ABSTRACT
Stem cells are undifferentiated cells that can be distinguished from others by their ability to self-renew and to differentiate into new specific cell types. Mesenchymal stem cells (MSC) are adult stem cells that can be obtained from different sources, such as adipose tissue, bone marrow, dental pulp, and umbilical cord. They can either replicate, originating new identical cells, or differentiate into cells of mesodermal origin and from other germ layers. MSC have been studied as new tools for regenerative therapy. Although encouraging results have been demonstrated, MSC-based therapies still face a great barrier: the difficulty of isolating these cells from heterogeneous environments. MSC are currently characterized by immunolabelling through a set of multiple surface membrane markers, including CD29, CD73, CD90 and CD105, which are also expressed by other cell types. Hence, the present work aimed to identify new specific biomarkers for the characterization of human MSC using DNA aptamers produced by the SELEX (Systematic Evolution of Ligands by EXponential Enrichment) technique. Our results showed that MSC from different origins bound to DNA candidate aptamers, that is, DNA or RNA oligonucleotides selected from random libraries that bind specifically to biological targets. Aptamer-bound MSC could be isolated by fluorescenceactivated cell sorting (FACS) procedures, enhancing the induction of differentiation into specific phenotypes (chondrocytes, osteocytes and adipocytes) when compared to the whole MSC population. Flow cytometry analyses revealed that candidate aptamers bound to 50% of the MSC population from dental pulp and did not present significant binding rates to human fibroblasts or lymphocytes, both used as negative control. Moreover, immunofluorescence images and confocal analyses revealed staining of MSC by aptamers localized in the surfacemembrane of these cells. The results also showed internal staining of human monocytes by our investigated aptamers. A non-specific control aptamer (CNTR APT) obtained from the random pool was then utilized to compare the specificity of the aptamers bound to the analyzed non-apoptotic cells, showing no staining for MSC. However, 40% of the monocytes bound to the CNTR APT. Normalized data based on the cells bound to candidate aptamers compared to those bound to the CNTR APT, revealed a 10 to 16-fold higher binding rate for MSC against 2-fold for monocytes. Despite its low specificity, monocyte-aptamer binding occurs probably due to the expression of shared markers with MSC, since monocytes are derived from hematopoietic stem cells and are important for the immune system ability to internalize/phagocyte external molecules. Given that, we performed a pull-down assay followed by mass spectrometry analysis to detect which MSC-specific protein or other target epitope not coexpressed by monocytes or the CNTR APT would bind to the candidate aptamer. Distinguishing between MSC and monocyte epitopes is important, as both cells are involved in immunomodulatory effects after MSC transplantations. ADAM17 was found to be a target of the APT10, emerging as a possible biomarker of MSC, since its involvement in the inhibition of the TGF signaling cascade, which is responsible for the differentiation of MSC. Thus, MSC with a higher stemness profile should overexpress the protein ADAM17, which presents a catalytic site with affinity to APT10. Another target of Apt 10 is VAMP3, belonging to a transmembrane protein complex that is involved in endocytosis and exocytosis processes during immune and inflammatory responses. Overall, proteins identified as targets of APT10 may be cell surface MSC biomarkers, with importance for MSC-based cell and immune therapies
Células tronco são células indiferenciadas que podem ser distinguidas de outros tipos celulares por meio da habilidade de se auto renovarem e de se diferenciarem em novos tipos celulares. Células tronco mesenquimais (MSC) são células tronco adultas encontradas em diferentes tecidos como tecido adiposo, polpa de dente e cordão umbilical. Estas células podem se autodividir em células idênticas ou se diferenciarem em células de origem mesodermal. Estas células têm sido estudadas em novas aplicações que envolvem terapia regenerativas. Embora resultados encorajadores tenham sido demonstrados, terapias que utilizam MSC ainda encontram uma grande barreira: a dificuldade no isolamento destas células a partir de um ambiente heterogêneo. MSC são caracterizadas por populações positivas em ensaios de imunomarcação para os epítopos membranares CD29, CD73, CD90 e CD105, presentes também em outros tipos celulares. Assim, o presente trabalho tem o objetivo de identificar novos biomarcadores de MSC de origem humana, utilizando aptâmeros de DNA produzidos pela técnica SELEX (Systematic Evolution of Ligands by EXponential Enrichment) como ferramenta. Nossos resultados mostraram que MSC de diferentes origens ligam-se a aptâmeros (oligonucleotídeos de DNA ou RNA que atuam como ligantes específicos de alvos moleculares) de DNA candidatos que atuam no isolamento de MSC por meio da técnica FACS de separação celular, promovendo uma maior indução de diferenciação em