The Role of Constitutive Nitric-oxide Synthase in Ultraviolet B Light-induced Nuclear Factor κB Activity*

Background: Early activation of NF-κB upon UVB irradiation is through a noncanonical eIF2-dependent IκB reduction pathway. Results: Inhibition of constitutive nitric-oxide synthase inhibited UVB-induced NF-κB activation. Conclusion: Constitutive nitric-oxide synthase is required for NF-κB activation. Significance: Learning the regulation of NF-κB upon UVB irradiation is critical for understanding the initiation and development of UVB-induced tumorigenesis. NF-κB is a transcription factor involved in many signaling pathways that also plays an important role in UV-induced skin tumorigenesis. UV radiation can activate NF-κB, but the detailed mechanism remains unclear. In this study, we provided evidence that the activation of constitutive nitric-oxide synthase plays a role in regulation of IκB reduction and NF-κB activation in human keratinocyte HaCaT cells in early phase (within 6 h) post-UVB. Treating the cells with l-NAME, a selective inhibitor of constitutive nitric-oxide synthase (cNOS), can partially reverse the IκB reduction and inhibit the DNA binding activity as well as nuclear translocation of NF-κB after UVB radiation. A luciferase reporter assay indicates that UVB-induced NF-κB activation is totally diminished in cNOS null cells. The cNOS-mediated reduction of IκB is likely due to the imbalance of nitric oxide/peroxynitrite because treating the cells with lower (50 μm), but not higher (100–500 μm), concentration of S-nitroso-N-acetylpenicillamine (SNAP) can reverse the effect of l-NAME in partial restore IκB level post-UVB. Our data also showed that NF-κB activity was required for maintaining a stable IκB kinase α subunit (IKKα) level because treating the cells with NF-κB or cNOS inhibitors could reduce IKKα level upon UVB radiation. In addition, our data demonstrated that although NF-κB protects cells from UVB-induced death, its pro-survival activity was likely neutralized by the pro-death activity of peroxynitrite after UVB radiation.

warmed up for 5 min. The dose rate for 8 or 50 mJ/cm 2 of UVB radiation was 0.8 or 3.8 milliwatts/s, respectively. Medium was removed before exposing cells to UVB. After UVB radiation, fresh medium was added to the culture plates with or without drugs, and the cells were continuously incubated at 37°C with 5% CO 2 until further analysis.
Drug Treatments-L-N G -Nitro-arginine methyl ester (L-NAME, Sigma) was added to cells to a final concentration of 1 mM at 1 h before UVB radiation. After irradiation, cells were either continuously incubated with L-NAME (1 mM) for 1 h and then replaced with fresh medium (acute treatment) or continuously incubated for the whole period until further analysis (continuous treatment). S-Nitroso-N-acetylpenicillamine (SNAP, Invitrogen) was added to cells to the indicated final concentration at 1 h before UVB irradiation. After irradiation, the cells were continuously incubated with the same concentration of SNAP until further analysis. BAY11-7085 (5 M, Sigma), JSH-23 (10 M, Sigma), MG132 (10 M, Sigma), or Ro106 (10 M, Sigma) were added to cells immediately after UVB radiation and kept in the medium for the whole period until further analysis.
ELISA for NF-B Activity-Cells were harvested with 0.25% trypsin-EDTA, and nuclear extracts were separated from cytoplasmic extracts by NE-PER nuclear and cytoplasmic extraction reagents (Thermo Scientific) following the manufacturer's protocol. NF-B activity in the nuclear extract was detect by the ELISA-based transcription factor assay kits for NF-B p50 and p65 (Thermo Scientific) or EMSA. For ELISA, NF-B binding buffer and poly(dI⅐dC) were added into the wells followed by nuclear extracts and incubated for 1 h with mild agitation. After washing three times with washing buffer, antibody against p65 was added and incubated for 1 h without agitation. After washing three times, the secondary antibody was added and incubated for 1 h without agitation. Then the chemiluminescent substrates were added and chemiluminescence was measured by luminometer (Molecular Devices Spectra Max M2).
