Transactivating Effect Research Articles

Functional characterization of HNF4A gene variants identify promoter and cell line specific transactivation effects.

Hepatocyte nuclear factor-4 alpha (HNF-4A) regulates genes with roles in glucose metabolism and β-cell development. Although pathogenic HNF4A variants are commonly associated with maturity-onset diabetes of the young (MODY1; HNF4A-MODY), rare phenotypes also include hyperinsulinemic hypoglycemia, renal Fanconi syndrome and liver disease. While the association of rare functionally damaging HNF1A variants with HNF1A-MODY and type 2 diabetes is well established owing to robust functional assays, the impact of HNF4A variants on HNF-4A transactivation in tissues including the liver and kidney is less known, due to lack of similar assays. Our aim was to investigate the functional effects of seven HNF4A variants, located in the HNF-4A DNA binding domain and associated with different clinical phenotypes, by various functional assays and cell lines (transactivation, DNA binding, protein expression, nuclear localization) and in silico protein structure analyses. Variants R85W, S87N and R89W demonstrated reduced DNA binding to the consensus HNF-4A binding elements in the HNF1A promoter (35, 13 and 9%, respectively) and the G6PC promoter (R85W ~10%). While reduced transactivation on the G6PC promoter in HepG2 cells was shown for S87N (33%), R89W (65%) and R136W (35%), increased transactivation by R85W and R85Q was confirmed using several combinations of target promoters and cell lines. R89W showed reduced nuclear levels. In silico analyses supported variant induced structural impact. Our study indicates that cell line specific functional investigations are important to better understand HNF4A-MODY genotype-phenotype correlations, as our data supports ACMG/AMP interpretations of loss-of-function variants and propose assay-specific HNF4A control variants for future functional investigations.

Transcriptional activation of Mink enteritis virus VP2 by the C-terminal of its NS1 protein.

Mink enteritis virus (MEV) NS1 is a multidomain and multifunctional protein containing origin binding, helicase, and transactivation domains. In particular, parvoviral NS1 proteins are transactivators of the viral capsid protein promoter although the manner by which they exert these transactivation effects remained unclear. In this study, the region of the transactivation domain of the NS1 C-terminal was found located at aa 557 ~ 668 and any deletion within this region reduced the transactivation activity. A dominant negative mutation of the 63 aa deletion in the C-terminal of NS1 protein resulted in loss of ability to activate P38 and VP2-5'UTR in a dual-luciferase reporter assay system, a VP2 protein expression system, and within the whole MEV genome, independent of downstream genes. Additionally, a full-length MEV clone deficient in its NS1 C-terminal failed to rescue the virus, possibly due to the loss of integrity of DNA sequences interacting with NS1 protein, and expression of VP2 was also inhibited even when normal NS1 protein was supplied in trans.

STRONG STAYGREEN inhibits DNA binding of PvNAP transcription factors during leaf senescence in switchgrass.

Fine tuning the progression of leaf senescence is important for plant fitness in nature, while the "staygreen" phenotype with delayed leaf senescence has been considered a valuable agronomic trait in crop genetic improvement. In this study, a switchgrass (Panicum virgatum L.) CCCH-type Zinc finger gene, Strong Staygreen (PvSSG), was characterized as a suppressor of leaf senescence as overexpression or suppression of the gene led to delayed or accelerated leaf senescence, respectively. Transcriptomic analysis marked that chlorophyll (Chl) catabolic pathway genes were involved in the PvSSG-regulated leaf senescence. PvSSG was identified as a nucleus-localized protein with no transcriptional activity. By yeast two-hybrid screening, we identified its interacting proteins, including a pair of paralogous transcription factors, PvNAP1/2 (NAC-LIKE, ACTIVATED BY AP3/PI). Overexpression of PvNAPs led to precocious leaf senescence at least partially by directly targeting and transactivating Chl catabolic genes to promote Chl degradation. PvSSG, through protein-protein interaction, repressed the DNA-binding efficiency of PvNAPs and alleviated its transactivating effect on downstream genes, thereby functioning as a "brake" in the progression of leaf senescence. Moreover, overexpression of PvSSG resulted in up to 47% higher biomass yield and improved biomass feedstock quality, reiterating the importance of leaf senescence regulation in the genetic improvement of switchgrass and other feedstock crops.

