The palliative effects of folic acid on retinal microvessels in diabetic retinopathy via regulating the metabolism of DNA methylation and hydroxymethylation.

Diabetic retinopathy (DR) is one of the severe microvascular complications of diabetes. The protective effects of FA on retinal vascular endothelial cells against high glucose levels involve in multiple aspects in DR; however, the underlying mechanism is not fully elucidated. In present study, we investigated the transcriptome as well as genome-wide DNA methylation and hydroxymethylation signature in human retinal microvascular endothelial ACBRI 181 cells cultured within high glucose (HG) medium supplemented with or without FA by RNA-seq, MeDIP-seq, and hMeDIP-seq. Total 3308 differential expressed genes (DEGs) were involved in multiple biological processes and molecular functions containing angiogenesis, inflammation, S-adenosyl methionine metabolism, and hypoxia response. Moreover, the global DNA methylation and hydroxymethylation in ACBRI 181 cells with FA treatment were both compromised compared to HG. Combined with transcriptome data, four subclusters of DEGs with hyper- or hypomethylated promoters were further verified. Unexpectedly, promoters of these 487 genes all displayed a pattern of increased DNA hydroxymethylation. Furthermore, hyperglycemia rat model was established and administered with FA. The DNA methylation and hydroxymethylation changes of selected target genes COL1A1, ITGA7, MMP-14, and VEGFB confirmed by MeDIP-qPCR were consistent with the results in human ACBRI 181 cells. Finally, the presence of activated DNMT1 and TET2 induced by FA was determined in ACBRI 181 cells and hyperglycemia rat. Taken together, this research provided a resource of expression and epigenetic profiles in retinal microvascular endothelial cell, emphasizing a pharmacological mechanism of FA on DNA methylation and hydroxymethylation regulation in retinal microvessel cells of DR.

Screening of small molecules attenuating biofilm formation of Acinetobacter baumannii by inhibition of ompA promoter activity.

Anti-virulence therapeutic strategies are promising alternatives against drug-resistant pathogens. Outer membrane protein A (OmpA) plays a versatile role in the pathogenesis and antimicrobial resistance of Acinetobacter baumannii. Therefore, OmpA is an innovative target for anti-virulence therapy against A. baumannii. This study aimed to develop a high-throughput screening (HTS) system to discover small molecules inhibiting the ompA promoter activity of A. baumannii and screen chemical compounds using the bacterial growth-based HTS system. The ompA promoter and open reading frame of nptI fusion plasmids that controlled the expression of nptI encoding resistance to kanamycin by the ompA promoter were constructed and then transformed into A. baumannii ATCC 17978. This reporter strain was applied to screen small molecules inhibiting the ompA promoter activity in a chemical library. Of the 7,520 chemical compounds, 15 exhibited ≥ 70% growth inhibition of the report strain cultured in media containing kanamycin. Three compounds inhibited the expression of ompA and OmpA in the outer membrane of A. baumannii ATCC 17978, which subsequently reduced biofilm formation. In conclusion, our reporter strain is useful for large-scale screening of small molecules inhibiting the ompA expression in A. baumannii. Hit compounds identified by the HTS system are promising scaffolds to develop novel therapeutics against A. baumannii.

Discovery of an Orally Efficacious MYC Inhibitor for Liver Cancer Using a GNMT-Based High-Throughput Screening System and Structure-Activity Relationship Analysis.

Glycine-N-methyl transferase (GNMT) downregulation results in spontaneous hepatocellular carcinoma (HCC). Overexpression of GNMT inhibits the proliferation of liver cancer cell lines and prevents carcinogen-induced HCC, suggesting that GNMT induction is a potential approach for anti-HCC therapy. Herein, we used Huh7 GNMT promoter-driven screening to identify a GNMT inducer. Compound K78 was identified and validated for its induction of GNMT and inhibition of Huh7 cell growth. Subsequently, we employed structure-activity relationship analysis and found a potent GNMT inducer, K117. K117 inhibited Huh7 cell growth in vitro and xenograft in vivo. Oral administration of a dosage of K117 at 10 mpk (milligrams per kilogram) can inhibit Huh7 xenograft in a manner equivalent to the effect of sorafenib at a dosage of 25 mpk. A mechanistic study revealed that K117 is an MYC inhibitor. Ectopic expression of MYC using CMV promoter blocked K117-mediated MYC inhibition and GNMT induction. Overall, K117 is a potential lead compound for HCC- and MYC-dependent cancers.

