1.Unveiling E2F4, TEAD1 and AP-1 as regulatory transcription factors of the replicative senescence program by multi-omics analysis.
Yuting WANG ; Liping LIU ; Yifan SONG ; Xiaojie YU ; Hongkui DENG
Protein & Cell 2022;13(10):742-759
Senescence, a stable state of growth arrest, affects many physiological and pathophysiological processes, especially aging. Previous work has indicated that transcription factors (TFs) play a role in regulating senescence. However, a systematic study of regulatory TFs during replicative senescence (RS) using multi-omics analysis is still lacking. Here, we generated time-resolved RNA-seq, reduced representation bisulfite sequencing (RRBS) and ATAC-seq datasets during RS of mouse skin fibroblasts, which demonstrated that an enhanced inflammatory response and reduced proliferative capacity were the main characteristics of RS in both the transcriptome and epigenome. Through integrative analysis and genetic manipulations, we found that transcription factors E2F4, TEAD1 and AP-1 are key regulators of RS. Overexpression of E2f4 improved cellular proliferative capacity, attenuated SA-β-Gal activity and changed RS-associated differentially methylated sites (DMSs). Moreover, knockdown of Tead1 attenuated SA-β-Gal activity and partially altered the RS-associated transcriptome. In addition, knockdown of Atf3, one member of AP-1 superfamily TFs, reduced Cdkn2a (p16) expression in pre-senescent fibroblasts. Taken together, the results of this study identified transcription factors regulating the senescence program through multi-omics analysis, providing potential therapeutic targets for anti-aging.
Aging
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Animals
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Cellular Senescence/genetics*
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E2F4 Transcription Factor/genetics*
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Fibroblasts/metabolism*
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Mice
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TEA Domain Transcription Factors/metabolism*
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Transcription Factor AP-1/metabolism*
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Transcriptome
2.Activated protein 1-cyclin D1/E2F 1 pathways involved in cell cycle changes induced by benzo (a) pyrene.
Meng YE ; Bing-ci LIU ; Xiao-wei JIA ; Ai GAO ; Shi JIAO ; Feng-mei ZHANG
Chinese Journal of Industrial Hygiene and Occupational Diseases 2008;26(2):72-76
OBJECTIVETo investigate the roles of activated protein 1 (AP-1) in cell cycle changes on human embryo lung fibroblasts (HELF) induced by benzo (a) pyrene [B (a) P], and relationships between AP-1 and cyclin D1/CDK4-E2F-1/4.
METHODSCells transfected with AP-1 luciferase reporter plasmid (AP-H) were cultured with serum-free RPMI1640 for 48 h, and treated with 2 micromol/L B (a) P for 24 h. AP-1 relative activity was detected by luciferase assay. Changes of cell cycle and the expression of cyclin D1, CDK4 and E2F-1/4 were checked using the flow cytometer and Western blot assay.
RESULTSAfter B (a) P was treated for 24 h, the ratio of G1 phase cells (71 +/- 2)% was decreased to (48 +/- 3)% (P < 0.05), and an increase was observed in the ratio of S phase. AP-1 activity and cyclin D1/E2F-1 expression were increased significantly, but CDK4/E2F-4 expression did not change after B (a) P treatment. When AP-1 activity was inhibited by curcumin, decreases of G1 phase in response to B (a) P treatment were blocked, and overexpression of cyclin D1/E2F-1 was attenuated, but CDK4/E2F-4 expression was not changed significantly.
CONCLUSIONAP-1 is involved in B (a) P induced cell cycle changes, and is the upstream signals of cyclin D1/E2F-1, but not CDK4/E2F-4.