An Anthocyanin-Related Glutathione S-Transferase, MrGST1, Plays an Essential Role in Fruit Coloration in Chinese Bayberry (Morella rubra).

Chinese bayberry (Morella rubra) is a fruit tree economically important in China and accumulates abundant amounts of anthocyanins in fruit as it ripens. Owing to the fact that all anthocyanin containing fruit tissues in Chinese bayberry are edible and anthocyanins can provide various health benefits in human body, the mechanisms underpinning anthocyanin accumulation in this fruit are worthy of investigation. It has been known that in plants anthocyanins are synthesized in the cytoplasmic surface of the endoplasmic reticulum and subsequently transported into the vacuole for storage, and glutathione S-transferases (GSTs) have been verified to be involved in this process. But the characterization and functionalization of the GST counterpart in Chinese bayberry is not available. The GST anthocyanin transporter MrGST1 was discovered to be related with anthocyanin accumulation in fruit from distinct developmental stages of “Biqi,” a staple cultivar that accumulates over 1 mg/g anthocyanins in ripe fruit. The expression of MrGST1 was well associated with anthocyanin accumulation either in fruit collected at six developmental stages or in ripe fruit from 12 cultivars. MrGST1 was found to be responsible for the transport of anthocyanins but not proanthocyanidins when the Arabidopsis tt19 mutant was functionally complemented. Transient ectopic expression of MrGST1 in combination with MrMYB1.1 and MrbHLH1 dramatically boosted pigmentation in Nicotiana tabacum leaves in contrast to MrMYB1.1 and MrbHLH1. The promoter of MrGST1 comprised eight MYB binding sites (MBSs) according to cis-element analysis. Data from yeast one-hybrid assay and dual-luciferase tests demonstrated that MrMYB1.1 exerted considerable transactivation effect on the MrGST1 promoter by recognizing the MBS4, the fourth MBS from the ATG start site. Our results together provided molecular evidence for the contribution of MrGST1 in regulating anthocyanin accumulation in Chinese bayberry fruit.

Convergent and divergent regulations of ethylene and abscisic acid biosynthesis during persimmon fruit postharvest ripening

Phytohormones are signaling molecules produced by plants and play pivotal roles in various physiological and developmental processes. Ethylene and abscisic acid (ABA) are usually considered to have separate roles in regulating fruit ripening and co-regulation involving both hormones have rarely been investigated. Here, similar changes in ethylene and ABA were observed underlying postharvest persimmon ripening. High CO2 (95% CO2, 1% O2) and high CO2 + 1-methylcyclopropene (CO2 + 1-MCP) are two widely used commercial deastringency treatments for persimmon, which accumulate acetaldehyde by anaerobic metabolism to precipitate astringent soluble tannins. Unexpectedly, CO2 treatment triggered the accumulation of both ethylene and ABA. This could be inhibited by CO2 + 1-MCP, which also prevented CO2-driven rapid softening. Three differentially expressed genes, DkACS1, DkNCED1 + 3′ and DkNCED2 + 3′ involved in ethylene and ABA synthesis were identified, but none of the differentially expressed transcription factors tested showed any obvious transactivation effects on their gene promoters. RNA binding protein DkRBM24–1 was shown to regulate the transcription of DkACS1 and DkNCED2 + 3′ by binding to the 3′UTR region of their mRNA. Further analysis indicated that DkbHLH11 was able to activate the expression of DkNCED1 + 3′ after phosphorylation by protein kinase DkMAPKKK1. Overall, the present study indicated the ethylene and ABA production in ripening fruit have both shared (DkRBM24–1) and distinct (DkbHLH11) regulators.

A New Fungal Triterpene from the Fungus Aspergillus flavus Stimulates Glucose Uptake without Fat Accumulation.

