1.Construction and identification of antisense c-Jun N-terminal kinase 1 eukaryotic fluorescent expressing plasmids and JNK1-/- human embryo lung fibroblasts cell line.
Hui XU ; Xiao-qing HE ; Rui CHEN ; Shi-wei YIN ; Lei PENG ; Guo-qiang WANG ; Ai-ping LI ; Jian-wei ZHOU ; Qi-zhan LIU
Chinese Journal of Industrial Hygiene and Occupational Diseases 2008;26(9):538-541
OBJECTIVETo construct antisense c-Jun N-terminal kinase 1 (JNK1) eukaryotic fluorescent expressing vector and JNK1-/- human embryo lung fibroblasts cell line.
METHODSTrizol reagent was used to extract total RNA in HELF. The proper primers of JNK1 were chosen and synthesized. RT-PCR and gene recombinant techniques were used to construct the fragment of JNK1. After purification, the PCR products were cut, and JNK1 were inserted reversely into eukaryotic fluorescent expressing vector pEGFP-C1. Enzyme-cutting and DNA auto-sequencing were used to prove the successful construction of JNK1 eukaryotic expressing vector. Then plasmids were extracted and transfected into HELF cells and screen by G418 24 h later. Monoclone was chosen and cultured. Fluorescent imaging and Western blot were used to identify the JNK1-/- HELF cell line.
RESULTSSequence analysis of pEGFP-C1-as JNK1 plasmids was same as expected. The expression level of JNK1 was inhibited markedly.
CONCLUSIONConstruction of antisense JNK1 eukaryotic fluorescent expressing vectors and JNK1-/- HELF cell line is successful.
Cell Line ; DNA, Antisense ; genetics ; Fibroblasts ; metabolism ; Genetic Vectors ; Humans ; Mitogen-Activated Protein Kinase 8 ; genetics ; metabolism ; Transfection
2.Effect of ERK1/2 on low shear stress-induced expression of IL-8 mRNA in human endothelial cells.
Min CHENG ; Yi LI ; Huaiqing CHEN ; Yongmei NIE ; Yi ZHANG ; Xiaoqing LIU
Journal of Biomedical Engineering 2005;22(2):230-234
Fluid shear stress plays an important role in many physiological and pathophysiological processes of the cardiovascular system. It modulates vascular function and structure via stimulating mechanosensitive endothelial cell signal events. Previous studies have identified that the exposure of vascular endothelial cells to fluid mechanical forces can modulate the expressions of many genes, including IL-8 gene. In order to gain an insight into the role of extracellular signal regulated kinase (ERK1/2) signal pathway in the expression of IL-8 mRNA in human umbilical vein endothelial cells (HUVECs) under the stimulation by low shear stress (4.20 dyne/cm2), we employed Western blot to measure phosphorylation of ERK1/2 and used quantitative reversal transcription-polymerase chain reaction (qRT-PCR) to assay the expression of IL-8 mRNA. The results showed: (1) Shear stress could activate ERK1/2 with a rapid, biphasic time course (maximum by 10 min and basal by 2 h); the treatment of HUVECs with Genistein (a highly specific inhibitor of tyrosine protein kinase, TPK) or PD98059 (the inhibitor of mitogen-activated protein/extracellular signal regulated kinase kinase, MEK) culd prevent shear-dependent activation of ERK1/2; (2) When treated with Genistein or PD98059, significant inhibition of IL-8 mRNA expression induced by low shear stress was observed in HUVECs. This in vitro study demonstrates that ERK1/2 plays an important role in IL-8 mRNA expression induced by low shear stress.
Cells, Cultured
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Endothelium, Vascular
;
cytology
;
metabolism
;
Humans
;
Interleukin-8
;
biosynthesis
;
genetics
;
Mitogen-Activated Protein Kinase 1
;
physiology
;
Mitogen-Activated Protein Kinase 3
;
physiology
;
RNA, Messenger
;
biosynthesis
;
genetics
;
Signal Transduction
;
Stress, Mechanical
;
Umbilical Veins
;
cytology
3.The role of Smad4 and MAPK proteins in signal transduction pathway in non-small cell lung cancer.