Electrophoretic Mobility Shift Assay-A 22-bp synthetic oligonucleotide 5Ј-AGTTGAGGGGACTTTCCCAGGC-3Ј containing the specific NF-B-binding site was annealed and labeled with [␥-32 P]ATP using T4 polynucleotide kinase. A DNA binding reaction mixture of total 20 l containing poly(dI⅐dC), labeled probe, binding buffer (10 mM Tris HCl, pH 8.0, 150 mM KCl, 0.5 mM EDTA, 0.1% Triton X-100, 12.5% glycerol, and 0.2 mM DTT), and 10 g of cell nuclear extract was incubated at room temperature for 30 min and loaded onto a 5% nondenaturing polyacrylamide gel for electrophoresis. The gel was run in 0.5ϫ Tris borate-EDTA buffer at 120 V, transferred to a double layer of Whatman paper, and dried on a gel dryer for 45-60 min at 76°C. The dried gel was used to expose an autoradiography film (Denville Scientific) at Ϫ80°C, the NF-B bound 32 P-labeled DNA was detected, and the band intensity was analyzed by ImageJ.
Reporter Transfection and Luciferase Activity Assay-HeLa and HEK293 cells seeded in 96-well plate were cotransfected with NF-B luciferase reporter containing 3ϫ binding sites of NF-B (kindly provided by Dr. Jian Jian Li, University of California, Davis) together with CMV-Renilla plasmid (Promega) using Lipofectamine 2000 (Life Technologies). At 24 h posttransfection, cells were exposed to UVB radiation with or without L-NAME treatment. Luciferase activity was measured at 6 h post-UVB using Dual-Glo luciferase assay kit (E2920, Promega). The reading of luciferase signal was normalized to the reading of Renilla following the manufacturer's instructions.
Immunofluorescence Staining of NF-B-Cells were fixed with 3.6% formaldehyde for 10 min at room temperature, rinsed with PBS three times, and permeabilized with 0.1% Triton X-100 in PBS for 5 min. Cells were then blocked with blocking buffer (2 mg/ml BSA in PBS) for 1 h before incubating with mouse anti-p65 monoclonal antibody (sc-8008, Santa Cruz Biotechnology) for 1 h. After washing three times with PBS, cells were incubated with a fluorescein-conjugated horse antimouse antibody (DI-2488, Vector Laboratories) for 1 h, washed with PBS, and mounted with ProLong Gold antifade reagent with DAPI (P36931, Life Technologies). The pictures were taken by NIKON Eclipse E600 with an exposure time of 0.1 s and analyzed with NIS-Elements Basic Research 3.2 imaging software. Three cells of each group were randomly picked for quantification analysis.
cNOS Silencing Using the RNA Interference Method-Lipofectamine RNAiMAX reagent (13778030), scrambled siRNA (AM4611), nNOS (human) siRNA (AM16708 siRNA ID 117855), and eNOS (human) siRNA (AM16708 siRNA ID 106158) were purchased from Life Technologies. 2.5 ϫ 10 6 cells were seeded in a 6-well tissue culture plate in antibiotic-free medium the day before transfection. 4 l of Lipofectamine RNAiMAX and 2 l (10 M) siRNA were prepared separately in 100 l of DMEM medium (free of FBS and antibiotics) and then mixed and incubated for 5 min at room temperature. The mixture was then added to 1 ml of the medium with cells, and the medium volume was added up to 2 ml at 8 h after transfection. The cells were then incubated at 37°C with 5% CO 2 for 16 h before UV radiation.
