Alcohol Consumption in Combination with an Atherogenic Diet Increased Indices of Atherosclerosis in Apolipoprotein E/Low-Density Lipoprotein Receptor Double-Knockout Mice.

BACKGROUND: Alcohol abuse and adherence to atherogenic diet (AD; a low-carbohydrate-high-protein diet) have been positively associated with cardiovascular disease. In addition, it has been demonstrated clinically that dietary intake is increased on days when alcohol is consumed. Here, the additive effects of ethanol (EtOH) and AD on atherosclerosis, a major underlying cause of cardiovascular disease, were investigated in apolipoprotein E/low-density lipoprotein receptor double-knockout (KO) mice. The mechanisms, especially aortic oxidative stress damage, were highlighted. METHODS: Twelve-week-old male KO mice on AD with or without EtOH treatment were bred for 4 months. Age-matched male C57BL/6J mice on a standard chow diet without EtOH treatment served as controls. Analyses were conducted using ultrasound biomicroscopy, histopathological and fluorescence immunohistochemical examinations, Western blots, and polymerase chain reaction. RESULTS: KO mice on AD with EtOH treatment showed increases in aortic maximum intima media thickness, hypoechoic plaque formation, and mean Oil-Red-O content. These results were associated with enhanced ratio of aortic 8-hydroxy-2'-deoxyguanosine (8-OHdG)-immunopositive area to the metallothionein (MT) immunopositive area and suppression of AD-induced up-regulated aortic Mt1, Mt2, and upstream stimulatory factor 1 mRNA expressions. Moreover, 8-OHdG was expressed in the nuclei of CD31- and alpha smooth muscle actin-immunopositive cells, and the up-regulated mRNA expressions of aortic nitric oxide synthase 3 and platelet-derived growth factors were only observed in the KO mice on AD with EtOH treatment. CONCLUSIONS: Alcohol abuse and adherence to AD may promote the shift of aortic oxidative stress and antioxidative stress balance toward oxidative stress predominance and reduced antioxidative stress, which may be partly due to the decrease in MT at the cell biological level and down-regulation of Mt at the gene level, which in turn could play a role in the up-regulation of endothelial dysfunction-related and vascular smooth muscle cell proliferation-related gene expression and the progression of atherosclerosis in mice with hyperlipidemia.

Suppression of metastasis through inhibition of chitinase 3-like 1 expression by miR-125a-3p-mediated up-regulation of USF1.

Rationale: Chitinase 3-like 1 (Chi3L1) protein is up-regulated in various diseases including solid cancers. According to Genome-Wide Association Study (GWAS)/Online Mendelian Inheritance in Man (OMIM)/Differentially Expressed Gene (DEG) analyses, Chi3L1 is associated with 38 cancers, and more highly associated with cancer compared to other oncogenes such as EGFR, TNFα, etc. However, the mechanisms and pathways by which Chi3L1 is associated with cancer are not clear. In current study, we investigated the role of Chi3L1 in lung metastasis. Methods: We performed the differentially expressed gene analysis to explore the genes which are associated with Chi3L1 using the web-based platform from Biomart. We investigated the metastases in lung tissues of C57BL/6 mice injected with B16F10 melanoma following treatment with Ad-shChi3L1. We also investigated the expression of USF1 and Chi3L1 in Chi3L1 KD mice lung tissues by Western blotting and IHC. We also analyzed lung cancer cells metastases induced by Chi3L1 using migration and cell proliferation assay in human lung cancer cell lines. The involvement of miR-125a-3p in Chi3L1 regulation was determined by miRNA qPCR and luciferase reporter assay. Results: We showed that melanoma metastasis in lung tissues was significantly reduced in Chi3L1 knock-down mice, accompanied by down-regulation of MMP-9, MMP-13, VEGF, and PCNA in Chi3L1 knock-down mice lung tissue, as well as in human lung cancer cell lines. We also found that USF1 was conversely expressed against Chi3L1. USF1 was increased by knock-down of Chi3L1 in mice lung tissues, as well as in human lung cancer cell lines. In addition, knock-down of USF1 increased Chi3L1 levels in addition to augmenting metastasis cell migration and proliferation in mice model, as well as in human cancer cell lines. Moreover, in human lung tumor tissues, the expression of Chi3L1 was increased but USF1 was decreased in a stage-dependent manner. Finally, Chi3L1 expression was strongly regulated by the indirect translational suppressing activity of USF1 through induction of miR-125a-3p, a target of Chi3L1. Conclusion: Metastases in mice lung tissues and human lung cancer cell lines were decreased by KD of Chi3L1. USF1 bound to the Chi3L1 promoter, however, Chi3L1 expression was decreased by USF1, despite USF1 enhancing the transcriptional activity of Chi3L1. We found that USF1 induced miR-125a-3p levels which suppressed Chi3L1 expression. Ultimately, our results suggest that lung metastasis is suppressed by knock-down of Chi3L1 through miR-125a-3p-mediated up-regulation of USF1.

