Cervical cancer stem cells manifest radioresistance: Association with upregulated AP-1 activity.

Transcription factor AP-1 plays a central role in HPV-mediated cervical carcinogenesis. AP-1 has also been implicated in chemo-radio-resistance but the mechanism(s) remained unexplored. In the present study, cervical cancer stem-like cells (CaCxSLCs) isolated and enriched from cervical cancer cell lines SiHa and C33a demonstrated an elevated AP-1 DNA-binding activity in comparison to non-stem cervical cancer cells. Upon UV-irradiation, CaCxSLCs showed a UV exposure duration-dependent higher proliferation and highly increased AP-1 activity whereas it was completely abolished in non-stem cancer cells. CaCxSLCs also showed differential overexpression of c-Fos and c-Jun at transcript as well as in protein level. The loss of AP-1 activity and expression was accompanied by decrease in cell viability and proliferation in UV-irradiated non-stem cancer cells. Interestingly, CaCxSLCs treated with curcumin prior to UV-irradiation abolished AP-1 activity and a concomitant reduction in SP cells leading to abrogation of sphere forming ability, loss of proliferation, induction of apoptosis and the cells were poorly tumorigenic. The curcumin pre-treatment abolished the expression of c-Fos and c-Jun but upregulated Fra-1 expression in UV-irradiated CaCxSLCs. Thus, the study suggests a critical role of AP-1 protein in the manifestation of radioresistance but targeting with curcumin helps in radiosensitizing CaCxSLCs through upregulation of Fra-1.

Tiron Inhibits UVB-Induced AP-1 Binding Sites Transcriptional Activation on MMP-1 and MMP-3 Promoters by MAPK Signaling Pathway in Human Dermal Fibroblasts.

Recent research found that Tiron was an effective antioxidant that could act as the intracellular reactive oxygen species (ROS) scavenger or alleviate the acute toxic metal overload in vivo. In this study, we investigated the inhibitory effect of Tiron on matrix metalloproteinase (MMP)-1 and MMP-3 expression in human dermal fibroblast cells. Western blot and ELISA analysis revealed that Tiron inhibited ultraviolet B (UVB)-induced protein expression of MMP-1 and MMP-3. Real-time quantitative PCR confirmed that Tiron could inhibit UVB-induced mRNA expression of MMP-1 and MMP-3. Furthermore, Tiron significantly blocked UVB-induced activation of the MAPK signaling pathway and activator protein (AP)-1 in the downstream of this transduction pathway in fibroblasts. Through the AP-1 binding site mutation, it was found that Tiron could inhibit AP-1-induced upregulation of MMP-1 and MMP-3 expression through blocking AP-1 binding to the AP-1 binding sites in the MMP-1 and MMP-3 promoter region. In conclusion, Tiron may be a novel antioxidant for preventing and treating skin photoaging UV-induced.

What do we know about sulforaphane protection against photoaging?

Sulforaphane (SFN), a natural compound occurring in cruciferous vegetables, has been known for years as a chemopreventive agent against many types of cancer. Recently, it has been investigated as an antioxidant and anti-aging agent, and interesting conclusions have been made over the last decade. SFN demonstrated protective effects against ultraviolet (UV)-induced skin damage through several mechanisms of action, for example, decrease of reactive oxygen species production, inhibition of matrix metalloproteinase expression, and induction of phase 2 enzymes. SFN used as a protective agent against UV damage is a whole new matter, and it seems to be a very promising ingredient in upcoming anti-aging drugs and cosmetics.

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.

Activator protein 1 shows a biphasic induction and transcriptional activity after low dose X-irradiation in EA.hy.926 endothelial cells.

Low dose radiotherapy (LD-RT) is known to exert an anti-inflammatory effect. The underlying molecular mechanisms, however, are still a matter of actual research. We have recently shown that LD-RT of stimulated EA.hy.926 endothelial cells (EC) resulted in a biphasic DNA-binding and transcriptional activity of NF-kappaB in parallel with a biphasic course of leukocyte adhesion. Here we report, that following low dose X-irradiation, an increased activator protein 1 (AP-1) DNA-binding activity was observed in EC with a first relative maximum at 0.3 Gy as analysed by electrophoretic mobility shift assay. AP-1 activity then decreased at doses between 0.5 and 1 Gy and subsequently increased again at 3 Gy. This biphasic profile was confirmed on the transcriptional level by an AP-1 specific chemoluminescence reporter assay. In conclusion, the discontinuous dose response of AP-1 activation may add a further facet to the plethora of mechanisms contributing to the anti-inflammatory efficacy of LD-RT.