Vitamin D decreases silencer methylation to downregulate renin gene expression.

Renin, encoded by REN, is an essential enzyme in the renin-angiotensin aldosterone system (RAAS) which is responsible for the maintenance of blood pressure homeostasis. Transcriptional regulation of REN has been linked to enhancer-promoter crosstalk, cAMP response element-binding protein (CREB), the active metabolite of vitamin D, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), and a less well-characterized intronic silencer element. We hypothesized that in addition to these, differential DNA methylation is linked to REN expression and influenced by 1,25(OH)2D3. REN expressing cells (HEK293) were used to elucidate the effect of 1,25(OH)2D3 on REN methylation and expression as quantified by methylation-sensitive qPCR and RT-qPCR, respectively. In vitro 1,25(OH)2D3 supplementation (10 nM) induced significant hypomethylation of the REN silencer (P < 0.050), which was linked to a significant reduction in REN expression (P < 0.010) but had no effect on enhancer methylation. In addition, 1,25(OH)2D3 increased VDR (P < 0.05), as well as TET1 (P < 0.05) expression, suggesting an association between 1,25(OH)2D3 and DNA methylation. Thus, it appears that the silencer element, which is controlled by DNA methylation and influenced by 1,25(OH)2D3, plays an essential role in regulating REN expression.

KDM6A promotes imatinib resistance through YY1-mediated transcriptional upregulation of TRKA independently of its demethylase activity in chronic myelogenous leukemia.

Rationale: Despite landmark therapy of chronic myelogenous leukemia (CML) with tyrosine kinase inhibitors (TKIs), drug resistance remains problematic. Cancer pathogenesis involves epigenetic dysregulation and in particular, histone lysine demethylases (KDMs) have been implicated in TKI resistance. We sought to identify KDMs with altered expression in CML and define their contribution to imatinib resistance. Methods: Bioinformatics screening compared KDM expression in CML versus normal bone marrow with shRNA knockdown and flow cytometry used to measure effects on imatinib-induced apoptosis in K562 cells. Transcriptomic analyses were performed against KDM6A CRISPR knockout/shRNA knockdown K562 cells along with gene rescue experiments using wildtype and mutant demethylase-dead KDM6A constructs. Co-immunoprecipitation, luciferase reporter and ChIP were employed to elucidate mechanisms of KDM6A-dependent resistance. Results: Amongst five KDMs upregulated in CML, only KDM6A depletion sensitized CML cells to imatinib-induced apoptosis. Re-introduction of demethylase-dead KDM6A as well as wild-type KDM6A restored imatinib resistance. RNA-seq identified NTRK1 gene downregulation after depletion of KDM6A. Moreover, NTRK1 expression positively correlated with KDM6A in a subset of clinical CML samples and KDM6A knockdown in fresh CML isolates decreased NTRK1 encoded protein (TRKA) expression. Mechanistically, KDM6A was recruited to the NTRK1 promoter by the transcription factor YY1 with subsequent TRKA upregulation activating down-stream survival pathways to invoke imatinib resistance. Conclusion: Contrary to its reported role as a tumor suppressor and independent of its demethylase function, KDM6A promotes imatinib-resistance in CML cells. The identification of the KDM6A/YY1/TRKA axis as a novel imatinib-resistance mechanism represents an unexplored avenue to overcome TKI resistance in CML.

Differential Methylation Pattern of Schizophrenia Candidate Genes in Tetrahydrocannabinol-Consuming Treatment-Resistant Schizophrenic Patients Compared to Non-Consumer Patients and Healthy Controls.

