Ultraviolet Light Activates NF (cid:1) B through Translational Inhibition of I (cid:1) B (cid:2) Synthesis*

UV light induces a delayed and prolonged (3–20 h) activation of NF (cid:1) B when compared with the immediate and acute (10–90 min) activation of NF (cid:1) B in response to tumor necrosis factor (cid:2) treatment. In the early phase (3–12 h) of NF (cid:1) B activation, UV light reduces inhibitor of NF (cid:1) B (I (cid:1) B) through an I (cid:1) B kinase-independent, but polyubiquitin-dependent, pathway. However, the mechanism for the UV light-induced reduction of I (cid:1) B and activation of NF (cid:1) B is not known. In this report, we show that UV light down-regulates the total amount of I (cid:1) B through decreasing I (cid:1) B mRNA translation. Our data show that UV light inhibits translation of I (cid:1) B in wild-type mouse embryo fibroblasts (MEF S/S ) and that this inhibition is prevented in MEF A/A cells in which the phosphorylation site, Ser-51 in the eukaryotic translation initiation factor 2 (cid:2) -subunit, is replaced with a non-phosphorylatable Ala (S51A). Our data also show that UV light-induced NF (cid:1) B activation is delayed in MEF A/A cells

Ultraviolet light activates the nuclear factor B (NFB) 1 (1,2). One well established mechanism for NFB activation is that, upon stimulation such as with tumor necrosis factor (TNF)␣ or lipopolysaccharide treatment, an inhibitor of NFB (IB) kinase (IKK) is activated and phosphorylates IB␣ at Ser-32 and Ser-36 (3,4). The phosphorylated IB␣ dissociates fromtheNFBandisrapidlydegradedthroughthepolyubiquitindependent proteasomal pathway. The free NFB then translocates to the nucleus and activates target genes (5)(6)(7)(8). However this mechanism is not applicable to UV light-induced early phase (within 12 h) activation of NFB (2,9). Compared with TNF␣, UV light activates NFB in a delayed and prolonged manner. The UV light-induced early phase activation of NFB is dependent upon the degradation of IB through the polyubiquitin-degradation pathway (9); however, the mechanism is not understood. It was reported that UV light does not induce IKK activation nor N-terminal serine phosphorylation of IB␣ during this time (2). It has also been reported that IKK activity is required and that the IKK-targeted serine phosphorylation sites on IB␣ are critical for UV light-induced NFB activation, even though IKK activation is not detected above the basal level after UV light irradiation (10). In this report, we elucidate the role of translational regulation in early phase activation of NFB after UV light irradiation. Previously, we reported that UV light irradiation activates an RNA-dependent protein kinase-like endoplasmic reticulum (ER)-stress activated kinase (PERK) that phosphorylates eukaryotic translation initiation factor 2 ␣ (eIF2␣) and inhibits protein synthesis (11). Now we show that UV light-induced early phase activation of NFB is due to the translational inhibition of new IB synthesis through the phosphorylation of Ser-51 in eIF2␣.

MATERIALS AND METHODS
UV Light Irradiation-Ultraviolet C light was generated from a 15 W ultraviolet C light source (UVP Inc., Upland, CA). The intensity of ultraviolet C light was standardized by using a UV light meter (UVP Inc., Upland, CA) set at 3 W/m 2 . The media was withdrawn during the ultraviolet C irradiation. After UV light irradiation, fresh medium was added to each plate.
Analysis of Total IB-Cells were harvested with Nonidet P-40 lysis buffer (2% Nonidet P-40, 80 mM NaCl, 100 mM Tris-HCl, and 0.1% SDS), and the protein concentrations were measured with the Bio-Rad protein assay kit (Bio-Rad). Equal amounts of protein samples were subjected to SDS-PAGE and electroblotted to nitrocellulose membranes. The membranes were probed with either an anti-IB antibody or a mouse monoclonal anti-␤-actin antibody (Sigma). Two anti-IB antibodies were used in the experiments. A rabbit polyclonal anti-IB antibody (sc-371; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) was used for mouse IB, and a mouse monoclonal anti-IB antibody (sc-1643; Santa Cruz Biotechnology) was used for human IB. After extensively washing with Tris-buffered saline plus Tween 20, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies. Signals were detected by using a SuperSignal TM chemiluminescent kit (Pierce).