Eckol protects V79-4 lung fibroblast cells against gamma-ray radiation-induced apoptosis via the scavenging of reactive oxygen species and inhibiting of the c-Jun NH(2)-terminal kinase pathway.

The radioprotective effect of eckol against gamma-ray radiation-induced oxidative stress and its possible protective mechanisms were investigated. Eckol was found to reduce the intracellular reactive oxygen species generated by gamma-ray radiation. Moreover, eckol also protected against radiation-induced cellular DNA damage and membrane lipid peroxidation, which are the main targets of radiation-induced damage. In addition, eckol recovered the cell viability damaged by radiation via the inhibition of apoptosis. Irradiated cells with eckol treatment reduced the expression of bax, the activation of caspase 9 and caspase 3, which were induced by radiation. However, irradiated cells with eckol recovered the expression of bcl-2 and mitochondrial cytochrome c which were decreased by radiation. The anti-apoptotic effect of eckol exerted via the inhibition of mitogen-activated protein kinase kinase-4 (MKK4/SEK1)-c-Jun NH(2)-terminal kinase (JNK)-activator protein 1 (AP-1) cascades induced by radiation. In summary, the results suggest that eckol protects cells against the oxidative stress induced by radiation via the reduction of reactive oxygen species and the attenuation of activation in SEK1-JNK-AP-1 pathway.

SAG/ROC2/RBX2 E3 ligase promotes UVB-induced skin hyperplasia, but not skin tumors, by simultaneously targeting c-Jun/AP-1 and p27.

Sensitive to apoptosis gene (SAG)/regulator of cullins-2/RING box protein 2 is a stress-responsive RING component of Skp-1/Cullins/F-box protein E3 ubiquitin ligase. When overexpressed, SAG inhibits apoptosis induced by reactive oxygen species or hypoxia. Here, we report that SAG overexpression inhibits ultraviolet (UV) B-induced apoptosis in mouse JB6 epidermal cells. Using a transgenic mouse model, in which SAG expression was targeted primarily to epidermis by a K14 promoter, we showed that, at the early stage of UVB skin carcinogenesis (10 weeks post-UVB exposure), c-Jun, p27, p53, c-Fos and cyclin D1 were strongly induced. While having no effect on UVB-induced p53, c-Fos and cyclin D1, SAG-transgenic expression reduced the levels of c-Jun and p27 and inhibited AP-1 activity. The net outcome of SAG-mediated inhibition of c-Jun/AP-1 (pro-tumor promotion) and of p27 (antiproliferation) increased skin hyperplasia, with no apparent effect on apoptosis, as evidenced by increased skin thickness, and increased rate of DNA synthesis, but hardly any apoptosis. Although skin hyperplasia was promoted, SAG-transgenic expression had no significant effect on tumor formation in the later stage of UVB carcinogenesis. Thus, by simultaneously targeting c-Jun and p27, SAG accelerates UVB-induced skin hyperplasia, but not carcinogenesis.

Differential inhibition of UV-induced activation of NF kappa B and AP-1 by extracts from black raspberries, strawberries, and blueberries.

