Hepatitis B virus X protein activates the p38 mitogen-activated protein kinase pathway in dedifferentiated hepatocytes

Hepatitis B virus X protein (pX) is implicated in hepatocarcinogenesis by an unknown mechanism. Employing a cellular model linked to pX-mediated transformation, we investigated the role of the previously reported Stat3 activation by pX in hepatocyte transformation. Our model is composed of a differentiated hepatocyte (AML12) 3pX-1 cell line that undergoes pX-dependent transformation and a dedifferentiated hepatocyte (AML12) 4pX-1 cell line that does not exhibit transformation by pX. We report that pX-dependent Stat3 activation occurs only in non-pX-transforming 4pX-1 cells and conclude that Stat3 activation is not linked to pX-mediated transformation. Maximum Stat3 transactivation requires Ser727 phosphorylation, mediated by mitogenic pathway activation. Employing dominant negative mutants and inhibitors of mitogenic pathways, we demonstrate that maximum, pX-dependent Stat3 transactivation is inhibited by the p38 mitogen-activated protein kinase (MAPK)-specific inhibitor SB 203580. Using transient-transreporter and in vitro kinase assays, we demonstrate for the first time that pX activates the p38 MAPK pathway only in 4pX-1 cells. pX-mediated Stat3 and p38 MAPK activation is Ca(2+) and c-Src dependent, in agreement with the established cellular action of pX. Importantly, pX-dependent activation of p38 MAPK inactivates Cdc25C by phosphorylation of Ser216, thus initiating activation of the G(2)/M checkpoint, resulting in 4pX-1 cell growth retardation. Interestingly, pX expression in the less differentiated hepatocyte 4pX-1 cells activates signaling pathways known to be active in regenerating hepatocytes. These results suggest that pX expression in the infected liver effects distinct mitogenic pathway activation in less differentiated versus differentiated hepatocytes.     

Hepatitis B virus X protein differentially regulates cell cycle progression in X-transforming versus nontransforming hepatocyte (AML12) cell lines.

Hepatitis B virus (HBV) X protein (pX) is implicated in hepatocarcinogenesis of chronically infected HBV patients. To understand mechanism(s) of pX-mediated cellular transformation, we employed two tetracycline-regulated, pX-expressing cell lines, constructed in AML12 immortalized hepatocytes: one a differentiated (3pX-1) and the other a de-differentiated (4pX-1) hepatocyte cell line. Only 3pX-1 cells undergo pX-mediated transformation, via sustained Ras-Raf-mitogen-activated protein kinase pathway activation. pX-nontransforming 4pX-1 cells display sustained, pX-dependent JNK pathway activation. To understand how pX mediates different growth characteristics in 3pX-1 and 4pX-1 cells, we report, herein, comparative cell cycle analyses. pX-transforming 3pX-1 cells display pX-dependent G(1), S, and G(2)/M progression evidenced by cyclin D(1), A, and B(1) induction, and Cdc2 kinase activation. pX-nontransforming 4pX-1 cells display pX-dependent G(1) and S phase entry, followed by S phase pause and absence of Cdc2 kinase activation. Interestingly, 4pX-1 cells exhibit selective pX-induced expression of cyclin-dependent kinase inhibitor p21(Cip1), tumor suppressor p19(ARF), and proapoptotic genes bax and IGFBP-3. Despite the pX-mediated induction of growth arrest and apoptotic genes and the absence of pX-dependent Cdc2 activation, 4pX-1 cells do not undergo pX-dependent G(2)/M arrest or apoptosis. Nocodazole-treated, G(2)/M-arrested 4pX-1 cells exhibit pX-dependent formation of multinucleated cells, similar to human T-cell lymphotropic virus type I Tax-expressing cells. We propose that in 4pX-1 cells, pX deregulates the G(2)/M checkpoint, thus rescuing cells from pX-mediated apoptosis.     

