Induction of apoptosis in ovarian carcinoma cells by AHPN/CD437 is mediated by retinoic acid receptors

Retinoids have great promise in the area of cancer therapy and chemoprevention. These natural and synthetic derivatives of vitamin A have been shown to play an important role in regulating cell differentiation and proliferation. While all-trans-retinoic acid (ATRA) has been demonstrated to inhibit the growth of several ovarian tumor cell lines, other ovarian carcinoma cell lines have been found to be resistant to retinoid dependent growth suppression. Interestingly, a novel synthetic retinoid, CD437 or AHPN, has been demonstrated to inhibit the growth of both ATRA-sensitive (CA-OV3) and ATRA-resistant (SK-OV3) ovarian tumor cell lines as well as to induce apoptosis. The overall goal of this research was to understand the mechanism by which AHPN/CD437 induces apoptosis in ovarian tumor cell lines. Since a number of studies have demonstrated the importance of nuclear receptors (RARs and RXRs) in mediating cellular responses to retinoids, we wished to determine the role of RARs in mediating the AHPN/CD437 response. We modulated RAR level and function by overexpressing either wild type RAR-gamma or a pan dominant negative mutant of all RAR subtypes called RAR-beta (R269Q), or through the use of an RAR-gamma antagonist, MM11253. We found that inhibition of RAR function reduced but did not eliminate induction of apoptosis in both CA-OV3 and SK-OV3 cells by AHPN/CD437. Likewise, overexpression of wild type RAR-gamma was found to increase apoptosis after treatment with AHPN/CD437. Our results suggest that in ovarian carcinomas, AHPN/CD437 induced apoptosis is mediated at least in part via an RAR pathway.     

Injury-induced osteopontin gene expression in rat arterial smooth muscle cells is dependent on mitogen-activated protein kinases ERK1/ERK2.

Previous work shows that osteopontin has a role during matrix reorganization after tissue injury including vascular conditions such as atherosclerosis and restenosis following angioplasty. In vitro, osteopontin promotes activities such as adhesion and migration but the mechanisms that regulate the expression of this matrix protein remain essentially unknown. This study examined if the ERK signaling pathway is involved in injury-induced osteopontin expression in cultured rat aortic smooth muscle cells. Northern and Western blotting demonstrated a marked activation of osteopontin expression in response to injury. Treating the cells with PD98059, a specific MEK1 inhibitor, prior to injury, blocked this upregulation. MEK1 phosphorylates ERK1/ERK2, which belong to the family of mitogen-activated protein kinases. We conclude that ERK1/ERK2 are involved in the regulation of osteopontin expression in cultured vascular smooth muscle cells.     

Ets1 was significantly activated by ERK1/2 in mutant K-ras stably transfected human adrenocortical cells

In our previous study on the tumorigenesis of human functional adrenal tumors, we observed a high frequency of point mutation in the K-ras gene in clinical adrenal tumors. Therefore, we analyzed gene profiles of mutant K-ras transfected adrenocortical cells by DNA microarray to determine the expression pattern of genes related to cell cycle, signal transduction, apoptosis, tumorigenesis, steroidogenesis, and other expressed sequence tags (ESTs). Then we analyzed all of the significant differentially expressed genes by bioinformatics tools, "Matchminer" and "Gominer." The results revealed that expression of mutant K-ras gene induced by IPTG upregulated Ets1, which was mainly related to cell proliferation. After carefully being analyzed by software "DAVID" and "Pathart," Ets1 was found to be activated by being phosphorylated at theronine 38 by ERK1/2, and in turn, to regulate the following genes: uPA, MMP-3, and prolactin (Ling et al., 2003; Duffy and Daggan, 2004; Maupas-Schwalm et al., 2004; van Themsche et al., 2004). The result of Western blotting analysis confirmed that Ets1 was really phosphorylated when mutant K-ras was activated. On the other hand, the membrane blotting analyses indicated that the expression levels of uPA, MMP-3, and prolactin in human adrenocortical cells stably transfected with the mutant K-ras gene were significantly higher than those in normal control cells. Compared to control cells, the level of prolactin raised 1.4-fold, the level of MMP-3 raised 1.8-fold, and the level of uPA raised 2.1-fold in the transfected cells. From the results of this study, we proposed a mechanism of Ets1 in human adrenocortical cells expressing a mutated K-ras gene.     

