Luciferase Activity Of Promoter Research Articles

POU2F1 inhibits miR-29b1/a cluster-mediated suppression of PIK3R1 and PIK3R3 expression to regulate gastric cancer cell invasion and migration.

The transcription factor POU2F1 regulates the expression levels of microRNAs in neoplasia. However, the miR-29b1/a cluster modulated by POU2F1 in gastric cancer (GC) remains unknown. Gene expression in GC cells was evaluated using reverse-transcription polymerase chain reaction (PCR), western blotting, immunohistochemistry, and RNA in situ hybridization. Co-immunoprecipitation was performed to evaluate protein interactions. Transwell migration and invasion assays were performed to investigate the biological behavior of GC cells. MiR-29b1/a cluster promoter analysis and luciferase activity assay for the 3'-UTR study were performed in GC cells. In vivo tumor metastasis was evaluated in nude mice. POU2F1 is overexpressed in GC cell lines and binds to the miR-29b1/a cluster promoter. POU2F1 is upregulated, whereas mature miR-29b-3p and miR-29a-3p are downregulated in GC tissues. POU2F1 promotes GC metastasis by inhibiting miR-29b-3p or miR-29a-3p expression in vitro and in vivo. Furthermore, PIK3R1 and/or PIK3R3 are direct targets of miR-29b-3p and/or miR-29a-3p, and the ectopic expression of PIK3R1 or PIK3R3 reverses the suppressive effect of mature miR-29b-3p and/or miR-29a-3p on GC cell metastasis and invasion. Additionally, the interaction of PIK3R1 with PIK3R3 promotes migration and invasion, and miR-29b-3p, miR-29a-3p, PIK3R1, and PIK3R3 regulate migration and invasion via the phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/Akt/mTOR) pathway in GC cells. In addition, POU2F1, PIK3R1, and PIK3R3 expression levels negatively correlated with miR-29b-3p and miR-29a-3p expression levels in GC tissue samples. The POU2F1-miR-29b-3p/miR-29a-3p-PIK3R1/PIK3R1 signaling axis regulates tumor progression and may be a promising therapeutic target for GC.

Macrophage Migration Inhibitory Factor (MIF) Upregulates CXCR7 and Contributes to Chemotherapy Resistance in Colorectal Cancer.

Colorectal cancer is one of the most common malignant tumors worldwide, with high incidence and mortality rates making it a focus of research. Chemotherapy is a primary treatment modality for colon cancer, but chemotherapy resistance severely impacts treatment efficacy. MIF has been found to promote tumor progression and resistance in various cancers. This study aims to investigate the role of MIF in chemotherapy resistance in colon cancer and its potential mechanisms, particularly through the upregulation of CXCR7 expression, affecting the metabolism and drug sensitivity of colon cancer cells. The expression levels of MIF in colon cancer tissues and its association with patient prognosis were evaluated by analyzing TCGA and HPA data. Subsequently, the expression levels of MIF in colon cancer cell lines and resistant cell lines were detected by qRT-PCR and immunohistochemistry, and the effect of MIF on oxaliplatin sensitivity was assessed. The impact of MIF on the metabolic activity of colon cancer cells was measured using a cellular energy metabolism analyzer. Further experiments explored the mechanism by which MIF affects the metabolic activity of colon cancer cells through the upregulation of CXCR7 expression, and the role of CTCF in regulating CXCR7 transcription was validated by silencing CTCF. Finally, the effect of MIF on drug sensitivity of colon cancer cells was verified in a mouse xenograft tumor model. In this study, we found that the expression of MIF in colon cancer tissues was significantly higher than in normal tissues, and high MIF expression was associated with poor prognosis in patients. The expression levels of MIF in resistant colon cancer cell lines were significantly higher than in parental cell lines, and MIF overexpression significantly increased the resistance of colon cancer cells to oxaliplatin. Conversely, silencing MIF significantly reduced the IC50 value of resistant cells and increased apoptosis. MIF overexpression significantly increased the ECAR and OCR levels of colon cancer cells, while MIF knockdown significantly reduced these metabolic indicators. Further studies indicated that MIF affects the metabolic activity of colon cancer cells by upregulating CXCR7 expression. CTCF binding peaks at the CXCR7 promoter region and luciferase activity assays indicated that CTCF regulates CXCR7 transcription, and silencing CTCF significantly enhanced the sensitivity of colon cancer cells to oxaliplatin. In vivo experiments in mice showed that MIF silencing combined with oxaliplatin treatment significantly inhibited tumor growth and increased the necrotic area of tumor tissues. In conclusion, this study reveals the crucial role of MIF in chemotherapy resistance in colon cancer through the upregulation of CXCR7 expression, with CTCF playing an important regulatory role in this process. Our findings provide new theoretical insights and potential therapeutic targets for overcoming chemotherapy resistance in colon cancer. Future research should further explore the roles of MIF and CXCR7 in other types of cancers and the potential of MIF and CXCR7 as therapeutic targets.

