Stalled Hox promoters as chromosomal boundaries.

Many developmental control genes contain stalled RNA Polymerase II (Pol II) in the early Drosophila embryo, including four of the eight Hox genes. Here, we present evidence that the stalled Hox promoters possess an intrinsic insulator activity. The enhancer-blocking activities of these promoters are dependent on general transcription factors that inhibit Pol II elongation, including components of the DSIF and NELF complexes. The activities of conventional insulators are also impaired in embryos containing reduced levels of DSIF and NELF. Thus, promoter-proximal stalling factors might help promote insulator-promoter interactions. We propose that stalled promoters help organize gene complexes within chromosomal loop domains.

B cell translocation gene 2 (Btg2) is regulated by Stat3 signaling and inhibits adipocyte differentiation.

Btg2, a member of a family of antiproliferative proteins, is involved in downregulation of the JAK2-Stat3 signaling pathway. Here, we present evidence that the inhibitory effect of Btg2 on adipogenesis is suppressed by the proadipogenic activity of the Stat3 signaling pathway. Btg2 expression fluctuates during adipogenic differentiation of preadipocytes. Btg2 is also expressed at different levels in fat tissues from lean and obese mice. Furthermore, knockdown of Btg2 expression enhanced lipid accumulation and upregulated the expression of adipogenic marker genes. To gain insights into the molecular mechanisms of Btg2 action in adipocytes, adipocytes were treated with previously identified bioactive compounds and the expression of Btg2 was assessed. This effort identified the small molecule WP1066, a known Stat3 inhibitor, as an inducer of Btg2 expression. In line with this observation, siRNA-mediated silencing of Stat3 resulted in upregulated Btg2 expression and decreased lipid accumulation. Furthermore, siRNA-mediated silencing of Btg2 attenuated WP1066-mediated inhibition of adipocyte differentiation. We discuss a model for the role of Btg2 in adipogenesis and propose that Btg2 and Stat3 act in a functional hierarchy.

Sulfuretin induces osteoblast differentiation through activation of TGF-ß signaling.

The identification and examination of potential determinants controlling the progression of cell fate toward osteoblasts can be intriguing subjects. In this study, the effects of sulfuretin, a major compound isolated from Rhus verniciflua Stokes, on osteoblast differentiation were investigated. Treatments of sulfuretin induced alkaline phosphatase (ALP) activity in mesenchymal C3H10T1/2 cells and mineralization in preosteoblast MC3T3-E1 cells. Pro-osteogenic effects of sulfuretin were consistently observed in freshly isolated primary bone marrow cells. In mechanical studies, sulfuretin specifically induced expression of TGF-ß target genes, such as SMAD7 and PAI-1, but not other signaling pathway-related genes. Similar to the results of gene expression analysis, reporter assays further demonstrated TGF-ß-specific induction by sulfuretin. Furthermore, disruption of TGF-ß signaling using treatment with TGF-ß-specific inhibitor, SB-431542, and introduction of SMAD2/3 small interfering RNA impaired the effects of sulfuretin in inducing ALP activity and expression of ALP mRNA. Together, these data indicate that the pro-osteogenic effects of sulfuretin are mediated through activation of TGF-ß signaling, further supporting the potential of sulfuretin in the prevention of bone-related diseases such as bone fracture and osteoporosis.

Temporal regulation of single-minded target genes in the ventral midline of the Drosophila central nervous system.

Differentiation of a specific organ or tissue requires sequential activation of regulatory genes. However, little is known about how serial gene expression is temporally regulated. Here, we present evidence that differential expression of single-minded (sim) target genes can be attributed, in part, to the number of Sim and Tango (Tgo) heterodimer binding sites within their enhancer regions. The Sim, termed a master regulator, directs ventral midline differentiation of Drosophila central nervous system (CNS). According to data on the onset timing of ventral midline gene expression, sim target genes are classified into at least 2 groups (early and late). The sim and rhomboid (rho) genes are activated during early midline differentiation whereas orthodenticle (otd), CG10249, and slit (sli) genes undergo activation during later stages of midline differentiation. Germline transformation and in situ hybridization with transgenic embryos demonstrate that enhancers activating sim and rho expression contain 4 Sim-Tgo binding sites whereas only 1 Sim-Tgo binding site is found in an enhancer of sli. A mutagenized version of the rho enhancer lacking either 1, 2, or 3 Sim-Tgo binding sites mediated progressively more delayed expression of a lacZ reporter gene in the ventral midline. In contrast, a modified sli enhancer displayed progressively earlier onset of lacZ expression when 1, 2, or 3 more Sim-Tgo binding sites were added. Taken together, these results suggest that the number of Sim-Tgo-binding sites is decisive in determining the timing of gene expression in the developing ventral midline. We also discuss a combinatorial model accounting for the sequential expression of sim target genes.