Positive and negative regulators of the metallothionein gene (review).

Metallothioneins (MTs) are metal-binding proteins involved in diverse processes, including metal homeostasis and detoxification, the oxidative stress response and cell proliferation. Aberrant expression and silencing of these genes are important in a number of diseases. Several positive regulators of MT genes, including metal-responsive element-binding transcription factor (MTF)-1 and upstream stimulatory factor (USF)-1, have been identified and mechanisms of induction have been well described. However, the negative regulators of MT genes remain to be elucidated. Previous studies from the group of the present review have revealed that the hematopoietic master transcription factor, PU.1, directly represses the expression levels of MT genes through its epigenetic activities, and upregulation of MT results in the potent inhibition of myeloid differentiation. The present review focuses on PU.1 and several other negative regulators of this gene, including PZ120, DNA methyltransferase 3a with Mbd3 and Brg1 complex, CCAAT enhancer binding protein α and Ku protein, and describes the suppression of the MT genes through these transcription factors.

Expressions of heparanase and upstream stimulatory factor in hepatocellular carcinoma.

BACKGROUND: The expression of heparanase (HPSE) was associated with postoperative metastatic recurrence in patients with hepatocellular carcinoma (HCC). The six E-box binding sites in the core promoter of the HPSE gene suggested that transcription factors of E-box such as upstream stimulatory factor (USF) might regulate the transcription of the HPSE gene. The aim of our study is to measure the levels of HPSE and USF expression and investigate the relationship between USF expression and clinicopathological parameters in patients with HCC. METHODS: HPSE, USF1 and USF2 expressions in human HCC cell lines (BEL-7402, HepG2 and HCCLM3) and 15 fresh human HCC tissue samples were measured by real-time reverse transcriptase-PCR and Western blot analysis. The normal liver cell line QSG7701 or fresh normal liver tissue samples obtained from 15 additional surgical patients with hepatic rupture was used as a control. The protein expressions were determined by immunohistochemistry in paraffin-embedded human HCC tissues and corresponding non-neoplastic tumor surrounding tissues (NTST) of 57 patients. RESULTS: HPSE, USF1 and USF2 mRNA expressions were increased in HCC cell lines and HCC tissues compared with normal liver cell line and normal liver tissue. The protein expressions of HPSE, USF1 and USF2 in HCC cell lines and HCC tissues were also increased. Both USF1 and USF2 expressions were positively correlated with HPSE. USF1 and USF2 expressions were increased in patients with liver cirrhosis, worse tissue differentiation, advanced HCC stages and metastatic recurrence. CONCLUSIONS: Increased USF in HCC is associated with HPSE expression. USF might be an important factor in regulating HPSE expression and act as a novel marker of metastatic recurrence of HCC patients.

The basic helix-loop-helix/leucine zipper transcription factor USF2 integrates serum-induced PAI-1 expression and keratinocyte growth.

Plasminogen activator inhibitor type-1 (PAI-1), a major regulator of the plasmin-dependent pericellular proteolytic cascade, is prominently expressed during the tissue response to injury although the factors that impact PAI-1 induction and their role in the repair process are unclear. Kinetic modeling using established biomarkers of cell cycle transit (c-MYC; cyclin D1; cyclin A) in synchronized human (HaCaT) keratinocytes, and previous cytometric assessments, indicated that PAI-1 transcription occurred early after serum-stimulation of quiescent (G0) cells and prior to G1 entry. It was established previously that differential residence of USF family members (USF1→USF2 switch) at the PE2 region E box (CACGTG) characterized the G0 → G1 transition period and the transcriptional status of the PAI-1 gene. A consensus PE2 E box motif (5'-CACGTG-3') at nucleotides -566 to -561 was required for USF/E box interactions and serum-dependent PAI-1 transcription. Site-directed CG → AT substitution at the two central nucleotides inhibited formation of USF/probe complexes and PAI-1 promoter-driven reporter expression. A dominant-negative USF (A-USF) construct or double-stranded PE2 "decoy" attenuated serum- and TGF-ß1-stimulated PAI-1 synthesis. Tet-Off induction of an A-USF insert reduced both PAI-1 and PAI-2 transcripts while increasing the fraction of Ki-67(+) cells. Conversely, overexpression of USF2 or adenoviral-delivery of a PAI-1 vector inhibited HaCaT colony expansion indicating that the USF1 → USF2 transition and subsequent PAI-1 transcription are critical events in the epithelial go-or-grow response. Collectively, these data suggest that USF2, and its target gene PAI-1, regulate serum-stimulated keratinocyte growth, and likely the cadence of cell cycle progression in replicatively competent cells as part of the injury repair program.

