Activated ras oncogene collaborates with HBx gene of hepatitis B virus to transform cells by suppressing HBx-mediated apoptosis

The hepatitis B virus HBx protein is a promiscuous transactivator implicated in the development of hepatocellular carcinoma. The ectopic expression of HBx fails to transform both primary and immortalized rodent cells, but rather induces apoptosis. Furthermore, most transgenic mice harboring HBx do not develop liver tumors. Thus, it remains unclear whether and how HBx contributes to oncogenesis. Here, we show that HBx collaborates with activated H-ras to transform immortalized rodent cells. Indeed, REF52 cells transfected by both HBx and activated H-ras were morphologically transformed and were able to grow in soft agar. Remarkably, nude mice injected with REF52 cells transfected by both HBx and activated H-ras developed tumors, whereas the mice injected with REF52 cells transfected by either gene alone did not. Thus, we concluded that HBx could contribute to neoplastic transformation of cells in collaboration with other oncogenes, such as H-ras, that renders cells to overcome the HBx-mediated apoptosis. Further, we found that HBx mediated apoptosis was suppressed by activated H-ras through activation of the phosphatidylinositol-3 kinase and Akt pathway. Data presented here firmly established the oncogenic potential of HBx during multistage carcinogenesis.ope

cAMP-responding element-binding protein and c-Ets1 interact in the regulation of ATP-dependent MUC5AC gene expression.

Exogenous ATP activates purinoreceptors on the cell surface that regulate diverse cellular functions, including mucous cell secretion in the respiratory epithelium. In this study, ATP increased MUC5AC mRNA in primary human nasal epithelial cells and in NCI-H292 pulmonary adenocarcinoma cells in vitro. ATP-induced MUC5AC mRNA was mediated by phospholipase Cbeta3. A dominant-negative mutation in the PDZ binding domain of PLCbeta3 inhibited ATP-induced MUC5AC gene expression. ATP sequentially activated the phosphorylation of Akt, ERK1/2, p38, RSK1, and cAMP-responding element-binding protein (CREB) in a protein kinase C-independent manner. ATP-induced MUC5AC mRNA levels were regulated by CREB via direct interaction with c-Ets1 on the MUC5AC gene promoter (located -938 to -930). Effects of CREB and c-Ets1 were additive. Inhibition of either CREB or c-Ets1 inhibited ATP-induced MUC5AC gene expression. Stimulation with ATP caused the direct binding of CREB and c-Ets1 to the MUC5AC promoter, increasing the phosphorylation of c-Ets1. Chromatin immunoprecipitation assays demonstrated that in the presence of ATP, both c-Ets1 and CREB bound to the MUC5AC promoter. The effects of exogenous ATP on MUC5AC gene expression are mediated by a complex regulatory cascade controlling interactions between CREB and c-Ets1 that bind to a promoter element in the MUC5AC gene enhancing MUC5AC gene transcription. ATP-dependent activation of MUC5AC gene expression via CREB-c-Ets1 may contribute to mucous cell hypersecretion associated with common respiratory disorders.ope

Suppression of prostaglandin E2-induced MUC5AC overproduction by RGS4 in the airway.

The mechanism by which E-prostanoid (EP) receptor is critically involved in PGE(2)-induced mucin 5AC (MUC5AC) gene expression in the airway has been unclear. Furthermore, there have been little reports regarding the negative regulatory mechanism and/or proteins that affect PGE(2)-induced MUC5AC overproduction. In the present study, we found that PGE(2) induced MUC5AC gene expression in a dose-dependent manner (EC(50): 73.31 +/- 3.13 nM) and that the EP(2/4)-specific agonist, misoprostol, increased MUC5AC mRNA level, whereas the EP(1/3)-specific agonist, sulprostone, had no effect. Interestingly, the cAMP concentration (685.1 +/- 14.9 pM) of the EC(50) value of EP(4)-mediated cAMP production was much higher than that of EP(2) (462.33 +/- 23.79 pM), suggesting that EP(4) has higher sensitivity to PGE(2) compared with EP(2). Moreover, PGE(2)-induced Muc5ac overproduction was much increased in regulator of G protein signaling (Rgs) 4 knockout (KO) mice compared with wild-type mice at both transcriptional and translational levels, and it was dramatically suppressed in Rgs4 KO mice that had been infected with lentivirus expressing RGS4 (lenti::RGS4) compared with lentivirus expressing enhanced green fluorescent protein (lenti::eGFP). Finally, we demonstrate that PGE(2) can induce MUC5AC overproduction via the EP(4) receptor and that RGS4 may have suppressive effects in controlling MUC5AC overexpression in the airway. These findings may provide a molecular paradigm for the development of novel drugs for respiratory diseases.ope