Butein is a novel anti-adipogenic compound.

Rhus verniciflua Stokes (RVS) has been used as a traditional herbal medicine for its various biological activities including anti-adipogenic effects. Activity-guided separation led to the identification of the anti-adipogenic functions of butein. Butein, a novel anti-adipogenic compound, robustly suppressed lipid accumulation and inhibited expression of adipogenic markers. Molecular studies showed that activated transforming growth factor-ß (TGF-ß) and suppressed signal transducer and activator of transcription 3 (STAT3) signaling pathways were mediated by butein. Analysis of the temporal expression profiles suggests that TGF-ß signaling precedes the STAT3 in the butein-mediated anti-adipogenic cascade. Small interfering RNA-mediated silencing of STAT3 or SMAD2/3 blunted the inhibitory effects of butein on adipogenesis indicating that an interaction between two signaling pathways is required for the action of butein. Upon butein treatments, stimulation of TGF-ß signaling was still preserved in STAT3 silenced cells, whereas regulation of STAT3 signaling by butein was significantly impaired in SMAD2/3 silenced cells, further showing that TGF-ß acts upstream of STAT3 in the butein-mediated anti-adipogenesis. Taken together, the present study shows that butein, a novel anti-adipogenic compound from RVS, inhibits adipocyte differentiation through the TGF-ß pathway followed by STAT3 and peroxisome proliferator-activated receptor γ signaling, further implicating potential roles of butein in TGF-ß- and STAT3-dysregulated diseases.

Paeoniflorin, a monoterpene glycoside, attenuates lipopolysaccharide-induced neuronal injury and brain microglial inflammatory response.

Chronic activation of microglial cells endangers neuronal survival through the release of various proinflammatory and neurotoxic factors. Paeoniflorin (PF), a water-soluble monoterpene glycoside found in the root of Paeonia lactiflora Pall, has a wide range of pharmacological functions, such as anti-oxidant, anti-inflammatory, and anti-cancer effects. Neuroprotective potential of PF has also been demonstrated in animal models of neuropathologies. Here, we have examined the efficacy of PF in the repression of inflammation-induced neurotoxicity and microglial inflammatory response. In organotypic hippocampal slice cultures, PF significantly blocked lipopolysaccharide (LPS)-induced hippocampal cell death and productions of nitric oxide (NO) and interleukin (IL)-1ß. PF also inhibited the LPS-stimulated productions of NO, tumor necrosis factor-α, and IL-1ß from primary microglial cells. These results suggest that PF possesses neuroprotective activity by reducing the production of proinflammatory factors from activated microglial cells.

Anti-inflammatory activity of cinnamon water extract in vivo and in vitro LPS-induced models.

BACKGROUND: Cinnamon bark is one of the most popular herbal ingredients in traditional oriental medicine and possesses diverse pharmacological activities including anti-bacterial, anti-viral, and anti-cancer properties. The goal of this study is to investigate the in vivo and in vitro inhibitory effect of cinnamon water extract (CWE) on lipopolysaccharide (LPS)-induced tumor necrosis factor (TNF)-α and its underlying intracellular mechanisms. METHODS: CWE was orally administrated to mice for 6 days prior to intraperitoneal injection of LPS. Serum levels of TNF-α and interleukin (IL)-6 were determined 1 hour after LPS stimulation. Peritoneal macrophages from thioglycollate-injected mice were isolated and assayed for viability, cytokine expression and signaling molecules upon LPS stimulation. CWE was further fractioned according to molecular size, and the levels of total polyphenols and biological activities of each fraction were measured. RESULTS: The oral administration of CWE to mice significantly decreased the serum levels of TNF-α and IL-6. CWE treatment in vitro decreased the mRNA expression of TNF-α. CWE blocked the LPS-induced degradation of IκBα as well as the activation of JNK, p38 and ERK1/2. Furthermore, size-based fractionation of CWE showed that the observed inhibitory effect of CWE in vitro occurred in the fraction containing the highest level of total polyphenols. CONCLUSIONS: Treatment with CWE decreased LPS-induced TNF-α in serum. In vitro inhibition of TNF-α gene by CWE may occur via the modulation of IκBα degradation and JNK, p38, and ERK1/2 activation. Our results also indicate that the observed anti-inflammatory action of CWE may originate from the presence of polyphenols.

