Abstract: Background: Epidemiological statistics has shown that there are approximately 1.2 million new cases of
lung cancer diagnosed every year and the death rate of these patients is 17.8%. Earlier diagnosis is key to
promote the five-year survival rate of these cancer patients. Some tumor markers have been found to be
valuable for earlier diagnosis, but a single marker has limitation in its sensitivity and specificity of cancer
diagnosis. To improve the efficiency of diagnosis, several distinct tumor marker groups are combined
together using a mathematical evaluation model, called artificial neural network (ANN). Lung cancer
markers have been identified to include carcinoembryonic antigen, carcinoma antigen 125, neuron specific
enolase, b2-microglobulin, gastrin, soluble interleukin-6 receptor, sialic acid, pseudouridine, nitric
oxide, and some metal ions.
Methods: These tumor markers were measured through distinct experimental procedures in 50 patients
with lung cancer, 40 patients with benign lung diseases, and 50 cases for a normal control group. The
most valuable were selected into an optimal tumor marker group by multiple logistic regression analysis.
The optimal marker group-coupled ANN model was employed as an intelligent diagnosis system.
Results: We have presented evidence that this system is superior to a traditional statistical method, its
diagnosis specificity significantly improved from 72.0% to 100.0% and its accuracy increased from
71.4% to 92.8%.
Conclusions: The ANN-based system may provide a rapid and accurate diagnosis tool for lung cancer
Abstract: It is clear that solar UV irradiation is a crucial environmental factor resulting in skin diseases partially through activation of cell signaling toward altered gene expression and reprogrammed protein translation. Such a key translational control mechanism is executed by the eukaryotic initiation factor 2α subunit (eIF2α) and the downstream events provoked by phosphorylation of eIF2α at Ser(51) are clearly understood, but the upstream signaling mechanisms on the eIF2α-Ser(51) phosphorylation responses to different types of UV irradiations, namely UVA, UVB and UVC, are still not well elucidated. Herein, our evidence reveals that UVA, UVB and UVC all induce a dose- and time-dependent phosphorylation of eIF2α-Ser(51) through distinct signaling mechanisms. UVA-induced eIF2α phosphorylation occurs through MAPKs, including ERKs, JNKs and p38 kinase, and phosphatidylinositol (PI)-3 kinase. By contrast, UVB-induced eIF2α phosphorylation is through JNKs and p38 kinase, but not ERKs or PI-3 kinase, whereas UVC-stimulated response to eIF2α phosphorylation is via JNKs alone. Furthermore, we have revealed that ATM is involved in induction of the intracellular responses to UVA and UVB, rather than UVC. These findings demonstrate that wavelength-specific UV irradiations activate differential response signaling pathways converged on the eIF2α phosphorylation. Importantly, we also show evidence that a direct eIF2α kinase PKR is activated though phosphorylation by either RSK1 or MSK1, two downstream kinases of MAPKs/PI-3 kinase-mediated signaling pathways.
Abstract: Nrf1 [NF-E2 (nuclear factor-erythroid 2)-related factor 1] is a CNC (cap'n'collar) bZIP (basic-region leucine zipper) transcription factor that is tethered to ER (endoplasmic reticulum) and nuclear envelope membranes through its N-terminal signal peptide (residues 1-30). Besides the signal peptide, amino acids 31-90 of Nrf1 also negatively regulate the CNC-bZIP factor. In the present study we have tested the hypothesis that amino acids 31-90 of Nrf1, and the overlapping NHB2 (N-terminal homology box 2; residues 82-106), inhibit Nrf1 because they control its topology within membranes. This region contains three amphipathic alpha-helical regions comprising amino acids 31-50 [called the SAS (signal peptide-associated sequence)], 55-82 [called the CRACs (cholesterol-recognition amino acid consensus sequences)] and 89-106 (part of NHB2). We present experimental data showing that the signal peptide of Nrf1 contains a TM1 (transmembrane 1) region (residues 7-24) that is orientated across the ER membrane in an N(cyt)/C(lum) fashion with its N-terminus facing the cytoplasm and its C-terminus positioned in the lumen of the ER. Once Nrf1 is anchored to the ER membrane through TM1, the remaining portion of the N-terminal domain (NTD, residues 1-124) is transiently translocated into the ER lumen. Thereafter, Nrf1 adopts a topology in which the SAS is inserted into the membrane, the CRACs are probably repartitioned to the cytoplasmic side of the ER membrane, and NHB2 may serve as an anchor switch, either lying on the luminal surface of the ER or traversing the membrane with an N(cyt)/C(lum) orientation. Thus Nrf1 can adopt several topologies within membranes that are determined by its NTD.
