Cell fate regulated by nuclear factor-κB- and activator protein-1-dependent signalling in human melanocytes exposed to ultraviolet A and ultraviolet B.

BACKGROUND: Ultraviolet (UV) radiation constitutes an important risk factor for malignant melanoma, but the wavelength responsible for the initiation of this disease is not fully elucidated. Solar UV induces multiple signalling pathways that are critical for initiation of apoptotic cell death as a cellular defence against malignant transformation. OBJECTIVES: To evaluate the involvement of the transcription factors nuclear factor (NF)-κB and activator protein (AP)-1 in the signalling pathways induced by UVA or UVB irradiation in human melanocytes. METHODS: Primary cultures of normal human melanocytes were irradiated with UVA or UVB, and the concomitant DNA damage and redox alterations were monitored. The resulting activation of the NF-κB and AP-1 signalling pathways and subsequent apoptosis were studied. RESULTS: UVB irradiation causes DNA damage detected as formation of cyclobutane pyrimidine dimers, while UVA induces increased levels of 8-hydroxydeoxyguanosine and lipid peroxidation. UVA and UVB initiate phosphorylation of c-Jun N-terminal protein kinase and extracellular signal-regulated kinase, and the apoptosis signalling pathways converge into a common mechanism. Downregulation of c-Jun suppresses AP-1-mediated signalling and prevents apoptosis upstream of lysosomal and mitochondrial membrane permeabilization, whereas inhibition of NF-κB by SN50 increases apoptosis. CONCLUSIONS: We conclude that AP-1 induces proapoptotic signalling, whereas NF-κB is a key antiapoptotic/prosurvival factor in both UVA- and UVB-induced cellular damage in human melanocytes, which might in turn impact melanoma development and progression.

Transcriptional repression of c-Jun's E3 ubiquitin ligases contributes to c-Jun induction by UV.

UV radiation is a major environmental carcinogen. The oncoprotein c-Jun that is required for development of skin cancer is stabilized by UV radiation. The mechanism leading to its stabilization after exposure to UV is not known. The lack of knowledge was particularly sharpened, after the discovery that JNK, the most potent positive regulator of c-Jun, activates Itch, an E3-ligase of c-Jun and JunB. In this study we demonstrate that the expression of all three E3 ubiquitin ligases of c-Jun is down-regulated by UV. The levels of Itch/AIP4 and Fbw7alpha transcripts are reduced following UV exposure in every cell line examined. Repression of hCOP1 and its associated protein hDET1, which is required for c-Jun degradation, is cell type dependent. Expression of Fbw7alpha is down-regulated by UVC or UVB, independently of the p53, MAPK and the PKC pathways but the repression is inhibited in the absence of active Fbw7 proteins suggesting that a target protein of Fbw7 is involved in Fbw7 expression/repression. The repression does not require protein synthesis and UV does not change Fbw7 mRNA stability. The characteristics of Fbw7alpha repression perfectly match with those of c-Jun induction. Unlike UV, ionizing radiation does not repress Fbw7alpha and does not induce c-Jun. In addition, the repression kinetics correlates tightly with the kinetics of c-Jun induction by UV. Moreover, abrogation of Fbw7 UV-responsiveness abolishes c-Jun induction by UV, and knockdown of Fbw7 results in elevated basal expression of c-Jun but reduced UV-dependent induction thus, proving the essential role of this repression in c-Jun induction by UV.

Photoprotective and anti-skin-aging effects of eicosapentaenoic acid in human skin in vivo.