How the Dorsal gradient works: insights from postgenome technologies.

Gradients of extracellular signaling molecules and transcription factors are used in a variety of developmental processes, including the patterning of the Drosophila embryo, the establishment of diverse neuronal cell types in the vertebrate neural tube, and the anterior-posterior patterning of vertebrate limbs. Here, we discuss how a gradient of the maternal transcription factor Dorsal produces complex patterns of gene expression across the dorsal-ventral (DV) axis of the early Drosophila embryo. The identification of 60-70 Dorsal target genes, along with the characterization of approximately 35 associated regulatory DNAs, suggests that there are at least six different regulatory codes driving diverse DV expression profiles.

Promoter elements associated with RNA Pol II stalling in the Drosophila embryo.

RNA Polymerase II (Pol II) is bound to the promoter regions of many or most developmental control genes before their activation during Drosophila embryogenesis. It has been suggested that Pol II stalling is used to produce dynamic and rapid responses of developmental patterning genes to transient cues such as extracellular signaling molecules. Here, we present a combined computational and experimental analysis of stalled promoters to determine how they come to bind Pol II in the early Drosophila embryo. At least one-fourth of the stalled promoters contain a shared sequence motif, the "pause button" (PB): KCGRWCG. The PB motif is sometimes located in the position of the DPE, and over one-fifth of the stalled promoters contain the following arrangement of core elements: GAGA, Inr, PB, and/or DPE. This arrangement was used to identify additional stalled promoters in the Drosophila genome, and permanganate footprint assays were used to confirm that the segmentation gene engrailed contains paused Pol II as seen for heat-shock genes. We discuss different models for Pol II binding and gene activation in the early embryo.

Ginsenoside rb1 and rg3 attenuate glucocorticoid-induced neurotoxicity.

Glucocorticoid (GC) hormones, increased in response to stress, can cause neuronal loss. We tested the effect of GC hormone on cell viability of neural SHSY-5Y cells and protective effects of ginsenoside Rb1 and Rg3 on the action of GC. We treated SHSY-5Y cells with increasing concentrations of synthetic GC dexamethasone (DEX; 10, 25, 50, and 100 nM) for 24 and 48 h, and then determined cell viability by using MTT assay. We then treated SHSY-5Y cells with DEX (100 nM) with or without the ginsenosides to examine their preventive effects on the cytotoxicity. To explore the underlying molecular mechanisms, we measured mRNA expression of bax and bcl-2 by using reverse transcriptase real-time PCR. SHSY-5Y cells treated with DEX significantly reduced cell viability as compared with control cells. In the presence of Rb1 or Rg3, DEX-induced cytotoxicity was effectively blocked. DEX considerably increased pro-apoptotic bax mRNA expression as compared with control cells. However, Rb1 and Rg3 completely blocked DEX-mediated up-regulation of bax expression. DEX significantly increased neuronal death in organotypic hippocampal slice cultures of rat brain with enhanced generation of ROS, which was effectively inhibited by ginsenoside Rb1 and Rg3. This suggests a potential role of the ginsenosides to target GC action in the brain.

Macrophages from Mice Administered Rhus verniciflua Stokes Extract Show Selective Anti-Inflammatory Activity.

