Resveratrol protects against high glucose-induced steroidogenesis and apoptosis in murine granulosa cells.

Resveratrol (Res) is a polyphenolic compound that exhibits a diverse array of biological effects. Herein, we detected the ability of Res on murine granulosa cells (GCs) against impaired steroidogenesis and apoptotic death in response to high glucose levels. Ovarian GCs were harvested from C57BL/6 mice and cultured in steroidogenic media supplemented with follicle-stimulating hormone (FSH, 30 ng/mL), Res (50 µmol/L), and low or high glucose concentrations (5 mM or 30 mM). After culture for 24 h, cell supernatants were harvested and the levels of progesterone and estradiol therein were measured. Also, caspase-3 activity and the expression of genes associated with apoptosis and steroidogenesis were assessed. High-glucose treatment suppressed steroidogenesis in this assay system, resulting in the impaired expression of steroidogenesis-related genes including Cyp11a1, Cyp19a1, 3ßHSD, and StAR and a concomitant decrease in progesterone and estradiol production. Cells exposed to high glucose also exhibited apoptotic phenotypes characterized by Bax upregulation, Bcl-2 downregulation, and increased caspase-3 activity levels. However, Res treatment was sufficient to reverse this high glucose level-induced apoptotic and steroidogenic phenotypes with improving progesterone and estradiol production, and these maybe related the effects of Res on Cyp11a1, Cyp19a1, 3ßHSD, and StAR expressions. These data suggested that Res is well suited to overcoming the negative effects of hyperglycemia of GC functionality.

Discovery of Thieno[3,2-d]pyrimidine derivatives as potent and selective inhibitors of ataxia telangiectasia mutated and Rad3 related (ATR) kinase.

The ataxia telangiectasia mutated and rad3-related (ATR) kinase regulates the DNA damage response (DDR), which plays a critical role in the ATR-Chk1 signaling pathway. ATR inhibition can induce synthetic lethality (SL) with several DDR deficiencies, making it an attractive drug target for cancers with DDR defects. In this study, we developed a series of selective and potent ATR inhibitors with a thieno[3,2-d]pyrimidine scaffold using a hybrid design. We identified compound 34 as a representative molecule that inhibited ATR kinase with an IC50 value of 1.5 nM and showed reduced potency against other kinases tested. Compound 34 also exhibited potent antiproliferative effects against LoVo cells and SL effects against HT-29 cells. Moreover, compound 34 demonstrated good pharmacokinetic properties, in vivo antitumor efficacy, and no obvious toxicity in the LoVo xenograft tumor model. Therefore, compound 34 is a promising lead compound for drug development to combat specific DDR deficiencies in cancer patients.

Sperm-borne microRNA-34c regulates maternal mRNA degradation and preimplantation embryonic development in mice.

BACKGROUND: Studies have shown that sperm-borne microRNAs (miRNAs) are involved in mammalian preimplantation embryonic development. In humans, spermatozoan miR-34c levels are correlated with in vitro fertilization outcomes, such as embryo quality and the clinical pregnancy and live birth rates. In rabbits and cows, miR-34c improves the developmental competence of embryos generated by somatic cell nuclear transfer. However, the mechanisms underlying the regulation of embryonic development by miR-34c remain unknown. METHODS: Female C57BL/6 mice (6-8 weeks old) were superovulated, and pronucleated zygotes were collected and microinjected with an miR-34c inhibitor or a negative-control RNA. The embryonic development of the microinjected zygotes was evaluated, and the messenger RNA (mRNA) expression profiles of the embryos at the two-cell, four-cell and blastocyst stages (five embryos per group) were determined by RNA sequencing analysis. Gene expression levels were verified by reverse transcription-quantitative polymerase chain reaction. Cluster analysis and heat map visualization were performed to detect differentially expressed mRNAs. Pathway and process enrichment analyses were performed using ontology resources. Differentially expressed mRNAs were systematically analyzed using the Search Tool for the Retrieval of Interacting Genes/Proteins database to determine their biological functions. RESULTS: Embryonic developmental potential was significantly reduced in zygotes microinjected with the miR-34c inhibitor compared with those microinjected with a negative-control RNA. Two-cell stage embryos microinjected with an miR-34c inhibitor presented altered transcriptomic profiles, with upregulated expression of maternal miR-34c target mRNAs and classical maternal mRNAs. Differentially expressed transcripts were mainly of genes associated with lipid metabolism and cellular membrane function at the two-cell stage, with cell-cycle phase transition and energy metabolism at the four-cell stage; and with vesicle organization, lipid biosynthetic process and endomembrane system organization at the blastocyst stage. We also showed that genes related to preimplantation embryonic development, including Alkbh4, Sp1, Mapk14, Sin3a, Sdc1 and Laptm4b, were significantly downregulated after microinjection of an miR-34c inhibitor. CONCLUSIONS: Sperm-borne miR-34c may regulate preimplantation embryonic development by affecting multiple biological processes, such as maternal mRNA degradation, cellular metabolism, cell proliferation and blastocyst implantation. Our data demonstrate the importance of sperm-derived miRNAs in the development of preimplantation embryos.

