Molecular Mechanisms of Large-Conductance Ca2+-Activated Potassium Channel Activation by Ginseng Gintonin

Gintonin is a unique lysophosphatidic acid (LPA) receptor ligand found inPanax ginseng. Gintonin induces transient [Ca2+]ithrough G protein-coupled LPA receptors. Large-conductance Ca2+-activated K+(BKCa) channels are expressed in blood vessels and neurons and play important roles in blood vessel relaxation and attenuation of neuronal excitability. BKCachannels are activated by transient [Ca2+]iand are regulated by various Ca2+-dependent kinases. We investigated the molecular mechanisms of BKCachannel activation by gintonin. BKCachannels are heterologously expressed inXenopus oocytes. Gintonin treatment induced BKCachannel activation in oocytes expressing the BKCachannelαsubunit in a concentration-dependent manner (EC50= 0.71 ± 0.08 µg/mL). Gintonin-mediated BKCachannel activation was blocked by a PKC inhibitor, calphostin, and by the calmodulin inhibitor, calmidazolium. Site-directed mutations in BKCachannels targeting CaM kinase II or PKC phosphorylation sites but not PKA phosphorylation sites attenuated gintonin action. Mutations in the Ca2+bowl and the regulator of K+conductance (RCK) site also blocked gintonin action. These results indicate that gintonin-mediated BKCachannel activations are achieved through LPA1 receptor-phospholipase C-IP3-Ca2+-PKC-calmodulin-CaM kinase II pathways and calcium binding to the Ca2+bowl and RCK domain. Gintonin could be a novel contributor against blood vessel constriction and over-excitation of neurons.

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The Anti-Atherogenic Properties of Sesamin are Mediated via Improved Macrophage Cholesterol Efflux Through PPARγ1-LXRα and MAPK Signaling

International Journal for Vitamin and Nutrition Research ◽

10.1024/0300-9831/a000195 ◽

2014 ◽

Vol 84 (1-2) ◽

pp. 79-91 ◽

Cited By ~ 7

Author(s):

Amin F. Majdalawieh ◽

Hyo-Sung Ro

Keyword(s):

Cholesterol Efflux ◽

Transcriptional Activity ◽

Molecular Mechanisms ◽

Foam Cell ◽

Mapk Signaling ◽

Luciferase Reporter ◽

Foam Cell Formation ◽

Dependent Manner ◽

Concentration Dependent Manner ◽

Macrophage Cholesterol Efflux

Background: Foam cell formation resulting from disrupted macrophage cholesterol efflux, which is triggered by PPARγ1 and LXRα, is a hallmark of atherosclerosis. Sesamin and sesame oil exert anti-atherogenic effects in vivo. However, the exact molecular mechanisms underlying such effects are not fully understood. Aim: This study examines the potential effects of sesamin (0, 25, 50, 75, 100 μM) on PPARγ1 and LXRα expression and transcriptional activity as well as macrophage cholesterol efflux. Methods: PPARγ1 and LXRα expression and transcriptional activity are assessed by luciferase reporter assays. Macrophage cholesterol efflux is evaluated by ApoAI-specific cholesterol efflux assays. Results: The 50 μM, 75 μM, and 100 μM concentrations of sesamin up-regulated the expression of PPARγ1 (p< 0.001, p < 0.001, p < 0.001, respectively) and LXRα (p = 0.002, p < 0.001, p < 0.001, respectively) in a concentration-dependent manner. Moreover, 75 μM and 100 μM concentrations of sesamin led to 5.2-fold (p < 0.001) and 6.0-fold (p<0.001) increases in PPAR transcriptional activity and 3.9-fold (p< 0.001) and 4.2-fold (p < 0.001) increases in LXR transcriptional activity, respectively, in a concentration- and time-dependent manner via MAPK signaling. Consistently, 50 μM, 75 μM, and 100 μM concentrations of sesamin improved macrophage cholesterol efflux by 2.7-fold (p < 0.001), 4.2-fold (p < 0.001), and 4.2-fold (p < 0.001), respectively, via MAPK signaling. Conclusion: Our findings shed light on the molecular mechanism(s) underlying sesamins anti-atherogenic effects, which seem to be due, at least in part, to its ability to up-regulate PPARγ1 and LXRα expression and transcriptional activity, improving macrophage cholesterol efflux. We anticipate that sesamin may be used as a therapeutic agent for treating atherosclerosis.