The death domain kinase RIP1 is essential for tumor necrosis factor alpha signaling to p38 mitogen-activated protein kinase

The cytokine tumor necrosis factor alpha (TNF-alpha) stimulates the NF-kappaB, SAPK/JNK, and p38 mitogen-activated protein (MAP) kinase pathways by recruiting RIP1 and TRAF2 proteins to the tumor necrosis factor receptor 1 (TNFR1). Genetic studies have revealed that RIP1 links the TNFR1 to the IkappaB kinase (IKK) complex, whereas TRAF2 couples the TNFR1 to the SAPK/JNK cascade. In transfection studies, RIP1 and TRAF2 stimulate p38 MAP kinase activation, and dominant-negative forms of RIP1 and TRAF2 inhibit TNF-alpha-induced p38 MAP kinase activation. We found TNF-alpha-induced p38 MAP kinase activation and interleukin-6 (IL-6) production impaired in rip1(-/-) murine embryonic fibroblasts (MEF) but unaffected in traf2(-/-) MEF. Yet, both rip1(-/-) and traf2(-/-) MEF exhibit a normal p38 MAP kinase response to inducers of osmotic shock or IL-1alpha. Thus, RIP1 is a specific mediator of the p38 MAP kinase response to TNF-alpha. These studies suggest that TNF-alpha-induced activation of p38 MAP kinase and SAPK/JNK pathways bifurcate at the level of RIP1 and TRAF2. Moreover, endogenous RIP1 associates with the MAP kinase kinase kinase (MAP3K) MEKK3 in TNF-alpha-treated cells, and decreased TNF-alpha-induced p38 MAP kinase activation is observed in Mekk3(-/-) cells. Taken together, these studies suggest a mechanism whereby RIP1 may mediate the p38 MAP kinase response to TNF-alpha, by recruiting the MAP3K MEKK3.     

Hepatitis B virus X protein differentially activates RAS-RAF-MAPK and JNK pathways in X-transforming versus non-transforming AML12 hepatocytes.

The hepatitis B virus (HBV) X protein (pX) is implicated in hepatocarcinogenesis of chronic HBV patients by an unknown mechanism. Activities of pX likely relevant to hepatocyte transformation include activation of the mitogenic RAS-RAF-MAPK and JNK pathways. To assess the importance of mitogenic pathway activation by pX in transformation, we employed a cellular model system composed of two tetracycline-regulated, pX-expressing cell lines, constructed in AML12-immortalized hepatocytes. This system includes the differentiated 3pX-1 and the de-differentiated 4pX-1 hepatocytes. Our studies have demonstrated that conditional pX expression transforms only 3pX-1 cells. Here, comparative in vitro kinase assays and various in vivo analyses demonstrate that pX affects an inverse activation of RAS-RAF-MAPK and JNK pathways in 3pX-1 versus 4pX-1 cells. Sustained pX-dependent RAS-RAF-MAPK pathway activation is observed in pX-transforming 3pX-1 cells, whereas sustained pX-dependent JNK pathway activation is observed in pX non-transforming 4pX-1 cells. This differential, pX-dependent mitogenic pathway activation affects differential activation of cAMP-response element-binding protein and c-Jun and determines the proliferative response of 3pX-1 and 4pX-1 cells. Furthermore, tetracycline-regulated, pX-NLS-expressing cell lines demonstrate that expression of the nuclear pX-NLS variant minimally activates the RAS-RAF-MAPK pathway and results in markedly reduced transformation. These results link sustained, pX-mediated activation of RAS-RAF-MAPK pathway to hepatocyte transformation.     

Activation of c-Jun NH2-terminal kinase/stress-activated protein kinase (JNK/SAPK) is critical for hypoxia-induced apoptosis of human malignant melanoma.

