Transient Transfection Assays Research Articles

HOTAIR-mediated reciprocal regulation of EZH2 and DNMT1 contribute to polyphyllin I-inhibited growth of castration-resistant prostate cancer cells in vitro and in vivo

BackgroundPolyphyllin I (PPI), one of the steroidal saponins in paris polyphylla, has been reported to exhibit antitumor effects. However, the detailed molecular mechanism underlying this has not been elucidated. MethodsCell viability and cell cycle distribution were measured using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) and Flow cytometry assays, respectively. Cell invasion and migration were examined by Transwell invasion and wound healing assays. Western blot analysis was performed to examine the protein expressions of zeste homolog 2 (EZH2), DNA methyltransferase 1 (DNMT1). QRT-PCR was used to examine the levels of long non-coding RNA (lncRNA) HOX transcript antisense RNA (HOTAIR). Small interfering RNAs (siRNAs) method was used to knockdown HOTAIR. Exogenously expressions of HOTAIR, DNMT1 and EZH2 were carried out by Transient transfection assays. EZH2 promoter activity was measured by Secrete-Pair Dual Luminescence Assay Kit. A nude mice xenograft model was used to confirm the findings in vitro. ResultsWe showed that PPI significantly inhibited growth, induced cell cycle arrest of castration-resistant prostate cancer (CRPC) cells. In addition, PPI also reduced the migration and invasion in CRPC cells. In mechanism, we found that PPI decreased the protein expressions of EZH2, DNMT1 and levels of HOTAIR. Interestingly, silenced HOTAIR reduced EZH2 and DNMT1 protein expressions. On the contrary, exogenously expressed HOTAIR resisted PPI-inhibited EZH2 and DNMT1 protein expressions, EZH2 promoter activity and cell growth. Moreover, excessive EZH2 antagonized PPI-suppressed DNMT1 protein expression or vice versa. Consistent with this, PPI inhibited tumor growth, HOTAIR, the protein expressions of DNMT1 and EZH2 in vivo. ConclusionOur results show that PPI inhibits growth of CRPC cells through inhibition of HOTAIR expression, subsequently; this results in the repression of DNMT1 and EZH2 expressions. The interactions among HOTAIR, DNMT1 and EZH2, and reciprocal regulation of DNMT1 and EZH2 contribute to the overall responses of PPI. This study reveals a novel mechanism for HOTAIR-mediated regulating DNMT1 and EZH2 in response to PPI in inhibition of the growth of CRPC cells.

Hairy and enhancer of split 6 prevents hepatic lipid accumulation through inhibition of Pparg2 expression.

Peroxisome proliferator‐activated receptor gamma (PPARγ) is a master regulator for white adipocyte differentiation and lipid storage. The increased level of hepatic PPARγ2 isoform reprograms liver for lipid storage and causes abnormal fat accumulation in certain pathophysiologic conditions. The current study aimed to investigate a role of transcriptional repressor hairy and enhancer of split 6 (HES6) in the regulation of Pparg2 expression and hepatic steatosis induced by diet. Liver‐specific overexpression of Hes6 using adenovirus reduced Pparg2 messenger RNA levels by 90% and hepatic triglyceride accumulation by 22% compared to the levels in mice injected with an adenoviral empty vector with Western diet feeding. In sharp contrast, silencing Hes6 gene expression using short hairpin RNA increased hepatic lipid accumulation and Pparg2 messenger RNA levels by 70% and 4‐fold, respectively. To locate hepatocyte nuclear factor 4 alpha (HNF4α) binding site(s), through which repressional activity of HES6 is mediated, a 2.5‐kb Pparg2 promoter‐driven luciferase reporter was constructed for transient transfection assays. Subsequently, chromatin immunoprecipitation and electrophoretic mobility shift assays were performed. An HNF4α binding consensus sequence was identified at 903 base pairs upstream from the transcription start site of Pparg2. Deletion or point mutation of the sequence in a luciferase reporter containing the Pparg2 promoter abolished HNF4α‐mediated activation in HeLa cells. Chromatin immunoprecipitation and electrophoretic mobility shift assays further confirmed direct recruitment and binding of HNF4α to the site. Gene expression analysis with liver samples from subjects with nonalcoholic steatohepatitis suggested that the axis of the Hes6–Hnf4a–Pparg2 transcriptional cascade is also responsible for hepatic fat accumulation in humans. Conclusion: HES6 represses Pparg2 gene expression, thereby preventing hepatic lipid accumulation induced by chronic Western diet feeding or pathophysiologic conditions. (Hepatology Communications 2017;1:1085–1098)

Streptococcus gordonii lipoproteins induce IL-8 in human periodontal ligament cells