Cell Survival Analysis-Total cell death was analyzed by determination of the loss of membrane phospholipid symmetry and membrane integrity using a FITC-conjugated-annexin V (ANX5)/propidium iodide (PI) apoptosis detection kit (BD Biosciences) following the manufacturer's protocol. Briefly, the cells were harvested by 0.25% trypsin digestion, combined with the cells floating in the medium, and washed twice with ice-cold PBS. Cells were then suspended in 200 l of ANX5 binding buffer (10 mM Hepes/NaOH, pH 7.4, 140 mM NaCl, and 2.5 mM CaCl 2 ). The cell suspension was mixed with 5 l of ANX5-FITC and 5 l of PI. The cell mixture was incubated for 15 min in the dark at room temperature, and the ANX5/PI double-stained cells were analyzed by FACSort flow cytometer (BD Biosciences) equipped with CellQuest software (BD Biosciences). Total cell number of 1 ϫ 10 5 was used for each analysis. The number of cells positive for ANX5, PI, and both ANX5 and PI were counted. Cell survival rate (R) was calculated as: R ϭ (1 ϫ 10 5 Ϫ number of positive stained cells)/1 ϫ 10 5 .
Clonogenic Assay-Immediately after treatment, cells were harvested with 0.25% trypsin-EDTA and counted, and then 5 ϫ 10 3 cells/well were plated in 6-well plates. After 6 days, cells were fixed with cold methanol for 10 min at Ϫ20°C and then stained by 1% crystal violet in 25% methanol for 10 min at room temperature. Cells were then rinsed with distilled water, and colonies with size greater than 0.4 mm were counted by Kodak IS in vivo F system equipped with Kodak Molecular Imaging software (Eastman Kodak).

Both cNOS Activation and NO⅐ Elevation Mediate UVB-induced IB Reduction via Translation Pathway-
The phosphorylation of eIF2␣ and sequentially the inhibition of protein synthesis play an important role in regulation of UVC-induced IB reduction and NF-B activation (11,16). Because cNOSs mediate UVB-induced eIF2␣ phosphorylation via activating eIF2A kinases 3 (EIF2AK3; PERK) and 4 (EIF2AK4; GCN2) (15), we examined whether cNOS also regulates UVB-induced IB reduction and NF-B activation. First, the effect of acute (1 h post-UVB) or continuous (kept until cell collection) treatment of L-NAME (1 mM), a selective inhibitor of cNOS (18), on IB protein expression level was analyzed in UVB-irradiated HaCaT cells. Our data indicated that the acute or continuous treatment of L-NAME alone did not alter the IB level in the cells (Fig. 1A, lanes 2 and 3 versus lane 1) without UVB radiation. Although the acute treatment had no statistically significant effect on IB level (Fig. 1A, lane 5 versus lane 4), the continuous treatment partially protected the reduction of IB level at 6 h post-UVB radiation (Fig. 1A, lane 6 versus lane 4). Furthermore, the protective effect of L-NAME on IB at 6 h post-UVB was dependent on its concentration (Fig. 1B). The effect started at 0.1 M and appeared to be saturated at 1 M (Fig. 1B). These results indicated that cNOS activity plays a role in regulation of IB after UVB irradiation.
Activated cNOS mediates UVB-induced eIF2␣ phosphorylation via two pathways. Immediately after UVB radiation, in the first pathway, the coupled cNOS-catalyzed NO⅐ production depletes L-Arg, which leads to the activation of GCN2, and in the second pathway, the uncoupled cNOS-catalyzed O 2 . production rapidly reacts with NO⅐ to form ONOO Ϫ and activates PERK (15). To further determine the mechanism of cNOS-mediated NF-B activation, we examined the effect of SNAP, a NO⅐ donor (19), on the protective effect of L-NAME on UVBinduced IB reduction. Interestingly, the partial protection of L-NAME on the IB (Fig. 1C, lane 13 versus lane 8) was diminished with a low dose (50 M, Fig. 1C, lane 9 versus lane 13) but not affected by higher doses (100 -500 M, Fig. 1C, lanes 10 -12 versus lane 13) of SNAP treatment, indicating that NO⅐ level also plays a role in regulation of IB reduction after UVB irradiation. In addition, UVB did not induce a detectable amount of Ser-32/36 phosphorylated IB (p-IB) but induced a high level of Ser-52 phosphorylated eIF2␣ (p-eIF2␣) (Fig. 1D, lane 3). The UVB-induced eIF2␣ phosphorylation was decreased with L-NAME treatment (Fig. 1D, lane 4 versus lane 3), suggesting that cNOSs protect IB reduction via translation regulation pathway.
