Reduced DNA Binding Activity Research Articles

Empagliflozin mitigates ponatinib-induced cardiotoxicity by restoring the connexin 43-autophagy pathway

BackgroundEmpagliflozin (EMPA), a selective sodium-glucose cotransporter type 2 (SGLT2) inhibitor, has been shown to reduce major adverse cardiovascular events in patients with heart failure of different etiologies, although the underlying mechanism still remains unclear. Ponatinib (PON) is a multi-tyrosine kinase inhibitor successfully used against myeloid leukemia and other human malignancies, but its cardiotoxicity remains worrisome. Cardiac connexins (Cxs) are both substrates and regulators of autophagy and responsible for proper heart function. Alteration in connexin expression and localization have been described in patients with heart failure. AimsTo assess whether EMPA can mitigate PON-induced cardiac dysfunction by restoring the connexin 43-autophagy pathway. Methods and ResultsMale C57BL/6 mice, randomized into four treatment groups (CNTRL, PON, EMPA, PON+EMPA) for 28 days, showed increased autophagy, decreased Cx43 expression as well as Cx43 lateralization, and attenuated systo-diastolic cardiac dysfunction after treatment with EMPA and PON compared with PON alone. Compared with CNTRL (DMSO), cardiomyocyte-differentiated H9c2 (dH9c2) cells treated with PON showed significantly reduced cell viability to approximately 20 %, decreased autophagy, increased cell senescence and reduced DNA binding activity of serum response factor (SRF) to serum response elements (SRE), which were paralleled by reduction in cardiac actin expression. Moreover, PON induced a significant increase of Cx43 protein and its S368-phosphorylated form (pS368-Cx43), as well as their displacement from the plasma membrane to the perinuclear and nuclear cellular region. All these effects were reverted by EMPA. ConclusionEMPA attenuates PON-induced cardiotoxicity by reducing senescence, enhancing the SRE-SRF binding and restoring the connexin 43-autophagy pathway. This effect may pave the way to use of SGLT2 inhibitors in attenuating tyrosine-kinase inhibitor cardiotoxicity.

Targeted mutagenesis of the β-strand DNA binding region of African swine fever virus histone-like protein (pA104R) impairs DNA-binding activity and antibody recognition

African Swine Fever (ASF) is a highly contagious disease caused by a double-stranded DNA virus (ASFV). Despite significant advances made over the last decade, issues such as residual virulence and absence of differentiating infected from vaccinated animals (DIVA) capacity remain an obstacle in the development of live attenuated vaccines (LAVs) against ASFV. It is, therefore, necessary to identify novel strategies to improve vaccine safety, by rational mutagenesis of virulence associated genes and generation of DIVA markers. ASFV encodes a HU (histone-like protein from E. coli strain U93) homolog protein, pA104R, which is involved in viral genome assembly and host immune recognition. A phylogenetic analysis revealed that pA104R is highly conserved among ASFV isolates, suggesting that it can be a good target for vaccine design. Thus, we selectively mutated the β-strand DNA binding region (BDR) of pA104R to attenuate its enzymatic activity, and identified and mutated several B-cell epitopes present in pA104R to generate a negative marker. Residues K64, K66, and R69 in the BDR were identified as relevant for pA104R activity, with double mutation of the first two showing additive attenuation. pA104R-reactive IgM and IgG epitopes were also identified in the bottom of the BDR, with selective mutagenesis drastically reducing antibody recognition and, when combined with mutations in the arm of the BDR, leading to a further reduction of DNA-binding activity. Interestingly, the immunodominant pA104R-reactive IgG epitope was mainly recognized by IgG1 suggesting that pA104R induces a dominant Th2 response. In sum, the rational mutagenesis can reduce pA104R-DNA binding activity and immune reactivity, providing a rationale for the development of an ASFV pA104R-based DIVA vaccine.

Downregulation of miR-21 gene expression by CRE-Ter to modulate osteoclastogenesis: De Novo mechanism