A novel C-type lectin protein (BjCTL5) interacts with apoptosis stimulating proteins of p53 (ASPP) to activate NF-κB signaling pathway in primitive chordate

C-type lectin proteins (CTLs), a group of pattern recognition receptors (PRRs), play pivotal roles in immune responses. However, the signal transduction and regulation of CTLs in cephalochordates have yet to be explored. In this study, we examined the composition of CTLs in Branchiostoma japonicum, identifying a total of 272 CTLs. These CTLs underwent further analysis concerning domain arrangement, tandem and segmental duplication events. A multidomain C-type lectin gene, designated as BjCTL5, encompassing CLECT, KR, CUB, MAM, and SR domains, was the focal point of our investigation. BjCTL5 exhibits ubiquitous expression across all detected tissues and is responsive to stimulation by LPS, mannose, and poly (I:C). The recombinant protein of BjCTL5 can bind to Escherichia coli and Staphylococcus aureus, inducing their agglutination and inhibiting the proliferation of S. aureus. Yeast two-hybrid, CoIP, and confocal immunofluorescence experiments revealed the interaction between BjCTL5 and apoptosis-stimulating proteins of p53, BjASPP. Intriguingly, BjCTL5 was observed to induce the luciferase activity of the NF-κB promoter in HEK293T cells. These results suggested a potential interaction between BjCTL5 and BjASPP, implicating that they involve in the activation of the NF-κB signaling pathway, which provides an evolutionary viewpoint on NF-κB signaling pathway in primitive chordate.

Frequent Inactivation of Secreted Frizzled-Related Protein 2 during the Development of Cervical Carcinoma: Identification of Susceptible Alleles and Clinical Implications

ABSTRACT Background: In this study, importance of SFRP2, wnt stem cell renewal pathway antagonist, in the development of cervical cancer (CACX) was evaluated Aims and Objectives: Alterations (expression/ methylation/ deletion) of SFRP2 were analysed in primary cervical lesions of different clinical stages followed by their correlation with different clinicopathological parameters. Then, susceptible allele(s) of SFRP2 was identified through case control study followed by and in vitro validation. Results: The mRNA expression of SFRP2 was gradually reduced with progression of CACX. In immunohistochemistry, SFRP2 membrane expression was mainly present in the spinous layers of normal cervical epithelium and its reduced protein expression in CACX samples showed concordance with mRNA expression. Frequent deletion/ methylation of SFRP2 were seen to be associated with development of cervical cancer. Methylation of SFRP2 was prevalently associated with early invasive lesions (stage I/II) while, deletion with late invasive lesions (stage III/IV). Overall alterations (deletion/ methylation) of SFRP2 were significantly increased from premalignant CIN to stage-I/II samples followed by comparable change to the next stage (stage III/IV) samples. Moreover, deletion and/or methylation of SFRP2 were associated with poor prognosis of the patients. In a case control study, out of its seven microsatellite alleles infrequent SFRP_CA15/16 alleles along with frequent SFRP_CA17 allelewere found to be associated with CACX development. Comparatively reduced expression (mRNA/ protein) of SFRP2 was seen in the tumor adjacent normal cervical epithelium having SFRP_CA15/16 alleles than the other alleles. This has been further validated in in vitro luciferase promoter activity assay where SFRP_CA16 repeat showed high reduced activity followed by SFRP_CA15 repeat than the other repeats. Conclusion: Thus, our data showed that presence of the infrequent susceptible alleles along with deletion/methylation might have synergistic effect on frequent inactivation of SFRP2 during development of CACX.

Peroxisome proliferator-activated receptor γ coactivator 1α regulates downstream of tyrosine kinase-7 (Dok-7) expression important for neuromuscular junction formation

The neuromuscular junction (NMJ)—formed between a motor nerve terminal and skeletal muscle fiber—plays an important role in muscle contraction and other muscle functions. Aging and neurodegeneration worsen NMJ formation and impair muscle function. Downstream of tyrosine kinase-7 (Dok-7), expressed in skeletal muscle fibers, is essential for the formation of NMJ. Exercise increases the expression of the transcriptional coactivator peroxisome proliferator-activated receptor γ coactivator 1α (PGC1α) in skeletal muscles and restores NMJ formation. In this study, we used skeletal muscle-specific PGC1α knockout or overexpression mice to examine the role of PGC1α in regulating Dok-7 expression and NMJ formation. Our findings revealed that Dok-7 expression is regulated by PGC1α, and luciferase activity of the Dok-7 promoter is greatly increased by coexpressing PGC1α and estrogen receptor-related receptor α. Thus, we suggest PGC1α is involved in exercise-mediated restoration of NMJ formation.