Skin aging can be attributed to photoaging (extrinsic) and chronological (intrinsic) aging. Photoaging and intrinsic aging are induced by damage to human skin attributable to repeated exposure to ultraviolet (UV) irradiation and to the passage of time, respectively. In our previous report, eicosapentaenoic acid (EPA) was found to inhibit UV-induced matrix metalloproteinase-1 (MMP-1) expression in human dermal fibroblasts. Therefore, we investigated the effects of EPA on UV-induced skin damage and intrinsic aging by applying EPA topically to young and aged human skin, respectively. By immunohistochemical analysis and Western blotting, we found that topical application of EPA reduced UV-induced epidermal thickening and inhibited collagen decrease induced by UV light. It was also found that EPA attenuated UV-induced MMP-1 and MMP-9 expression by inhibiting UV-induced c-Jun phosphorylation, which is closely related to UV-induced activator protein-1 activation, and by inhibiting JNK and p38 activation. EPA also inhibited UV-induced cyclooxygenase-2 (COX-2) expression without altering COX-1 expression. Moreover, it was found that EPA increased collagen and elastic fibers (tropoelastin and fibrillin-1) expression by increasing transformin growth factor-beta expression in aged human skin. Together, these results demonstrate that topical EPA has potential as an anti-skin-aging agent.

Ultraviolet irradiation suppresses T cell activation via blocking TCR-mediated ERK and NF-kappa B signaling pathways.

UV irradiation is carcinogenic and immunosuppressive. Previous studies indicate that UV-mediated alteration of APCs and induction of suppressor T cells play a critical role in UV-induced immune suppression. In this study, we show that UV irradiation can directly (independently of APCs and suppressor T cells) inhibit T cell activation by blocking TCR-mediated phosphorylation of ERK and IkappaB via overactivation of the p38 and JNK pathways. These events lead to the down-modulation of c-Jun, c-Fos, Egr-1, and NF-kappaB transcription factors and thereby inhibit production of cytokines, e.g., IL-2, IL-4, IFN-gamma, and TNF-alpha, upon TCR stimulation. We also show that UV irradiation can suppress preactivated T cells, indicating that UV irradiation does not only impair T cell function in response to T cell activation, but can also have systemic effects that influence ongoing immune responses. Thus, our data provide an additional mechanism by which UV irradiation directly suppresses immune responses.

Differential inhibition of UVB-induced AP-1 and NF-kappaB transactivation by components of the jun bZIP domain.

Potential targets for chemoprevention of nonmelanoma skin cancer include UV-induced nuclear factor kappaB (NF-kappaB) and activator protein-1 (AP-1) activation in keratinocytes. Inhibition of both these ultraviolet light B (UVB)-induced transcription factors has been shown with the dominant-negative c-jun mutant, TAM67; however, its mechanism of action has not yet been determined. Here we demonstrated that transient transfection of a mouse keratinocyte cell line (308) with a dominant-negative phosphorylation mutant of c-Jun before exposure to 250 J/m(2) UVB inhibits transactivation mediated by both AP-1 and NF-kappaB transcription factors to levels below those of UVB exposed controls. Through the utilization of immunoprecipitation techniques, protein-protein interactions between NF-kappaB family members IkappaBalpha, IkappaBbeta, p50, and p65 (Rel-A) were identified with an Xpress tagged dominant-negative c-Jun (TAM67) protein. Expression of the leucine zipper domain of the TAM67 protein inhibited UVB-induced NF-kappaB transactivation but not AP-1 transactivation. Expression of the bZIP domain of the TAM67 protein was able to inhibit transactivation mediated by both transcription factors. These data demonstrate that TAM67 is able to inhibit two significant UVB-induced molecular targets AP-1 and NF-kappaB, and that the inhibition of these two transcription factor families is potentially due to protein-protein interactions between different regions of the dominant-negative c-Jun protein.

In situ demonstration of phosphorylated c-jun and p38 MAP kinase in epidermal keratinocytes following ultraviolet B irradiation of human skin.

