Wei Ya Ma

p38 Kinase Mediates UV-induced Phosphorylation of p53 Protein at Serine 389

The p53 tumor suppressor protein is a transcription factor that plays a key role in the process of apoptosis and the cell’s defense against tumor development. Activation of p53 occurs, at least in part, by phosphorylation of its protein. Very recently it has been reported that UV induced a functional activation of p53 via phosphorylation at serine 389. Here, we report that the UV-induced phosphorylation of p53 at serine 389 is mediated by p38 kinase. UVC-induced phosphorylation of p53 at serine 389 was markedly impaired by either pretreatment of cells with p38 kinase inhibitor, SB202190, or stable expression of a dominant negative mutant of p38 kinase. In contrast, there was no inhibition observed in cells treated with specific MEK1 inhibitor, PD98059, or with stable expression of a dominant negative mutant of ERK2 or JNK1. Most importantly, p38 kinase could be co-immunoprecipitated with p53 by using antibodies against p53. Incubation of active p38 kinase with p53 protein caused the phosphorylation of p53 protein at serine 389 in vitro, while no phosphorylation of p53 at serine 389 was observed when p53 was incubated with activated JNK2 or ERK2. Furthermore, pretreatment of cells with SB202190 blocked the p53 DNA binding activity and p53-dependent transcription. These results strongly suggest that the p38 kinase is at least one of the most important mediators of p53 phosphorylation at serine 389 induced by UVC radiation.

Requirement of Erk, but Not JNK, for Arsenite-induced Cell Transformation

Trivalent arsenic (arsenite, As3+) is a human carcinogen, which is associated with cancers of skin, lung, liver, and bladder. However, the mechanism by which arsenite causes cancer is not well understood. In this study, we found that exposure of Cl 41 cells, a well characterized mouse epidermal cell model for tumor promotion, to a low concentration of arsenite (<25 μm) induces cell transformation. Interestingly, arsenite induces Erk phosphorylation and increased Erk activity at doses ranging from 0.8 to 200 μm, while higher doses (more than 50 μm) are required for activation of JNK. Arsenite-induced Erk activation was markedly inhibited by introduction of dominant negative Erk2 into cells, while expression of dominant negative Erk2 did not show inhibition of JNK and MEK1/2. Furthermore, arsenite-induced cell transformation was blocked in cells expressing the dominant negative Erk2. In contrast, overexpression of dominant negative JNK1 was shown to increase cell transformation even though it inhibits arsenite-induced JNK activation. Our results not only show that arsenite induces Erk activation, but also for the first time demonstrates that activation of Erk, but not JNK, by arsenite is required for its effects on cell transformation.

Requirement for phosphatidylinositol 3-kinase in epidermal growth factor-induced AP-1 transactivation and transformation in JB6 P+ cells.

Phosphatidylinositol 3-kinase (PI 3-kinase) plays a role in a variety of biological processes, including regulation of gene expression, cell growth, and differentiation. However, little is known about its role in the cytoplasmic events involved in epidermal growth factor (EGF)-induced transduction of signals to the transcriptional machinery of the nucleus and in EGF-induced cell transformation. In this study, we examined whether PI 3-kinase is a mediator for the activation of AP-1 and neoplastic transformation by EGF in the murine epidermal cell line JB6. The results showed the following. (i) EGF not only induced a high level of PI 3-kinase activity by itself but also enhanced insulin-induced PI 3-kinase activity in JB6 P+ cells, the EGF-induced PI-3 kinase activity could be blocked by constitutive overexpression of a dominant negative P85 subunit of PI 3-kinase (deltaP85), and insulin could markedly promote EGF-induced AP-1 activity in a dose-dependent manner in JB6 P+ cells as well as promote EGF-induced JB6 P+ cell transformation. (ii) Inhibition of PI-3 kinase with wortmannin or LY294002 markedly decreased the AP-1 activity induced by insulin, EGF, or EGF and insulin in a dose-dependent manner, while wortmannin did not block UVB-induced AP-1 activity. (iii) AP-1 activation by insulin, EGF, or EGF and insulin could be completely inhibited by overexpression of deltaP85 in all the dose and time courses studied. (iv) Inhibitors of PI 3-kinase (wortmannin and LY294002) and stable overexpression of deltaP85 inhibited EGF-induced transformation but had no significant inhibitory effect on cell proliferation induced by EGF or EGF and insulin. These results demonstrate for the first time that PI 3-kinase appears to be required for EGF- or insulin-induced AP-1 transactivation and cell transformation but not cell proliferation in JB6 cells.

Involvement of the Acid Sphingomyelinase Pathway in UVA-induced Apoptosis

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 μm), induced apoptosis in JY and MS1418 cells, but the substrate of SMase, SM (20–80 μm), 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.