Downregulation of Wtap causes dilated cardiomyopathy and heart failure

RNA binding proteins have been shown to regulate heart development and cardiac diseases. However, the detailed molecular mechanisms is not known. In this study, we identified Wilms' tumor 1-associating protein (WTAP, a key regulatory protein of the m6A RNA methyltransferase complex) as a key regulator of heart function and cardiac diseases. WTAP is associated with heart development, and its expression is downregulated in both human and mice with heart failure. Cardiomyocyte-specific knockout of Wtap (Wtap-CKO) induces dilated cardiomyopathy, heart failure and neonatal death. Although WTAP deficiency in the heart decreases METTL3 (methyltransferase-like 3) protein levels, cardiomyocyte-specific overexpression of Mettl3 in Wtap-CKO mice does not rescue the phenotypes of Wtap-CKO mice. Instead, WTAP deficiency in the heart decreases chromatin accessibility in the promoter regions of Mef2a (myocyte enhancer factor-2α) and Mef2c, leading to reduced mRNA and protein levels of these genes and lower expression of their target genes. Conversely, WTAP directly binds to the promoter of the Mef2c gene and increases its promoter luciferase activity and expression. These data demonstrate that WTAP plays a key role in heart development and cardiac function by maintaining the chromatin accessibility of cardiomyocyte specific genes.

Pterostilbene targets the molecular oscillator RORγ to restore circadian rhythm oscillation and protect against sleep restriction induced metabolic disorders

BackgroundConsiderable researches have directed toward metabolic disorders caused by sleep restriction (SR). SR-induced disruption of circadian metabolic rhythmicity is identified as an important pathophysiological mechanism. The flavonoid pterostilbene (PTE) is abundant in the traditional Chinese medicine dragon's blood with protective efficacy against obesity-related metabolic dysfunctions. Our previous study found that PTE ameliorates exercise intolerance and clock gene oscillation in the skeletal muscles subjected to SR. PurposeThis study aimed to explore whether PTE improves SR-induced metabolic disorders and delineate the relationship between PTE and the circadian clock. Study design and methodsTwo hundred male C57/B6J mice were kept awake for 20 h/d over five consecutive days and concurrently gavaged with 50, 100, or 200 mg/kg·bw/d PTE. Food consumption and body weight were monitored, and the metabolic status of the mice was evaluated by performing OGTT and ITT, measuring the serum lipid profiles and liver histopathology in response to SR. Daily behavior was analyzed by Clocklab™. The circadian rhythms of the liver clock genes and metabolic output genes were evaluated by cosine analysis. Binding between PTE and RORα/γ or NR1D1/2 was investigated by molecular docking. A luciferase reporter assay was used to determine the impact of PTE on Bmal1 transcription in SR-exposed mice co-transfected with Ad-BMAL1-LUC plus Ad-RORγ-mCherry or Ad-NR1D1-EGFP. ResultsPTE significantly ameliorated abnormal glucose and lipid metabolism (p < 0.05) in SR-exposed mice. PTE improved circadian behavior (p < 0.05) and rescued the circadian rhythm oscillation of the liver clock (p < 0.05) and metabolic output genes (p < 0.05) under SR condition. Molecular docking disclosed that PTE might interact with RORs, and PTE was found to increase Bmal1 promoter luciferase activity with RORE elements in the presence of Ad-RORγ-mCherry (p < 0.05). ConclusionsPTE may protect against SR-induced metabolic disorders by directly modulating RORγ to maintain circadian metabolic rhythm. The findings provide valuable insights into the potential use of PTE in the treatment of metabolic disorders associated with disruptions in the circadian rhythm.

Ectopic expression of tumor suppressive miR-181c-5p downregulates oncogenic Notch signaling in MDA-MB-231 cells

Triple negative breast cancer (TNBC) is a very invasive subtype of breast cancer (BCa), this is accounted for 15–20% of all BCa cases. TNBC patients have very limited therapy option due to lack of effective targets and patients shows the worse survival. Therefore, present study has tried to introduce the target based therapy by studying the tumor suppressive role of miR-181c-5p on oncogenic Notch1 signaling. Transient transfection, bioinformatics, qRT-PCR, Notch1 luciferase assay and western blotting techniques were utilized to study the effect of induced expression of miR-181c-5p on oncogenic Notch1 signaling in MDA-MB-231 cells. Results shows that miR-181c-5p mimic increase the expression of miR-181c-5p by 45.26% and 75.96% in 24 and 48 h incubation, respectively (p < 0.0003) in transfected cells. The miR-181c-5p binds at NOTCH1 3′ UTR target binding site with a minimum free energy of − 26.0 kcal/mol. The AGO protein showed significant interaction with the miR-181c-5p and miR-181c-5p-NOTCH1 complex. Decreased expression of NOTCH1 by 32.88% and 45.87% (p < 0.0001); and HES1 expression by 14.06% and 53.24% (p < 0.0001) was observed in 24 and 48 h transfected cells respectively. Notch1 promoter luciferase activity was reduced by 25.72% and 46.98% in 24 and 48 h miRNA-mimic transfected cells. Western blot analysis also showed significant reduction in NOTCH1 and HES1 proteins expression. In conclusion, present study suggests that the forced expression of tumor suppressive miR-181c-5p negatively regulates oncogenic Notch1 signaling in TNBC. Negative regulation of Notch1 signaling via miR-181c-5p mimic could be a hopeful therapeutic strategy in TNBC patient treatment.