Recent studies from our laboratory have shown that the transactivation of nuclear factor kappa B (NF kappa B) and activator protein-1 (AP-1) plays an important mechanistic role in ultraviolet (UV)-induced skin carcinogenesis in mice. We also demonstrated that a methanol extract (ME) fraction from black raspberries (Rubus occidentalis) (RO; RO-ME) inhibits benzo[a]pyrene-7,8-diol-9,10-epoxide [B(a)PDE]-induced activation of NF kappa B and AP-1 in cultured mouse epidermal cells. In the present study, we determined if RO-ME might also inhibit the induction of NF kappa B and AP-1 in mouse epidermal cells exposed to mid UV radiation (UVB) and short UV radiation (UVC) and whether methanol fractions from strawberries and blueberries would also be effective. Our results showed that RO-ME inhibited UVB-induced activation of NF kappa B in mouse epidermal cells in a time- and dose-dependent manner; however, the methanol fractions from strawberries and blueberries were ineffective. Interestingly, none of the fractions from all 3 berry types inhibited UVB- or UVC-induced activation of AP-1, suggesting that inhibition of UV-induced signaling pathways is specific for black raspberries and NF kappa B. Cyanidin-3-rutinoside, an anthocyanin found in abundance in black raspberries and not in strawberries or high-bush blueberries, was found to contribute to the inhibition of UVB-induced activation of NF kappa B. These results suggest that berries differ in their ability to influence signaling pathways leading to activation of NF kappa B and AP-1 when using UV light as the inducer.

Flashlamp pulsed-dye laser suppressed TGF-beta1 expression and proliferation in cultured keloid fibroblasts is mediated by MAPK pathway.

BACKGROUND AND OBJECTIVES: Our previous clinical study indicated that transforming growth factor-beta1 (TGF-beta1) and mitogen-activated protein kinases (MAPK) are both involved in keloid regression following flashlamp pulsed-dye laser (PDL). To further characterize of this involvement, this work examined whether PDL suppression of TGF-beta1 expression was mediated through MAPK pathway in cultured keloid fibroblasts (KF). STUDY DESIGN/MATERIALS AND METHODS: Primary culture of KF harvested from keloid patients received various dosages of PDL treatment in 585-nm wavelength. TGF-beta1 expressions in KF following various dosages of PDL were assessed. Additionally, MAPK pathway activities were studied using the PD98059 (an ERK inhibitor), SB203580 (a p38 kinase inhibitor), and SP600125 (a JNK inhibitor), to determine the role in keloid following PDL treatment. Activator protein-1 (AP-1), a transcription factor of TGF-beta, was analyzed by electrophoretic mobility shift assay (EMSA). Phosphorylated c-Jun, one of the components of AP-1, was also detected. RESULTS: The observation results demonstrated that optimal dosages of PDL significantly suppressed KF proliferation and TGF-beta1 expression. EMSA study identified PDL downregulation of super-shift of AP-1. Three subtypes of MAPK cascades were augmented between 30 minutes and 4 hours following PDL treatment, particularly phosphorylation of ERK1/2 and p38. Pre-treatment with PD98059, SB203580, but not SP600125, markedly inhibited the downregulating effects of TGF-beta1 and phosphorylated c-Jun expression following PDL treatment. CONCLUSION: PDL induced keloid regression is mediated by triggering MAPK cascades and blockade of AP-1 transcription and TGF-beta expression. Modulation of TGF-beta and MAPK interaction in keloids may provide specific targets for therapeutic intervention.

Loss of tumor suppressor p53 decreases PTEN expression and enhances signaling pathways leading to activation of activator protein 1 and nuclear factor kappaB induced by UV radiation.

Transcription factor p53 and phosphatase PTEN are two tumor suppressors that play essential roles in suppression of carcinogenesis. However, the mechanisms by which p53 mediates anticancer activity and the relationship between p53 and PTEN are not well understood. In the present study, we found that pretreatment of mouse epidermal Cl41 cells with pifithrin-alpha, an inhibitor for p53-dependent transcriptional activation, resulted in a marked increase in UV-induced activation of activator protein 1 (AP-1) and nuclear factor kappaB (NF-kappaB). Consistent with activation of AP-1 and NF-kappaB, pifithrin-alpha was also able to enhance the UV-induced phosphorylation of c-Jun-NH2-kinases (JNK) and p38 kinase, whereas it did not show any effect on phosphorylation of extracellular signal-regulated kinases. Furthermore, the UV-induced signal activation, including phosphorylation of JNK, p38 kinase, Akt, and p70S6K, was significantly enhanced in p53-deficient cells (p53-/-), which can be reversed by p53 reconstitution. In addition, knockdown of p53 expression by its small interfering RNA also caused the elevation of AP-1 activation and Akt phosphorylation induced by UV radiation. These results show that p53 has a suppressive activity on the cell signaling pathways leading to activation of AP-1 and NF-kappaB in cell response to UV radiation. More importantly, deficiency of p53 expression resulted in a decrease in PTEN protein expression, suggesting that p53 plays a critical role in the regulation of PTEN expression. In addition, overexpression of wild-type PTEN resulted in inhibition of UV-induced AP-1 activity. Because PTEN is a well-known phosphatase involved in the regulation of phosphatidylinositol 3-kinase (PI-3K)/Akt signaling pathway, taken together with the evidence that PI-3K/Akt plays an important role in the activation of AP-1 and NF-kappaB during tumor development, we anticipate that inhibition of AP-1 and NF-kappaB by tumor suppressor p53 seems to be mediated via PTEN, which may be a novel mechanism involved in anticancer activity of p53 protein.