Abstract: In rat liver RL-34 cells, endogenous Nrf1 (nuclear factor-erythroid 2 p45 subunit-related factor 1) is localized in the ER (endoplasmic reticulum) where it exists as a glycosylated protein. Electron microscopy has demonstrated that ectopic Nrf1 in COS-1 cells is located in the ER and the NE (nuclear envelope). Subcellular fractionation, together with a membrane proteinase protection assay, revealed that Nrf1 is an integral membrane protein with both luminal and cytoplasmic domains. The N-terminal 65 residues of Nrf1 direct its integration into the ER and NE membranes and tether it to a Triton X-100-resistant membrane microdomain that is associated with lipid rafts. The activity of Nrf1 was increased by the electrophile tBHQ (t-butyl hydroquinone) probably through an N-terminal domain-dependent process. We found that the NST (Asn/Ser/Thr-rich) domain, along with AD1 (acidic domain 1), contributes positively to the transactivation activity of full-length Nrf1. Furthermore, the NST domain contains seven putative -Asn-Xaa-Ser/Thr- glycosylation sites and, when glycosylation was prevented by replacing all of the seven asparagine residues with either glutamine (Nrf1(1-7xN/Q)) or aspartic acid (Nrf1(1-7xN/D)), the former multiple point mutant possessed less activity than the wild-type factor, whereas the latter mutant exhibited substantially greater activity. Lastly, the ER stressors tunicamycin, thapsigargin and Brefeldin A were found to inhibit basal Nrf1 activity by approximately 25%, and almost completely prevented induction of Nrf1-mediated transactivation by tBHQ. Collectively, these results suggest that the activity of Nrf1 critically depends on its topology within the ER, and that this is modulated by redox stressors, as well as by its glycosylation status.
Abstract: Transcription factor Nrf3 (NF-E2 p45-related factor 3) is targeted to the endoplasmic reticulum (ER). Mouse Nrf3 is subject to proteolysis, Asn glycosylation, and deglycosylation reactions. It is synthesized as a approximately 96-kDa protein that is subsequently converted into isoforms of approximately 90, 80, and 70 kDa. In the ER, the approximately 90-kDa glycoprotein is predominant and gives rise to approximately 80- and approximately 70-kDa isoforms. The approximately 90- and approximately 80-kDa polypeptides were observed in the nuclear envelope, whereas the approximately 70-kDa isoform was detected primarily in the nucleoplasm. Our experiments showed the N-terminal homology box 1 (NHB1, residues 12-31) is part of a tripartite signal peptide sequence, comprising n, h, and c regions. The h region (residues 12-23) was demonstrated to target Nrf3 to the ER and is necessary for its Asn glycosylation. The n region (residues 1-11) controlled the abundance of the approximately 90-kDa glycoprotein. The c region (residues 24-39) was found to contain a signal peptidase cleavage site that is responsible for production of the approximately 90-kDa mature Nrf3 glycoprotein from a approximately 96-kDa precursor. We have found that Nrf3 is activated by the ER stressors tunicamycin and brefeldin A, and that NHB1 is required for this response. Amino acids between the c region and NHB2 (residues 76-100) controlled the proteolytic processing of mouse Nrf3 into cleavage products of approximately 80-kDa (glycated) and approximately 70-kDa (non-glycated); by contrast, human Nrf3 lacked a signal peptidase cleavage site between its c region and NHB2. Lastly, data are presented suggesting that the NHB2 sequence in mouse Nrf3 may regulate the topology of the transcription factor within the ER membrane.
Abstract: Nrf1 (nuclear factor-erythroid 2 p45 subunit-related factor 1) is negatively controlled by its NTD (N-terminal domain) that lies between amino acids 1 and 124. This domain contains a leucine-rich sequence, called NHB1 (N-terminal homology box 1; residues 11-30), which tethers Nrf1 to the ER (endoplasmic reticulum). Electrophoresis resolved Nrf1 into two major bands of approx. 95 and 120 kDa. The 120-kDa Nrf1 form represents a glycosylated protein that was present exclusively in the ER and was converted into a substantially smaller polypeptide upon digestion with either peptide:N-glycosidase F or endoglycosidase H. By contrast, the 95-kDa Nrf1 form did not appear to be glycosylated and was present primarily in the nucleus. NHB1 and its adjacent residues conform to the classic tripartite signal peptide sequence, comprising n-, h- and c-regions. The h-region (residues 11-22), but neither the n-region (residues 1-10) nor the c-region (residues 23-30), is required to direct Nrf1 to the ER. Targeting Nrf1 to the ER is necessary to generate the 120-kDa glycosylated protein. The n-region and c-region are required for correct membrane orientation of Nrf1, as deletion of residues 2-10 or 23-30 greatly increased its association with the ER and the extent to which it was glycosylated. The NHB1 does not contain a signal peptidase cleavage site, indicating that it serves as an ER anchor sequence. Wild-type Nrf1 is glycosylated through its Asn/Ser/Thr-rich domain, between amino acids 296 and 403, and this modification was not observed in an Nrf1(Delta299-400) mutant. Glycosylation of Nrf1 was not necessary to retain it in the ER.