Through activity-guided fractionation, a new triterpene (asperflagin, 1) was isolated as a PPAR-γ agonist from the jellyfish-derived fungus Aspergillus flavus. Asperflagin displayed selective and moderate transactivation effects on PPAR-γ in Ac2F rat liver cells. Based on further biological evaluation and molecular docking analysis, we postulated that asperflagin might function as a PPAR-γ partial agonist. This compound was calculated to display a typical PPAR-γ ligand–receptor interaction that is distinct from that of full agonistic antidiabetics such as rosiglitazone, and may retain the antidiabetic effect without accompanying weight gain. Weight gain and obesity are typical side effects of the PPAR-γ full agonist rosiglitazone, and lead to suboptimal outcomes in diabetic patients. Compared to rosiglitazone, asperflagin showed higher glucose uptake in HepG2 human liver cells at concentrations of 20 and 40 μM but induced markedly lower adipogenesis and lipid accumulation in 3T3-L1 preadipocytes. These results suggest that asperflagin may be utilized for further study on advanced antidiabetic leads.

Citrus heat shock transcription factor CitHsfA7-mediated citric acid degradation in response to heat stress.

Heat stress is a major abiotic stress for plants, which can generate a range of biochemical and genetic responses. In 'Ponkan' mandarin fruit, hot air treatment (HAT) accelerates the degradation of citric acid. However, the transcriptional regulatory mechanisms of citrate degradation in response to HAT remain to be elucidated. Here, 17 heat shock transcription factor sequences were isolated, and dual-luciferase assays were employed to investigate whether the encoded proteins that could trans-activate the promoters of key genes in the GABA shunt, involved in citrate metabolism. We identified four heat shock transcription factors (CitHsfA7, CitHsfA3, CitHsfA4b and CitHsfA8) that showed trans-activation effects on CitAco3, CitIDH3 and CitGAD4, respectively. Transient expression of the CitHsfs in citrus fruits indicated that CitHsfA7 was the only factor that resulted in a significant lowering of the citric acid content, and these results were confirmed by a virus-induced gene silencing system (VIGS). Sub-cellar localization showed that CitHsfA7 is located in the nucleus and is capable of binding directly to a putative HSE in the CitAco3 promoter and enhance its expression. We proposed that the induction of CitHsfA7 transcript level contributes to citric acid degradation in citrus fruit, via modulation of CitAco3 in response to HAT.

Regulation of the expression of human endogenous retroviruses: elements in fetal development and a possible role in the development of cancer and neurological diseases.

Human endogenous retroviruses (HERVs) are remnants of ancient retroviral germline infections. Most HERV sequences are silenced in somatic cells, but interest is emerging on the involvement of HERV derived transcripts and proteins in human physiology and disease. A HERV-W encoded protein, syncytin-1, has been co-opted into fetal physiology, where it plays a role in trophoblast formation. Altered HERV transcription and expression of HERV derived proteins are associated with various cancer types and neurological diseases such as multiple sclerosis (MS). The implication of HERVs as potential mediators of both health and disease suggests important roles of regulatory mechanisms and alterations of these in physiological and pathological processes. The regulation of HERV sequences is mediated by a wide variety of mechanisms, and the focus of this review is on selected aspects of these, including epigenetic mechanisms such as CpG methylation and histone modifications of the HP1-H3K9me axis, viral transactivation events, and regulatory perspectives of transient stimuli in the microenvironment. Increasing knowledge of the regulation of HERV sequences will not only contribute to the understanding of complex pathogeneses, but also may pinpoint potential targets for better diagnosis and treatment in complex diseases as MS.

AFP2 Coordinates the Activity of PIF7 for Thermomorphogenesis in Arabidopsis Seedlings