células específicas (condrócitos, osteócitos e adipócitos) comparada com a população total de MSC. Análises de citometria de fluxo mostraram que os aptâmeros candidatos se ligam a 50% das MSC de polpa de dente e não apresentam taxa de ligação significante para fibroblastos e linfócitos de origem humana - utilizados como controles negativo. Além domais, imagens de imunofluorescência e confocal mostraram ligação na superfície da membrana de MSC e a marcação interna de monócitos a estes aptâmeros. Portanto, um aptâmero controle (CNTR APT) foi utilizado para comparar a especificidade dos aptâmeros ligados a células viáveis, mostrando a não ligação deste aptâmero a MSC. Porém, 40% da população de monócitos ligou-se ao CNTR APT. Uma normalização baseada na comparação entre as taxas de ligação entre células ligadas com aptâmeros candidatos e o aptâmero controle gerou uma taxa de especificidade entre 10-16 vezes maior para MSC contra 2,5 vezes para os monócitos. Deste modo, embora os resultados tenham mostrado uma taxa de ligação entre monócitos e aptâmeros, as MSC ligadas aos aptâmeros candidatos possuem uma maior taxa de especificidade devido a uma maior presença de antígenos que são expressos em ambas as células. Um ensaio de Pull Down seguido de espectrometria de massas foi utilizado para a identificação de biomarcadores que se ligariam aos aptâmeros candidatos, e que não seriam co-expressos por monócitos e por antígenos ligados ao aptâmero controle. Deste modo, a proteína ADAM17 foi identificada nas amostras de APT10 ligadas às MSC. Tal proteína está relacionada à inibição de uma cascata de sinalização da família de proteínas TGF, responsável pela diferenciação de MSC. Assim, MSC com maior potencial tronco deveriam expressar ADAM17 em maior quantidade. Tal proteína apresenta um sítio catalítico que demonstra interagir com o APT10, de acordo com predição Docking entre proteína e DNA. Foi identificada também, a proteína VAMP3, que pertence a um complexo proteico transmembranar responsável pelos processos de endocitose e exocitose, e que podem ter um papel importante na liberação de citocinas e outras moléculas relacionadas às respostas imune e inflamatórias. Deste modo, o APT10 identificou proteínas importantes que devem estar relacionas com a melhora de imunoterapias que utilizam MSC
Subject(s)
Stem Cells , Biomarkers/analysis , SELEX Aptamer Technique/instrumentation , Mesenchymal Stem Cells/classification , ADAM17 Protein/pharmacology , Patient Isolation , Mass Spectrometry/methods , Staining and Labeling/methods , Transplantation/adverse effects , Umbilical Cord , DNA/agonists , Transforming Growth Factors/agonists , Cell Separation/instrumentation , Cytokines/adverse effects , Adipocytes/metabolism , Chondrocytes/classification , Scientists for Health and Research for Development , Adult Stem Cells/classification , Fibroblasts/chemistry , Flow Cytometry/instrumentation , Germ Layers , Antigens/adverse effectsABSTRACT
Alzheimer's disease(AD) patients in China have been surging, and the resultant medical burden and care demand have a huge impact on the development of individuals, families, and the society. The active component compound of Epimedii Folium, Astragali Radix, and Puerariae Lobatae Radix(YHG) can regulate the expression of iron metabolism-related proteins to inhibit brain iron overload and relieve hypofunction of central nervous system in AD patients. Hepcidin is an important target regulating iron metabolism. This study investigated the effect of YHG on the expression of a disintegrin and metalloprotease-17(ADAM17), a key enzyme in the hydrolysis of β amyloid precursor protein(APP) in HT22 cells, by mediating hepcidin. To be specific, HT22 cells were cultured in vitro, followed by liposome-mediated siRNA transfection to silence the expression of hepcidin. Real-time PCR and Western blot were performed to examine the silencing result and the effect of YHG on hepcidin in AD cell model. HT22 cells were randomized into 7 groups: control group, Aβ25-35 induction(Aβ) group, hepcidin-siRNA(siRNA) group, Aβ25-35 + hepcidin-siRNA(Aβ + siRNA) group, Aβ25-35+YHG(Aβ+YHG) group, hepcidin-siRNA+YHG(siRNA+YHG) group, Aβ25-35+hepcidin-siRNA+YHG(Aβ+siRNA+YHG) group. The expression of ADAM17 mRNA in cells was detected by real-time PCR, and the expression of ADAM17 protein by immunofluorescence and Western blot. Immunofluorescence showed that the ADAM17 protein expression was lower in the Aβ group, siRNA group, and Aβ+siRNA group than in the control group(P<0.05) and the expression was lower in the Aβ+siRNA group(P<0.05) and higher in the Aβ+YHG group(P<0.05) than in the Aβ group. Moreover, the ADAM17 protein expression was lower in the Aβ+siRNA group(P<0.05) and higher in the siRNA+YHG group(P< 0.05) than in the siRNA group. The expression was higher in the Aβ+siRNA+YHG group than in the Aβ+siRNA group(P<0.05). The results of Western blot and real-time PCR were consistent with those of immunofluorescence. The experiment showed that YHG induced hepcidin to up-regulate the expression of ADAM17 in AD cell model and promote the activation of non-starch metabolic pathways, which might be the internal mechanism of YHG in preventing and treating AD.
