Sulfuretin Prevents Obesity and Metabolic Diseases in Diet Induced Obese Mice

The global obesity epidemic and associated metabolic diseases require alternative biological targets for new therapeutic strategies. In this study, we show that a phytochemical sulfuretin suppressed adipocyte differentiation of preadipocytes and administration of sulfuretin to high fat diet-fed obese mice prevented obesity and increased insulin sensitivity. These effects were associated with a suppressed expression of inflammatory markers, induced expression of adiponectin, and increased levels of phosphorylated ERK and AKT. To elucidate the molecular mechanism of sulfuretin in adipocytes, we performed microarray analysis and identified activating transcription factor 3 (Atf3) as a sulfuretin-responsive gene. Sulfuretin elevated Atf3 mRNA and protein levels in white adipose tissue and adipocytes. Consistently, deficiency of Atf3 promoted lipid accumulation and the expression of adipocyte markers. Sulfuretin’s but not resveratrol’s anti-adipogenic effects were diminished in Atf3 deficient cells, indicating that Atf3 is an essential factor in the effects of sulfuretin. These results highlight the usefulness of sulfuretin as a new anti-obesity intervention for the prevention of obesity and its associated metabolic diseases.

Cardiac Fibroblast-Specific Activating Transcription Factor 3 Promotes Myocardial Repair after Myocardial Infarction / 中华医学杂志(英文版)

<p><b>Background</b>Myocardial ischemia injury is one of the leading causes of death and disability worldwide. Cardiac fibroblasts (CFs) have central roles in modulating cardiac function under pathophysiological conditions. Activating transcription factor 3 (ATF3) plays a self-protective role in counteracting CF dysfunction. However, the precise function of CF-specific ATF3 during myocardial infarction (MI) injury/repair remains incompletely understood. The aim of this study was to determine whether CF-specific ATF3 affected cardiac repair after MI.</p><p><b>Methods</b>Fifteen male C57BL/6 wild-type mice were performed with MI operation to observe the expression of ATF3 at 0, 0.5, 1.0, 3.0, and 7.0 days postoperation. Model for MI was constructed in ATF3TGfl/flCol1a2-Cre+ (CF-specific ATF3 overexpression group, n = 5) and ATF3TGfl/flCol1a2-Cre- male mice (without CF-specific ATF3 overexpression group, n = 5). In addition, five mice of ATF3TGfl/flCol1a2-Cre+ and ATF3TGfl/flCol1a2-Cre- were subjected to sham MI operation. Heart function was detected by ultrasound and left ventricular remodeling was observed by Masson staining (myocardial fibrosis area was detected by blue collagen deposition area) at the 28 day after MI surgery in ATF3TGfl/flCol1a2-Cre+ and ATF3TGfl/flCol1a2-Cre- mice received sham or MI operation. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect cell proliferation/cell cycle-related gene expression in cardiac tissue. BrdU staining was used to detect fibroblast proliferation.</p><p><b>Results</b>After establishment of an MI model, we found that ATF3 proteins were increased in the heart of mice after MI surgery and dominantly expressed in CFs. Genetic overexpression of ATF3 in CFs (ATF3TGfl/flCol1a2-Cre+ group) resulted in an improvement in the heart function as indicated by increased cardiac ejection fraction (41.0% vs. 30.5%, t = 8.610, P = 0.001) and increased fractional shortening (26.8% vs. 18.1%, t = 7.173, P = 0.002), which was accompanied by a decrease in cardiac scar area (23.1% vs. 11.0%, t = 8.610, P = 0.001). qRT-PCR analysis of CFs isolated from ATF3TGfl/flCol1a2-Cre+ and ATF3TGfl/flCol1a2-Cre- ischemic hearts revealed a distinct transcriptional profile in ATF3-overexpressing CFs, displaying pro-proliferation properties. BrdU-positive cells significantly increased in ATF3-overexpressing CFs than control CFs under angiotensin II stimuli (11.5% vs. 6.8%, t = 31.599, P = 0.001) or serum stimuli (31.6% vs. 20.1%, t = 31.599, P = 0.001). The 5(6)-carboxyfluorescein N-hydroxysuccinimidyl ester assay showed that the cell numbers of the P2 and P3 generations were higher in the ATF3-overexpressing CFs at 24 h (P2: 91.6% vs. 71.8%, t = 8.465, P = 0.015) and 48 h (P3: 81.6% vs. 51.1%, t = 9.029, P = 0.012) after serum stimulation. Notably, ATF3 overexpression-induced CF proliferation was clearly increased in the heart after MI injury.</p><p><b>Conclusions</b>We identify that CF-specific ATF3 might contribute to be MI repair through upregulating the expression of cell cycle/proliferation-related genes and enhancing cell proliferation.</p>