miR-21 expression stimulates osteoclast cells in the context of osteoclastogenesis. A previous report showed that NFκB-miR-21 pathway could serve as an innovative alternative to devise therapeutics for healing diabetic ulcers. Furthermore, our study demonstrated that a highly water-soluble curcuminoids-rich extract (CRE-Ter) inhibits osteoclastogenesis through NFκB pathway. The interplay between miR-21 and CRE-Ter in osteoclastogenesis has not yet been investigated. In this study, we examined the relation of CRE-Ter and miR-21 gene expression in receptor of the nuclear factor κB (NFκB) ligand (RANKL) - induced murine monocyte/macrophage RAW 264.7 cells, osteoclast cells, in osteoclastogenesis. Effect of CRE-Ter on generation of intracellular reactive oxygen species (ROS) was estimated by dichlorofluorescein diacetate (DCFH-DA). The results reveal that CRE-Ter reduced expression levels of miR-21 gene in osteoclasts. The inhibitory effects of CRE-Ter on in vitro osteoclastogenesis were evaluated by reduction in tartrate-resistant acid phosphatase (TRAP) content, and by reduction in expression levels of an osteoclast-specific gene, cathepsin K. Treatment of the osteoclast cells with CRE-Ter suppressed RANKL-induced NFκB activation including phospho-NFκB-p65, and phospho IκBα proteins. Western blot analysis revealed that NFκB inhibitor up-regulated CRE-Ter-promoted expression of phospho-NFκB-p65. In addition, CRE-Ter dose-dependently inhibited phospho-Akt expression. CRE-Ter also dose-dependently reduced DNA binding activity of NFκB and Akt as revealed by EMSA. ChIP assay revealed binding of NFκB-p65 to miR-21 promoters. In conclusion, our results demonstrate that CRE-Ter downregulates miR-21 gene expression in osteoclasts via a de novo mechanism, NFκB- Akt-miR-21 pathway.

Yulink, predicted from evolutionary analysis, is involved in cardiac function

BackgroundThe comparative evolutionary genomics analysis was used to study the functions of novel Ka/Ks-predicted human exons in a zebrafish model. The Yulink (MIOS, Entrez Gene: 54,468), a conserved gene from zebrafish to human with WD40 repeats at N-terminus, was identified and found to encode an 875 amino acid in human. The biological function of this Yulink gene in cardiomyocytes remains unexplored. The purpose of this study is to determine the involvement of Yulink in the functions of cardiomyocytes and to investigate its molecular regulatory mechanism.MethodsKnockdown of Yulink was performed using morpholino or shRNA in zebrafish, mouse HL-1 cardiomyocytes, and human iPSC-derived cardiomyocytes. The expression levels of mRNA and protein were quantified by qPCR and western blots. Other methods including DNA binding, ligand uptake, agonists treatment and Ca2+ imaging assays were used to study the molecular regulatory mechanism by Yulink. Statistical data were shown as mean ± SD or mean ± standard error.ResultsThe knockdown of yulink with three specific morpholinos in zebrafish resulted in cardiac dysfunctions with pericardial edema, decreased heart beats and cardiac output. The Yulink knockdown in mouse HL-1 cardiomyocytes disrupted Ca2+ cycling, reduced DNA binding activity of PPARγ (peroxisome proliferator-activated receptor gamma) and resulted in a reduction of Serca2 (sarcoplasmic reticulum Ca2+ ATPase 2) expression. Expression of Serca2 was up-regulated by PPARγ agonists and down-regulated by PPARγ-shRNA knockdown, suggesting that Yulink regulates SERCA2 expression through PPARγ in mouse HL-1 cardiomyocytes. On the other hand, YULINK, PPARγ or SERCA2 over-expression rescued the phenotypes of Yulink KD cells. In addition, knockdown of YULINK in human iPSC-derived cardiomyocytes also disrupted Ca2+ cycling via decreased SERCA2 expression.ConclusionsOverall, our data showed that Yulink is an evolutionarily conserved gene from zebrafish to human. Mechanistically Yulink regulated Serca2 expression in cardiomyocytes, presumably mediated through PPARγ nuclear entry. Deficiency of Yulink in mouse and human cardiomyocytes resulted in irregular Ca2+ cycling, which may contribute to arrhythmogenesis.

Transcriptional mutagenesis dramatically alters genome-wide p53 transactivation landscape

The transcriptional error rate can be significantly increased by the presence of DNA lesions that instruct mis-insertion during transcription; a process referred to as transcriptional mutagenesis (TM) that can result in altered protein function. Herein, we determined the effect of O6-methylguanine (O6-meG) on transcription and subsequent transactivation activity of p53 in human lung H1299 cells. Levels of TM and effects on transactivation were determined genome wide by RNA-seq. Results showed that 47% of all p53 transcripts contained an uridine misincorporation opposite the lesion at 6 h post transfection, which was decreased to 18% at 24 h. TM at these levels reduced DNA binding activity of p53 to 21% and 80% compared to wild type p53, respectively. Gene expression data were analysed to identify differentially expressed genes due to TM of p53. We show a temporal repression of transactivation of > 100 high confidence p53 target genes including regulators of the cell cycle, DNA damage response and apoptosis. In addition, TM repressed the transcriptional downregulation by p53 of several negative regulators of proliferation and differentiation. Our work demonstrates that TM, even when restricting its effect to an individual transcription factor, has the potential to alter gene expression programs and diversify cellular phenotypes.