Subject(s)
Humans , ADAM17 Protein , Alzheimer Disease/genetics , Amyloid beta-Peptides , Drugs, Chinese Herbal/pharmacology , Hepcidins/genetics , PuerariaABSTRACT
Abstract Although periodontitis is one of the commonest infectious inflammatory diseases in humans, the mechanisms involved with its immunopathology remain ill understood. Numerous molecules may induce inflammation and lead to bone resorption, secondary to activation of monocytes into osteoclasts. TACE (TNF-α converting enzyme) and DC-STAMP (dendritic cell-specific transmembrane protein) appear to play a role on bone resorption since TACE induces the release of sRANKL (soluble receptor activator of nuclear factor kappa-β ligand) whereas DC-STAMP is a key factor in osteoclast induction. The present study evaluated the levels of TACE and DC-STAMP in patients with and without periodontitis. Twenty individuals were selected: 10 periodontally healthy participants undergoing gingivectomy for esthetic reasons and 10 diagnosed with periodontitis. Protein levels of such molecules in gingival tissue were established using Western blotting. Protein levels of both TACE and DC-STAMP were higher in the periodontitis group than in the control group (p<0.05; Student t-test). In conclusion, TACE and DC-STAMP protein levels are elevated in patients with periodontitis, favoring progression of bone resorption.
Resumo Apesar de a periodontite ser uma das doenças infecto inflamatórias humanas mais comuns, os mecanismos que conduzem à imunopatologia não estão bem definidos. Inúmeras moléculas induzem atividade inflamatória que levam à perda óssea. Para que haja a reabsorção óssea, células monocíticas são ativadas e se transformam em osteoclastos. As moléculas TACE (Enzima conversora de TNF-α) e DC-STAMP (Proteína transmembrana específica de célula dendrítica) parecem atuar no processo de reabsorção óssea uma vez que a TACE induz a liberação de sRANKL (ativador do receptor do fator nuclear kappa-β ligante solúvel), enquanto a DC-STAMP é um fator chave na indução dos osteoclastos. Diante disso, o presente estudo avaliou a expressão gênica das moléculas TACE e DC-STAMP em pacientes com e sem periodontite uma vez que o papel destas moléculas no curso do desenvolvimento da periodontite ainda é pouco explorado. Foram selecionados 20 indivíduos, sendo 10 com saúde periodontal e com indicação para remoção de tecido gengival por motivos estéticos e 10 pacientes com periodontite. As análises da expressão das moléculas no tecido gengival foram realizadas por meio de western blotting. Os níveis proteicos tanto de TACE quanto de DC-STAMP, foram maiores nos tecidos do grupo com periodontite em comparação aos do grupo controle (p<0.05; Student' t-test). Portanto, os dados demonstram que a expressão protéica das moléculas TACE e DC-STAMP estão elevados em pacientes com periodontite, favorecendo a progressão da reabsorção óssea nesta patologia.
Subject(s)
Humans , Periodontitis , Bone Resorption , Adaptor Proteins, Signal Transducing/metabolism , ADAM17 Protein/metabolism , Membrane Proteins/metabolism , Osteoclasts , Cell DifferentiationABSTRACT
OBJECTIVE@#To explore the association between expression of ADAM17 and cetuximad resistance in human colorectal cancer SW480 cells.@*METHODS@#The expression of ADAM17 was detected using Western blotting in different human colorectal cancer cell lines, and the cells highly expressing ADAM17 were selected as the target cells. SW480 cells were transfected with ADAM17-siRNA 1 and ADAM17-siRNA 2 and the changes in the expression of ADAM17 protein were detected using Western blotting. SW480 cells were exposed to cetuximad for 24 h and the cell apoptosis was analyzed using flow cytometry. Transwell assay was used to examine the migration ability of SW480 cells with different expression levels of ADAM17; Western blotting was used to analyze the changes in the expressions of AKT signaling pathway-related proteins in the treated cells.@*RESULTS@#The baseline expressions of ADAM17 were significantly higher in SW480 cells than in the other human colorectal cancer cell lines tested ( < 0.05). Both ADAM17-siRNA 1 and 2 effectively reduced the expression of ADAM17 protein in SW480 cells. Knockdown of ADAM17 with siRNA 1 significantly increased the sensitivity of SW480 cells to tocetuximad ( < 0.05), obviously inhibited the cell proliferation, migration and invasion, and significantly reduced the expressions of p-EGFR and p-AKT in the cells ( < 0.001).@*CONCLUSIONS@#ADAM17 knockdown obviously inhibits EGFR-AKT signaling pathway and increases the sensitivity of SW480 cells to tocetuximad.