Design, synthesis, and biological evaluation of pyrido[3,2-d]pyrimidine derivatives as novel ATR inhibitors.

Targeting ataxia telangiectasia mutated and Rad3-related (ATR) kinase is being pursued as a new therapeutic strategy for the treatment of advanced solid tumor with specific DNA damage response deficiency. Herein, we report a series of pyrido[3,2-d]pyrimidine derivatives with potent ATR inhibitory activity through structure-based drug design. Among them, the representative compound 10q exhibited excellent potency against ATR in both biochemical and cellular assays. More importantly, 10q exhibited good liver microsomes stability in different species and also showed moderate inhibitory activity against HT-29 cells in combination treatment with the ATM inhibitor AZD1390. Thus, this work provides a promising lead compound against ATR for further study.

Engineered Bone Marrow Stem Cell-Sheets Alleviate Renal Damage in a Rat Chronic Glomerulonephritis Model.

Although mesenchymal stem cell (MSC)-based regenerative therapy is being developed for the treatment of kidney diseases, cell delivery and engraftment still need to be improved. Cell sheet technology has been developed as a new cell delivery method, to recover cells as a sheet form retaining intrinsic cell adhesion proteins, which promotes its transplantation efficiency to the target tissue. We thus hypothesized that MSC sheets would therapeutically reduce kidney disease with high transplantation efficiency. When the chronic glomerulonephritis was induced by two injections of the anti-Thy 1.1 antibody (OX-7) in rats, the therapeutic efficacy of rat bone marrow stem cell (rBMSC) sheet transplantation was evaluated. The rBMSC-sheets were prepared using the temperature-responsive cell-culture surfaces and transplanted as patches onto the surface of two kidneys of each rat at 24 h after the first injection of OX-7. At 4 weeks, retention of the transplanted MSC-sheets was confirmed, and the animals with MSC-sheets showed significant reductions in proteinuria, glomerular staining for extracellular matrix protein, and renal production of TGFß1, PAI-1, collagen I, and fibronectin. The treatment also ameliorated podocyte and renal tubular injury, as evidenced by a reversal in the reductions of WT-1, podocin, and nephrin and by renal overexpression of KIM-1 and NGAL. Furthermore, the treatment enhanced gene expression of regenerative factors, and IL-10, Bcl-2, and HO-1 mRNA levels, but reduced TSP-1 levels, NF-kB, and NAPDH oxidase production in the kidney. These results strongly support our hypothesis that MSC-sheets facilitated MSC transplantation and function, and effectively retarded progressive renal fibrosis via paracrine actions on anti-cellular inflammation, oxidative stress, and apoptosis and promoted regeneration.

Inhibition of IGF2BP1 attenuates renal injury and inflammation by alleviating m6A modifications and E2F1/MIF pathway.