Streptococcus gordonii, a Gram-positive oral bacterium, is a life-threatening pathogen that causes infective endocarditis. It is frequently isolated from the periapical lesions of patients with apical periodontitis and has thus been implicated in inflammatory responses. However, little is known about the virulence factors of S. gordonii responsible for the induction of inflammatory responses in the periapical areas. Here, we investigated the role of S. gordonii cell wall-associated virulence factors on interleukin (IL)-8 induction in human periodontal ligament (PDL) cells using ethanol-inactivated wild-type S. gordonii, a lipoteichoic acid (LTA)-deficient mutant (ΔltaS), and a lipoprotein-deficient mutant (Δlgt). Wild-type S. gordonii induced IL-8 expression at both the protein and mRNA levels in human PDL cells in a dose- and time-dependent manner. A transient transfection and reporter gene assay demonstrated that wild-type S. gordonii activated Toll-like receptor 2 (TLR2). Additionally, IL-8 production induced by wild-type S. gordonii was substantially inhibited by anti-TLR2-neutralizing antibodies. Both wild-type S. gordonii and the ΔltaS mutant induced IL-8 production; however, this response was not observed when cells were stimulated with the Δlgt mutant. Interestingly, lipoproteins purified from S. gordonii induced IL-8 production, whereas purified LTA did not. In addition, purified lipoproteins stimulated TLR2 more potently than LTA. Furthermore, S. gordonii-induced IL-8 expression was specifically inhibited by blocking p38 kinase, while lipoprotein-induced IL-8 expression was inhibited by blocking p38 kinase, ERK, or JNK. Of particular note, exogenous addition of purified S. gordonii lipoproteins enhanced Δlgt-induced IL-8 production in human PDL cells to an extent similar to that induced by the wild-type strain. Collectively, these results suggest that lipoproteins are an important component of S. gordonii for the induction of IL-8 production in human PDL cells through TLR2 activation. Therefore, lipoproteins potentially contribute to inflammatory apical periodontitis.

Spatiotemporally Controlled Ablation of Klf5 Results in Dysregulated Epithelial Homeostasis in Adult Mouse Corneas.

PurposeCorneal epithelial (CE) homeostasis requires coordination between proliferation and differentiation. Here we examine the role of cell proliferation regulator Krüppel-like factor 5 (Klf5) in adult mouse CE homeostasis.MethodsKlf5 was ablated in a spatiotemporally restricted manner by inducing Cre expression in 8-week-old ternary transgenic Klf5LoxP/LoxP/Krt12rtTA/rtTA/Tet-O-Cre (Klf5Δ/ΔCE) mouse CE by administering doxycycline via chow. Normal chow-fed ternary transgenic siblings served as controls. The control and Klf5Δ/ΔCE corneal (1) histology, (2) cell proliferation, and (3) Klf5-target gene expression were examined using (1) periodic acid Schiff reagent-stained sections, (2) Ki67 expression, and (3) quantitative PCR and immunostaining, respectively. The effect of KLF4, KLF5, and OCT1 on gastrokine-1 (GKN1) promoter activity was determined by transient transfection in human skin keratinocyte NCTC-2544 cells.ResultsKlf5 expression was decreased to 23% of the controls in Klf5Δ/ΔCE corneas, which displayed increased fluorescein uptake, downregulation of tight junction proteins Tjp1 and Gkn1, desmosomal Dsg1a, and basement membrane Lama3 and Lamb1, suggesting defective permeability barrier. In transient transfection assays, KLF5 and OCT1 synergistically stimulated GKN1 promoter activity. Klf5Δ/ΔCE CE displayed significantly fewer cell layers and Ki67+ proliferative cells coupled with significantly decreased cyclin-D1, and elevated phospho(Ser-10) p27/Kip1 expression. Expression of Krt12, E-cadherin, and β-catenin remained unaltered in Klf5Δ/ΔCE corneas.ConclusionsKlf5 contributes to adult mouse CE homeostasis by promoting (1) permeability barrier function through upregulation of Tjp1, Gkn1, Dsg1a, Lama3, and Lamb1, and (2) basal cell proliferation through upregulation of cyclin-D1 and suppression of phospho(Ser-10) p27/Kip1, without significantly affecting the expression of epithelial markers Krt12, E-cadherin, and β-catenin.

PPAR\u03b1-dependent Insig2a overexpression inhibits SREBP-1c processing during fasting

Peroxisome-proliferator-activated receptor alpha (PPARα) and sterol regulatory element-binding protein (SREBP) play a role in regulating cellular fatty acid and cholesterol homeostasis via fatty acid oxidation and lipogenesis. The control of SREBP processing is regulated by the insulin induced gene (INSIG)2a protein, which binds SREBP to prevent SREBP translocation to the Golgi apparatus during nutrient starvation in the liver. However, the regulation of SREBP-1c processing by INSIGs during fasting and the regulatory mechanisms of the mouse Insig2a gene expression have not been clearly addressed. In the present study, we found that Insig2a was upregulated by PPARα in mouse livers and primary hepatocytes during fasting, whereas Insig2a mRNA expression was decreased in the livers of refed mice. A PPAR-responsive element between −126 bp and −114 bp in the Insig2a promoter was identified by a transient transfection assay and a chromatin immunoprecipitation assay; its role in regulation by PPARα was characterised using Pparα-null mice. These results suggest that PPARα is a trans-acting factor that enhances Insig2a gene expression, thereby suppressing SREBP-1c processing during fasting.