Subject(s)
Humans , ADAM17 Protein , Genetics , Metabolism , Antineoplastic Agents, Immunological , Pharmacology , Apoptosis , Cell Line, Tumor , Cell Movement , Cell Proliferation , Cetuximab , Pharmacology , Colorectal Neoplasms , Drug Therapy , Genetics , Metabolism , Pathology , Drug Resistance, Neoplasm , Genetics , ErbB Receptors , Metabolism , Gene Knockdown Techniques , Neoplasm Invasiveness , Oncogene Protein v-akt , Metabolism , RNA, Small Interfering , Signal Transduction , Transfection , MethodsABSTRACT
BACKGROUND: Extracellular metolloproteases have been implied in different process such as cell death, differentiation and migration. Membrane-bound metalloproteases of the ADAM family shed the extracellular domain of many cytokines and receptor controlling auto and para/juxtacrine cell signaling in different tissues. ADAM17 and ADAM10 are two members of this family surface metalloproteases involved in germ cell apoptosis during the first wave of spermatogenesis in the rat, but they have other signaling functions in somatic tissues. RESULTS: In an attempt to further study these two enzymes, we describe the presence and localization in adult male rats. Results showed that both enzymes are detected in germ and Sertoli cells during all the stages of spermatogenesis. Interestingly their protein levels and cell surface localization in adult rats were stage-specific, suggesting activation of these enzymes at particular events of rat spermatogenesis. CONCLUSIONS: Therefore, these results show that ADAM10 and ADAM17 protein levels and subcellular (cell surface) localization are regulated during rat spermatogenesis.
Subject(s)
Animals , Male , Rats , Spermatogenesis/physiology , Spermatozoa/metabolism , ADAM Proteins/metabolism , Seminiferous Tubules/chemistry , Sertoli Cells/cytology , Sertoli Cells/metabolism , Spermatids/cytology , Spermatids/metabolism , Testis/anatomy & histology , RNA, Messenger/analysis , Immunohistochemistry , Cell Differentiation/physiology , Rats, Sprague-Dawley , Apoptosis/physiology , fas Receptor/analysis , Reverse Transcriptase Polymerase Chain Reaction , ADAM Proteins/analysis , ADAM10 Protein , ADAM17 ProteinABSTRACT
Ebosin is a novel exopolysaccharide produced by Streptomyces sp.139 with remarkable activity against rheumatic arthritis in vivo. In this paper, we reported effects of Ebosin on the inflammatory cytokines including interleukin-1beta (IL-1beta), interleukin-6 (IL-6) and tumor necrosis factor alpha (TNFalpha) in THP-1 cells. With the special fluorogenic peptide as substrates, the enzymatic activities of interleukin-1beta converting enzyme (ICE) and TNFalpha-converting enzyme (TACE) were inhibited by Ebosin separately. Using the real-time reverse transcription polymerase chain reaction (real-time PCR), the mRNA synthesis of the three cytokines were identified decline separately by Ebosin. The secretion quantum of three cytokines in THP-1 cells with Ebosin was lower than that of normal THP-1 cells determined by ELISA assay and Western blotting. All of these results showed that Ebosin has remarkably suppressed synthesis of the three cytokines in THP-1 cells through different pathways. The primary study of Ebosin on anti-inflammation mechanism was promoted developing the new drugs treating rheumatic arthritis.
Subject(s)
Humans , ADAM Proteins , Metabolism , ADAM17 Protein , Anti-Inflammatory Agents , Pharmacology , Caspase 1 , Metabolism , Cell Line, Tumor , Interleukin-1beta , Genetics , Metabolism , Interleukin-6 , Genetics , Metabolism , Leukemia, Monocytic, Acute , Metabolism , Pathology , Polysaccharides, Bacterial , Pharmacology , RNA, Messenger , Metabolism , Streptomyces , Metabolism , Tumor Necrosis Factor-alpha , Genetics , MetabolismABSTRACT
Glycoprotein (GP) Ibα ectodomain shedding has important implications for thrombosis and hemostasis. A disintegrin and metalloproteinase 17 (ADAM17) was identified to play an essential role in agonist induced GPIbα shedding. The relationship of GPIbα shedding and ADAM17 in the acute stage of atherosclerotic ischemic stroke (AIS) patients has not been thoroughly studied. A total of 306 patients and 230 controls matched for age, sex, race, history of hypertension and diabetes mellitus were enrolled in the study. GPIbα, ADAM17, glycocalicin were detected by flow cytometry, Western blotting, and enzyme-linked immunosorbent assay (ELISA) respectively. Compared with the control group, the expression of GPIbα in patients with acute ischemic stroke was significantly lower (P=0.000, P<0.01). Plasma glycocalicin and ADAM17 in AIS group were higher than those in control group (P=0.699, P=0.000). Pearson's analysis showed glycocalicin bore no correlation with GPIbα in AIS patients (r=0.095, P>0.05). GPIbα and National Institute of Health Stroke Scale (NIHSS) had negative correlation (r=-0.514, P<0.01). Our findings indicate that ADAM17 may be a risk factor for ischemic stroke in Chinese and the expression of GPIbα can serve as a measure for stroke severity.