Baicalein Inhibits Dextran Sulfate Sodium-induced Mouse Colitis.

BackgroundBaicalein is a bioactive flavone that is originally extracted from the root of Scutellaria baicalensis Georgi. This plant has long served as Chinese herbal medicine in the management of multiple diseases including inflammatory bowel diseases. Although it has been revealed that baicalein inhibits experimental colitis in mice, the molecular mechanisms still remain largely unrecognized.MethodsThe experimental colitis was induced in mice by 3% dextran sulfate sodium (DSS) in drinking water. The mice were given baicalein (10 or 25 mg/kg) by gavage for 7 days before and after DSS administration. Expression of COX-2 and inducible nitric oxide synthase (iNOS) and molecules involved in NF-κB signaling, such as inhibitor of κBα (IκBα), pIκBα, p65, and phospho-p65 was examined by Western blot analysis in the tissue of the mouse colon. Activity of IκB kinase β (IKKβ) was assessed by measuring the relative amount of radioactive γ-phosphate of ATP transferred to the IκBα substrate protein. The expression and phosphorylation of STAT3 and its target gene cyclin D1 were also measured.ResultsBaicalein prominently mitigated the severity of DSS-induced colitis in mice. It inhibited the expression of COX-2 and iNOS. Moreover, baicalein attenuated activity and phosphorylation of IKKβ and subsequent degradation of IκBα. Baicalein suppressed the phosphorylation and nuclear translocation of p65, resulting in a reduced DNA binding activity of NF-κB. Baicalein also suppressed the phosphorylation of STAT3 and expression of cyclin D1. Baicalein exhibited the synergistic effect on inhibition of COX-2 induced by DSS with curcumin, an ingredient of turmeric.ConclusionsProtective effects of baicalein on DSS-induced colitis are associated with suppression of NF-κB and STAT3 signaling pathways, which may contribute to its cancer preventive effects on colon carcinogenesis.

Phosphorylation of FANCD2 Inhibits the FANCD2/FANCI Complex and Suppresses the Fanconi Anemia Pathway in the Absence of DNA Damage

SummaryInterstrand crosslinks (ICLs) of the DNA helix are a deleterious form of DNA damage. ICLs can be repaired by the Fanconi anemia pathway. At the center of the pathway is the FANCD2/FANCI complex, recruitment of which to DNA is a critical step for repair. After recruitment, monoubiquitination of both FANCD2 and FANCI leads to their retention on chromatin, ensuring subsequent repair. However, regulation of recruitment is poorly understood. Here, we report a cluster of phosphosites on FANCD2 whose phosphorylation by CK2 inhibits both FANCD2 recruitment to ICLs and its monoubiquitination in vitro and in vivo. We have found that phosphorylated FANCD2 possesses reduced DNA binding activity, explaining the previous observations. Thus, we describe a regulatory mechanism operating as a molecular switch, where in the absence of DNA damage, the FANCD2/FANCI complex is prevented from loading onto DNA, effectively suppressing the FA pathway.

MMB triazole analogs are potent NF-κB inhibitors and anti-cancer agents against both hematological and solid tumor cells

Triazole derivatives of melampomagnolide B (MMB) have been synthesized via click chemistry methodologies and screened against a panel of 60 human cancer cell lines. Several derivatives showed promising anti-cancer activity, affording growth inhibition (GI50) values in the nanomolar range (GI50 = 0.02–0.99 μM). Lead compound 7h exhibited EC50 values of 400 nM and 700 nM, respectively, against two AML clinical specimens. Compound 7h was significantly more potent than parthenolide as an inhibitor of p65 phosphorylation in both hematological and solid tumor cell lines, indicating its ability to inhibit the NF-κB pathway. In TMD-231 breast cancer cells, treatment with 7h reduced DNA binding activity of NF-κB through inhibition of IKK-β mediated p65 phosphorylation and caused elevation of basal IκBα levels through inhibition of constitutive IκBα turnover and NF-κB activation. Molecular docking and dynamic modeling studies indicated that 7h interacts with the kinase domain of the monomeric IKKβ subunit, leading to inhibition of IKKβ activation, and compromising phosphorylation of downstream targets of the NF-κB pathway; dynamic modeling studies show that this interaction also causes unwinding of the α-helix of the NEMO binding site on IKKβ. Molecular docking studies with 10, a water-soluble analog of 7h, demonstrate that this analog interacts with the dimerization/oligomerization domain of monomeric IKKβ and may inhibit oligomer formation and subsequent autophosphorylation. Sesquiterpene lactones 7h and 10 are considered ideal candidates for potential clinical development.