Abstract: Double-stranded RNA-dependent protein kinase R (PKR) has been implicated in anti-viral (antitumor) and apoptotic responses. PKR is activated by extracellular stresses and phosphorylates the alpha subunit of protein synthesis initiation factor eIF2, thereby inhibiting protein synthesis and impeding virus multiplication. Phosphorylation of eIF2alpha in mammalian cells has been shown to be increased after ultraviolet (UV) stress and to be required for UV-induced repression of protein translation. UVA is an important etiological factor in skin carcinogenesis and we observed that UVA induced phosphorylation of PKR (Thr(451)) and eIF2alpha (Ser(51)) in mouse skin epidermal JB6 Cl41 cells. The induction was suppressed by the MEK1 inhibitor, PD 98059. UVA stimulation of PKR and eIF2alpha phosphorylation was also inhibited by a dominant-negative mutant (DNM) of ERK2- or RSK2-deficient cells (RSK2(-)). An inhibitor of p38, SB 202190 or a DNM of p38alpha kinase (DNM-p38alpha) suppressed UVA-induced phosphorylation of eIF2alpha (Ser(51)) but had no effect on phosphorylation of PKR (Thr(451)). Our data indicated that phosphorylation of PKR at Thr(451) is mediated through ERK2 and RSK2, but not through p38 kinase, and is involved in the regulation of Ser(51) phosphorylation of eIF2alpha in UVA-irradiated JB6 cells. In vitro and in vivo kinase assays indicated that phosphorylation of eIF2alpha at Ser(51) occurred indirectly through ERK2, RSK2 or p38 kinase in the cellular response to UVA. These data may lead to the use of these signaling molecules as targets to develop more effective chemopreventive agents with fewer side effects to control UV-induced skin cancer.
Abstract: The oncoprotein c-Jun is a component of the activator protein-1 transcription factor complex, which is involved in cellular proliferation, transformation, and death. The stabilization of c-Jun is critically important for its function. The phosphorylation of c-Jun by c-Jun NH(2)-terminal kinase 1 and extracellular signal-regulated protein kinases reduces c-Jun ubiquitination resulting in increased stabilization of c-Jun. In this report, we showed that COOH-terminal Src kinase (CSK) binds with and phosphorylates c-Jun at Y26 and Y170. Phosphorylation of c-Jun by CSK, in opposition to c-Jun NH(2)-terminal kinase 1 and extracellular signal-regulated protein kinases, promoted c-Jun degradation and reduced stability. By promoting c-Jun degradation, CSK helps to maintain a low steady-state level of c-Jun, thereby inhibiting activator protein-1 activity and cell transformation caused by c-Jun. These results indicated that this function of CSK controls cell proliferation under normal growth conditions and may have implications for CSK loss of function in carcinogenesis.
Abstract: Nrf1 (nuclear factor-erythroid 2 p45 subunit-related factor 1) and Nrf2 regulate ARE (antioxidant response element)-driven genes. At its N-terminal end, Nrf1 contains 155 additional amino acids that are absent from Nrf2. This 155-amino-acid polypeptide includes the N-terminal domain (NTD, amino acids 1-124) and a region (amino acids 125-155) that is part of acidic domain 1 (amino acids 125-295). Within acidic domain 1, residues 156-242 share 43% identity with the Neh2 (Nrf2-ECH homology 2) degron of Nrf2 that serves to destabilize this latter transcription factor through an interaction with Keap1 (Kelch-like ECH-associated protein 1). We have examined the function of the 155-amino-acid N-terminal polypeptide in Nrf1, along with its adjacent Neh2-like subdomain. Activation of ARE-driven genes by Nrf1 was negatively controlled by the NTD (N-terminal domain) through its ability to direct Nrf1 to the endoplasmic reticulum. Ectopic expression of wild-type Nrf1 and mutants lacking either the NTD or portions of its Neh2-like subdomain into wild-type and mutant mouse embryonic fibroblasts indicated that Keap1 controls neither the activity of Nrf1 nor its subcellular distribution. Immunocytochemistry showed that whereas Nrf1 gave primarily cytoplasmic staining that was co-incident with that of an endoplasmic-reticulum marker, Nrf2 gave primarily nuclear staining. Attachment of the NTD from Nrf1 to the N-terminus of Nrf2 produced a fusion protein that was redirected from the nucleus to the endoplasmic reticulum. Although this NTD-Nrf2 fusion protein exhibited less transactivation activity than wild-type Nrf2, it was nevertheless still negatively regulated by Keap1. Thus Nrf1 and Nrf2 are targeted to different subcellular compartments and are negatively regulated by distinct mechanisms.
