Activation of the c-Jun N-terminal kinase (JNK) signaling pathway is essential during PBOX-6-induced apoptosis in chronic myelogenous leukemia (CML) cells

The mitogen-activated protein (MAP) kinase family is activated in response to a wide variety of external stress signals such as UV irradiation, heat shock, and many chemotherapeutic drugs and leads to the induction of apoptosis. A novel series of pyrrolo-1,5-benzoxazepines have been shown to potently induce apoptosis in chronic myelogenous leukemia (CML) cells, which are resistant to many chemotherapeutic agents. In this study we have delineated part of the mechanism by which a representative compound known as PBOX-6 induces apoptosis. We have investigated whether PBOX-6 induces activation of MAP kinase signaling pathways in CML cells. Treatment of K562 cells with PBOX-6 resulted in the transient activation of two JNK isoforms, JNK1 and JNK2. In contrast, PBOX-6 did not activate the extracellular signal-regulated kinase (ERK) or p38. Apoptosis was found to occur independently of the small GTPases Ras, Rac, and Cdc42 but involved phosphorylation of the JNK substrates, c-Jun and ATF-2. Pretreatment of K562 cells with the JNK inhibitor, dicoumarol, abolished PBOX-6-induced phosphorylation of c-Jun and ATF-2 and inhibited the induced apoptosis, suggesting that JNK activation is an essential component of the apoptotic pathway induced by PBOX-6. Consistent with this finding, transfection of K562 cells with the JNK scaffold protein, JIP-1, inhibited JNK activity and apoptosis induced by PBOX-6. JIP-1 specifically scaffolds JNK, MKK7, and members of the mixed-lineage kinase (MLK) family, implicating these kinases upstream of JNK in the apoptotic pathway induced by PBOX-6 in K562 cells.     

Compression-induced senescence of nucleus pulposus cells by promoting mitophagy activation via the PINK1/PARKIN pathway

The current research aimed to explore the possible relationship between PINK1/PARKIN-mediated mitophagy and the compression-induced senescence of nucleus pulposus cells (NPCs). Therefore, the stages of senescence in NPCs were measured under compression lasting 0, 24 and 48 hours. The mitophagy-related markers, autophagosomes and mitochondrial membrane potential were tested to determine the levels of PINK1/PARKIN-mediated mitophagy under compression. The PINK1 and PARKIN levels were also measured by immunohistochemistry of human and rat intervertebral disc (IVD) tissues taken at different degenerative stages. A specific mitophagy inhibitor, cyclosporine A (CSA) and a constructed PINK1-shRNA were used to explore the relationship between mitophagy and senescence by down-regulating the PINK1/PARKIN-mediated mitophagy levels. Our results indicated that compression significantly enhanced the senescence of NPCs in a time-dependent manner. Also, PINK1/PARKIN-mediated mitophagy was found to be activated by the extended duration of compression on NPCs as well as the increased degenerative stages of IVD tissues. After inhibition of PINK1/PARKIN-mediated mitophagy by CSA and PINK1-shRNA, the senescence of NPCs induced by compression was strongly rescued. Hence, the excessive degradation of mitochondria in NPCs by mitophagy under continuous compression may accelerate the senescence of NPCs. Regulating PINK1/PARKIN-mediated mitophagy might be a potential therapeutic treatment for IVD degeneration.     

Activation of c-Jun N-terminal kinase (JNK) during mitosis in retinal progenitor cells

Most studies of c-Jun N-terminal Kinase (JNK) activation in retinal tissue were done in the context of neurodegeneration. In this study, we investigated the behavior of JNK during mitosis of progenitor cells in the retina of newborn rats. Retinal explants from newborn rats were kept in vitro for 3 hours and under distinct treatments. Sections of retinal explants or freshly fixed retinal tissue were used to detect JNK phosphorylation by immunohistochemistry, and were examined through both fluorescence and confocal microscopy. Mitotic cells were identified by chromatin morphology, histone-H3 phosphorylation, and location in the retinal tissue. The subcellular localization of proteins was analyzed by double staining with both a DNA marker and an antibody to each protein. Phosphorylation of JNK was also examined by western blot. The results showed that in the retina of newborn rats (P1), JNK is phosphorylated during mitosis of progenitor cells, mainly during the early stages of mitosis. JNK1 and/or JNK2 were preferentially phosphorylated in mitotic cells. Inhibition of JNK induced cell cycle arrest, specifically in mitosis. Treatment with the JNK inhibitor decreased the number of cells in anaphase, but did not alter the number of cells in either prophase/prometaphase or metaphase. Moreover, cells with aberrant chromatin morphology were found after treatment with the JNK inhibitor. The data show, for the first time, that JNK is activated in mitotic progenitor cells of developing retinal tissue, suggesting a new role of JNK in the control of progenitor cell proliferation in the retina.     