Assay for IB Synthesis-The mouse embryo fibroblasts (MEFs) were UV light-irradiated (30 J/m 2 ). At the indicated time points after irradiation ( Fig. 1), the cells were incubated with methionine/cysteine-free minimal essential medium (Invitrogen) for 15 min and then pulse-* This work was supported by National Institutes of Health Grants RO1 CA86926 (to S. W.) and RO1 DK42394 (to R. J. K.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Assay for Protein Synthesis-The UV light-irradiated MEFs were pulse-labeled with [ 35 S]-Met/Cys as described above. The [ 35 S]-incorporation was analyzed by SDS-PAGE or trichloroacetic acid precipitation. For electrophoresis, equal amounts of cell lysate were resolved on a SDS-12% PAGE. The gel was stained with Coomassie Blue R-250 and treated with En3Hance (PerkinElmer Life Sciences). The gel was then dried for autoradiography. For trichloroacetic acid precipitation, equal amounts of cell lysate were added to 0.5 ml of 0.1 mg/ml bovine serum albumin containing 0.02% sodium azide and placed on ice. An equal volume of ice-cold 20% trichloroacetic acid was added, subjected to a vigorous vortex, and incubated for 30 min on ice. The cell lysate was filtered through a Millipore filtration apparatus onto a glass microfiber disk (GF/C, Whatman, Clifton, NJ). Disks were washed twice with 1 ml of ice-cold 10% trichloroacetic acid and twice with 1 ml of 100% ethanol. Disks were air-dried, and the radioactivity was measured in a scintillation counter (Packard, Waltham, MA).
Assay for eIF2␣ Phosphorylation-MEFs were UV light-irradiated and harvested in the Nonidet P-40 lysis buffer containing proteinase inhibitor mixture (Complete TM , Roche Applied Science) at the indicated post-irradiation time points (Fig. 2). Equal amounts of proteins were subjected to SDS-PAGE, which was followed by Western blotting analysis with antibody against phosphorylated eIF2␣ (Research Genetics, Inc., Huntsville, AL) (12). The levels of eIF2␣ phosphorylation were visualized by the SuperSignal TM chemiluminescent system (Pierce).
Electrophoretic Mobility Shift Assay (EMSA)-A pair of 23-bp synthetic oligonucleotides (5Ј-GATCCAGAGGGGACTTTCCGAGA-3Ј) containing the NFB-binding site was annealed and labeled with 32 P using T4 polynucleotide kinase and [ 32 P-␥]ATP. A DNA-binding reaction mixture of 20 l contained 20 mM Tris-HCl, pH 7.5, 4% Ficoll-400, 2 mM EDTA, 0.5 mM dithiothreitol, 0.5 g of poly(dI⅐dC), 32 P-labeled oligonucleotide (20,000 cpm), and 8 g of nuclear extract from MCF-7 cells or MEFs after UV light treatment. In the cases indicated, 0.2 g of Rel A antibody or Rel B antibody and a 100-fold excess of mutated consensusbinding sequence (5Ј-GATCCAGACCATGGTATCCGAGA-3Ј) were also included. The mixture was incubated at room temperature for 45 min and loaded onto a 5.3% nondenaturing polyacrylamide gel. The gel was run in 0.5 ϫ TBE buffer (45 mM Tris-borate, 1 mM EDTA, pH 8) at 120 V, dried, and autoradiographed.
Immunofluorescence Staining-MCF-7 and MCF-7-PERK⌬C cells were UV light-irradiated and then, at the indicated time points (Fig. 4), were fixed with 4% paraformaldehyde and permeabilized with 0.1% saponin. The cells were incubated with a mouse anti-NFB monoclonal antibody. After washing with PBS, the cells were incubated with a fluorescein-conjugated goat anti-mouse antibody. The localization of NFB was visualized with an Olympus IX70 fluorescence microscope using a Kodak M290 camera with an exposure time of 0.25 s.