H. pylori-induced promoter hypermethylation downregulates USF1 and USF2 transcription factor gene expression.

Helicobacter pylori infection is associated with the development of gastric adenocarcinoma. Upstream stimulatory factors USF1 and USF2 regulate the transcription of genes related to immune response, cell cycle and cell proliferation. A decrease in their expression is observed in human gastric epithelial cells infected with H. pylori, associated to a lower binding to their DNA E-box recognition site as shown by electrophoretic mobility shift assay. DNA methylation leads to gene silencing. The treatment of cells with 5'-azacytidine, an inhibitor of DNA methylation, restored the USF1 and USF2 gene expression in the presence of infection. Using promoter PCR methylation assay, a DNA hypermethylation was shown in the promoter region of USF1 and USF2 genes, in infected cells. The inhibition of USF1 and USF2 expression by H. pylori and the DNA hypermethylation in their gene promoter region was confirmed in gastric tissues isolated from 12 to 18 months infected mice. Our study demonstrated the involvement of USF1 and USF2 as molecular targets of H. pylori and the key role of DNA methylation in their regulation. These mechanisms occurred in the context of metaplastic lesions, suggesting that alteration of USF1 and USF2 levels could participate in the promotion of neoplastic process during H. pylori infection.

USF1/2 transcription factor DNA-binding activity is induced during rat Sertoli cell differentiation.

Each Sertoli cell can support a finite number of developing germ cells. During development of the testis, the cessation of Sertoli cell proliferation and the onset of differentiation determine the final number of Sertoli cells and, hence, the number of sperm that can be produced. We hypothesize that the transition from proliferation to differentiation is facilitated by E-box transcription factors that induce the expression of differentiation-promoting genes. The relative activities of E-box proteins were studied in primary Sertoli cells isolated from 5-, 11-, and 20-day-old rats, representing proliferating, differentiating, and differentiated cells, respectively. E-box DNA-binding activity is almost undetectable 5 days after birth but peaks with initiation of differentiation 11 days after birth and remains elevated. Upstream stimulatory factors 1 and 2 (USF1 and USF2) were found to be the predominant E-box proteins present within DNA-protein complexes formed after incubating E-box-containing probes with nuclear extracts from developing Sertoli cells. The known potentiator of Sertoli cell differentiation, thyroxine, increases USF DNA-binding activity in Sertoli cells before differentiation (5-day-old Sertoli cells) but not after differentiation is initiated (11- and 20-day-old Sertoli cells). The developmental-specific increase in USF1 and USF2 DNA-binding activity may facilitate the switch from proliferation to differentiation and, thus, determine the ultimate number of Sertoli cells present within the testes and the upper limit of fertility.

Upstream stimulatory factor 2 is implicated in the progression of biliary atresia by regulation of hepcidin expression.

BACKGROUND: Hepcidin is downregulated during the progression of biliary atresia (BA), but the mechanism is still unknown. METHODS: We analyzed single nucleotide polymorphism of rs7251432 and 916145 within hepcidin and its upstream, USF2 gene, respectively, in 52 patients of BA and 96 healthy controls. Liver tissues were obtained from 10 patients with early and late stage of BA, 10 patients with choledochal cyst, and 4 normal controls to study upstream stimulatory factor 2 (USF2) messenger RNA (mRNA) and protein expressions. Chromatin immunoprecipitation assay and USF2-specific short interference RNA (siRNA) were used in human HepG2 cells to show that USF2 can regulate hepcidin expression. RESULTS: C and CC allele frequencies of rs916145 of USF2 were significantly higher in patients with BA than in healthy controls. There was also significantly higher USF2 protein nuclear translocation in the early stage of BA than in the late stage, which was compatible with higher hepcidin mRNA expression in the early stage of BA. Chromatin immunoprecipitation assay demonstrated physiologic bindings of USF2 to the hepcidin promoter in HepG2 cells. USF2 siRNA also significantly knocked down hepcidin mRNA expression. CONCLUSION: The study demonstrates that C allele of rs916145 in USF2 gene has more frequency for developing BA, and decreased USF2 protein nuclear translocation might partly play a role in the decreased hepcidin expression in the cholestatic liver injury of the late stage of BA.

Binding of upstream stimulatory factor 1 to the E-box regulates the 4G/5G polymorphism-dependent plasminogen activator inhibitor 1 expression in mast cells.