Inhibitory effect and mechanism of action of blue light irradiation on adipogenesis

Blue light can reach the epidermis, dermis, and hypodermis of the skin and affect the physiology of each cell type. However, thus far, most studies have focused on the effects of blue light on the epidermis and dermis; no studies have been conducted on the effects of blue light on the hypodermis. In this study, we attempted to elucidate the effects of blue light on the hypodermis. Specifically, we investigated the effects of blue light on 3T3-L1 adipogenesis and its mechanism of action. In this study, we found that blue light reduced lipid accumulation and GPDH activity, indicating its antiadipogenic properties. Furthermore, blue light inhibited the mRNA and protein levels of peroxisome proliferator-activated receptor γ (PPARγ) and its target genes, such as Fasn and FABP4, and the luciferase activity of the PPRE promoter, suggesting that blue light downregulates the expression of adipogenic genes, leading to suppression of 3T3-L1 adipogenesis. Furthermore, blue light increased the phosphorylation of PPARγ, indicating its anti-adipogenic effect through PPARγ degradation. Blue light upregulates the expression and phosphorylation of transient receptor potential vanilloid 1 (TRPV1). Furthermore, blue light restored calcium influx, which was attenuated by adipogenesis. These data suggest that TRPV1 is involved in the anti-adipogenic effects of blue light. Blue light induced the phosphorylation of AMPK, ACC, and MAPKs, whereas capsazepine, an antagonist of TRPV1, attenuated the effects of blue light. Capsazepine treatment also reduced the blue light-induced anti-adipogenic effects. These results indicate that TRPV1 operates upstream of AMPK or MAPKs in the blue light-induced anti-adipogenesis process, which is mediated by activating the TRPV1-AMPK/MAPK signaling pathways. Meanwhile, inhibition of AMPK and MAPKs reduced the phosphorylation level of PPARγ, indicating that AMPK/MAPK signaling contributes to the phosphorylation of PPARγ. These results suggest the possibility of using blue light as an antiadipogenic agent.

SPI1 exacerbates iron accumulation and promotes osteoclast formation through inhibiting the expression of Hepcidin

BackgroundOsteoporosis (OP) can be caused by an overactive osteoclastic function. Anti-osteoporosis considerable therapeutic effects in tissue repair and regeneration because bone resorption is a unique osteoclast function. In this study, we mainly explored the underlying mechanisms of osteoclasts' effects on osteoporosis. MethodsRAW264.7 cells were used and induced toward osteoclast and iron accumulation by M-CSF and RANKL administration. We investigated Hepcidin and divalent metal transporter 1 (DMT1) on iron accumulation and osteoclast formation in an ovariectomy (OVX)-induced osteoporosis. Osteoporosis was induced in mice by OVX, and treated with Hepcidin (10, 20, 40, 80 mg/kg, respectively) and overexpression of DMT1 by tail vein injection. Hepcidin, SPI1, and DMT1 were detected by immunohistochemical staining, western blot and RT-PCR. The bioinformatics assays, luciferase assays, and Chromatin Immunoprecipitation (ChIP) verified that Hepcidin was a direct SPI1 transcriptional target. Iron accumulation was detected by laser scanning confocal microscopy, Perl's iron staining and iron content assay. The formation of osteoclasts was assessed using tartrate-resistant acid phosphatase (TRAP) staining. ResultsWe found that RAW264.7 cells differentiated into osteoclasts when exposed to M-CSF and RANKL, which increased the protein levels of osteoclastogenesis-related genes, including c-Fos, MMP9, and Acp5. We also observed higher concentration of iron accumulation when M-CSF and RANKL were administered. However, Hepcidin inhibited the osteoclast differentiation cells and decreased intracellular iron concentration primary osteoclasts derived from RAW264.7. Spi-1 proto-oncogene (SPI1) transcriptionally repressed the expression of Hepcidin, increased DMT1, facilitated the differentiation and iron accumulation of mouse osteoclasts. Overexpression of SPI1 significantly declined luciferase activity of HAMP promoter and increased the enrichment of HAMP promoter. Furthermore, our results showed that Hepcidin inhibited osteoclast differentiation and iron accumulation in mouse osteoclasts and OVX mice. ConclusionTherefore, the study revealed that SPI1 could inhibit Hepcidin expression contribute to iron accumulation and osteoclast formation via DMT1 signaling activation in mouse with OVX.