Erratum to: Peroxisome proliferator-activated receptor gamma activates fas ligand gene promoter inducing apoptosis in human breast cancer cells.

In just over a decade, apart from established metabolic actions, peroxisome proliferator-activated receptor gamma (PPARγ) has evolved as key therapeutic target in cancer disease. Fas ligand (FasL), a trans-membrane protein, induces apoptosis by crosslinking with the Fas receptor. Despite the FasL relevance, little is available on the regulation of its expression. In the current study, we explored for the first time the potential role of PPARγ in triggering apoptotic events through the Fas/FasL pathway in breast cancer cells. In MCF7 cells, by reverse transcription-polymerase chain reaction and Western blotting, we showed that the synthetic PPARγ ligand rosiglitazone (BRL) enhanced FasL expression, that was abrogated by a specific PPARγ antagonist GW9662. Transient transfection assays demonstrated that BRL transactivated human FasL promoter gene in a PPARγ-dependent manner. Progressive 5′ deletion analysis has identified a minimal promoter fragment spanning nucleotides from −318 to −237 bp, which is still sensitive to BRL treatment. FasL promoter activity was abrogated by mithramycin, suggesting an involvement of Sp1 transcription factor in PPARγ action. Electrophoretic mobility shift and chromatin immuno-precipitation assays demonstrated that BRL increased the binding of PPARγ and Sp1 to the Sp1 sequence located within the FasL gene promoter. The role of PPARγ and Fas/FasL pathways in BRL-induced apoptotic events was assessed by caspase 8 cleavage in the presence of GW as well as PPARγ and FasL RNA interferences. Our results indicate that PPARγ positively regulates the FasL gene expression also in MDA-MB231 and in BT20, revealing a new molecular mechanism by which BRL induces apoptosis in breast cancer cells.

Identification of the 2-tridecanone cis-acting element in the promoter of cytochrome P450 CYP6B7 in Helicoverpa armigera.

The expression level of cytochrome P450 genes in insects can be induced by plant allelochemicals, which is important for insects to adapt to host plants. Cytochrome P450 CYP6B7 has been reported to be involved in pyrethroid insecticide resistance in Helicoverpa armigera, and its transcription level was induced by some inducers. Currently, the regulatory mechanism of the induced expression of CYP6B7 remains unknown, although it is very important for understanding the detoxification mechanism to allelochemicals in host plants. The objective of the present study was to investigate the cis-acting element in the promoter of CYP6B7 mediating the inducible up-regulation of CYP6B7 in H. armigera by 2-tridecanone. The promoter region of CYP6B7 was cloned by genome walking technique and analyzed by transient transfection assay. Progressive 5' deletion of the promoter region of CYP6B7 revealed that the relative luciferase activity of construct -320/+232 could be significantly induced by 2-tridecanone. Further stepwise deletion between -320 and -238 bp found that construct -292/+232 could also be significantly induced by 2-tridecanone, but the adjacent construct -256/+232 could not, suggesting the essential role of the sequence between -292 and -257 bp for 2-tridecanone induction. Nucleotide mutations between -292 and -281 bp had no influence on the induction effect by 2-tridecanone, but nucleotide mutations between -280 and -257 bp significantly decreased the induction effect. These results demonstrated that the cis-acting element for 2-tridecanone induction was between -280 and -257 bp in the promoter of CYP6B7.

Abstract 4489: Knockdown ARID5A suppresses proliferation of LNCaP prostate cancer cell through the inhibition of global protein synthesis