Ambient temperature induces the hypocotyl elongation of seedling, called as thermomorphogenesis. It has been reported that the bHLH transcriptional factor PIF7 acts as the critical component to modulate plant thermomorphogenesis, but the underlying mechanism remains elusive. The phytohormone abscisic acid (ABA) suppresses the hypocotyl elongation under high temperature (HT) stress. As the ABI5 binding protein, AFP2 acts as the negative factor to control ABA signaling. In this study, we first identified AFP2 as the interaction protein of PIF7 <i>in vitro</i> and <i>in vivo</i>. Phenotype analysis revealed that overexpressing <i>AFP2</i> reduced the hypocotyl elongation, while loss-of-function <i>afp2</i> mutant showed longer hypocotyl under HT. Consistently, overexpressing <i>AFP2</i> impaired the transactivation effect of PIF7 on auxin biosynthesis related genes <i>YUC8</i> and <i>IAA19</i>, which possibly resulted into the shorter hypocotyl in the transgenic line overexpressing <i>AFP2</i> or co-overexpressing <i>AFP2</i> and <i>PIF7</i>. Thus, these data suggest that AFP2 suppressed PIF7 activity to suppress hypocotyl elongation. Furthermore, we found that HT gradually induced the degradation of AFP2 that possibly released the inhibitory effect of AFP2 on PIF7, thus induced hypocotyl elongation under HT. Taken together, our result reveals the novel function of AFP2 in coordinating thermomorphogenesis through sophistically modulating PIF7 activity.

A novel role of kallikrein-related peptidase 8 in the pathogenesis of diabetic cardiac fibrosis.

Rationale: Among all the diabetic complications, diabetic cardiomyopathy, which is characterized by myocyte loss and myocardial fibrosis, is the leading cause of mortality and morbidity in diabetic patients. Tissue kallikrein-related peptidases (KLKs) are secreted serine proteases, that have distinct and overlapping roles in the pathogenesis of cardiovascular diseases. However, whether KLKs are involved in the development of diabetic cardiomyopathy remains unknown.The present study aimed to determine the role of a specific KLK in the initiation of endothelial-to-mesenchymal transition (EndMT) during the pathogenesis of diabetic cardiomyopathy.Methods and Results-By screening gene expression profiles of KLKs, it was found that KLK8 was highly induced in the myocardium of mice with streptozotocin-induced diabetes. KLK8 deficiency attenuated diabetic cardiac fibrosis, and rescued the impaired cardiac function in diabetic mice. Small interfering RNA (siRNA)-mediated KLK8 knockdown significantly attenuated high glucose-induced endothelial damage and EndMT in human coronary artery endothelial cells (HCAECs). Diabetes-induced endothelial injury and cardiac EndMT were significantly alleviated in KLK8-deficient mice. In addition, transgenic overexpression of KLK8 led to interstitial and perivascular cardiac fibrosis, endothelial injury and EndMT in the heart. Adenovirus-mediated overexpression of KLK8 (Ad-KLK8) resulted in increases in endothelial cell damage, permeability and transforming growth factor (TGF)-β1 release in HCAECs. KLK8 overexpression also induced EndMT in HCAECs, which was alleviated by a TGF-β1-neutralizing antibody. A specificity protein-1 (Sp-1) consensus site was identified in the human KLK8 promoter and was found to mediate the high glucose-induced KLK8 expression. Mechanistically, it was identified that the vascular endothelial (VE)-cadherin/plakoglobin complex may associate with KLK8 in HCAECs. KLK8 cleaved the VE-cadherin extracellular domain, thus promoting plakoglobin nuclear translocation. Plakoglobin was required for KLK8-induced EndMT by cooperating with p53. KLK8 overexpression led to plakoglobin-dependent association of p53 with hypoxia inducible factor (HIF)-1α, which further enhanced the transactivation effect of HIF-1α on the TGF-β1 promoter. KLK8 also induced the binding of p53 with Smad3, subsequently promoting pro-EndMT reprogramming via the TGF-β1/Smad signaling pathway in HCAECs. The in vitro and in vivo findings further demonstrated that high glucose may promote plakoglobin-dependent cooperation of p53 with HIF-1α and Smad3, subsequently increasing the expression of TGF-β1 and the pro-EndMT target genes of the TGF-β1/Smad signaling pathway in a KLK8-dependent manner.Conclusions: The present findings uncovered a novel pro-EndMT mechanism during the pathogenesis of diabetic cardiac fibrosis via the upregulation of KLK8, and may contribute to the development of future KLK8-based therapeutic strategies for diabetic cardiomyopathy.

Increase of the Intracellular Zinc Concentration Leads to an Activation and Internalisation of the Epidermal Growth Factor Receptor in A549 Cells.