Role of the JNK/c-Jun/AP-1 signaling pathway in galectin-1-induced T-cell death

Galectin-1 (gal-1), an endogenous β-galactoside-binding protein, triggers T-cell death through several mechanisms including the death receptor and the mitochondrial apoptotic pathway. In this study we first show that gal-1 initiates the activation of c-Jun N-terminal kinase (JNK), mitogen-activated protein kinase kinase 4 (MKK4), and MKK7 as upstream JNK activators in Jurkat T cells. Inhibition of JNK activation with sphingomyelinase inhibitors (20 μM desipramine, 20 μM imipramine), with the protein kinase C-δ (PKCδ) inhibitor rottlerin (10 μM), and with the specific PKCθ pseudosubstrate inhibitor (30 μM) indicates that ceramide and phosphorylation by PKCδ and PKCθ mediate gal-1-induced JNK activation. Downstream of JNK, we observed increased phosphorylation of c-Jun, enhanced activating protein-1 (AP-1) luciferase reporter, and AP-1/DNA-binding in response to gal-1. The pivotal role of the JNK/c-Jun/AP-1 pathway for gal-1-induced apoptosis was documented by reduction of DNA fragmentation after inhibition JNK by SP600125 (20 μM) or inhibition of AP-1 activation by curcumin (2 μM). Gal-1 failed to induce AP-1 activation and DNA fragmentation in CD3-deficient Jurkat 31-13 cells. In Jurkat E6.1 cells gal-1 induced a proapoptotic signal pattern as indicated by decreased antiapoptotic Bcl-2 expression, induction of proapoptotic Bad, and increased Bcl-2 phosphorylation. The results provide evidence that the JNK/c-Jun/AP-1 pathway plays a key role for T-cell death regulation in response to gal-1 stimulation.     

Hydrogen sulfide protects against acetaminophen-induced acute liver injury by inhibiting apoptosis via the JNK/MAPK signaling pathway

Acetaminophen (APAP) is a widely used over-the-counter analgesic and antipyretic. It can cause hepatotoxicity. Recent studies demonstrated that hydrogen sulfide (H₂S) exhibits cell protection in several cell types. This study was designed to investigate whether H ₂S ameliorated APAP-induced acute liver injury and to elucidate its mechanisms. In this study, we analyzed the detailed biological and molecular processes of APAP-induced hepatotoxicity using a bioinformatics analysis, which showed that apoptosis and the c-Jun N-terminal kinase (JNK)/mitogen-activated protein kinase pathway were confirmed to play critical roles in these processes. We further investigated the protective effects of H ₂S on APAP-induced hepatotoxicity. In vivo, we observed that the exogenous supplement of H ₂S ameliorated APAP-induced liver injury. Cystathionine-β-synthase (CBS) and cystathionine-γ-lyase (CSE) systems were the endogenous pathway of H ₂S. The expression of CBS/CSE was decreased in APAP-treated mice, while H ₂S could significantly restore it. In addition, APAP-induced JNK activation was inhibited by H ₂S in vivo. In vitro, H ₂S abolished the active effects of APAP on caspase3, Bax, and Bcl-2 expressions as well as JNK phosphorylation in hepatocytes. It was found through flow cytometry that the amount of APAP-induced apoptotic hepatocytes was decreased in the presence of H ₂S. In conclusion, our results suggested that H ₂S attenuated APAP-induced apoptosis in hepatocytes through JNK/MAPK siganaling pathway.     

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.     