Mitogen-activated protein kinase (MAPK) signaling was examined in malignant melanoma cells exposed to hypoxia. Here we demonstrate that hypoxia induced a strong activation of the c-Jun NH2-terminal kinase (JNK), also termed stress-activated protein kinase (SAPK), in the melanoma cell line 530 in vitro. Other members of the MAPK family, e.g., extracellular signal-regulated kinase and p38, remained unaffected by the hypoxic stimulus. Activated JNK/SAPK could also be observed in the vicinity of hypoxic tumor areas in melanoma metastases as detected by immunohistochemistry. Functional analysis of JNK/SAPK activation in the melanoma cell line 530 revealed that activation of JNK/SAPK is involved in hypoxia-mediated tumor cell apoptosis. Both a dominant negative mutant of JNK/SAPK (SAPKbeta K-->R) and a dominant negative mutant of the immediate upstream activator of JNK/SAPK, SEK1 (SEK1 K-->R), inhibited hypoxia-induced apoptosis in transient transfection studies. In contrast, overexpression of the wild-type kinases had a slight proapoptotic effect. Inhibition of extracellular signal-regulated kinase and p38 pathways by the chemical inhibitors PD98058 and SB203580, respectively, had no effect on hypoxiainduced apoptosis. Under normoxic conditions, no influence on apoptosis regulation was observed after inhibition of all three MAPK pathways. In contrast to recent findings, JNK/SAPK activation did not correlate with Fas or Fas ligand (FasL) expression, suggesting that the Fas/FasL system is not involved in hypoxia-induced apoptosis in melanoma cells. Taken together, our data demonstrate that hypoxia-induced JNK/SAPK activation appears to play a critical role in apoptosis regulation of melanoma cells in vitro and in vivo, independent of the Fas/FasL system.     

Role of p38 mitogen-activated protein kinase phosphorylation and Fas-Fas ligand interaction in morphine-induced macrophage apoptosis

In this study, we evaluated the molecular mechanisms involved in morphine-induced macrophage apoptosis. Both morphine and TGF-beta promoted P38 mitogen-activated protein kinase (MAPK) phosphorylation, and this phosphorylation was inhibited by SB 202190 as well as by SB 203580. Anti-TGF-beta Ab as well as naltrexone (an opiate receptor antagonist) inhibited morphine-induced macrophage P38 MAPK phosphorylation. Anti-TGF-beta Ab also attenuated morphine-induced p53 as well as inducible NO synthase expression; in contrast, N(G)-nitro-L-arginine methyl ester, an inhibitor of NO synthase, inhibited morphine-induced P38 MAPK phosphorylation and Bax expression. Morphine also enhanced the expression of both Fas and Fas ligand (FasL), whereas anti-FasL Ab prevented morphine-induced macrophage apoptosis. Moreover, naltrexone inhibited morphine-induced FasL expression. In addition, macrophages either deficient in FasL or lacking p53 showed resistance to the effect of morphine. Inhibitors of both caspase-8 and caspase-9 partially prevented the apoptotic effect of morphine on macrophages. In addition, caspase-3 inhibitor prevented morphine-induced macrophage apoptosis. These findings suggest that morphine-induced macrophage apoptosis proceeds through opiate receptors via P38 MAPK phosphorylation. Both TGF-beta and inducible NO synthase play an important role in morphine-induced downstream signaling, which seems to activate proteins involved in both extrinsic (Fas and FasL) and intrinsic (p53 and Bax) cell death pathways.     

Taiwan cobra phospholipase A2‐elicited JNK activation is responsible for autocrine fas‐mediated cell death and modulating Bcl‐2 and Bax protein expression in human leukemia K562 cells