Ultraviolet B (UVB) irradiation is known to induce activation of cellular stress response pathways in cultured cells or intact human skin, as demonstrated by phosphorylation of MAP kinase family members and up- or down-stream targets, using biochemical assays. This study demonstrates by immunohistochemistry that low-dose UVB irradiation of normal human skin induces rapid and reversible phosphorylation of c-jun (a target of c-jun N-terminal kinase) and p38 mitogen activated protein kinase (p38 MAP kinase). Phosphorylation was maximal at 4-8 h and returned to normal levels at 48 h after irradiation. Nuclear localization of these phosphorylated substrates was found using antisera against the epitope containing the phosphorylated serine-73 of c-jun, and the dually phosphorylated epitope (threonine-180 and tyrosine-182) of p38 MAP kinase. Nearly all epidermal cells were positive for c-jun phosphorylation, whereas p38 phosphorylation was seen predominantly in the differentiated layers. In contrast to the massive activation of c-jun and p38, only a small population of the suprabasal cells showed nuclear translocation of nuclear factor kappa B (NFkappaB), and a few scattered cells became apoptotic, as determined by TUNEL (TdT mediated dUTP nick end labelling) staining. The expression of involucrin and skin-derived anti-leukoproteinase (SKALP)/elafin, two genes putatively under control of the c-jun and p38 pathways, was found to be increased. These findings establish the first cellular localization of UVB-induced protein phosphorylation of stress response proteins in human epidermis, thereby providing a link between cellular activation and gene expression in defined cell populations.

Akt negatively regulates the cJun N-terminal kinase pathway in PC12 cells.

The protein serine/threonine kinase Akt is a target of phosphatidylinositol 3-kinase that mediates many of the trophic actions of growth factors on cells. In PC12 cells, complete removal of serum leads to rapid stimulation of the cJun N-terminal kinase (JNK) pathway. Inclusion of insulin-like growth factor-1, a stimulator of Akt in PC12 cells, inhibits JNK activation in this setting, whereas addition of wortmannin to PC12 cells in the presence of serum stimulates JNK activity, suggesting that growth factor-mediated signaling through the phosphatidylinositol 3-kinase/Akt pathway chronically inhibits the JNK pathway in PC12 cells. To explore the possible role of Akt as a negative regulator of JNK activity in PC12 cells, a myristoylated, gain-of-function Akt polypeptide (Myr-Akt) was expressed by retrovirus-mediated gene transfer. Stimulation of JNK activity by serum withdrawal or UV irradiation in PC12 cell clones stably expressing Myr-Akt was inhibited approximately 95% or 50%, respectively, relative to control transfected PC12 cells. Phosphorylation of both JNKs and a proximal activator, MAP kinase kinase 4 (MKK4), in response to UV irradiation was inhibited in Myr-Akt-expressing PC12 cells. Furthermore, transient expression of Myr-Akt strongly inhibited cJun transactivation mediated by MEKK1 or MKK7-JNK3, a gain-of-function MKK7-JNK fusion protein. Interestingly, inhibited JNK activation in the Myr-Akt-expressing PC12 cells is associated with marked induction of JNK-interacting protein-1 (JIP-1). We propose that negative regulation of the JNK pathway through Akt-dependent induction of specfic JIP proteins contributes to the antiapoptotic actions of Akt in neuronal cell types.

UV-Laser induced protein/DNA crosslinking reveals sequence variations of DNA elements bound by c-Jun in vivo.

Many proteins involved in the modulation of gene expression exert their function through direct interaction with DNA. The sequence specificity of these interactions provides the basis for many regulatory mechanisms. The sites that are utilized by a transcription factor are usually analyzed using in vitro binding studies. To detect true in vivo binding sites we developed a method, presented here, that allows construction of recognition element DNA (reDNA) libraries which represent in vivo binding sites plus flanking sequences. reDNA libraries can be constructed for any well-characterized transcription factor. Here we used this method for an in vivo study of genomic DNA elements that interact with the transcription factor c-Jun in rat cerebellum.

4-Aminoquinoline antimalarials enhance UV-B induced c-jun transcriptional activation.