Septic acute kidney injury (AKI) is characterized by inflammation. Pyroptosis often occurs during AKI and is associated with the development of septic AKI. This study found that induction of insulin-like growth factor 2 mRNA binding protein 1 (IGF2BP1) to a higher level can induce pyroptosis in renal tubular cells. Meanwhile, macrophage migration inhibitory factor (MIF), a subunit of NLRP3 inflammasomes, was essential for IGF2BP1-induced pyroptosis. A putative m6A recognition site was identified at the 3'-UTR region of E2F transcription factor 1 (E2F1) mRNA via bioinformatics analyses and validated using mutation and luciferase experiments. Further actinomycin D (Act D) chase experiments showed that IGF2BP1 stabilized E2F1 mRNA dependent on m6A. Electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) indicated that E2F1 acted as a transcription factor to promote MIF expression. Thus, IGF2BP1 upregulated MIF through directly upregulating E2F1 expression via m6A modification. Experiments on mice with cecum ligation puncture (CLP) surgery verified the relationships between IGF2BP1, E2F1, and MIF and demonstrated the significance of IGF2BP1 in MIF-associated pyroptosis in vivo. In conclusion, IGF2BP1 was a potent pyroptosis inducer in septic AKI through targeting the MIF component of NLRP3 inflammasomes. Inhibiting IGF2BP1 could be an alternate pyroptosis-based treatment for septic AKI.

Polydatin protects against gouty nephropathy by inhibiting renal tubular cell pyroptosis.

OBJECTIVE: To investigate the protective effect and mechanism of polydatin (PD) against gouty nephropathy (GN) in mice. METHODS: Twenty-four mice were randomly divided into three groups: the control group (no treatment), the GN group (300 mg/kg hypoxanthine + 150 mg/kg potassium oxonate), and the GN + PD group (300 mg/kg hypoxanthine + 150 mg/kg potassium oxonate + 50 mg/kg PD). Histological changes in the kidneys and the levels of uric acid (UA), blood urea nitrogen (BUN), and serum creatinine (SCr) in the sera were measured. In addition, the expression of gasdermin D (GSDMD) protein in renal tubular epithelial cells, and the expression of NOD-like receptor protein 3 (NLRP3), GSDMD, and caspase-1 proteins in the kidney tissues were determined by immunohistochemistry, immunofluorescence, and Western blot. RESULTS: In vitro, PD inhibited the expression of NLRP3, caspase-1, and GSDMD and protected the renal tubular epithelial cells from pyroptosis. In vivo, PD treatment significantly ameliorated the pathological changes in kidney tissue, and reversed the decrease of serum UA and BUN in GN model mice. The expression of NLRP3, GSDMD, and caspase-1 proteins was also decreased in the PD-treated GN mice. CONCLUSION: The results suggest that PD has a protective effect on mice with GN, which may be related to the downregulation of NLRP3, GSDMD, and caspase-1 proteins and the inhibition of renal tubular epithelial cells pyroptosis.

Blocking GSDME-mediated pyroptosis in renal tubular epithelial cells alleviates disease activity in lupus mice.

An increase in apoptosis and/or defects in the clearance of apoptotic cells resulting in massive secondary necrosis have been recognized as the main causes of systemic lupus erythematosus (SLE). Recent findings have revealed that gasdermin E (GSDME)-mediated pyroptosis is a mechanism associated with secondary necrosis. We aimed to investigate the effects of GSDME-mediated pyroptosis on disease activity in lupus mice. In vivo, high levels of GSDME expression were observed in the renal tubules of pristane-induced lupus (PIL) mice and SLE patients. In lupus mice, GSDME knockout or SP600125 administration effectively ameliorated lupus-like features by inhibiting GSDME-mediated renal tubular epithelial cell pyroptosis. In vitro, treatment with tumour necrosis factor-α (TNF-α) plus cycloheximide (CHX) or SLE sera induced HK2 cells to undergo pyroptosis in a caspase-3- and GSDME-dependent manner. Likewise, SP600125 significantly reduced GSDME expression and decreased pyroptosis in HK2 cells. GSDME-mediated pyroptosis may be associated with SLE pathogenesis, and targeting GSDME may be a potential strategy for treating SLE.

Inhibitors of cell cycle checkpoint target Wee1 kinase - a patent review (2003-2022).