Abstract AT-Rich Interaction Domain-containing protein 5a (ARID5A; also known as Modulator Recognition Factor 1(MRF1) ) was first cloned in our laboratory by screening a cDNA library of Tera-2 mRNA with an oligonucleotide probe from the major immediate early gene of human cytomegalo virus. It has been reported that ARID5A forms a complex with androgen receptor (AR) in yeast two hybrid assays and is a cosuppressor of AR in transient transfection assays. In this study, we report that the knockdown of ARID5a expression inhibits proliferation of LNCaP cells on the contrary to the expectation from the transient transfection assay. We found that proliferation of cells stimulated by the treatment with dihydro-testosterone (DHT) was suppressed when ARID5a expression was down-regulated by siRNA technology. Androgen stimulates cell proliferation through activation of mammalian target of rapamycin complex 1 (mTORC1). However, the suppression of ARID5a does not affect androgen-activated mTOR activity. Furthermore, the expression of androgen-inducible and androgen-suppressive genes were not affected by suppression of ARID5a. We concluded that ARID5a is not a cosuppressor of AR. Meanwhile, flow cytometer analysis showed that DHT-stimulated cell cycle was arrested at G1 phase after the knockdown of ARID5a expression. Oil Red O staining assays showed that the inhibition of ARID5a expression led to decreased DHT-induced lipid accumulation. Our data further proved that the knockdown of ARID5a expression inhibited the protein expression of DHT-induced sterol regulatory element binding transcription factor 1 (SREBP-1). In addition, Western blot analysis revealed that the down-regulation of ARID5a decreased the expression of cell survival / proliferation and cell cycle regulation proteins, such as hypoxia inducible factor 1 alpha (HIF1a), cyclin D1 and cyclin D3. Real time PCR data revealed that the knockdown of ARID5a expression did not affect the steady level of HIF1a, cyclin D1 and cyclin D3 mRNA. These suggested that knockdown of ARID5a expression affected protein expression at translational and/or post-translational level. Our data suggested that knockdown of ARID5a expression increased the phosphorylation of eukaryotic translation initiation factor 2a (eIF2a) which inhibited translation initiation. S35 labeling assay confirmed that the knockdown of ARID5a expression suppressed the global protein synthesis. We showed that one of the four eIF2a kinases, general control non-derepressible 2 (GCN2) was activated by the suppression of ARID5a expression. We are currently studying on the mechanisms by which ARID5A regulates GCN2 kinase. Citation Format: Chao Sun, Viktor Chesnokov, Keiichi Itakura. Knockdown ARID5A suppresses proliferation of LNCaP prostate cancer cell through the inhibition of global protein synthesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4489. doi:10.1158/1538-7445.AM2017-4489

Abstract 2145: Regulation of YAP1 during hypoxia and its novel role in vascular mimicry and angiogenesis

Abstract Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related mortality. As in other solid tumors, angiogenesis is necessary for the growth and progression of NSCLC. Vascular mimicry is a phenomenon in which non-endothelial tumor cells form angiogenic vascular structures. We have shown in the past that NSCLC cancer stem-like cells (CSCs) have unique ability to undergo vascular mimicry. The present study shows that transcriptional co-activator YAP1, the oncogenic component of the Hippo pathway, enables the NSCLC CSCs to form angiogenic tubule-like structures in matrigel. Inhibition of YAP1 or depletion of YAP1 suppressed vascular mimicry of CSCs as well as angiogenic tubule formation by HUVECs in matrigel as well as in a fibrin gel bead assay (FIBA) assay. The NSCLC CSCs were found to have higher mRNA expression of VEGF receptor II (KDR) and Angiopoietin-2 (AngPT-2) and depletion of YAP1 reduced the expression of VEGF, KDR and AngPT-2 mRNA; these promoters were induced by YAP1 in transient transfection assays, suggesting a possible molecular mechanism by which YAP1 regulates angiogenesis. Hypoxia is a strong inducer of tumor angiogenesis, cancer progression and metastasis. Our experiments showed that NSCLC cells grown in hypoxic conditions or treated with hypoxia mimetic DMOG have higher YAP1 mRNA and protein expression. However, such a change was not observed in the expression of YAP1 orthologue, TAZ, nor in the canonical Hippo pathway proteins like LATS1&2, Mst1&2, Sav or Mob. We identified a novel regulation of YAP1 by prolyl hydroxylase PHD2, which is mainly known to regulate HIF1α. PHD2 was found to hydroxylate proline residue(s) in YAP1 in a region between aa 284 to aa 289 as seen by mutational analysis. YAP1 was found to directly bind to PHD2 and depletion of PHD2 or treatment with DMOG which is an inhibitor of prolyl hydroxylases, elevated YAP1 protein levels in the nucleus. Further, YAP1 was found to associate with HIF1α as detected by co-immunoprecipitation experiments and could enhance HIF1α-mediated induction of the VEGF promoter. Proximity ligation assays performed on TMA showed enhanced YAP1 and HIF1α interaction in lung tumor tissues compared to normal cells. Our data suggest a novel regulation of YAP1 in hypoxic environment that supports angiogenesis and tumor growth. Citation Format: Namrata Bora Singhal, Srikumar Chellappan. Regulation of YAP1 during hypoxia and its novel role in vascular mimicry and angiogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2145. doi:10.1158/1538-7445.AM2017-2145

Leptin induces CREB-dependent aromatase activation through COX-2 expression in breast cancer cells

Leptin plays a key role in the control of adipocyte formation, as well as in the associated regulation of energy intake and expenditure. The goal of this study was to determine if leptin-induced aromatase enhances estrogen production and induces tumor cell growth stimulation. To this end, breast cancer cells were incubated with leptin in the absence or presence of inhibitor pretreatment, and changes in aromatase and cyclooxygenase-2 (COX-2) expression were evaluated at the mRNA and protein levels. Transient transfection assays were performed to examine the aromatase and COX-2 gene promoter activities and immunoblot analysis was used to examine protein expression. Leptin induced aromatase expression, estradiol production, and promoter activity in breast cancer cells. Protein levels of phospho-STAT3, PKA, Akt, ERK, and JNK were increased by leptin. Leptin also significantly increased cAMP levels, cAMP response element (CRE) activation, and CREB phosphorylation. In addition, leptin induced COX-2 expression, promoter activity, and increased the production of prostaglandin E2. Finally, a COX-2 inhibitor and aromatase inhibitor suppressed leptin-induced cell proliferation in MCF-7 breast cancer cells. Together, our data show that leptin increased aromatase expression in breast cancer cells, which was correlated with COX-2 upregulation, mediated through CRE activation and cooperation among multiple signaling pathways.