Xiang-Dong TONG ; Hong-Xu LIU ; Hui-Ru ZHAO ; Shi-Guang XU ; Yu LI ; Li-Bo HAN ; Lin ZHANG
Chinese Journal of Oncology 2006;28(10):741-745
OBJECTIVETo investigate the expression of Smad4 in non-small cell lung cancer (NSCLC), its correlation with MAPK (mitogen activated protein kinase) and their clinical significance in NSCLC.
METHODSWestern blotting and RT-PCR were employed to test 42 resected lung cancers and normal lung tissues for the expression of Smad4. Imunohistochemistry was used to detect Smad4 and subtribes of MAPK in 71 paraffin samples.
RESULTSThe level of protein and mRNA expression of Smad4 in lung cancer tissues were 0.2092 +/- 0.1308 and 0.3986 +/- 0. 1982, respectively, lower than those in normal tissues (0.7852 +/- 0.4386 and 1.1206 +/- 0.6772, P < 0.05). The expression of p38, ERK1 and Smad4 was associated with TNM staging (P = 0.000, 0.000 and 0.005, respectively) and JNK1 with tumor location (P = 0.028) and staging (P = 0.000). There was a correlation between p38 and Smad4 (P = 0.000). The expression of Smad4 (P = 0.0001), p38 (P = 0.0000) and JNK1 (P = 0.0208), tumor differentiation (P = 0.0059) and staging (P = 0.0000) were significantly correlated with prognosis of NSCLC by univariate analysis. Smad4 (P = 0.019), p38 (P = 0.044), tumor differentiation (P = 0.003), and staging (P = 0.020) were correlated with prognosis tested by multivariable analysis. Taking p38 and Smad4 together, we found that the negative expression of p38 and positive expression of Smad4 were associated with a better prognosis of NSCLC (P = 0.000).
CONCLUSIONSmad4 could be of importance for the initiation and development of NSCLC. There is a significant correlation between main proteins of TGF-beta/smad4 and those of ras-MAPK signal transduction pathways. The expression of Smad4 is inhibited by p38. Smad4, as well as p38, tumor differentiation and staging can be used as prognostic factors of NSCLC.
Adult ; Aged ; Blotting, Western ; Carcinoma, Non-Small-Cell Lung ; metabolism ; pathology ; Cell Differentiation ; Female ; Humans ; Lung Neoplasms ; genetics ; metabolism ; pathology ; Male ; Middle Aged ; Mitogen-Activated Protein Kinase 3 ; genetics ; metabolism ; Mitogen-Activated Protein Kinase 8 ; genetics ; metabolism ; Mitogen-Activated Protein Kinases ; genetics ; metabolism ; Neoplasm Staging ; Prognosis ; RNA, Messenger ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Signal Transduction ; Smad4 Protein ; genetics ; metabolism ; physiology ; p38 Mitogen-Activated Protein Kinases ; genetics ; metabolism
4.CSN1 inhibits c-Jun phosphorylation and down-regulates ectopic expression of JNK1.
Tomohiko TSUGE ; Suchithra MENON ; Yingchun TONG ; Ning WEI
Protein & Cell 2011;2(5):423-432
CSN1 is a component of the COP9 signalosome (CSN), a conserved protein complex with pleiotropic functions in many organs and cell types. CSN regulates ubiquitinproteasome dependent protein degradation via the deneddylation and the associated deubiquitination activities. In addition, CSN associates with protein kinases and modulates cell signaling, particularly the activator protein 1 (AP-1) pathway. We have shown previously that CSN1 suppresses AP-1 transcription activity and inhibits ultraviolet (UV) and serum activation of c-fos expression. Here we show that CSN1 can inhibit phosphorylation of proto-oncogene c-Jun product and repress c-Jun dependent transcription. Further, CSN1 dramatically downregulates ectopic expression of c-Jun N-terminal kinase 1 (JNK1) in cultured cells. The decline in JNK1 is not caused by excessive proteolysis or by 3' UTR-dependent mRNA instability, but by CSN1-dependent repression of one or multiple steps in transcriptional and posttranscriptional mechanisms. Thus, in contrast to CSN5/Jab1, which promotes AP-1 activity, CSN1 displays a negative effect on the AP-1 pathway. Finally, we discuss about the dynamic equilibrium of the CSN complexes in regulation of the AP-1 pathway.