The bark of Rhus verniciflua Stokes (RVS) is used as a food additive and herbal medicine for various inflammatory disorders and cancer in Eastern Asia. RVS has been shown to exert anti-inflammatory effects in lipopolysaccharide (LPS)-stimulated macrophages in vitro, but whether oral administration of RVS affects the inflammatory response of macrophage needs to be verified. RVS was given orally to mice for ten days. For isolation of macrophages, intraperitoneal injection of thioglycollate was performed. For determination of serum inflammatory response, intraperitoneal injection of LPS was applied. RVS stimulated monocyte differentiation in thioglycollate-induced peritonitis by increasing the population of cells expressing CD11b and class A scavenger receptors. These monocyte-derived macrophages showed an increased uptake of acetylated low-density lipoprotein. When peritoneal macrophages from the RVS group were stimulated with LPS, the levels of tumor necrosis factor (TNF)-α and interleukin (IL)-6 in the supernatant decreased, but the level of IL-12 increased. The surface expression of CD86 was reduced, but surface expression of class II major histocompatibility complex molecules was increased. RVS suppressed the serum levels of LPS-induced TNF-α and IL-6. Collectively, RVS promoted monocyte differentiation upon inflammatory insults and conferred selective anti-inflammatory activity without causing overall inhibitory effects on immune cells.

The short gastrulation shadow enhancer employs dual modes of transcriptional synergy.

It remains unclear how a limited amount of maternal transcription factor Dorsal (Dl) directs broad expression of short gastrulation (sog) throughout the presumptive neurogenic ectoderm in the Drosophila early embryo. Here, we present evidence that the sog shadow enhancer employs dual modes of transcriptional synergy to produce this broad pattern. Bioinformatics analyses indicated that a minimal enhancer region, systematically mapped in vivo, contains five Dl-, three Zelda (Zld)-, and three Bicoid (Bcd)-binding sites; four of these five Dl-binding sites are closed linked to two Zld- and two Bcd-binding sites. Mutations of either the linked Zld- or Bcd-binding sites led to severe reduction in lacZ expression width, length, and/or strength in transgenic embryos. In addition, alteration of the helical phasing in this enhancer region by insertion of spacer sequences between linked sites also resulted in aberrant lacZ expression. These results suggest that synergistic interactions between Dl and Zld and between DI and Bcd are required for broad sog expression.

Structural characterization of the gene encoding the large zinc finger protein ZAS3: implication to the origin of multiple promoters in eukaryotic genes.

ZAS3 is a large zinc finger protein that regulates kappaB-mediated transcription and TNF-driven signal transduction pathway. Herein, we have characterized the mouse ZAS3 gene that spans 400 kb and splits into 16 exons. Four ZAS3 exons, ranging from 676 to 3956 nucleotides, are significantly larger than the average size of mammalian internal exons. Intron 10, when retained in mRNAs, encodes N-terminal DNA binding domain, called ZASN. As predicted from cDNAs, 5' untranslated region composed of the 2317 nucleotides is extremely long and contains upstream open reading frames, suggesting that translation initiation of ZAS3 transcripts by conventional cap-dependent ribosome scanning mechanism may be inefficient. Additionally, cDNA data analysis followed by reporter gene assays shows that the ZAS3 locus harbors two promoters that are 80 kb apart. The data suggest that the expression of ZAS3 is controlled by a combination of differential promoter usage, alternative splicing, and possible intergenic splicing. The distribution and degree of conservation of exons within the ZAS3 locus, together with the complex alternative splicing events and upstream open reading frame in 5' untranslated exons, lead us to speculate that multiple promoters of an eukaryotic gene might be residual traces of regulatory regions of other genes lost in evolution.

Transcriptional activity of the short gastrulation primary enhancer in the ventral midline requires its early activity in the presumptive neurogenic ectoderm.

The short gastrulation (sog) shadow enhancer directs early and late sog expression in the neurogenic ectoderm and the ventral midline of the developing Drosophila embryo, respectively. Here, evidence is presented that the sog primary enhancer also has both activities, with the late enhancer activity dependent on the early activity. Computational analyses showed that the sog primary enhancer contains five Dorsal (Dl)-, four Zelda (Zld)-, three Bicoid (Bcd)-, and no Single-minded (Sim)-binding sites. In contrast to many ventral midline enhancers, the primary enhancer can direct lacZ expression in the ventral midline as well as in the neurogenic ectoderm without a canonical Simbinding site. Intriguingly, the impaired transcriptional synergy between Dl and either Zld or Bcd led to aberrant and abolished lacZ expression in the neurogenic ectoderm and in the ventral midline, respectively. These findings suggest that the two enhancer activities of the sog primary enhancer are functionally consolidated and geographically inseparable. [BMB Reports 2016; 49(10): 572-577].