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.

Ultraviolet irradiation represses PATCHED gene transcription in human epidermal keratinocytes through an activator protein-1-dependent process.

Basal cell carcinoma (BCC) is one of the major types of skin cancer arising from keratinocytes. The SONIC HEDGEHOG pathway is deregulated in 100% of sporadic BCCs, as indicated by the overexpression of PATCHED, whose product encodes the receptor of SONIC HEDGEHOG, in 100% of analyzed BCCs. Reverse transcription-PCR analysis revealed that exposure to UVB irradiation, which is a risk factor known to contribute to BCC development, induces a strong and sharp decrease of PATCHED mRNA level both in vitro and ex vivo. Transcription of a reporter gene driven by the 4.4-kb 5'-regulatory region of the human PATCHED gene was shown to be down-regulated after UVB irradiation. Furthermore, overexpression of c-JUN, a member of the activator protein (AP)-1 family, induced repression of the PATCHED promoter. The role of AP-1 in UVB-induced PATCHED repression was confirmed in mouse embryonic fibroblasts knocked out for c-JUN NH(2)-terminal protein kinase. This study thus provides the first evidence of UV-induced down-regulation at the transcriptional level of the BCC-associated tumor suppressor PATCHED relying on activation of the AP-1 oncogenic pathway.

Site-directed mutagenesis of the ATM promoter: consequences for response to proliferation and ionizing radiation.

Although ATM, the protein defective in ataxia-telangiectasia (A-T), is activated primarily by radiation, there is also evidence that expression of the protein can be regulated by both radiation and growth factors. Computer analysis of the ATM promoter proximal 700-bp sequence reveals a number of potentially important cis-regulatory sequences. Using nucleotide substitutions to delete putative functional elements in the promoter of ATM, we examined the importance of some of these sites for both the basal and the radiation-induced activity of the promoter. In lymphoblastoid cells, most of the mutations in transcription factor consensus sequences [Sp1(1), Sp1(2), Cre, Ets, Xre, gammaIre(2), a modified AP1 site (Fse), and GCF] reduced basal activity to various extents, whereas others [gammaIre(1), NF1, Myb] left basal activity unaffected. In human skin fibroblasts, results were generally the same, but the basal activity varied up to 8-fold in these and other cell lines. Radiation activated the promoter approximately 2.5-fold in serum-starved lymphoblastoid cells, reaching a maximum by 3 hr, and all mutated elements equally blocked this activation. Reduction in Sp1 and AP1 DNA binding activity by serum starvation was rapidly reversed by exposure of cells to radiation. This reduction was not evident in A-T cells, and the response to radiation was less marked. Data provided for interaction between ATM and Sp1 by protein binding and co-immunoprecipitation could explain the altered regulation of Sp1 in A-T cells. The data described here provide additional evidence that basal and radiation-induced regulation of the ATM promoter is under multifactorial control.

New unusual iridoids from the leaves of noni (Morinda citrifolia L.) show inhibitory effect on ultraviolet B-induced transcriptional activator protein-1 (AP-1) activity.

A novel iridoid dimer in whose structure the two iridoid units are connected by a rare ether group, together with two new unusual iridoids showing significant inhibition of UVB-induced Activator Protein-1 (AP-1) activity in cell cultures, have been isolated from the leaves of noni (Morinda citrifolia L.). Their structures were determined on the basis of detailed high-field 1D and 2D spectral analysis. Their inhibitory effect on UVB-induced transcriptional Activator Protein-1 (AP-1) activity are also discussed.