3' Untranslated Regions
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Animals
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COP9 Signalosome Complex
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Cell Line
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Down-Regulation
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Humans
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Intracellular Signaling Peptides and Proteins
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genetics
;
metabolism
;
Mice
;
Mitogen-Activated Protein Kinase 8
;
metabolism
;
Phosphorylation
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Proto-Oncogene Proteins c-jun
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antagonists & inhibitors
;
metabolism
;
Transcription Factor AP-1
;
metabolism
5.Regulation of expression of matrix metalloproteinase-9 by JNK in Raw 264.7 cells: presence of inhibitory factor(s) suppressing MMP-9 induction in serum and conditioned media.
Yun Song LEE ; Huong Thi LAN TRAN ; Quang VAN TA
Experimental & Molecular Medicine 2009;41(4):259-268
Matrix metalloproteinase-9 (MMP-9) secreted from macrophages plays an important role in tissue destruction and inflammation through degradation of matrix proteins and proteolytic activation of cytokines/chemokines. Whereas the MEK-ERK and PI3K-Akt pathways up-regulate MMP-9 expression, regulation of MMP-9 by JNK remains controversial. Presently, we aimed to determine the role of JNK in MMP-9 regulation in Raw 264.7 cells. Inhibition of JNK by the JNK inhibitor SP600125 induced MMP-9 in the absence of serum and suppressed the expression of TNF-alpha, IL-6 and cyclooxygenase-2 in LPS-treated Raw 264.7 cells. In a knockdown experiment with small interfering RNA, suppression of JNK1 induced MMP-9 expression. Interestingly, mouse serum suppressed SP600125-mediated MMP-9 induction, similar to IFN-gamma. However, the inhibitory activity of mouse serum was not affected by pyridone 6, which inhibits Janus kinase downstream to IFN-gamma. In addition to mouse serum, conditioned media of Raw 264.7 cells contained the inhibitory factor(s) larger than 10 kDa, which suppressed SP600125- or LPS-induced MMP-9 expression. Taken together, these data suggest that JNK1 suppresses MMP-9 expression in the absence of serum. In addition, the inhibitory factor(s) present in serum or secreted from macrophages may negatively control MMP-9 expression.
Animals
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Anthracenes/metabolism
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Cell Line
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Culture Media, Conditioned/*chemistry
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Enzyme Activation
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Enzyme Induction
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Enzyme Inhibitors/metabolism
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Extracellular Signal-Regulated MAP Kinases/genetics/metabolism
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*Gene Expression Regulation, Enzymologic
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MAP Kinase Signaling System/physiology
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Macrophages/cytology/*metabolism
;
Matrix Metalloproteinase 9/genetics/*metabolism
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Mice
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Mitogen-Activated Protein Kinase 8/genetics/*metabolism
;
NF-kappa B/genetics/metabolism
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Proto-Oncogene Proteins c-akt/genetics/metabolism
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Tumor Necrosis Factor-alpha/metabolism
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p38 Mitogen-Activated Protein Kinases/genetics/metabolism
6.The effect of noise exposure on insulin sensitivity in mice may be mediated by the JNK/IRS1 pathway.
Lijie LIU ; Cong FANG ; Jing YANG ; Hongyu ZHANG ; Yi HUANG ; Chuanying XUAN ; Yongfang WANG ; Shengwei LI ; Jun SHA ; Mingming ZHA ; Min GUO
Environmental Health and Preventive Medicine 2018;23(1):6-6
BACKGROUND:
Epidemiological studies have suggested that noise exposure may increase the risk of type 2 diabetes mellitus (T2DM), and experimental studies have demonstrated that noise exposure can induce insulin resistance in rodents. The aim of the present study was to explore noise-induced processes underlying impaired insulin sensitivity in mice.
METHODS:
Male ICR mice were randomly divided into four groups: a control group without noise exposure and three noise groups exposed to white noise at a 95-dB sound pressure level for 4 h/day for 1, 10, or 20 days (N1D, N10D, and N20D, respectively). Systemic insulin sensitivity was evaluated at 1 day, 1 week, and 1 month post-noise exposure (1DPN, 1WPN, and 1MPN) via insulin tolerance tests (ITTs). Several insulin-related processes, including the phosphorylation of Akt, IRS1, and JNK in the animals' skeletal muscles, were examined using standard immunoblots. Biomarkers of inflammation (circulating levels of TNF-α and IL-6) and oxidative stress (SOD and CAT activities and MDA levels in skeletal muscles) were measured via chemical analyses.