BACKGROUND: Plasminogen activator inhibitor (PAI)-1 is a key regulator of the fibrinolytic system. PAI-1 levels are markedly elevated in the asthmatic airways. The 4G/5G polymorphism of the PAI-1 gene is associated with allergic asthma. OBJECTIVE: To characterize the mechanisms of the 4G/5G-dependent PAI-1 expression in mast cells (MCs), a major source of PAI-1 and key effector cells in asthma. METHODS: Transcription of PAI-1 was assessed by transiently transfecting human MC line (HMC-1) cells with the luciferase-tagged PAI-1 promoters containing the 4G or 5G allele (4G-PAI-1 or 5G-PAI-1 promoter). Upstream stimulatory factor (USF)-1 and the E-box interactions were studied by electrophoretic mobility shift assays and supershift assays. Expression of USF-1 was determined by Western blot analysis. RESULTS: The 4G-PAI-1 promoter has higher promoter activity than the 5G-PAI-1 promoter in stimulated HMC-1 cells, and the E-box adjacent to the 4G/5G site (E-4G/5G) regulates the genotype-specific PAI-1 transcription. USF-1 binds to the E-4G with greater affinity than to the E-5G. USF-1 level is increased in HMC-1 cells after stimulation, and elevated USF-1 enhances PAI-1 transcription. Overexpression of wild-type USF-1 or dominant-negative USF remedies the 4G/5G-dependent PAI-1 transcription. CONCLUSION: Binding of USF-1 to the E-4G/5G regulates the 4G/5G polymorphism-dependent PAI-1 expression in MCs.

High glucose levels upregulate upstream stimulatory factor 2 gene transcription in mesangial cells.

Previously, we demonstrated that upstream stimulatory factor 2 (USF2) mediates high glucose-induced thrombospondin1 (TSP1) gene expression and TGF-beta activity in glomerular mesangial cells and plays a role in diabetic renal complications. In the present studies, we further determined the molecular mechanisms by which high glucose levels regulate USF2 gene expression. In primary rat mesangial cells, we found that glucose treatment time and dose-dependently up-regulated USF2 expression (mRNA and protein). By using cycloheximide to block the de novo protein synthesis, similar rate of USF2 degradation was found under either normal glucose or high glucose conditions. USF2 mRNA stability was not altered by high glucose treatment. Furthermore, high glucose treatment stimulated USF2 gene promoter activity. By using the luciferase-promoter deletion assay, site-directed mutagenesis, and transactivation assay, we identified a glucose-responsive element in the USF2 gene promoter (-1,740 to -1,620, relative to the transcription start site) and demonstrated that glucose-induced USF2 expression is mediated through a cAMP-response element-binding protein (CREB)-dependent transactivation of the USF2 promoter. Furthermore, siRNA-mediated CREB knock down abolished glucose-induced USF2 expression. Taken together, these data indicate that high glucose levels up-regulate USF2 gene transcription in mesangial cells through CREB-dependent transactivation of the USF2 promoter.

Gene regulation profiles by progesterone and dexamethasone in human endometrial cancer Ishikawa H cells.

OBJECTIVE: Progesterone and glucocorticoids such as dexamethasone mediate distinct biological functions, yet they bind to receptors that recognize the same consensus DNA response element. In breast cancer, progestins are associated with the incidence and progression of tumors, whereas glucocorticoids are growth-suppressive in mammary cancer cells; the differential effects of these two steroids are less well understood in the hormone-dependent disease cancer of the uterine endometrium. We set out to identify genes that are regulated by progesterone through progesterone receptors and dexamethasone through glucocorticoid receptors in a well-differentiated human endometrial cancer cell line. METHODS: PR- and GR-positive Ishikawa H endometrial cancer cells were treated with vehicle, dexamethasone (100 nM) or progesterone (100 nM) for 2 h, 6 h, 12 h and 24 h, and RNA was isolated. Affymetrix microarrays were performed using the human HG-U133A chip, querying the expression of 22,000 genes. Expression of genes of particular interest was confirmed by real-time RT-PCR. RESULTS: Expression analysis demonstrated that dexamethasone and progesterone regulate overlapping but distinct sets of genes and presumably exert many similar but also unique biological effects. Using real-time RT-PCR, we confirmed three particular genes of interest: the transcript for cysteine 1 (legumain), a gene associated with metastasis, that is strongly downregulated by progesterone, upstream c-fos relating transcription factor-2 (USF-2), an anti-proliferative factor that is induced by both progesterone and dexamethasone and N-cadherin, a cellular adhesion molecule downregulated by dexamethasone. CONCLUSION: These studies provide new insight into the effects of progesterone and dexamethasone in endometrial cancer cells and provide an extensive list of regulated pathways which can be assessed in the future as biomarkers and molecular targets for new therapies. Taken together, our findings indicate that progesterone and dexamethasone are primarily growth inhibitors in Ishikawa H endometrial cancer cells.