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 ; Animals ; Cellular Senescence/genetics* ; E2F4 Transcription Factor/genetics* ; Fibroblasts/metabolism* ; Mice ; TEA Domain Transcription Factors/metabolism* ; Transcription Factor AP-1/metabolism* ; Transcriptome

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

OBJECTIVETo investigate the mechanism of silicosis by observing the effects of silica on the expression of plasminogen activator inhibitor-1 (PAI-1) and activator protein-1 (AP-1) in human alveolar epithelial cells type II (A549).

METHODSA549 cell and SiO(2) (200 microg /ml) were co-cultured for 0, 4, 8, 16 and 24 h respectively. The reverse transcriptase polymerase chain reaction (RT-PCR), Western blotting and SP immunocytochemistry were used for detections of the PAI-1 mRNA and protein expression. The nucleoprotein and total protein expression of AP-1 were investigated by Western blotting.

RESULTSThe expression levels of PAI-1 mRNA and protein were increased in a time-dependent manner(r(mRNA) = 0.911, r(protein) = 0.902, P < 0.05). The expressions of PAI-1 mRNA and protein in experimental groups were higher than that in control group (P < 0.05) and was the highest in 24 h group [(0.73 +/- 0.01) vs (0.36 +/- .03)]. The nucleoprotein expressions of c-jun/c-fos in experimental groups were also higher than in control group (P < 0.05), and the nucleoprotein expression level of c-jun was the highest in 4 h group [(1.54 +/- 0.02) vs (0.56 +/- 0.03)]; the nucleoprotein expression level of c-fos was the highest in 8 h group [(0.36 +/- 0.01) vs (0.15 +/- 0.01)]. Both c-jun and c-fos expression were decreased after 16 h, but the total protein expression of c-jun/c-fos had no difference in all experimental groups. The positive signal of PAI-1 was located in cytoplasm and nucleus.

CONCLUSIONSiO(2) could induce PAI-1 expression of A549 in a time-dependent manner, and AP-1 activation can be observed in early time.

Alveolar Epithelial Cells ; drug effects ; metabolism ; Cell Line ; Humans ; Plasminogen Activator Inhibitor 1 ; metabolism ; Silicon Dioxide ; toxicity ; Transcription Factor AP-1 ; metabolism

OBJECTIVE: To investigate the role of AP-1 in the secretion of Type I collagen in TGF-beta1-stimulated human lung fibroblasts. METHODS: Human lung fibroblasts cell line (HLF-02) was cultured, and then stimulated with 10 microg/L TGF-beta1 at different time points. Curcumin was added into the culture medium to inhibit the AP-1 activity before incubating with TGF-beta1. AP-1 DNA binding activity was assayed by electrophoretic mobility shift assay (EMSA), and the expression of Type I collagen was detected by Western blot and RT-PCR. RESULTS: TGF-beta1 could induce the transcription and secretion of Type I collagen in HLF-02 cells(P<0.05). TGF-beta1 could upregulate the AP-1 DNA binding activity ( P<0.05). Curcumin ( 5, 10, 15, and 20 micromol/L) could inhibit the AP-1 DNA binding activity in TGF-beta1-stimulated cells (the inhibition ratio was 17.1%, 17.6%, 24.2%, and 31.3%; P<0.05). Curcumin (5, 10, 15, and 20 micromol/L) could also inhibit the secretion of Type I collagen significantly (the inhibition ratio was 62.1%, 58.8%, 62.1%, and 59.6%; P<0.05). CONCLUSION AP-1 is responsible for the secretion of TGF-beta1-induced Type I collagen in human lung fibroblasts.
Cell Line ; Collagen Type I ; metabolism ; Fibroblasts ; cytology ; drug effects ; metabolism ; Humans ; Lung ; cytology ; Signal Transduction ; Transcription Factor AP-1 ; metabolism ; Transforming Growth Factor beta1 ; pharmacology

OBJECTIVETo evaluate the effect of low salt (LS) on the expression of cyclooxygenase-2 (COX-2) and the activity of nuclear factor kappa B (NF-kappaB) and activator protein-1 (AP-1) in the mouse macula densa derived (MMDD1) cell line.