Both nNOS and eNOS Are Involved in Regulation of UVBinduced NF-B Activation-To determine whether the cNOSmediated IB reduction is correlated to NF-B activation after UVB irradiation, we examined the effect of L-NAME on the DNA binding activity of NF-B using an ELISA-based assay ( Fig. 2A) and EMSA assay (Fig. 2B). Our data indicated that the acute or continuous treatment of L-NAME alone had no statistically significant effect on the NF-B activity (Fig. 2, A and B,  lanes 2 and 3 versus lane 1). For the ELISA assay, our data showed that the DNA binding activity of NF-B was increased to 1.8 Ϯ 0.2-fold at 6 h after UVB irradiation ( Fig. 2A, lane 4 versus lane 1). While the acute treatment of L-NAME did not have a statistically significant effect on NF-B activity ( Fig. 2A,  lanes 4 and 5), the continuous treatment of L-NAME inhibited the UVB-induced NF-B activity to 0.8 Ϯ 0.1-fold at 6 h post-UVB irradiation (Fig. 2A, lane 6 versus lane 4). Similar results were observed with EMSA assay, which showed that NF-B activity increased 2.7 Ϯ 0.4-fold with UVB alone, and continuous treatment of L-NAME reduced the induction to 1.4 Ϯ 0.2fold, whereas acute treatment of L-NAME showed no statisti-cally significant change (Fig. 2B). To confirm the role of cNOS in regulation of UVB-induced NF-B, we determined NF-B activity post-UVB in HeLa and HEK293 cells using an NF-Bdriven luciferase assay. Although HeLa cells express both cNOSs, HEK293 is known to be null for both nNOS and eNOS (20). Our data demonstrated that NF-B activity was increased to 1.4 Ϯ 0.1-fold at 6 h post-UVB, and the UVB-induced NF-B activation could be totally inhibited by the continuous treatment of L-NAME in HeLa cells (Fig. 2C). However, the inducibility of NF-B by UVB was totally diminished in cNOS null FIGURE 1. The effect of cNOS on IB reduction in the early phase after UVB radiation. HaCaT cells were exposed to 50 mJ/cm 2 UVB radiation with or without drug treatment as indicated and collected at 6 h post-UVB radiation. The expression levels of indicated proteins were measured by Western blot analysis. A, cells were treated with acute (1 h post-UVB) or continuous (6 h post-UVB) incubation of L-NAME (1 mM), and statistical analysis is shown. *, p Ͻ 0.05 versus corresponding control; **, p Ͻ 0.05 versus UVB alone. B, dose-dependent treatment of L-NAME and its effect on IB. C, cells were treated with L-NAME and different dose of SNAP. D, the effect of L-NAME on phosphorylated IB (p-IB), phosphorylated eIF2␣ (p-eIF2␣), total IB (T-IB), and total eIF2␣ (T-eIF2␣) protein level.

cNOS Regulates UVB-induced NF-B Activity
HEK293 cells (Fig. 2D). The result confirms that the early activation of NF-B upon UVB irradiation is cNOS-dependent. In addition to DNA binding activity of NF-B, the continuous treatment of L-NAME also had a stronger inhibitory effect on nuclear translocation of NF-B at 6 h post-UVB than the acute treatment of L-NAME had (Fig. 3). As shown by the semiquantitative analysis, nuclear NF-B increased from 31 Ϯ 2% to 75 Ϯ 2.5% with UVB alone and decreased to 48 Ϯ 3% with continuous treatment of L-NAME (Fig. 3, bottom panel, bar 1 versus bar  4 versus bar 6). Meanwhile the acute treatment of L-NAME did not statistically affect nuclear translocation of NF-B (Fig. 3,  bottom panel, bar 4 versus bar 5). These results were correlated to the IB reduction and NF-B activity with the same treatment ( Figs. 1 and 2).