INTRODUCTION: DNA damage repair in most malignancies with mutation of p53 is more dependent on the G2/M checkpoint. Wee1 kinase is a key regulator of the G2/M checkpoint. If Wee1 is inhibited, it results in cells with unrepaired DNA damage entering mitosis prematurely, leading to mitotic catastrophe and subsequent cell death via the apoptotic program. Therefore, inhibition of Wee1 kinase which overexpressed in several cancer cell lines has emerged as a promising therapy for cancer treatment. AREAS COVERED: This review summarizes for the first time the structures of small-molecule inhibitors of Wee1 reported in patents published from 2003 to 2022 and the recent clinical developments. It also provides perspectives on the challenges and the future directions. We used different methods to search different databases (PubMed, Reaxys, clinicaltrials.gov)for the literature we needed. EXPERT OPINION: Although the small-molecule inhibitors of Wee1, Adavosertib, and ZN-C3 have entered the clinical phase II, the clinical toxicity exhibited by Adavosertib remains the subject of greater concern. The use of Wee1 inhibitors as monotherapy or in combination therapy remains the main trend in Wee1 inhibitors at present.

[Determination of nine N-nitrosamines in animal derived foods by QuEChERS-isotope dilution combined with gas chromatography-tandem mass spectrometry].

In this study, a comprehensive analytical method based on gas chromatography-tandem mass spectrometry (GC-MS/MS) was developed for the determination of nine N-nitrosamines in animal derived foods. There are many kinds of N-nitrosamines in foods that are harmful to human health. However, the national standard GB 5009.26-2016 pertains only to the detection of N-dimethylnitrosamine; there are many drawbacks of this method, such as complicated sample preparation, low recovery rate, and poor reproducibility. Hence, it is of practical significance to establish a method for the simultaneous determination of a variety of N-nitrosamines. The optimal extraction conditions for the developed method were as follows: 10.0 g aliquots of the sample were placed in a 50 mL centrifuge tube, followed by the addition of 10 mL acetonitrile and 200 µL internal working standard solutions. After 30 min of freezing treatment, 4 g magnesium sulfate and 1 g sodium chloride were added for dehydration, and the tube was centrifuged at 9000 r/min for 5 min. After vortex centrifugation, 5 mL of the clear supernatant was purified using 150 mg polystyrene divinylbenzene (PLS-A). The purified extracts were dewatered using 1.6 g MgSO4 and 0.4 g NaCl, and then filtered through a 0.22 µm membrane filter unit prior to GC-MS/MS analysis. Temperature-programmed was applied at an initial temperature of 50 ℃. After 0.16 min, the temperature was raised to 220 ℃ at the rate of 900 ℃/min for 5 min. N-Nitrosamines were separated on an HP-Innowax column (30 m×0.25 mm×0.25 µm). Identification and quantification were achieved using an electron impact ion (EI) source in positive ion mode with multiple reaction monitoring (MRM). The internal standard method was used to quantify the N-nitrosamines. Under the optimal conditions, the correlation coefficients of the standard calibration curves were not less than 0.99 in the range of 0.1-50.0 µg/L. The limits of detection were 0.03-0.30 µg/kg (S/N=3), and limits of quantification were 0.15-1.00 µg/kg (S/N=10). At spiked levels of 0.5, 1.0, and 3.0 µg/kg, the average recoveries of N-nitrosamines in spiked samples ranged from 80.4% to 98.5%, with relative standard deviations between 2.41% and 12.50%. This method was used to determine animal derived food products, except N-itrosomethylethylamine and N-nitrosomorpholine, others were founded. The results showed that N-nitrosamines levels in salted aquatic products were generally higher than those of the other samples. The method established in this study is simple to operate, and it does not require any time-consuming distillation extraction. Furthermore, there is minimal consumption of samples and reagents; consequently, the experiment cost is reduced, and the method is environmentally friendly. This method has theoretical and practical significance for the control of N-nitrosamines residues in animal derived foods, establishment of detection standards, and corresponding management measures.

Beneficial phytoestrogenic effects of resveratrol on polycystic ovary syndromein rat model.