Retracted: HO-1 overexpression alleviates senescence by inducing autophagy via the mitochondrial route in human nucleus pulposus cells

Intervertebral disc degeneration (IDD) is closely associated with aging. Our previous studies have confirmed that heme oxygenase-1 (HO-1) can inhibit nucleus pulposus (NP) cell apoptosis. However, whether or not HO-1 is involved in NP cell senescence and autophagy is unclear. Our results indicated that HO-1 expression was reduced in IDD tissues and replicative senescent NP cells. HO-1 overexpression using a lentiviral vector reduced the NP cell senescence level, protected mitochondrial function, and promoted NP cell autophagy through the mitochondrial pathway. Autophagy inhibitor 3-MA pretreatment reversed the anti-senescent and protective effects on the mitochondrial function of HO-1, which promoted the degradation of the extracellular matrix (ECM) in the intervertebral disc. In vivo, HO-1 overexpression inhibited IDD and enhanced autophagy. In summary, these results suggested that HO-1 overexpression alleviates NP cell senescence by inducing autophagy via the mitochondrial route.     

Periplocin mediates TRAIL-induced apoptosis and cell cycle arrest in human myxofibrosarcoma cells via the ERK/p38/JNK pathway

BackgroundPeriploca sepium is traditionally used in Chinese medicine to treat particularly rheumatic disorders and as a tonic. Periplocin was found as the most cytotoxic compound of its root bark and induced death receptor mediated apoptosis in liposarcoma cells. Sarcomas are a rare type of cancer with only a few treatment options. The five-year survival rate of advanced tumors is low.PurposeIn this study, we investigated the effects of periplocin in two myxofibrosarcoma (MFS)cell lines, MUG-Myx2a and MUG-Myx2b, which are subclones of the same tumor and reflect the tumor´s heterogeneity, and in T60 primary myxofibrosarcoma cells.MethodsThe xCELLigence system and the CellTiter 96® AQ_ueous assay were used for studying cell viability. FACS and Western blot experiments were used to investigate the effects of periplocin on apoptosis induction, cell cycle distribution, and the expression of cleaved PARP, caspase 3, p53, phospho-histone γH2AX, ERK/phospho ERK, p38/phospho p38, and, finally, JNK/phospho JNK. Additionally, the expression of the apoptotic markers Bim, NOXA, Bak, Bcl-2, Bcl-xl, and the death receptors IGFR, FADD, TRADD, TNFR1A, TRAIL-R1, and TRAIL-R2 were evaluated using reversed real-time PCR.ResultsPeriplocin decreased dose-dependently the viability of all MFS cell lines and was more effective than the standard chemotherapeutic doxorubicin. It arrested the cells in the G2/M phase and led to caspase activation. Moreover, periplocin increased the mRNA expression of NOXA, Bak, Bcl-2, and death receptors such as TRAIL-R1 and TRAIL-R2 and the protein expression of ERK/phospho ERK, p38/phospho p38, and JNK/phospho JNK. In all cases, differences in the effects in the different subclones were observed.ConclusionPeriplocin showed promising effects in MFS cells. The higher effectiveness compared to doxorubicin is an important aspect for further research with regard as a treatment option. The different effects of periplocin in the two subclones showed the great importance of intratumoral heterogeneity in MFS therapy.     

Magnolol induces apoptosis via inhibiting the EGFR/PI3K/Akt signaling pathway in human prostate cancer cells

We observed that treatment of prostate cancer cells for 24 h with magnolol, a phenolic component extracted from the root and stem bark of the oriental herb Magnolia officinalis, induced apoptotic cell death in a dose‐ and time‐dependent manner. A sustained inhibition of the major survival signal, Akt, occurred in magnolol‐treated cells. Treatment of PC‐3 cells with an apoptosis‐inducing concentration of magnolol (60 µM) resulted in a rapid decrease in the level of phosphorylated Akt leading to inhibition of its kinase activity. Magnolol treatment (60 µM) also caused a decrease in Ser(¹³⁶) phosphorylation of Bad (a proapoptotic protein), which is a downstream target of Akt. Protein interaction assay revealed that Bcl‐xL, an anti‐apoptotic protein, was associated with Bad during treatment with magnolol. We also observed that during treatment with magnolol, translocation of Bax to the mitochondrial membrane occurred and the translocation was accompanied by cytochrome c release, and cleavage of procaspase‐8, ‐9, ‐3, and poly(ADP‐ribose) polymerase (PARP). Similar results were observed in human colon cancer HCT116Bax^+/? cell line, but not HCT116Bax^?/? cell line. Interestingly, at similar concentrations (60 µM), magnolol treatment did not affect the viability of normal human prostate epithelial cell (PrEC) line. We also observed that apoptotic cell death by magnolol was associated with significant inhibition of pEGFR, pPI3K, and pAkt. These results suggest that one of the mechanisms of the apoptotic activity of magnolol involves its effect on epidermal growth factor receptor (EGFR)‐mediated signaling transduction pathways. J. Cell. Biochem. 106: 1113–1122, 2009. © 2009 Wiley‐Liss, Inc.     