UV Light Inhibits Translation of IB through Phosphorylation of Ser-51
Residue of eIF2␣-Because the half-life of IB is short (2), inhibition of new IB synthesis can have a significant impact upon the total amount of intracellular IB. Indeed, treating cells with a protein synthesis inhibitor such as cycloheximide can induce NFB activation (13,14). Because UV light irradiation inhibits protein synthesis through induction of eIF2␣ phosphorylation (11), we examined whether eIF2␣ phosphorylation plays a role in UV light-induced NFB activation. MEFs with wild-type eIF2␣ (MEF S/S ) or with an Ser-51 3 Ala mutation at the phosphorylation site in eIF2␣ (MEF A/A ) (15) were used in the experiments. We first measured the total amount of IB in UV light-irradiated MEF cells. Western blot analysis demonstrated that UV light irradiation significantly reduced IB in MEF S/S cells by 40 -97% within 4 -24 h (Fig. 1A, whereas IB remained at a high level in MEF A/A cells (Fig. 1A, lanes 6 -8). We then analyzed IB translation in MEF S/S and MEF A/A cells. The cells were UV light-irradiated and then metabolically pulse-labeled for 30 min with [ 35 S]-Met/ Cys at the indicated time points (Fig. 1). The synthesis of IB was monitored by autoradiography after immunoprecipitation with anti-IB antibody. IB synthesis was reduced by 70% in MEF S/S cells at 4 h post-UV light irradiation (Fig. 1B, lane 2), whereas IB synthesis was almost undetectable at 8 -24 h post-UV light irradiation (Fig. 1B, lanes 3 and 4). In contrast, IB synthesis in MEF A/A cells was maintained at 90 -60% at 4 -24 h post-UV light irradiation (see Fig. 3B, lanes 6 -8). Our data show that in the early phase (4 -8 h) post-UV light irradiation, the translation level of IB correlated with the total amount of IB in the cells (Fig. 1A, lanes 1-3 and 5-7 versus B,  lanes 1-3 and 5-7). These data suggest that in the early phase after UV light irradiation, reduction of IB in the cells is due to UV light-induced translational inhibition of IB synthesis.
We further analyzed protein synthesis levels and eIF2␣ phosphorylation in these cell lines after UV light treatment. Analysis of 35 S-Met/Cys incorporation at 4 h after UV light irradiation (30 J/m 2 ) demonstrated that protein synthesis was reduced to 56% in wild-type MEF S/S , but protein synthesis remained at 98% in homozygous S51A eIF2␣ mutant MEF A/A (Fig. 2, A and B). At 8 -24 h post-UV light irradiation, expression of eIF2␣ (S51A) partially reversed UV light-induced translation inhibition (Fig. 2, A and B). The increased translational inhibition paralleled the increased level of eIF2␣ phosphorylation in MEF S/S cells (Fig. 2C, lanes 1-4). The UV light-induced eIF2␣ phosphorylation was not detected in MEF A/A cells (Fig.  2C, lanes 5-8). The increased eIF2␣ phosphorylation correlated with the reduction in IB synthesis and level (Fig. 2C versus Fig. 1, A and B). This result suggests that the reduction of IB upon UV light irradiation is due to the phosphorylation of eIF2␣ and is translationally regulated. UV Light-induced NFB Activation Requires Phosphorylation of eIF2␣-To confirm that UV light induces early phase NFB activation through a translational regulation pathway, we examined NFB activation further in the MEFs using an electrophoretic mobility shift assay (EMSA). UV light treatment for 4 -24 h increased the activation of NFB in wild-type MEFs (Fig. 3A, lanes 2-4). In contrast, NFB was not significantly activated in MEF A/A cells at 4 or 8 h (Fig. 3A, lanes 6 and  7). However, at 24 h post-UV light irradiation, there was an increase in the activation of the NFB (Fig. 3A, lane 8). This delayed activation is consistent with a previous report showing that in the late stage of UV light irradiation, IKK is involved in UV light-induced activation of NFB (2).