(1) Background: Zinc is suggested to play a major role in epidermal growth factor (EGF)-induced cell regeneration and proliferation. To deepen the knowledge on the underlying mechanisms zinc’s effects on the epidermal growth factor receptor (EGFR) activation and its endocytosis was investigated in the alveolar carcinoma cell line A549. (2) Methods: An increase of intracellular zinc was generated by adding zinc extracellularly compared to the intracellular release of zinc from zinc-binding proteins by stimulation with a nitric oxide donor. Zinc-initiated EGFR phosphorylation was checked by Western blotting and receptor endocytosis assays were performed by using flow cytometry. (3) Results: Besides a dose-dependent EGFR phosphorylation, a dose- and time dependent significant receptor internalisation was initiated by both types of zinc increases. In addition, both increased intracellular zinc levels further promoted EGF-induced EGFR phosphorylation and internalisation. (4) Conclusion: This report confirms a transactivating effect of zinc on the EGFR for A549 cells and is the first describing an influence of zinc on the EGFR endocytosis. The transferability of the fine-tuning of EGFR-induced signalling by zinc needs to be verified in vivo, but the presented data underline that zinc might be helpful during treatment of disturbed regeneration and tissue repair.

Lysophosphatidic acid receptor 5 transactivation of TGFBR1 stimulates the mRNA expression of proteoglycan synthesizing genes XYLT1 and CHST3

Lysophosphatidic acid (LPA) via transactivation dependent signalling pathways contributes to a plethora of physiological and pathophysiological responses. In the vasculature, hyperelongation of glycosaminoglycan (GAG) chains on proteoglycans leads to lipid retention in the intima resulting in the early pathogenesis of atherosclerosis. Therefore, we investigated and defined the contribution of transactivation dependent signalling in LPA mediated GAG chain hyperelongation in human vascular smooth muscle cells (VSMCs).LPA acting via the LPA receptor 5 (LPAR5) transactivates the TGFBR1 to stimulate the mRNA expression of GAG initiation and elongation genes xylosyltransferase-1 (XYLT1) and chondroitin 6-sulfotransferase-1 (CHST3), respectively. We found that LPA stimulates ROS and Akt signalling in VSMCs, however they are not associated in LPAR5 transactivation of the TGFBR1. We observed that LPA via ROCK dependent pathways transactivates the TGFBR1 to stimulate genes associated with GAG chain elongation. We demonstrate that GPCR transactivation of the TGFBR1 occurs via a universal biochemical mechanism and the identified effectors represent potential therapeutic targets to inhibit pathophysiological effects of GPCR transactivation of the TGFBR1.

Neuroprotective effects of direct activation and transactivation of PDGFβ receptors

Neuroprotective effects of direct activation and transactivation of PDGFβ receptors

Famotidine with Celecoxib Adjuvant Therapy on Hospitalized COVID-19 Patients: A Case Series