Benzo(a)pyrene ; toxicity ; Cell Cycle ; drug effects ; Cells, Cultured ; Cyclin D1 ; metabolism ; Cyclin-Dependent Kinase 4 ; metabolism ; E2F1 Transcription Factor ; metabolism ; E2F4 Transcription Factor ; metabolism ; Fibroblasts ; cytology ; metabolism ; Humans ; Transcription Factor AP-1 ; genetics ; metabolism ; Transfection
3.Aldosterone stimulates alpha1-(1) procollagen mRNA expression in HSC via activation of ERK1/2 and AP-1.
Xu LI ; Ying MENG ; Shao-xi CAI ; Xi-shan YANG ; Ping-sheng WU
Chinese Journal of Hepatology 2005;13(11):815-818
OBJECTIVEIt has been known that the intrahepatic renin-angiotensin-aldosterone system (RAAS) plays a key role in the fibrogenesis in livers. Aldosterone (Aldo), the principal effector molecule of the RAAS, exerts local effects on cell growth and fibrogenesis. However, the signal transduction mechanisms underlying the effects of Aldo on hepatic fibrogenesis remain to be fully elucidated. The present study aims to investigate the signal transduction mechanism underlying the effects of Aldo on the signal passageway of active protein-1 (AP-1).
METHODSIn vitro, HSCs-T6 cell line was treated with Aldo for 10 min, 30 min, 60 min, 120 min and 180 min, and protein expression of Phospho-P42/44 was detected by Western blot. In addition, HSCs-T6 cell line was preincubated for 60 min or not with U0126 (an inhibitor of the MAPK/ERK kinase), and also with antioxidant-N-acetylcysteine (NAC) prior to exposure to Aldo for the indicated times. Protein expression of Phospho-P42/44 was measured by Western blot. DNA biding activity of AP-1 was analyzed by electrophoretic gel mobility shift assay (EMSA). By means of RT-PCR, expression of alpha1(1) procollagen mRNA was detected.
RESULTSAldo stimulated HSC via extracellular signal-regulated kinase (ERK1/2) pathway. Time course experiments showed that Aldo induced Phospho-P42/44 expression, which was abrogated by U0126, reaching a maximum at 10 minutes, and then declined progressively. NAC inhibited the Phospho-P42/44 expression. EMSA showed that stimulation of HSC by Aldo markedly increased AP-1 DNA binding activity. U0126 markedly reduced AP-1 DNA binding activity induced by Aldo; NAC partly decreased AP-1 activity induced by Aldo. Aldo up-regulated expression of alpha1(1) procollagen mRNA, which was attenuated by U0126 and NAC.
CONCLUSIONStimulation of HSC by Aldo results in activation of AP-1 via ERK1/2 pathway, leading to up-regulation of AP-1 target gene alpha1(1) procollagen mRNA expression.
Aldosterone ; pharmacology ; Cell Line ; Collagen Type I ; biosynthesis ; genetics ; Hepatocytes ; cytology ; metabolism ; Humans ; Mitogen-Activated Protein Kinase 3 ; metabolism ; RNA, Messenger ; biosynthesis ; genetics ; Transcription Factor AP-1 ; metabolism
4.An experimental study of inhibiting effect of decoy oligodeoxynucleotides on the gene expression of collagen in NIH3T3 cell.
Tian-zhi HAO ; Huan-ping LIANG ; Feng-lin LV ; Xiang XU ; Hua GUO ; Fu-long WANG ; Wen-jun YANG ; Yan LUO ; Lei LI
Chinese Journal of Burns 2003;19(3):175-178
OBJECTIVETo investigate the effect of activator protein-1 (AP-1) decoy-oligodeoxynucleotides (Decoy-ODNs) on the expression of fibroblast alpha2 type I collagen, so as to explore the gene therapy of pathologic scar.
METHODSDecoy-ODNs targeting AP-1 were designed and synthesized. NIH3T3 cells were transfected by cationic liposomes. The distribution of Decoy-ODNs in the cells was investigated. The inhibiting effects of Decoy-ODNs on AP-1 were determined by electrophoretic mobility shift assay (EMSA). And the effects of Decoy-ODNs on the collagen synthesis in the cells were analyzed by RT-PCR.