IL-2/IL-18 prevent the down-modulation of NKG2D by TGF-beta in NK cells via the c-Jun N-terminal kinase (JNK) pathway

TGF-beta is known to play a major role for the reduced NKG2D expression seen in cancer patients. However, the mechanisms for reduced TGF-beta-induced down-regulation of NKG2D are unclear. In this study, we observed that IL-2/IL-18 increased the NKG2D expression in the TGF-beta treated NK cell line in a dose-dependent manner. Incubation with the JNK inhibitor SP600125 inhibited the NKG2D expression induced by IL-2/IL-18 in the TGF-beta treated human NK cell line. Moreover, the NK cytotoxicity assay showed that the reduced NK cytotoxicity by TGF-beta was recovered by IL-2/IL-18 treatment. The results indicate that IL-2/IL-18 strongly prevented the TGF-beta-induced NKG2D down-regulation in NK cells via the JNK pathway. Taken together, the protected expression of NKG2D by IL-2/IL-18 provides insight into the mechanism of NKG2D regulation and it also supplied useful information for creating a novel therapeutic approach to treat TGF-beta-secreting cancer cells.     

Arrestin-3 Binds c-Jun N-terminal Kinase 1 (JNK1) and JNK2 and Facilitates the Activation of These Ubiquitous JNK Isoforms in Cells via Scaffolding

Non-visual arrestins scaffold mitogen-activated protein kinase (MAPK) cascades. The c-Jun N-terminal kinases (JNKs) are members of MAPK family. Arrestin-3 has been shown to enhance the activation of JNK3, which is expressed mainly in neurons, heart, and testes, in contrast to ubiquitous JNK1 and JNK2. Although all JNKs are activated by MKK4 and MKK7, both of which bind arrestin-3, the ability of arrestin-3 to facilitate the activation of JNK1 and JNK2 has never been reported. Using purified proteins we found that arrestin-3 directly binds JNK1α1 and JNK2α2, interacting with the latter comparably to JNK3α2. Phosphorylation of purified JNK1α1 and JNK2α2 by MKK4 or MKK7 is increased by arrestin-3. Endogenous arrestin-3 interacted with endogenous JNK1/2 in different cell types. Arrestin-3 also enhanced phosphorylation of endogenous JNK1/2 in intact cells upon expression of upstream kinases ASK1, MKK4, or MKK7. We observed a biphasic effect of arrestin-3 concentrations on phosphorylation of JNK1α1 and JNK2α2 both in vitro and in vivo. Thus, arrestin-3 acts as a scaffold, facilitating JNK1α1 and JNK2α2 phosphorylation by MKK4 and MKK7 via bringing JNKs and their activators together. The data suggest that arrestin-3 modulates the activity of ubiquitous JNK1 and JNK2 in non-neuronal cells, impacting the signaling pathway that regulates their proliferation and survival.     

USP15 promotes the apoptosis of degenerative nucleus pulposus cells by suppressing the PI3K/AKT signalling pathway

ObjectivesDegenerative disc disease is characterized by an enhanced breakdown of its existing nucleus pulposus (NP) matrix due to the dysregulation of matrix enzymes and factors. Ubiquitin‐specific protease 15 (USP15) is reported to be abnormal in certain human diseases. However, its role in NP degeneration remains unclear. Therefore, we aimed to explore the function of USP15 in degenerative NP cell specimens.MethodsWe induced gene silencing and overexpression of USP15 in degenerative NP cells using RNA interference (RNAi) and a lentiviral vector, respectively. qRT‐PCR and Western blotting were used to determine gene and protein expression levels. Cell apoptosis was analysed via flow cytometry. Protein interaction was examined by performing a co‐immunoprecipitation assay. Furthermore, the PI3K inhibitor LY294002 and agonist IGF‐1 were used to investigate the link between USP15 and AKT in NP degeneration.ResultsWe found that USP15 was up‐regulated in degenerative NP cells and that its overexpression accelerated the process of apoptosis. Moreover, USP15 expression levels negatively correlated with AKT phosphorylation in degenerative NP cells. Furthermore, targeting and silencing USP15 with miR‐338‐3p and studying its interaction with FK506‐binding protein 5 (FKBP5) revealed enhancement of FKBP5 ubiquitination, indicating that USP15 is a component of the FKBP5/AKT signalling pathway in degenerative NP cells.ConclusionsOur results show that USP15 exacerbates NP degradation by deubiquitinating and stabilizing FKBP5. This in turn results in the suppression of AKT phosphorylation in degenerative NP cells. Therefore, our study provides insights into the understanding of USP15 function as a potential molecule in the network of NP degeneration.     