Signal transduction pathways: targets for green and black tea polyphenols.

Tea is one of the most popular beverages consumed in the world and has been demonstrated to have anti-cancer activity in animal models. Research findings suggest that the polyphenolic compounds, (-)-epigallocatechin-3-gallate found primarily in green tea, and theaflavin-3,3'-digallate, a major component of black tea, are the two most effective anti-cancer factors found in tea. Several mechanisms to explain the chemopreventive effects of tea have been presented but others and we suggest that tea components target specific cell-signaling pathways responsible for regulating cellular proliferation or apoptosis. These pathways include signal transduction pathways leading to activator protein-1 (AP-1) and/or nuclear factor kappa B (NF-kappaB). AP-1 and NF-kappaB are transcription factors that are known to be extremely important in tumor promoterinduced cell transformation and tumor promotion, and both are influenced differentially by the MAP kinase pathways. The purpose of this brief review is to present recent research data from other and our laboratory focusing on the tea-induced cellular signal transduction events associated with the MAP kinase, AP-1, and NF-kappaB pathways.

Increase of intracellular glutathione by low-dose gamma-ray irradiation is mediated by transcription factor AP-1 in RAW 264.7 cells.

The mechanism of the elevation of intracellular glutathione induced by low-dose gamma-rays was examined in RAW 264.7 cells. The expression of mRNA for gamma-glutamylcysteine synthetase (gamma-GCS) increased soon after gamma-ray (0.5 Gy) irradiation, and peaked between 3 h and 6 h post-irradiation. A dose of 0.25 to 0.5 Gy was optimum for induction of gamma-GCS mRNA expression at 3 h post-irradiation. The effect of inhibitors of activator protein-1 (AP-1) and nuclear factor kappaB (NF-kappaB) on the radiation-induced gamma-GCS gene expression was then examined. The induction of gamma-GCS mRNA expression was significantly suppressed when AP-1 DNA binding, but not NF-kappaB DNA binding, was inhibited. Finally, electrophoretic mobility shift assay showed that the low-dose radiation markedly increased the DNA binding of AP-1, but not NF-kappaB, soon after irradiation. These results suggest that the increase of glutathione levels in RAW 264.7 cells by low-dose gamma-ray irradiation is mediated by transcriptional regulation of the gamma-GCS gene, predominantly through the AP-1 binding site in its promoter.

Nordihydroguaiaretic acid-mediated inhibition of ultraviolet B-induced activator protein-1 activation in human keratinocytes.

Nordihydroguaiaretic acid (NDGA) is a polyphenolic compound from the Larrea tridentata bush that has been identified as a chemopreventive drug in animal studies. Topically applied NDGA has been shown to prevent phorbol ester promotion of tumors in mouse skin, suggesting that NDGA may be a candidate drug for the chemoprevention of skin cancer. Ultraviolet (UV) B irradiation from sunlight exposure is the major cause of human skin cancer. UVB irradiation causes epigenetic alterations in target keratinocytes, such as the upregulation of signal transduction pathways that induce the expression of transcription factors. Specifically, UVB induces activator protein-1 (AP-1), a transcription factor complex that alters normal cellular gene expression. A component of the UVB-induced AP-1 complex, c-fos, also was identified as a mediator of the signaling pathway that leads to AP-1 activation. Thus, NDGA was investigated as a potential inhibitory agent for UVB-induced signaling pathways in the human keratinocyte cell line HaCaT. NDGA significantly inhibited UVB-induced c-fos and AP-1 transactivation. In addition, NDGA was found to inhibit activity of phosphatidylinositol 3-kinase (PI 3-kinase), a UVB-inducible enzyme that contributes to the induced expression of c-fos and AP-1. Therefore, NDGA prevents UVB-induced c-fos expression and AP-1 transactivation by inhibiting the PI 3-kinase signaling pathway. Effective skin chemoprevention strategies may incorporate NDGA to inhibit components of the UVB-induced cell signaling pathways that increase AP-1 activity.