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R2D5 antigen: a calcium-binding phosphoprotein predominantly expressed in olfactory receptor neurons.

The Journal of Cell Biology ◽

10.1083/jcb.123.4.963 ◽

1993 ◽

Vol 123 (4) ◽

pp. 963-976 ◽

Cited By ~ 17

Author(s):

Y Nemoto ◽

J Ikeda ◽

K Katoh ◽

H Koshimoto ◽

Y Yoshihara ◽

...

Keyword(s):

Signal Transduction ◽

Calcium Binding ◽

Olfactory Receptor ◽

Olfactory Receptor Neurons ◽

Ependymal Cells ◽

Cam Kinase Ii ◽

Cam Kinase ◽

Dependent Protein Kinase ◽

Dependent Protein ◽

Receptor Neurons

R2D5 is a mouse monoclonal antibody that labels rabbit olfactory receptor neurons. Immunoblot analysis showed that mAb R2D5 recognizes a 22-kD protein with apparent pI of 4.8, which is abundantly contained in the olfactory epithelium and the olfactory bulb. We isolated cDNA for R2D5 antigen and confirmed by Northern analysis and neuronal depletion technique that R2D5 antigen is expressed predominantly, but not exclusively, in olfactory receptor neurons. Analysis of the deduced primary structure revealed that R2D5 antigen consists of 189 amino acids with calculated M(r) of 20,864 and pI of 4.74, has three calcium-binding EF hands, and has possible phosphorylation sites for Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) and cAMP-dependent protein kinase (A kinase). Using the bacterially expressed protein, we directly examined the biochemical properties of R2D5 antigen. R2D5 antigen binds Ca2+ and undergoes a conformational change in a manner similar to calmodulin. R2D5 antigen is phosphorylated in vitro by CaM kinase II and A kinase at different sites, and 1.81 and 0.80 mol of Pi were maximally incorporated per mol of R2D5 antigen by CaM kinase II and A kinase, respectively. Detailed immunohistochemical study showed that R2D5 antigen is also expressed in a variety of ependymal cells in the rabbit central nervous system. Aside from ubiquitous calmodulin, R2D5 antigen is the first identified calcium-binding protein in olfactory receptor neurons that may modulate olfactory signal transduction. Furthermore our results indicate that olfactory receptor neurons and ependymal cells have certain signal transduction components in common, suggesting a novel physiological process in ependymal cells.

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Methanol Extract of Mongolian Iris bungei Maxim. Stimulates 3T3-L1 Adipocyte Differentiation

Journal of Nanoscience and Nanotechnology ◽

10.1166/jnn.2021.19160 ◽

2021 ◽

Vol 21 (7) ◽

pp. 3943-3949

Author(s):

Jaegoo Yeon ◽

Sung-Suk Suh ◽

Ui-Joung Youn ◽

Badamtsetseg Bazarragchaa ◽

Ganbold Enebish ◽

...