Antiadipogenic and proosteogenic effects of luteolin, a major dietary flavone, are mediated by the induction of DnaJ (Hsp40) Homolog, Subfamily B, Member 1.

Luteolin (3,4,5,7-tetrahydroxyflavones), a major dietary flavone, regulates a variety of biological effects including cancer progression, insulin resistance and inflammation. However, its exact actions on adipogenesis and osteogenesis and the underlying molecular mechanisms are yet to be clarified. In this study, we show that luteolin suppresses lipid accumulation but increases osteoblast differentiation. In mechanism studies, luteolin increases the expression of the heat shock proteins (Hsp) 40 (Dnajb1) and Hsp90 (Hsp90b1), but not those of other heat shock proteins including Hsp20, Hsp27, Hsp47, Hsp70, Hsp72, and Hsp90, and another type of Hsp40 (Dnaja1). Silencing Dnajb1 by using small interfering RNAs (siRNAs), but not against Hsp90b1, recapitulates the effects of luteolin in adipocyte and osteoblast differentiation. Consistently, the forced expression of Dnajb1 decreases the lipid accumulation and stimulates alkaline phosphatase (ALPL) activity. The antiadipogenic and proosteogenic effects of luteolin are significantly blunted in Dnajb1-deficient cells, further suggesting that Dnajb1 is, at least in part, required for luteolin's dual actions in adipogenesis and osteogenesis. Together, our data implicate luteolin as an ingredient and Dnajb1 as a molecular target for the development of functional foods and drugs in metabolic and bone-related diseases.

Inactivation of brain myo-inositol monophosphate phosphatase by pyridoxal-5'-phosphate.

Myo-inositol monophosphate phosphatase (IMPP) is a key enzyme in the phosphoinositide cell-signaling system. This study found that incubating the IMPP from a porcine brain with pyridoxal-5'-phosphate (PLP) resulted in a time-dependent enzymatic inactivation. Spectral evidence showed that the inactivation proceeds via the formation of a Schiff's base with the amino groups of the enzyme. After the sodium borohydride reduction of the inactivated enzyme, it was observed that 1.8 mol phosphopyridoxyl residues per mole of the enzyme dimer were incorporated. The substrate, myo-inositol-1-phosphate, protected the enzyme against inactivation by PLP. After tryptic digestion of the enzyme modified with PLP, a radioactive peptide absorbing at 210 nm was isolated by reverse-phase HPLC. Amino acid sequencing of the peptide identified a portion of the PLP-binding site as being the region containing the sequence L-Q-V-S-Q-Q-E-D-I-T-X, where X indicates that phenylthiohydantoin amino acid could not be assigned. However, the result of amino acid composition of the peptide indicated that the missing residue could be designated as a phosphopyridoxyl lysine. This suggests that the catalytic function of IMPP is modulated by the binding of PLP to a specific lysyl residue at or near its substrate-binding site of the protein.

Midline enhancer activity of the short gastrulation shadow enhancer is characterized by three unusual features for cis-regulatory DNA.

The shadow enhancer of the short gastrulation (sog) gene directs its sequential expression in the neurogenic ectoderm and the ventral midline of the developing Drosophila embryo. Here, we characterize three unusual features of the shadow enhancer midline activity. First, the minimal regions for the two different enhancer activities exhibit high overlap within the shadow enhancer, meaning that one developmental enhancer possesses dual enhancer activities. Second, the midline enhancer activity relies on five Single-minded (Sim)-binding sites, two of which have not been found in any Sim target enhancers. Finally, two linked Dorsal (Dl)- and Zelda (Zld)-binding sites, critical for the neurogenic ectoderm enhancer activity, are also required for the midline enhancer activity. These results suggest that early activation by Dl and Zld may facilitate late activation via the noncanonical sites occupied by Sim. We discuss a model for Zld as a pioneer factor and speculate its role in midline enhancer activity.