Phospholipase A₂ (PLA₂) from Naja naja atra venom induced apoptotic death of human leukemia K562 cells. Degradation of procaspases, production of tBid, loss of mitochondrial membrane potential, Bcl‐2 degradation, mitochondrial translocation of Bax, and cytochrome c release were observed in PLA₂‐treated cells. Moreover, PLA₂ treatment increased Fas and FasL protein expression. Upon exposure to PLA₂, activation of p38 MAPK (mitogen‐activated protein kinase) and JNK (c‐Jun NH₂‐terminal kinase) was found in K562 cells. SB202190 (p38 MAPK inhibitor) pretreatment enhanced cytotoxic effect of PLA₂ and led to prolonged JNK activation, but failed to affect PLA₂‐induced upregulation of Fas and FasL protein expression. Sustained JNK activation aggravated caspase8/mitochondria‐dependent death pathway, downregulated Bcl‐2 expression and increased mitochondrial translocation of Bax. SP600125 (JNK inhibitor) abolished the cytotoxic effect of PLA₂ and PLA₂‐induced autocrine Fas death pathway. Transfection ASK1 siRNA and overexpression of dominant negative p38α MAPK proved that ASK1 pathway was responsible for PLA₂‐induced p38 MAPK and JNK activation and p38α MAPK activation suppressed dynamically persistent JNK activation. Downregulation of FADD abolished PLA₂‐induced procaspase‐8 degradation and rescued viability of PLA₂‐treated cells. Taken together, our results indicate that JNK‐mediated autocrine Fas/FasL apoptotic mechanism and modulation of Bcl‐2 family proteins are involved in PLA₂‐induced death of K562 cells. J. Cell. Biochem. 109: 245–254, 2010. © 2009 Wiley‐Liss, Inc.     

Involvement of p38 MAP kinase during iron chelator-mediated apoptotic cell death

Iron is an essential element for the neoplastic cell growth, and iron chelators have been tested for their potential anti-proliferative and cytotoxic effects. To determine the mechanism of cell death induced by iron chelators, we explored the pathways of the three structurally related mitogen-activated protein (MAP) kinase subfamilies during apoptosis induced by iron chelators. We report that the chelator deferoxamine (DFO) strongly activates both p38 MAP kinase and extracellular signal-regulated kinase (ERK) at an early stage of incubation, but slightly activates c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) at a late stage of incubation. Among three MAP kinase blockers used, however, the selective p38 MAP kinase inhibitor SB203580 could only protect HL-60 cells from chelator-induced cell death, indicating that p38 MAP kinase serves as a major mediator of apoptosis induced by iron chelator. DFO also caused release of cytochrome c from mitochondria and induced activation of caspase 3 and caspase 8. Interestingly, treatment of HL-60 cells with SB203580 greatly abolished cytochrome c release, and activation of caspase 3 and caspase 8. Collectively, the current study reveals that p38 MAP kinase plays an important role in iron chelator-mediated cell death of HL-60 cells by activating downstream apoptotic cascade that executes cell death pathway.     

The involvement of reactive oxygen species (ROS) and p38 mitogen-activated protein (MAP) kinase in TRAIL/Apo2L-induced apoptosis

To determine the apoptotic signaling pathway which tumor necrosis factor-related apoptosis-inducing ligand (TRAIL/Apo2L) induced, we investigated the contribution of reactive oxygen species (ROS), p38 mitogen-activated protein (MAP) kinase and caspases in human adenocarcinoma HeLa cells. Here we show that upon TRAIL/Apo2L exposure there was pronounced ROS accumulation and activation of p38 MAP kinase, and that activation of caspases and apoptosis followed. Pretreatment with antioxidants such as glutathione or estrogen attenuated TRAIL/Apo2L-induced apoptosis through a reduction of ROS generation and diminished p38 MAP kinase and caspase activation. The p38 MAP kinase inhibitor SB203580 prevented apoptosis through a blockage of caspase activation, although ROS generation was not attenuated. Furthermore, the pan-caspase inhibitor Z-Val-Ala-DL-Asp-fluoromethyl ketone fully prevented apoptosis, while neither ROS accumulation nor p38 MAP kinase activation were affected. Therefore, our results suggest that TRAIL/Apo2L-induced apoptosis is mediated by ROS-activated p38 MAP kinase followed by caspase activation in HeLa cells.     