Phorbol myristate acetate-induced Egr-1 expression is suppressed by phospholipase D isozymes in human glioma cells

Early growth response-1 (Egr-1) is involved in the regulation of cell growth. Here, we found that overexpression of phospholipase D (PLD) isozymes decreased tumor promoter phorbol myristate acetate (PMA)-induced Egr-1 expression and transactivation in glioma cells. Suppression of PMA-induced Egr-1 was dependent on the expression level of PLD isozymes. Overexpression of catalytically inactive PLD, treatment with PA, and prevention of PA dephosphorylation by 1-propranolol significantly suppressed PMA-induced Egr-1 expression. PLD-induced suppression of Egr-1 was reversed by inhibition of phosphatidylinositol 3-kinase (PI3K). Taken together, these results suggest that elevated expression and activity of PLD attenuate PMA-induced Egr-1 expression via PI3K pathway.     

Discoidin domain receptor 1 expression in activated T cells is regulated by the ERK MAP kinase signaling pathway

The expression and function of discoidin domain receptor 1 (DDR1) in T cells are still poorly explored. We have recently shown that activation of primary human T cells via their T cell receptor leads to increased expression of DDR1, which promoted their migration in three-dimensional collagen. In the present study, we provide evidence that activated T cells bind collagen through DDR1. We found that the DDR1:Fc blocking molecule significantly reduced the ability of activated T cells to bind soluble biotinylated collagen. However, DDR1:Fc had no impact on the adhesion of activated T cells to collagen and overexpression of DDR1 in Jurkat T cells did not enhance their adhesion. Together, our results indicate that DDR1 can promote T cell migration without enhancing adhesion to collagen, suggesting that it can contribute to the previously described amoeboid movement of activated T cells in collagen matrices. Our results also show that CD28, in contrast to IL-2 expression, did not costimulate the expression of DDR1 in primary human T cells. Using specific inhibitors, we demonstrated that TCR-induced expression of DDR1 in T cells is regulated by the Ras/Raf/ERK MAP Kinase and PKC pathways but not by calcium/calcineurin signaling pathway or the JNK and P38 MAP Kinases. Thus, our study provides additional insights into the physiology of DDR1 in T cells and may therefore further our understanding of the regulatory mechanisms of T cell migration.     

ERK/Egr-1 signaling pathway is involved in CysLT2 receptor-mediated IL-8 production in HEK293 cells

The CysLT₂ receptor is involved in myocardial ischemia/reperfusion injury, differentiation of colorectal cancers, bleomycin-induced pulmonary inflammation and fibrosis. However, the signal transduction of cysteinyl leukotriene receptor 2 (CysLT₂) in inflammatory responses remains to be clarified. In HEK293 cells stably expressing hCysLT₁, hCysLT₂ and rGPR17, we determined the signaling pathways for interleukin-8 (IL-8) production after CysLT₂ receptor activation. HEK293 cells were stably transfected with the recombinant plasmids of pcDNA3.1(+)-hCysLT₁, pcDNA3.1(+)-hCysLT₂ and pcDNA3.1-rGPR17. Leukotriene C₄ (LTC₄) and LTD₄ were used as the agonists to induce IL-8 production and the related changes in signal molecules. We found that LTC₄ and LTD₄ significantly induced IL-8 promoter activation in the HEK293 cells stably expressing hCysLT₂, but not in those expressing hCysLT₁ and rGPR17. In hCysLT₂-HEK293 cells, LTC₄ induced elevation of intracellular calcium, ERK1/2 phosphorylation and Egr-1 expression, and stimulated IL-8 expression and release. These responses were blocked by the selective CysLT₂ receptor antagonist HAMI3379. The ERK1/2 inhibitor U0126 inhibited Egr-1 and IL-8 expression as well as IL-8 release, but the JNK and p38 inhibitors did not have the inhibitory effects. Down-regulation of Egr-1 by RNA interference with its siRNA inhibited the LTC₄-induced IL-8 expression and release. In conclusion, these findings indicate the ERK-Egr-1 pathway of CysLT₂ receptors mediates IL-8 production induced by the pro-inflammatory mediators LTC₄ and LTD₄.     