Characterization of STING from common carp (Cyprinus carpio L.) involved in spring viremia of carp virus infection

Stimulator of interferon genes (STING) serve as an endoplasmic reticulum (ER) protein and modulates innate immune responses to viral contagion. Most investigations involving teleost STING antiviral immunity have examined DNA viruses. Therefore, fish STING signaling events against RNA viruses require additional exploration. Here, common carp STING (named CcSTING) was cloned and characterized. The bioinformatics analyses of CcSTING showed evolutionary conservations and were most closely related to other cyprinid STINGs. Immunofluorescence staining discovered that the CcSTING was chiefly placed in the cytoplasm, specifically within the ER. CcSTING was ubiquitously generated in all analyzed organs, with especially strong expression in the gills and head kidney. Spring viremia of carp virus (SVCV) stimulation and poly(I:C) infection induced the generation of CcSTING in immune-associated organs, as well as in peripheral blood leukocytes. Additional investigations revealed that CcSTING overexpression strongly suppressed SVCV replication in EPC cells. Mechanistically, CcSTING enhanced IFN-1 and ISGs expression following SVCV infection. CcSTING also substantially increased both IFN and NF-κB promoter luciferase activity via a dosage-dependent fashion. Lastly, CcSTING significantly up-regulated both TBK1 and p65 phosphorylation. Collectively, these findings demonstrated the critical role and underlying mechanism of fish STING in response to RNA virus.

FRI625 Diabetic Ischemic Skeletal Muscle Injury Occurs Through Pkc-beta/sp1 Regulation Of Bag3 Expression

Abstract Disclosure: A. Mani: None. V. Lamin: None. T. Wong: None. M.V. Singh: None. A.O. Dokun: None. Peripheral arterial disease (PAD) affects more than 200 million people worldwide and is characterized by impaired blood flow to the lower extremities. Individuals with diabetes (DM) and PAD have more severe ischemic limb injuries and increased limb amputations, but the mechanisms involved are poorly understood. Following experimental PAD, C57Bl/6 mice develop limited ischemic limb injury largely due to protective effect of genes within the limb salvage QTL-1 (LSq-1) a genetic locus on chromosome 7. However, when diabetes is induced in C57Bl/6 mice they develop severe ischemic limb injuries following experimental PAD, akin to the findings in humans. Previously, we identified BAG3 as a gene within LSq-1 that is protective against ischemic limb injuries. BAG3 is upregulated in ischemic limbs from non-DM C57Bl/6 mice but poorly expressed in ischemic limbs of DM mice. The mechanism of BAG3 upregulation in ischemia is not known however the BAG3 promoter has a Specificity protein 1 (Sp1) transcription factor binding site suggesting its possible role. Moreover, how DM impairs BAG3 expression in ischemic skeletal muscles in not known. PKCβ regulates expression of transcription factors, and its phosphorylation has been implicated in the vascular pathology in DM but a role in ischemic diabetic skeletal muscle injury has not been established. Here we assessed the role of Sp1 in BAG3 upregulation in ischemic skeletal muscle cells (HSMC), we found Sp1 is upregulated in ischemic HSMCs and overexpression of Sp1 enhanced BAG3 promoter luciferase activity while knock down of Sp1 impaired BAG3 upregulation in ischemia. However, in vitro, ischemic HSMCs under DM conditions (High glucose or HG, 25mM + Palmitic acid or PA, 200µM) express lower BAG3 (fold change: Vehicle, 1.0±0.02 vs HG+PA, 0.57±0.04; n=4; p&amp;lt;0.05) and Sp1 (fold change: Vehicle, 1.0±0.15 vs HG+PA, 0.22±0.03; n=4; p&amp;lt;0.05). Overexpression of BAG3 improved survival of ischemic HSMCs in DM conditions in vitro (fold change: Vehicle, 1.0±0.02 vs HG+PA, 1.3±0.02 vs BAG3+HG+PA, 1.03±0.03; n=4-5; p&amp;lt;0.05) and improved extent of limb injury in vivo (Necrosis Score; DM mice without BAG3 2.67±0.29 vs DM mice + BAG3, 0.87±0.29; n=8-9; p&amp;lt;0.05). Additionally, under DM conditions ischemic HSMCs showed increased phosphorylation of PKCβ on the Ser661. Blocking PKCβ activation with Ruboxistaurin (Rbx), a specific inhibitor of PKCβ phosphorylation prevented the DM induced phosphorylation of PKCβ on the Ser661 (p-PKCβ/PKCβ ratio: Vehicle, 0.08±0.006 vs HG±PA, 0.19±0.03 vs Rbx+HG+PA, 0.10±0.006; n=4; p&amp;lt;0.05) and rescued BAG3 and Sp1 expression (BAG3, fold change: Vehicle, 1.0±0.06 vs HG±PA, 0.50±0.02 vs Rbx+HG+PA, 0.76±0.03; SP1, fold change: Vehicle, 1.0±0.02 vs HG±PA, 0.57±0.03 vs Rbx+HG+PA, 0.85±0.02; n=4; p&amp;lt;0.05). We conclude that impaired BAG3 expression in ischemic HSMC is mediated by impaired Sp1 expression due to increased PKCβ phosphorylation in DM. Presentation: Friday, June 16, 2023

