Phosphorylation Levels Of c-Jun Research Articles

CREB1 promotes cholangiocarcinoma metastasis through transcriptional regulation of the LAYN-mediated TLN1/β1 integrin axis: CREB1 promotes cholangiocarcinoma metastasis through regulating LAYN/TLN1/β1 integrin axis

BackgroundLayilin (LAYN) plays an important role in tumor progression, invasion, and metastasis; however, its role in cholangiocarcinoma (CHOL) has not been elucidated. MethodsWe utilized the GEPIA, STRING, and hTFtarget databases for bioinformatics analysis. Overexpression or knockdown cell lines were constructed by transfecting the cells with different plasmids. Western blot (WB) was performed to detect LAYN, TLN1, and CREB1 expression. Cell proliferation, migration, and invasiveness were assessed using CCK-8 and Transwell assays. Immunofluorescence and WB were used to detect epithelial-mesenchymal transition (EMT) markers. The CHOL metastasis model was established by injecting RBE cells into the tail veins of nude mice. Metastatic lesions were identified using hematoxylin and eosin staining. Co-immunoprecipitation and Chromatin immunoprecipitation were used to validate the interactions. ResultsLAYN was highly expressed in the CHOL cells. Knockdown of LAYN significantly inhibited proliferation, migration, invasion, and EMT in both QBC-939 and RBE human CHOL cells, while overexpression of LAYN had the opposite effect. Furthermore, in a CHOL metastasis model using nude mice, knocking down LAYN expression markedly suppressed CHOL liver and lung metastases. LAYN interacts with TLN1, and CREB1 binds to the LAYN promoter, with all three showing a positive correlation. Additionally, bioinformatics analysis revealed high expression of both TLN1 and CREB1 in CHOL. Knockdown of TLN1 or CREB1 in QBC-939 and RBE cells inhibited cell proliferation, migration, invasion, and EMT, reversing the effects of LAYN overexpression. Moreover, knockdown of TLN1 or CREB1 also suppressed the expression of ITGB1 and the phosphorylation levels of c-Jun, p38 MAPK, and ERK, further reversing the effects of LAYN overexpression. ConclusionOur results suggest that CREB1 promotes CHOL metastasis through transcriptional regulation of the LAYN-mediated TLN1/β1 integrin axis.

Antiphotoaging Effects of Damiana (Turnera diffusa) Leaves Extract via Regulation AP-1 and Nrf2/ARE Signaling Pathways.

Damiana (Turnera diffusa), of the family Passifloraceae, has been widely studied for its pharmacological effects, especially for antioxidant and antibacterial actions. However, there are limited scientific findings describing its antiphotoaging effects on the skin. In the present study, the underlying molecular mechanisms of the protective effect of Damiana were investigated in keratinocytes (HaCaTs) and normal human dermal fibroblasts (HDFs) subject to UVB irradiation. The mRNA expression of matrix metalloproteinases (MMPs) and procollagen type I was determined by reverse transcription-polymerase chain reaction. The protein expression of antiphotoaging-related signaling molecules in the activator protein-1 (AP-1) and nuclear factor erythroid 2-related factor 2 (NRF2)/antioxidant response element (ARE) pathways was assessed by Western blotting. We observed that Damiana blocked the upregulated production of reactive oxygen species induced in UVB-irradiated HaCaTs and HDFs in a dose-dependent manner. Treatment with Damiana also significantly ameliorated the mRNA expression of MMPs and procollagen type I. In addition, the phosphorylation level of c-Jun and c-Fos was also decreased through the attenuated expression of p-38, p-ERK, and p-JNK after treatment with Damiana. Furthermore, the treatment of cells with Damiana resulted in the inhibition of Smad-7 expression in the TGF-β/Smad pathway and upregulated the expression of the Nrf2/ARE signaling pathway. Hence, the synthesis of procollagen type I, a precursor of collagen I, was promoted. Collectively, these results provide us with the novel insight that Damiana is a potential source of antiphotoaging compounds.