Overexpression of Par-4 enhances thapsigargin-induced apoptosis via down-regulation of XIAP and inactivation of Akt in human renal cancer cells

The prostate-apoptosis-response-gene-4 (Par-4) protein has been shown to function as an effector of cell death in response to various apoptotic stimuli that trigger mitochondria and membrane receptor-mediated cell death pathways. We found that overexpressing Par-4 by stable transfection sensitizes Caki cells to induction of apoptosis by TRAIL and drugs that induce endoplasmic reticulum (ER) stress [thapsigargin (TG), tunicamycin (TU) and etoposide]. Ectopic expression of Par-4 is associated with decreased levels of XIAP protein in TG-treated cells, caused in part by XIAP protein instability and caspase activation. Levels of phospho-Akt are decreased in Caki/Par-4 cells to a significantly greater extent than in Caki/Vector cells by treatment with TG, and this is in turn associated with decreased levels of phospho-PDK1, the kinase upstream of Akt. In conclusion, we provide evidence that ectopic expression of Par-4 sensitizes Caki cells to TG and that XIAP protein instability and inactivation of Akt are important in cellular pathways affected by Par-4ope

Expression of MUC5AC mRNA in the goblet cells of human nasal mucosa.

OBJECTIVES/HYPOTHESIS: Mucus hypersecretion is a common feature in chronic sinusitis with polyps. Because mucus hypersecretion is commonly accompanied by goblet cell hyperplasia, it is important to identify which mucin gene mRNAs are expressed in the goblet cells of the surface epithelium in the human airway. This study aims to investigate the pattern of expression of MUC5AC messenger RNA (mRNA) in the goblet cells of human nasal mucosa. METHODS: Six nasal polyps, five inferior turbinate mucosa specimens, and three normal-appearing mucosa specimens of the posterior ethmoid sinus were obtained. Each of the specimens was cut into 10-microm-thick serial frozen sections, and in situ hybridization of MUC5AC mRNA was performed with an oligonucleotide probe. Alcian blue (pH 2.5)-periodic acid-Schiff (AB-PAS) staining was performed on the serial sections. RESULTS: In human nasal polyps, MUC5AC mRNA was expressed in the cytoplasm of most of the goblet cells. However, in the inferior turbinate, MUC5AC mRNA was expressed in only some of the goblet cells. On the contrary, in the normal-appearing mucosa of the posterior ethmoid sinus, MUC5AC mRNA was barely expressed in the goblet cells. Furthermore, MUC5AC mRNA was mainly expressed in some of the PAS-positive goblet cells. CONCLUSIONS: Only a portion of the goblet cells in the human nasal mucosa expressed MUC5AC mRNA. This result suggests that surface goblet cells might have other mucin genes, in addition to MUC2 and MUC5AC.restrictio

Upregulation of MUC8 and downregulation of MUC5AC by inflammatory mediator in human nasal polyps cultured nasal epithelium

Polyps are believed to be the source of mucus hypersecretion in chronic inflammation of the sinus. However, it is not clear which mucins are responsible for the hypersecretion of mucus by nasal polyps. We describe the over-expression of MUC8 mRNA in nasal polyps and the upregulation of MUC8 mRNA expression and downregulation of MUC5AC mRNA expression by inflammatory mediators. We found that the level of MUC8 mRNA, but not the level of MUC5AC mRNA, increased in nasal polyps. We also found that there was an increase in intracellular mucin in nasal polyps, compared to the normal nasal inferior turbinate. A mixture of inflammatory mediators increased MUC8 mRNA expression and decreased MUC5AC mRNA expression in cultured normal human nasal epithelial cells. Among inflammatory mediators, IL-4 is responsible for the decrease in MUC5AC mRNA and MUC5AC mucin secretion. These results indicate that MUC8 may be one of the major mucins secreted from the polyp epithelium and that it may play an important role in the pathogenesis of mucus hypersecretion in chronic sinusitis with polyps.ope