Keyword(s):

Bacterial Infections ◽

Fatty Acid Synthase ◽

Molecular Mechanisms ◽

Adipocyte Differentiation ◽

Methanol Extract ◽

Regulatory Element ◽

Peroxisome Proliferator ◽

Dependent Manner ◽

Concentration Dependent Manner ◽

Peroxisome Proliferator Activated Receptor

Iris bungei Maxim. (IB), which is native to China and Mongolia, is used as a traditional medicine for conditions such as inflammation, cancer, and bacterial infections. However, the effects of Iris bungei Maxim. on adipocyte differentiation have not been studied. In the present study, we first demonstrated the molecular mechanisms underlying the adipogenic activity of the methanol extract of Mongolian I. bungei Maxim. (IB). IB significantly enhanced intracellular lipid accumulation and adipocyte differentiation in 3T3-L1 preadipocytes in a concentration-dependent manner. Moreover, IB markedly stimulated the expression of genes related to adipogenesis such as peroxisome proliferator-activated receptor γ, adiponectin, and aP2. In addition, we also observed that IB induces lipogenic genes such as fatty acid synthase, sterol regulatory element binding protein 1c, stearoyl-CoA desaturase, and acetyl-CoA carboxylase. Interestingly IB regulated adipocyte differentiation in both the early and middle stages. Taken together, these adipogenic and lipogenic effects of IB suggest its efficacy for the prevention and/or treatment of type 2 diabetes.

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Betulinic Acid Suppresses Ovarian Cancer Cell Proliferation through Induction of Apoptosis

Biomolecules ◽

10.3390/biom9070257 ◽

2019 ◽

Vol 9 (7) ◽

pp. 257 ◽

Cited By ~ 1

Author(s):

Dahae Lee ◽

Seoung Rak Lee ◽

Ki Sung Kang ◽

Yuri Ko ◽

Changhyun Pang ◽

...

Keyword(s):

Ovarian Cancer ◽

Cancer Cell ◽

Betulinic Acid ◽

Molecular Mechanisms ◽

Underlying Mechanism ◽

Nuclear Staining ◽

Dependent Manner ◽

Meoh Extract ◽

Concentration Dependent Manner ◽

Induction Of Apoptosis

Ovarian cancer is one of the leading causes of cancer deaths worldwide in women, and the most malignant cancer among the different gynecological cancers. In this study, we explored potentially anticancer compounds from Cornus walteri (Cornaceae), the MeOH extract of which has been reported to show considerable cytotoxicity against several cancer cell lines. Phytochemical investigations of the MeOH extract of the stem and stem bark of C. walteri by extensive application of chromatographic techniques resulted in the isolation of 14 compounds (1–14). The isolated compounds were evaluated for inhibitory effects on the viability of A2780 human ovarian carcinoma cells and the underlying molecular mechanisms were investigated. An 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was employed to assess the anticancer effects of compounds 1–14 on A2780 cells, which showed that compound 11 (betulinic acid) reduced the viability of these cells in a concentration-dependent manner and had an half maximal (50%) inhibitory concentration (IC50) of 44.47 μM at 24 h. Nuclear staining and image-based cytometric assay were carried out to detect the induction of apoptosis by betulinic acid. Betulinic acid significantly increased the condensation of nuclei and the percentage of apoptotic cells in a concentration-dependent manner in A2780 cells. Western blot analysis was performed to investigate the underlying mechanism of apoptosis. The results indicated that the expression levels of cleaved caspase-8, -3, -9, and Bax were increased in A2780 cells treated with betulinic acid, whereas those of Bcl-2 were decreased. Thus, we provide the experimental evidence that betulinic acid can induce apoptosis in A2780 cells through both mitochondria-dependent and -independent pathways and suggest the potential use of betulinic acid in the development of novel chemotherapeutics for ovarian cancer therapy.

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Vascular Protective Effects of Xanthotoxin and Its Action Mechanism in Rat Aorta and Human Vascular Endothelial Cells

Journal of Vascular Research ◽

10.1159/000509112 ◽

2020 ◽

Vol 57 (6) ◽

pp. 313-324

Author(s):

Li-Hua Cao ◽

Ho Sub Lee ◽

Zhe-Shan Quan ◽

Yun Jung Lee ◽

Yu Jin

Keyword(s):