Abstract: The tumor suppressor p53 protein is one of the most highly connected nodes in cellular signal transduction pathways and acts as a central regulatory switch in networks controlling cell proliferation and apoptosis. It is involved in the activation of genes that maintain control over cellular responses to DNA errors such as DNA repair, chromosomal recombination, and chromosome segregation. Here we show that ribosomal S6 kinase 2 (RSK2) activates and phosphorylates p53 (Ser15) in vitro and in vivo and colocalizes with p53 in the nucleus. Deficiency of p53 diminishes RSK2-mediated phosphorylation of histone H3 (Ser10) and adding back p53 to p53-/- embryonic fibroblasts restored phosphorylation of histone H3 at Ser10. These results show that the p53 protein is an important substrate of RSK2 and a critical intermediary in the RSK2 and histone H3 interaction. The RSK2-p53-histone H3 complex may likely contribute to chromatin remodeling and cell cycle regulation.
Abstract: Signal transducers and activators of transcription (STATs) play a critical role in signal transduction pathways. STATs are a family of cytoplasmic proteins with roles as signal messengers and transcription factors that participate in normal cellular responses to cytokines and growth factors. Phosphorylation of STAT1 at Ser727 is essential for its activation and occurs in response to stress signals, inflammation or infection. We observed that UVB induced phosphorylation of STAT1 (Ser727) in mouse epidermal JB6 Cl41 cells. This stimulation was inhibited by PD98059 and UO126, wortmannin, LY294002, SB202190 and SP600125 and dominant negative mutants of ERK2 (DNM-ERK2), p38 (DNM-p38) and JNK1 (DNM-JNK1). The response was absent in Jnk1(-/-) or Jnk2(-/-) knockout cells, but was unaffected by a dominant negative mutant of the phosphatidylinositol-3 kinase (PI-3K) p85 subunit (DNM-Deltap85). STAT1 (Ser727) phosphorylation was also blocked in a Rsk2(-) cell line. In Pdk1(-/-) cells STAT1 was not activated by UVB stimulation compared with strong activation in Pdk1(+/+) cells. Our data indicate that phosphorylation of STAT1 (Ser727) occurs through PI-3K, ERKs, p38 kinase, JNKs, PDK1 and p90RSK2 in the cellular response to UVB. We also show an inhibitory effect of theaflavins and EGCG on UVB-induced STAT1 (Ser727), ERKs, JNKs, PDK1 and p90RSK2 phosphorylation.
Abstract: Phosphorylation at Ser727 in signal transducer and activator of transcription 1 (STAT1) is essential for its activation and signal transduction. However, the upstream kinases responsible for phosphorylating Ser727 are still elusive. Here, we provide evidence showing that UVA-induced mitogen-activated protein kinase (MAPK) signaling pathways lead to STAT1 Ser727 phosphorylation. Our experimental results show that UVA-induced Ser727 phosphorylation of STAT1 was, to different degrees, diminished by PD98059 and U0126, two specific inhibitors of MEKs, and SB202190 and PD169316, inhibitors of p38 kinase and c-Jun N-terminal kinases (JNKs), respectively. STAT1 phosphorylation was also blocked by a dominant negative mutant of p38beta kinase or JNK1, JNK1- or JNK2-deficiency, or an N-terminal or C-terminal kinase-dead mutant of mitogen- and stress-activated protein kinase 1 (MSK1), a downstream kinase closer to p38 kinase and extracellular signal-regulated kinases (ERKs). In vitro kinase assays using the combined STAT1 proteins as substrates from immunoprecipitation and glutathione S-transferase pull down show that active ERK1, JNK1, p38 kinase, MEK1 and MSK1 stimulated phosphorylation of STAT1 (Ser727) indirectly through an unidentified factor or a downstream kinase. Overall, our data indicate that phosphorylation of STAT1 at Ser727 occurs through diverse MAPK cascades including MEK1, ERKs, p38 kinase, JNKs and MSK1 in the cellular response to UVA.
Abstract: Activating transcription factor 2 (ATF2) has been shown to regulate gene expression in the cellular response to environmental stresses such as ultraviolet (UV) irradiation. However, the signal transduction mechanism of ATF2 activation by UV is not as yet completely understood. In the present study, we provide evidence showing that UVC-stimulated phosphorylation of ATF2 (Thr71) was to varying degrees prevented by a dominant negative mutant of p38beta kinase, c-Jun N-terminal kinase 1 (JNK1) or extracellular signal-regulated kinase 2 (ERK2). The phosphorylation was also suppressed by PD98059, an MEK inhibitor, or H89, a potent inhibitor of mitogen- and stress-activated protein kinase 1 (MSK1), and a C- or N-terminal 'kinase-dead' mutant of MSK1 (MSK1-Cd or MSK1-Nd). Furthermore, co- immunoprecipitation experiments revealed a potential intracellular signaling complex consisting of ATF2 and ERKs and/or MSK1. In vitro kinase assays revealed that ERK1, ERK2 and MSK1, like p38 kinase and JNK2, directly phosphorylate ATF2 at Thr71, but addition of RSK2 or Akt1 had almost no effect. Active kinase immunoprecipitated by an MSK1, ERKs or p38 antibody from an extract of JB6 cells irradiated by UVC can directly phosphorylate ATF2 at Thr71, suggesting UVC induces a direct phosphorylation of ATF2 by ERKs or MSK1. Overall, our results reveal that MSK1 and ERKs, like p38 kinase and JNKs, are required for ATF2 phosphorylation (Thr71) in the UVC response.