METHODSMMDD1 cells were transfected with luciferase reporter plasmid containing AP-1 or NF-kappaB. Luciferase reporter assay was used to evaluate the effect of normal salt (NS) and low salt (LS) on the activities of NF-kappaB and AP-1. The changes of COX-2 expression were examined by RT-PCR. The expression of p-p38 MAPK, p-p44/42, c-Jun, c-Fos, and COX-2 in MMDD1 cells were analyzed by Western blot.

RESULTSThe expressions of COX-2 mRNA and protein in MMDD1 cells were significantly increased by LS (P < 0.01). Phosphorylated p38 and p44/42 MAP kinase were significantly increased by treatment at 180 min (P < 0.01). The up-regulated COX-2 protein expression with LS were significantly reduced with SB 203580 (p38 inhibitors) and PD-98059 (p44/42 inhibitors) (P < 0.01). The expressions of c-Jun and c-Fos were increased by LS. The luciferase activities of AP-1 and NF-kappaB were stimulated in LS (P < 0.01), the up-regulated luciferase activities were attenuated by PDTC at 25 micromol/L (NF-kappaB inhibitor) and curcumin at 20 micromol/L (AP-1 inhibitor) (P < 0.01). LS altered COX-2 mRNA abundance and protein expression were decreased in treatment with PDTC at 25 micromol/L, curcumin at 20 micromol/L (P < 0.01).

CONCLUSIONLS can induce the expression of COX-2 in MMDD1 cells, which may be involved in the activation of p38 MAP kinase, p44/42 kinase, AP-1, and NF-kappaB pathways.

Animals ; Cell Line ; Cyclooxygenase 2 ; metabolism ; Kidney ; cytology ; metabolism ; Mice ; NF-kappa B ; metabolism ; Signal Transduction ; Transcription Factor AP-1 ; metabolism

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

OBJECTIVETo investigate the role of angiotensin II (AngII)-angiotensin II type 1 receptor (AT1R) pathway in the production of proinflammatory cytokines in macrophage, and to analyze its mechanisms.

METHODSRAW264.7 macrophages were cultured in vitro in DMEM nutrient medium containing 10% FBS, and then they were divided into control group (ordinary culture for 6 hours without any stimulation), ZD7155 group (pretreated with 38 µmol/L AT1R-specific inhibitor ZD7155 for 1 hour, then cultured with fresh nutrient solution for 6 hours), AngII group (cultured with 0.01 µmol/L AngII for 6 hours), and ZD7155 + AngII group (pretreated with 38 µmol/L AT1R-specific inhibitor ZD7155 for 1 hour, then cultured with 0.01 µmol/L AngII for 6 hours) according to the random number table. Contents of TNF-α and IL-1β in the supernatant were measured by ELISA. Expressions of TNF-α mRNA and IL-1β mRNA were determined by RT-PCR. Activity of NF-κB and AP-1 were examined by electrophoretic mobility shift assay. Data were processed with one-way analysis of variance.