To identify the subunit of NFB that is activated after UV light irradiation, we performed a super-gel-shift assay using antibodies against Rel A (p65) and Rel B of NFB family. Only anti-Rel A was able to produce a supershift of the DNA-protein complex ( Fig. 3B; Ref. 16), supporting the idea that UV light specifically activates Rel A. To further elucidate whether the translational inhibition mechanism is unique for UV light irradiation, we measured NFB activation in the same cell lines after TNF␣ treatment. The results demonstrate that there is no difference in TNF␣-induced activation of NFB between MEF S/S and MEF A/A cells (Fig. 3C, lanes 1-6 versus 7-12). These results suggest that translational inhibition of IB is critical for UV light-induced early phase activation of NFB.
PERK Mediates IB Depletion in Response to UV Light-PERK is an ER-stress-induced eIF2␣ kinase (17,18) that mediates UV light-induced eIF2␣ phosphorylation and protein synthesis inhibition (11). We examined whether PERK mediates UV light-induced down-regulation of IB by studying a parental control MCF-7 cell line and a line that stably expresses a trans-dominant negative mutant PERK (PERK⌬C) mutant that has a carboxyl-terminal truncation (11). Where IB was reduced 20 -40% within 4 -24 h post-UV light irradiation in parental MCF-7 cells (Fig. 4A, lanes 1-4), IB was stable in MCF-7-PERK⌬C cells, even at 24 h post-UV light irradiation (Fig. 4A, lanes 5-8). These results agree with our previous report that UV light-induced eIF2␣ phosphorylation and that translational inhibition is mediated by PERK (11).
To confirm that PERK-mediated translational inhibition results in the activation of NFB, we analyzed NFB activation in MCF-7 and MCF-7-PERK⌬C cells using an EMSA. NFB activation was observed in MCF-7 cells at 8 h post-UV light irradiation and reached a high level at 24 h post-UV light irradiation (Fig. 4B, lanes 3 and 4). These results are consistent with the findings on NFB activation in the MEF cells (Fig. 3A,  lane 4). The NFB sequence-specificity in binding was confirmed by competition with unlabeled oligonucleotides containing wild-type or mutated NFB consensus binding sequences (Fig. 4B, lanes 5 and 6). NFB activation at 8 h and 24 h post-UV light irradiation was significantly lower in MCF-7-PERK⌬C cells (Fig. 4B, lanes 9 and 10).
To determine whether the deactivation of NFB was due to the inhibition of NFB translocation caused by the elevated IB level in MCF-7-PERK⌬C cells, we analyzed NFB translocation in the cells. NFB translocated into the nucleus at 9 h post-UV light irradiation in MCF-7 cells (Fig. 4C, top row). However, this translocation was delayed in MCF-7-PERK⌬C cells (Fig. 4C, bottom row). DISCUSSION Cells respond to environmental stimuli through rapid and reversible covalent modification of translation initiation factors to mediate immediate increases or decreases in protein synthesis (19,20). Stress conditions that inhibit initiation of protein synthesis decrease the activity of eIF2 through phosphorylation of its ␣-subunit at Ser-51 (21). Four protein kinases are known to phosphorylate Ser-51 in eIF2␣ in response to different stress stimuli as follows: (i) the dsRNA-activated protein kinase PKR that is activated by dsRNA produced during viral infection (20); (ii) the general control of nitrogen metabolism kinase GCN2 that responds to amino acid depletion (22); (iii) the heme-regulated inhibitor kinase that responds to heme deprivation (23); and (iv) PERK that responds to the accumulation of unfolded proteins in the endoplasmic reticulum as well as to glucose depletion (24,25). Previous studies demonstrated that PERK and GCN2 can mediate translational inhibition through the phosphorylation of eIF2␣ in response to UV light irradiation (11,26,27). However, the roles of PERK and GCN2 in UV light-induced translation inhibition are a source of controversy. This may be because of the different cell lines, assay conditions, and times used in the experiments. We now demonstrate that UV light irradiation signals through PERK-mediated translational inhibition of IB, depletion of IB, and activation of NFB upon UV light irradiation.