Subject(s)
Female , Humans , Male , Middle Aged , ADAM Proteins , Blood , ADAM17 Protein , Biomarkers , Blood , Blood Platelets , Metabolism , Brain Ischemia , Blood , Diagnosis , China , Intracranial Arteriosclerosis , Blood , Diagnosis , Platelet Glycoprotein GPIb-IX Complex , Metabolism , Reproducibility of Results , Sensitivity and Specificity , Severity of Illness Index , Stroke , Blood , DiagnosisABSTRACT
<p><b>OBJECTIVE</b>To investigate the expression of A disintegrin and metalloproteinase 17 (ADAM17) mRNA and ADAM17 protein in esophageal squamous cell carcinoma (ESCC), and to evaluate their correlation with clinicopathological features and prognosis.</p><p><b>METHODS</b>The expression of ADAM17 mRNA in 50 ESCC and 50 normal esophageal tissues was detected by RT-PCR. The expression of ADAM17 protein in 80 ESCC and 80 normal esophageal tissues was detected with immunohistochemioal staining (SP). Log rank test and Cox proportional hazards analysis were used to analyze the prognosis of ESCC.</p><p><b>RESULTS</b>The expression of ADAM17 mRNA in 50 ESCC and 50 normal esophageal tissues was 0.937 ± 0.241 and 0.225 ± 0.077, respectively. The positive expression rates of ADAM17 protein in 80 ESCC and 80 normal esophageal tissues was 66.2% and 6.2%, respectively. The expressions of ADAM17 mRNA and ADAM17 protein in the ESCC were significantly higher than those in normal esophageal tissues (P < 0.01). The expressions of ADAM17 mRNA and protein were positively correlated with lymph node metastasis and TNM staging (P < 0.05). There were no correlations between the expressions of ADAM17 mRNA and protein and sex, age and histological grade (P > 0.05) . The expression of ADAM17 protein was positively correlated with EGFR protein (P < 0.01). The lymph node metastasis, TNM staging and the expression of ADAM17 and EGFR protein were independent prognostic factors.</p><p><b>CONCLUSIONS</b>ADAM17 mRNA and protein are highly expressed in ESCC than in normal esophageal tissues and may play an important role in the development, invasion and metastasis of ESCC. They may be used as prognostic factors of ESCC.</p>
Subject(s)
Aged , Female , Humans , Male , Middle Aged , ADAM Proteins , Genetics , Metabolism , ADAM17 Protein , Biomarkers, Tumor , Metabolism , Carcinoma, Squamous Cell , Metabolism , Pathology , General Surgery , Esophageal Neoplasms , Metabolism , Pathology , General Surgery , Follow-Up Studies , Kaplan-Meier Estimate , Lymphatic Metastasis , Neoplasm Staging , Proportional Hazards Models , RNA, Messenger , Metabolism , ErbB Receptors , Metabolism , Reverse Transcriptase Polymerase Chain Reaction , Survival RateABSTRACT
<p><b>OBJECTIVE</b>To investigate the main proteinases responsible for CD16b shedding under different stimulators.</p><p><b>METHODS</b>HEK293 cell line stably expressing CD16b was constructed by lentivirus system. The cell line was then overexpressed with a disintegrin and metalloproteinase 10 (ADAM10) or ADAM17, suppressed with short hairpin RNA of ADAM10 or ADAM17, and reconstituted with ADAM10 or ADAM17, respectively. After each treatment, the cell line was stimulated with ionomycin or phorbol 12-myristate- 13-acetate (PMA) for 12 hours. The soluble CD16b released from cell membrane was detected by immunoprecipition and immunoblot. Quantitation was then implemented to compare the amount of soluble CD16b in cell supernatant after stimulation.</p><p><b>RESULTS</b>HEK293 cell line stably expressing CD16b was successfully established. When CD16b expressing cell line was overexpressed with ADAM10, shedding of CD16b was increased after stimulation with ionomycin but not PMA; when the cell line overexpressed with ADAM17, shedding of CD16b was increased after stimulation with PMA but not ionomycin. Similarly, when ADAM10 was suppressed by short hairpin RNA, CD16b shedding was decreased after stimulation with ionomycin; when ADAM17 was suppressed by short hairpin RNA, CD16b shedding was decreased after stimulation with PMA. The shedding of CD16b was increased again when CD16b expressing cell line was reconstituted with ADAM10 and stimulated by ionomycin or reconstituted with ADAM17 and stimulated by PMA.</p><p><b>CONCLUSIONS</b>Both ADAM10 and ADAM17 could shed CD16b, but they possess differed preferences. ADAM10 is the main sheddase under stimulation of ionomycin, while ADAM17 is the main sheddase under stimulation of PMA.</p>