Transcriptional activation of membrane-related Toll-like receptor 5 through the NF-κB subunit p65 and Vibrio parahaemolyticus flagellin in orange-spotted grouper (Epinephelus coioides)

Toll-like receptor 5 (TLR5) is an important pattern recognition receptor (PRR) for bacterial flagellin. There are two kinds of subtypes in fish, the membrane-associated Toll-like receptor 5 (TLR5M) and the soluble form of TLR5 (TLR5S). Among them, TLR5M is a homolog of mammalian TLR5, and TLR5S is fish-specific. TLR5M in orange-spotted grouper (Epinephelus coioides) was involved in recognizing flagellin and activating the nuclear factor (NF)-κB signalling pathway while EcTLR5S negatively regulated the NF-κB pathway. In mammals, the NF-κB pathway is critical to the expression of inflammatory cytokines and effectors. To better understand the activity of the TLR5M gene, we characterized the TLR5M 5′-flanking sequence region from E. coioides and assayed the TLR5M promoter luciferase reporter activity in HEK 293 T cells and grouper spleen cells (GS). The 5′-flanking region of TLR5M included three NF-κB binding sites. To elucidate the molecular basis of TLR5M gene expression, the activity of the TLR5M gene promoter was characterized and the deleted or site-directed mutants were generated to explore the functional significance of these kinds of binding sites. The luciferase activity of the TLR5M promoter in HEK 293 T cells was detected after flagellin was treated. The overexpression of NF-κB/p65 greatly increased the wild-type TLR5M promoter luciferase activity higher than the mutants. TLR5M promoter luciferase activity was increased more in the presence of p65 overexpression and Vibrio parahaemolyticus Flagellin C (vpFlaC) stimulation. These results suggest that NF-κB may be the important transcription factor and vpFlaC may act as a ligand, they could maximally increase the TLR5M promoter luciferase activity.

YAP promotes AP-1 expression in tubular epithelial cells in the kidney.

Chronic kidney disease (CKD) is a major health problem. Kidney fibrosis is a hallmark and final common pathway of CKD. The Hippo/yes-associated protein (YAP) pathway regulates organ size, inflammation, and tumorigenesis. Our previous study demonstrated tubular YAP activation by tubule-specific double knockout of mammalian STE20-like protein kinase 1/2 (Mst1/2) induced CKD in mice, but the underlying mechanisms remain to be fully elucidated. Activator protein (AP)-1 activation was found to promote tubular atrophy and tubulointerstitial fibrosis. Therefore, we studied whether YAP regulates AP-1 expression in the kidney. We found that expression of various AP-1 components was induced in kidneys subjected to unilateral ureteric obstruction and in Mst1/2 double knockout kidneys, and these inductions were blocked by deletion of Yap in tubular cells, with Fosl1 being most affected compared with other AP-1 genes. Inhibition of Yap also most highly suppressed Fosl1 expression among AP-1 genes in HK-2 and IMCD3 renal tubular cells. YAP bound to the Fosl1 promoter and promoted Fosl1 promoter-luciferase activity. Our results suggest that YAP controls AP-1 expression and that Fosl1 is the primary target of YAP in renal tubular cells.NEW & NOTEWORTHY Yes-associated protein (YAP) activation leads to tubular injury, renal inflammation, and fibrosis, but the underlying mechanisms are not fully understood. We now provide genetic evidence that YAP promotes activator protein-1 expression and that Fosl1 is the primary target of YAP in renal tubular cells.

Anti-Pollutant Activity of Porphyra yezoensis Water Extract and Its Active Compound, Porphyra 334, against Urban Particulate Matter-Induced Keratinocyte Cell Damage