Endothelial Cells ◽

Cell Adhesion ◽

Adhesion Molecule ◽

Molecular Mechanisms ◽

Umbilical Vein ◽

Neuroprotective Effect ◽

Intercellular Adhesion Molecule ◽

Dependent Manner ◽

Protective Effects ◽

Concentration Dependent Manner

Objective: Xanthotoxin (XAT) is a linear furanocoumarin mainly extracted from the plants Ammi majus L. XAT has been reported the apoptosis of tumor cells, anti-convulsant, neuroprotective effect, antioxidative activity, and vasorelaxant effects. This study aimed to investigate the vascular protective effects and underlying molecular mechanisms of XAT. Methods: XAT’s activity was studied in rat thoracic aortas, isolated with aortic rings, and human umbilical vein endothelial cells (HUVECs). Results: XAT induced endothelium-dependent vasodilation in a concentration-dependent manner in the isolated rat thoracic aortas. Removal of endothelium or pretreatment of aortic rings with L-NAME, 1H-[1,2,4]-oxadiazolo-[4,3-a]-quinoxalin-1-one, and wortmannin significantly inhibited XAT-induced relaxation. In addition, treatment with thapsigargin, 2-aminoethyl diphenylborinate, Gd3+, and 4-aminopyridine markedly attenuated the XAT-induced vasorelaxation. XAT increased nitric oxide production and Akt- endothelial NOS (eNOS) phosphorylation in HUVECs. Moreover, XAT attenuated the expression of TNF-α-induced cell adhesion molecules such as intercellular adhesion molecule, vascular cell adhesion molecule-1, and E-selectin. However, this effect was attenuated by the eNOS inhibitors L-NAME and asymmetric dimethylarginine. Conclusions: This study suggests that XAT induces vasorelaxation through the Akt-eNOS-cGMP pathway by activating the KV channel and inhibiting the L-type Ca2+ channel. Furthermore, XAT exerts an inhibitory effect on vascular inflammation, which is correlated with the observed vascular protective effects.

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Suppression of the TGF-β pathway by a macrolide antibiotic decreases fibrotic responses by ocular fibroblasts in vitro

Royal Society Open Science ◽

10.1098/rsos.200441 ◽

2020 ◽

Vol 7 (9) ◽

pp. 200441

Author(s):

Thomas Stahnke ◽

Beata Gajda-Deryło ◽

Anselm G. Jünemann ◽

Oliver Stachs ◽

Katharina A. Sterenczak ◽

...

Keyword(s):

Molecular Mechanisms ◽

Transforming Growth Factor ◽

Macrolide Antibiotic ◽

Drug Repositioning ◽

Dependent Manner ◽

Glaucoma Filtration Surgery ◽

Concentration Dependent Manner ◽

Device Implantation

To elucidate and to inhibit post-surgical fibrotic processes after trabeculectomy in glaucoma therapy, we measured gene expression in a fibrotic cell culture model, based on transforming growth factor TGF-β induction in primary human tenon fibroblasts (hTFs), and used Connectivity Map (CMap) data for drug repositioning. We found that specific molecular mechanisms behind fibrosis are the upregulation of actins, the downregulation of CD34, and the upregulation of inflammatory cytokines such as IL6, IL11 and BMP6 . The macrolide antibiotic Josamycin (JM) reverses these molecular mechanisms according to data from the CMap, and we thus tested JM as an inhibitor of fibrosis. JM was first tested for its toxic effects on hTFs, where it showed no influence on cell viability, but inhibited hTF proliferation in a concentration-dependent manner. We then demonstrated that JM suppresses the synthesis of extracellular matrix (ECM) components. In hTFs stimulated with TGF-β1, JM specifically inhibited α-smooth muslce actin expression, suggesting that it inhibits the transformation of fibroblasts into fibrotic myofibroblasts. In addition, a decrease of components of the ECM such as fibronectin, which is involved in in vivo scarring, was observed. We conclude that JM may be a promising candidate for the treatment of fibrosis after glaucoma filtration surgery or drainage device implantation in vivo .