Previous work has documented that the earliest observable response in mammalian cells following ultraviolet (UV) irradiation is the activation of plasma membrane-associated Src tyrosine kinases. These molecules then trigger a signalling cascade that results in activation of the transcription factor AP-1 which subsequently transactivates the early immediate genes including c-jun. This pathway has been postulated to play a protective role against UV damage. As aminoquinoline antimalarials such as chloroquine are known to downregulate several photoinduced cutaneous disorders including LE-specific skin disease, we asked whether chloroquine might be capable of modulating this early limb of the UV light response. A431 cells (a human epidermal keratinocyte cell line) that had been transfected with a c-jun luciferase reporter gene construct were then treated with physiologically relevant concentrations of chloroquine followed by exposure to 0-125 J/m2 of UV-B from a bank of unfiltered FS20 lamps. Chloroquine pretreatment resulted in a dose-dependent increase in luciferase activity in permanently transfected A431 cells (luciferase activity was increased by 45% at 2.5 x 10(-5) M chloroquine and 125 J/m2 of UV-B). Hydroxychloroquine pretreatment also resulted in an increase in luciferase activity. Primaquine, an 8-aminoquinoline, did not influence the UV-B induced c-jun activity. Furthermore, chloroquine did not have a similar impact on HSP-70 gene activity during heat shock. These studies suggest that the beneficial effect of the 4-aminoquinoline antimalarials in various photodermatoses including cutaneous LE might result in part from the capacity of these drugs to enhance the protective early limb of the UV response.

Influences of 50-Hz magnetic fields and ionizing radiation on c-jun and c-fos oncoproteins.

The effect of magnetic fields (50 Hz, 100 microT[rms] sinusoidal magnetic field combined with a 55 microT geomagnetic-like field) and/or gamma rays of 60 Cobalt on the expression of the c-jun and c-fos proteins was investigated in primary rat tracheal epithelial cells and two related immortalized cell lines. Quite similar patterns and amplitudes of induction of these proteins were evidenced after either ionizing radiation or magnetic field exposure. No synergism after both treatments was observed. These findings suggest that magnetic fields explored in the present study may be considered as an insult at the cellular level.

Radiation-induced apoptosis in developing rats and kainic acid-induced excitotoxicity in adult rats are associated with distinctive morphological and biochemical c-Jun/AP-1 (N) expression.

Ionizing radiation produces apoptosis in the developing rat brain. Strong c-Jun immunoreactivity, as revealed with the antibody c-Jun/AP-1 (N) which is raised against the amino acids 91-105 mapping with the amino terminal domain of mouse c-Jun p39, is simultaneously observed in the nucleus and cytoplasm of apoptotic cells. Western blotting of total brain homogenates, using the same antibody, shows a p39 band in control rats which is accompanied by a strong, phosphorylated p62 double-band in irradiated animals. In addition, increased c-Jun N-terminal kinase 1 expression, as found on western blots, is found in irradiated rats when compared with controls. Intraperitoneal injection of kainic acid at convulsant doses to the adult rat produces cell death with morphological features of necrosis, together with the appearance of cells with fine granular chromatin degeneration and small numbers of apoptotic-like cells, in the entorhinal and piriform cortices, basal amygdala, certain thalamic nuclei, and CA1 region of the hippocampus. c-Jun expression in kainic acid-treated rats, as revealed with the c-Jun/AP-1 (N) antibody, is found in the nuclei of a minority of cells in the same areas. The vast majority of c-Jun-immunoreactive cells have normal nuclear morphology, whereas necrotic cells are negative and only a few cells with fine granular chromatin condensation and apoptotic cells following kainic acid injection are stained with c-Jun antibodies. Western blotting, using the same antibody, shows a p39 band in control rats, which is accompanied by a band at about p26 from 6 h onwards following kainic acid injection. Decreased c-Jun N-terminal kinase 1 expression, as revealed on western blots, is observed in kainic acid-treated rats. These results show that the antibody c-Jun/AP-1 (N) recognizes three different forms of c-Jun-related immunoreactivity in normal and pathological states, which are associated with the different outcome of cells. These results stress the necessity of examining in detail the composition of c-Jun-immunoreactive bands and the metabolic state of c-Jun(s) in different paradigms of cell death and survival.

Induction of c-fos, c-jun, junB and junD mRNA and AP-1 by alkylating mutagens in cells deficient and proficient for the DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT) and its relationship to cell death, mutation induction and chromosomal instability.