Urban particulate matter (UPM) causes skin aging and inflammatory reactions by influencing skin cells through the aryl hydrocarbon receptor (AhR) signaling pathway. Porphyra yezoensis (also known as Pyropia yezoensis), a red alga belonging to the Bangiaceae family, is an edible red seaweed. Here, we examined the anti-pollutant effect of P. yezoensis water extract. While UPM treatment induced xenobiotic response element (XRE) promoter luciferase activity, P. yezoensis water extract reduced UPM-induced XRE activity. Next, we isolated an active compound from P. yezoensis and identified it as porphyra 334. Similar to the P. yezoensis water extract, porphyra 334 attenuated UPM-induced XRE activity. Moreover, although UPM augmented AhR nuclear translocation, which led to an increase in cytochrome P450 1A1 (CYP1A1) mRNA levels, these effects were reduced by porphyra 334. Moreover, UPM induced the production of reactive oxygen species (ROS) and reduced cell proliferation. These effects were attenuated in response to porphyra 334 treatment. Furthermore, our results revealed that the increased ROS levels induced by UPM treatment induced transient receptor potential vanilloid 1 (TRPV1) activity, which is related to skin aging and inflammatory responses. However, porphyra 334 treatment reduced this reaction by inhibiting ROS production induced by CYP1A1 activation. This indicates that porphyra 334, an active compound of P. yezoensis, attenuates UP-induced cell damage by inhibiting AhR-induced ROS production, which results in a reduction in TRPV1 activation, leading to cell proliferation. This also suggests that porphyra 334 could protect the epidermis from harmful pollutants.

Human cytomegalovirus pUL97 upregulates SOCS3 expression via transcription factor RFX7 in neural progenitor cells

Congenital human cytomegalovirus (HCMV) infection causes severe damage to the fetal brain, and the underlying mechanisms remain elusive. Cytokine signaling is delicately controlled in the fetal central nervous system to ensure proper development. Here we show that suppressor of cytokine signaling 3 (SOCS3), a negative feedback regulator of the IL-6 cytokine family signaling, was upregulated during HCMV infection in primary neural progenitor cells (NPCs) with a biphasic expression pattern. From viral protein screening, pUL97 emerged as the viral factor responsible for prolonged SOCS3 upregulation. Further, by proteomic analysis of the pUL97-interacting host proteins, regulatory factor X 7 (RFX7) was identified as the transcription factor responsible for the regulation. Depletion of either pUL97 or RFX7 prevented the HCMV-induced SOCS3 upregulation in NPCs. With a promoter-luciferase activity assay, we demonstrated that the pUL97 kinase activity and RFX7 were required for SOCS3 upregulation. Moreover, the RFX7 phosphorylation level was increased by either UL97-expressing or HCMV-infection in NPCs, suggesting that pUL97 induces RFX7 phosphorylation to drive SOCS3 transcription. We further revealed that elevated SOCS3 expression impaired NPC proliferation and migration in vitro and caused NPCs migration defects in vivo. Taken together, these findings uncover a novel regulatory mechanism of sustained SOCS3 expression in HCMV-infected NPCs, which perturbs IL-6 cytokine family signaling, leads to NPCs proliferation and migration defects, and consequently affects fetal brain development.

Parkinson’s disease-associated 18 bp promoter variant of DJ-1 alters REST binding and regulates its expression

Parkinson’s disease (PD) is a progressive neurodegenerative disorder with a complex etiology. Presence of autosomal mutations in PARK7/DJ-1 gene has been associated with early-onset PD. Growing evidence has suggested that DJ-1 acts as a putative sensor of oxidative stress. Reduced levels of DJ-1 protein have been reported in the cerebrospinal fluid of sporadic PD patients. Several case-control association studies have identified DJ-1 g.168_185del (rs200968609) variants conferring susceptibility towards PD pathogenesis. Similarly, among the PD patients in eastern India, the deletion allele (g.168_185) of this DJ-1 promoter polymorphism was found to be associated with PD. Hence, we aimed to find out the functional contribution of this promoter variant of DJ-1 in PD pathogenesis. The expression of DJ-1 was observed to be significantly reduced in the presence of both deletion and duplication sequences as identified from the luciferase promoter activity assay. The transcription factor binding prediction tool identified DJ-1 promoter 18 bp insertion polymorphism as the only binding partner of REST (RE1 Silencing Transcription Factor). Transient Chromatin Immuno-precipitation (ChIP) assay further confirmed this prediction. Previous reports have highlighted the role of REST in regulating the expression of stress-responsive genes. Our study has identified the functional involvement of DJ-1 promoter variants and REST-mediated regulation of DJ-1 expression in PD pathogenesis.

Identification of a 5-Methylcytosine Site (mC-7) That May Inhibit CXCL11 Expression and Regulate E. coli F18 Susceptibility in IPEC-J2 Cells.