The novel retinoid AHPN/CD437 induces a rapid but incomplete apoptotic response in human myeloma cells

The synthetic retinoid 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid (AHPN/CD437) appears to possess an apoptotic activity superior to classical retinoids in vitro as in vivo. Numerous studies have shown that CD437-induced apoptosis is independent of its nuclear receptor activity, suggesting that CD437 might have a unique mechanism of action. The purpose of this study was to compare CD437- and all-trans retinoic acid (atRA)-induced cell death. CD437 provoked a rapid apoptotic phenotype immediately followed by secondary necrosis in RPMI 8226, U266 and L363 human myeloma cell lines. Nuclear apoptotic features were observed upon both CD437 and atRA treatments. In contrast, membrane blebbing and the subsequent formation of apoptotic bodies, a classical apoptotic event, was only observed upon atRA treatment. In addition, CD437, contrary to atRA, was unable to induce tissue transglutaminase (tTG), an intracellular enzyme involved in the formation of cross-linked protein polymers contributing to apoptotic morphological changes. Taken together, these data suggest that CD437 induces rapid but incomplete apoptotic phenotype in human myeloma cells.     

Induction or suppression of expression of cytochrome C oxidase subunit II by heregulin beta 1 in human mammary epithelial cells is dependent on the levels of ErbB2 expression

The ErbB family of receptor kinases is composed of four members: epidermal growth factor receptor (EGFR/ErbB1), ErbB2/neu, ErbB3, and ErbB4. Amplification of the ErbB2/neu is found in about 30% of breast cancer patients and is associated with a poor prognosis. Heregulin (HRG) activates the ErbB2 via induction of heterodimerization with ErbB3 and ErbB4 receptors. With suppression subtractive hybridization, we demonstrated that the expression of cytochrome c oxidase subunit II (COXII) is HRG-responsive. Two nontransformed human mammary epithelial cell lines, the HB2 and the HB2(ErbB2) (the HB2 engineered to overexpress ErbB2), displayed an opposite response to HRG-mediated regulation. HRG upregulated mRNA expression of COXII in the HB2 cells, but suppressed COXII expression in the HB2(ErbB2) cells. A human breast cancer cell line (T47D), which expresses ErbB2 at a level similar to that of the HB2 cells, also responded to HRG by increasing COXII mRNA levels. Therefore, HRG regulation of COXII expression depends on the levels of ErbB2 expression. Furthermore, the expression of COXII was inversely correlated to the levels of ErbB2, i.e., the cells overexpressing ErbB2 exhibited lower COXII levels. HRG-evoked signal transduction differed between the cells with normal ErbB expression and the cells overexpressing ErbB2. The activation of both ERK and PI3-K was essential for HRG regulation of COXII, i.e., blockage of either pathway eliminated HRG-mediated alteration. This is the first report demonstrating that the expression of mitochondria-encoded COXII is HRG-responsive. The levels of ErbB2 expression are decisive for the diverse biological activities of HRG.     

Induction of Egr-1 is associated with anti-metastatic and anti-invasive ability of beta-lapachone in human hepatocarcinoma cells