BACKGROUND: Patients suffering from schizophrenic psychosis show reduced synaptic connectivity compared to healthy individuals. Furthermore, the use of cannabis often precedes the onset of schizophrenic psychosis. Therefore, we investigated whether consumption of cannabis has an impact on the methylation pattern of schizophrenia candidate genes concerned with the development and preservation of synapses and synaptic function. METHODS: Fifty blood samples of outpatients affected by treatment-resistant schizophrenic psychosis were collected in the outpatient department of Ch Ste Anne/INSERM (Paris, France). Extracted DNA was sent to the LMN/MHH (Hanover, Germany) where DNA samples were bisulfite converted. The methylation patterns of the promoter region of neuregulin 1 (NRG1), neurexin (NRXN1), disrupted in schizophrenia 1 (DISC1), and microtubule-associated-protein tau (MAPT) were then analysed by sequencing according to Sanger. RESULTS: In NRXN1 the group of non-consumer patients showed a methylation rate slightly lower than controls. In patients with preliminary use of tetrahydrocannabinol (THC) the NRXN1 promoter turned out to be methylated almost two times higher than in non-consumer patients. In MAPT, non-consumer patients showed a significant lower mean methylation rate in comparison to controls. In THC-consuming patients the difference compared with controls became less. NRG1 and DISC1 showed no significant differences between groups, whereas DISC1 appeared to be not methylated at all. CONCLUSION: In MAPT and NRXN1 mean methylation rates were lower in non-consumer patients compared with controls, which seems to be a compensatory mechanism. With consumption of THC, mean methylation rates were increased: in the case of MAPT compared with controls, and in NRXN1 even significantly beyond that. Methylation of NRG1 and DISC1 seems not to be affected by the psychiatric disorder or by consumption of THC.

A G-quadruplex nanoswitch in the SGK1 promoter regulates isoform expression by K+/Na+ balance and resveratrol binding.

BACKGROUND: High sodium intake can up-regulate the level of renal serum- and glucocorticoid-inducible kinase-1 (SGK1), which plays a pivotal role in controlling blood pressure via activation of the epithelial sodium channel (ENaC), which can lead to salt-sensitive hypertension. Increased potassium intake, or a vegetarian diet, counteracts salt-sensitive hypertension, but the underlying mechanisms are not fully understood. METHODS: Bioinformatics and molecular modeling were used to identify G-quadruplex (G4) and their conformations in the SGK1 promoter. CD spectra and UV melting dynamics were measured to study the stability of G4 as influenced by potassium/sodium balance and resveratrol. RT-PCR and Western blot were employed to study the effects of potassium and resveratrol on the SGK1 isoform expression. RESULTS: The SGK1 gene encodes a G4 structure in the proximal upstream of promoter-2; the G4 structure is stabilized by potassium or resveratrol, but destabilized by sodium. Super-physiological levels of sodium stimulate the transcription of all SGK1 isoforms, whereas resveratrol or potassium supplementation inhibits the transcription of iso-2 and iso-3, but not iso-1. CONCLUSIONS: Stabilizing the G4 by potassium or resveratrol induces alternative promoter usage and/or pre-mRNA splicing in the transcription of SGK1. GENERAL SIGNIFICANCE: Potassium/sodium ion balance or resveratrol binding can act to regulate G4 molecular switches for controlling SGK1 gene expression, thereby presenting a new avenue for drug development.

Anti-proliferative activity of A. Oxyphylla and its bioactive constituent nootkatone in colorectal cancer cells.