An early and immediate response of cells upon irradiation with UV light and various other forms of genotoxic stress is the induction of the proto-oncogenes c-fos and c-jun. To address the questions of whether (a) methylating agents that are powerful carcinogens are effective in induction of fos and jun mRNAs, (b) induction is affected by the repair capacity of the cells, and (c) induction is accompanied by genotoxic effects, the levels of c-fos, c-jun, junB and junD mRNA were analysed in isogenic Chinese hamster cell lines deficient (phenotypically Mex-) and proficient (Mex+) for the DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT) after treatment with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and methyl methanesulfonate (MMS). Both methylating agents were very effective in inducing fos and jun mRNAs, although they differ markedly in their potency to induce O6-methylguanine in DNA. Most responsive were c-fos and c-jun (up to 80-fold increases in mRNA level) whereas junB (up to ninefold) and junD (up to twofold) displayed an intermediate and weak response, respectively. No difference in the dose-dependence of induction of these mRNAs was observed between Mex- and Mex+ cells indicating that the critical genotoxic and mutagenic lesion induced by MNNG, i.e. O6-methylguanine, which is rapidly repaired by MGMT, does not act as a trigger for this response. Induction of fos and jun mRNAs by MNNG and MMS was accompanied by a dose-dependent increase in the activity of the transcription factor AP-1. To induce fos and jun mRNAs as well as AP-1, doses of MNNG were required which were more than 50-fold higher than those inducing gene mutations, recombination events (SCEs) and reproductive cell death, and fivefold higher than those inducing chromosomal aberrations in Mex cells. Therefore, the immediate induction of fos and jun mRNAs and AP-1 in Mex- cells upon their exposure to MNNG appears not to be essential for the generation of MNNG-induced mutagenic and genotoxic effects, which is possibly due to the high genotoxic potential of non-repaired O6-methylguanine. However, for MMS and UV light, which was included in this study for comparison, c-fos, c-jun, junB and junD mRNA as well as AP-1 induction paralleled the dose-response for induction of cell killing effects, recombination and chromosomal breakage indicating that increased expression of Fos and Jun is related to the generation of MMS and UV-induced genetic changes. These data are in line with a model according to which the induced c-Fos and Jun proteins are involved in defense against UV radiation and other DNA damaging agents.

Spontaneous and light-evoked expression of JunB-like protein in the hamster suprachiasmatic nucleus near subjective dawn.

Some cells in the hamster suprachiasmatic nucleus (SCN) show a circadian rhythm of expression of junB mRNA in constant darkness, while others show junB mRNA only in response to light at night. We found that both the light-induced and spontaneous expressions of junB mRNA are translated into protein in SCN cells. In constant darkness, JunB-like immunoreactivity (lir) appears spontaneously in cells in the dorsal SCN around subjective dawn and persists for at least 4 h into the subjective day. During the subjective night, there is no spontaneous expression, but a light pulse can induce JunB-lir in cells throughout the SCN, and especially in the ventrolateral portion. As a component of AP-1 proteins, JunB may play a role both in mediating circadian responses to photic stimuli and in spontaneous oscillation of elements of the SCN circadian pacemaker.

Ionizing radiation-induced apoptosis is associated with c-Jun expression and c-Jun/AP-1 activation in the developing cerebellum of the rat.

Previous studies have shown that ionizing radiation-induced cell death in the developing brain has morphological characteristics of apoptosis and is associated with internucleosomal DNA fragmentation. In the present study, we have observed c-Jun induction in cells sensitive to ionizing radiation during the whole process of radiation-induced cell death, and that this expression is accompanied by modifications in the composition of AP-1 complexes: c-Jun/AP-1 activity is highly increased whereas Jun D/AP-1 is slightly decreased. These results show that c-Jun expression and c-Jun/AP-1 activity are induced in the developing brain following ionizing radiation.

Neurotransmitter modulation of Fos- and Jun-like proteins in the turtle retina.