Kahweol from Coffee Induces Apoptosis by Upregulating Activating Transcription Factor 3 in Human Colorectal Cancer Cells

Kahweol as a coffee-specific diterpene has been reported to induce apoptosis in human cancer cells. Although some molecular targets for kahweol-mediated apoptosis have been elucidated, the further mechanism for apoptotic effect of kahweol is not known. Activating transcription factor 3 (ATF3) has been reported to be associated with apoptosis in colorectal cancer. The present study was performed to investigate the molecular mechanism by which kahweol stimulates ATF3 expression and apoptosis in human colorectal cancer cells. Kahweol increased apoptosis in human colorectal cancer cells. It also increased ATF3 expression through the transcriptional activity. The responsible cis-element for ATF3 transcriptional activation by kahweol was CREB located between −147 to −85 of ATF3 promoter. ATF3 overexpression increased kahweol-mediated cleaved PARP, while ATF3 knockdown attenuated the cleavage of PARP by kahweol. Inhibition of ERK1/2 and GSK3β blocked kahweol-mediated ATF3 expression. The results suggest that kahweol induces apoptosis through ATF3-mediated pathway in human colorectal cancer cells.

Transcription Factors Regulating Inflammatory Cytokine Production Are Differentially Expressed in Peripheral Blood Mononuclear Cells of Behçet Disease Depending on Disease Activity

BACKGROUND: Behçet disease (BD) is a relapsing inflammatory disease with increased production of inflammatory cytokines in peripheral blood mononuclear cells (PBMCs); however, the underlying molecular mechanisms are not well known. OBJECTIVE: To analyze whether the differential expression of transcription factors is involved in the increased tumor necrosis factor (TNF)-α and interleukin (IL)-6 production by PBMCs of BD patients compared to healthy controls (HCs). METHODS: Expression of transcription factors was examined by real-time reverse transcriptase-polymerase chain reaction and western blotting. Cytokine production by CD11b+ cells transfected with siRNAs against transcription factors was measured by enzyme-linked immunosorbent assay. RESULTS: In the absence of lipopolysaccharide stimulation, the transcript level of CCAAT-enhancer-binding proteins (C/EBP) β was increased in PBMCs from patients with active BD compared to that in PBMCs from patients with stable BD. The C/EBPδ transcript level was higher in PBMCs from patients with active BD than in those from HCs. The activating transcription factor 3 (ATF3) transcript level was increased in PBMCs from patients with stable BD compared to that in PBMCs from HCs. siRNAs targeting C/EBPβ and C/EBPδ significantly reduced the production of IL-6 and TNF-α in lipopolysaccharide-stimulated CD11b+ cells from patients with BD as well as from HCs. CONCLUSION: We found differential expression of C/EBPβ, C/EBPδ, and ATF3 in PBMCs from patients with BD depending on disease activity, indicating the involvement of these molecules in BD pathogenesis.

Activating transcription factor-3 induction is involved in the anti-inflammatory action of berberine in RAW264.7 murine macrophages

Berberine is an isoquinoline alkaloid found in Rhizoma coptidis, and elicits anti-inflammatory effects through diverse mechanisms. Based on previous reports that activating transcription factor-3 (ATF-3) acts as a negative regulator of LPS signaling, the authors investigated the possible involvement of ATF-3 in the anti-inflammatory effects of berberine. It was found berberine concentration-dependently induced the expressions of ATF-3 at the mRNA and protein levels and concomitantly suppressed the LPS-induced productions of proinflammatory cytokines (TNF-α, IL-6, and IL-1β). In addition, ATF-3 knockdown abolished the inhibitory effects of berberine on LPS-induced proinflammatory cytokine production, and prevented the berberine-induced suppression of MAPK phosphorylation, but had little effect on AMPK phosphorylation. On the other hand, the effects of berberine, that is, ATF-3 induction, proinflammatory cytokine inhibition, and MAPK inactivation, were prevented by AMPK knockdown, suggesting ATF-3 induction occurs downstream of AMPK activation. The in vivo administration of berberine to mice with LPS-induced endotoxemia increased ATF-3 expression and AMPK phosphorylation in spleen and lung tissues, and concomitantly reduced the plasma and tissue levels of proinflammatory cytokines. These results suggest berberine has an anti-inflammatory effect on macrophages and that this effect is attributable, at least in part, to pathways involving AMPK activation and ATF-3 induction.