The dependence of eIF2␣ phosphorylation upon IB reduction was studied by using MEF S/S or MEF A/A (15). Our results show that UV light irradiation reduced the total amount of IB in the MEF S/S cells but not in the MEF A/A cells (Fig. 1A). The total amounts of IB correlated with the rates of IB translation in the cells (Fig. 1B). In addition, a reduced amount and a lower synthesis rate of IB were observed in the MEF A/A cells (Fig. 1, A and B). This may be because of the lower background activity of NFB in the MEF A/A cells (Fig. 3A), which regulates the expression of IB (28). The requirement for PERK was studied in MCF-7 and MCF-7-PERK⌬C cell lines (11). Our data show that NFB was activated when IB levels were reduced to ϳ60% in MCF-7 cells (Fig. 4, A and B). This result agrees with the results from the MEF S/S cells (Figs. 1 and 3). In MCF-7-PERK⌬C cell lines, however, UV light-induced IB reduction and NFB activation were significantly inhibited (Fig. 4, A and  B). These results suggest that PERK mediates UV light-induced IB reduction and NFB activation. Interestingly, whereas UV light-induced IB reduction and NFB activation correlated very well at all time points in both wild-type and mutant cell lines, they did not correlate in the late-phase (24 h) post-UV light irradiation in the mutant cell lines. In MEF A/A cells, NFB activation was independent of IB reduction at 24 h post-UV light irradiation (Figs. 1A and 3A). However, in MCF-7-PERK⌬C, although the IB level was high and NFB was not activated at 24 h post-UV light irradiation, NFB significantly translocated to the nucleus, in a manner similar to the parental cells (Fig. 4C). These results suggest that eIF2␣ phosphorylation and PERK activation may also play other roles in UV light-induced IB degradation and NFB activation. It will be interesting to identify the factors and signaling pathways that affect IB degradation and NFB activation in these mutated cell lines. FIG. 4. PERK mediates UV light-induced early phase NFB activation. A, immunoblot analysis of total amounts of IB and ␤-actin in UV light-irradiated MCF-7 and MCF-7-PERK⌬C cells. The intensities of the bands were quantified by ImageJ version 1.31 (Mac OS X, NIH). The expression levels of IB were normalized by the expression levels of ␤-action and were expressed as a percentage of IB expression at 0 h post-UV light irradiation. B, EMSA for NFB activation in UV light-irradiated MCF-7 and MCF-7-PERK⌬C cells. The autoradiograph shows the mobility shift of NFB at the indicated post-UV light irradiation time points. As indicated, non-labeled oligonucleotide containing wild-type or mutated NFB consensus-binding sequences were used in the competition analysis. C, NFB nuclear localization assay for UV light-irradiated MCF-7 and MCF-7-PERK⌬C cells. NFB was stained with a mouse anti-NFB monoclonal antibody and a secondary fluorescein-conjugated goat anti-mouse antibody. Samples were analyzed by fluorescence microscopy.
In summary, our data show that UV light-induced early phase IB degradation and NFB activation can be prevented by the expression of a non-phosphorylatable eIF2␣ (S51A) mutant or a trans-dominant negative mutant PERK. Our data also show that UV light inhibits IB translation, which can be reversed by expression of an eIF2␣ (S51A) mutant. Based on these results, we propose a novel mechanism for the UV light-induced early phase activation of NFB (Fig. 5). We propose that UV light irradiation activates PERK, which phosphorylates eIF2␣ and inhibits new IB synthesis. The existing IB is depleted by natural degradation through the phosphorylation of Ser-32/Ser-36 by background IKK activity (10) and the polyubiquitin-dependent proteasomal pathway. Depletion of IB leads to NFB activation. These studies have identified a novel mechanism by which translational control can regulate gene transcription. Upon UV light irradiation, eIF2␣ is phosphorylated by PERK and inhibits IB mRNA translation. The existing IB is depleted by natural degradation through the polyubiquitin pathway. Subsequently, NFB is released from IB to translocate to the nucleus.