The primary pathogen causing post-weaning diarrhea in piglets is Escherichia coli F18 (E. coli F18), hence it is essential to investigate the mechanism governing E. coli F18 resistance in native pig breeds. Based on the previous RNA-seq results of the duodenum from E. coli F18-resistant and -susceptible Meishan piglets, CXCL11, an important functional gene, was preliminarily screened. In this investigation, in order to further examine the expression regulation mechanism of E. coli F18 in intestinal porcine epithelial cells (IPEC-J2) against E. coli F18 infection, CXCL11 gene expression on IPEC-J2 cells infected by E. coli F18 was detected, which was significantly downregulated (p &lt; 0.01). Secondly, the overexpression on the IPEC-J2 cell line was successfully structured, and a relative quantification method of the PILIN, bacteria enumeration, and immunofluorescence assay indicated that the CXCL11 overexpression significantly reduced the ability of E. coli F18 to interact with IPEC-J2 in vitro. The promoter region of the CXCL11 gene was predicted to contain a CpG island (-619 ~ -380 bp) of which 13 CpG sites in the sequencing region were methylated to varying degrees, and the methylation level of one CPG site (mC-7) positively linked negatively with the expression of the CXCL11 gene (p &lt; 0.05). Meanwhile, a dual luciferase assay detected the mutation of the mC-7 site that significantly inhibited the luciferase activity of the CXCL11 gene promoter (p &lt; 0.01). Transcription factor prediction and expression verification indicated that mC-7 is located in the OSR1-binding domain, and that its expression level is related to E. coli F18 susceptibility. We speculated that methylation modification of the mC-7 site of the CpG island in the promoter region of the CXCL11 gene might inhibit the binding of transcription factor OSR1 with the mC-7 site, and then affect its expression level to regulate the susceptibility to E. coli F18.

PAF1 cooperates with YAP1 in metaplastic ducts to promote pancreatic cancer

Acinar-to-ductal metaplasia (ADM) is a precursor lesion of pancreatic ductal adenocarcinoma (PDAC); however, the regulators of the ADM-mediated PDAC development and its targeting are poorly understood. RNA polymerase II-associated factor 1 (PAF1) maintains cancer stem cells leading to the aggressiveness of PDAC. In this study, we investigated whether PAF1 is required for the YAP1-mediated PDAC development and whether CA3 and verteporfin, small molecule inhibitors of YAP1/TEAD transcriptional activity, diminish pancreatic cancer (PC) cell growth by targeting the PAF1/YAP1 axis. Here, we demonstrated that PAF1 co-expresses and interacts with YAP1 specifically in metaplastic ducts of mouse cerulein- or KrasG12D-induced ADM and human PDAC but not in the normal pancreas. PAF1 knockdown (KD) reduced SOX9 in PC cells, and the PC cells showed elevated PAF1/YAP1 complex recruitment to the promoter of SOX9. The PAF1 KD reduced the 8xTEAD and SOX9 promoter-luciferase reporter activities in the mouse KC (KrasG12D; Pdx-1 Cre) cells and human PC cells, indicating that the PAF1 is required for the YAP1-mediated development of ADM and PC. Moreover, treatment with CA3 or verteporfin reduced the expressions of PAF1, YAP1, TEAD4, and SOX9 and decreased colony formation and stemness in KC and PC cells. CA3 treatment also reduced the viability and proliferation of PC cells and diminished the duct-like structures in KC acinar explants. CA3 or verteporfin treatment decreased the recruitment of the PAF1/YAP1 complex to the SOX9 promoter in PC cells and reduced the 8xTEAD and SOX9 promoter-luciferase reporter activities in KC and PC cells. Overall, PAF1 cooperates with YAP1 during ADM and PC development, and verteporfin and CA3 inhibit ADM and PC cell growth by targeting the PAF1/YAP1/SOX9 axis in vitro and ex vivo models. This study identified a regulatory axis of PDAC initiation and its targeting, paving the way for developing targeted therapeutic strategies for pancreatic cancer patients.

Signaling Pathway of Taurine-Induced Upregulation of TXNIP

Taurine, a sulfur-containing β-amino acid, is present at high concentrations in mammalian tissues and plays an important role in several essential biological processes. However, the genetic mechanisms involved in these physiological processes associated with taurine remain unclear. In this study, we investigated the regulatory mechanism underlying the taurine-induced transcriptional enhancement of the thioredoxin-interacting protein (TXNIP). The results showed that taurine significantly increased the luciferase activity of the human TXNIP promoter. Further, deletion analysis of the TXNIP promoter showed that taurine induced luciferase activity only in the TXNIP promoter region (+200 to +218). Furthermore, by employing a bioinformatic analysis using the TRANSFAC database, we focused on Tst-1 and Ets-1 as candidates involved in taurine-induced transcription and found that the mutation in the Ets-1 sequence did not enhance transcriptional activity by taurine. Additionally, chromatin immunoprecipitation assays indicated that the binding of Ets-1 to the TXNIP promoter region was enhanced by taurine. Taurine also increased the levels of phosphorylated Ets-1, indicating activation of Ets-1 pathway by taurine. Moreover, an ERK cascade inhibitor significantly suppressed the taurine-induced increase in TXNIP mRNA levels and transcriptional enhancement of TXNIP. These results suggest that taurine enhances TXNIP expression by activating transcription factor Ets-1 via the ERK cascade.