Alanine substitution mutations in the DNA binding region of a global staphylococcal virulence regulator affect its structure, function, and stability

SarA, a winged-helix DNA binding protein, is a global virulence regulator in Staphylococcus aureus. The putative DNA binding region of SarA is located between amino acid residues Leu 53 and Gln 97. Previous studies have demonstrated that residues at positions 84, 88, 89, and 90 are critical for its function. To precisely understand the roles of the DNA binding residues, we have investigated nine mutants of a recombinant SarA (rSarA) along with the rSarA mutants carrying mutations at the above four positions. Of the thirteen mutants, eleven mutants show weaker DNA binding activity in vitro compared to rSarA. As noted earlier, the DNA binding affinity of rSarA was maximally affected due to the mutation at position 84 or 90. Each of the functionally-defective mutants also possesses an altered structure and stability. Additionally, the mutations at positions 84 and 90 have severely affected the formation of hydrogen (H) bonds at the interface between SarA and the cognate DNA. The mutation at position 64 also has perturbed the generation of some interface H-bonds. Therefore, the disruption of H-bonds in the protein-DNA interface and the structural alteration in the protein may be responsible for the reduced DNA binding activity of the mutants.

Impaired Nrf2 regulation of mitochondrial biogenesis in rostral ventrolateral medulla on hypertension induced by systemic inflammation

Oxidative stress in rostral ventrolateral medulla (RVLM), where sympathetic premotor neurons reside, is involved in the development of hypertension under systemic inflammation. Mitochondrial dysfunction contributes to tissue oxidative stress. In this study, we sought to investigate whether hypertension developed under systemic inflammation is attributable to impaired mitochondrial biogenesis in RVLM. In normotensive Sprague-Dawley rats, intraperitoneal infusion of a low dose Escherichia coli lipopolysaccharide (LPS) for 7 days promoted a pressor response, alongside a decrease in mitochondrial DNA (mtDNA) copy number, reductions in protein expression of nuclear DNA-encoded transcription factors for mitochondrial biogenesis, including mitochondrial transcription factor A (TFAM) and nuclear factor erythroid-derived 2-like 2 (Nrf2), and suppression of nuclear translocation of the phosphorylated Nrf2 (p-Nrf2) in RVLM neurons; all of which were abrogated by treatment with intracisternal infusion of an interleukin-1β (IL-1β) blocker, IL-1Ra, or a mobile mitochondrial electron carrier, coenzyme Q10 (CoQ10). Microinjection into RVLM of IL-1β suppressed the expressions of p-Nrf2 and TFAM, and evoked a pressor response; conversely, the Nrf2 inducer, tert-butylhydroquinone, lessened the LPS-induced suppression of TFAM expression and pressor response. At cellular level, exposure of neuronal N2a cells to IL-1β decreased mtDNA copy number, increased protein interaction of Nrf2 to its negative regulator, kelch-like ECH-associated protein 1 (Keap1), and reduced DNA binding activity of p-Nrf2 to Tfam gene. Together these results indicate that defect mitochondrial biogenesis in RVLM neurons entailing redox-sensitive and IL-1β-dependent suppression of TFAM because of the increase in the formation of Keap1/Nrf2 complex, reductions in nuclear translocation of the activated Nrf2 and its binding to the Tfam gene promoter may underlie hypertension developed under the LPS-induced systemic inflammation.

NF-κB pathway inhibition by anthrocyclic glycoside aloin is key event in preventing osteoclastogenesis in RAW264.7 cells