Subject(s)
Humans , ADAM Proteins , Genetics , Metabolism , Physiology , ADAM10 Protein , ADAM17 Protein , Amyloid Precursor Protein Secretases , Genetics , Metabolism , Physiology , Calcium Ionophores , Pharmacology , Carcinogens , Pharmacology , Cells, Cultured , Drug Evaluation, Preclinical , GPI-Linked Proteins , Metabolism , Gene Knockdown Techniques , HEK293 Cells , Ionomycin , Pharmacology , Membrane Proteins , Genetics , Metabolism , Physiology , Protein Processing, Post-Translational , Protein Transport , Proteolysis , Receptors, IgG , Metabolism , Tetradecanoylphorbol Acetate , Pharmacology , TransfectionABSTRACT
<p><b>OBJECTIVE</b>To study the effect of siRNA targeting ADAM17 (ADAM17-siRNA) on the proliferation of prostate cancer PC-3 cells.</p><p><b>METHODS</b>After transfecting PC-3 cells with ADAM17-siRNA 1 and ADAM17-siRNA 2, we detected the expressions of ADAM17 mRNA and protein by RT- PCR and Western blotting, respectively. We measured the changes in the proliferation and DNA synthesis of PC-3 cells by MTT and bromodeoxyuridine (BrdU) incorporation assay, examined the cell cycle profile by flow cytometry, and determined the expressions of the genes associated with PC-3 cell proliferation by Western blotting.</p><p><b>RESULTS</b>Both ADAM17-siRNA 1 and 2 effectively reduced the expressions of ADAM17 mRNA and protein in the PC-3 cells. Knockdown of ADAM17 with the two siRNAs significantly inhibited cell proliferation as compared with the control group (0.43 +/- 0.57 and 0.44 +/- 0.64 vs 0.80 +/- 0.51, P < 0.05) and down-regulated DNA synthesis (0.48 +/- 0.43 and 0.54 +/- 0.59 vs 0.79 +/- 0.72, P < 0.05). The cell cycle profile showed that the cell population of the G1 phase was markedly higher in both the ADAM17-siRNA groups than in the control ([61.83 +/- 2.41]% and [59.78 +/- 1.92]% vs [41.38 +/- 1.53]%, P < 0.05), but that of the S phase remarkably lower in the former two than in the latter ([23.64 +/- 2.56]% and [25.24 +/- 1.86]% vs [33.51 +/- 1.47]%, P < 0.05), with a concomitant decrease in the expression of the cell cycle protein cyclin D1 and increase in the cyclin-dependent kinase inhibitor p21.</p><p><b>CONCLUSION</b>ADAM17-siRNA can effectively inhibit the proliferation of PC-3 cells by up-regulating cyclin D1 and down-regulating p21 protein, and ADAM17 has a potential value in the gene therapy of prostate cancer.</p>
Subject(s)
Humans , Male , ADAM Proteins , Genetics , Metabolism , ADAM17 Protein , Cell Line, Tumor , Cell Proliferation , Cyclin D1 , Metabolism , Cyclin-Dependent Kinase Inhibitor p21 , Metabolism , Down-Regulation , Prostatic Neoplasms , Genetics , Metabolism , Pathology , RNA Interference , RNA, Messenger , Genetics , RNA, Small Interfering , Genetics , Signal Transduction , TransfectionABSTRACT
The sigma-1 receptor is a molecular chaperone protein highly enriched in the brain. Recent studies linked it to many diseases, such as drug addition, Alzheimer's disease, stroke, depression, and even cancer. Sigma-1 receptor is enriched in lipid rafts, which are membrane microdomains essential in signaling processes. One of those signaling processes is ADAM17- and ADAM10-dependent ectodomain shedding. By using an alkaline phosphatase tagged substrate reporter system, we have shown that ADAM10-dependent BTC shedding was very sensitive to both membrane lipid component change and sigma-1 receptor agonist DHEAS treatment while ADAM17-dependent HB-EGF shedding was not; and overexpression of sigma-1 receptor diminished ADAM17- and ADAM10-dependent shedding. Our results indicate that sigma-1 receptor plays an important role in modifying the function of transmembrane proteases.
