• Journal of Life Science
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• v.28 no.7
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• pp.765-771
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• 2018

Peroxisome proliferator-activated receptor gamma ($PPAR{\gamma}$) is a key transcription factor that regulates adipogenesis, and epigenetic control of $PPAR{\gamma}$ is of great interest in obesity-inhibition research. Our previous study showed that CACUL1 (CDK2-associated cullin domain 1) acts as a corepressor that inhibits $PPAR{\gamma}$ transcriptional activity and adipocyte differentiation. Here, we investigated the roles of protein arginine methyltransferase 5 (PRMT5), a novel binding partner of CACUL1, in regulating $PPAR{\gamma}$. The interaction between PRMT5 and CACUL1 was shown by immunoprecipitation assay in vivo and GST pulldown assay in vitro. As shown by luciferase reporter assay, PRMT5 and CACUL1 cooperated to inhibit the transcriptional activity of $PPAR{\gamma}$. The suppressive role of PRMT5 in adipogenesis was examined by Oil Red O staining using 3T3-L1 cells, which stably overexpress or deplete PRMT5. Overexpression of PRMT5 suppresses $PPAR{\gamma}$-mediated adipogenesis, whereas PRMT5 knockdown increases lipid accumulation in 3T3-L1 cells. Consistently, PRMT5 attenuates the expression of Lpl and aP2, the target genes of $PPAR{\gamma}$, as demonstrated by RT-qPCR analysis. Overall, these results suggest that PRMT5 interacts with CACUL1 to impair the transcriptional activity of $PPAR{\gamma}$, leading to the inhibition of adipocyte differentiation. Therefore, the regulation of PRMT5 enzymatic activity may provide a clue to develop an anti-obesity drug.

• International Journal of Oral Biology
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• v.44 no.4
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• pp.165-172
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• 2019

Berberine has been used clinically for more than a decade to treat various diseases, has been shown to be effective in osteoblast differentiation, and is a potential treatment option for osteoporosis. However, compared with existing osteoporosis drugs, berberine is somewhat less effective. This study aimed to identify a new compound with efficacy superior to that of berberine. The osteogenic activities of various berberine derivatives were evaluated via cell differentiation in C2C12 preosteoblast cell lines. Alkaline phosphatase (ALP) staining assay and structure-activity relationship demonstrated that compound 2b had a potent osteogenic effect. Furthermore, compound 2b dose dependently increased ALP activity and showed no toxicity at the effective concentration, indicating its efficacy. Additionally, compound 2b upregulated BMP2-induced transcriptional activity in a promoter activity assay using ALP, BSP, and OC promoters.

• Journal of Microbiology and Biotechnology
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• v.17 no.8
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• pp.1353-1360
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• 2007

Three csp-like genes were identified in the Corynebacterium glutamicum genome and designated cspA, cspB, and cspA2. The genes cspA and cspA2 encode proteins, comprising of 67 amino acid residues, respectively. They share 83% identity with each other. Identity of those proteins with Escherichia coli Csp proteins was near 50%. The cspB gene encodes a protein composed of 127 amino acids, which has 40% and 35% sequence identity with CspA and CspA2, respectively, especially at its N-terminal region. Analysis of the gene expression profiles was done using transcriptional cat fusion, which identified not only active expression of the three genes at the physiological growth temperature of $30^{\circ}C$ but also growth phase-dependent expression with the highest activity at late log phase. The promoters of cspA and cspA2 were more active than that of cspB. The expression of the two genes increased by 30% after a temperature downshift to $15^{\circ}C$, and such stimulation was more evident in the late growth phase. In addition, the cspA gene appeared to show DNA-binding activity in vivo, and the activity increased at lower temperatures. Interestingly, the presence of cspA in multicopy hindered the growth of the host C. glutamicum cells at $20^{\circ}C$, but not at $30^{\circ}C$. Altogether, these data suggest that cspA, cspB, and cspA2 perform functions related to cold shock as well as normal cellular physiology. Moreover, CspA and its ortholog CspA2 may perform additional functions as a transcriptional regulator.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.10
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• pp.4255-4259
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• 2014

Lactobacilli are probiotics shown to have antitumor activities. In addition, they can regulate gene expression through epigenetic mechanisms. In this study, we aimed to assess anti tumor activities of Lactobacillus acidophilus and Lactobacillus crispatus on the MDA-MB-231 breast cancer cell line. The effects of culture supernatants were determined by MTT [3-(4,5-dimethylthiazol-2-y-2,5-diphenyltetrazolium bromide] assay. Changes in expression of 5 cancer-testis antigens (CTAs), namely AKAP4, ODF4, PIWIL2, RHOXF2 and TSGA10, were analyzed by quantitative real time RT-PCR. The culture supernatants of the 2 lactobacilli inhibited MDA-MB-231 cell proliferation. In addition, transcriptional activity of all mentioned CTAs except AKAP4 was significantly decreased after 24 hour treatment with culture supernatants. This study shows that Lactobacillus acidophilus and Lactobacillus crispatus have antiproliferative activity against MDA-MB-231 cells. In addition, these lactobacilli could decrease transcriptional activity of 4 CTAs. Previous studies have shown that expression of CTAs is epigenetically regulated, so it is possible that lactobacilli cause this expression downregulation through epigenetic mechanisms. As expression of CTAs in cancers is usually associated with higher grades and poor prognosis, downregulation of their expression by lactobacilli may have clinical implications.