We previously showed that both nNOS and eNOS are expressed in HaCaT cells (14). To determine the contribution of each isoform of cNOS in regulation of UVB-induced reduction of IB, we analyzed the extent of the effect of nNOS and/or eNOS knockdown on IB expression after UVB irradiation. Our data showed that treating the cells with nNOS and/or eNOS siRNA partially reduced the expression level of both cNOSs (Fig. 4A). Our data also showed that although it did not alter the background level of IB (Fig. 4B, lanes 3-5 versus lanes  1 and 2), the siRNA knockdown of nNOS and/or eNOS could

cNOS Regulates UVB-induced NF-B Activity
partially protect IB from UVB-induced reduction (Fig. 4B,  lanes 8 -10 versus lanes 6 and 7). The increased level of IB correlated to an increased retention of NF-B in cytosol post-UVB (Fig. 5), indicating that the activity of both cNOSs contributes to UVB-induced IB reduction and NF-B nucleus translocation.
Cross-regulation among cNOS, IB, NF-B, and IKK␣ after UVB Radiation-Previous studies suggested that IB reduction in the early phase (within 12 h) post-UVC is dependent on the background activity of IKK␣, but independent of induced activation of IKK␣ (3,11). Previous studies also suggested that UVC-induced NF-B activation contributes to IB synthesis (11). To further determine whether UVB-induced cNOS activation is involved in regulation of IB level via upstream and/or downstream signaling pathways, we compared the effect of L-NAME with two commonly used NF-B inhibitors, BAY11-7085 and JSH-23 on ubiquitin or proteasomal degradation pathway-mediated IB degradation. BAY11-7085 inhibits IB phosphorylation, and JSH-23 interferes with the binding of NF-B to its target DNA (21,22). Our data showed that L-NAME and BAY11-7085, but not JSH-23, had the same effect on partially protecting IB from UVB-induced reduction (Fig.   SEPTEMBER 19, 2014 • VOLUME 289 • NUMBER 38 6, lanes 5, 8, and 11 versus lane 2). Interestingly, in combined treatments, the effect of L-NAME and JSH-23, but not BAY11-7085, could be added on top of the effect of a proteasome inhibitor MG132 or an ubiquitin ligase inhibitor Ro106 in protecting IB from UVB-induced reduction (Fig. 6, lanes 6 and 7, 9 and  10, and 12 and 13 versus lanes 3 and 4). These results indicated that cNOS is independent of ubiquitin and proteasome pathway in protecting IB reduction after UVB radiation.

cNOS Regulates UVB-induced NF-B Activity
Because IKK␣ plays a critical role in regulation of IB degradation through ubiquitin and proteasome pathway (23)(24)(25) and IKK expression is regulated by NF-B (26), we examined whether UVB-induced cNOS-mediated NF-B activation would have an effect on IKK␣ expression. Again, L-NAME, BAY11-7085, and JSH-23 were used in the study. Our data showed that the three inhibitors and UVB alone had no effect on IKK␣ expression by themselves (Fig. 7A, lanes 2-5 versus  lane 1). However, the combinational treatment of UVB with each inhibitor significantly reduced IKK␣ expression (Fig. 7A,  lanes 6 -9 versus lane 5). Moreover, the continuous treatment was more effective than the acute treatment of L-NAME on the inhibition of IKK␣ expression (Fig. 7A, lane 9 versus lane 8). To determine the mechanism for the NF-B mediated IKK␣ expression post-UVB, we examined the mRNA level of IKK␣ under the same treatments. Our data indicated that the mRNA of IKK␣ decreased to ϳ30% at 6 h post-UVB, whereas none of the three inhibitors could rescue the reduction (Fig. 7B), indicating that cNOS-mediated NF-B activation is critical in preventing IKK␣ degradation post-UVB.