RESULTSAP-1 Decoy-ODNs could competitively inhibit the AP-1 in vitro activity. Cationic liposomes could play roles by effectively transfecting Decoy-ODNs into the plasma and nucleus. The mRNA expression of fibroblast alpha2 type I collagen decreased evidently after 24 hours of Decoy-ODNs action.
CONCLUSIONDecoy-ODNs could inhibit the mRNA expression of fibroblast alpha2 type I collagen by antagonizing AP-1.
Animals ; Collagen Type I ; biosynthesis ; Fibroblasts ; drug effects ; metabolism ; Mice ; NIH 3T3 Cells ; Oligodeoxyribonucleotides ; genetics ; pharmacology ; RNA, Messenger ; metabolism ; Transcription Factor AP-1 ; genetics ; Transfection
5.Regulatory mechanism of activator protein-1 on the expression of MUC5AC induced by cigarette smoke extract.
Journal of Central South University(Medical Sciences) 2010;35(11):1150-1155
OBJECTIVE:
To investigate the mechanism of activator protein-1 (AP-1) on cigarette smoke-induced airway mucous hypersecretion and to explore the possible signal transduction pathway that activates AP-1.
METHODS:
The airway epithelial cell line (BEAS-2B) was cultured in vivo and treated with cigarette smoke extract (CSE). The DNA binding activity of AP-1 was blocked by the transfection of c-Jun dominant negative mutant TAM67 into the cells. SP600125 and PD98059 were used to block the activation of c-Jun terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) respectively. MUC5AC protein was detected by enzyme-linked immunosorbent assay, MUC5AC mRNA level was analyzed by RT-PCR, while the protein contents of p-JNK, p-ERK and p-P38 were detected by Western blot, and the DNA binding activity of AP-1 was determined by electrophoretic mobility shift assay.
RESULTS:
The MUC5AC protein production and mRNA expression in the CSE group were significantly higher than those in the control group, and the DNA binding activity of AP-1 was also higher than that in the control group (P<0.01). The protein contents of p-ERK and p-JNK in the CSE group were higher than those in the control group (P<0.01), but the p-P38 level was not significantly different from that in the control group (P>0.05). After the transfection of TAM67 into the cells, the expression levels of MUC5AC protein and mRNA and the binding activity of AP-1 decreased significantly (P<0.01). The DNA binding activity of AP-1 and the expression levels of MUC5AC protein and mRNA were lower in the SP600125 group and in the PD98059 group than those in the CSE group (P<0.05).
CONCLUSION
After being activated by JNK and ERK which are phosphorylated by cigarette smoke, AP-1 binds to its DNA binding elements on the promoter of MUC5AC gene and up-regulates the MUC5AC expression at the transcriptional level.
Bronchi
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cytology
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Cells, Cultured
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Epithelial Cells
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cytology
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metabolism
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Humans
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Mucin 5AC
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genetics
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metabolism
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Smoke
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adverse effects
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Smoking
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adverse effects
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Tobacco
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chemistry
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Transcription Factor AP-1
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pharmacology
6.Effect of AP-1 on expression of plasminogen activator inhibitor-1 in human vascular endothelial cells.
Xiao-dong LI ; Shu-yu ZU ; Wen WANG ; Guang-jin ZHU
Acta Academiae Medicinae Sinicae 2003;25(3):307-311
OBJECTIVETo study the effect of nuclear transcription factor AP-1 on tumor necrosis factor alpha (TNF-alpha) or minimal modified low density lipoprotein (mmLDL)-induced expression of plasminogen activator inhibitor-1 (PAI-1) in human vascular endothelial cells.
METHODSUsing gene recombination techniques, four luciferase reporter gene plasmids containing different length of human PAI-1 gene promoter were constructed. Through the transient transfection analysis, the roles of AP-1 element (from -823 bp to -553 bp) in PAI-1 promoter have been determined. In order to further verify the role of AP-1 element, the three site-directed mutants were recovered using PCR and sequencing assay.