Execution of macrophage apoptosis by Mycobacterium avium through apoptosis signal-regulating kinase 1/p38 mitogen-activated protein kinase signaling and caspase 8 activation

Macrophage apoptosis is an important component of the innate immune defense machinery (against pathogenic mycobacteria) responsible for limiting bacillary viability. However, little is known about the mechanism of how apoptosis is executed in mycobacteria-infected macrophages. Apoptosis signal-regulating kinase 1 (ASK1) was activated in Mycobacterium avium-treated macrophages and in turn activated p38 mitogen-activated protein (MAP) kinase. M. avium-induced macrophage cell death could be blocked in cells transfected with a catalytically inactive mutant of ASK1 or with dominant negative p38 MAP kinase arguing in favor of a central role of ASK1/p38 MAP kinase signaling in apoptosis of macrophages challenged with M. avium. ASK1/p38 MAP kinase signaling was linked to the activation of caspase 8. At the same time, M. avium triggered caspase 8 activation, and cell death occurred in a Fas-associated death domain (FADD)-dependent manner. The death signal induced upon caspase 8 activation linked to mitochondrial death signaling through the formation of truncated Bid (t-Bid), its translocation to the mitochondria and release of cytochrome c. Caspase 8 inhibitor (z-IETD-FMK) could block the release of cytochrome c as well as the activation of caspases 9 and 3. The final steps of apoptosis probably involved caspases 9 and 3, since inhibitors of both caspases could block cell death. Of foremost interest in the present study was the finding that ASK1/p38 signaling was essential for caspase 8 activation linked to M. avium-induced death signaling. This work provides the first elucidation of a signaling pathway in which ASK1 plays a central role in innate immunity.     

ATR-dependent activation of p38 MAP kinase is responsible for apoptotic cell death in cells depleted of Cdc7

Cdc7 is a serine/threonine kinase that plays essential roles in the initiation of eukaryotic DNA replication and checkpoint response. In previous studies, depletion of Cdc7 by small interfering RNA was shown to induce an abortive S phase that led to the cell cycle arrest in normal human fibroblasts and apoptotic cell death in various cancer cells. Here we report that stress-activated p38 MAP kinase was activated and responsible for apoptotic cell death in Cdc7-depleted HeLa cells. The activation of p38 MAP kinase in the Cdc7-depleted cells was shown to depend on ATR, a major sensor kinase for checkpoint or DNA damage responses. Only the p38 MAP kinase, and not the other stress-activated kinases such as JNK or ERK, was activated, and both caspase 8 and caspase 9 were activated for the induction of apoptosis. Activation of apoptosis in Cdc7-depleted cells was completely abolished in cells treated with small interfering RNA or an inhibitor of the p38 MAP kinase, suggesting that p38 MAP kinase activation was responsible for apoptotic cell death. Taken together, we suggest that the ATR-dependent activation of the p38 MAP kinase is a major signaling pathway that induces apoptotic cell death after depletion of Cdc7 in cancer cells.     

Induction of apoptosis in the lung tissue from rats exposed to cigarette smoke involves p38/JNK MAPK pathway

Smoking is a major cause of human lung cancer. Past studies suggest that apoptosis might influence the malignant phenotype, but little is known about the association between apoptosis and cigarette smoke (CS)-induced lung pathogenesis. Using an in situ cell death detection kit (TA300), the association of CS with apoptosis was determined in a concentration-dependent manner. Furthermore, the expression of related proteins were investigated in the terminal bronchiole areas of the lung tissue from rats exposed to CS. Results showed that the expression of phosphotyrosine proteins was increased significantly in lung tissue of rats exposed to CS from 5 to 15 cigarettes. Using Western blotting and immunoprecipitation assay, Fas, a death receptor, was proved just be one of these phosphotyrosine proteins. CS triggered activation of MAP kinase (p38/JNK or ERK2) pathway, which led to Jun or p53 phosphorylation and FasL induction links Fas phosphorylation. Further, smoke treatment produced an increase in the level of proapoptotic proteins (Bax, t-Bid, cytochrome c and caspase-3), but a decline in Bcl-2, procaspase-8 and procaspase-9 proteins. Thus, CS-induced apoptosis may result from two main mechanisms, one is the activation of p38/JNK-Jun-FasL signaling, and the other is stimulated by the stabilization of p53, increase in the ratio of Bax/Bcl-2, release of cytochrome c; thus, leading to activation of caspase cascade.     