AIMS: The effective treatment of polycystic ovary syndrome (PCOS)-related hormonal disorders necessitates the development of novel treatment strategies. Resveratrol is found in certain food products, and is known to exhibit phytoestrogen properties. The present study was to assess whether resveratrol exhibits beneficial phytoestrogenic effects and associated hormonal modulation in a rat model of PCOS. MATERIALS AND METHODS: This model was established by administering oral letrozole to female Sprague-Dawley (SD) rats prior to randomizing them into control, model and resveratrol treatment groups (40, 80, or 160 mg/kg). Animals were treated for 30 days, after which time ovarian tissues were collected and evaluated via hematoxylin and eosin staining. In addition, serum levels of estradiol and adiponectin were assessed via ELISA, and ovarian expression of nesfatin-1 and aromatase was assessed through RT-PCR and western blotting. RESULTS: We found that resveratrol administration was associated with increased levels of plasma adiponectin and estradiol levels and restoration of normal ovarian morphology in PCOS model animals. In addition, this treatment was linked to the increased ovarian expression of nesfatin-1 and aromatase at the RNA and protein levels. CONCLUSIONS: Together things findings suggest that resveratrol may represent an effective tool for treating PCOS owing to its phytoestrogenic properties.

[Expression of CREB in children with recurrent wheezing and its effect on ORMDL3 gene expression].

OBJECTIVE: To study the expression level of cAMP response element-binding protein (CREB) in children with recurrent wheezing under three years of age and its effect on the expression of the serum orosomucoid 1-like protein 3 (ORMDL3) gene. METHODS: Thirty-six children with recurrent wheezing under three years of age who visited the hospital from June 2017 to June 2019 were selected as the recurrent wheezing group. Twenty-four healthy children from physical examination were selected as the control group. The CREB expression level in peripheral blood was measured by quantitative real-time PCR. Human bronchial epithelial cells (BEAS-2B) were cultured, and dual-luciferase reporter assay and quantitative real-time PCR were used to investigate the effects of overexpression and siRNA interference of CREB on the promoter activity and mRNA expression of the ORMDL3 gene in the BEAS-2B cells. RESULTS: The expression level of CREB in the recurrent wheezing group was significantly higher than that in the control group (P<0.001). In BEAS-2B cells, overexpression of CREB significantly up-regulated the promoter activity and mRNA expression of the ORMDL3 gene (P<0.05), while siRNA interference of CREB significantly reduced the promoter activity and mRNA expression of the ORMDL3 gene (P<0.05). CONCLUSIONS: The expression of CREB is increased in children with recurrent wheezing, and CREB may be involved in the pathogenesis of recurrent wheezing by regulating expression of the ORMDL3 gene.

Bronchial epithelial pyroptosis promotes airway inflammation in a murine model of toluene diisocyanate-induced asthma.

Airway epithelial injury in response to allergens such as toluene diisocyanate (TDI) leads to persistent airway inflammation. Pyroptosis is recognized as a strong proinflammatory cell death process. However, the role of pyroptosis in bronchial epithelial injury and airway inflammation in TDI-induced asthma remains unknown. In this study, cytotoxic effect of TDI on 16HBE cells (a human bronchial epithelial cell line) was detected. Then a TDI-induced experimental asthma mouse model was established for in vivo study. Here we found that TDI induced pyroptosis in 16HBE cells, as evidenced by enhanced expressions of caspase-1 and elevated levels of LDH, IL-1ß and HMGB1. As expected, TDI-induced inflammatory cell death was significantly blocked by a specific NLRP3 inflammasome inhibitor. Intriguingly, in asthmatic mice, the increased cleavages of caspase-1 and pyroptotic executioner gasdermin D (GSDMD) in bronchial epithelial cells were decreased by NLRP3 inflammasome inhibitor. Furthermore, inhibition of NLRP3 inflammasome attenuated airway hyper-responsiveness and airway inflammation, accompanied by lower levels of IL-1ß, IgE and Th2-related cytokines. Our data suggest that bronchial epithelial pyroptosis exacerbates airway inflammation and hyper-responsiveness in TDI-induced asthma via NLRP3 inflammasome activation and GSDND cleavage. Therefore, NLRP3 inflammasome-mediated pyroptosis may be a potential treatment target for TDI-induced asthma.

Berberine decreases insulin resistance in a PCOS rats by improving GLUT4: Dual regulation of the PI3K/AKT and MAPK pathways.