RESULTSThe induction by TNF-alpha or mmLDL were decreased markedly when the three AP-1 elements in PAI-1 promoter had been mutated respectively.
CONCLUSIONSThese results indicate that the AP-1 element in PAI-1 promoter may have important role in PAI-1 gene transcriptions in endothelial cells induced by TNF-alpha or mmLDL.
Base Sequence ; Cells, Cultured ; Endothelium, Vascular ; cytology ; metabolism ; Humans ; Molecular Sequence Data ; Plasminogen Activator Inhibitor 1 ; biosynthesis ; genetics ; Promoter Regions, Genetic ; Transcription Factor AP-1 ; genetics ; metabolism ; Tumor Necrosis Factor-alpha ; genetics ; metabolism ; Umbilical Veins ; cytology
7.Studies on the changes in the interleukin-13 expression and on activator protein-1 activity in rat pulmonary tissue with acute lung injury induced by endotoxin.
Qi LI ; Gui-Sheng QIAN ; Nan YANG ; Chang-Zheng WANG ; Jian-Cheng XU ; Qing ZHANG
Chinese Journal of Burns 2004;20(3):148-150
OBJECTIVETo investigate the changes in plasma level of interleukin-13 (IL-13) and the changes in the pulmonary IL-13 mRNA content and the pulmonary activator protein-1 (AP-1) activity of the rats inflicted with acute lung injury (ALI) induced by lipopolysaccharide (LPS), so as to explore the relationship between IL-13 expression and AP-1 activity.
METHODSOne hundred and twenty Wistar rats were employed in the study and were randomly divided into A (2 mg/kg), B (4 mg/kg), C (6 mg/kg) and D (8 mg/kg) groups according to different dosage of LPS administration and a control group (NS group) at each observing time point. The rats were observed at 1, 2, 4 and 6 postburn hours (PBHs) and every 6 rats were deployed in every group and each time points. A model of systemic inflammatory response syndrome-acute lung injury (SIRS-ALI) was replicated in Wistar rats. The plasma content of IL-13 was assayed by enzyme-linked immunosorbent assay (ELISA), and the pulmonary tissue content of IL-13 mRNA and AP-1 activity by reverse transcriptase-polymerase chain reaction (RT-PCR) and electrophoretic mobility shift assays (EMSA).
RESULTSThe plasma content of IL-13, pulmonary content of IL-13 mRNA and AP-1 activity increased simultaneously after LPS administration. All the above indices were significantly different statistically between the LPS groups and the control group (P < 0.05 - 0.01). The plasma level of IL-13 and pulmonary tissue mRNA content and AP-1 activity in A, B, C and D groups were increased significantly with peak levels at 2 PBHs.
CONCLUSIONThe pulmonary AP-1 activity increased with the enhanced expression of IL-13, which was related to the development of SIRS-ALI.
Acute Lung Injury ; metabolism ; Animals ; Endotoxins ; toxicity ; Female ; Interleukin-13 ; blood ; genetics ; physiology ; Lung ; chemistry ; Male ; Rats ; Rats, Wistar ; Transcription Factor AP-1 ; analysis ; physiology
8.Three transcription factors and the way immune cells affected by different plasma change in opposite ways in the development of the syndrome of pre-eclampsia.
Zhou LIANG ; Jing ZHU ; Yunfei WANG ; You WANG ; Yu ZHANG ; Jianhua LIN ; Wen DI ;
Chinese Medical Journal 2014;127(12):2252-2258
BACKGROUNDHow the transcriptional factors regulated the innate and adaptive immune system in pregnancy and pre-eclampsia are less understood. Nevertheless, what the plasma work in the development of this disease was not sure. The present study was design to evaluate what the transcriptional factors change in innate and adaptive immune system and what the plasma do in this filed.