Aldehyde dehydrogenase 3B2 promotes the proliferation and invasion of cholangiocarcinoma by increasing Integrin Beta 1 expression

Aldehyde dehydrogenases (ALDHs) play an essential role in regulating malignant tumor progression; however, their role in cholangiocarcinoma (CCA) has not been elucidated. We analyzed the expression of ALDHs in 8 paired tumor and peritumor perihilar cholangiocarcinoma (pCCA) tissues and found that ALDH3B1 and ALDH3B2 were upregulated in tumor tissues. Further survival analysis in intrahepatic cholangiocarcinoma (iCCA, n = 27), pCCA (n = 87) and distal cholangiocarcinoma (dCCA, n = 80) cohorts have revealed that ALDH3B2 was a prognostic factor of CCA and was an independent prognostic factor of iCCA and pCCA. ALDH3B2 expression was associated with serum CEA in iCCA and dCCA, associated with tumor T stage, M stage, neural invasion and serum CA19-9 in pCCA. In two cholangiocarcinoma cell lines, overexpression of ALDH3B2 promoted cell proliferation and clone formation by promoting the G1/S phase transition. Knockdown of ALDH3B2 inhibited cell migration, invasion, and EMT in vitro, and restrained tumor metastasis in vivo. Patients with high expression of ALDH3B2 also have high expression of ITGB1 in iCCA, pCCA, and dCCA at both mRNA and protein levels. Knockdown of ALDH3B2 downregulated the expression of ITGB1 and inhibited the phosphorylation level of c-Jun, p38, and ERK. Meanwhile, knockdown of ITGB1 inhibited the promoting effect of ALDH3B2 overexpression on cell proliferation, migration, and invasion. ITGB1 is also a prognostic factor of iCCA, pCCA, and dCCA and double-positive expression of ITGB1 and ALDH3B2 exhibits better performance in predicting patient prognosis. In conclusion, ALDH3B2 promotes tumor proliferation and metastasis in CCA by regulating the expression of ITGB1 and upregulating its downstream signaling pathway. The double-positive expression of ITGB1 and ALDH3B2 serves as a better prognostic biomarker of CCA.

Abstract 6380: Suppression of the solar ultraviolet-induced skin carcinogenesis by TOPK inhibitor HI-TOPK-032

Abstract Non-melanoma skin cancer (NMSC) such as cutaneous squamous cell carcinoma (cSCC) is caused by solar ultraviolet (SUV) exposure and is the most common cancer in the United States. T-LAK cell-originated protein kinase (TOPK), a serine-threonine kinase is activated by SUV irradiation and involved in skin carcinogenesis. Strategies with research focusing on the TOPK signaling pathway and targeted therapy in skin carcinogenesis may helpful for the discovery of additional treatments against skin cancer. In this study, we found that TOPK can directly bind to and phosphorylate c-Jun (as one of the core member of AP-1) at Ser63 and Ser73 after SSL exposure in a JNKs-independent manner. TOPK knocking down, or HI-TOPK-032 (TOPK specific inhibitor) attenuated colony formation and cell proliferation of skin cancer cells. Phosphorylated levels of c-Jun were overexpressed in human AK and cSCC compared to normal skin tissues, and HI-TOPK-032 inhibited the phosphorylation of c-Jun in SCC cell line in a dose-dependent manner. Furthermore, HI-TOPK-032 decreased SSL-induced AP-1 transactivation activity. Moreover, acute SSL-induced inflammation was attenuated by the topical application of HI-TOPK-032 in SKH1 hairless mice. Importantly, HI-TOPK-032 suppressed chronic SSL-induced skin carcinogenesis and c-Jun phosphorylation levels in SKH1 hairless mice. Our results demonstrate that TOPK can phosphorylate and activate c-Jun at Ser63 and Ser73 in the process of skin carcinogenesis and HI-TOPK-032 could be used as a potential chemopreventive drug against cSCC development. Citation Format: Eunmiri Roh, Yaping Han, Kanamata Reddy, Tatyana A. Zykova, Mee Hyun Lee, Ke Yao, Ruihua Bai, Clara Curiel-Lewandrowski, Zigang Dong. Suppression of the solar ultraviolet-induced skin carcinogenesis by TOPK inhibitor HI-TOPK-032 [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6380.

Suppression of the solar ultraviolet-induced skin carcinogenesis by TOPK inhibitor HI-TOPK-032.

Nonmelanoma skin cancer (NMSC) such as cutaneous squamous cell carcinoma (cSCC) is caused by solar ultraviolet (SUV) exposure and is the most common cancer in the United States. T-LAK cell-originated protein kinase (TOPK), a serine-threonine kinase is activated by SUV irradiation and involved in skin carcinogenesis. Strategies with research focusing on the TOPK signaling pathway and targeted therapy in skin carcinogenesis may helpful for the discovery of additional treatments against skin cancer. In this study, we found that TOPK can directly bind to and phosphorylate c-Jun (as one of the core member of AP-1) at Ser63 and Ser73 after SSL exposure in a JNKs-independent manner. TOPK knocking down, or HI-TOPK-032 (TOPK specific inhibitor) attenuated colony formation and cell proliferation of skin cancer cells. Phosphorylated levels of c-Jun were overexpressed in human AK and cSCC compared with normal skin tissues, and HI-TOPK-032 inhibited the phosphorylation of c-Jun in SCC cell line in a dose-dependent manner. Furthermore, HI-TOPK-032 decreased SSL-induced AP-1 transactivation activity. Moreover, acute SSL-induced inflammation was attenuated by the topical application of HI-TOPK-032 in SKH1 hairless mice. Importantly, HI-TOPK-032 suppressed chronic SSL-induced skin carcinogenesis and c-Jun phosphorylation levels in SKH1 hairless mice. Our results demonstrate that TOPK can phosphorylate and activate c-Jun at Ser63 and Ser73 in the process of skin carcinogenesis and HI-TOPK-032 could be used as a potential chemopreventive drug against cSCC development.