Subject(s)
Animals , Humans , ADAM Proteins , Metabolism , ADAM10 Protein , ADAM17 Protein , Amyloid Precursor Protein Secretases , Metabolism , Betacellulin , COS Cells , Chlorocebus aethiops , Gene Expression , HEK293 Cells , Heparin-binding EGF-like Growth Factor , Intercellular Signaling Peptides and Proteins , Metabolism , Membrane Microdomains , Metabolism , Membrane Proteins , Metabolism , Receptors, sigma , MetabolismABSTRACT
We constructed prokaryotic expression vectors for different domains of TACE gene and expressed the fusion proteins, so as to explore their effects on the proliferation, adhesion and invasion potential of tumor cells in vitro. The total RNA was isolated from THP1 cell. TACE cDNA was amplified by RT-PCR and subcloned into pMD18-T vector to construct pMD-18T-TACE vector. The different cDNA fragment of TACE were amplified from plasmid pMD-18T-TACE and then cloned into pET-28a( + ) to construct expression vector pET28a( + )- 300, pET28a( + )-T800, and pET28a( + )-T1300, which respectively transformed into E. coli BL21 (DE3). The expression of His-tagged fusion proteins were induced with IPTG and purified through BBST NTA resin. The proliferation ability was examined by MTT assay. The adhesive and invasive ability were examined by plated adhesion model and Transwell assay. The protein pET28a( + )-T300 and pET28a( + )-T1300 can reduce the proliferation, adhesion and invasion ability of human lung carcinoma cell A549 in vitro, but otherwise the protein pET28a( + )-T800 had not shown the inhibitive function. The fusion protein of disintegrin domain of TACE have the similar biological function to other disintegrins, which can be used for further research on function of TACE in inflammation and tumor.
Subject(s)
Humans , ADAM Proteins , Genetics , Pharmacology , ADAM17 Protein , Adenocarcinoma , Pathology , Cell Adhesion , Cell Line, Tumor , Cell Proliferation , Escherichia coli , Genetics , Metabolism , Genetic Vectors , Lung Neoplasms , Pathology , Neoplasm Invasiveness , Prokaryotic Cells , Metabolism , Recombinant Fusion Proteins , Genetics , PharmacologyABSTRACT
<p><b>OBJECTIVE</b>To construct the bait vector pGBKT7-TACEc (cytoplasmic tail of tumor necrosis factor-alpha converting enzyme) of Macthmaker GAL4 Two-hybrid System 3, and to test whether it has self-activation and toxic action.</p><p><b>METHODS</b>TACEc gene was amplified by RT-PCR from the mouse testis, and the EcoRI and BamHI sites were introduced into it. The TACEc gene, after sequenced, was cloned into pGBKT7. Self-activation and toxic action of the recombination vector pGBKT7-TACEc was tested.</p><p><b>RESULTS</b>The pGBKT7-TACEc vector was successfully constructed and proved of no self-activation and toxic action.</p><p><b>CONCLUSION</b>The pGBKT7-TACEc can be applied to the screening of the mouse testis cDNA library in the yeast two-hybrid system.</p>
Subject(s)
Animals , Male , Mice , ADAM Proteins , Genetics , Metabolism , ADAM17 Protein , Cloning, Molecular , DNA, Complementary , Mice, Inbred BALB C , Plasmids , RNA , Reverse Transcriptase Polymerase Chain Reaction , Testis , Metabolism , Two-Hybrid System Techniques , Yeasts , MetabolismABSTRACT
<p><b>BACKGROUND</b>Chronic obstructive pulmonary disease (COPD) is associated not only with airway inflammation characterized by mucin hypersecretion but also with systemic inflammation. Tumor necrosis factor alpha (TNF-alpha) is found to take part in systemic inflammation, and ErbB3 plays an important role in mucin hypersecretion of COPD. Since TNF-alpha converting enzyme (TACE) is involved in the activation of both TNF-alpha and ErbB3, we established rat models of COPD to investigate the expressions of TACE, TNF-alpha and ErbB3 and to explore the correlations among TACE, TNF-alpha and ErbB3 respectively.</p><p><b>METHODS</b>Thirty Wistar male rats were randomly divided into COPD group (group C, n = 10), saline solution parallel group (group P, n = 8), and normal control group (group N, n = 8). Group C was challenged with passive cigarette smoking and intratracheal instillation of lipopolysaccharide. Six weeks later pulmonary functions were tested, bronchoalveolar fluid and arterial blood gases were assayed, and histopathological evaluations were performed in turn. The expressions of TACE, TNF-alpha and ErbB3 in lungs of all rats were determined histochemically.</p><p><b>RESULTS</b>The expressions of TACE, TNF-alpha and ErbB3 were significantly higher in group C than in group N (P < 0.01). The contents of TNF-alpha in serum (P < 0.01) and bronchoalveolar lavage fluid (BALF) (P < 0.01) were elevated more significantly in group C than in group N. A positive correlation existed between TACE and TNF-alpha (r = 0.784, P < 0.01) and between TACE and ErbB3 (r = 0.526, P < 0.01) respectively.</p><p><b>CONCLUSIONS</b>TNF-alpha and ErbB3 are involved in the pathogenesis of COPD. TACE contributes to the progress of COPD indirectly through the function of TNF-alpha and ErbB3.</p>
Subject(s)