METHODSPeripheral blood mononuclear cells (PBMC) from non-pregnant women (n = 18), women with clinically normal pregnancies (n = 23) and women with pre-eclampsia (n = 20) were separated from peripheral blood to isolate monocytes and T cells. The purity of monocytes and T cells were analysed by flow cytometry. Monocytes and T cells were stimulated in either lipopolysaccharides (LPS) or phorbol-myristate-acetate (PMA), respectively. Transcription Factor Arrays were used to screen the transcription factors of interest in comparing of different groups. PBMC were isolated from another 8 non-pregnant samples were co-incubated with different groups of plasma. Polymerase chain reaction (PCR) was performed using whole cell extractions of the samples.
RESULTSNuclear factor of activated T-cells-1 (NFAT-1), signal transducers and activators of transcription-1 (STAT-1) and activator protein-1 (AP-1) are up-regulated in monocytes in pregnancy and more so in pre-eclampsia. On the the contrary, NFAT-1, STAT-1 and AP-1 are down-regulated in T cells in pregnancy and more so in pre-eclampsia. A reduction was observed in interferon (IFN)-γ, interleukin (IL)-12 and IL-4 expression in T cells incubated with pre-eclamptic plasma. An elevation was observed in tumor necrosis factor (TNF)-α, IL-1 and IL-12 expression in monocytes incubated with pre-eclamptic plasma.
CONCLUSIONSInnate immunity is over activated and adaptive immunity is over suppressed in the development of pre-eclampsia. NFAT-1, STAT-1 and AP-1 might be the central transcription factors in the pathogenesis of pre-eclampsia. They induced some changes in plasma and "educate" the monocytes and T cells for relevant cytokine production. Successful completion of this study will enhance our understanding of pre-eclampsia and will discover new knowledge beyond pregnancy. The work will inform future therapies for the treatment of a wide range of condition such as transplantation immunology and a wide range of immune and inflammatory conditions.
Adult ; Female ; Humans ; Immunity, Innate ; physiology ; Interferon-gamma ; metabolism ; Interleukin-12 ; metabolism ; Interleukin-4 ; metabolism ; Leukocytes, Mononuclear ; metabolism ; Male ; NFATC Transcription Factors ; genetics ; metabolism ; Pre-Eclampsia ; immunology ; metabolism ; Pregnancy ; STAT1 Transcription Factor ; genetics ; metabolism ; Transcription Factor AP-1 ; genetics ; metabolism ; Transcription Factors ; genetics ; metabolism ; Young Adult
9.All-trans retinoic acid diminishes collagen production in a hepatic stellate cell line via suppression of active protein-1 and c-Jun N-terminal kinase signal.
Journal of Huazhong University of Science and Technology (Medical Sciences) 2010;30(6):726-733
Following acute and chronic liver injury, hepatic stellate cells (HSCs) become activated to undergo a phenotypic transformation into myofibroblast-like cells and lose their retinol content, but the mechanisms of retinoid loss and its potential roles in HSCs activation and liver fibrosis are not understood. The influence of retinoids on HSCs and hepatic fibrosis remains controversial. The purpose of this study was to evaluate the effects of all-trans retinoid acid (ATRA) on cell proliferation, mRNA expression of collagen genes [procollagen α1 (I), procollagen α1 (III)], profibrogenic genes (TGF-β(1), CTGF, MMP-2, TIMP-1, TIMP-2, PAI-1), fibrolytic genes (MMP-3, MMP-13) and the upstream element (JNK and AP-1) in the rat hepatic stellate cell line (CFSC-2G). Cell proliferation was evaluated by measuring BrdU incorporation. The mRNA expression levels of collagen genes [procollagen α1 (I), procollagen α1 (III)], profibrogenic genes (TGF-β(1), CTGF, MMP-2, TIMP-1, TIMP-2, PAI-1), and fibrolytic genes (MMP-3, MMP-13) were quantitatively detected by using real-time PCR. The mRNA expression of JNK and AP-1 was quantified by RT-PCR. The results showed that ATRA inhibited HSCs proliferation and diminished the mRNA expression of collagen genes [procollagen α1 (I), procollagen α1 (III)] and profibrogenic genes (TGF-β(1), CTGF, MMP-2, TIMP-1, TIMP-2, PAI-1), and significantly stimulated the mRNA expression of MMP-3 and MMP-13 in HSCs by suppressing the mRNA expression of JNK and AP-1. These findings suggested that ATRA could inhibit proliferation and collagen production of HSCs via the suppression of active protein-1 and c-Jun N-terminal kinase signal, then decrease the mRNAs expression of profibrogenic genes (TGF-β(1), CTGF, MMP-2, TIMP-1, TIMP-2, PAI-1), and significantly induce the mRNA expression of MMP-3 and MMP-13.