Inhibition of ultraviolet B-induced activator protein-1 (AP-1) activity by aspirin in AP-1-luciferase transgenic mice

Aspirin is under consideration as a promising chemopreventative agent for human cancers. To study the usefulness of aspirin as a chemopreventative agent for UV-induced human skin cancer, we investigated the effect of aspirin on UVB-induced activator protein-1 (AP-1) activity. In the JB6 cell culture system, aspirin or sodium salicylate (SA) inhibited UVB-induced AP-1 activity in a dose-dependent manner; this inhibitory effect occurred only in cells pretreated with aspirin or SA before UVB irradiation but not cells treated with aspirin or SA after UVB irradiation. Furthermore, these inhibitory effects on UVB-induced AP-1 activity appeared to be mediated through blocking of activation of MAP kinase family members, including extracellular signal-regulated protein kinases, c-Jun N-terminal kinases, and p38. It was not due to absorption of UVB light by aspirin. In the skin of AP-1-luciferase transgenic mice, UVB irradiation induced a rapid increase in AP-1 activity, which reached the peak at 48 h post-UVB irradiation. The topical pretreatment of mouse skin with aspirin markedly blocked the UVB-induced AP-1 transactivation in vivo. These data provide the first evidence that aspirin and SA are inhibitors of UV-induced signal transduction and thus could be used as a chemopreventative agent for skin cancer.

Translocation of Protein Kinase Cε and Protein Kinase Cδ to Membrane Is Required for Ultraviolet B-induced Activation of Mitogen-activated Protein Kinases and Apoptosis

UV-induced signal transduction may be involved in tumor promotion and induction of apoptosis. The role of protein kinase C (PKC) in UVB-induced signal transduction is not well understood. This study showed that UVB markedly induced translocation of membrane-associated PKCε and PKCδ, but not PKCα, from cytosol to membrane. Dominant negative mutant (DNM) PKCε or PKCδ inhibited UVB-induced translocation of PKCε and PKCδ, respectively. UVB-induced activation of extracellular signal-regulated protein kinases (Erks) and c-Jun NH2-terminal kinases (JNKs) was strongly inhibited by DNM PKCε and PKCδ, whereas the DNM of PKCα was less effective on the UVB-induced phosphorylation of Erks and JNKs. Among the PKC inhibitors used only rottlerin, a selective inhibitor of PKCδ, markedly inhibited the UVB-induced activation of Erks and JNKs, but not p38 kinases. Safingol, a selective inhibitor for PKCα, did not show any inhibitory effect on UVB-induced mitogen-activated protein kinase activation. GF109203X is a stronger inhibitor of classical PKC than novel PKC. Lower concentrations of GF109203X (<10 μm) had no effect on UVB-induced activation of Erks or JNKs. However, at higher concentrations (over 20 μm), GF109203X inhibited UVB-induced activation of JNKs, Erks, and even p38 kinases. Meanwhile, rottlerin and GF109203X markedly inhibited UVB-induced apoptosis of JB6 cells, whereas safingol had little inhibitory effect. DNM-Erk2 cells and PD98059, a selective inhibitor for mitogen-activated protein kinase/extracellular signal-regulated kinase 1 that directly activates Erks, inhibited UVB-induced apoptosis. DNM-JNK1 cells also blocked UVB-induced apoptosis, whereas SB202190, a specific inhibitor for p38 kinases, did not produce the inhibitory effect. These data demonstrate that PKCδ and PKCε, but not PKCα, mediate UVB-induced signal transduction and apoptosis in JB6 cells through activation of Erks and JNKs.

Omega 3 but not omega 6 fatty acids inhibit AP-1 activity and cell transformation in JB6 cells.

Epidemiological and animal-based investigations have indicated that the development of skin cancer is in part associated with poor dietary practices. Lipid content and subsequently the derived fatty acid composition of the diet are believed to play a major role in the development of tumorigenesis. Omega 3 (ω3) fatty acids, including docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), can effectively reduce the risk of skin cancer whereas omega 6 (ω6) fatty acids such as arachidonic acid (AA) reportedly promote risk. To investigate the effects of fatty acids on tumorigenesis, we performed experiments to examine the effects of the ω3 fatty acids EPA and DHA and of the ω6 fatty acid AA on phorbol 12-tetradecanoate 13-acetate (TPA)-induced or epidermal growth factor (EGF)-induced transcription activator protein 1 (AP-1) transactivation and on the subsequent cellular transformation in a mouse epidermal JB6 cell model. DHA treatment resulted in marked inhibition of TPA- and EGF-induced cell transformation by inhibiting AP-1 transactivation. EPA treatment also inhibited TPA-induced AP-1 transactivation and cell transformation but had no effect on EGF-induced transformation. AA treatment had no effect on either TPA- or EGF-induced AP-1 transactivation or transformation, but did abrogate the inhibitory effects of DHA on TPA- or EGF-induced AP-1 transactivation and cell transformation in a dose-dependent manner. The results of this study demonstrate that the inhibitory effects of ω3 fatty acids on tumorigenesis are more significant for DHA than for EPA and are related to an inhibition of AP-1. Similarly, because AA abrogates the beneficial effects of DHA, the dietary ratio of ω6 to ω3 fatty acids may be a significant factor in mediating tumor development.

