Autophagic feedback-mediated degradation of IKKα requires CHK1- and p300/CBP-dependent acetylation of p53.

In our previous report, we demonstrated that one of the catalytic subunits of the IκB kinase (IKK) complex, IKKα (encoded by CHUK), performs an NF-κB-independent cytoprotective role in human hepatoma cells under the treatment of the anti-tumor therapeutic reagent arsenite. IKKα triggers its own degradation, as a feedback loop, by activating p53-dependent autophagy, and therefore contributes substantially to hepatoma cell apoptosis induced by arsenite. Interestingly, IKKα is unable to interact with p53 directly but plays a critical role in mediating p53 phosphorylation (at Ser15) by promoting CHK1 activation and CHK1-p53 complex formation. In the current study, we found that p53 acetylation (at Lys373 and/or Lys382) was also critical for the induction of autophagy and the autophagic degradation of IKKα during the arsenite response. Furthermore, IKKα was involved in p53 acetylation through interaction with the acetyltransferases for p53, p300 (also known as EP300) and CBP (also known as CREBBP) (collectively p300/CBP), inducing CHK1-dependent p300/CBP activation and promoting p300-p53 or CBP-p53 complex formation. Therefore, taken together with the previous report, we conclude that both IKKα- and CHK1-dependent p53 phosphorylation and acetylation contribute to mediating selective autophagy feedback degradation of IKKα during the arsenite-induced proapoptotic responses.

MAPK/AP-1 pathway activation mediates AT1R upregulation and vascular endothelial cells dysfunction under PM2.5 exposure.

Acute and chronic exposure to particulate matter (PM) 2.5 is associated with adverse health effect upon the cardiovascular (CV) system. However, the molecular mechanism by which PM2.5 evokes CV injuries has not been fully clarified. In our recent report, we demonstrate that exposure to PM2.5 leads to elevation of circulating angiotensin II (ANGII) levels and local expressions of angiotensinogen (AGT, the precursor of ANGII), angiotensin-converting enzyme (ACE) and ANGII type 1 receptor (AT1R) in the vascular endothelial cells, which subsequently instigates the oxidative stress and proinflammatory response in the vascular endothelium. In the present study, we disclosed that PM2.5 exposure induced the activation of the transcriptional factor AP-1 and its components, c-Jun and ATF2, in the human vascular endothelial cells. Although the DNA-binding sites for AP-1 were identified within the promoter regions of AGT, ACE and AT1R genes, RT-PCR and immunoblot assays indicated that AP-1 transactivation was only involved in AT1R upregulation, but did not affect the induction of AGT and ACE expression under the same conditions. Furthermore, ERKs and p38K functioned as the upstream protein kinases involving in AP-1 transactivation and AT1R upregulation under PM2.5 stimulation. In addition, the oxidative stress and proinflammatory responses in the PM2.5-treated vascular endothelial cells were significantly reduced when MAPKs and AP-1 activation were inhibited. Therefore, we conclude that PM2.5 exposure induces MAPK/AP-1 cascade activation, which contributes to AT1R upregulation and vascular endothelial dysfunction. Identifying novel therapeutic targets to alleviate AP-1 transactivation and restore AT1R expression may be helpful for the management of PM2.5-induced CV burden.

Src/STAT3-dependent heme oxygenase-1 induction mediates chemoresistance of breast cancer cells to doxorubicin by promoting autophagy.