Naringenin-Mediated ATF3 Expression Contributes to Apoptosis in Human Colon Cancer

Naringenin (NAR) as one of the flavonoids observed in grapefruit has been reported to exhibit an anti-cancer activity. Activating transcription factor 3 (ATF3) is associated with apoptosis in human colon cancer cells. This study was performed to investigate the molecular mechanism by which NAR stimulates ATF3 expression and apoptosis in human colon cancer cells. NAR reduced the cell viability and induced an apoptosis in human colon cancer cells. ATF3 overexpression increased NAR-mediated cleaved PARP, while ATF3 knockdown attenuated the cleavage of PARP by NAR. NAR increased ATF3 expression in both protein and mRNA level, and increased the luciferase activity of ATF3 promoter in a dose-dependent manner. The responsible region for ATF3 transcriptional activation by NAR is located between -317 and -148 of ATF3 promoter. p38 inhibition blocked NAR-mediated ATF3 expression, its promoter activation and apoptosis. The results suggest that NAR induces apoptosis through p38-dependent ATF3 activation in human colon cancer cells.

ATF3 Mediates Anti-Cancer Activity of Trans-10, cis-12-Conjugated Linoleic Acid in Human Colon Cancer Cells

Conjugated linoleic acids (CLA) are a family of isomers of linoleic acid. CLA increases growth arrest and apoptosis of human colorectal cancer cells through an isomer-specific manner. ATF3 belongs to the ATF/CREB family of transcription factors and is associated with apoptosis in colorectal cancer. The present study was performed to investigate the molecular mechanism by which t10, c12-CLA stimulates ATF3 expression and apoptosis in human colorectal cancer cells. t10, c12-CLA increased an apoptosis in human colorectal cancer cells in dose dependent manner. t10, c12-CLA induced ATF3 mRNA and luciferase activity of ATF3 promoter in a dose-dependent manner. The responsible region for ATF3 transcriptional activation by t10, c12-CLA is located between -147 and -1850 of ATF3 promoter. mRNA stability of ATF3 was not affected by t10, c12-CLA treatment. t10, c12-CLA increases GSK3beta expression and suppresses IGF-1-stimulated phosphorylation of Akt. The knockdown of ATF3 suppressed expression of GSK3beta and NAG-1 and PARP cleavage. The results suggest that t10, c12-CLA induces apoptosis through ATF3-mediated pathway in human colorectal cancer cells.

The Role of ATF3 Expression and Calcineurin Inhibition in the Putative Cancer Stem Cell Fraction of Human Cutaneous Squamous Cell Carcinoma / 대한피부과학회지

BACKGROUND: A previous study reported that calcineurin inhibition by cyclosporin A (CsA) showed tumor-enhancing effects through the induction of the ATF3 transcription factor and the associated suppression of p53. The development and aggressiveness of cutaneous squamous cell carcinoma (SCC) may be determined by cancer stem cell populations, which have self-renewing potential. OBJECTIVE: To determine the role of ATF3 and calcineurin inhibition in the proliferation of SCC and evaluate the existence of putative SCC stem cells. METHODS: We performed real-time PCR, fluorescence activated cell sorting, and clonogenicity assays in SCC13 cells under conditions of calcineurin inhibition by CsA or ATF3 and p53 overexpression. The relationships amongst calcineurin inhibition, p53, and ATF3 were demonstrated by western blot analysis and transient transfection assays in SCC13 cells. RESULTS: In putative stem cell populations of SCC13 cells enriched in self-renewal potential, p53 expression was lower than that in differentiated SCC13 cells. CsA treatment or ATF3 overexpression caused an expansion of stem cell populations. Additionally, p53 overexpression inhibited cellular proliferation and reduced clonogenicity in SCC13 cells. CsA treatment led to a decrease in p53 expression and an increase in ATF3 in SCC13 cells on western blots. SCC13 cells with CsA and small interfering RNA against ATF3 demonstrated lower cell viability than SCC13 cells with CsA only and SCC13 cells with CsA and small interfering control RNA after 14 days. CONCLUSION: Putative cancer stem cell populations and differentiated cell populations in SCCs are positively regulated by ATF3 and p53, respectively.