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Resveratrol inhibits Kv2.2 currents through the estrogen receptor GPR30-mediated PKC pathway

AJP Cell Physiology ◽

10.1152/ajpcell.00146.2013 ◽

2013 ◽

Vol 305 (5) ◽

pp. C547-C557 ◽

Cited By ~ 22

Author(s):

Wen-Hao Dong ◽

Jia-Chen Chen ◽

Yan-Lin He ◽

Jia-Jie Xu ◽

Yan-Ai Mei

Keyword(s):

Molecular Mechanisms ◽

Cerebellar Granule Cells ◽

Dependent Manner ◽

Patch Clamp Technique ◽

Concentration Dependent Manner ◽

Α Subunit ◽

Inactivation Curve ◽

Hek 293 ◽

Nongenomic Action ◽

Α Subunits

Resveratrol (REV) is a naturally occurring phytoalexin that inhibits neuronal K+ channels; however, the molecular mechanisms behind the effects of REV and the relevant α-subunit are not well defined. With the use of patch-clamp technique, cultured cerebellar granule cells, and HEK-293 cells transfected with the Kv2.1 and Kv2.2 α-subunits, we investigated the effect of REV on Kv2.1 and Kv2.2 α-subunits. Our data demonstrated that REV significantly suppressed Kv2.2 but not Kv2.1 currents with a fast, reversible, and mildly concentration-dependent manner and shifted the activation or inactivation curve of Kv2.2 channels. Activating or inhibiting the cAMP/PKA pathway did not abolish the inhibition of Kv2.2 current by REV. In contrast, activation of PKC with phorbol 12-myristate 13-acetate mimicked the inhibitory effect of REV on Kv2.2 by modifying the activation or inactivation properties of Kv2.2 channels and eliminated any further inhibition by REV. PKC and PKC-α inhibitor completely eliminated the REV-induced inhibition of Kv2.2. Moreover, the effect of REV on Kv2.2 was reduced by preincubation with antagonists of GPR30 receptor and shRNA for GPR30 receptor. Western blotting results indicated that the levels of PKC-α and PKC-β were significantly increased in response to REV application. Our data reveal, for the first time, that REV inhibited Kv2.2 currents through PKC-dependent pathways and a nongenomic action of the oestrogen receptor GPR30.

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Evaluation of Anti-Inflammatory Activities of Qingre-Qushi Recipe (QRQS) against Atopic Dermatitis: Potential Mechanism of Inhibition of IL-33/ST2 Signal Transduction

Evidence-based Complementary and Alternative Medicine ◽

10.1155/2017/2489842 ◽

2017 ◽

Vol 2017 ◽

pp. 1-11 ◽

Cited By ~ 3

Author(s):

Mengjiao Chen ◽

Peijun Ding ◽

Lili Yang ◽

Xufeng He ◽

Chunjie Gao ◽

...

Keyword(s):

Signal Transduction ◽

Molecular Mechanisms ◽

Histological Study ◽

High Dose ◽

Dependent Manner ◽

Hacat Cells ◽

Concentration Dependent Manner ◽

Dermal Thickness ◽

Significant Difference ◽

Anti Inflammatory

To evaluate the anti-inflammatory activities of QRQS against AD and the inhibitory molecular mechanisms of IL-33/ST2 signal transduction, BALB/c mice were divided into six groups (normal control, OVA control, low-dose of QRQS, middle-dose of QRQS, high-dose of QRQS, and cetirizine) and epicutaneously exposed to ovalbumin or PBS for 3 weeks and treated with QRQS for 2 weeks. Skin biopsies and blood samples were obtained for histological study, antibody analysis, and RNA isolation. HaCaT cells, stimulated by TNF-α and IFN-γ, were treated with QRQS to evaluate mRNA and protein expression by RT-PCR and ELISA. QRQS decreased both epidermal and dermal thickness, alleviated dermatitis, and reduced IL-33 and ST2 positive cell numbers. The concentration of specific IgE, IgG, IgG1, and IgG2a antibodies in serum and the expression of IL-33, ST2, IL-1RAcP, IL-4, and IL-13 mRNA in the skin were suppressed. No significant difference exists in TNF-α or IFN-γ. QRQS decreased IL-33 mRNA and protein secretion in HaCaT cells exposed to TNF-α and IFN-γ in a time- and concentration-dependent manner. QRQS regulates related molecule expression of ovalbumin-induced dermatitis involved in the IL-33/ST2 signaling axis in the treatment of acute AD.