• Development and Reproduction
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• v.15 no.1
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• pp.25-30
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• 2011

Embryonic stem (ES) cells can self-renew maintaining the undifferentiated state. Self renewal requires many factors such as Oct4, Sox2, FoxD3, and Nanog. NF-${\kappa}B$ is a transcription factor involved in many biological activities. Expression and activity of NF-${\kappa}B$ increase upon differentiation of ES cells. Reportedly, Nanog protein directly binds to NF-${\kappa}B$ protein and inhibits its activity in ES cells. Here, we found a potential binding site of NF-${\kappa}B$ in the human Nanog promoter and postulated that NF-${\kappa}B$ protein may regulate expression of the Nanog gene. We used human embryonic carcinoma (EC) cells as a model system of ES cells and confirmed decrease of Nanog and increase of NF-${\kappa}B$ upon differentiation induced by retinoic acid. Although deletion analysis on the DNA fragment including NF-${\kappa}B$ binding site suggested involvement of NF-${\kappa}B$ in the negative regulation of the promoter, site-directed mutation of NF-${\kappa}B$ binding site had no effect on the Nanog promoter activity. Furthermore, no direct association of NF-${\kappa}B$ with the Nanog promoter was detected during differentiation. Therefore, we conclude that NF-${\kappa}B$ protein may not be involved in transcriptional regulation of Nanog gene expression in EC cells and possibly in ES cells.

• The Korean Journal of Physiology and Pharmacology
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• v.20 no.1
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• pp.91-99
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• 2016

(E)-3-(3-methoxyphenyl)-1-(2-pyrrolyl)-2-propenone (MPP) is an aldol condensation product resulting from pyrrole-2-carbaldehyde and m- and p- substituted acetophenones. However, its biological activity has not yet been evaluated. Since it has been reported that some propenone-type compounds display anti-inflammatory activity, we investigated whether MPP could negatively modulate inflammatory responses. To do this, we employed lipopolysaccharide (LPS)-stimulated macrophage-like RAW264.7 cells and examined the inhibitory levels of nitric oxide (NO) production and transcriptional activation, as well as the target proteins involved in the inflammatory signaling cascade. Interestingly, MPP was found to reduce the production of NO in LPS-treated RAW264.7 cells, without causing cytotoxicity. Moreover, this compound suppressed the mRNA levels of inflammatory genes, such as inducible NO synthase (iNOS) and tumor necrosis factor (TNF)-${\alpha}$. Using luciferase reporter gene assays performed in HEK293 cells and immunoblotting analysis with nuclear protein fractions, we determined that MPP reduced the transcriptional activation of nuclear factor (NF)-${\kappa}B$. Furthermore, the activation of a series of upstream signals for NF-${\kappa}B$ activation, composed of Src, Syk, Akt, and $I{\kappa}B{\alpha}$, were also blocked by this compound. It was confirmed that MPP was able to suppress autophosphorylation of overexpressed Src and Syk in HEK293 cells. Therefore, these results suggest that MPP can function as an anti-inflammatory drug with NF-${\kappa}B$ inhibitory properties via the suppression of Src and Syk.

• The Korean Journal of Physiology and Pharmacology
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• v.22 no.4
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• pp.457-465
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• 2018

The expression of BCL-2 interacting cell death suppressor (BIS), an anti-stress or anti-apoptotic protein, has been shown to be regulated at the transcriptional level by heat shock factor 1 (HSF1) upon various stresses. Recently, HSF1 was also shown to bind to BIS, but the significance of these protein-protein interactions on HSF1 activity has not been fully defined. In the present study, we observed that complete depletion of BIS using a CRISPR/Cas9 system in A549 non-small cell lung cancer did not affect the induction of heat shock protein (HSP) 70 and HSP27 mRNAs under various stress conditions such as heat shock, proteotoxic stress, and oxidative stress. The lack of a functional association of BIS with HSF1 activity was also demonstrated by transient downregulation of BIS by siRNA in A549 and U87 glioblastoma cells. Endogenous BIS mRNA levels were significantly suppressed in BIS knockout (KO) A549 cells compared to BIS wild type (WT) A549 cells at the constitutive and inducible levels. The promoter activities of BIS and HSP70 as well as the degradation rate of BIS mRNA were not influenced by depletion of BIS. In addition, the expression levels of the mutant BIS construct, in which 14 bp were deleted as in BIS-KO A549 cells, were not different from those of the WT BIS construct, indicating that mRNA stability was not the mechanism for autoregulation of BIS. Our results suggested that BIS was not required for HSF1 activity, but was required for its own expression, which involved an HSF1-independent pathway.