Co-localization of activating transcription factor 3 and phosphorylated c-Jun in axotomized facial motoneurons / 대한해부학회지

Activating transcription factor 3 (ATF3) and c-Jun play key roles in either cell death or cell survival, depending on the cellular background. To evaluate the functional significance of ATF3/c-Jun in the peripheral nervous system, we examined neuronal cell death, activation of ATF3/c-Jun, and microglial responses in facial motor nuclei up to 24 weeks after an extracranial facial nerve axotomy in adult rats. Following the axotomy, neuronal survival rate was progressively but significantly reduced to 79.1% at 16 weeks post-lesion (wpl) and to 65.2% at 24 wpl. ATF3 and phosphorylated c-Jun (pc-Jun) were detected in the majority of ipsilateral facial motoneurons with normal size and morphology during the early stage of degeneration (1-2 wpl). Thereafter, the number of facial motoneurons decreased gradually, and both ATF3 and pc-Jun were identified in degenerating neurons only. ATF3 and pc-Jun were co-localized in most cases. Additionally, a large number of activated microglia, recognized by OX6 (rat MHC II marker) and ED1 (phagocytic marker), gathered in the ipsilateral facial motor nuclei. Importantly, numerous OX6- and ED1-positive, phagocytic microglia closely surrounded and ingested pc-Jun-positive, degenerating neurons. Taken together, our results indicate that long-lasting co-localization of ATF3 and pc-Jun in axotomized facial motoneurons may be related to degenerative cascades provoked by an extracranial facial nerve axotomy.

Is Activating Transcription Factor 3 Up-Regulated in Patients with Hypospadias?

PURPOSE: Even though hypospadias is one of the most common congenital anomalies, the cause of hypospadias is largely unknown. With regard to molecular biology and microarray technology, it appears that hypospadias is potentially related to disrupted gene expression. Genomic analysis of hypospadiac tissue indicated a potential role for activating transcription factor 3 (ATF3) in the development of this anomaly. This study prospectively examined the expression of ATF3 in tissues from 20 children with hypospadias compared with 26 normal penile skin tissue samples from elective circumcision. MATERIALS AND METHODS: Prepucial tissue was obtained from children who underwent repair of hypospadias for comparison with tissue samples from children who underwent elective circumcision. Skin specimens were evaluated for the expression of ATF3 protein by immunohistochemical staining. RESULTS: Immunohistochemical staining for ATF3 in samples from children who underwent repair of hypospadias was significantly greater than in samples from children who underwent elective circumcision (80% vs. 11%, respectively; p<0.05). CONCLUSIONS: Our results indicate that ATF3 is up-regulated in the penile skin tissue of boys with hypospadias, which suggests a role for this transcription factor in the development of this abnormality.

Expression of the signal transducer and activator of transcription factor 3 in colorectal carcinomas, colonic adenomas and ulcerative colitis

Despite the growing understanding of the involvement of protooncogenes and tumour suppressor genes in the oncogenesis of CRC, the exact biological and molecular mechanisms underpinning this process remain poorly understood. The signal transducer and activator of transcription [STAT3] has been implicated in the regulation of growth and malignant transformation. Accumulating evidences have come to indicate that abnormalities in the Janus kinase [JAK]/STAT pathway are involved in oncogenesis of several cancers. The aim of this study was to investigate the expression of JAK3 and STAT3 in both normal and activated forms by immunohistochemistry in adenomas of the colon, ulcerative colitis and CRC compared to normal colonic mucosa. Tissues from 30 cases with primary CRC and seven cases with ulcerative colitis [UC], removed by colectomy, were included. In addition, tissues from 10 colonic adenomas, 15 CRC and eight cases with UC, obtained by endoscopic biopsies, were examined histopathologically. Immuno-histochemical evaluation of STAT3, p-STAT3, JAK3 and p-JAK3 expression in tissue sections was completed. Statistical analysis and correlation of data were then performed. Normal colonic mucosa showed expression of STAT3 only. Immunoreactivity of p-JAK3 increased significantly [p < 0.05] and correlated with the degree of dysplasia in colonic adenomas. Immunoreactivity of p-STAT3 increased significantly [p < 0.05] and correlated with the degree of dysplasia in cases with UC. In CRC a significant positive correlation was found between p-STAT3 expression and grading, STAT3, JAK3 and p-JAI<3 and TNM or Dukes' staging, and p-STAT3 and nodal status excluding distant metastasis [p<0.05]. JAK3 and STAT3, and particularly their activated forms, were found to correlate significantly with the degree of dysplasia in adenomas and UC, indicating their potential role in colorectal carcinogenesis. They also correlate with anaplasia and invasion, suggesting a definitive role in progression of CRC