BackgroundOsteoporosis is a bone pathology leading to increased fracture risk and challenging the quality of life. As current treatments can exhibit deleterious side effects, the use of phyto-compounds with therapeutic and preventive activities against orthopaedic related problems represents a promising alternative. PurposeWe investigated the effect of aloin, an anthrocyclic compound, on inhibition of osteoclastogenesis using receptor of the nuclear factorκB (NF-κB) ligand (RANKL)-induced RAW264.7 macrophage cells. Study design/methodsThe inhibitory effect of aloin on in vitro osteoclastogenesis was evaluated by reduction in tartrate-resistant acid phosphatase (TRAP) content and expression levels of osteoclast-specific gene, cathepsin K. Multinuclear formation of osteoclast was assessed with haematoxylin and eosin staining. F4/80 content the marker of the murine monocyte/macrophage cells, was evaluated by immunocytochemistry. The underlining mechanisms were assessed by Western blots and EMSA. Effect of aloin on generation of intracellular reactive oxygen species (ROS) was estimated by dichlorofluorescein diacetate (DCFH-DA). Bone degradation effect was evaluated by bone pit assay. The bone pit culture supernatant was studied by Fluorescein assay. ResultsWe demonstrated that aloin reduced TRAP content and levels of osteoclast-specific gene and protein, cathepsin K. Treatment with aloin (0.75µM) prevented multinuclear formation (haematoxylin and eosin staining), reduced intracellular TRAP content (TRAP Staining) and increased F4/80 content (F4/80 immunohistochemistry) in RANKL (20ng/ml) treated RAW cells. Treatment of the RAW cells with aloin suppressed RANKL-induced NF-κB pathway components like IKKα, IKKβ, Phospho.IKK α/β, NF-κB-p65, Phospho NF-κB-p65 and IκBα. EMSA studies showed aloin dose dependently reduced DNA binding activity of NF-κB. Additionally, in vitro bone pit assay revealed that aloin prevented bone degradation and also decreased the fluorescence content in cells, thus confirming the role of aloin in inhibition of osteoclastogenesis . ConclusionCollectively, this study identifies aloin as a potent inhibitor of osteoclastogenesis and bone resorption. The action of aloin was in par with alendronate sodium trihydrate and may provide evidence for its therapeutic potential to treat diseases involving abnormal bone lysis.

Expression of PIM Kinases in Reed-Sternberg Cells Fosters Immune Privilege and Tumor Cell Survival in Classical Hodgkin Lymphoma

The classical Hodgkin lymphoma (cHL) is characterized by a presence of rare malignant Reed-Sternberg (RS) cells surrounded by abundant reactive infiltrate. Tumor RS cells express multiple cytokines, chemokines and immunoregulatory proteins, such as PD-L1 or galectin-1, that enhance recruitment of the infiltrating cells and allow malignant cells to escape from host immune surveillance. Since targeting these immunomodulatory molecules is a highly promising therapeutic strategy in cHL, identification of pathways and mechanisms orchestrating tumor immune evasion and supporting RS cell survival can further reveal targetable vulnerabilities of cHL. Expression of immunoregulatory proteins in cHL is modulated by tumor-specific activity of certain pro-survival transcription factors, such as NFκB and STATs. Since the activity of these transcription factors is modulated by oncogenic PIM1/2/3 serine/threonine kinases, we hypothesized that PIMs may support RS cell survival and foster their immune privilege. For these reasons, we investigated PIM1/2/3 expression in cHL and determined mechanisms underlying their expression. Furthermore, we assessed impact of PIM inhibition on expression of key factors engaged in development of the immunosuppressive microenvironment and HRS cell survival.Our analyses revealed that PIM1/2/3 are ubiquitously expressed in primary and cultured RS cell lines. At least one PIM isoform was expressed in each cell line and in 97% of 67 primary cHL biopsies. RS cells treated with JAK1/2/3 inhibitor exhibited reduced PIM1 and PIM2 levels. Genetic inhibition of canonical NFkB activity with IkB super-repressor or shRNA-mediated ablation of alternative NFkB pathway led to decrease of PIM2 and PIM3, suggesting that PIM-1/2/3 expresion in cHL depends at least in part on JAK-STAT and NFkB activity.To assess the role of PIM kinases in cell viability, we silenced expression of each PIM isoform (individually or simultaneously) in HDLM-2 cells. Knockdowns of individual PIM isoforms were associated with marked increased in remaining isoforms expression and were not associated with toxicity. In marked contrast, downregulation of all three isoforms increased cellular apoptosis by 17%. For this reason, for subsequent studies we used a newly developed pan-PIM inhibitor (SEL24-B489). The inhibitor was toxic to all cells with IC50 ranging from 3-5 µM. To determine mechanisms underlying toxicity, we assessed the activities of specific PIM substrates: 4EBP1, S6, and p65 (RelA). SEL24-B489 rapidly decreased PIM-dependent phosphorylation of these molecules in all tested cell lines. Furthermore, it reduced DNA binding activity of the NFκB-p65 complexes, indicating that PIM kinases modulate NFκB activity in cHL. For this reason, we next assessed the consequences of PIM inhibition on NFκB-dependent transcription. SEL24-B489 significantly downregulated mRNA of NFkB target genes associated with HRS cell survival and proliferation, such as Bfl-1, RelB and CD40. In cells treated with PIM inhibitor SEL24-B489 we also found markedly decreased expression NFkB-dependent cytokines and chemokines specifically shaping pro-tumoral microenvironment, such as IL-8, CCL5 and IL-13. In addition, cell lines exposed to SEL24-B489 treatment exhibited decreased expression of immunomodulatory PD-L1 and Gal-1 proteins. Finally, we investigated the efficacy of SEL24-B489 in vivo in the murine xenograft model using HDLM-2 cells. In contrast to animals exposed to vehicle alone, we observed inhibition of tumor growth by 95,8% in SEL24-B489-treated animals (p=0,0002). Consistent with the in vitro data, we observed strong downregulation of phospho-S6, GAL-1 and PD-L1 proteins in tumor sections from PIM inhibitor-treated animals.Taken together, we demonstrated that the oncogenic PIM-1/2/3 kinases are expressed in RS cells and their activity can be specifically blocked using a pan-PIM inhibitor SEL24-B489. PIM inhibition significantly reduced activity of specific PIM substrates and decreased the expression of NFκB-dependent pro-survival genes and key immunomodulatory proteins. These results highlight the pleiotropic activity of SEL24-B489 and indicate that PIM kinases are promising therapeutic targets in cHL. DisclosuresCzardybon:Selvita S.A.: Employment. Galezowski:Selvita S.A.: Employment. Windak:Selvita S.A.: Employment. Brzozka:Selvita S.A.: Employment.