beta-lapachone, a quinone compound obtained from the bark of the lapacho tree (Tabebuia avellanedae), was reported to have anti-inflammatory and anti-cancer activities. In this study, we investigated novel functions of beta-lapachone in terms of anti-metastasis and anti-invasion abilities using human hepatocarcinoma cell lines, HepG2 and Hep3B. beta-lapachone dose-dependently inhibited cell viability and migration of both HepG2 and Hep3B cells, as determined by methylthiazoletetrazolium (MTT) assay and wound healing assay. RT-PCR and Western blot data revealed that beta-lapachone dramatically increased the levels of protein, as well as mRNA expression of early growth response gene-1 (Egr-1) and throbospondin-1 (TSP-1) at an early point in time, and then decreased in a time-dependent manner. In addition, down-regulation of Snail and up-regulation of E-cadherin expression were observed in beta-lapachone-treated HepG2 and Hep3B cells, and this the associated with decreased invasive ability as measured by matrigel invasion assay. Taken together, our results strongly suggest that beta-lapachone may be expected to inhibit the progression and metastasis of hepatoma cells, at least in part by inhibiting the invasive ability of the cells via up-regulation of the expression of the Egr-1, TSP-1, and E-cadherin.     

Lipopolysaccharide stimulation of ERK1/2 increases TNF-alpha production via Egr-1

Lipopolysaccharide (LPS) is a potent activator of tumor necrosis factor-alpha (TNF-alpha) production by macrophages. LPS stimulates the phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 and increases TNF-alpha mRNA and protein accumulation in RAW 264.7 murine macrophages. However, the role of ERK1/2 activation in mediating LPS-stimulated TNF-alpha production is not well understood. Inhibition of ERK1/2 activation with PD-98059 or overexpression of dominant negative ERK1/2 decreased LPS-induced TNF-alpha mRNA quantity. LPS rapidly increased early growth response factor (Egr)-1 binding to the TNF-alpha promoter; this response was blunted in cells treated with PD-98059 or transfected with dominant-negative ERK1/2. Using a chloramphenicol acetyltransferase reporter gene linked to the Egr-1 promoter, we show that LPS increased Egr-1 promoter activity via an ERK1/2-dependent mechanism. These results delineate the role of ERK1/2 activation of Egr-1 activity in mediating LPS-induced increases in TNF-alpha mRNA expression in macrophages.     

Egr-1 is activated by 17beta-estradiol in MCF-7 cells by mitogen-activated protein kinase-dependent phosphorylation of ELK-1

Early growth response-1 (Egr-1) is an immediate-early gene induced by E2 in the rodent uterus and breast cancer cells. E2 induces Egr-1 mRNA and protein levels in MCF-7 human breast cancer cells and reporter gene activity in cells transfected with pEgr-1A, a construct containing the -600 to +12 region of the Egr-1 promoter linked to the firefly luciferase gene. Deletion analysis of the Egr-1 promoter identified a minimal E2-responsive region of the promoter that contained serum response element (SRE)3 (-376 to -350) which bound Elk-1 and serum response factor (SRF) in gel mobility shift assays. Hormone-responsiveness of Egr-1 in MCF-7 cells was specifically inhibited by PD98059, a mitogen-activated protein kinase kinase inhibitor, but not by LY294002, an inhibitor of phosphatidylinositol-3-kinase (PI3-K). These results contrasted with hormone-dependent activation of the SRE in the c-fos promoter, which was inhibited by both PD98059 and LY294002. Differences in activation of the SREs in Egr-1 and c-fos were related to promoter sequence, which defines the affinities of Elk-1 and SRF to their respective binding sites. Thus, Egr-1, like c-fos, is activated through non-genomic (extranuclear) pathways of estrogen action in breast cancer cells.     

Troglitazone induction of COX-2 expression is dependent on ERK activation in keratinocytes

Cyclooxygenase-2 (COX-2) plays an important role in tumorigenesis of several tissues, including skin. We report here that troglitazone, a thiazolidinedione class of antidiabetic drug, induced COX-2 expression at both the protein and mRNA levels and increased production of prostaglandin E2 (PGE2) in cultured keratinocytes. Troglitazone-induced COX-2 expression in keratinocytes was likely peroxisome proliferator-activated receptor gamma (PPARgamma)-independent. Troglitazone treatment of these cells also resulted in a sustained increase in phosphorylation of ERK. We show that induction of COX-2 by troglitazone was almost completely inhibited by specific inhibitors of ERK activation. These data suggest that troglitazone is capable of inducing COX-2 expression through an ERK-dependent mechanism in mouse skin keratinocytes.     