RESULTSCompared with those in AngII group [(119 ± 14), (105 ± 17) pg/mL, respectively], the levels of TNF-α and IL-1β in the supernatant in control group [(24 ± 11), (24 ± 6) pg/mL, with F value respectively 1.62, 8.03, P values all below 0.01], ZD7155 group [(22 ± 11), (25 ± 8) pg/mL, with F value respectively 1.62, 4.52, P values all below 0.01], and ZD7155 + AngII group [(45 ± 13), (62 ± 11) pg/mL, with F value respectively 1.16, 2.29, P < 0.05 or P < 0.01] were all obviously decreased. The expressions of TNF-α mRNA and IL-1β mRNA, and activity of NF-κB and AP-1 showed the similar changes as above: (1) The levels of TNF-α mRNA and IL-1β mRNA in AngII group were all higher than those in control group (with F value respectively 7.59, 3.38, P < 0.05 or P < 0.01), ZD7155 group (with F value respectively 10.66, 2.24, P values all below 0.05), and ZD7155 + AngII group (with F value respectively 5.10, 5.09, P values all below 0.01). (2) Activity of NF-κB and AP-1 was respectively 69 027 ± 2502, 36 752 ± 2055 in AngII group, all higher than those in control group (45 709 ± 1203, 20 325 ± 2695, with F value respectively 4.32, 1.72, P values all below 0.01), ZD7155 group (46 303 ± 1897, 21 951 ± 2517, with F value respectively 1.74, 1.50, P values all below 0.01), and ZD7155 + AngII group (38 271 ± 690, 22 365 ± 3797, with F value respectively 13.13, 3.41, P values all below 0.01).

CONCLUSIONSAngII can mediate activation of transcription factor NF-κB and AP-1 via combination of AT1R, thereby contributing to the production and release of proinflammatory cytokines TNF-α and IL-1β in macrophage.

Angiotensin II ; metabolism ; Animals ; Cell Line ; Interleukin-1beta ; metabolism ; Macrophages ; metabolism ; Mice ; NF-kappa B ; metabolism ; Receptor, Angiotensin, Type 1 ; metabolism ; Transcription Factor AP-1 ; metabolism ; Tumor Necrosis Factor-alpha ; metabolism

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

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 ; cytology ; Cells, Cultured ; Epithelial Cells ; cytology ; metabolism ; Humans ; Mucin 5AC ; genetics ; metabolism ; Smoke ; adverse effects ; Smoking ; adverse effects ; Tobacco ; chemistry ; Transcription Factor AP-1 ; pharmacology

OBJECTIVETo investigate the roles of cyclin D1 and CDK4 in the cell cycle changes of human embryonic lung fibroblasts (HELFs) exposed to silica.

METHODSHELFs were divided into 4 groups: control group, curcumin (20 µmol/L for 1 h) group, silica (200 µg/ml for 24 h) group and curcumin plus silica group, i.e. after exposure to 20 µmol/L curcumin for 1h, the HELFs were treated with 200 µg/ml silica for 24 h. Western blot and Immunofluorescence assays were utilized to detect the expression levels of cyclin D1, CDK4 and E2F1/4. Flow cytometry was used to detect the cell cycle progression, the RNA transfection technique was used to investigate the silica-induced signal pathway and the roles of which in silica-induced cell cycle changes.

RESULTSThe expression levels of cyclin D1 and CDK4 significantly increased and the expression level of E2F-4 decreased obviously, but the expression level of E2F-1 did not significantly change in silica group. The proportion of G1 phase cells obviously decreased and the proportion of S phase cells significantly increased in silica group, as compared with control group (P < 0.05). When suppressing the expression of cyclin D1 or CDK4, the proportions of cells in G1 phase in anti-D1 plus silica group and anti-K4 plus silica group did not obviously change, as compared with control group. When suppressing AP-1 activity, the cyclin D1 and CDK4 expression levels decreased and the E2F-4 expression level increased in curcumin plus silica group, as compared with silica group.

CONCLUSIONThe results of present suggested that 200 µg/ml silica could induce the high expression of cyclin D1 and CDK4 and the low expression of E2F-4, resulting in the cell cycle changes by AP-1/cyclin D1 pathway in HELFs.

Cell Cycle ; drug effects ; Cells, Cultured ; Cyclin D1 ; metabolism ; Cyclin-Dependent Kinase 4 ; metabolism ; E2F4 Transcription Factor ; metabolism ; Fibroblasts ; cytology ; metabolism ; G1 Phase ; Humans ; Quartz ; adverse effects ; Transcription Factor AP-1 ; metabolism ; Transfection