Expressions of ATF3 and CTGF and their regulation by estradiol in the prepuce of hypospadias patients / 中华男科学杂志

<p><b>OBJECTIVE</b>Estrogen is closely associated with hypospadias. The present study was to explore the molecular mechanism of hypospadias caused by estradiol.</p><p><b>METHODS</b>Fibroblasts obtained from the prepuce of hypospadiac and normal children were cultured in vitro and treated with 17-beta ethinyl estradiol (17-EE) at the concentrations of 1 micromol/L to 0.1 nmol/L for 2 hours, or at 0.1 micromol/L for 0.5, 1, 2, 4, 8, 16 and 24 hours. MTT assay was used to evaluate the effect of 17-EE on the proliferation of the cells, and RT-PCR was employed to detect the expressions of the activating transcription factor-3 (ATF3) and connective tissue growth factor (CTGF) in the hypospadiac tissue. The results were compared with those obtained from the nonhypospadiac tissue.</p><p><b>RESULTS</b>The expressions of ATF3 and CTGF were significantly upregulated in the hypospadiac tissue as compared with the nonhypospadiac group. At the concentration of 1 micromol/L, 17-EE significantly inhibited the proliferation of the cells. ATF3 mRNA was elevated at 1-2 hours, while CTGF mRNA showed no significant changes in 24 hours.</p><p><b>CONCLUSION</b>ATF3 and CTGF are two candidate genes involved in the etiology of hypospadias. And estradiol may induce hypospadias by upregulating the expressions of ATF3 and CTGF.</p>

Isolation of Id1 interaction protein in human prostate cancer / 中华男科学杂志

<p><b>OBJECTIVE</b>To isolate the inhibitor of DNA binding 1 (Id1) interaction protein and to determine the role and action mechanism of Id1 in human prostate cancer.</p><p><b>METHODS</b>The expression vector pET-28a/Id1 was established and used as a bait to prey the interaction protein by pull-down assay.</p><p><b>RESULTS</b>A clear interaction protein band was observed by SDS-PAGE, which was found to be activating transcription factor 3 (ATF3) by Western blotting.</p><p><b>CONCLUSION</b>Id1 may play a role in human prostate cancer by interacting with ATF3.</p>

Expression of Activating Transcription Factor 3 in Ischemic Penumbra Region Following Focal Cerebral Ischemia / 대한해부학회지

It has been demonstrated that some of immediate early genes (IEGs) such as c-Jun or fos are induced immediately following neuronal injury and they play an important role in determining the fate of the injured neurons. Of IEGs, the activating transcription factor 3 (ATF3) is focused by many investigators, because they are expressed in various types of neural insults and have been known to serve a diverse function in both neuronal survival and death. However, little is known about the functional role of ATF3 in ischemic brain injury. So in this study, the authors examined the expression pattern of the activating transcription factor 3 (ATF3) following middle cerebral artery (MCA) occlusion-reperfusion injury. According to the findings obtained by triphenyltetrazolium chloride (TTC) stains, the authors have classified the infarcted area into two regions, the ischemic core region and the ischemic penumbra region. In both regions, many neurons underwent neuronal degeneration, characterized by the shrunken nuclei with eosinophilic perikaryon. The H & E stain also demonstrated the increased number of probable activated astrocytes and microglia in the ischemic brain regions and this was confirmed by GFAP- and OX42-immunohistochemistry. Immunohistochemical study for ATF3 also demonstrated the specific upregulation of ATF3 in the nuclei of neurons under ischemic injury, but not in those of the contralateral regions. Interestingly, the number of the ATF3 positive neurons in the ischemic penumbra regions outnumbered that of the ischemic core regions. Based on many reports that the neuronal death in ischemic penumbra region is caused by programed cell death rather than by necrosis which is main cause of neuronal death in ischemic core region, our results could suggest that the ATF3 is an important IEGs which determine the fate of the ischemic neurons.