Chemotherapeutic resistance in breast cancer, whether acquired or intrinsic, remains a major clinical obstacle. Thus, increasing tumor cell sensitivity to chemotherapeutic agents will be helpful in improving the clinical management of breast cancer. In the present study, we found an induction of HO-1 expression in doxorubicin (DOX)-treated MDA-MB-231 human breast adenocarcinoma cells, which showed insensitivity to DOX treatment. Knockdown HO-1 expression dramatically upregulated the incidence of MDA-MB-231 cell death under DOX treatment, indicating that HO-1 functions as a critical contributor to drug resistance in MDA-MB-231 cells. We further observed that DOX exposure induced a cytoprotective autophagic flux in MDA-MB-231 cells, which was dependent on HO-1 induction. Moreover, upregulation of HO-1 expression required the activation of both signal transducer and activator of transcription (STAT)3 and its upstream regulator, protein kinase Src. Abrogating Src/STAT3 pathway activation attenuated HO-1 and autophagy induction, thus increasing the chemosensitivity of MDA-MB-231 cells. Therefore, we conclude that Src/STAT3-dependent HO-1 induction protects MDA-MB-231 breast cancer cells from DOX-induced death through promoting autophagy. In the following study, we further demonstrated the contribution of Src/STAT3/HO-1/autophagy pathway activation to DOX resistance in another breast cancer cell line, MDA-MB-468, which bears a similar phenotype to MDA-MB-231 cells. Therefore, activation of Src/STAT3/HO-1/autophagy signaling pathway might play a general role in protecting certain subtypes of breast cancer cells from DOX-induced cytotoxicity. Targeting this signaling event may provide a potential approach for overcoming DOX resistance in breast cancer therapeutics.

p85α mediates NFAT3-dependent VEGF induction in the cellular UVB response.

Exposure to solar ultraviolet B (UVB) radiation is known to induce several pathological reactions in the skin. In these processes, upregulation of VEGF expression has been demonstrated to be important in angiogenesis-associated photodamage and even skin cancers. However, the signaling events that are responsible for VEGF induction under UVB exposure have not been fully defined. Here, we demonstrate that the regulatory subunit of the phosphoinositide 3-kinase (PI3K), p85α, plays a role in mediating UVB-induced VEGF expression in mouse embryonic fibroblasts (MEFs) and mouse epithermal cells, the effect of which is unrelated to the PI3K activity. The transcriptional factor NFAT3 functions as a downstream target of p85α to mediate the induction of VEGF expression in the UVB response. Although lacking NFAT3-binding ability, p85α is required for the recruitment of NFAT3 to the NFAT-response element within the vegf promoter. Furthermore, by identifying the adjacent NFAT- and AP-1-binding sites within the vegf promoter, we also found an induced interaction between NFAT3 and one of the AP-1 components, c-Fos, after UVB irradiation. Without the aid of c-Fos, NFAT3 lost its vegf-promoter-binding ability. Taken together, our results reveal a novel PI3K-independent role for p85α in controlling VEGF induction during the cellular UVB response by regulating NFAT3 activity. Targeting p85α might be helpful for preventing UVB-induced angiogenesis and the associated photodamage.

Catecholamine-Induced ß2-adrenergic receptor activation mediates desensitization of gastric cancer cells to trastuzumab by upregulating MUC4 expression.

Trastuzumab is currently used for patients with Her2(+) advanced gastric cancer. However, the response rate to trastuzumab among the patients is low. The molecular mechanisms underlying trastuzumab resistance in gastric cancer are unknown. Our in vitro data show that activation of ß2-adrenergic receptor (ß2-AR) triggered by catecholamine caused "targeting failure" of trastuzumab in gastric cancer cells. The antitumor activities of trastuzumab were significantly impeded by chronic catecholamine stimulation in gastric cancer cells and in the mice bearing human gastric cancer xenografts. Mechanistically, catecholamine induced upregulation of the MUC4 expression at both transcription and protein levels via activating STAT3 and ERK. The effects of catecholamine could be effectively blocked by ß2-AR antagonist ICI-118,551, indicating that ß2-AR-mediated signaling pathway plays a key role in upregulation of MUC4, which was previously demonstrated to interfere with the recognition and physical binding of trastuzumab to Her2 molecules. Moreover, a significant elevation of the MUC4 level was observed in the xenograft tissues in nude mice chronically treated with isoproterenol. Knockdown of MUC4 restored the binding activities of trastuzumab to Her2-overexpressing gastric cancer cells. In addition, coexpression of ß2-AR and MUC4 were observed in gastric cancer tissues. Our data indicated a novel trastuzumab resistance mechanism, by which catecholamine-induced ß2-AR activation mediates desensitization of gastric cancer cells to trastuzumab through upregulating the MUC4 expression.