• Molecules and Cells
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• v.22 no.2
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• pp.163-167
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• 2006

Histone deacetylase (HDAC) activity is commonly associated with transcriptional repression. However, there is also evidence for a function in transcriptional activation. Previous studies have demonstrated a fundamental role of deacetylase activity in $IFN{\alpha}$-responsive gene transcription. In the case of type II IFN ($IFN{\gamma}$) results are controversial: some genes require HDAC activity, while transcription of others is repressed by HDAC. To investigate the effect of HDAC on transcription of an $IFN{\gamma}$-activated gene, real-time PCR was used to measure CXCL10 mRNA in Hela cells stimulated with $IFN{\gamma}$ in the presence or absence of the HDAC inhibitor TSA. Chromatin imunoprecipitation combined with real-time PCR was used to check acetylation of histone H4 and recruitment of the STAT1 complex to the ISRE locus of the CXCL10 gene. Activation of CXCL10 transcription in response to $IFN{\gamma}$ was paralleled by a decrease in histone H4 acetylation and an increase in recruitment of the STAT1 complex to the CXCL10 ISRE locus. The transcription of CXCL10 and histone H4 deacetylation were blocked by TSA, but the latter had no obvious affect on recruitment of the STAT1 complex. Our data indicate that $IFN{\gamma}$ and STAT-dependent gene transcription requires the participation of HDAC, as does the $IFN{\alpha}$-STAT pathway.

• Proceedings of the Korea Environmental Mutagen Society Conference
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• 2002.05a
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• pp.25-35
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• 2002

Transcription factor Nrf2 is essential for the antioxidant responsive element (ARE)-mediated induction of phase II detoxifying and oxidative stress enzyme genes. Detailed analysis of differential Nrf2 activity displayed in transfected cell lines ultimately led to the identification of a new protein, which we named Keap1, that suppresses Nrf2 transcriptional activity by specific binding to its evolutionarily conserved amino-terminal regulatory domain. The closest homolog of Keap1 is a Drosophila actin-binding protein called Kelch, implying that Keap1 might be a Nrf2 cytoplasmic effector. We then showed that electrophilic agents antagonize Keap1 inhibition of Nrf2 activity in vivo, allowing Nrf2 to traverse from the cytoplasm to the nucleus and potentiate the ARE response. We postulate that Keap1 and Nrf2 constitute a crucial cellular sensor for oxidative stress, and together mediate a key step in the signaling pathway that leads to transcriptional activation by this novel Nrf2 nuclear shuttling mechanism. The activation of Nrf2 leads in turn to the induction of phase II enzyme and antioxidative stress genes in response to electrophiles and reactive oxygen species.

• Microbiology and Biotechnology Letters
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• v.49 no.3
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• pp.305-315
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• 2021

The cotton leafworm, Spodoptera littoralis, is a major pest in Egypt and many countries worldwide, and causes heavy economic losses. As a result, management measures to control the spread of the worm are required. S. littoralis nucleopolyhedrovirus (SpliNPV) is one of the most promising bioagents for the efficient control of insect pests. In this study, a chitinase gene (chitA) of a 1.8 kb DNA fragment was cloned and fully characterized from SpliNPV-EG1, an Egyptian isolate. A sequence of 601 amino acids was deduced when the gene was completely sequenced with a predicted molecular mass of 67 kDa for the preprotein. Transcriptional analyses using reverse transcription polymerase chain reaction (RT-PCR) revealed that chitA transcripts were detected first at 12 h post infection (hpi) and remained detectable until 168 hpi, suggesting their transcriptional regulation from a putative late promoter motif. In addition, quantitative analysis using quantitative RT-PCR showed a steady increase of 7.86-fold at 12 hpi in chitA transcription levels, which increased up to 71.4-fold at 120 hpi. An approximately 50 kDa protein fragment with chitinolytic activity was purified from ChitA-induced bacterial culture and detected by western blotting with an anti-recombinant SpliNPV chitinase antibody. Moreover, purification of the expressed ChitA recombinant protein showed in vitro growth inhibition of two different fungi species, Fusarium solani and F. oxysporum, confirming that the enzyme assembly and activity was correct. The results supported the potential role and application of the SpliNPV-ChitA protein as a synergistic agent in agricultural fungal and pest control programs.