Mutual Interplay between the Human Cytomegalovirus Terminase Subunits pUL51, pUL56, and pUL89 Promotes Terminase Complex Formation.

Human cytomegalovirus (HCMV) genome encapsidation requires several essential viral proteins, among them pUL56, pUL89, and the recently described pUL51, which constitute the viral terminase. To gain insight into terminase complex assembly, we investigated interactions between the individual subunits. For analysis in the viral context, HCMV bacterial artificial chromosomes carrying deletions in the open reading frames encoding the terminase proteins were used. These experiments were complemented by transient-transfection assays with plasmids expressing the terminase components. We found that if one terminase protein was missing, the levels of the other terminase proteins were markedly diminished, which could be overcome by proteasome inhibition or providing the missing subunit in trans These data imply that sequestration of the individual subunits within the terminase complex protects them from proteasomal turnover. The finding that efficient interactions among the terminase proteins occurred only when all three were present together is reminiscent of a folding-upon-binding principle leading to cooperative stability. Furthermore, whereas pUL56 was translocated into the nucleus on its own, correct nuclear localization of pUL51 and pUL89 again required all three terminase constituents. Altogether, these features point to a model of the HCMV terminase as a multiprotein complex in which the three players regulate each other concerning stability, subcellular localization, and assembly into the functional tripartite holoenzyme.IMPORTANCE HCMV is a major risk factor in immunocompromised individuals, and congenital CMV infection is the leading viral cause for long-term sequelae, including deafness and mental retardation. The current treatment of CMV disease is based on drugs sharing the same mechanism, namely, inhibiting viral DNA replication, and often results in adverse side effects and the appearance of resistant virus strains. Recently, the HCMV terminase has emerged as an auspicious target for novel antiviral drugs. A new drug candidate inhibiting the HCMV terminase, Letermovir, displayed excellent potency in clinical trials; however, its precise mode of action is not understood yet. Here, we describe the mutual dependence of the HCMV terminase constituents for their assembly into a functional terminase complex. Besides providing new basic insights into terminase formation, these results will be valuable when studying the mechanism of action for drugs targeting the HCMV terminase and developing additional substances interfering with viral genome encapsidation.

Functional characterization of rs2229094 (T>C) polymorphism in the tumor necrosis factor locus and lymphotoxin alpha expression in human retina: the Retina 4 project.

PurposeThe objective of this study is to determine the expression and localization of lymphotoxin alpha (LTA) in human retinas and the functionality of one of its polymorphisms rs2229094 (C13R) (T>C), previously associated with proliferative vitreoretinopathy (PVR) development.Materials and methodsTotal RNA from three healthy human retinas were extracted and subjected to reverse transcription-polymerase chain reaction (RT-PCR) analysis, using flanking primers of LTA cDNA. In addition, three human eyes with retinal detachment (RD) and three healthy control eyes were subjected to immunohistochemistry (IHC) with a specific antibody against LTA. The functionality of T and C alleles was assessed by using pCEFL-Flag expression vector and transient transfection assays in COS-1 cell line. In addition, expression analysis by RT-PCR, Western blot and subcellular localization of both alleles and by immunofluorescence assay was performed.ResultsRT-PCR analysis revealed no significant levels of messenger RNA (mRNA) LTA in healthy human retinas. Sequential IHC staining showed differences between healthy human and RD retinas. No differences in mRNA and protein expression levels and in subcellular localization between both alleles were found. Both alleles were located in the cytoplasm of COS-1 cells.ConclusionAlthough results suggest lack of functionality, the differences found in IHC study and its strong association with PVR and its relationship with tumor necrosis factor locus, warrant further studies and could justify the use of this polymorphism as a valid biomarker to identify high-risk patients to develop PVR after RD.