Prunella vulgaris L. Exerts a Protective Effect Against Extrinsic Aging Through NF-κB, MAPKs, AP-1, and TGF-β/Smad Signaling Pathways in UVB-Aged Normal Human Dermal Fibroblasts.

Prunella vulgaris L., a well-known traditional Chinese herbal medicine, has anti-inflammatory and antioxidant activities. In the present study, the underlying molecular mechanisms of the protective effect of P. vulgaris extract (PVE) were investigated in UVB-irradiated normal human dermal fibroblasts (NHDFs). The mRNA expression of matrix metalloproteinases (MMPs), procollagen type I, and cytokines, such as interleukin-6 (IL-6) and tumor necrosis factor (TNF-α), was determined by reverse transcription–polymerase chain reaction. The expression of anti-photoaging-related signaling molecules in the NF-κB, MAPK/AP-1, and TGF/Smad pathways was assessed by western blot. We observed that PVE blocked the upregulated production of radical oxygen species induced in UVB-irradiated NHDFs in a dose-dependent manner. Treatment with PVE also significantly ameliorated the mRNA levels of MMPs, procollagen type I, TNF-α, and IL-6. In addition, the phosphorylation level of c-Jun and c-Fos was decreased through the attenuated expression levels of p-ERK and p-JNK after treatment with PVE. Furthermore, cells treated with PVE showed inhibited Smad 7 and increased Smad 2/3 expression in the TGF-β/Smad signaling pathway. Hence, synthesis of procollagen type I, a precursor of collagen I, was promoted. These findings indicate that treatment with PVE has a potential protective effect against UVB-induced photoaging and photoinflammation.

Abstract 1456: Integrin beta4-induced mitophagy promotes the lactate production of cancer-associated fibroblasts in breast cancer

Abstract Integrin beta4 neoexpression is induced in cancer-associated fibroblasts (CAFs) by some triple negative breast cancer cells (MDA-MB-231 and BT-20) through a contact-dependent manner. Integrin beta4 expression is well known to contribute to the migration and survival of cancer cells. However, there is very little known about the role of integrin beta4 expression in CAFs, especially in the aspect of tumor progression. In our study, we found that the exogenous expression of integrin beta4 increased the lactate production of CAFs. The observation made us to hypothesize that integrin beta4 neoexpression is associated with the mitophagy, a cause for aerobic glycolysis, in CAFs. To confirm the assumption, the expression alteration of mitophagy-related genes was first screened in the CAF transfected with integrin beta4 overexpression plasmid. BCL2 interacting protein 3 like (BNIP3L) and microtubule associated protein 1 light chain 3 alpha-II (LC3-II) were significantly upregulated in the integrin beta4 overexpressing CAFs, which was reversed both in the knockdown of integrin beta4 expression and the treatment with an integrin beta4 inhibitor. Autophagosome maturation and lysosomal fusion were also increased in the integrin beta4 overexpressing CAFs compared to the control. Phosphorylation level of c-Jun was upregulated by integrin beta4 overexpression in CAFs and was able to bind to the putative promoter regions of BNIP3L and LC3-II. Taken together, these results demonstrate that integrin beta4 neoexpression induces upregulation of BNIP3L and LC3-II ultimately leading to mitophagy in CAFs, contributing to cancer metabolism via mitophagy-induced lactate production, namely reverse Warburg effect. Citation Format: Jin Sol Sung, Suki Kang, Joo Hyun Lee, Seong Gyeong Mun, Baek Gil Kim, Nam Hoon Cho. Integrin beta4-induced mitophagy promotes the lactate production of cancer-associated fibroblasts in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1456.

MAPK/c-Jun signaling pathway contributes to the upregulation of the anti-apoptotic proteins Bcl-2 and Bcl-xL induced by Epstein-Barr virus-encoded BARF1 in gastric carcinoma cells.