BACKGROUND: Up to 80% of SARS-CoV-2 positive patients are asymptomatic and do not appear to progress to COVID-19. SARS-CoV-2 infection is not sufficient for development of COVID-19 disease; this may reflect differences in host inflammatory responses to infection. COX-2 expression is transcriptionally upregulated by SARS nucleocapsid N protein, and COVID-19 is associated with markedly elevated levels of prostaglandin E2 (PGE2). PGE2 modulates a wide variety of innate and adaptive inflammatory responses including mast cell activation. SARS-CoV-2 infection-associated mast cell degranulation may play a role in development of COVID-19. A randomized trial of hospitalized COVID-19 cases has demonstrated that COX-2 protein antagonist celecoxib dampened systemic PGE2 levels, prevented clinical deterioration, and was associated with rapid pulmonary CT-chest improvement. High doses of famotidine, a histamine H2 receptor antagonist/inverse agonist reduces severity of COVID-19 symptoms. We hypothesize that adjuvant therapy with a combination of celecoxib and high dose (HD) famotidine may improve COVID-19 outcomes.METHODS: We report a single institution, consecutive case series of 25 COVID-19 hospitalized patients treated with celecoxib and HD famotidine as adjuvant therapy. Outcome measurements include time to discharge, changes in supplemental oxygen requirements, pulmonary CT-chest findings, and laboratory changes in peripheral blood lactate dehydrogenase (LDH), C-reactive protein (CRP), ferritin, neutrophil counts, lymphocyte levels, neutrophil/lymphocyte ratio, creatinine, glomerular filtration rate, Interleukin 6 (IL6) and D-dimer concentrations. Nucleocapsid N protein sequence analysis was performed to compare COX-2 promoter activation domains of SARS, SARS-CoV-2, and related Sarbecovirus betacoronaviruses.RESULTS: All 25 patients in the series survived hospitalized COVID-19 without mechanical ventilation or renal replacement therapy (RRT) and were discharged on room air within a median of 3 days (range 1-16 days). Statistically significant improvements from admission to discharge were observed for all aggregated outcome measures. In general, progressive improvement was noted in supplemental oxygen requirements and ground-glass CT findings. Amino acid sequence analysis demonstrated high levels of conservation of N2 protein COX-2 promoter transactivation domains and nuclear localization sequences between SARS, SARS-CoV-2, and closely related Pangolin, Civet cat, and Horseshoe bat coronaviruses, but not MERS or other human or animal Betacoronaviruses.DISCUSSION: In this series, combined treatment with oral celecoxib and HD famotidine in an adjuvant setting was associated with 100% survival and improved radiographic outcomes, as well as statistically significant improvements in clinical, biomarker, and renal function measurements. The 9 patients with extremely high LDH (>365, Wuhan model prediction of 98% mortality) survived and were discharged on room air. Conservation of SARS and SARS-CoV-2 nucleocapsid N protein N2 sequences support that celecoxib treatment may mitigate the effects of direct viral transactivation of COX-2 expression in infected cells. HD famotidine acts to reduce cellular effects of local histamine via H2 receptor antagonism. Acute kidney injury was either prevented or mitigated by treatment; HD famotidine contributes to this effect. In contrast to these findings, previously published COVID-19 clinical intervention studies have not shown consistent laboratory or radiographic improvement. The data support performing randomized placebo-controlled trials to further examine the hypothesis that celecoxib with HD famotidine as adjuvant therapy to standard of care may reverse and prevent clinical deterioration in adult hospitalized COVID-19 patients.

Novel Mechanisms for IGF-I Regulation by Glucagon in Carp Hepatocytes: Up-Regulation of HNF1α and CREB Expression via Signaling Crosstalk for IGF-I Gene Transcription.

Glucagon, a key hormone for glucose homeostasis, can exert functional crosstalk with somatotropic axis via modification of IGF-I expression. However, its effect on IGF-I regulation is highly variable in different studies and the mechanisms involved are largely unknown. Using grass carp as a model, the signal transduction and transcriptional mechanisms for IGF-I regulation by glucagon were examined in Cyprinid species. As a first step, the carp HNF1α, a liver-enriched transcription factor, was cloned and confirmed to be a single-copy gene expressed in the liver. In grass carp hepatocytes, glucagon treatment could elevate IGF-I, HNF1α, and CREB mRNA levels, induce CREB phosphorylation, and up-regulate HNF1α and CREB protein expression. The effects on IGF-I, HNF1α, and CREB gene expression were mediated by cAMP/PKA and PLC/IP3/PKC pathways with differential coupling with the MAPK and PI3K/Akt cascades. During the process, protein:protein interaction between HNF1α and CREB and recruitment of RNA Pol-II to IGF-I promoter also occurred with a rise in IGF-I primary transcript level. In parallel study to examine grass carp IGF-I promoter activity expressed in αT3 cells, similar pathways for post-receptor signaling were also confirmed in glucagon-induced IGF-I promoter activation and the trans-activating effect by glucagon was mediated by the binding sites for HNF1α and CREB located in the proximal region of IGF-I promoter. Our findings, as a whole, shed light on a previously undescribed mechanism for glucagon-induced IGF-I gene expression by increasing HNF1α and CREB production via functional crosstalk of post-receptor signaling. Probably, by protein:protein interaction between the two transcription factors and subsequent transactivation via their respective cis-acting elements in the IGF-I promoter, IGF-I gene transcription can be initiated by glucagon at the hepatic level.

Lignans from Schisandra sphenanthera protect against lithocholic acid-induced cholestasis by pregnane X receptor activation in mice.