Abstract: Recently, blockage of p53-dependent transcriptional activation and apoptosis by pifithrin-alpha (PFTalpha) has been reported to be useful for reducing the side effects of cancer therapy and the compound is thus thought to be a specific inhibitor of p53 [Komarov et al., Science 1999;285:1733-1737]. Here, we found that PFTalpha did not inhibit UVB- or doxorubicin (Dox)-stimulated p53-mediated transcriptional activation and apoptosis in JB6 cells. Instead, p53-dependent activation and apoptosis were not only induced by PFTalpha itself but were also enhanced by a combination of PFTalpha with UVB or Dox. Furthermore, PFTalpha-induced apoptosis was mediated through p53-dependent and -independent signaling pathways. Extracellular signal-regulated kinases and p38 kinase, but not c-jun N-terminal kinases (JNKs), were activated, and these activations were required for phosphorylation and accumulation of p53 in the cellular apoptotic response to PFTalpha. Thus, we conclude that PFTalpha is not a specific p53 inhibitor in JB6 cells but is a potential activator of p53-mediated signaling and apoptosis.
Abstract: Phosphorylation at Ser(727) is known to be required for complete activation of STAT3 by diverse stimuli including UV irradiation, but the kinase(s) responsible for phosphorylating STAT3 (Ser(727)) is still not well discerned. In the present study, we observed that activation of ATM is required for a UVA-stimulated increase in Ser(727) phosphorylation of STAT3 as well as in activation and phosphorylation of p90 ribosomal protein S6 kinases (RSKs). Moreover, UVA-stimulated activation of upstream kinases, such as c-Jun N-terminal kinases (JNKs) and ERKs, involved in mediating phosphorylation of RSKs and STAT3 was defective or delayed in ATM-deficient cells. Furthermore, we provide evidence that RSK2-deficient cells were defective for UV-induced Ser(727) phosphorylation of STAT3, and the defect was restored after ectopic expression of transfected full-length RSK2. In vitro experiments showed that active RSK2 and JNK1 induce the phosphorylation of STAT3 precipitates from immunoprecipitation but not from glutathione S-transferase (GST) pull-down. Interestingly, the GST fusion STAT3 proteins mixed together with STAT3 immunoprecipitates can be phosphorylated by JNK. However, the in vitro phosphorylation of STAT3 was reduced by the GST-STAT3 beta protein, a dominant negative form of STAT3. Taken together, our results demonstrate that the STAT3 phosphorylation at Ser(727) is triggered by active RSK2 or JNK1 in the presence of a downstream kinase or a cofactor, and thereby the intracellular phosphorylation process is stimulated through a signaling pathway involving ATM, MAPKs, RSK2, and an as yet unidentified kinase or cofactor. Additionally, RSK2-mediated phosphorylation of STAT3 (Ser(727)) was further determined to be required for basal and UVA-stimulated STAT3 transcriptional activities.
Abstract: Arsenite is known to be an environmental human carcinogen. However, the mechanism of action of this compound in skin carcinogenesis is not completely clear. Here, we provide evidence that arsenite can induce phosphorylation of histone H3 at serine 10 in a time- and dose-dependent manner in JB6 Cl 41 cells. Arsenite induces phosphorylation of Akt1 at serine 473 and increases Akt1 activity. A dominant-negative mutant of Akt1 inhibits the arsenite-induced phosphorylation of histone H3 at serine 10. Additionally, active Akt1 kinase strongly phosphorylates histone H3 at serine 10 in vitro. The arsenite-induced phosphorylation of histone H3 at serine 10 was almost completely blocked by a dominant-negative mutant of extracellular signal-regulated kinase 2 and the mitogen-activated protein kinase/extracellular signal-regulated kinase inhibitor PD98059. N- or C-terminal mutant mitogen- and stress-activated protein kinase 1 or its inhibitor H89 had no effect on arsenite-induced phosphorylation of histone H3 at serine 10 in JB6 Cl 41 cells. However, cells deficient in p90 ribosomal S6 kinase 2 (Rsk2(-/-)) totally block this phosphorylation in a dose- and time-dependent manner. Taken together, these results suggested that arsenite-induced phosphorylation of histone H3 at serine 10 is mediated by Akt1, extracellular signal-regulated kinase 2 and p90 ribosomal S6 kinase 2 but not mitogen- and stress-activated protein kinase 1.