Induction of COX-2 and reactive gliosis by P2Y receptors in rat cortical astrocytes is dependent on ERK1/2 but independent of calcium signalling

The present study has been aimed at characterizing the ATP/P2 receptor (and transductional pathways) responsible for the morphological changes induced in vitro by alphabetamethyleneATP on rat astrocytes obtained from cerebral cortex, a brain area highly involved in neurodegenerative diseases. Exposure of cells to this purine analogue resulted in elongation of cellular processes, an event reproducing in vitro a major hallmark of in vivo reactive gliosis. alphabetamethyleneATP-induced gliosis was prevented by the P2X/P2Y blocker pyridoxalphosphate-6-azophenyl-2'-4'-disulfonic acid, but not by the selective P2X antagonist 2',3'-O-(2,4,6-trinitrophenyl)-ATP, ruling out a role for ligand-gated P2X receptors. Conversely, the Gi/Go protein inactivator pertussis toxin completely prevented alphabetamethyleneATP-induced effects. No effects were induced by alphabetamethyleneATP on intracellular calcium concentrations. RT-PCR and western blot analysis showed that alphabetamethyleneATP-induced gliosis involves up-regulation of cyclooxygenase-2 (but not lipooxygenase). Also this effect was fully prevented by pyridoxalphosphate-6-azophenyl-2'-4'-disulfonic acid. Experiments with inhibitors of mitogen-activated protein kinases (MAPK) suggest that extracellular signal regulated protein kinases (ERK)1/2 mediate both cyclooxygenase-2 induction and the associated in vitro gliosis. These findings suggest that purine-induced gliosis involves the activation of a calcium-independent G-protein-coupled P2Y receptor linked to ERK1/2 and cyclooxygenase-2. Based on the involvement of cyclooxygenase-2 and inflammation in neurodegenerative diseases, these findings open up new avenues in the identification of novel biological targets for the pharmacological manipulation of neurodegeneration.     

Egr-1 inhibits the expression of extracellular matrix genes in chondrocytes by TNFα-induced MEK/ERK signalling

Introduction  

TNFα is increased in the synovial fluid of patients with rheumatoid arthritis and osteoarthritis. TNFα activates mitogen-activated kinase kinase (MEK)/extracellular regulated kinase (ERK) in chondrocytes; however, the overall functional relevance of MEK/ERK to TNFα-regulated gene expression in chondrocytes is unknown.     

Induction of tissue factor expression in human endothelial cells by CD40 ligand is mediated via activator protein 1, nuclear factor kappa B, and Egr-1

Induction of tissue factor expression in endothelial cells via ligation of CD40 probably figures prominently in the pathogenesis of prevalent inflammatory diseases, including atherosclerosis. However, the molecular mechanisms of tissue factor gene expression triggered by CD40 ligand (CD40L) in this cell type remain unknown. We demonstrate here that the tissue factor promoter region -278 bp to +121 bp contains the CD40L-responsive elements, consisting of activator protein 1 (AP-1)+/-, nuclear factor (NF) kappaB-, and Egr-1-binding sites. Mutations of either the AP-1- or NF-kappaB-binding sites markedly reduced the CD40L-dependent promoter activation. The AP-1 and NF-kappaB sites displayed constitutive and CD40L-enhanceable DNA binding activity, respectively. Of note, mutation of the Egr-1-binding sites, previously not associated with CD40 signaling, impaired activation of the tissue factor promoter. Accordingly, CD40L strongly induced Egr-1 protein expression and DNA binding activity to all three bindings sites. In contrast to CD40L, other established inducers of tissue factor in endothelial cells, interleukin-1beta or tumor necrosis factor alpha, did not increase the expression of Egr-1. In conclusion, induction of tissue factor gene expression in human endothelial cells by CD40L involves AP-1 and NF-kappaB as well as Egr-1, a pathway previously not implicated in CD40 signaling and distinct from that employed by certain other proinflammatory cytokines.