Glucocorticoid receptor represses brain-derived neurotrophic factor expression in neuron-like cells

Brain-derived neurotrophic factor (BDNF) is involved in many functions such as neuronal growth, survival, synaptic plasticity and memorization. Altered expression levels are associated with many pathological situations such as depression, epilepsy, Alzheimer’s, Huntington’s and Parkinson’s diseases. Glucocorticoid receptor (GR) is also crucial for neuron functions, via binding of glucocorticoid hormones (GCs). GR actions largely overlap those of BDNF. It has been proposed that GR could be a regulator of BDNF expression, however the molecular mechanisms involved have not been clearly defined yet. Herein, we analyzed the effect of a GC agonist dexamethasone (DEX) on BDNF expression in mouse neuronal primary cultures and in the newly characterized, mouse hippocampal BZ cell line established by targeted oncogenesis. Mouse Bdnf gene exhibits a complex genomic structure with 8 untranslated exons (I to VIII) splicing onto one common and unique coding exon IX. We found that DEX significantly downregulated total BDNF mRNA expression by around 30%. Expression of the highly expressed exon IV and VI containing transcripts was also reduced by DEX. The GR antagonist RU486 abolished this effect, which is consistent with specific GR-mediated action. Transient transfection assays allowed us to define a short 275 bp region within exon IV promoter responsible for GR-mediated Bdnf repression. Chromatin immunoprecipitation experiments demonstrated GR recruitment onto this fragment, through unidentified transcription factor tethering. Altogether, GR downregulates Bdnf expression through direct binding to Bdnf regulatory sequences. These findings bring new insights into the crosstalk between GR and BDNF signaling pathways both playing a major role in physiology and pathology of the central nervous system.

Fatty acid-binding protein 4 regulates fatty infiltration after rotator cuff tear by hypoxia-inducible factor 1 in mice.

BackgroundFatty infiltration in skeletal muscle is directly linked to loss of muscle strength and is associated with various adverse physical outcomes such as muscle atrophy, inflammation, insulin resistance, mobility impairments, and even mortality in the elderly. Aging, mechanical unloading, muscle injury, and hormonal imbalance are main causes of muscle fat accumulation, and the fat cells are derived from muscle stem cells via adipogenic differentiation. However, the pathogenesis and molecular mechanisms of fatty infiltration in muscles are still not fully defined. Fatty acid‐binding protein 4 (FABP4) is a carrier protein for fatty acids and is involved in fatty acid uptake, transport, and lipid metabolism. Rotator cuff tear (RCT) usually occurs in the elderly and is closely related with fatty infiltration in injured muscle. To investigate potential mechanisms for fatty infiltration other than adipogenic differentiation of muscle stem cells, we examined the role of FABP4 in muscle fatty infiltration in an RCT mouse model.MethodsIn the RCT model, we evaluated the expression of FABP4 by qRT‐PCR, western blotting, and immunohistochemical analyses. Histological changes such as inflammation and fat accumulation in the injured muscles were examined immunohistochemically. To evaluate whether hypoxia induces FABP4 expression, the levels of FABP4 mRNA and protein in C3H10T1/2 cells after hypoxia were examined. Using a transient transfection assay in 293T cells, we assessed the promoter activity of FABP4 by hypoxia‐inducible factors (HIFs). Additionally, we evaluated the reduction in FABP4 expression and fat accumulation using specific inhibitors for HIF1 and FABP4, respectively.ResultsFABP4 expression was significantly increased after RCT in mice, and its expression was localized in the intramuscular fatty region. Rotator cuff tear‐induced FABP4 expression was up‐regulated by hypoxia. HIF1α, which is activated by hypoxia, augmented the promoter activity of FABP4, together with HIF1β. Hypoxia‐induced FABP4 expression was significantly decreased by HIF1 inhibitor treatment. Furthermore, in RCT model mice, fat accumulation was remarkably reduced by FABP4 inhibitor treatment.ConclusionsThis study shows that RCT induces FABP4 expression, leading to fat accumulation in injured muscle. FABP4 transcription is regulated by the direct binding of HIF1 to the FABP4 promoter in the hypoxic condition induced by RCT. Fat accumulation in injured muscle was reduced by the inhibition of FABP4. Ultimately, in the RCT model, we identified a novel mechanism for fatty infiltration by FABP4, which differs from adipogenic differentiation of muscle stem cells, and we found that fatty infiltration might be regulated by inhibition of HIF1 or FABP4.

Determination of the Bioactive Components in a Grape Seed Procyanidin Extract Responsible for Enhanced Farnesoid X Receptor Transactivation