Abstract: Cyclooxygenases (COX) are rate-limiting enzymes that catalyze the conversion of arachidonic acid to prostaglandins, which are involved in many physiological and pathophysiological responses. COX-2, one of two isoforms of COX, was recently found to play an important role in carcinogenesis in many cell and tissue types. COX-2 inhibitors, which belong to the family of nonsteroidal anti-inflammatory drugs, are believed to be effective in many biological activities such as tumor chemoprevention because of their inhibition of COX-2. However, in the present study we found that both piroxicam, a general COX inhibitor, and NS-398, a COX-2 selective inhibitor, effectively suppressed the activation of transcription factor activator protein 1 (AP-1) induced by ultraviolet B (UVB) or 12-O-tetradecanoylphorbol-13-acetate in mouse epidermal JB6 cells. These COX-2 inhibitors could also inhibit 12-O-tetradecanoylphorbol-13-acetate-induced cell transformation. UVB significantly increased AP-1 activity in Cox-2(-/-) fibroblasts transfected with an AP-1 luciferase reporter gene, and this increase was blocked by NS-389 or piroxicam. In JB6, Cox-2(-/-), or wild-type Cox-2(+/+) cells, both NS-398 and piroxicam inhibited UVB-induced phosphorylation of c-Jun NH(2)-terminal kinases, the kinases that activate the AP-1/c-Jun complex. Based on our results, we propose that the inhibition of AP-1 activity by COX-2 inhibitors NS-398 or piroxicam may occur by a mechanism that is independent of COX-2.
Abstract: Resveratrol, a constituent of grapes and other foods, is one of the most promising agents for cancer prevention. In a previous study, we showed that the antitumor activity of resveratrol occurs through extracellular signal-regulated protein kinases (ERKs) and p38 kinase-mediated p53 activation. In this study, we also determined that c-jun NH(2)-terminal kinases (JNKs) are involved in resveratrol-induced p53 activation and induction of apoptosis. In the JB6 mouse epidermal cell line, resveratrol activated JNKs dose-dependently within a dose range of 10-40 microM, the same dosage responsible for the inhibition of tumor promoter-induced cell transformation. Stable expression of a dominant negative mutant of JNK1 or disruption of the Jnk1 or Jnk2 gene markedly inhibited resveratrol-induced p53-dependent transcription activity and induction of apoptosis. Furthermore, resveratrol-activated JNKs were shown to phosphorylate p53 in vitro, but this activity was repressed in the cells expressing a dominant negative mutant of JNK1 or in Jnk1 or Jnk2 knockout (Jnk1(-/-) or Jnk2(-/-)) cells. These data suggested that JNKs act as mediators of resveratrol-induced activation of p53 and apoptosis, which may occur partially through p53 phosphorylation.
Abstract: Solar UVA, but not UVC, reaches the earth's surface and therefore is an important etiological factor for the induction of human skin cancer. ATM kinase is an important regulator of cell survival and cell cycle checkpoints. Here, we observe that UVA, unlike UVC, triggers ATM kinase activity, and the activation may occur through reactive oxygen species produced after irradiation of cells with UVA. We also show that ATM activation is involved in the apoptotic response to UVA but not UVC. Furthermore, we provide evidence that ATM-dependent p53 and c-Jun N-terminal kinase (JNK) pathways are linked to UVA-induced apoptosis. On the other hand, UVC-induced apoptosis occurs through ATR-dependent p53 phosphorylation as well as the JNK pathway. Therefore, these results suggest that ATM, like p53, is involved in the UVA-induced apoptosis to suppress carcinogenesis.
Abstract: In resting cells, eIF4E-binding protein 1 (4E-BP1) binds to the eukaryotic initiation factor-4E (eIF-4E), preventing formation of a functional eIF-4F complex essential for cap-dependent initiation of translation. Phosphorylation of 4E-BP1 dissociates it from eIF-4E, relieving the translation block. Studies suggested that insulin- or growth factor-induced 4E-BP1 phosphorylation is mediated by phosphatidylinositol 3-kinase (PI3-kinase) and its downstream protein kinase, Akt. In the present study we demonstrated that UVB induced 4E-BP1 phosphorylation at multiple sites, Thr-36, Thr-45, Ser-64, and Thr-69, leading to dissociation of 4E-BP1 from eIF-4E. UVB-induced phosphorylation of 4E-BP1 was blocked by p38 kinase inhibitors, PD169316 and SB202190, and MSK1 inhibitor, H89, but not by mitogen-activated protein kinase kinase inhibitors, PD98059 or U0126. The PI3-kinase inhibitor, wortmannin, did not block UVB-induced 4E-BP1 phosphorylation, but blocked both UVB- and insulin-induced activation of PI3-kinase and phosphorylation of Akt. 4E-BP1 phosphorylation was blocked in JB6 Cl 41 cells expressing a dominant negative p38 kinase or dominant negative MSK1, but not in cells expressing dominant negative ERK2, JNK1, or PI3-kinase p85 subunit. Our results suggest that UVB induces phosphorylation of 4E-BP1, leading to the functional dissociation of 4E-BP1 from eIF-4E. The p38/MSK1 pathway, but not PI3-kinase or Akt, is required for mediating the UVB-induced 4E-BP1 phosphorylation.