BARF1, encoded by Epstein-Barr virus (EBV), has been hypothesized to function as an oncogene, which was expressed in gastric carcinoma cells. Additionally, it has been reported that the anti-apoptotic function is closely associated with the expression of the B-cell lymphoma-2 (Bcl-2) protein. In addition, the signaling pathway has been reported to be involved in numerous diseases, including the mitogen-activated protein kinase (MAPK) cascade. In order to study the specific mechanism of anti-apoptotic function, BARF1-stably-expressing immortalized normal human embryo gastric epithelial cell line GES1 (GES-BARF1), and well-, moderately- and poorly-differentiated gastric carcinoma cell lines, MKN28 (which has been reported to be contaminated with the moderately-differentiated MKN74 gastric carcinoma cell line), SGC7901 and BGC823 (MKN-BARF1, SGC-BARF1 and BGC-BARF1, respectively) (GCC-BARF1) were constructed, with transfection of cells with the empty vector pSG5 acting as controls. Western blot analysis was performed to analyze the protein expression and the phosphorylation levels. Compared with the controls, it was found that the protein expression levels of c-Jun, Bcl-2 and B-cell lymphoma-extra large (Bcl-xL), as well as the phosphorylation levels of c-Jun, c-Jun N-terminal kinase (JNK) 1/2/3, p38 and extracellular signal-regulated kinase (ERK) 1/2 proteins were upregulated in 3 GCC-BARF1 but not significantly changed in GES-BARF1. The expression levels of the c-Jun, Bcl-2 and Bcl-xL proteins, and levels of c-Jun protein phosphorylation were significantly decreased in SGC-BARF1 cells subsequent to treatment with SP600125, SB203580, and U0126, which were the specific inhibitors of JNK1/2/3, p38 and ERK1/2 respectively. In addition, there was a gradual increase in the protein expression and phosphorylation levels between normal gastric epithelial cells, and well-differentiated, moderately-differentiated and poorly-differentiated gastric carcinoma cells, but this was not statistically significant. Therefore, the present study hypothesized that JNK1/2/3-, p38- and ERK1/2-MAPK/c-Jun cascade signaling pathways may contribute to the upregulation of the expression of the anti-apoptotic proteins Bcl-2 and Bcl-xL induced by BARF1 in gastric carcinoma cells. This mechanism may mainly work in the progressive phase of the development in EBV-associated gastric carcinoma.

Berberine Suppresses Fibronectin Expression through Inhibition of c-Jun Phosphorylation in Breast Cancer Cells

PurposeThe exact mechanism regulating fibronectin (FN) expression in breast cancer cells has not been fully elucidated. In this study, we investigated the pharmacological mechanism of berberine (BBR) with respect to FN expression in triple-negative breast cancer (TNBC) cells.MethodsThe clinical significance of FN mRNA expression was analyzed using the Kaplan-Meier plotter database (http://kmplot.com/breast). FN mRNA and protein expression levels were analyzed by real-time polymerase chain reaction and western blotting, respectively.ResultsUsing publicly available clinical data, we observed that high FN expression was associated with poor prognosis in patients with breast cancer. FN mRNA and protein expression was increased in TNBC cells compared with non-TNBC cells. As expected, recombinant human FN significantly induced cell spreading and adhesion in MDA-MB231 TNBC cells. We also investigated the regulatory mechanism underlying FN expression. Basal levels of FN mRNA and protein expression were downregulated by a specific activator protein-1 (AP-1) inhibitor, SR11302. Interestingly, FN expression in TNBC cells was dose-dependently decreased by BBR treatment. The level of c-Jun phosphorylation was also decreased by BBR treatment.ConclusionOur findings demonstrate that FN expression is regulated via an AP-1–dependent mechanism, and that BBR suppresses FN expression in TNBC cells through inhibition of AP-1 activity.

Effect of ophiopogonin D in resisting vascular endothelial cell apoptosis induced by AngⅡthrough up-regulating CYP2J2/EETs