NF-B Activation Protects Cell Death Upon UVB Radiation-Because both cNOS and NF-B have dual roles in regulation of apoptosis and cell survival (27)(28)(29)(30), we examined the short term (4 -6 h) and long term (6 days) effects of L-NAME, BAY11-7085, and JSH-23 on cell survival and recovery after UVB irradiation using apoptotic and clonogenic assays, respectively. For the short term effect of 50 mJ/cm 2 UVB irradiation, the irradiation alone decreased the cell survival rate to ϳ85 and 71% at 4 and 6 h post-irradiation, respectively (Fig. 8A, lanes 4 and 7). Although the drug alone did not have statistically significant effect on cell survival (Fig. 8A, lanes 2 and 3 versus lane 1), inhibition of NF-B activity by BAY11-7085 and JSH-23 further decreased the cell survival rate from 71 to ϳ66% at 6 h post-UVB (Fig. 8A, lanes 8 and 9). Interestingly, although the continuous treatment of L-NAME increased the cell survival rate from 81 to 85% at 4 h post-UVB but had no statistically significant effect at 6 h post-UVB (Fig. 8B, lane 5 versus lane 3 and  lane 8 versus lane 6), the acute treatment of L-NAME increased cell survival rate from approximately 81 to 88% at 4 h post-UVB and from 69 to 74% at 6 h post-UVB (Fig. 8B, lane 4 versus lane  3 and lane 7 versus lane 6). Again, the treatment of drug alone   2, 5, 8, or 11); ∧, p Ͻ 0.05 versus UVB with corresponding MG132 or Ro106 (lanes 3 or 4).
had no significant effect on the cell survival rate (Fig. 8B, lane 2  versus lane 1).
For the long term effect of UVB, a lower dose (8 mJ/cm 2 ) of UVB was used. UVB irradiation alone reduced the colony formation to 30 Ϯ 5% (Fig. 9, A-C). The 6-and 24-h treatment of JSH-23 or L-NAME alone did not show a statistically significant effect on colony formation, but the 144-h (6-day) treatment of either JSH-23 or L-NAME alone reduced colony formation (Fig.  9, A and B). With UVB irradiation, the treatment of JSH-23 further reduced colony formation from 30 to 16 -9% depending on the length of drug treatment (Fig. 9A, lanes 6 -8 versus lane  5). However, the treatment of L-NAME increased colony formation from 30 to 54 -33% depending on the length of drug treatment (Fig. 9B, lanes 6 -8 versus lane 5). Further analysis revealed that the double treatment with JSH-23 and L-NAME had no statistically significant effect on colony formation after UVB irradiation (Fig. 9C, lane 5 versus lane 1), indicating that JSH-23 and L-NAME could cancel each other's effect on UVBreduced colony formation.