Ribosomal protein S7 regulates arsenite-induced GADD45α expression by attenuating MDM2-mediated GADD45α ubiquitination and degradation.

The stress-responding protein, GADD45α, plays important roles in cell cycle checkpoint, DNA repair and apoptosis. In our recent study, we demonstrate that GADD45α undergoes a dynamic ubiquitination and degradation in vivo, which process can be blocked by the cytotoxic reagent, arsenite, resulting in GADD45α accumulation to activate JNKs cell death pathway, thereby revealing a novel mechanism for the cellular GADD45α functional regulation. But the factors involved in GADD45α stability modulations are unidentified. Here, we demonstrated that MDM2 was an E3 ubiquitin ligase for GADD45α. One of MDM2-binding partner, ribosomal protein S7, interacted with and stabilized GADD45α through preventing the ubiquitination and degradation of GADD45α mediated by MDM2. This novel function of S7 is unrelated to p53 but seems to depend on S7/MDM2 interaction, for the S7 mutant lacking MDM2-binding ability lost its function to stabilize GADD45α. Further investigations indicated that arsenite treatment enhanced S7-MDM2 interaction, resulting in attenuation of MDM2-dependent GADD45α ubiquitination and degradation, thereby leading to GADD45α-dependent cell death pathway activation. Silencing S7 expression suppressed GADD45α-dependent cytotoxicity induced by arsenite. Our findings thus identify a novel function of S7 in control of GADD45α stabilization under both basal and stress conditions and its significance in mediating arsenite-induced cellular stress.

IKKα contributes to UVB-induced VEGF expression by regulating AP-1 transactivation.

Exposure to ultraviolet B (UVB) irradiation from sunlight induces the upregulation of VEGF, a potent angiogenic factor that is critical for mediating angiogenesis-associated photodamage. However, the molecular mechanisms related to UVB-induced VEGF expression have not been fully defined. Here, we demonstrate that one of the catalytic subunits of the IκB kinase complex (IKK), IKKα, plays a critical role in mediating UVB-induced VEGF expression in mouse embryonic fibroblasts (MEFs), which requires IKKα kinase activity but is independent of IKKß, IKKγ and the transactivation of NF-κB. We further show that the transcriptional factor AP-1 functions as the downstream target of IKKα that is responsible for VEGF induction under UVB exposure. Both the accumulation of AP-1 component, c-Fos and the transactivation of AP-1 by UVB require the activated IKKα located within the nucleus. Moreover, nuclear IKKα can associate with c-Fos and recruit to the vegf promoter regions containing AP-1-responsive element and then trigger phosphorylation of the promoter-bound histone H3. Thus, our results have revealed a novel independent role for IKKα in controlling VEGF expression during the cellular UVB response by regulating the induction of the AP-1 component and phosphorylating histone H3 to facilitate AP-1 transactivation. Targeting IKKα shows promise for the prevention of UVB-induced angiogenesis and the associated photodamage.

AP-1 activation attenuates the arsenite-induced apoptotic response in human bronchial epithelial cells by up-regulating HO-1 expression.

Arsenite is a cytotoxic reagent that has been used clinically to treat certain cancers. Although the cytotoxic mechanisms of arsenite have been investigated, the cellular mechanisms that act against arsenite damage are poorly understood. Heme oxygenase 1 (HO-1) has been implicated in cellular survival under other multiple stress conditions. Here, we show that a significant induction of HO-1 expression is present in human bronchial epithelial cells (Beas-2B) treated with lethal doses of arsenite treatment. This induction depends on the known ERK/AP1 signaling pathway. As expected, HO-1 RNAi knockdown, or ERK/AP1 inhibition, renders the Beas-2B cells more sensitive to arsenite damage. Our data thus suggest that transcriptional upregulation of HO-1 expression via a putative ERK/AP-1 pathway constitutes an inherent mechanism by which arsenite-induced apoptosis is attenuated.

Modulation of IFN-γ receptor 1 expression by AP-2α influences IFN-γ sensitivity of cancer cells.