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Molecular Bases of Human Malformation Syndromes Involving the SHH Pathway: GLIA/R Balance and Cardinal Phenotypes

International Journal of Molecular Sciences ◽

10.3390/ijms222313060 ◽

2021 ◽

Vol 22 (23) ◽

pp. 13060

Author(s):

Yo Niida ◽

Sumihito Togi ◽

Hiroki Ura

Keyword(s):

Molecular Mechanisms ◽

Brain Size ◽

Balance Model ◽

Dependent Manner ◽

Loss Of Function ◽

Concentration Dependent Manner ◽

Shh Pathway ◽

Embryonic Morphogenesis ◽

Malformation Syndromes ◽

Molecular Bases

Human hereditary malformation syndromes are caused by mutations in the genes of the signal transduction molecules involved in fetal development. Among them, the Sonic hedgehog (SHH) signaling pathway is the most important, and many syndromes result from its disruption. In this review, we summarize the molecular mechanisms and role in embryonic morphogenesis of the SHH pathway, then classify the phenotype of each malformation syndrome associated with mutations of major molecules in the pathway. The output of the SHH pathway is shown as GLI activity, which is generated by SHH in a concentration-dependent manner, i.e., the sum of activating form of GLI (GLIA) and repressive form of GLI (GLIR). Which gene is mutated and whether the mutation is loss-of-function or gain-of-function determine in which concentration range of SHH the imbalance occurs. In human malformation syndromes, too much or too little GLI activity produces symmetric phenotypes affecting brain size, craniofacial (midface) dysmorphism, and orientation of polydactyly with respect to the axis of the limb. The symptoms of each syndrome can be explained by the GLIA/R balance model.

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DDK regulates replication initiation by controlling the multiplicity of Cdc45-GINS binding to Mcm2-7

10.1101/2020.12.09.417428 ◽

2020 ◽

Author(s):

Lorraine De Jesus Kim ◽

Larry J Friedman ◽

Christian Ramsoomair ◽

Jeff Gelles ◽

Stephen P Bell

Keyword(s):

Single Molecule ◽

Replication Origin ◽

Molecular Mechanisms ◽

Replication Initiation ◽

Phosphorylation Sites ◽

Dependent Manner ◽

Multiplicity Results ◽

Biochemical Assays ◽

Eukaryotic Dna ◽

Two Stages

The committed step of eukaryotic DNA replication occurs when the replicative Mcm2-7 helicase pairs that license each replication origin are activated. Helicase activation requires the recruitment of Cdc45 and GINS to Mcm2-7, forming Cdc45-Mcm2-7-GINS complexes (CMGs). Using single-molecule biochemical assays to monitor CMG formation, we found that Cdc45 and GINS are recruited to loaded Mcm2-7 in two stages. Initially, Cdc45 and GINS are individually recruited to unstructured Mcm2-7 N-terminal tails in a Dbf4-dependent kinase (DDK)-dependent manner, forming Cdc45-tail-GINS intermediates (CtGs). The multiple phosphorylation sites on the Mcm2-7 tails promote DDK-dependent modulation of the number of CtGs formed per Mcm2-7. In a second, inefficient event, a subset of CtGs transfer their Cdc45 and GINS components to form CMGs. Importantly, higher CtG multiplicity results in increased frequency of CMG formation. Our findings reveal molecular mechanisms sensitizing helicase activation to DDK levels with implications for the control of replication origin efficiency and timing.

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