Cholestasis is a clinical syndrome caused by toxic bile acid retention that will lead to serious liver diseases. Ursodeoxycholic acid (UDCA) and obeticholic acid (OCA) are the only two FDA-approved drugs for its treatment. Thus, there is a clear need to develop new therapeutic approaches for cholestasis. Here, anti-cholestasis effects of the lignans from a traditional Chinese herbal medicine, Schisandra sphenanthera, were investigated as well as the involved mechanisms. Adult male C57BL/6J mice were randomly divided into 9 groups including the control group, LCA group, LCA with specific lignan treatment of Schisandrin A (SinA), Schisandrin B (SinB), Schisandrin C (SinC), Schisandrol A (SolA), Schisandrol B (SolB), Schisantherin A (StnA) and Schisantherin B (StnB), respectively. Mice were treated with each drug (qd) for 7 days, while the administration of lithocholic acid (LCA) (bid) was launched from the 4th day. Twelve hours after the last LCA injection, mice were sacrificed and samples were collected. Serum biochemical measurement and histological analysis were conducted. Metabolomics analysis of serum, liver, intestine and feces were performed to study the metabolic profile of bile acids. RT-qPCR and Western blot analysis were conducted to determine the hepatic expression of genes and proteins related to bile acid homeostasis. Dual-luciferase reporter gene assay was performed to investigate the transactivation effect of lignans on human pregnane X receptor (hPXR). RT-qPCR analysis was used to detect induction effects of lignans on hPXR-targeted genes in HepG2 cells. Lignans including SinA, SinB, SinC, SolA, SolB, StnA, StnB were found to significantly protect against LCA-induced intrahepatic cholestasis, as evidenced by significant decrease in liver necrosis, serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP) activity. More importantly, serum total bile acids (TBA) and total bilirubin (Tbili) were also significantly reduced. Metabolomics analysis revealed these lignans accelerated the metabolism of bile acids and increased the bile acid efflux from liver into the intestine or feces. Gene analysis revealed these lignans induced the hepatic expressions of PXR-target genes such as Cyp3a11 and Ugt1a1. Luciferase reporter gene assays illustrated that these bioactive lignans can activate hPXR. Additionally, they can all upregulate hPXR-regulate genes such as CYP3A4, UGT1A1 and OATP2. These results clearly demonstrated the lignans from Schisandra sphenanthera exert hepatoprotective effects against LCA-induced cholestasis by activation of PXR. These lignans may provide an effective approach for the prevention and treatment of cholestatic liver injury.

Ginsenoside Rb1 inhibits vascular calcification as a selective androgen receptor modulator

Ginsenoside Rb1 (Rb1), a major component of ginseng, has a steroidal chemical structure, implying that it exerts sex hormone-like actions. Recent studies have been suggested cardioprotective actions of Rb1. However, the actions of Rb1 in vascular calcification, one of the significant pathological features associated with aging and atherosclerosis, have not been examined. In the present study, we examined the effects of Rb1 on vascular calcification, focusing on its androgen-like actions. Using inorganic phosphate (Pi)-induced calcification of vascular smooth muscle cells (VSMC), we found that Rb1, like testosterone, significantly inhibited calcium deposition in a concentration-dependent manner. Further, this inhibition of Rb1 was abolished by bicalutamide, an androgen receptor antagonist, but not by MPP or PHTPP, estrogen receptor α or β antagonists. Rb1 significantly inhibited apoptosis, one of the regulatory mechanisms of calcification, and restored growth arrest-specific gene 6 (Gas6) expression that was suppressed by Pi. Moreover, Rb1 transactivated Gas6, and proximal androgen-responsive element (ARE) of the promoter region was found to be crucial for Gas6 transactivation. In contrast, in a human prostate cancer cell line, testosterone-induced ARE activity was abrogated by Rb1. This antagonistic effect was also confirmed by the transrepression and downregulation of prostate-specific antigen in the presence of testosterone and Rb1 together. Thus, these findings provide a novel mechanistic insight into the vasculoprotective actions of Rb1 as a selective androgen receptor modulator, i.e., inhibitory effects on VSMC calcification through androgen receptor-mediated Gas6 transactivation and antagonistic effects in prostate cancer cells.