RESULTS:
The data obtained in this study showed the following: (1) The impairment of systemic insulin sensitivity was transient in the N1D group but prolonged in the N10D and N20D groups. (2) Noise exposure led to enhanced JNK phosphorylation and IRS1 serine phosphorylation as well as reduced Akt phosphorylation in skeletal muscles in response to exogenous insulin stimulation. (3) Plasma levels of TNF-α and IL-6, CAT activity, and MDA concentrations in skeletal muscles were elevated after 20 days of noise exposure.
CONCLUSIONS
Impaired insulin sensitivity in noise-exposed mice might be mediated by an enhancement of the JNK/IRS1 pathway. Inflammation and oxidative stress might contribute to insulin resistance after chronic noise exposure.
Animals
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Biomarkers
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metabolism
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Inflammation
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physiopathology
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Insulin Receptor Substrate Proteins
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genetics
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metabolism
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Insulin Resistance
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genetics
;
immunology
;
MAP Kinase Signaling System
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physiology
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Male
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Mice
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Mice, Inbred ICR
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Mitogen-Activated Protein Kinase 8
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genetics
;
metabolism
;
Noise
;
adverse effects
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Oxidative Stress
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physiology
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Proto-Oncogene Proteins c-akt
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genetics
;
metabolism
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Random Allocation
;
Time Factors
7.27-O-(E)-p-coumaric acyl ursolic acid via JNK/SAPK signal pathway regulates apoptosis of human breast cancer MDA-MB-231 cell line.
China Journal of Chinese Materia Medica 2015;40(4):722-726
27-O-(E)-p-coumaric acyl ursolic acid( DY-17) from Ilex latifolia is a compound of the monomer. To investigate the DY-17 inducing apoptosis in the human breast cancer cell line, the MDA-MB-231 cells were used as research object in this experiment. The proliferation activity of the MDA-MB-231 cells stimulated with the different concentrations of DY-17 (20, 40 µmol · L(-1)) was detected at different time( 12, 24, 36, 48, 60,72 h) . We surveyed the DY-17 inducing apoptosis of the MDA-MB-231 cells with the fluorescent staining technology. The rate of MDA-MB-231 cells apoptosis and necrosis was determined by flow cell cytometry (FCC). Moreover, expression of JNK, phosphorylated JNK, Bax, PARP shear and caspase-3 shear related to JNK/SAPK pathways were investigated in every group ( control group, EGF group, EGF + DY-17 40 µmol · L(1) group and EGF + SP600125 group) with Western blot. The MTT results showed that, in the presence of DY-17, the proliferation activity of MDA-MB-231 cells decreased in a dose-dependent and time-dependent manner. The apoptosis and necrosis rates of MDA-MB-231 cells with DY-17(20, 40 µmol · L(-1)) groups was respectively 31.86%, 49.91% by flow cytometry and significantly increased compared with control group under Fluores- cence microscopy. Up-regulation of the JNK phosphorylation protein expression was observed in EGF group compared with control group. In addition, markedly decreased the expression of JNK phosphorylation protein were also surveyed in EGF + DY-17 40 µmol · L(-1) group compared with EGF group. The expression of Bax, shear PARP and shear caspase-3 protein in EGF + DY-17 40 µmol · L(-1) group were significantly increased in comparison with EGF group. The results showed DY-17 induced apoptosis of human breast cancer MDA-MB-231 cell line related to down-regulating JNK/SAPK signal pathways.
Apoptosis
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drug effects
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Breast Neoplasms
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drug therapy
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enzymology
;
genetics
;
Cell Line, Tumor
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Cell Proliferation
;
drug effects
;
Drugs, Chinese Herbal
;
pharmacology
;
Female
;
Humans
;
MAP Kinase Kinase 4
;
genetics
;
metabolism
;
Mitogen-Activated Protein Kinase 8
;
genetics
;
metabolism
;
Signal Transduction
;
drug effects
;
Triterpenes
;
chemistry
;
pharmacology
8.alpha-Lipoic Acid Inhibits Expression of IL-8 by Suppressing Activation of MAPK, Jak/Stat, and NF-kappaB in H. pylori-Infected Gastric Epithelial AGS Cells.