BACKGROUND: A. oxyphylla extract is known to possess a wide range of pharmacological activites. However, the molecular mechanism of A. oxyphylla and its bioactive compound nootkatone in colorectal cancer is unknown. METHODS: Our study aims to examine the role of A. oxyphylla and its bioactive compound nootkatone, in tumor suppression using several in vitro assays. RESULTS: Both A. oxyphylla extract and nootkatone exhibited antiproliferative activity in colorectal cancer cells. A. oxyphylla displayed antioxidant activity in colorectal cancer cells, likely mediated via induction of HO-1. Furthermore, expression of pro-apoptotic protein NAG-1 and cell proliferative protein cyclin D1 were increased and decreased respectively in the presence of A. oxyphylla. When examined for anticancer activity, nootkatone treatment resulted in the reduction of colony and spheroid formation. Correspondingly, nootkatone also led to increased NAG-1 expression and decreased cyclin D1 expression. The mechanism by which nootkatone suppresses cyclin D1 involves protein level regulation, whereas nootkatone increases NAG-1 expression at the transcriptional level. In addition to having PPARγ binding activity, nootkatone also increases EGR-1 expression which ultimately results in enhanced NAG-1 promoter activity. CONCLUSION: In summary, our findings suggest that nootkatone is an anti-tumorigenic compound harboring antiproliferative and pro-apoptotic activity.

Maternal betaine supplementation decreases hepatic cholesterol deposition in chicken offspring with epigenetic modulation of SREBP2 and CYP7A1 genes.

Maternal betaine was reported to regulate offspring hepatic cholesterol metabolism in mammals. However, it is unclear whether and how feeding betaine to laying hens affects hepatic cholesterol metabolism in offspring chickens. Rugao yellow-feathered laying hens (n = 120) were fed basal or 0.5% betaine-supplemented diet for 28 D before the eggs were collected for incubation. Maternal betaine significantly decreased the hepatic cholesterol content (P < 0.05) in offspring chickens. Accordingly, the cholesterol biosynthetic enzymes, sterol regulator element-binding protein 2 (SREBP2) and 3-hydroxy-3-methylglutaryl coenzyme A reductase, were decreased, while cholesterol-7alpha-hydroxylase (CYP7A1), which converts cholesterol to bile acids, was increased at both mRNA and protein levels in betaine-treated offspring chickens. Hepatic mRNA and protein expression of low-density lipoprotein receptor was significantly (P < 0.05) increased, while the mRNA abundance of cholesterol acyltransferase 1 (ACAT1) that mediates cholesterol esterification was significantly (P < 0.05) decreased in the betaine group. Meanwhile, hepatic protein contents of DNA methyltransferases 1 and betaine homocysteine methyltransferase were increased (P < 0.05), which was associated with modifications of CpG methylation on affected cholesterol metabolic genes. Furthermore, the level of CpG methylation on gene promoters was increased (P < 0.05) for sterol regulator element-binding protein 2 and abundance of cholesterol acyltransferase 1 yet decreased (P < 0.05) for cholesterol-7alpha-hydroxylase. These results indicate that maternal betaine supplementation significantly decreases hepatic cholesterol deposition through epigenetic regulation of cholesterol metabolic genes in offspring juvenile chickens.

Vitamin B12 is neuroprotective in experimental pneumococcal meningitis through modulation of hippocampal DNA methylation.

BACKGROUND: Bacterial meningitis (BM) causes apoptotic damage to the hippocampus and homocysteine (Hcy) accumulation to neurotoxic levels in the cerebrospinal fluid of children. The Hcy pathway controls bioavailability of methyl, and its homeostasis can be modulated by vitamin B12, a cofactor of the methionine synthase enzyme. Herein, the neuroprotective potential and the underlying mode of action of vitamin B12 adjuvant therapy were assessed in an infant rat model of BM. METHODS: Eleven-day old rats were intracysternally infected with Streptococcus pneumoniae serotype 3, or saline, treated with B12 or placebo, and, 24 h after infection, their hippocampi were analyzed for apoptosis in the dentate gyrus, sulfur amino acids content, global DNA methylation, transcription, and proximal promoter methylation of candidate genes. Differences between groups were compared using 2-way ANOVA followed by Bonferroni post hoc test. Correlations were tested with Spearman's test. RESULTS: B12 attenuated BM-induced hippocampal apoptosis in a Hcy-dependent manner (r = 0.80, P < 0.05). BM caused global DNA hypomethylation; however, B12 restored this parameter. Accordingly, B12 increased the methylation capacity of hippocampal cells from infected animals, as inferred from the ratio S-adenosylmethionine (SAM):S-adenosylhomocysteine (SAH) in infected animals. BM upregulated selected pro-inflammatory genes, and this effect was counteracted by B12, which also increased methylation of CpGs at the promoter of Ccl3 of infected animals. CONCLUSION: Hcy is likely to play a central role in hippocampal damage in the infant rat model of BM, and B12 shows an anti-inflammatory and neuroprotective action through methyl-dependent epigenetic mechanisms.

