results obtained with this method the percent content of berberine hydrochloride in some Angong Niuhuang Wan from different factories and different batches was 0.331-0.456% and the average recovery was 97.23%; coefficient of variation was 1.2%.
Assuntos
Alcaloides de Berberina/análise , Berberina/análise , Medicamentos de Ervas Chinesas/análise , Cromatografia Líquida de Alta Pressão , Combinação de MedicamentosRESUMO
Berberine (1) and palmatine (2) in cortex phellodendron and Chinese patent medicines were determined by HPLC. The optimal composition of mobile phase CH3COOEt-HCOOH-EtOH (15:3:2) for HPLC separation of berberine and palmatine was successfully determined by using window diagram technique. Detection wavelength was 346nm. Flow rate: 1.5 ml/min. Calibration graphs for 1 and 2 were rectilinear for 0.06-0.32 micrograms and 0.12-0.32 micrograms respectively. The average recoveries of the two corresponding components were found to be over 96% and their coefficients of variation were below 5%.
Assuntos
Alcaloides de Berberina/análise , Berberina/análise , Medicamentos de Ervas Chinesas/análise , Cromatografia Líquida de Alta PressãoRESUMO
We investigated stem cell factor (SCF) expression in osteoblasts because mast cells, which occur ectopically in hyperparathyroid bone, are induced by SCF. Nontransformed osteoblasts and Saos2 or MG63 cells expressed SCF in response to PTH. Western analysis showed only large, cell-associated isoforms, Mrs approximately 40-48 kD. Transfection of MG63 cells with plasmids expressing antisense SCF mRNA eliminated immunoreactive SCF. Sequencing osteoblast SCF cDNAs showed that exon 6 was omitted. mRNAs without exon 6 produce membrane-associated SCF isoforms in rodents, suggesting that human SCFs are processed similarly. The major osteoblastic SCF mRNA, approximately 5 kB, was augmented by PTH. Neither protein or mRNA was increased by vitamin D, however, 6-7 kB transcripts were predominant in other tissues but not detectable in osteoblasts. We conclude that osteoblasts express SCF in response to PTH, with mRNA and protein processing differences relative to other cells. SCF stimulates osteoclasts, suggesting that PTH-induced osteoblastic SCF functions to accelerate bone turnover. Mast cells may occur due to SCF overexpression at extreme PTH levels.
Assuntos
Osteoblastos/metabolismo , Hormônio Paratireóideo/farmacologia , Fator de Células-Tronco/genética , Sequência de Aminoácidos , Sequência de Bases , Osso e Ossos/metabolismo , Linhagem Celular , DNA Antissenso/genética , DNA Antissenso/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Dados de Sequência Molecular , RNA Mensageiro/metabolismo , Análise de Sequência de DNA , Deleção de Sequência/genética , Fator de Células-Tronco/metabolismo , Transfecção/genética , Vitamina D/farmacologiaRESUMO
Bone resorption by osteoclasts is modified by agents that affect cyclic guanosine monophosphate (cGMP), but their relative physiological roles, and what components of the process are present in osteoclasts or require accessory cells such as osteoblasts, are unclear. We studied cGMP regulation in avian osteoclasts, and in particular the roles of nitric oxide and natriuretic peptides, to clarify the mechanisms involved. C-type natriuretic peptide drives a membrane guanylate cyclase, and increased cGMP production in mixed bone cells. However, C-type natriuretic peptide did not increase cGMP in purified osteoclasts. By contrast, osteoclasts did produce cGMP in response to nitric oxide (NO) generators, sodium nitroprusside or 1-hydroxy-2-oxo-3,3-bis(3-aminoethyl)-1-triazene. These findings indicate that C-type natriuretic peptide and NO modulate cGMP in different types of bone cells. The activity of the osteoclast centers on HCI secretion that dissolves bone mineral, and both NO generators and hydrolysis-resistant cGMP analogues reduced bone degradation, while cGMP antagonists increased activity. NO synthase agonists did not affect activity, arguing against autocrine NO production. Osteoclasts express NO-activated guanylate cyclase and cGMP-dependent protein kinase (G-kinase). G-kinase reduced membrane HCI transport activity in a concentration-dependent manner, and phosphorylated a 60-kD osteoclast membrane protein, which immunoprecipitation showed is not an H+-ATPase subunit. We conclude that cGMP is a negative regulator of osteoclast activity. cGMP is produced in response to NO made by other cells, but not in response to C-type natriuretic peptide. G-kinase modulates osteoclast membrane HCI transport via intermediate protein(s) and may mediate cGMP effects in osteoclasts.
Assuntos
GMP Cíclico/biossíntese , Óxido Nítrico/fisiologia , Osteoclastos/metabolismo , Ácidos/antagonistas & inibidores , Animais , Células da Medula Óssea/metabolismo , Células Cultivadas , Galinhas , Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , Relação Dose-Resposta a Droga , Ativação Enzimática , Concentração de Íons de Hidrogênio , Proteínas de Membrana/metabolismo , Peptídeo Natriurético Tipo C/fisiologia , Óxido Nítrico Sintase/metabolismo , Óxido Nítrico Sintase Tipo II , Osteoclastos/enzimologia , FosforilaçãoRESUMO
Osteoclasts are macrophage-derived polykaryons that degrade bone in an acidic extracellular space. This differentiation includes expression of proteinases and acid transport proteins, cell fusion, and bone attachment, but the sequence of events is unclear. We studied two proteins expressed at high levels only in the osteoclast, cathepsin K, a thiol proteinase, and tartrate-resistant acid phosphatase (TRAP), and compared this expression with acid transport and bone degradation. Osteoclastic differentiation was studied using human apheresis macrophages cocultured with MG63 osteosarcoma cells, which produce cytokines including RANKL and CSF-1 that mediate efficient osteoclast formation. Immunoreactive cathepsin K appeared at 3-5 days. Cathepsin K activity was seen on bone substrate but not within cells, and cathepsin K increased severalfold during further differentiation and multinucleation from 7 to 14 days. TRAP also appeared at 3-5 d, independently of cell fusion or bone attachment, and TRAP activity reached much higher levels in osteoclasts attached to bone fragments. Two proteinases that occur in the precursor macrophages, cathepsin B, a thiol proteinase related to cathepsin K, and an unrelated lysosomal aspartate proteinase, cathepsin D, were also studied to determine the specificity of the differentiation events. Cathepsin B occurred at all times, but increased two- to threefold in parallel with cathepsin K. Cathepsin D activity did not change with differentiation, and secreted activity was not significant. In situ acid transport measurements showed increased acid accumulation after 7 days either in cells on osteosarcoma matrix or attached to bone, but bone pit activity and maximal acid uptake required 10-14 days. We conclude that TRAP and thiol proteinase expression begin at essentially the same time, and precede cell fusion and bone attachment. However, major increases in acid secretion and proteinases expression continue during cell fusion and bone attachment from 7 to 14 days.