Abstract: Most of the signal pathways involved in ultraviolet (UV)-induced skin carcinogenesis are thought to originate at plasma membrane receptors. However, UVA-induced signal transduction to downstream ribosomal protein S6 kinases, p70(S6K) and p90(RSK), is not well understood. In this report, we show that UVA stimulation of the epidermal growth factor receptor (EGFR) may lead to activation of p70(S6K)/p90(RSK) through phosphatidyl isositol (PI)-3 kinase and extracellular receptor-activated kinases (ERKs). Evidence is provided that phosphorylation and activation of p70(S6K)/p90(RSK) induced by UVA were prevented in Egfr(-/-) cells and were also markedly inhibited by the EGFR-specific tyrosine kinase inhibitors AG1478 and PD153035. Furthermore, EGFR tyrosine kinase inhibitors and EGFR deficiency significantly suppressed activation of PI-3 kinase and ERKs in regulating activation of p90(RSK)/p70(S6K) but had no effect on activation of c-Jun NH(2)-terminal kinases (JNKs) and p38 kinase in response to UVA. Thus, our results suggest that UVA-induced EGFR signaling may be required for activation of p90(RSK)/p70(S6K), PI-3 kinase, and ERKs but not JNKs or p38 kinase.
Abstract: Phosphorylation of Tyr(705) and Ser(727) of signal transducer and activator of transcription 3 (STAT3) are known to be required for maximal activation by diverse stimuli. Tyr(705) phosphorylation is generally accepted to be mediated by the Janus kinase family. But the mechanism for STAT3 (Ser(727)) phosphorylation is not well understood. Here, we provide evidence that UVA-induced phosphorylation of STAT3 at Ser(727) is inhibited by pretreatment of JB6 cells with PD98059 or SB202190. Phosphorylation of STAT3 (Ser(727)) is also markedly prevented by a dominant negative mutant of ERK2, c-Jun N-terminal kinase 1 (JNK1), or p38 kinase and in knockout Jnk1(-/-) or Jnk2(-/-) cells. Furthermore, STAT3 (Ser(727)) phosphorylation is suppressed by C- or N-terminal "kinase-dead" mutants of mitogen- and stress-activated protein kinase 1 (MSK1), a downstream kinase of ERKs and p38 kinase, and H89, a potential MSK1 inhibitor. In vitro experiments showed that active MSK1 and JNKs, but not ERKs or p38 kinase, phosphorylate STAT3 (Ser(727)). Additionally, the role of MAPKs in mediating UVA-stimulated DNA binding activity of STAT3 was investigated. Overall, these results suggest that UVA-induced Ser(727) phosphorylation of STAT3 may occur through MSK1 and JNKs.
Abstract: Activator protein-1 (AP-1), a dimeric complex consisting of proteins encoded by the jun and fos gene families, is a transcription factor induced by a variety of signals including those eliciting proliferation, differentiation, and neoplastic transformation. Although AP-1 has been widely studied in the last decade, physiological levels of AP-1 in different tissues are unclear. In the present study, we analyzed AP-1 activity in several organs (liver, kidney, brain, lung, spleen, heart, skin) of AP-1-luciferase transgenic mice of various ages. Results of these studies indicate that the level of AP-1 in young mice is much higher than that in older mice, and, second, that the skin contains considerably higher levels of AP-1 than other organs. The level of phosphorylated extracellular signal-regulated protein kinase (ERK) in skin was higher in 1- and 2-day-old mice than in mice of other ages. In addition, phosphorylated p38 kinase was high in 2-day-old and 1-wk-old mice, but phosphorylated c-Jun NH(2)-terminal kinase was not detected at any age. AP-1 activity and level of phosphorylated ERKs declined with maturation. These results imply that AP-1 activity mediated through an ERKs-dependent pathway may be involved in skin development.
Abstract: Histone H3 phosphorylation is related closely to chromatin remodeling and chromosome condensation. H3 phosphorylation at serine 28 is coupled with mitotic chromosome condensation in diverse mammalian cell lines. However, the pathway that mediates phosphorylation of H3 at serine 28 is unknown. In the present study, ERK1, ERK2, or p38 kinase strongly phosphorylated H3 at serine 28 in vitro. JNK1 or JNK2 was able also to phosphorylate H3 at serine 28 in vitro but to a lesser degree. UVB irradiation markedly induced phosphorylation of H3 at serine 28 in JB6 Cl 41 cells. PD 98059, a MEK1 inhibitor, and SB 202190, a p38 kinase inhibitor, efficiently repressed UVB-induced H3 phosphorylation at serine 28. Expression of dominant negative mutant (DNM) ERK2 in JB6 Cl 41 cells totally blocked UVB-induced phosphorylation of H3 at serine 28. Additionally, DNM p38 kinase or DNM JNK1 partially blocked UVB-induced H3 phosphorylation at serine 28. Furthermore, UVB-induced H3 phosphorylation at serine 28 was inhibited in Jnk1(-/-) cells but not in Jnk2(-/-) cells. These results suggest that UVB-induced H3 phosphorylation at serine 28 may be mediated by mitogen-activated protein kinases.