This study aimed to investigate the effect and mechanism of ophiopogonin D (OP-D) on Ang Ⅱ-induced HUVECs apoptosis, in order to provide a reliable basis for the safety and efficacy of traditional Chinese medicines. The effect of Ang Ⅱ on survival and total proteins content of HUVECs were measured by MTT and Western blotting. The effect of OP-D on Ang Ⅱ-induced lactate dehydrogenase (LDH) release rate in HUVECs was measured by enzyme standard instrument. The effects of OP-D and 11,12-EET on phosphorylation of JNK/c-Jun induced by Ang Ⅱ were measured by Western blot and RT-PCR with the help of JNK specific inhibitor SP600125 and CYP450 isozymes selective inhibitor 6-(2-propargyloxyphenyl) hexanoic acid (PPOH). The cell apoptosis was assayed by flow cytometry. According to the results, different doses of Ang Ⅱ had no significant effect on cell survival; treatment with Ang Ⅱ at 1×10⁻⁶ mol·L⁻¹ could increase the release of LDH (P</i><0.001), improve the JNK and c-Jun phosphorylation levels(P</i><0.01, P</i><0.001), increase the expression of caspase-3(P</i><0.01), and promote the apoptosis of HUVECs(P</i><0.001). The phosphorylation of JNK and c-Jun could be inhibited by the pre-treatment with SP600125, 11,12-EET and OP-D. Pre-treatment with OP-D could significantly reduce the release of LDH induced by Ang Ⅱ stimulation, decrease the expression of caspase-3, and diminish the apoptosis of cells. The protective effect of OP-D was suppressed, when being pretreated with PPOH. The experimental results showed that the apoptosis of HUVECs induced by Ang Ⅱ may be associated with JNK/c-Jun signaling pathway. OP-D-mediated CYP2J2 expression increased 11,12-EET levels, and could remarkably resist Ang Ⅱ-induced injury and apoptosis of cells, which is associated with the maintenance of endothelium homeostasis.

Zinc-finger protein 418 overexpression protects against cardiac hypertrophy and fibrosis.

BackgroundThis study aimed to investigated the effect and mechanism of zinc-finger protein 418 (ZNF418) on cardiac hypertrophy caused by aortic banding (AB), phenylephrine (PE) or angiotensin II (Ang II) in vivo and in vitro.MethodsThe expression of ZNF418 in hearts of patients with dilated cardiomyopathy (DCM) or hypertrophic cardiomyopathy (HCM) and AB-induced cardiac hypertrophy mice, as well as in Ang II- or PE-induced hypertrophic primary cardiomyocytes was detected by western blotting. Then, the expression of ZNF418 was up-regulated or down-regulated in AB-induced cardiac hypertrophy mice and Ang II -induced hypertrophic primary cardiomyocytes. The hypertrophic responses and fibrosis were evaluated by echocardiography and histological analysis. The mRNA levels of hypertrophy markers and fibrotic markers were detected by RT-qPCR. Furthermore, the phosphorylation and total levels of c-Jun were measured by western blotting.ResultsZNF418 was markedly down-regulated in hearts of cardiac hypertrophy and hypertrophic primary cardiomyocytes. Down-regulated ZNF418 exacerbated the myocyte size and fibrosis, moreover increased the mRNA levels of ANP, BNP, β-MHC, MCIP1.4, collagen 1a, collagen III, MMP-2 and fibronection in hearts of AB-treated ZNF418 knockout mice or Ang II-treated cardiomyocytes with AdshZNF418. Conversely, these hypertrophic responses were reduced in the ZNF418 transgenic (TG) mice treated by AB and the AdZNF418-transfected primary cardiomyocytes treated by Ang II. Additionally, the deficiency of ZNF418 enhanced the phosphorylation level of c-jun, and overexpression of ZNF418 suppressed the phosphorylation level of c-jun in vivo and in vitro.ConclusionZNF418 maybe attenuate hypertrophic responses by inhibiting the activity of c-jun/AP-1.

MicroRNA-126 Inhibits Proliferation, Migration, Invasion, and EMT in Osteosarcoma by Targeting ZEB1.

Osteosarcoma (OS) is a tumor with rapid progression, high metastatic potential and poor clinical prognosis. This study was aimed to investigate the function of miR-126 in OS cells. The miR-126 expression in OS cell lines and OS tissues were explored by qRT-PCR. Then, the effects of miR-126 on proliferation, cycle, migration, invasion, and transforming growth factor (TGF)-β1 induced epithelial to mesenchymal transition (EMT) were assessed. Predicted by TargetScan, one of target genes for miR-126 was verified by luciferase activity assay. Meantime, the mRNA and protein expressions of ZEB1 were assessed by qRT-PCR and Western blot assay. Subsequently, the effect of ZEB1 silence on miR-126 down-regulated cells was also evaluated. Finally, the expressions of key kinases involved in c-Jun N-terminal kinase (JNK) and Janus-activated kinase (JAK)-1/signal transducer and activator of transcription (STAT)-3 pathways were detected by Western blot analysis. Result showed that miR-126 was down-regulated in OS tissues and cell lines. Overexpression of miR-126 significantly inhibited cell proliferation, migration, invasion, and TGF-β1 induced EMT. The effect of miR-126 knockdown was just the opposite. ZEB1 was predicted and verified as a target gene of miR-126. Meantime, the influence of miR-126 knockdown was abrogated by ZEB1 silence. Additionally, the phosphorylation levels of c-Jun, JNK, JAK1, and STAT3 were down-regulated in miR-126 over-expressed cells, and the effect of miR-126 knockdown was reversed by ZEB1 silence. In conclusion, miR-126 inhibits proliferation, migration, invasion and EMT in OS by targeting ZEB1 through inactivation of JNK and JAK1/STAT3 pathways. J. Cell. Biochem. 118: 3765-3774, 2017. © 2017 Wiley Periodicals, Inc.