Cross-talk between the pathways leading to the induction of apoptosis and the secretion of tumor necrosis factor-alpha in ricin-treated RAW 264.7 cells

Ricin induced apoptotic nuclear morphological changes in mouse macrophage cell line RAW264.7 cells at concentrations sufficient to cause severe protein synthesis inhibition. Ricin also induced the release of tumor necrosis factor-alpha (TNF-alpha) from this cell line in a dose-dependent manner but the profile was bell-shaped. However, the isolated galactose-specific ricin B-chain had no such effects. These results suggest that the receptor-binding of ricin through the B-chain is not enough, and subsequent attack on the intracellular target, i.e., the 28S ribosomal RNA (rRNA), by the A-chain of internalized ricin is required for the effects of ricin. Z-D-CH2-DCB, a caspase family inhibitor, showed potent inhibition of the release of TNF-alpha from RAW264.7 cells as well as blockage of the induction of apoptosis by ricin. Furthermore, SB202190, a specific P38 mitogen-activated protein (MAP) kinase inhibitor that strongly inhibits the release of TNF-alpha, also showed significant inhibition of ricin-induced apoptosis. These results suggest that there may be cross-talk between the pathways leading to the release of TNF-alpha and apoptosis. Time course analysis revealed that the activation of p38 MAP kinase started prior to the induction of TNF-alpha release and apoptosis. Since the activation of p38 MAP kinase in ricin-treated RAW264.7 cells was not prevented by Z-D-CH2-DCB, the activation of p38 MAP kinase may occur upstream of the caspase cascade. Among the other protein synthesis inhibitors examined, modeccin and anisomycin, which can trigger a ribotoxic stress response similar to ricin, induced the release of TNF-alpha, but emetine and cycloheximide did not. These results suggest that the specific attack on the 28S ribosomal RNA and the resulting ribotoxic stress response may trigger the multiple signal transduction pathways through the activation of p38 MAP kinase, which in turn leads to TNF-alpha release and apoptosis.     

Modulated kinase activities in cells undergoing tumour necrosis factor-induced apoptotic cell death

Tumour necrosis factor-alpha (TNF) has a variety of cellular effects including apoptotic and necrotic cytotoxicity. TNF activates a range of kinases, but their role in cytotoxic mechanisms is unclear. HeLa cells expressing elevated type II 75 kDa TNF receptor (TNFR2) protein, analysed by flow cytometry and Western analysis, showed altered c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (p38MAPK; but not MAPK) protein content and activation. There was greater JNK activation, but reduced p38MAPK activation in dying cells compared to those still to enter TNF-induced apoptosis. Moreover, cells displaying more rapid apoptosis possess higher levels of type I 55 kDa TNFR1 receptor isoform, but less TNFR2. These findings reveal differential kinase activation in TNF-induced apoptotic death.     

The role of p38 MAP kinase and c-Jun N-terminal protein kinase signaling in the differentiation and apoptosis of immortalized neural stem cells

The two distinct members of the mitogen-activated protein (MAP) kinase family c-Jun N-terminal protein kinase (JNK) and p38 MAP kinase, play an important role in central nervous system (CNS) development and differentiation. However, their role and functions are not completely understood in CNS. To facilitate in vitro study, we have established an immortal stem cell line using SV40 from fetal rat embryonic day 17. In these cells, MAP kinase inhibitors (SP600125, SB202190, and PD98059) were treated for 1, 24, 48, and 72 h to examine the roles of protein kinases. Early inhibition of JNK did not alter phenotypic or morphological changes of immortalized cells, however overexpression of Bax and decrease of phosphorylated AKT was observed. The prolonged inhibition of JNK induced polyploidization of immortalized cells, and resulted in differentiation and inhibition of cell proliferation. Moreover, JNK and p38 MAP kinase but not ERK1/2 was activated, and p21, p53, and Bax were overexpressed by prolonged inhibition of JNK.

These results indicate that JNK and p38 MAP kinase could play dual roles on cell survival and apoptosis. Furthermore, this established cell line could facilitate study of the role of JNK and p38 MAP kinase on CNS development or differentiation/apoptosis.