Activation by phosphorylation and purification of human c-Jun N-terminal kinase (JNK) isoforms in milligram amounts

c-Jun N-terminal kinases (JNKs) are part of the mitogen-activated protein kinase (MAPK) signaling cascade. They are activated through dual phosphorylation of two residues in the activation loop, a threonine and a tyrosine, by MAP2 kinases (MKK4 and 7) in response to various extracellular stresses such as UV or osmotic shock, as well as by cytokines and growth factors. Only small amounts of phosphorylated, active JNKs have previously been produced because of difficulties in expressing these phosphorylated kinases in Escherichia coli, which lack the appropriate upstream kinases. We have now established a novel activation and purification method that allows for reproducible production of milligram amounts of active, phosphorylated JNKs suitable for a variety of enzymatic, biophysical and structural characterizations. We utilize N-terminally His-tagged MKK4 that is coexpressed in E. coli with a constitutively active form of MEKK1. This phosphorylated, active His-MKK4 is purified by Ni–NTA chromatography and used to phosphorylate milligram amounts of three different isoforms of human JNKs (JNK1α1, JNK1α2 and JNK2α2) that had separately been expressed and purified from E. coli in their inactive forms. These in vitro activated JNKs are phosphorylated on both residues (T183, Y185) in their activation loops and are active towards their substrate, ATF2.     

Activation of non-canonical Wnt/JNK pathway by Wnt3a is associated with differentiation fate determination of human bone marrow stromal (mesenchymal) stem cells

The canonical Wnt signaling pathway can determine human bone marrow stromal (mesenchymal) stem cell (hMSC) differentiation fate into osteoblast or adipocyte lineages. However, its downstream targets in MSC are not well characterized. Thus, using DNA microarrays, we compared global gene expression patterns induced by Wnt3a treatment in two hMSC lines: hMSC-LRP5^T253 and hMSC-LRP5^T244 cells carrying known mutations of Wnt co-receptor LRP5 (T253I or T244M) that either enhances or represses canonical Wnt signaling, respectively. Wnt3a treatment of hMSC activated not only canonical Wnt signaling, but also the non-canonical Wnt/JNK pathway through upregulation of several non-canonical Wnt components e.g. naked cuticle 1 homolog (NKD1) and WNT11. Activation of the non-canonical Wnt/JNK pathway by anisomycin enhanced osteoblast differentiation whereas its inhibition by SP600125 enhanced adipocyte differentiation of hMSC. In conclusion, canonical and non-canonical Wnt signaling cooperate in determining MSC differentiation fate.     

Ligation of IFN-gamma-induced HLA-DR molecules on fibroblasts induces RANTES expression via c-Jun N-terminal kinase (JNK) pathway

The role of human leukocyte antigen (HLA) class II molecules on non-antigen presenting cells has been a matter of controversy. We recently reported that ligation of HLA-DR molecule with anti-HLA-DR antibodies (L243) and/or antigenic peptide/T cell receptor complex resulted in a secretion of several chemokines such as RANTES. In the present study, we aimed to detect putative signal transduction pathway leading to RANTES production from fibroblasts when the DR molecules were ligated with L243. Protein tyrosine kinase inhibitor (GF109203X) suppressed RANTES expression in a dose dependent manner for up to 50% from gingival fibroblasts (GF), while protein kinase C inhibitor (genistein) had no inhibitory effect. Ligation of DR molecules with L243 resulted in tyrosine phosphorylation of 54 kDa cellular protein. Thus, we suspected that either Jun N-terminal kinase-2 (JNK-2) or Src family proteins were involved in HLA-DR-mediated signaling. JNK inhibitor (SP600125), but not Src inhibitor (PP2), suppressed both L243 stimulated RANTES mRNA expression and protein secretion. The maximum inhibition for RANTES production by SP600125 was more than 80%. Additionally, JNK inhibitor nearly completely blocked tumor necrosis factor-alpha (TNF-alpha)-induced RANTES production in GF. Furthermore, ligation of GF HLA-DR with L243 induced selective phosphorylation of JNK-2. We concluded that JNK-2 was one of the HLA-DR-mediated signal transduction pathways.     