RETRACTED ARTICLE: MicroRNA-30d promotes angiogenesis and tumor growth via MYPT1/c-JUN/VEGFA pathway and predicts aggressive outcome in prostate cancer

BackgroundEven though aberrant expression of microRNA (miR)-30d has been reported in prostate cancer (PCa), its associations with cancer progression remain contradictory. The aim of this study was to investigate clinical significance, biological functions and underlying mechanisms of miR-30d deregulation in PCa.MethodsInvolvement of miR-30d deregulation in malignant phenotypes of PCa was demonstrated by clinical sample evaluation, and in vitro and in vivo experiments. The mechanisms underlying its regulatory effect on tumor angiogenesis were determined.ResultsmiR-30d over-expression was observed in both PCa cells and clinical specimens. High-miR-30d was distinctly associated with high pre-operative PSA and Gleason score, advanced clinical and pathological stages, positive metastasis and biochemical recurrence (BCR), and reduced overall survival of PCa patients. Through gain- and loss-of-function experiments, we found that miR-30d promoted PCa cell proliferation, migration, invasion, and capillary tube formation of endothelial cells, as well as in vivo tumor growth and angiogenesis in a mouse model. Simulation of myosin phosphatase targeting subunit 1 (MYPT1), acting as a direct target of miR-30d, antagonized the effects induced by miR-30d up-regulation in PCa cells. Notably, miR-30d/MYPT1 combination was identified as an independent factor to predict BCR of PCa patients. Furthermore, miR-30d exerted its pro-angiogenesis function, at least in part, by inhibiting MYPT1, which in turn, increased phosphorylation levels of c-JUN and activated VEGFA-induced signaling cascade in endothelial cells.ConclusionsmiR-30d and/or its target gene MYPT1 may serve as novel prognostic markers of PCa. miR-30d promotes tumor angiogenesis of PCa through MYPT1/c-JUN/VEGFA pathway.

Synergistic effects of c-Jun and SP1 in the promotion of TGFβ1-mediated diabetic nephropathy progression

Diabetic nephropathy (DN) is a major complication of diabetes mellitus. Transforming growth factor beta 1 (TGFβ1) is a well-distinguished mediator of progressive renal fibrosis in DN. However, the molecular mechanisms contributing to enhanced TGFβ1 expression in the progression of DN are not fully understood. Herein, we reported that c-Jun and specificity protein 1 (SP1) were critical upstream regulators of TGFβ1 expression in DN. The increase in c-Jun and SP1 expressions was positively correlated with TGFβ1 in both high glucose-treated human renal mesangial cells (HRMCs) and diabetic kidneys. Furthermore, c-Jun dose-dependently promoted SP1-mediated TGFβ1 transcription and vice versa. The synergistic effects of c-Jun and SP1 were attributed to their auto-regulation and cross-activation. Moreover, enhanced phosphorylation levels of c-Jun and SP1 were accompanied with increased TGFβ1 expression in diabetic kidneys. Accordingly, dephosphorylation of c-Jun and SP1 by the specific c-Jun N-terminal kinase (JNK) inhibitor SP600125 prevented the increase in TGFβ1 expression. These results suggested that c-Jun and SP1 synergistically activated profibrotic TGFβ1 expression in the development of DN by auto-regulation, cross-activation and phospho-modification.

Cathelicidin-BF, a Novel Antimicrobial Peptide from Bungarus fasciatus, Attenuates Disease in a Dextran Sulfate Sodium Model of Colitis