Berberine has been found to exhibit an array of pharmacological activities relating to the lowering of blood glucose and the treatment of polycystic ovarian syndrome (PCOS). The mechanism underlying these activites, however, is poorly understood. In the present study, female Sprague-Dawley (SD) rats were given oral letrozole to establish a model of insulin-resistant PCOS, and animals were then randomized into untreated or berberine-treated groups (400, 200, or 100 mg/kg). After 28 days, we measured homeostasis model assessment of insulin resistance (HOMA-IR) and insulin sensitivity index (ISI) values in these animals. We further conducted H&E staining of ovarian tissues, assessed mRNA expression of glucose transporter 4 (GLUT4) via real time PCR, and used Western blotting to measure GLUT4 and PI3K/AKT and MAPK pathway protein levels. Berberine treatment was able to help restore HOMA-IR and ISI values to normal levels while simultaneously bolstering the expression of GLUT4. Normal ovarian morphology was also restored upon berberine treatment. We further found that 400 mg/kg berberine treatment was associated with activation of PI3K/AKT signaling and suppression of the MAPK pathway. In conclusion, berberine has the potential to reduce PCOS pathology and IR values in a rat model system through a mechanism linked to GLUT4 upregulation via PI3K/AKT activation and MAPK pathway suppression.

MiR-181c affects estrogen-dependent endometrial carcinoma cell growth by targeting PTEN.

MicroRNAs (miRNAs), which is a type of non-coding and single-stranded small molecule RNA, bind either completely or incompletely to 3'-UTR of the target gene mRNA to inhibit mRNA translation or degradation. In our study, we aimed to explore the roles and mechanisms of miR-181c in the apoptosis of RL95-2 human endometrial carcinoma cells. Cell activity and apoptosis were detected by cell counting Kit-8 (CCK-8) assay and flow cytometry (FCM), respectively. Related mRNAs and proteins expression was determined by quantitative real-time reverse transcription PCR (qRT-PCR) and western blot assays, respectively. The binding capacity of PTEN-3'-UTR and miR-181c was assessed by luciferase reporter assay. The obtained results suggested that E2 evidently increased the cell activity of RL95-2 cells. In addition, miR-181c inhibitor suppressed the cell viability and enhanced the apoptosis capacity of E2-induced RL95-2 cells and distinctly reduced the miR-181c expression. We also found that miR-181c could bind to PTEN-3'-UTR and miR-181c inhibitor up-regulated the expression level of PTEN in E2-induced RL95-2 cells. Besides, overexpression of PTEN markedly promoted the apoptosis of E2-induced RL95-2 cells through regulating the Bax and Bcl-2 expression, and modulated the expression of AKT pathway, p53 and Cyclin D. In conclusion, our findings revealed that miR-181c affected the estrogen-dependent endometrial carcinoma cell growth by targeting PTEN. The potential effects of miR-181c on the apoptosis of E2-induced RL95-2 cells suggest that miR-181c could be an effective target for endometrial carcinoma therapies.

Methylation status of ORMDL3 regulates cytokine production and p-ERK/MMP9 pathway expression.

Orosomucoid like-3 (ORMDL3) has been identified to be associated with the development of asthma according to previous studies. However, the definite role of ORMDL3 in the pathogenesis of asthma remains unclear. In this study, we found ORMDL3 was highly expressed in PBMC specimens from childhood asthma patients. Cytokines production and p-ERK/MMP-9 pathway expression was also increased in childhood asthma patients compared with controls. In addition, ORMDL3 overexpression induced IL-6 and IL-8 release and activated p-ERK/MMP-9 pathway in vitro. Increased ORMDL3 expression was observed after treated with 5-Aza-CdR. 5-Aza-CdR decreased the percentage of the CpG island in the ORMDL3 promoter region and increased its promoter activity. In addition, 5-Aza-CdR significantly increased IL-6 and IL-8 levels in NHBE cells while there was no obvious alteration after knocking down ORMDL3. Knockdown of ORMDL3 also significantly decreased the expression of p-ERK/MMP-9 pathway in the presence or absence of 5-Aza-CdR. In conclusion, our study provided novel evidence for the association between ORMDL3 and asthma-associated cytokines. Moreover, DNA methylation plays an important role in ORMDL3-mediated increased IL-6 and IL-8 levels and p-ERK/MMP-9 pathway expression.