The expression of the Fos and Jun families of nuclear phosphoproteins can be induced by a variety of extracellular stimuli and is known to participate in the transcriptional regulation of target genes. To examine the role of these transcription factors in retinal function, we used polyclonal antisera to localize these protein families in the turtle retina. Fos-like immunoreactivity was in many somata in the inner nuclear and ganglion cell layers. In contrast, Jun-like immunoreactivity was in a smaller number of amacrine cells and many somata in the ganglion cell layer. The monostratified dendritic arbors of one prominent amacrine cell type with Jun-like immunoreactivity were also labeled. There were no dramatic differences in the levels of Fos-like immunoreactivity or Jun-like immunoreactivity between light- or dark-adapted retinas. We examined the effects of excitatory amino acids and gamma-aminobutyric acid (GABA) on the expression of these proteins in vitro. In some experiments, cobalt was used to block synaptic transmission. The excitatory amino acids increased both Fos- and Jun-like immunoreactivity, while GABA generally showed no such stimulatory effect. In cobalt-treated retinas, the same cell types had Jun-like immunoreactivity as seen in the controls, but overall levels of immunoreactivity were increased. In cobalt-treated dark-adapted retinas, some excitatory amino acids increased cytoplasmic Fos-like immunoreactivity in the somata and processes of large cells in the ganglion cell layer. Our results suggest that Fos- and Jun-related proteins may play an important role in the postsynaptic responses to amino acid transmitters in a wide variety of amacrine and ganglion cells.

Radiation signaling mediated by Jun activation following dissociation from a cell type-specific repressor.

The promoter regions of several radiation-inducible genes contain AP-1 cis-acting regulatory elements that are dependent upon protein kinase C signaling. We analyzed nuclear protein from irradiated human tumor cell lines for binding to the AP-1 consensus sequence. The increase in nuclear protein binding following irradiation was specific for the AP-1 sequence and was reduced by antibodies to c-Jun and c-Fos. The AP-1 DNA binding sequence was found to regulate transcription in irradiated cells and mutation of the AP-1 site within the c-jun promoter abolished transcriptional induction by radiation. The gene encoding the chimeric transcription factor Gal4-Jun5-253, which includes the DNA binding region of Gal4 and the transcriptional regulatory region of c-Jun, was cotransfected with the reporter plasmid with Gal4 binding sequences (G5B-CAT). Transfection of RIT-3 and HeLa cells revealed that the regulatory region of Jun was sufficient to activate transcription following irradiation. Conversely, Hep G2 cells, which do not contain the cell type-specific Jun repressor, were not responsive to radiation-induced Jun activation. The c-Jun repressor was found to regulate Jun activation by experiments using the expression vector CMV-jun, which competes for Jun inhibitor and eliminates radiation-induction of Jun. We propose transcription factor dissociation from inhibitor proteins may participate in the initiation of cellular responses to ionizing radiation.

Regulation of c-jun/AP-1 expression in rat L6 myoblasts.

Myogenic differentiation of skeletal myoblasts in culture is triggered by withdrawal of serum mitogens. Expression of one of the serum-inducible, immediate early genes, the protooncogene c-jun, is maintained under low-serum conditions during myogenic differentiation of L6 myoblasts. In this report we have used agents that modulate protein kinases and Ca2+ levels to investigate how the expression of c-jun and myogenin mRNA and also the activator protein 1 (AP-1) DNA-binding activity are regulated in differentiating L6 cells. Our results show that expression of c-jun and myogenin are regulated independent of each other. Furthermore, down regulation of c-jun expression does not cause an increase in myogenin expression, suggesting that c-jun does not suppress myogenin expression in these cells. Electrophoretic mobility shift and ultraviolet cross-linking analyses revealed that the AP-1 complexes of proliferating myoblasts and differentiating myotubes are formed of similar set of polypeptides, and the AP-1 binding activity is probably modulated by posttranslational modifications in differentiating L6 cells.

UV-induced activation of AP-1 involves obligatory extranuclear steps including Raf-1 kinase.

Irradiation of cells with ultraviolet light (UV) leads to modifications of c-Jun resembling those elicited by phorbol esters or oncogenes, and to enhanced transcription of AP-1-dependent genes. The UV-induced signal also triggers activation of Raf-1 and MAP-2 kinases. A dominant-negative Raf-1 kinase mutant strongly interferes with both phorbol ester and UV-induced AP-1 activation, indicating obligatory involvement of identical components in cytoplasmic signal transduction. Thus, from a presumably nuclear site of energy absorption, a signal needs to be transmitted to the cytoplasm in order to achieve activation of a nuclear transcription factor. Further, signals elicited from different primary sites merge prior to or at the level of activation of Raf-1 kinase.