ABSTRACT
Objective To explore the role of cytidine/uridine monophosphate kinase 2( CMPK2) in the immune-mediated antitumor effect of IFNα in hepatocellular carcinoma. Methods RT-qPCR and Western blot were used to analyze the expression of CMPK2 in Huh7 after the treatment of IFNα. The CMPK2 overexpressing Huh7 cells were generated by stably infecting with lentivirus. The ATP level in the cells and the supernatant of CMPK2 overexpress-ing Huh7 cells were measured by CellTiter-Glo ATP fluorescence assay. RT-qPCR was applied to test the expression of inflammatory cytokines in macrophages under the treatment of the supernatant of CMPK2 overexpress-ing Huh7 cells. Results The transcription and protein level of CMPK2 were significantly enhanced after the treat-ment of IFNα for 6 hours ( P＜0.01) . CMPK2 increased the ATP level in the cells and supernatant of Huh7 cells ( P＜0.01) . The supernatant of CMPK2 overexpressing Huh7 cells activated the expression of IL1β, IL6 and CCL5 in macrophages( P＜0.01) . Conclusions IFNα increases the expression of CMPK2 in Huh7 cells to activate the expression of inflammatory cytokines in macrophages.
ABSTRACT
<p><b>OBJECTIVE</b>To study the role of sirtuin 1 (SIRT1) in Fas ligand (FasL) expression regulation during vascular lesion formation and to elucidate the potential mechanisms.</p><p><b>METHODS</b>SIRT1 and FasL protein levels were detected by Western blotting in either mouse arteries extract or the whole rat aortic vascular smooth muscle cell (VSMC) lysate. Smooth muscle cell (SMC)-specific human SIRT1 transgenic (Tg) C57BL/6 mice and their littermate wild-type (WT) controls underwent complete carotid artery ligation (ligation groups) or the ligation-excluded operation (sham groups). The carotid arteries were collected 1 day after operation. Reverse transcription-polymerase chain reaction was performed to detect the mRNA levels of SIRT1 and FasL. Luciferase reporter assays were performed to detect the effect of WT-SIRT1, a dominant-negative form of SIRT1 (SIRT1H363Y), and GATA-6 on the promoter activity of FasL. Flow cytometry assay was applied to measure the hypodiploid DNA content of VSMC so as to monitor cellular apoptosis.</p><p><b>RESULTS</b>SIRT1 was expressed in both rat aortic VSMCs and mouse arteries. Forced SIRT1 expression increased FasL expression both in injured mouse carotid arteries 1 day after ligation (P<0.001) and VSMCs treated with serum (P<0.05 at the transcriptional level, P<0.001 at the protein level). No notable apoptosis was observed. Furthermore, transcription factor GATA-6 increased the promoter activity of FasL (P<0.001). The induction of FasL promoter activity by GATA-6 was enhanced by WT-SIRT1 (P<0.001), while SIRT1H363Y significantly relieved the enhancing effect of WT-SIRT1 on GATA-6 (P<0.001).</p><p><b>CONCLUSIONS</b>Overexpression of SIRT1 up-regulates FasL expression in both flow-restricted mouse carotid arteries and serum-stimulated VSMCs. The transcription factor GATA-6 participates in the transcriptional regulation of FasL expression by SIRT1.</p>
Subject(s)
Animals , Male , Rats , Apoptosis , Carotid Arteries , Physiology , Fas Ligand Protein , Genetics , GATA6 Transcription Factor , Physiology , Muscle, Smooth, Vascular , Cell Biology , Metabolism , Myocytes, Smooth Muscle , Metabolism , RNA, Messenger , Rats, Sprague-Dawley , Sirtuin 1 , Physiology , Up-RegulationABSTRACT
The silent information regulator protein 2 (Sir2) and its homologues play an important role in the regulation of cellular physiological processes such as survival, apoptosis, and aging. SIRT1, the mammalian Sir 2 homologue, has been shown to deacetylate a wide range of non-histone substrates and histone substrates. It has been constantly reported that SIRT1 may be associated with the occurrence of metabolic syndrome, genomic homeostasis, tumors, and neurodegenerative diseases. Calorie restriction may mitigate many major diseases in rodent models by SIRT1-mediated deacetylase activity and prolong the life expectancies in these animals. Therefore, SIRT1 may be emphasized as a new therapy target for many different diseases.