Interferon (IFN)-γ plays crucial roles in regulating both innate and adaptive immunity. The existence of IFN-γ receptor 1 (IFNGR1) molecules on the cell surface is a prerequisite to the initiation of IFN-γ signaling; low expression of IFNGR1 leads to a functional blockade of IFN-γ signaling. However, the molecular mechanisms by which IFNGR1 expression is controlled are unclear. In the present study, we demonstrated that IFNGR1 expression was reduced or lost in breast cancer. Heterogeneous IFNGR1 immunoreactivity appeared to be associated with the morphological heterogeneity of breast cancer, and loss of IFNGR1 expression was predominantly observed in poorly differentiated areas. We identified the functional activating protein (AP)-2 and specificity protein (SP)-1 sites within the IFNGR1 promoter. Ectopic expression of AP-2α drastically repressed the expression of IFNGR1 and hindered IFN-γ signaling, whereas AP-2α gene silencing elevated IFNGR1 levels. Overexpression of SP-1 effectively antagonized the repressive effects of AP-2α. Simultaneous recruitment of both transcription factors to the AP-2 and SP-1 motifs, respectively, in the IFNGR1 promoter was demonstrated, implying that AP-2α and SP-1 may synergistically modulate IFNGR1 transcription. Moreover, AP-2α overexpression in AP-2-deficient SW480 cells remarkably inhibited Stat1 phosphorylation and the anti-proliferative effects of IFN-γ, whereas knockdown of the AP-2α expression dramatically enhanced the sensitivities of HeLa cells highly expressing AP-2 to IFN-γ, indicating that dysregulation of AP-2α expression is associated with impaired IFN-γ actions in cancer cells.

Glucocorticoid regulation of a novel HPV-E6-p53-miR-145 pathway modulates invasion and therapy resistance of cervical cancer cells.

Glucocorticoids are stress-responsive neuroendocrine mediators and play an important role in malignant progression, especially in solid tumours. We demonstrate a novel mechanism by which glucocorticoids modulate p53-dependent miR-145 expression in HPV-positive cervical cancer cells through induction of E6 proteins. We found that expression of miR-145 was reduced in cervical cancer tissues. Cortisol induced HPV-E6 expression and suppressed p53 and miR-145 in cervical cancer cells. MiR-145 expression in cervical cancer cells was wild-type p53-dependent, and cortisol-induced down-regulation of miR-145 expression prevented chemotherapy-induced apoptosis, whereas over-expression of miR-145 enhanced sensitivity to mitomycin and reversed the chemoresistance induced by glucocorticoids. We also show that miR-145 augments the effects of p53 by suppressing the inhibitors of p53 in cervical cancer cells, suggesting that miR-145 plays a role in p53 tumour suppression. Finally, we demonstrate that miR-145 inhibits both the motility and invasion of cervical cancer cells. Our findings identify a novel pathway through which the neuroendocrine macroenvironment affects cervical tumour growth, invasion and therapy resistance and show that miR-145 may serve as a target for cervical cancer therapy. Copyright © 2012 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

A novel role of IKKalpha in the mediation of UVB-induced G0/G1 cell cycle arrest response by suppressing Cyclin D1 expression.

Exposure to ultraviolet B (UVB) irradiation (290-320nm wavelength) from sunlight induces a variety of medical problems, including sunburn, immunosuppression and skin cancers. However, the molecular mechanisms related to UVB-induced cell damage and/or mutagenic effects have not been fully defined. Here, we demonstrate that one of the catalytic subunits of the IkappaB kinase complex (IKK), IKKalpha, plays a critical role in mediation of the UVB-induced G0/G1 cell cycle arrest response by suppressing Cyclin D1 expression. Notably, IKKa-dependent Cyclin D1 regulation is unrelated to IKKbeta/NF-kappaB activity. We further show that IKKalpha-dependent downregulation of Cyclin D1 expression in the UVB response results from the reduction of ERK1/2-dependent Cyclin D1 transcription coupled with an increase of p38 kinase-dependent Cyclin D1 proteolysis. Thus, our results have identified the novel role of IKKalpha in regulating cell cycle progression during the cellular UVB response. Targeting IKKalpha might be promising for the prevention of UVB-induced cell damage and tumorigenic effects.