Transgenerational Inheritance of Betaine-Induced Epigenetic Alterations in Estrogen-Responsive IGF-2/IGFBP2 Genes in Rat Hippocampus.

SCOPE: Betaine serves as a methyl donor for DNA methylation. Here, the effects of betaine on hippocampal expression of neurogenesis genes and their DNA methylation status across three generations are investigated. METHODS AND RESULTS: Pregnant rats (F0) are fed control and betaine-supplemented diets throughout gestation and lactation. Female F1 and F2 offspring at weaning, together with the F0 dams, are used in the study. Hippocampal expression of aromatase, estrogen receptor α, and estrogen-related receptor ß is downregulated in F1, together with the estrogen-responsive insulin-like growth factor 2/insulin-like growth factor binding protein 2 (IGF-2/IGFBP2) genes. However, all these genes are upregulated in F2, which follows the same pattern of F0. In agreement with changes in mRNA expression, the imprinting control region (ICR) of IGF-2 gene is hypomethylated in F1 but hypermethylated in F2 and F0. In contrast, the promoter DNA methylation status of all the affected genes is hypermethylated in F1 but hypomethylated in F2 and F0. Methyl transfer enzymes, such as betaine homocysteine methyltransferase and DNA methyltransferase 1, follow the same pattern of transgenerational inheritance. CONCLUSION: These results indicate that betaine exerts a transgenerational effect on hippocampal expression of estrogen-responsive genes in rat offspring, which is associated with corresponding alterations in DNA methylation on ICR of IGF-2 gene and the promoter of affected genes.

Reduction of nuclear Y654-p-ß-catenin expression through SH3GL2-meditated downregulation of EGFR in chemotolerance TNBC: Clinical and prognostic importance.

Triple negative breast cancer (TNBC) originates from a less differentiated ductal cell of breast, which is less sensitive to chemotherapy. The chemotolerance mechanism of TNBC has not yet been studied in detail. For this reason, molecular profiles (expression/genetic/epigenetic) of Y654-p-ß-catenin (active) and its kinase epidermal growth factor receptor (EGFR) along with SH3GL2 (regulator of EGFR homeostasis) were compared between neoadjuvant chemotherapy treated (NACT) and pretherapeutic TNBC samples. Reduced nuclear expression of Y654-p-ß-catenin protein with low proliferation index and CD44 prevalence showed concordance with reduced expression of EGFR/Y1045-p-EGFR proteins in the NACT samples than the pretherapeutic TNBC samples. Infrequent messenger RNA expression, gene amplification (10-32.5%), and mutation (1%) of EGFR were seen in the TNBC samples irrespective of therapy, suggesting the importance of EGFR protein stabilization in this tumor. The upregulation of SH3GL2 seen in the NACT samples in contrast to the pretherapeutic samples might be due to its promoter hypomethylation, as seen in the quantitative methylation assay. A similar trend of upregulation of SH3GL2 and downregulation of EGFR, Y1045-p-EGFR, Y654-p-ß-catenin were seen in the MDA-MB-231 cell line using antharacycline antitumor drugs (doxorubicin/nogalamycin). The NACT patients with reduced expression of Y654-p-ß-catenin and/or EGFR and high expression of SH3GL2 showed comparatively better prognosis than the pretherapeutic patients. Thus, our study showed that reduced nuclear expression of Y654-p-ß-catenin in NACT samples due to downregulation of EGFR protein through promoter hypomethylation-mediated upregulation of SH3GL2, resulting in low proliferation index/CD44 prevalence with better prognosis of the NACT patients, might have an important role in the chemotolerance of TNBC.