Animals
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Cell Line
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Cell Proliferation
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drug effects
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Collagen
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metabolism
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Collagen Type I
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genetics
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metabolism
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Hepatic Stellate Cells
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cytology
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metabolism
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JNK Mitogen-Activated Protein Kinases
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genetics
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metabolism
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Rats
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Transcription Factor AP-1
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genetics
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metabolism
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Tretinoin
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pharmacology
10.Intracellular CMTM2 negatively regulates human immunodeficiency virus type-1 transcription through targeting the transcription factors AP-1 and CREB.
Hong-shuo SONG ; Shuang SHI ; Xiao-zhi LU ; Feng GAO ; Ling YAN ; Ying WANG ; Hui ZHUANG
Chinese Medical Journal 2010;123(17):2440-2445
BACKGROUNDThe CKLF-like MARVEL transmembrane domain-containing family (CMTM) is a novel family of proteins linking chemokines and TM4SF. Different members exhibit diverse biological functions. In this study, the effect of intracellular CMTM2 on regulating human immunodeficiency virus type-1 (HIV-1) transcription was evaluated.
METHODSThe effects of CMTM2 on regulating full-length HIV-1 provirus and the HIV-1 long terminal repeat (LTR)-directed transcription were assessed by luciferase assay. Transcription factor assays, using the luciferase reporter plasmids of AP-1, CRE, and NF-κB were conducted to explore the signaling pathway(s) that may be regulated by CMTM2. The potential relationship between CMTM2 and the transcription factor AP-1 was further analyzed by Western blotting analyses to investigate the effect of CMTM2 on PMA-induced ERK1/2 phosphorylation.
RESULTSThe results from the current study revealed that CMTM2 acts as a negative regulator of HIV-1 transcription. CMTM2 exerted a suppressive action on both full-length HIV-1 provirus and HIV-1 LTR-directed transcription. Transcription factor assays showed that CMTM2 selectively inhibited basal AP-1 and CREB activity. Co-expression of HIV-1 Tat, a potent AP-1 and CREB activator, can not reverse CMTM2-mediated AP-1 and CREB inhibition, suggesting a potent and specific effect of CMTM2 on negatively regulating these two signaling pathways.
CONCLUSIONIntracellular CMTM2 can negatively regulate HIV-1 transcription, at least in part, by targeting the AP-1 and CREB pathways. Exploring the mechanisms further may lead to new ways to control HIV-1 replication.
Chemokines ; physiology ; Cyclic AMP Response Element-Binding Protein ; antagonists & inhibitors ; HIV Long Terminal Repeat ; HIV-1 ; genetics ; Humans ; Intracellular Space ; metabolism ; Jurkat Cells ; MARVEL Domain-Containing Proteins ; Tetradecanoylphorbol Acetate ; pharmacology ; Transcription Factor AP-1 ; antagonists & inhibitors ; Transcription, Genetic ; U937 Cells