Dietary procyanidins are beneficial for maintaining health. Grape seed procyanidin extract (GSPE) in particular has been shown to regulate cholesterol and triglyceride (TG) homeostasis in vivo (1–5). We have identified several underlying molecular mechanisms by which GSPE decreases serum TG levels, including farnesoid x receptor (FXR)‐dependent decreased hepatic triglyceride synthesis, inhibition of enterohepatic bile acid recirculation and increased hepatic TG catabolism (3–5). Furthermore, we identified GSPE as a histone deacetylase (HDAC) inhibitor, leading to enhanced peroxisome proliferator‐activated receptor alpha (PPARα) target‐gene transcription and increased fatty acid β‐oxidation (6). During our initial investigations we discovered that GSPE acts as a co‐agonist ligand for FXR (2). The present study was designed as a follow‐up to this initial observation and aimed to identify which component (or components) within GSPE are responsible for the observed FXR‐dependent co‐agonist activity. GSPE was fractionated by column chromatography to separate compounds based on molecular size (n=3). The degree of polymerization for each fraction was determined by high performance liquid chromatography. Using a cell‐based transient transfection assay, each fraction was tested in combination with the bile acid, chenodeoxycholic acid (CDCA), to assess its ability to increase FXR transactivation (n=3, analyzed in triplicate), and compared to the effect seen with GSPE. Five different fractions from each column consistently enhanced FXR transactivation and were subsequently analyzed by mass spectrometry (MS). The MS results show that the fractions that enhance FXR transactivation are enriched in dimers and dimer gallates. Ultimately, this study facilitates enhanced understanding regarding the observed GSPE‐mediated FXR‐dependent regulatory effects on metabolic homeostasis observed in vivo.Support or Funding InformationUSDA NIFA Hatch Project NEV0738; USDA NIFA Multi‐state Project W‐3122: Beneficial and adverse effects of natural chemicals on human health and food safety; USDA NIFA Hatch Project NEV0749; A grant from the National Institute of General Medical Sciences (P20GM103440) from the National Institutes of Health.

Efficient Vpu-Mediated Tetherin Antagonism by an HIV-1 Group O Strain.

Simian immunodeficiency viruses (SIVs) use their Nef proteins to counteract the restriction factor tetherin. However, a deletion in human tetherin prevents antagonism by the Nef proteins of SIVcpz and SIVgor, which represent the ape precursors of human immunodeficiency virus type 1 (HIV-1). To promote virus release from infected cells, pandemic HIV-1 group M strains evolved Vpu as a tetherin antagonist, while the Nef protein of less widespread HIV-1 group O strains acquired the ability to target a region adjacent to this deletion. In this study, we identified an unusual HIV-1 group O strain (RBF206) that evolved Vpu as an effective antagonist of human tetherin. While both RBF206 Vpu and Nef exert anti-tetherin activity in transient-transfection assays, mainly Vpu promotes RBF206 release in infected CD4+ T cells. Although mutations distinct from the adaptive changes observed in group M Vpus (M-Vpus) were critical for the acquisition of its anti-tetherin activity, RBF206 O-Vpu potently suppresses NF-κB activation and reduces CD4 cell surface expression. Interestingly, RBF206 Vpu counteracts tetherin in a largely species-independent manner, degrading both the long and short isoforms of human tetherin. Downmodulation of CD4, but not counteraction of tetherin, by RBF206 Vpu was dependent on the cellular ubiquitin ligase machinery. Our data present the first example of an HIV-1 group O Vpu that efficiently antagonizes human tetherin and suggest that counteraction by O-Nefs may be suboptimal.IMPORTANCE Previous studies showed that HIV-1 groups M and O evolved two alternative strategies to counteract the human ortholog of the restriction factor tetherin. While HIV-1 group M switched from Nef to Vpu due to a deletion in the cytoplasmic domain of human tetherin, HIV-1 group O, which lacks Vpu-mediated anti-tetherin activity, acquired a Nef protein that is able to target a region adjacent to the deletion. Here we report an unusual exception, identifying a strain of HIV-1 group O (RBF206) whose Vpu protein evolved an effective antagonism of human tetherin. Interestingly, the adaptive changes in RBF206 Vpu are distinct from those found in M-Vpus and mediate efficient counteraction of both the long and short isoforms of this restriction factor. Our results further illustrate the enormous flexibility of HIV-1 in counteracting human defense mechanisms.

A novel role of CKIP-1 in promoting megakaryocytic differentiation.

Casein kinase 2-interacting protein-1 (CKIP-1) is a known regulator of cardiomyocytes and macrophage proliferation. In this study, we showed that CKIP-1 was involved in the process of megakaryocytic differentiation. During megakaryocytic differentiation of K562 cells, CKIP-1 was dramatically upregulated and this upregulation induced by PMA was mediated through downregulation of transcription factor GATA-1. By transient transfection, oligonucleotide-directed mutagenesis and chromatin immunoprecipitation assays, we identified the transcriptional regulation of CKIP-1 by GATA-1. Overexpression of CKIP-1 initiated events of spontaneous megakaryocytic differentiation in K562 cells. Conversely, knockdown of CKIP-1 in cell lines suppressed megakaryocytic differentiation. Mechanistically, overexpression of CKIP-1 changed the expression levels of transcription factors that have been shown to be critical in erythro-megakaryocytic differentiation such as Fli-1, c-Myb and c-Myc. In vivo analysis confirmed that CKIP-1−/− mice had decreased number of CD41+ cells harvested from bone marrow, and lower platelet levels when compared to wild-type littermates. This is the first direct evidence suggesting that CKIP-1 is a novel regulator of megakaryocytic differentiation.