Involvement of Erks activation in cadmium-induced AP-1 transactivation in vitro and in vivo.

Cadmium is a potent and effective carcinogen in rodents and has recently been accepted by IARC (International Agency for Research on Cancer) as a category I carcinogen. Cadmium-induced up-regulation of intracellular signaling pathways leading to increased mitogenesis is thought to be a major mechanism for the carcinogenic activity following chronic cadmium exposure. In the present study, we found that exposure of cells to cadmium induced significant activation of AP-1 and all three members of the MAP kinase family in mouse epidermal JB6 cells. The induction of AP-1 activity by cadmium appears to involve activation of Erks, since the induction of AP-1 activity by cadmium was blocked by pretreatment of cells with PD98058. Interestingly, the induction of AP-1 by cadmium was greatly enhanced by the chemical tumor promoter, TPA and the growth factor EGF, but not by ultraviolet C radiation. In vivo studies demonstrated that cadmium could also induce transactivation of AP-1 in AP-1-luciferase report transgenic mice. Considering the role of AP-1 activation in tumor promotion, the results presented in this study provide a possible molecular mechanism for cadmium-induced carcinogenesis.

UVA-340 as energy source, mimicking natural sunlight, activates the transcription factor AP-1 in cultured fibroblasts: evidence for involvement of protein kinase-C.

Ultraviolet radiation (UVR) is known to affect a variety of cellular functions, including gene expression. A number of signaling pathways have been suggested to mediate these effects, including the participation of activator protein-1 (AP-1), activator protein-2 (AP-2) and nuclear factor-kappa B (NF-kappa B). The divergent results from previous studies could be explained, at least in part, by the source of UVR with different spectral characteristics as well as the type of cells employed as targets. In this study we have utilized UVA-340 as an energy source with output which closely matches the spectrum of natural sunlight over the range of 295-350 nm for irradiation of cultured fibroblasts. Combination of electrophoretic mobility shift assays and Northern analyses revealed activation of AP-1 but not NF-kappa B or AP-2. Inhibition studies further suggested the participation of protein kinase-C, but not protein kinase-A, and that an inhibitor of mitogen-activated protein kinase (MEK-1/2) did not alter the AP-1 activation. Free radical quenchers, sodium azide and N-acetylcysteine, did not affect the AP-1 binding activity. Finally, UVA-340 was shown to enhance transcriptional expression of the type-VII collagen gene (COL7A1), which is endogenously expressed in dermal fibroblast in an AP-1 dependent manner. Introduction of a mutation into the AP-1 site of the COL7A1 promoter abolished this activation. Thus, our results obtained by utilizing a novel energy source, UVA-340, mimicking natural sunlight at UVB and lower UVA range indicate a role for AP-1 in mediating the enhanced gene expression by UVR. Collectively, these results suggest that AP-1 is an important mediator of UVR action in fibroblasts.

Proteinase inhibitors I and II from potatoes block UVB-induced AP-1 activity by regulating the AP-1 protein compositional patterns in JB6 cells.

Proteinase inhibitor I (Inh I) and proteinase inhibitor II (Inh II) from potato tubers are effective proteinase inhibitors of chymotrypsin and trypsin. Inh I and Inh II were shown to suppress irradiation-induced transformation in mouse embryo fibroblasts suggesting that they possess anticarcinogenic characteristics. We have previously demonstrated that Inh I and Inh II could effectively block UV irradiation-induced activation of transcription activator protein 1 (AP-1) in mouse JB6 epidermal cells, which mechanistically may explain their anticarcinogenic actions. In the present study, we investigated the effects of Inh I and Inh II on the expression and composition pattern of the AP-1 complex following stimulation by UV B (UVB) irradiation in the JB6 model. We found that Inh I and Inh II specifically inhibited UVB-induced AP-1, but not NFkappaB, activity in JB6 cells. Both Inh I and Inh II up-regulated AP-1 constituent proteins, JunD and Fra-2, and suppressed c-Jun and c-Fos expression and composition in bound AP-1 in response to UVB stimulation. This regulation of the AP-1 protein compositional pattern in response to Inh I or Inh II may be critical for the inhibition of UVB-induced AP-1 activity by these agents found in potatoes.