Antimicrobial peptides are molecules of innate immunity. Cathelicidin-BF is the first cathelicidin peptide found in reptiles. However, the immunoregulatory and epithelial barrier protective properties of C-BF have not been reported. Inflammatory bowel diseases, including ulcerative colitis and Crohn's disease, can lead to colon cancer, the third most common malignant tumor. The objective is to develop the new found cathelicidin-BF as a therapeutic to patients of ulcerative colitis. The morphology of the colon epithelium was observed by H&E staining; apoptosis index and infiltration of inflammatory cells in colonic epithelium were measured by TUNEL and immunohistochemistry; the expression level of endogenous mCRAMP was analyzed by immunofluorescence; and phosphorylation of the transcription factors c-jun and NF-κB in colon were analyzed by Western blot. Our results showed that the morphology of the colon epithelium in the C-BF+DSS group was improved compared with the DSS group. Apoptosis and infiltration of inflammatory cells in colonic epithelium were also significantly attenuated in the C-BF+DSS group compared with the DSS group, and the expression level of endogenous mCRAMP in the DSS group was significantly higher than other groups. DSS-induced phosphorylation level of c-jun and NF-κB while C-BF effectively inhibited phosphorylation of NF-κB (p65). The barrier protective effect of C-BF was still excellent. In conclusion, C-BF effectively attenuated inflammation and improved disrupted barrier function. Notably, this is the first report to demonstrate that C-BF attenuates DSS-induced UC both through the regulation of intestinal immune and retention of barrier function, and the exact pathway was through NF-κB.

Inhibition of Vav3 could reverse the drug resistance of gastric cancer cells by downregulating JNK signaling pathway.

This study aims to investigate the effect and mechanism of Vav3 on the multidrug resistance of gastric cancer. Fluorescence quantitative RT-PCR and western blot assay were used to detect Vav3 and drug resistance genes in gastric cancer tissues as well as gastric cell lines such as SGC7901, SGC7901/adriamycin (ADR) and GES-1. Besides, Vav3-specific small interfering RNA (Vav3-siRNA) was applied to inhibit Vav3 in SGC7901/ADR, and SRB assay was used to determine chemosensitivity. After that, drug resistance genes and proteins in MAPK and PI3K/AKT signaling pathway were detected after Vav3-siRNA transfection. The results showed that overexpressed Vav3 was found in gastric cancer tissues and SGC7901 and SGC7901/ADR cells. Activity of SGC7901/ADR cells transfected with Vav3-siRNA combined with 5-fluorouracil/oxaliplatin was much lower than that of control groups, and MDR1/P-gp, GST-π and Bcl-2, Bax genes were significantly downregulated in Vav3-siRNA transfection group. AKT, ERK and p38 total protein and their phosphorylation levels showed no significant change in Vav3-siRNA-transfected SGC7901/ADR cells, whereas the ratio of C-Jun phosphorylation levels to total C-Jun protein was significantly downregulated. The results suggested that Vav3 may play a role in drug resistance of gastric cancer by inhibiting drug resistance genes MDR1/P-gp, GST-π and Bcl-2 through regulating the JNK signaling pathway.

Mechanisms regulating p21 gene expression by retinoic acid-induced gene G protein

To investigate the mechanisms by which retinoic acid-induced gene G (RIG-G) protein regulates p21 gene expression. Western blot was used to detect the effects of RIG-G protein overexpression on p21 protein expression level in leukemia cell line NB4 cells and the phosphorylation of both c-Jun and JNK in U937 cells. The c-Jun expression plasmid and p21 gene promoter-containing reporter plasmid were co-transfected into 293T cells, to explore the regulatory effect of c-Jun protein on p21 gene expression by luciferase reporter assay. Western blot showed that the overexpression of RIG-G protein significantly upregulated p21 protein level in the NB4 cells, and the level of p21 protein largely increased along with the induction of endogenous RIG-G protein during the differentiation of NB4 cells treated by all-trans retinoic acid (ATRA). Moreover, the phosphorylation of both c-Jun and JNK decreased in RIG-G-overexpressing U937 cells while total c-Jun and JNK proteins remained unchanged. After using the JNK inhibitor SP600125 to block JNK phosphorylation, the level of c-Jun phosphorylation was still dramatically reduced in the RIG-G-overexpressing U937T-RIG-G cells, compared with the control U937T-pTRE cells. These results indicated that the inhibitory effect of Rig-G protein on c-Jun phosphorylation could not only be through the JNK pathway, but also via some JNK-independent pathways. Luciferase reporter assay showed that when 0.1, 0.5, 1.0 and 2.0 µg c-Jun-expressing plasmids were respectively transfected into 293T cells, compared with the empty vector-transfected group, the relative luciferase activities were (83.0 ± 1.7)%, (73.7 ± 0.7)%, (68.9 ± 0.9)% and (64.1 ± 0.9)%, indicating that the transcriptional activity of p21 gene could be inhibited by c-Jun protein. RIG-G protein may suppress the phosphorylation of c-Jun protein through different signal pathways, thereby increasing the expression of p21 gene, arresting the cell cycle and inhibiting the cell growth in U937 cells.