Hyperbaric oxygen activates visfatin expression and angiogenesis via angiotensin II and JNK pathway in hypoxic human coronary artery endothelial cells.

Visfatin is an adipocytokine with important roles in endothelial angiogenesis. Hyperbaric oxygen (HBO) has been widely used to treat various medical illness with enhanced angiogenesis. The molecular effects of HBO on visfatin under hypoxia are poorly understood. This study aimed to investigate the effect of HBO on visfatin in hypoxic human coronary arterial endothelial cells (HCAECs). HCAECs under chemical hypoxia (antimycin A, 0.01 mmol/L) were exposed to HBO (2.5 atmosphere absolute; ATA) for 2-4 hours. Western blot, real-time polymerase chain reaction, electrophoretic mobility shift assay, luciferase promoter activity, migration and tube formation assay, and in vitro glucose uptake were measured. Visfatin protein expression increased in hypoxic HCAECs with earlier angiotensin II (AngII) secretion and c-Jun N-terminal kinase (JNK) phosphorylation, which could be effectively suppressed by the JNK inhibitor (SP600125), AngII antibody or AngII receptor blocker (losartan). In hypoxic HCAECs, HBO further induced earlier expression of visfatin and AngII. Hypoxia significantly increased DNA-protein binding activity of hypoxia-inducible factor-1α (HIF-1α) and visfatin. Hypoxia, hypoxia with HBO and exogenous addition of AngII also increased promoter transcription to visfatin; SP600125 and losartan blocked this activity. In HCAECs, glucose uptake, migration and tube formation were increased in the presence of hypoxia with HBO, but were inhibited by visfatin small interfering RNA, SP600125 and losartan. In conclusion, HBO activates visfatin expression and angiogenesis in hypoxic HCAECs, an effect mediated by AngII, mainly through the JNK pathway.

CPF impedes cell cycle re-entry of quiescent lung cancer cells through transcriptional suppression of FACT and c-MYC.

Blockade of cell cycle re-entry in quiescent cancer cells is a strategy to prevent cancer progression and recurrence. We investigated the action and mode of action of CPF mixture (Coptis chinensis, Pinellia ternata and Fructus trichosanthis) in impeding a proliferative switch in quiescent lung cancer cells. The results indicated that CPF impeded cell cycle re-entry in quiescent lung cancer cells by reduction of FACT and c-MYC mRNA and protein levels, with concomitant decrease in H3K4 tri-methylation and RNA polymerase II occupancy at FACT and c-MYC promoter regions. Animals implanted with quiescent cancer cells that had been exposed to CPF had reduced tumour volume/weight. Thus, CPF suppresses proliferative switching through transcriptional suppression of FACT and the c-MYC, providing a new insight into therapeutic target and intervention method in impeding cancer recurrence.

Grandmaternal betaine supplementation enhances hepatic IGF2 expression in F2 rat offspring through modification of promoter DNA methylation.