Abstract: The sphingomyelin-ceramide pathway is an evolutionarily conserved ubiquitous signal transduction system that regulates many cell functions including apoptosis. Sphingomyelin (SM) is hydrolyzed to ceramide by different sphingomyelinases. Ceramide serves as a second messenger in mediating cellular effects of cytokines and stress. In this study, we find that acid sphingomyelinase (SMase) activity was induced by UVA in normal JY lymphoblasts but was not detectable in MS1418 lymphoblasts from Niemann-Pick type D patients who have an inherited deficiency of acid SMase. We also provide evidence that UVA can induce apoptosis by activating acid SMase in normal JY cells. In contrast, UVA-induced apoptosis was inhibited in MS1418 cells. Exogenous SMase and its product, ceramide (10-40 micrometer), induced apoptosis in JY and MS1418 cells, but the substrate of SMase, SM (20-80 micrometer), induced apoptosis only in JY cells. These results suggest that UVA-induced apoptosis by SM is dependent on acid SMase activity. We also provide evidence that induction of apoptosis by UVA may occur through activation of JNKs via the acid SMase pathway.
Abstract: UVA exposure plays an important role in the etiology of skin cancer. The family of p90-kDa ribosomal S6 kinases (p90(RSK)/MAPKAP-K1) are activated via phosphorylation. In this study, results show that UVA-induced phosphorylation of p90(RSK) at Ser(381) through ERKs and JNKs, but not p38 kinase pathways. We provide evidence that UVA-induced p90(RSK) phosphorylation and kinase activity were time- and dose-dependent. Both PD98059 and a dominant negative mutant of ERK2 blocked ERKs and p90(RSK) Ser(381) phosphorylation, as well as p90(RSK) activity. A dominant negative mutant of p38 kinase blocked UVA-induced phosphorylation of p38 kinase, but had no effect on UVA-induced Ser(381) phosphorylation of p90(RSK) or kinase activity. UVA-induced p90(RSK) phosphorylation and kinase activity were markedly attenuated in JnK1(-/-) and JnK2(-/-) cells. A dominant negative mutant of JNK1 inhibited UVA-induced JNKs and p90(RSK) phosphorylation and kinase activity, but had no effect on ERKs phosphorylation. PD169316, a novel inhibitor of JNKs and p38 kinase, inhibited phosphorylation of p90(RSK), JNKs, and p38 kinase, but not ERKs. However, SB202190, a selective inhibitor of p38 kinase, had no effect on p90(RSK) or JNKs phosphorylation. Significantly, ERKs and JNKs, but not p38 kinase, immunoprecipitated with p90(RSK) when stimulated by UVA and p90(RSK) was a substrate for ERK2 and JNK2, but not p38 kinase. These data indicate clearly that p90(RSK) Ser(381) may be phosphorylated by activation of JNKs or ERKs, but not p38 kinase.
Abstract: Ultraviolet light A (UVA) plays an important role in the etiology of human skin cancer, and UVA-induced signal transduction has a critical role in UVA-induced skin carcinogenesis. The upstream signaling pathways leading to p70(S6K) phosphorylation and activation are not well understood. Here, we observed that UVA induces phosphorylation and activation of p70(S6K). Further, UVA-stimulated p70(S6K) activity and phosphorylation at Thr(389) were blocked by wortmannin, rapamycin, PD98059, SB202190, and dominant negative mutants of phosphatidylinositol (PI) 3-kinase p85 subunit (DNM-Deltap85), ERK2 (DNM-ERK2), p38 kinase (DNM-p38), and JNK1 (DNM-JNK1) and were absent in Jnk1-/- or Jnk2-/- knockout cells. The p70(S6K) phosphorylation at Ser(411) and Thr(421)/Ser(424) was inhibited by rapamycin, PD98059, or DNM-ERK2 but not by wortmannin, SB202190, DNM-Deltap85, or DNM-p38. However, Ser(411), but not Thr(421)/Ser(424) phosphorylation, was suppressed in DNM-JNK1 and abrogated in Jnk1-/- or Jnk2-/- cells. In vitro assays indicated that Ser(411) on immunoprecipitated p70(S6K) proteins is phosphorylated by active JNKs and ERKs, but not p38 kinase, and Thr(421)/Ser(424) is phosphorylated by ERK1, but not ERK2, JNKs, or p38 kinase. Moreover, p70(S6K) co-immunoprecipitated with PI 3-kinase and possibly PDK1. The complex possibly possessed a partial basal level of phosphorylation, but not at MAPK sites, which was available for its activation by MAPKs in vitro. Thus, these results suggest that activation of MAPKs, like PI 3-kinase/mTOR, may be involved in UVA-induced phosphorylation and activation of p70(S6K).