The ß2-adrenergic receptor and Her2 comprise a positive feedback loop in human breast cancer cells.

In this study, ß2-AR level was found to be up-regulated in MCF-7 cells overexpressing Her2 (MCF-7/Her2). Correlation of ß2-AR level with Her2 status was demonstrated in breast cancer tissue samples. Constitutive phosphorylation of ERK, mRNA expression up-regulation of catecholamine-synthesis enzymes, and increased epinephrine release were detected in MCF-7/Her2 cells. ß2-AR expression induced by epinephrine and involvement of ERK signaling were validated. The data indicate that Her2 overexpression and excessive phosphorylation of ERK cause epinephrine autocrine release from breast cancer cells, resulting in up-regulation of ß2-AR expression. The data also showed that catecholamine prominently stimulated Her2 mRNA expression and promoter activity. The activation and nuclear translocation of STAT3 triggered by isoproterenol were observed. Enhanced binding activities of STAT3 to the Her2 promoter after isoproterenol stimulation were verified. Using STAT3 shRNA and dominant negative STAT3 mutant, the role of STAT3 in isoproterenol-induced Her2 expression was further confirmed. The data support a model where ß2-AR and Her2 comprise a positive feedback loop in human breast cancer cells.

IKKß downregulation is critical for triggering JNKs-dependent cell apoptotic response in the human hepatoma cells under arsenite exposure.

Arsenite has a long history in treating leukemia, which might be also effective in the therapy of other cancers. Our previous published data have demonstrated that arsenite exposure induces apoptosis in the HepG2 human hepatoma cells via activating JNKs/AP-1 pathway, but the upstream signaling events responsible for JNKs (c-Jun N-terminal kinase) cascade activation have not been fully discovered. Since cross-talk between IKK/NF-κB and JNKs pathways under stress conditions is a hot topic, in this article, we investigate the potential roles of IKKα and IKKß, the catalytic subunits of IKK complexes, in the arsenite-induced JNKs pathway activation in the HepG2 cells. We found that arsenite exposure induced JNKs and AP-1 activation accompanying with a significant reduction of both IKKα and IKKß expressions. Overexpression of IKKß, but not of IKKα, inhibited arsenite-induced MKK7/JNKs/AP-1 pathway activation as well as the apoptotic response. Therefore, we conclude that the downregulation of IKKß expression is the prerequisite signaling event for mediating JNKs pathway activation and the cellular apoptotic response in the HepG2 cells under arsenite exposure. Targeting IKKß might be helpful to enhance the tumor therapeutic effect of arsenite.

Down-regulation of MHC class II expression through inhibition of CIITA transcription by lytic transactivator Zta during Epstein-Barr virus reactivation.

The presentation of peptides to T cells by MHC class II molecules is of critical importance in specific recognition to a pathogen by the immune system. The level of MHC class II directly influences T lymphocyte activation. The aim of this study was to identify the possible mechanisms of the down-regulation of MHC class II expression by Zta during EBV lytic cycle. The data in the present study demonstrated that ectopic expression of Zta can strongly inhibit the constitutive expression of MHC class II and CIITA in Raji cells. The negative effect of Zta on the CIITA promoter activity was also observed. Scrutiny of the DNA sequence of CIITA promoter III revealed the presence of two Zta-response element (ZRE) motifs that have complete homology to ZREs in the DR and left-hand side duplicated sequence promoters of EBV. By chromatin immunoprecipitation assays, the binding of Zta to the ZRE(221) in the CIITA promoter was verified. Site-directed mutagenesis of three conserved nucleotides of the ZRE(221) substantially disrupted Zta-mediated inhibition of the CIITA promoter activity. Oligonucleotide pull-down assay showed that mutation of the ZRE(221) dramatically abolished Zta binding. Analysis of the Zta mutant lacking DNA binding domain revealed that the DNA-binding activity of Zta is required for the trans repression of CIITA. The expression of HLA-DRalpha and CIITA was restored by Zta gene silencing. The data indicate that Zta may act as an inhibitor of the MHC class II pathway, suppressing CIITA transcription and thus interfering with the expression of MHC class II molecules.