Animals , Male , Rats , ADAM Proteins , Physiology , ADAM17 Protein , Bronchoalveolar Lavage Fluid , Chemistry , Immunohistochemistry , Lung , Pulmonary Disease, Chronic Obstructive , Metabolism , Pathology , Rats, Wistar , Receptor, ErbB-3 , Physiology , Tumor Necrosis Factor-alphaABSTRACT
Tumour necrosis factor-alpha converting enzyme (TACE) is the major protease responsible for processing proTNF from membrane-anchored precursor into secreted TNF-alpha. It was validated that TACE is involved in many diseases such as arthritis, multiple sclerosis and Alzheimers, therefore it represents a novel and significant target for therapeutic intervention in a variety of inflammatory and neuroimmunological diseases. To obtain the recombinant TACE ectodomain and use it as a selective molecule for the screening of TACE peptide inhibitors, the cDNA coded for catalytic domain (T800) and full-length ectodomain (T1300) of TACE were amplified by RT-PCR, the expression plasmid was constructed by inserting T800/T1300 into plasmid pET-28a/pET-28c and transformed into E. coli BL21 (DE3). SDS-PAGE and Western blotting analysis revealed that T800/T1300 was highly expressed in the form of inclusion body being induced by IPTG. After Ni2+ -NTA resin affinity chromatography, the purity of the recombinant T800/T1300 protein was more than 90%. T800 and T1300 protein were used in the screening of TACE-binding peptides from the phage display random 15-peptide library. After four rounds of biopanning, the positive phage clones were analyzed by ELISA, competitive inhibition assay and DNA sequencing. A common amino acid sequence-TRWLVYFSRPYLVAT was found and synthesized. The synthetic peptide was shown to bind to TACE and inhibit the TNF-alpha release from LPS-stimulated human peripheral blood mononuclear cells (PBMC) up to 60.3%. FACS analysis revealed that the peptide mediated the accumulation of TNF-alpha on LPS-stimulated PBMC surface. These results demonstrate that the TACE-binding peptide is an effective antagonist of TACE and the deduced motif might be applied to molecular design of anti-inflammation drugs.
Subject(s)
Animals , Humans , Mice , ADAM Proteins , Genetics , ADAM17 Protein , Amyloid Precursor Protein Secretases , Peptide Library , Peptides , Chemistry , Recombinant Proteins , Genetics , Tumor Necrosis Factor-alphaABSTRACT
The effects of inhibitors of TNF alpha converting enzyme (TACE) on TNF alpha secretion were studied to develop an approach to interfere inflammation processes. The HL-60 cell lines were stimulated in vitro with LPS intravenously for different time to establish the cellular model of inflammation and simultaneously induce in vivo inflammation animal model by LPS The cytotoxic effects of soluble TNF alpha were checked using MTT colorimetric method to determine the rate of cell proliferation. The level of expression of TACE was detected by using RT-PCR, FCM and immuno-histochemical technique respectively. It was found Chinese medicine Reduqing (RDQ) could inhibit the transcription of TNF alpha mRNA induced by LPS stimulation (P < 0.01, compared with the control). The antioligodeoxyribonucleotide (anti-ODN) of TNF alpha mRNA could inhibit 78.9% of TNF alpha secretion. The mimic peptides of TACE substrates with hydroxamine group showed potency in vivo and in vitro against converting of pro-TNF alpha. It was concluded that all the three types of TACE inhibitors can regulate the expression of TACE at different levels and inhibit sTNF alpha secretion, indicating TACE is a novel target for inflammation therapy.
Subject(s)
Humans , ADAM Proteins , ADAM17 Protein , Drugs, Chinese Herbal , Pharmacology , HL-60 Cells , Inflammation , Metabolism , Lipopolysaccharides , Metalloendopeptidases , Oligodeoxyribonucleotides , Protein Precursors , RNA, Messenger , Genetics , Metabolism , Reverse Transcriptase Polymerase Chain Reaction , Transcription, Genetic , Tumor Necrosis Factor-alpha , Genetics , Metabolism , Bodily SecretionsABSTRACT
In order to investigate the effects of LPS on the TACE gene transcription and expression and its regulating effect on the TM-TNF secretion, in vitro studies were carried out on HL-60 cells stimulated by LPS. TACE, TNF-alpha mRNA levels were detected by Dot-Elisa and the distribution of membrane molecules determined by flow cytometry assay and indirect immunofluorescence. The results showed that: (1) TACE was detected in or on HL-60 cells and it is predominantly localized on cell surface and to a perinuclear compartment. (2) LPS induced a time dependent increasement of TNF-alpha mRNA and enhanced TNF conversion with decreasing distribution of TNF in cell surface and increasing secretion of TNF protein. Such conversion could be inhibited by TACE ODN. (3) LPS also induced time-dependently increased expression of TACE gene and activation of its function. On the other hand, TACE protein in cell lysate and on cell surface was decreased. It was suggested that TACE molecular structure might change following its mediating membrane-anchored molecular secretion.