Ji Hyun CHOI ; Soon Ok CHO ; Hyeyoung KIM
Yonsei Medical Journal 2016;57(1):260-264
The epithelial cytokine response, associated with reactive oxygen species (ROS), is important in Helicobacter pylori (H. pylori)-induced inflammation. H. pylori induces the production of ROS, which may be involved in the activation of mitogen-activated protein kinases (MAPK), janus kinase/signal transducers and activators of transcription (Jak/Stat), and oxidant-sensitive transcription factor, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kappaB), and thus, expression of interleukin-8 (IL-8) in gastric epithelial cells. alpha-lipoic acid, a naturally occurring thiol compound, is a potential antioxidant. It shows beneficial effects in treatment of oxidant-associated diseases including diabetes. The present study is purposed to investigate whether alpha-lipoic acid inhibits expression of inflammatory cytokine IL-8 by suppressing activation of MAPK, Jak/Stat, and NF-kappaB in H. pylori-infected gastric epithelial cells. Gastric epithelial AGS cells were pretreated with or without alpha-lipoic acid for 2 h and infected with H. pylori in a Korean isolate (HP99) at a ratio of 300:1. IL-8 mRNA expression was analyzed by RT-PCR analysis. IL-8 levels in the medium were determined by enzyme-linked immunosorbent assay. NF-kappaB-DNA binding activity was determined by electrophoretic mobility shift assay. Phospho-specific and total forms of MAPK and Jak/Stat were assessed by Western blot analysis. ROS levels were determined using dichlorofluorescein fluorescence. As a result, H. pylori induced increases in ROS levels, mRNA, and protein levels of IL-8, as well as the activation of MAPK [extracellular signal-regulated kinase 1/2 (ERK1/2), c-Jun NH2-terminal kinase 1/2 (JNK1/2), p38], Jak/Stat (Jak1/2, Stat3), and NF-kappaB in AGS cells, which was inhibited by alpha-lipoic acid. In conclusion, alpha-lipoic acid may be beneficial for prevention and/or treatment of H. pylori infection-associated gastric inflammation.
Enzyme-Linked Immunosorbent Assay
;
Epithelial Cells/metabolism
;
Gastric Mucosa/*drug effects/metabolism/microbiology
;
Gene Expression Regulation, Bacterial
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Helicobacter Infections/immunology/*metabolism
;
Helicobacter pylori/drug effects/*pathogenicity
;
Humans
;
Interleukin-8/genetics/*metabolism
;
JNK Mitogen-Activated Protein Kinases
;
Janus Kinase 1
;
Mitogen-Activated Protein Kinases/*biosynthesis
;
NF-kappa B/*metabolism
;
RNA, Messenger/isolation & purification/metabolism
;
Reactive Oxygen Species/metabolism
;
STAT3 Transcription Factor
;
Stomach/metabolism/*microbiology
;
Thioctic Acid/*pharmacology
9.Transcription of the protein kinase C-delta gene is activated by JNK through c-Jun and ATF2 in response to the anticancer agent doxorubicin.
Byong Wook MIN ; Chang Gun KIM ; Jesang KO ; Yoongho LIM ; Young Han LEE ; Soon Young SHIN
Experimental & Molecular Medicine 2008;40(6):699-708
Expression of protein kinase C-delta (PKC delta) is up-regulated by apoptosis-inducing stimuli. However, very little is known about the signaling pathways that control PKC delta gene transcription. In the present study, we demonstrate that JNK stimulates PKC delta gene expression via c-Jun and ATF2 in response to the anticancer agent doxorubicin (DXR) in mouse lymphocytic leukemia L1210 cells. Luciferase reporter assays showed that DXR-induced activation of the PKC delta promoter was enhanced by ectopic expression of JNK1, c-Jun, or ATF2, whereas it was strongly reduced by expression of dominant negative JNK1 or by treatment with the JNK inhibitor SP600125. Furthermore, point mutations in the core sequence of the c-Jun/ATF2 binding site suppressed DXR-induced activation of the PKC delta promoter. Our results suggest an additional role for a JNK signaling cascade in DXR-induced PKC delta gene expression.