Brown Pine Leaf Extract and Its Active Component Trans-Communic Acid Inhibit UVB-Induced MMP-1 Expression by Targeting PI3K.

Japanese red pine (Pinus densiflora) is widely present in China, Japan, and Korea. Its green pine leaves have traditionally been used as a food as well as a coloring agent. After being shed, pine leaves change their color from green to brown within two years, and although the brown pine leaves are abundantly available, their value has not been closely assessed. In this study, we investigated the potential anti-photoaging properties of brown pine leaves for skin. Brown pine leaf extract (BPLE) inhibited UVB-induced matrix metalloproteinase-1 (MMP-1) expression to a greater extent than pine leaf extract (PLE) in human keratinocytes and a human skin equivalent model. HPLC analysis revealed that the quantity of trans-communic acid (TCA) and dehydroabietic acid (DAA) significantly increases when the pine leaf color changes from green to brown. BPLE and TCA elicited reductions in UVB-induced MMP-1 mRNA expression and activator protein-1 (AP-1) transactivation by reducing DNA binding activity of phospho-c-Jun, c-fos and Fra-1. BPLE and TCA also inhibited UVB-induced Akt phosphorylation, but not mitogen activated protein kinase (MAPK), known regulators of AP-1 transactivation. We additionally found that BPLE and TCA inhibited phosphoinositide 3-kinase (PI3K), the upstream kinase of Akt, in vitro. In summary, both BPLE and its active component TCA exhibit protective effects against UVB-induced skin aging. Taken together, these findings underline the potential for BPLE and TCA to be utilized as anti-wrinkling agents and cosmetic ingredients, as they suppress UVB-induced MMP-1 expression.

Valproic Acid Downregulates NF-κB p50 Activity and IRAK-1 in a Progressive Thyroid Carcinoma Cell Line

Histone deacetylase inhibitor (HDACI) valproic acid (VPA) is a promising drug, currently in clinical phase 2, for the therapy of advanced/poorly differentiated thyroid cancer. The nuclear factor-κB (NF-κB) pathway is constitutively activated in most tumors, including thyroid carcinomas; this often contributes to aggressive tumor growth and therapeutic resistance. We hypothesized that VPA could be useful to decrease NF-κB activity in human thyroid cancer cells. To clarify this, we treated the highly progressive thyroid cancer cell line BHT-101 with VPA (1.0-3.0 mM) for 48 h. Real-time polymerase chain reaction (PCR) and Western blot were used to measure expression of NF-κB-regulatory genes and proteins. NF-κB p50 activity was measured using an ELISA-based colorimetric transcription factor assay kit. We found that VPA significantly and dose-dependently impaired NF-κB activity reducing DNA binding activity of NF-κB p50 subunit by 30% at 1 mM, 40% at 1.5 mM, and 70% at 3 mM. Expression of interleukin-1 receptor-associated kinase-1 (IRAK-1) protein, an upstream mediator of NF-κB activation, was reduced by ̴30% at 1 and 1.5 mM. Furthermore, 3 mM VPA treatment significantly decreased expressions of IRAK-1, phospho-IκBα and NF-κB p50 subunit protein by ̴ 50%. This is the first study to demonstrate that VPA decreases NF-κB activity in a progressive thyroid cancer cell line. Intriguingly, 1mM of VPA, a clinically safe dose in the therapeutic range for epilepsy, was sufficient to reduce NF-κB activity. Thus, VPA may be a promising agent to overcome chemoresistance in cancer therapy and to improve therapeutic efficiency.