Activation of caspase-3 and c-Jun NH2-terminal kinase signaling pathways involving heroin-induced neuronal apoptosis

Heroin has been shown to cause spongiform leukoencephalopathy (SLE) in heroin addicts. In this study, we found that heroin could induce apoptosis of primary cultured cerebellar granule cells (CGC) and c-Jun N-terminal kinase (JNK) pathway is activated during CGCs apoptosis. Inhibiting JNK with a specific inhibitor, SP600125, reduced the levels of c-Jun phosphorylation and caspase-3 activation. We also showed that use the JNK inhibitor SP600125, caspase inhibitor z-VAD, or use SP600125 and z-VAD together significantly suppressed cell death induced by heroin. These results indicate that JNK pathway is an important mediator of the neurotoxic effects of heroin and inhibiting JNK activity may represent a new and effective strategy to treat heroin-induced SLE.

The Cancer/Testis Antigen CAGE with Oncogenic Potential Stimulates Cell Proliferation by Up-regulating Cyclins D1 and E in an AP-1- and E2F-dependent Manner

A cancer/testis antigen, CAGE, is widely expressed in various cancer tissues and cancer cell lines but not in normal tissues except the testis. In the present study, ectopic expression of CAGE in fibroblast cells resulted in foci formation, suggesting its cell-transforming ability. Using stable HeLa transfectant clones with the tetracycline-inducible CAGE gene, we found that CAGE overexpression stimulated both anchorage-dependent and -independent cell growth in vitro and promoted tumor growth in a xenograft mouse model. Cell cycle analysis showed that CAGE augments the levels of cyclin D1 and E, thereby activating cyclin-associated cyclin-dependent kinases and subsequently accelerating the G(1) to S progression. Moreover, increased cyclin D1 and E levels in CAGE-overexpressing cells were observed even in a growth arrested state, indicating a direct effect of CAGE on G(1) cyclin expression. CAGE-induced expression of cyclins D1 and E was found to be mediated by AP-1 and E2F-1 transcription factors, and among the AP-1 members, c-Jun and JunD appeared to participate in CAGE-mediated up-regulation of cyclin D1. CAGE overexpression also enhanced retinoblastoma phosphorylation and subsequent E2F-1 nuclear translocation. In contrast, small interfering RNA-mediated knockdown of CAGE suppressed the expression of G(1) cyclins, activation of AP-1 and E2F-1, and cell proliferation in both HeLa cervical cancer cells and Malme-3M melanoma cells. These results suggest that the cancer/testis antigen CAGE possesses oncogenic potential and promotes cell cycle progression by inducing AP-1- and E2F-dependent expression of cyclins D1 and E.

Abstract 4371: PI3K/Akt/JNK/c-Jun signaling pathway is a mediator for arsenite-induced cyclin D1 expression and cell growth in human bronchial epithelial cells

Abstract Arsenite exposure is associated with an increased risk of lung cancers. Although efforts have been made, the precise mechanisms of the arsenite-induced lung carcinogenesis remain enigmatic. Aberrant cyclin D1 expression is an early event in carcinogenesis often accompanies human malignancies in the lung. Our previous study has shown that arsenite was able to induce cyclin D1 expression in human keratinocyte HaCat and mouse epidermal Cl41 cells to promote cell cycle transition, cell proliferation and transformation, which accounts for its ability to induce skin carcinogenesis. However, the role of Cyclin D1 underlying the arsenite-induced human lung carcinogenesis remain elusive. In the present study, arsenite exposure induced the cell cycle transition, resulting in growth promotion of human bronchial epithelial cells. The promoter activity and the expression of cyclin D1 were augmented in response to arsenite treatment. To further evaluate the effect of cyclin D1 in arsenite-induced cell proliferation, a cyclin D1 siRNA construct was stably introduced into Beas-2B cells. The siRNA blocked the induction/expression of cyclin D1. It has been reported that JNKs mediate the cyclin D1 expression in hematopoietic cells. Therefore, we investigated the potential contribution of JNKs to cyclin D1 expression induced by arsenite. Our results strongly suggest that JNK1 is a major kinase responsible for maintaining the basal level of C-Jun phosphorylation, and both JNK1 and JNK2 are critical for arsenite-induced C-Jun phosphorylation. Moreover, using a loss of function mutant Dp85 or DN-Akt, we demonstrated that the PI-3K/Akt pathway was involved in arsenite-induced upregulation of cyclin D1 and cell proliferation. The introduction of Dp85 or DN-Akt into the cells also inhibited JNKs activation as well as c-Jun phosphorylation, while the growth promotion induced by arsenite was significantly inhibited by the PI3K inhibitor Ly294002 and JNK inhibitor, or by over expression of DN-Akt. Overall, our data suggest a signaling pathway of PI-3K/Akt/JNK/c-Jun/cylin D1 signaling in response to arsenite in human bronchial epithelial cells, which leading to the growth promotion. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4371.