정상 호흡기 상피 세포에서 IL-1

Dept. of Medical Science/박사[한글] 점액과분비는 호흡기에서 염증을 수반하는 질환에서 보통 관찰되어진다. 인간 정상 호흡기의 goblet 세포에서 매우 많이 발현되는 mucin이 MUC5AC이기 때문에 MUC5AC는 호흡기에서 매우 중요한 mucin으로 알려져 있다. 더욱이 이러한 MUC5AC의 발현은 다양한 염증 매개체에 의해 조절되어진다고 알려져 있다. 그러나 정상 코 상피 세포에서 중요한 염증 매개체로 알려진 IL-1beta나 TNF-alpha에 의한 MUC5AC 발현 신호전달은 아직까지 정확히 밝혀지지 않았다. 본 연구에서는 IL-1beta나 TNF-alpha가 어떠한 기전으로 MUC5AC 발현을 조절하는지 알아보기 위하여 생화학적인 방법과 유전학적인 방법을 사용하여 조사하였다. IL-1beta나 TNF-alpha에 의해 MAP kinase 중에서 ERK와 p38 MAP kinase 신호 전달 단백질이 관여함을 알았고 이러한 단백질을 억제하였을 때 MUC5AC 발현이 억제 되는 것으로 관찰되었다. 이러한 결과는 ERK와 p38 MAP kinase가 IL-1beta나 TNF-alpha에 의한 MUC5AC 발현신호전달에 필수적임을 알았다. 또한 ERK와 p38 MAP kinase의 downstream에 MSK1과 CREB이 관여함을 유전학적인 방법으로 증명하였고, IL-1beta나 TNF-alpha에 의해 활성화된 CREB은 MUC5AC promoter에 존재하는 CRE에 결합하는 것을 관찰하였다. 더욱이 MUC5AC promoter의 CRE (-878)에 의해 전사가 일어남을 관찰하였다. 이러한 결과들에 의해서 IL-1beta나 TNF-alpha에 의해 MUC5AC 발현 기전은 순차적으로 ERK p38 MAP kinase-MSK1-CREB-CRE 기전임을 증명하였다. 이러한 연구는 IL-1beta나 TNF-alpha에 의한 MUC5AC 발현신호전달을 증명함으로써 염증반응 동안에 mucin 과분비를 이해하는데 중요한 자료를 제시한다. [영문] Mucin hypersecretion is commonly observed in many inflammatory diseases of the respiratory tract. MUC5AC is generally recognized to be a major airway mucin because MUC5AC is highly expressed in the goblet cells of human airway epithelium. Moreover, it is regulated by various inflammatory cytokines. However, the mechanisms by which the interleukin (IL)-1beta and tumor necrosis factor (TNF)-alpha induce MUC5AC gene expression in normal nasal epithelial cells, and the signal molecules involved, especially in the downstream signaling of mitogen-activated protein (MAP) kinases, remain unclear. Here we show that pharmacologic or genetic inhibition of either ERK or p38 MAP kinase pathway abolished IL-1beta- and TNF-alpha-induced MUC5AC gene expression in normal human nasal epithelial cells. Our results also indicate that the activation of mitogen- and stress-activated protein kinase 1 (MSK1) and cAMP response element-binding protein (CREB) and CRE signaling cascades via ERK and p38 MAP kinases are crucial aspects of the intracellular mechanisms that mediate MUC5AC gene expression. Taken together, these studies give additional insights into the molecular mechanism of IL-1beta- and TNF-alpha-induced MUC5AC gene expression and will enhance our understanding on mucin hypersecretion during inflammation.ope

KATP channel controls LPS-induced MUC5AC overexpression in vivo

Glybenclamide; Hypersecretion; Inflammation; KATP channel; MUC5AC; Mucou

Chrysin inhibited stem cell factor (SCF)/c-Kit complex-induced cell proliferation in human myeloid leukemia cells

Stem cell factor (SCF) has important roles in the proliferation and differentiation of hematopoietic stem cells. The complex of c-Kit and its ligand SCF induce hematopoiesis, melanogenesis, and gametogenesis. However, the mechanism by which SCF induces cell proliferation in the human megakaryoblastic leukemia cell line, MO7e, and the signaling molecules involved, especially in downstream signaling of c-Kit, remain unclear. Here, we show that pharmacological inhibition of the PI3K pathway inhibits SCF/c-Kit signaling and cell proliferation. In addition, we find that the Shc/PDK1/PKC/Akt/c-raf signaling cascade is essential for SCF/c-Kit signal pathway. Our results also suggest that ERK5 is activated and translocated to the nucleus, activating CREB and STAT3. Interestingly, chrysin shuts down the SCF/c-Kit complex-induced signaling cascade. Taken together, these studies give additional insight into the molecular mechanism of SCF/c-Kit-induced cell proliferation and its inverse agonist, chrysin. Finally, these findings enhance our understanding of MO7e cell proliferation.ope