Ultraviolet B-induced Activated Protein-1 Activation Does Not Require Epidermal Growth Factor Receptor but Is Blocked by a Dominant Negative PKCλ/ι

The exposure of mammalian cells to UV irradiation leads to the activation of transcription factors such as activated protein-1 (AP-1) and NFκB. It is postulated that epidermal growth factor (EGF) receptor, but not protein kinase C (PKC), is the major membrane mediator in UV-induced signal transduction. Since UVB is responsible for most of the carcinogenic effects of sun exposure, we investigated the role of EGF receptors and PKC in UVB-induced AP-1 activation. Our results indicated that while the down-regulation of novel PKC (nPKC) and conventional PKC (cPKC) by pretreatment of cells with 12-O-tetradecanoyl phorbol-13-actetate cannot block UVB-induced AP-1 activity, it can block 12-O-tetradecanoyl phorbol-13-acetate-induced AP-1 activity. Further, the dominant negative mutant PKCλ/ι blocked UVB-induced AP-1 activity in all doses and time courses studied. In contrast, UVB-induced AP-1 activity from cells devoid of EGF receptor (B82) was not significantly different from that of the stable transfectants with a kinase-deficient EGF receptor (B82M721) or those with a wild-type EGF receptor (B82L) at all UVB irradiation doses and time courses studied. All of this evidence indicated that aPKC, but not EGF receptor, is involved in UVB-induced AP-1 activation.

Direct evidence for an important role of sphingomyelinase in ultraviolet-induced activation of c-Jun N-terminal kinase.

Sphingomyelinase (SMase) and its product ceramide have recently attracted a great deal of attention because of their possible role in the signal transduction pathway. However, the role of sphingomyelinase in UV-induced c-June N-terminal kinase (JNK) activation is still unclear. Thus, we investigated this issue directly using a genetic SMase-deficient (2 ∼3% residual acid SMase activity) lymphoblast cell line, MS1418. The results showed that while UV irradiation markedly induces JNK activation in a normal human lymphoblast cell line, JY, it induces only weak JNK activation in MS1418 cells. This difference of JNK response to UV irradiation between these two cell lines was further observed in time course and dose-response studies. In contrast, 12-O-tetradecanoylphorbol-13-acetate-induced JNK activation could be observed in both JY and MS1418 cells. Furthermore, significant JNK activation can be observed in MS1418 cells by exposure of the cells to SMase or C2-ceramide, whereas phospholipase A2 or phospholipase C did not show significant induction of JNK activity, and C2-dihydroceramide and sphingosine induce only much weaker JNK activation in MS1418 cells than that by C2-ceramide. These data demonstrated that SMase plays an essential role in UV-induced JNK activation.

Phosphatidylinositol-3 Kinase Is Necessary for 12-O-Tetradecanoylphorbol-13-acetate-induced Cell Transformation and Activated Protein 1 Activation

Phorbol esters, which activate isoforms of protein kinase C, are general activators of the transcription factor activated protein 1 (AP-1). The pathway involved in this signal transduction is not very clear. Currently, little is known about whether phosphatidylinositol-3 (PI-3) kinase plays any role in phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced signal transduction. We demonstrate here that TPA not only has markedly synergistic effects on insulin-induced PI-3 kinase activity, but it also can induce PI-3 kinase activity and the PI-3 phosphates by itself. We also found that insulin, a PI-3 kinase activator, enhanced TPA-induced AP-1 trans-activation and transformation in JB6 promotion-sensitive cells. Furthermore, wortmannin and LY294002, two PI-3 kinase inhibitors, markedly decreased AP-1 activity induced by insulin, TPA, or TPA and insulin and inhibited JB6 promotion-sensitive cell transformation induced by TPA or TPA and insulin. Most importantly, constitutive overexpression of the dominant negative PI-3 kinase P85 mutants completely blocked insulin- or TPA-induced AP-1 trans-activation and TPA-induced cell transformation. All evidence from present studies suggests that PI-3 kinase acts as a mediator in TPA-induced AP-1 activation and transformation in JB6 cells.