BACKGROUND: We reported previously that maternal betaine promotes hepatic insulin-like growth factor (IGF2) expression in F1 offspring rats through hypermethylation of the IGF2/H19 imprinting control region (ICR). It remains unknown whether this acquired trait can be transmitted to the F2 generation. This study aimed to determine whether dietary betaine supplementation to grand dams affects the hepatic IGF2 expression in F2 rat offspring and how it is related to alterations in DNA methylation. F2 rat offspring derived from grand dams fed basal or betaine-supplemented diet (10 g kg-1 ) were examined at weaning. Serum IGF2 concentration was measured with enzyme-linked immunosorbent assay (ELISA). Hepatic expression of IGF2, together with other proliferation and apoptosis markers, was determined by using quantitative polymerase chain reaction (qPCR), western blot, and immunohistochemistry. The methylation status of the IGF2/H19 ICR and the promoters of IGF2 gene were detected by methylated DNA immunoprecipitation quantitative polymerase chain reaction (MeDIP-qPCR). RESULTS: The maternal betaine-induced up-regulation of hepatic IGF2 expression in F1 rat offspring was transmitted to the F2 generation. The F2 rats from the betaine group demonstrated enhanced hepatic IGF2 expression at both mRNA and protein levels, in association with higher serum IGF2 concentration. No alterations were observed in the ICR methylation of the IGF2/H19 locus, and hypomethylation was detected in promoters of IGF2 gene in betaine group. CONCLUSION: These results indicate that maternal betaine enhances hepatic IGF2 expression in F2 rat offspring through modification of DNA methylation on IGF2 promoters. © 2019 Society of Chemical Industry.

The serum amyloid A3 promoter-driven luciferase reporter mice is a valuable tool to image early renal fibrosis development and shows the therapeutic effect of glucosyl-hesperidin treatment.

Tubulointerstitial fibrosis is a progressive process affecting the kidneys, causing renal failure that can be life-threatening. Thus, renal fibrosis has become a serious concern in the ageing population; however, fibrotic development cannot be diagnosed early and assessed noninvasively in both patients and experimental animal models. Here, we found that serum amyloid A3 (Saa3) expression is a potent indicator of early renal fibrosis; we also established in vivo Saa3/C/EBPß-promoter bioluminescence imaging as a sensitive and specific tool for early detection and visualization of tubulointerstitial fibrosis. Saa3 promoter activity is specifically upregulated in parallel with tumor necrosis factor α (TNF-α) and fibrotic marker collagen I in injured kidneys. C/EBPß, upregulated in injured kidneys and expressed in tubular epithelial cells, is essential for the increased Saa3 promoter activity in response to TNF-α, suggesting that C/EBPß plays a crucial role in renal fibrosis development. Our model successfully enabled visualization of the suppressive effects of a citrus flavonoid derivative, glucosyl-hesperidin, on inflammation and fibrosis in kidney disease, indicating that this model could be widely used in exploring therapeutic agents for fibrotic diseases.

Isoform specific editing of the coxsackievirus and adenovirus receptor.

The Coxsackievirus and adenovirus receptor (CAR) is both a viral receptor and cell adhesion protein. CAR has two transmembrane isoforms that localize distinctly in polarized epithelial cells. Whereas the seven exon-encoded isoform (CAREx7) exhibits basolateral localization, the eight exon-encoded isoform (CAREx8) can localize to the apical epithelial surface where it can mediate luminal adenovirus infection. To further understand the distinct biological functions of these two isoforms, CRISPR/Cas9 genomic editing was used to specifically delete the eighth exon of the CXADR gene in a Madine Darby Canine Kidney (MDCK) cell line with a stably integrated lentiviral doxycycline-inducible CAREx8 cDNA. The gene-edited clone demonstrated a significant reduction in adenovirus susceptibility when both partially and fully polarized, and doxycycline-induction of CAREx8 restored sensitivity to adenovirus. These data reinforce the importance of CAREx8 in apical adenovirus infection and provide a new model cell line to probe isoform specific biological functions of CAR.

Kurarinone from Sophora Flavescens Roots Triggers ATF4 Activation and Cytostatic Effects Through PERK Phosphorylation.