Capicua is involved in Dorsal-mediated repression of zerknüllt expression in Drosophila embryo.

The maternal transcription factor Dorsal (Dl) functions as both an activator and a repressor in a context-dependent manner to control dorsal-ventral patterning in the Drosophila embryo. Previous studies have suggested that Dl is an intrinsic activator and its repressive activity requires additional corepressors that bind corepressor-binding sites near Dl-binding sites. However, the molecular identities of the corepressors have yet to be identified. Here, we present evidence that Capicua (Cic) is involved in Dl-mediated repression in the zerknüllt (zen) ventral repression element (VRE). Computational and genetic analyses indicate that a DNA-binding consensus sequence of Cic is highly analogous with previously identified corepressor-binding sequences and that Dl failed to repress zen expression in lateral regions of cic mutant embryos. Furthermore, electrophoretic mobility shift assay (EMSA) shows that Cic directly interacts with several corepressor-binding sites in the zen VRE. These results suggest that Cic may function as a corepressor by binding the VRE.

Dichloromethane extracts of Sophora japonica L. stimulate osteoblast differentiation in mesenchymal stem cells.

Sophora japonica L. fruit prevents bone loss by inhibiting osteoclast activity. We hypothesized that S japonica L. extracts could promote osteoblast differentiation. To test this hypothesis, we investigated the effect of S japonica L. on osteoblast differentiation and identified the bioactive compound(s) from S japonica L. The mature fruit of S japonica L. was partitioned with ethanol, hexane, dichloromethane (DCM), ethyl acetate, and butanol, and their effects were tested on osteoblast differentiation of C3H10T1/2 cells. DCM fractionated extracts were identified as the most osteogenic fractions. DCM fractionated extracts dose-dependently stimulated alkaline phosphatase activity and matrix mineralization. The DCM fractions also induced expression of osteoblast markers such as alkaline phosphatase, osterix, and osteocalcin in C3H10T1/2 and primary bone marrow cells. Genistein was found abundantly in the DCM fractions. Furthermore, the genistein and DCM fractions similarly modulated the expression of estrogen target genes and were both active in transfection assays that measured estrogen agonistic activity. Finally, pharmacological inhibition by treatment with an estrogen receptor antagonist or specific inhibition of gene expression by small interference RNAs targeted to estrogen receptor-ß abolished the effects of the DCM extracts, further supporting the idea that the genistein in the DCM extracts mediated the pro-osteogenic effects. Taken together, we identified genistein as the key phytoestrogen responsible for the effects of S japonica L. on osteoblast differentiation.

Silk proteins stimulate osteoblast differentiation by suppressing the Notch signaling pathway in mesenchymal stem cells.

Silk fibroins are biomaterials that have been applied to surgical sutures, drug delivery systems, food supplements, and tissue engineering. Studies have shown the antiadipogenic effects of silk proteins in 3T3-L1 cells and obese mice. Furthermore, other studies have shown that silk proteins increase osteogenic marker expression in osteoblast-like cells. Because osteogenic and adipogenic differentiation from common mesenchymal progenitor cells are often regulated reciprocally, we hypothesized that silk proteins would stimulate osteoblast differentiation. The objective of this study was to evaluate the effects of silk proteins on promoting osteoblast differentiation and identify the underlying mechanism. We showed that silk proteins dose dependently stimulated alkaline phosphatase (ALP) activity, osteoblast differentiation, and induced expression of osteoblast markers in C3H10T1/2 and M2-10B4 multipotent cells. In addition, silk proteins also induced the expression of osteoblast markers in primary rat bone marrow cells isolated from tibiae. Molecular studies showed that silk proteins suppressed the expression of Notch-activated genes and blocked activation of the Notch-specific reporter. Similarly, inhibiting Notch signaling with pharmacologic inhibitors and by small interfering RNA-mediated Notch1 silencing also induced ALP activity and messenger RNA expression. Finally, induction of ALP activity and messenger RNA expression by silk proteins was blunted in Notch1 knock-downed cells, further demonstrating Notch signaling as an important mediator for the pro-osteogenic effects of silk proteins. Taken together, our data suggest that silk proteins may serve as functional foods to promote bone healing and therapeutic interventions for bone fractures and osteoporosis.