Activating Transcription Factor 2/*physiology
;
Animals
;
Anthracenes/pharmacology
;
Antibiotics, Antineoplastic/*pharmacology
;
Apoptosis
;
Cell Line, Tumor
;
Doxorubicin/*pharmacology
;
Mice
;
Mitogen-Activated Protein Kinase 8/*physiology
;
Mutation
;
Promoter Regions, Genetic
;
Protein Kinase C-delta/genetics/*metabolism
;
Proto-Oncogene Proteins c-jun/antagonists & inhibitors/*physiology
;
Signal Transduction/physiology
;
Transcription, Genetic
10.Mechanism of Shaofu Zhuyu Decoction in treatment of endometriosis-associated dysmenorrhea with syndrome of cold coagulation and blood stasis based on MSK1/2.
Yuan-Huan CHEN ; Hai-Yan MAO ; Quan-Sheng WU ; Xiao-Hua ZHANG ; Jian SHEN ; Peng FENG ; Can-Can HUANG ; Xiu-Jia JI
China Journal of Chinese Materia Medica 2022;47(17):4674-4681
This study aims to decipher the mechanism underlying the effect of Shaofu Zhuyu Decoction on endometriosis(EMT)-associated dysmenorrhea in rats with the syndrome of cold coagulation and blood stasis based on mitogen-and stress-activated protein kinase 1/2(MSK1/2).We employed a random number table to randomly assign SPF female non-pregnant rats into the sham group, and treated the rest rats with autologous transplantation+refrigerator freezing for the modeling of the syndrome of cold coagulation and blood stasis.The modeled rats were then randomly assigned into the control group and high-, medium-and low-dose Shaofu Zhuyu Decoction groups.The rats in the low-, medium-, and high-dose decoction groups were respectively administrated with 9, 4.5, and 2.3 g·kg~(-1) decoction through gavage once a day for 2 consecutive weeks, and those in the control group were administrated with 0.24 mg·kg~(-1) gestrinone through gavage once every 3 days for 2 weeks.After that, the size of ectopic focus in each rat was measured via laparotomy.Enzyme-linked immunosorbent assay(ELISA) was adopted to determine the expression of interleukin(IL)-6, IL-10, prostaglandin E2(PGE2), tumor necrosis factor-α(TNF-α).Western blot was employed to determine the protein levels of MSK1/2 and dual-specificity phosphatase 1(DUSP1) and real-time quantitative polymerase chain reaction(RT-PCR) to determine the mRNA levels of the two genes in rat eutopic endometrial tissue.Compared with the sham group, the model group showed increased levels of IL-6, PGE2, and TNF-α while decrease level of IL-10 in the serum(P<0.01).Compared with the model group, the high-and medium-dose decoction groups and the gestrinone group had declined levels of IL-6, PGE2, and TNF-α while risen level of IL-10 in the serum(P<0.01).The model group had lower protein levels and mRNA levels of MSK1/2 and DUSP1 in the eutopic endometrial tissue than the sham group(P<0.01). The high-and medium-dose decoction groups and the gestrinone group had higher protein and mRNA levels of MSK1/2 and DUSP1 in the eutopic endometrial tissue than the model group(P<0.01).The results indicated that Shaofu Zhuyu Decoction can regulate the abnormal expression of pro-inflammatory cytokines TNF-α, IL-6, and PGE2 and anti-inflammatory cytokines IL-10 and DUSP1 via MSK1/2 to alleviate EMT-associated dysmenorrhea in rats with the syndrome of cold coagulation and blood stasis.
Animals
;
Female
;
Rats
;
Anti-Inflammatory Agents/therapeutic use*
;
Cytokines
;
Dinoprostone
;
Drugs, Chinese Herbal/therapeutic use*
;
Dual-Specificity Phosphatases
;
Dysmenorrhea/genetics*
;
Endometriosis/genetics*
;
Gestrinone/therapeutic use*
;
Interleukin-10
;
Interleukin-6
;
Mitogen-Activated Protein Kinase 8/therapeutic use*
;
Mitogens/therapeutic use*
;
RNA, Messenger
;
Tumor Necrosis Factor-alpha/metabolism*