Involvement of ROS/ASMase/JNK signalling pathway in inhibiting UVA-induced apoptosis of HaCaT cells by polypeptide from Chlamys farreri

Polypeptide from Chlamys farreri (PCF), a novel marine active material isolated from gonochoric Chinese scallop C. farreri, has potential antioxidant activity and protective effect against ultraviolet (UV) irradiation. The aim was to investigate whether PCF protects HaCaT cells from apoptosis induced by UVA and explore related molecular mechanisms. The results showed that PCF significantly prevented UVA-induced apoptosis of HaCaT cells. PCF not only strongly reduced the intracellular reactive oxygen species (ROS) production, but also diminished expression of acid sphingomyelinase (ASMase) and phosphorylated JNK in HaCaT cells radiated by UVA in a dose-dependent manner. Pre-treatment with ROS scavenger NAC, ASMase inhibitor Desipramine or JNK inhibitor SP600125 was found to effectively prohibit UVA-induced apoptosis and Desipramine markedly blocked phosphorylation of JNK. So it is concluded that PCF obviously protects HaCaT cells from apoptosis induced by UVA and protective effects may attribute to decreasing intracellular ROS level and blocking ASMase/JNK apoptotic signalling pathway.     

Involvement of ROS/ASMase/JNK signalling pathway in inhibiting UVA-induced apoptosis of HaCaT cells by polypeptide from Chlamys farreri

Polypeptide from Chlamys farreri (PCF), a novel marine active material isolated from gonochoric Chinese scallop C. farreri, has potential antioxidant activity and protective effect against ultraviolet (UV) irradiation. The aim was to investigate whether PCF protects HaCaT cells from apoptosis induced by UVA and explore related molecular mechanisms. The results showed that PCF significantly prevented UVA-induced apoptosis of HaCaT cells. PCF not only strongly reduced the intracellular reactive oxygen species (ROS) production, but also diminished expression of acid sphingomyelinase (ASMase) and phosphorylated JNK in HaCaT cells radiated by UVA in a dose-dependent manner. Pre-treatment with ROS scavenger NAC, ASMase inhibitor Desipramine or JNK inhibitor SP600125 was found to effectively prohibit UVA-induced apoptosis and Desipramine markedly blocked phosphorylation of JNK. So it is concluded that PCF obviously protects HaCaT cells from apoptosis induced by UVA and protective effects may attribute to decreasing intracellular ROS level and blocking ASMase/JNK apoptotic signalling pathway.     

Daxx deletion mutant (amino acids 501-625)-induced apoptosis occurs through the JNK/p38-Bax-dependent mitochondrial pathway

Death-associated protein (Daxx) deletion mutant (aa 501-625) has been known to be an inducer of apoptosis. In this study, we observed that the Bax-dependent mitochondrial death signaling pathway plays an important role in Daxx501-625-induced apoptosis. Daxx fragment-induced activation of caspase-9 and -3 was mediated through the apoptosis signal-regulating kinase 1 (ASK1)-MEK-c-Jun-N-terminal kinase (JNK)/p38-Bax pathway. By overexpressing JNK-binding domain (JBD) of JIP1, a JNK-inhibitory protein, and treatment with SB203580, a specific p38 inhibitor, DU-145 cells were made resistant to Daxx501-625-induced apoptosis. Capase-3 deficiency, Bax deficiency, or overexpression of a dominant-negative caspase-9 mutant prevented apoptosis, even though the Daxx501-625 fragment still activated the ASK1-MEK-MAPK pathway. Interestingly, Daxx501-625-induced Bcl-2 interacting domain (Bid) cleavage was suppressed in the dominant-negative caspase-9 mutant cells, whereas Bim was still phosphorylated in these cells. These results suggest that cleavage of Bid occurs downstream of caspase-9 activation. In contrast, phosphorylation of Bim is upstream of caspase-9 activation. Taken together, our results suggest that Daxx501-625-induced apoptosis is mediated through the ASK1-MEK-JNK/p38-Bim-Bax-dependent caspase pathway.