The pepper AP2 domain-containing transcription factor CaDRAT1 plays a negative role in response to dehydration stress

APETALA2 (AP2) domain-containing transcription factors play critical regulatory roles in plant growth, development, and response to abiotic stresses, including dehydration stress. In Capsicum annuum (pepper), data are lacking regarding AP2 domain-containing transcription factors and their precise functions. Here, we identified six putative AP2 domain-containing transcription factors in pepper plants; the expression levels of these transcription factors were induced or repressed by dehydration stress. We named the gene having the highest fold change as Capsicum annuumDehydration Responsive AP2 domain-containing Transcription factor 1 (CaDRAT1). CaDRAT1 belongs to the ERF subfamily B-4, but the roles of these ERF proteins in dehydration responses are not well known. CaDRAT1 transcript levels were significantly induced after exposure to abscisic acid (ABA), mannitol, low temperature, and H2O2. The acidic domain in the CaDRAT1 C-terminal region had a transactivation effect on the reporter gene in the yeast GAL4 system. CaDRAT1-silenced pepper plants displayed a dehydration-resistant phenotype characterized by enhanced stomatal closure and low levels of transpirational water loss. Moreover, under dehydration stress conditions, expression levels of stress-related genes including CaNCED3 were higher in CaDRAT1-silenced pepper plants than in control plants and ABA content was also higher. Taken together, our findings suggest that CaDRAT1 functions as a negative regulator of dehydration resistance through modulation of ABA biosynthesis and signaling.

Sumoylation of PPARγ contributes to vascular endothelium insulin resistance through stabilizing the PPARγ-NcoR complex.

Sumoylation of peroxisome proliferator-activated receptorγ (PPARγ) affects its stabilization, sublocalization, and transcriptional activity. However, it remains largely unknown whether PPARγ sumoylation inhibits the transactivation effect, leading to endothelium insulin resistance (IR). To test this possibility, human umbilical vascular endothelial cells (HUVECs) with a 90% confluence were randomly allocated to two batches. One batch was first pretreated with or without vitamin E for 24 hr and the other infected with adenoviruses containing either PIAS1-shRNA (protein inhibitor of activated STAT1-short hairpin RNA) or scramble shRNA. Cells were suffered from high glucose and palmitic acid (PA) exposure for further 48 hr. The levels of PPARγ, p-IKK, IKK, and NcoR (nuclear corepressors) were measured by western blot analysis. The interaction of IKK and PIAS1, as well as the PPARγ sumoylation, were examined by coimmunoprecipitation. The results showed that the exposure of high glucose and PAinduced reactive oxygen species (ROS) production and IKK activation in HUVECs, promoting the interaction of IKK and PIAS1 and the sumoylation of PPARγ. However, vitamin E and PIAS1-shRNA partially decreased ROS production and IKK activation induced by high glucose and PA exposure. These data indicate that ROS-IKK-PIAS1 pathway mediates PPARγ sumoylation, leading to endothelium IR via stabilizing PPARγ-NcoR complex. These findings benefit understanding of regulatory networks of insulin signaling, which might provide a potential target to prevent and cure IR-related diseases.

SREBP-dependent lipidomic reprogramming as a broad-spectrum antiviral target

Viruses are obligate intracellular microbes that exploit the host metabolic machineries to meet their biosynthetic demands, making these host pathways potential therapeutic targets. Here, by exploring a lipid library, we show that AM580, a retinoid derivative and RAR-α agonist, is highly potent in interrupting the life cycle of diverse viruses including Middle East respiratory syndrome coronavirus and influenza A virus. Using click chemistry, the overexpressed sterol regulatory element binding protein (SREBP) is shown to interact with AM580, which accounts for its broad-spectrum antiviral activity. Mechanistic studies pinpoint multiple SREBP proteolytic processes and SREBP-regulated lipid biosynthesis pathways, including the downstream viral protein palmitoylation and double-membrane vesicles formation, that are indispensable for virus replication. Collectively, our study identifies a basic lipogenic transactivation event with broad relevance to human viral infections and represents SREBP as a potential target for the development of broad-spectrum antiviral strategies.