In response to cellular stresses, activating transcriptional factor 4 (ATF4) regulates the expression of both stress-relieving genes and apoptosis-inducing genes, eliciting cell fate determination. Since pharmacological activation of ATF4 exerts potent anti-tumor effects, modulators of ATF4 activation may have potential in cancer therapy. We herein attempted to identify small molecules that activate ATF4. A cell-based screening to monitor TRB3 promoter activation was performed using crude drugs used in traditional Japanese Kampo medicine. We found that an extract from Sophora flavescens roots exhibited potent TRB3 promoter activation. The activity-guided fractionation revealed that kurarinone was identified as the active ingredient. Intriguingly, ATF4 activation in response to kurarinone required PKR-like endoplasmic reticulum kinase (PERK). Moreover, kurarinone induced the cyclin-dependent kinase inhibitor p21 as well as cytostasis in cancer cells. Importantly, the cytostatic effect of kurarinone was reduced by pharmacological inhibition of PERK. These results indicate that kurarinone triggers ATF4 activation through PERK and exerts cytostatic effects on cancer cells. Taken together, our results suggest that modulation of the PERK-ATF4 pathway with kurarinone has potential as a cancer treatment.

Assessment of adipogenic, antioxidant, and anti-inflammatory properties of whole and whey bovine colostrum.

Bovine colostrum (BC) has been used for nutraceutical purposes for animals and humans. Bovine colostrum is a complex heterogeneous product and its antimicrobial activity, antioxidant potential, and growth factors can vary depending on age and species of the cow as well as their environment. Bovine colostrum preparation in skimmed or whey fractions can also alter properties of BC. Our goal was to compare cumulative anti-inflammatory, antioxidant, and adipogenic properties of natural (whole) versus whey BC. We compared properties of whole and whey BC in 3T3-L1 preadipocytes permanently transfected with reporters responding to changes in inflammatory (NfκbRE/green fluorescent protein), anti-inflammatory (Nrf2/YFP), and adipogenic (Fabp4/cyan fluorescent protein) status in cells. Interleukin-6 secretion in these cells was measured by ELISA. Whole and whey BC induce IL-6 secretion from 3T3-L1 fibroblasts; however, whey preparation stimulated less IL-6 secretion. Cumulative inflammatory nuclear factor (NF)κB activation in the presence of lipopolysaccharide was reduced by both whole (-27%) and whey BC (-22%) compared with lipopolysaccharide-treated cells (100%). Treatment with whole BC was more effective in the reduction of NFκB activation compared with whey BC and occurred in a dose-dependent manner. In consonance with decreased NFκB activation, the Nrf2 promoter activity was also reduced in response to whole (-27%) and whey (-13%) treatments compared with nontreated cells (100%). Whole and whey BC suppressed adipogenesis, measured as induction of Fabp4, by -27 and -13%, respectively, compared with nontreated 3T3-L1 fibroblasts (100%). Our results showed distinct differences in properties of whey and whole BC that could be used to attain reduced adipogenic or cumulative inflammatory responses.

Tanshinone prevents alveolar bone loss in ovariectomized osteoporosis rats by up-regulating phosphoglycerate dehydrogenase.

Osteoporosis is manifested by reduced bone mass. Tanshinone has been shown to affect osteoclast differentiation, but its role in osteoporosis remains less clear. This study aimed to investigate the effects and molecular mechanisms of tanshinone on osteoporosis. Osteoporosis was induced by bilateral ovariectomy (OVX) in adult female rats treated with or without tanshinone. Trabecular bone structure was assessed by micro-computed tomography (micro-CT). Bone marrow stromal cells (BMSCs) were isolated for analysis of stemness and senescence. mRNA levels of age related genes were examined and the role of the gene that was upregulated by tanshinone treatment was suppressed to determine its involvement in tanshinone mediated effects. Finally, the mechanism underlying tanshinone induced gene upregulation was explored. We found that tanshinone treatment restored alveolar bone structure in OVX rats as well as the stemness and senescence status of BMSCs isolated from OVX rats. Tanshinone upregulated Phgdh mRNA levels and inhibition of phosphoglycerate dehydrogenase Phgdh, the protein encoded by the Phgdh gene, abolished the effects of tanshinone on BMSC stemness and senescence. Finally, we found that OVX lead to hypermethylation of the promoter region of Phgdh which was suppressed by tanshinone treatment. Our study shows that tanshinone potently suppress OVX induced osteoporosis and BMSC senescence through upregulation of PHGDH.