Identification and Functional Characterization of P159L Mutation in HNF1B in a Family with Maturity-Onset Diabetes of the Young 5 (MODY5).

Mutation in HNF1B, the hepatocyte nuclear factor-1β (HNF-1β) gene, results in maturity-onset diabetes of the young (MODY) 5, which is characterized by gradual impairment of insulin secretion. However, the functional role of HNF-1β in insulin secretion and glucose metabolism is not fully understood. We identified a family with early-onset diabetes that fulfilled the criteria of MODY. Sanger sequencing revealed that a heterozygous P159L (CCT to CTT in codon 159 in the DNA-binding domain) mutation in HNF1B was segregated according to the affected status. To investigate the functional consequences of this HNF1B mutation, we generated a P159L HNF1B construct. The wild-type and mutant HNF1B constructs were transfected into COS-7 cells in the presence of the promoter sequence of human glucose transporter type 2 (GLUT2). The luciferase reporter assay revealed that P159L HNF1B had decreased transcriptional activity compared to wild-type (p < 0.05). Electrophoretic mobility shift assay showed reduced DNA binding activity of P159L HNF1B. In the MIN6 pancreatic β-cell line, overexpression of the P159L mutant was significantly associated with decreased mRNA levels of GLUT2 compared to wild-type (p < 0.05). However, INS expression was not different between the wild-type and mutant HNF1B constructs. These findings suggests that the impaired insulin secretion in this family with the P159L HNF1B mutation may be related to altered GLUT2 expression in β-cells rather than decreased insulin gene expression. In conclusion, we have identified a Korean family with an HNF1B mutation and characterized its effect on the pathogenesis of diabetes.

Anti‐inflammatory Activities of an Active Fraction Isolated from the root of Astragalus membranaceus in RAW 264.7 Macrophages

The root of Astragalus membranaceus (AR), which has been widely used in Traditional Chinese herbal formulae for treating foot ulcer, was found to exhibit anti-inflammatory property, but its molecular mechanism still remains unknown. We previously identified the anti-inflammatory sub-fraction using bioassay-guided fractionation. The objective of the present study was to investigate the anti-inflammatory mechanism of the major active fraction (MAF) (0.039 to 0.156 mg/mL) using lipopolysaccharide (LPS)-stimulated mouse macrophage RAW 264.7 cells. MAF was shown to inhibit LPS-induced mRNA and protein expression of inducible nitric oxide synthase by 54.7% and 65.1%, respectively. Additionally, MAF down-regulated the protein expression of cyclooxygenase-2 and MAPK regulator by 45.0% to 74.6%, as well as the reduction of DNA binding activity of nuclear factor kappa B (NFκB) by 66.5%. It also attenuated the production of prostaglandin E2 , interleukin-1 beta (IL-1β), IL-6 and tumor necrosis factor alpha by 21.2% to 86.2%. Furthermore, the chemical constituents of MAF were identified. A total of 13 known chemical compounds were found in MAF, including five isoflavonoids and eight saponins. In conclusion, a bioactive fraction of AR was identified which possessed anti-inflammatory property by reducing the release of inflammatory mediators and inactivation of NFκB through MAPK signalling pathway.

A properly configured ring structure is critical for the function of the mitochondrial DNA recombination protein, Mgm101.

Mgm101 is a Rad52-type recombination protein of bacteriophage origin required for the repair and maintenance of mitochondrial DNA (mtDNA). It forms large oligomeric rings of ∼14-fold symmetry that catalyze the annealing of single-stranded DNAs in vitro. In this study, we investigated the structural elements that contribute to this distinctive higher order structural organization and examined its functional implications. A pair of vicinal cysteines, Cys-216 and Cys-217, was found to be essential for mtDNA maintenance. Mutations to the polar serine, the negatively charged aspartic and glutamic acids, and the hydrophobic amino acid alanine all destabilize mtDNA in vivo. The alanine mutants have an increased propensity of forming macroscopic filaments. In contrast, mutations to aspartic acid drastically destabilize the protein and result in unstructured aggregates with severely reduced DNA binding activity. Interestingly, the serine mutants partially disassemble the Mgm101 rings into smaller oligomers. In the case of the C216S mutant, a moderate increase in DNA binding activity was observed. By using small angle x-ray scattering analysis, we found that Mgm101 forms rings of ∼200 Å diameter in solution, consistent with the structure previously established by transmission electron microscopy. We also found that the C216A/C217A double mutant tends to form broken rings, which likely provide free ends for seeding the growth of the super-stable but functionally defective filaments. Taken together, our data underscore the importance of a delicately maintained ring structure critical for Mgm101 activity. We discuss a potential role of Cys-216 and Cys-217 in regulating Mgm101 function and the repair of damaged mtDNA under stress conditions.