Association evidence of CCTTT repeat polymorphism in the iNOS promoter and the risk of atrial fibrillation in Taiwanese

Inducible nitric oxide synthase (iNOS) plays an important role in the pathogenesis of atrial fibrillation (AF). The iNOS promoter has a CCTTT-repeat length polymorphism that can determine the level of gene transcription. This study enrolled 200 AF patients and 240 controls. The length of CCTTT-repeat polymorphism in the iNOS promoter region was examined by polymerase chain reactions, with the alleles with ≤11 repeats designated as S and alleles with ≥12 repeats designated as L alleles. AF patients carried significantly higher frequencies of the LL genotype than control subjects (40.0% versus 28.3%, P = 0.010). Multivariate analysis showed that the presence of LL genotype was significantly associated with AF (odds ratio: 1.87, 95% CI = 1.10–3.17, P = 0.021). In vitro, transient transfection assay in HL-1 atrial myocytes showed that the responsiveness of iNOS transcriptional activity to tachypacing was correlated with the length of the CCTTT-repeats. Right atrial tissues from patients with chronic AF were investigated with immunoconfocal microscopy. Patients with LL genotype exhibited greater oxidative stress and substrate remodeling in their atria than those with non-LL genotypes. Our results suggest that the iNOS microsatellite polymorphism may contribute to the genetic background of AF in Chinese-Taiwanese patients.

Identification of microRNAs expressed in the midgut of Aedes albopictus during dengue infection

BackgroundThe midgut is the first barrier to dengue virus (DENV) infections of mosquitoes and therefore is a major bottleneck for the subsequent development of vector competence. However, the molecular mechanisms responsible for this barrier are unknown.ResultsWe constructed three small RNA libraries from the midguts of adult Aedes albopictus females that had been fed on either sugar solution, an uninfected blood meal, or a blood meal infected with DENV-2, and112 conserved microRNAs represented by 173 miRNA sequences were identified, with 34 novel microRNAs predicted by Mireap, RNAfold and Sfold software. In addition, the expression of aal-miR-1174, aal-miR-2951 and aal-miR-956 was confirmed via stem-loop quantitative real-time PCR (qRT-PCR). Compared with microRNA expression profiles of mosquitoes that had ingested a regular blood meal, 43 microRNAs were upregulated and 4were downregulated in mosquitoes that had ingested a DENV-2-infected blood meal. Among the differentially expressed microRNAs, miR-1767, miR-276-3p, miR-4448 and miR-4728-5p were verified via stem-loop qRT-PCR.ConclusionsAnalyses indicated that the changing patterns in miRNA expression during DENV-2 infection were significant and varied at different time points post infection. Most miRNA were upregulated at 24 h but were downregulated at 48 h post DENV-2 intake. The aal-miR-4728-5p was chosen for an in vitro transient transfection assay, and the results show that this miRNA enhances DENV replication in C6/36 cells. This study provides the first information on microRNAs expressed in the midgut of Ae. albopictus and describes species-specific changes in their expression levels following infection by DENV-2.

Characterization of a thienylcarboxamide derivative that inhibits the transactivation functions of cytomegalovirus IE2 and varicella zoster virus IE62

Previously we established reporter cell lines for human cytomegalovirus (HCMV) and varicella zoster virus (VZV) and identified several antiviral compounds against these viruses using the reporter cells. In this study, we found that one of the identified anti-HCMV compounds, a thienylcarboxamide derivative (coded as 133G4), was effective against not only HCMV but also VZV. The following findings indicate that 133G4 inhibits the activation of early gene promoters by HCMV IE2 and VZV IE62: i) 133G4 decreased the expression of HCMV early and late genes but not that of HCMV IE1/IE2 in HCMV-infected cells, ii) 133G4 inhibited the activation of several HCMV early gene promoters of transiently-transfected plasmids in HCMV-infected cells, and iii) in transient transfection assays, 133G4 decreased the activation of HCMV (or VZV) early gene promoters by HCMV IE2 (or VZV IE62) in the absence of other viral protein expression. The inhibition of early gene activation was observed in the human and African green monkey cell lines but not in the rodent cell lines, and the compound was not effective against murine CMV. In addition, VZV IE62 activated HCMV early promoters, and 133G4 still inhibited such promoter activation. Therefore, we hypothesized that 133G4 targets a cellular factor used commonly in activation of human herpesvirus promoters and examined whether 133G4 affects the functions of cellular proteins USF1, TBP, Med25 and EAP, the involvement of which in VZV IE62-dependent viral gene activation has been well characterized. Our experimental results using one-hybrid and bimolecular fluorescence complementation assays demonstrated that 133G4 did not inhibit the recruitment of USF1 or TBP to their binding sites, nor inhibited the direct interactions of VZV IE62 with Med25 and EAP. Thus, 133G4 is a unique anti-VZV and -HCMV compound, which warrants further studies to find out its inhibitory mechanism.