Sleep disturbance induces depressive behaviors and neuroinflammation by altering the circadian oscillations of clock genes in rats.

Sleep loss leads to a spectrum of mood disorders such as anxiety disorders, bipolar disorder and depression in many individuals. However, the underlying mechanisms are largely unknown. In this study, sleep-disturbed animals were tested for anxiety and depressive behaviors. We then studied the effects of SD on hypothalamic-pituitary-adrenal (HPA) axis function by measuring serum and CSF levels of corticosterone (CORT), and at the end of the experiment, brains were collected to measure the circadian oscillations of clock genes expression in the hypothalamus, glial cell activation and inflammatory cytokine alterations. Our results indicated that SD for 3 days resulted in anxiety- and depressive-like behaviors. SD exaggerated cortisol response to HPA axis, significantly altered the circadian oscillations of clock genes, decreased the expression of tight junction protein ZO-1 and Claudin 5 and increased the number of GFAP-positive cells and Iba-1-positive cells and caused subsequent elevation of pro-inflammatory cytokines IL-6, IL-1ß and TNFα. These findings demonstrated that SD for 3 days induced anxiety- and depression-like behaviors in rats in company with altering the circadian oscillations of clock genes and inducing neuroinflammation, indicating the underlying mechanism of sleep loss induced neuronal dysfunction.

GADD45alpha mediates arsenite-induced cell apoptotic effect in human hepatoma cells via JNKs/AP-1-dependent pathway.

Arsenite (As(III)), an effective chemotherapeutic agent for the acute promyelocytic leukemia (APL) and multiple myeloma (MM), might be also a promise for the therapy of other cancers, including the solid tumors. However, the molecular bases of arsenite-induced cytotoxicity in the tumor cells have not been fully defined. In this study, we have disclosed that arsenite effectively induces the apoptotic response in the HepG2 human hepatoma cells by triggering GADD45alpha induction and the subsequent activation of JNKs/AP-1 cell death pathway. However, signaling events relating to GADD45alpha/JNKs/AP-1 pathway activation have not been observed in HL7702 human diploid hepatic cells under the same arsenite exposure condition. Our results thus have illustrated the selective pro-apoptotic role of arsenite in the hepatoma cells by activating GADD45alpha-dependent cell death pathway whereas with little effect on the normal hepatic cells. The approaches to up-regulate GADD45alpha levels might be helpful in improving the chemotherapeutic action of arsenite on certain solid tumors including hepatoma.

Catecholamine up-regulates MMP-7 expression by activating AP-1 and STAT3 in gastric cancer.

BACKGROUND: Stress, anxiety and depression can cause complex physiological and neuroendocrine changes, resulting in increased level of stress related hormone catecholamine, which may constitute a primary mechanism by which physiological factors impact gene expression in tumors. In the present study, we investigated the effects of catecholamine stimulation on MMP-7 expression in gastric cancer cells and elucidated the molecular mechanisms of the up-regulation of MMP-7 level by catecholamine through an adrenergic signaling pathway. RESULTS: Increased MMP-7 expression was identified at both mRNA and protein levels in the gastric cancer cells in response to isoproterenol stimulation. ß2-AR antigonist effectively abrogated isoproterenol-induced MMP-7 expression. The activation of STAT3 and AP-1 was prominently induced by isoproterenol stimulation and AP-1 displayed a greater efficacy than STAT3 in isoproterenol-induced MMP-7 expression. Mutagenesis of three STAT3 binding sites in MMP-7 promoter failed to repress the transactivation of MMP-7 promoter and silencing STAT3 expression was not effective in preventing isoproterenol-induced MMP-7 expression. However, isoproterenol-induced MMP-7 promoter activities were completely disappeared when the AP-1 site was mutated. STAT3 and c-Jun could physically interact and bind to the AP-1 site, implicating that the interplay of both transcriptional factors on the AP-1 site is responsible for isoproterenol-stimulated MMP-7 expression in gastric cancer cells. The expression of MMP-7 in gastric cancer tissues was found to be at the site where ß2-AR was overexpressed and the levels of MMP-7 and ß2-AR were the highest in the metastatic locus of gastric cancer. CONCLUSIONS: Up-regulation of MMP-7 expression through ß2-AR-mediated signaling pathway is involved in invasion and metastasis of gastric cancer.