Activity of the tetrapyrrole regulator CrtJ is controlled by oxidation of a redox active cysteine located in the DNA binding domain

CrtJ from Rhodobacter capsulatus is a regulator of genes involved in the biosynthesis of haem, bacteriochlorophyll, carotenoids as well as structural proteins of the light harvesting-II complex. Fluorescence anisotropy-based DNA-binding analysis demonstrates that oxidized CrtJ exhibits ~20-fold increase in binding affinity over that of reduced CrtJ. Liquid chromatography electrospray tandem ionization mass spectrometric analysis using DAz-2, a sulfenic acid (-SOH)-specific probe, demonstrates that exposure of CrtJ to oxygen or to hydrogen peroxide leads to significant accumulation of a sulfenic acid derivative of Cys420 which is located in the helix-turn-helix (HTH) motif. In vivo labelling with 4-(3-azidopropyl)cyclohexane-1,3-dione (DAz-2) shows that Cys420 also forms a sulfenic acid modification in vivo when cells are exposed to oxygen. Moreover, a Cys420 to Ala mutation leads to a ~60-fold reduction of DNA binding activity while a Cys to Ser substitution at position 420 that mimics a cysteine sulfenic acid results in a ~4-fold increase in DNA binding activity. These results provide the first example where sulfenic acid oxidation of a cysteine in a HTH-motif leads to differential effects on gene expression.

Aloe emodin inhibits colon cancer cell migration/angiogenesis by downregulating MMP-2/9, RhoB and VEGF via reduced DNA binding activity of NF-κB

Aloe emodin (AE), a natural anthraquinone, is reported to have antiproliferative activity in various cancer cell lines. In this study we analyzed molecular mechanisms involved in the antimigratory and antiangiogenic activity of this hydroxy anthraquinone in colon cancer cell, WiDr. Our results show that a relatively non toxic concentration of AE suppressed the phorbol-12-myristyl-13-acetate (PMA) induced migration and invasion of tumor cells. On analysis for the molecules involved in the migration/invasion, we found AE downregulated mRNA expression and promoter/gelatinolytic activity of Matrix Metalloproteinase (MMP)-2/9, as well as the RhoB expression at gene and protein level. It was also a strong inhibitor of Vascular Endothelial Growth Factor (VEGF) expression, promoter activity and endothelial cell migration/invasion and in vitro angiogenesis. AE suppressed the nuclear translocation and DNA binding of NF-κB, which is an important transcription factor for controlling MMP-2/9 and VEGF gene expression. Taken together these data indicate that AE target multiple molecules responsible for cellular invasion, migration and angiogenesis. Inhibitory effect on angiogenic and metastatic regulatory processes make AE a sensible candidate as a specific blocker of tumor associated events.

Cntnap2 expression in the cerebellum of Foxp2(R552H) mice, with a mutation related to speech-language disorder

Foxp2(R552H) knock-in (KI) mice carrying a mutation related to human speech-language disorder exhibit impaired ultrasonic vocalization and poor Purkinje cell development. Foxp2 is a forkhead domain-containing transcriptional repressor that associates with its co-repressor CtBP; Foxp2(R552H) displays reduced DNA binding activity. A genetic connection between FOXP2 and CNTNAP2 has been demonstrated in vitro, but not in vivo. Here we show that Cntnap2 mRNA levels significantly increased in the cerebellum of Foxp2(R552H) KI pups, although the cerebellar population of Foxp2-positive Purkinje cells was very small. Furthermore, Cntnap2 immunofluorescence did not decrease in the poorly developed Purkinje cells of Foxp2(R552H) KI pups, although synaptophysin immunofluorescence decreased. Cntnap2 and CtBP were ubiquitously expressed, while Foxp2 co-localized with CtBP only in Purkinje cells. Taken together, these observations suggest that Foxp2 may regulate ultrasonic vocalization by associating with CtBP in Purkinje cells; Cntnap2 may be a target of this co-repressor.