Author Correction: Down-regulated GATA-1 up-regulates interferon regulatory factor 3 in lung adenocarcinoma.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Up-regulation of IRF-3 expression through GATA-1 acetylation by histone deacetylase inhibitor in lung adenocarcinoma A549 cells.

Interferon regulatory factor 3 (IRF-3) is an important transcription factor for interferon genes. Although its functional activation by viral infection has been widely explicated, the regulatory mechanism of IRF-3 gene expression in cancer cells is poorly understood. In this study, we demonstrated treatment of lung adenocarcinoma A549 cells with trichostatin A (TSA) and valproic acid (VPA), two different classes of histone deacetylase inhibitors, strongly stimulated IRF-3 gene expression. Truncated and mutated IRF-3 promoter indicated that a specific GATA-1 element was responsible for TSA-induced activation of IRF-3 promoter. Chromatin immunoprecipitation and electrophoretic mobility shift assay showed that TSA treatment increased the binding affinity of GATA-1 to IRF-3 promoter. Using immunoprecipitation assay and immunoblotting, we demonstrated that TSA increased the level of acetylated GATA-1 in A549 cells. In summary, our study implied that TSA enhanced IRF-3 gene expression through increased GATA-1 recruitment to IRF-3 promoter and the acetylation level of GATA-1 in lung adenocarcinoma A549 cells.

Down-regulated GATA-1 up-regulates interferon regulatory factor 3 in lung adenocarcinoma.

Interferon regulatory factor 3 (IRF-3) is widely known for its prompt response against viral infection by activating the interferon system. We previously reported that E2F1, Sp1 and Sp3 regulated transcriptional activity of IRF-3. Recently, different expression patterns of IRF-3 were found in lung cancer, leading to the alternation of the immunomodulatory function in tumorigenesis. However, the mechanism of transcriptional regulation of IRF-3 in lung cancer has not been extensively studied. Here, we investigated the characterization of IRF-3 promoter and found that GATA-1 bound to a specific domain of IRF-3 promoter in vitro and in vivo. We found elevated IRF-3 and decreased GATA-1 gene expression in lung adenocarcinoma in Oncomine database. Additionally, higher IRF-3 gene expression was observed in human lung adenocarcinoma, accompanied by aberrant GATA-1 protein expression. We further analyzed the relationship of GATA-1 and IRF-3 expression in lung adenocarcinoma cell lines and found that inhibition of GATA-1 by siRNA increased the promoter activity, mRNA and protein levels of IRF-3, while over-expression of GATA-1 down-regulated IRF-3 gene expression. Taken together, we conclude that reduced GATA-1 could be responsible for the upregulation of IRF-3 in lung adenocarcinoma cells through binding with a specific domain of IRF-3 promoter.

All-trans retinoic acid upregulates the expression of ciliary neurotrophic factor in retinal pigment epithelial cells.

Retinopathy of prematurity, a leading cause of visual impairment in low birth-weight infants, remains a crucial therapeutic challenge. Ciliary neurotrophic factor (CNTF) is a promyelinating trophic factor that promotes rod and cone photoreceptor survival and cone outer segment regeneration in the degenerating retina. Ciliary neurotrophic factor expression is regulated by many factors such as all-trans retinoic acid (ATRA). In this study, we found that ATRA increased CNTF expression in mouse retinal pigment epithelial (RPE) cells in a dose- and time-dependent manner, and PKA signaling pathway is necessary for ATRA-induced CNTF upregulation. Furthermore, we showed that ATRA promoted CNTF expression through CREB binding to its promoter region. In addition, CNTF levels were decreased in serum of retinopathy of prematurity children and in retinal tissue of oxygen-induced retinopathy mice. In mouse RPE cells cultured with high oxygen, CNTF expression and secretion were decreased, but could be recovered after treatment with ATRA. In conclusion, our data suggest that ATRA administration upregulates CNTF expression in RPE cells.