Construction and expression of a spliced variant of Epstein-Barr virus bzlf1 and preparation of its polyclonal antibody.

The BZLF1 gene-encoded protein, Zta (EB1, ZEBRA), is a key transcriptional activator of induction of the lytic cycle of EBV. Zta; it contains a basic region with homology to the DNA binding domains of the AP-1 family. In this study, an alternatively spliced BZLF1 (Delta BZLF1) cDNA lacking exon 2, which encodes the DNA-binding domain of Zta, was isolated from B95-8 marmoset cell line releasing EBV. The cDNA was inserted into a prokaryotic expression vector pET-28a+. The His-tagged recombinant protein was overproduced in E. coli BL21(DE3) and purified by nickel affinity chromatography. The purified fraction was characterized by Western blot and MALDI-TOF-MS analysis and used as an antigen to immunize mice. The antibody against Delta Zta can recognize both denatured and natural Zta protein. The Delta Zta protein and its antibody can be used to further investigate its unknown functions.

Selective degradation of IKKα by autophagy is essential for arsenite-induced cancer cell apoptosis.

Two catalytic subunits of the IKK complex, IKKα and IKKß, trigger NF-κB activation as well as NF-κB-independent signaling events under both physiological and pathological conditions. Here we identified the NF-κB-unrelated cytoprotective function of IKKα in promoting autophagy by triggering p53 transactivation and upregulation of its downstream autophagic mediator, DRAM1, in the arsenite-treated hepatoma cells, which responses depended on IKKα kinase activity. Furthermore, IKKα triggered p53/DRAM1-dependent autophagy by inducing CHK1 activation and CHK1/p53 interaction. Interestingly, after provoking autophagy, IKKα could be specifically recognized by the autophagic machinery via directly binding with LC3B, resulting in selective degradation of IKKα by autophagy. Unexpectedly, the selectivity of autophagic sequestration towards IKKα was mediated by novel mechanism independent of the classical LC3-interacting regions (LIRs) within IKKα, while C-terminal arm of LIR was involved in mediating IKKα/LC3B interaction. Taken together, we conclude that IKKα attenuates arsenite-induced apoptosis by inducing p53-dependent autophagy, and then selective feedback degradation of IKKα by autophagy contributes to the cytotoxic response induced by arsenite.

HER2-dependent MMP-7 expression is mediated by activated STAT3.

MMP-7 expression is highly regulated at the level of transcription. An understanding of how the MMP-7 gene is regulated is critical to elucidate the mechanisms of MMP-7 overexpression in the early tumor development. In the present study, increased mRNA and protein expressions of MMP-7 were observed in MCF-7 cells stably overexpressing HER2 (MCF-7/HER2). The promoter activity of MMP-7 gene was upregulated in MCF-7/HER2 cells and significantly enhanced by HRG induction. Examination of the MMP-7 promoter sequence revealed three potential STAT3 binding sites within the proximal region. MMP-7 promoter activity was remarkably induced in MCF-7 cells expressing the constitutively activated STAT3 (MCF-7/STAT3C). RT-PCR and Western blot analysis confirmed the expression upregulation of mRNA and protein of MMP-7 in the MCF-7/STAT3C cells. Binding of STAT3 to MMP-7 promoter was verified by ChIP and the critical STAT3 element within the MMP-7 promoter identified by the mutagenesis of the core STAT3 recognition sequence. Increased STAT3 phosphorylation was observed in either HER2 overexpressing cells or HRG-induced cells. The data indicate that HRG-induced HER2-dependent transcriptional upregulation and protein secretion of MMP-7 are mediated by activated STAT3. The expression of MMP-7 may be attributed to HER2/STAT3 activation.