DISCUSSION
Previous studies indicated that UVC-induced eIF2␣ phosphorylation played an important role in regulation of NF-B activation in the early phase (within 12 h) of radiation (11,16,31,32). Our previous studies also showed that UVB radiation induced an immediate activation of cNOS, which mediated the activation of eIF2␣ kinases PERK and GCN2 (15,33). In this study, we demonstrated that cNOS activation contributed to the activation of NF-B post-UVB irradiation. Inhibition of cNOS with a continuous, but not acute, treatment of L-NAME led to a partial inhibition of IB reduction (Fig. 1) and NF-B activation (Figs. 2 and 3). The role of cNOS in regulation of UVB-induced NF-B activation was confirmed by an NF-B luciferase reporter assay, indicating that L-NAME could inhibit the UVB-induced luciferase expression, and the inducibility of luciferase expression by UVB was diminished in cNOS null HEK293 cells (Fig. 2, C and D). In addition, it appears that both eNOS and nNOS are involved in the regulation of UVB-induced NF-B activation because siRNA knockdown nNOS or eNOS can partially inhibit IB reduction and NF-B nuclear translocation post-UVB irradiation (Figs. 4 and 5). However, HaCaT cells were treated with L-NAME 1 h prior to UVB radiation, and BAY11-7085 and JSH-23 were added immediately into medium after UVB radiation. Cells were collected at 6 h post-UVB radiation. A, cells were lysed with Nonidet P-40 lysis buffer, and the amount of IKK␣ was measured by Western blot analysis. B, total RNA was extracted for the cells, and the mRNA level of IKK␣ was determined by quantitative PCR. The error bars represent S.D. of three sets independent experiments. *, p Ͻ 0.05 versus corresponding control; **, p Ͻ 0.05 versus UVB alone. FIGURE 8. UVB-induced cell apoptosis assay with or without drug treatments. HaCaT cells were exposed to UVB irradiation with different drug treatments and were collected 4 and 6 h post-UVB. Annexin V/PI apoptosis detection kit was used to detect cell apoptosis. The numbers of cells positive for ANX5, PI, and both ANX5 and PI were counted. Cell survival rate (R) was calculated as: R ϭ (1 ϫ 10 5 Ϫ numbers of positive stained cells)/1 ϫ 10 5 . The error bars represent S.D. of three sets of independent experiments. A, HaCaT cells were exposed to UVB irradiation with and without JSH-23 or BAY11-7082 treatment. B, cells were exposed to UVB irradiation and treated with acute or continuous L-NAME treatment. *, p Ͻ 0.05 versus corresponding control; **, p Ͻ 0.05 versus corresponding UVB alone.
unlike the cNOS null HEK293 cells, the effects of siRNAs on IB expression and NF-B activation were limited even with double cNOS/nNOS knockdown, which suggests that cNOS activity might be more critical than its quantity in regulation of NF-B activation after UVB irradiation and that knockdown of one NOS may lead to the activation of other NOSs as reported previously (34).
The translational inhibition of IB synthesis as well as ubiquitin and proteasome-mediated IB degradation coordinately regulate the IB reduction after UVC irradiation (11,16). Our data showed that inhibition of the ubiquitin and proteasome pathway by MG132 and Ro106 could restore the IB level after UVB irradiation (Fig. 6, lanes 3 and 4), indicating that the pathway plays a critical role in regulation of UVB-induced IB reduction. Our data also showed that the inhibition of IB phosphorylation by BAY11-7085 or cNOS activation by L-NAME, but not the inhibition of NF-B activity by JSH-23, increased IB level after UVB irradiation (Fig. 6, lanes 5, 8, and  11, versus lane 2). Interestingly, the protective effect of L-NAME was additive to the effect of MG132 or Ro106 (Fig. 6,  lanes 11-13 versus lanes 5-7). These results agreed with our previous study suggesting that cNOS-mediated IB reduction after UVB irradiation is independent of ubiquitin and proteasome pathway (15). IKK␣ phosphorylates IB and promotes its degradation via ubiquitin and proteasome pathway (23)(24)(25)35). Previous studies suggested that only background activity but not activation of IKK␣ is required for UVC-induced reduction of IB (3,11,36). Our data indicated that although the protein status of IKK␣ was not statistically significantly changed after UVB irradiation (Fig. 7A, lane 5 versus lane 1), inhibition of IB phosphorylation, NF-B activity, or cNOS could significantly reduce the protein level of IKK␣ after UVB irradiation (Fig. 7A,  lanes 6 -9). On the other hand, the mRNA status of IKK␣ was substantially reduced upon UVB irradiation, and none of the drug treatment resulted in a notable change of the mRNA level of IKK␣ (Fig. 7B). Because the only common function of the treatments was to inhibit NF-B activation after UVB irradiation, these results suggest that NF-B activation post-UVB stabilizes IKK␣.
Both cNOS and NF-B play dual roles in regulation of apoptosis (27,29,30,33,(37)(38)(39). To better understand the roles of cNOS and NF-B in regulation of cell fate after UVB irradiation, we determined the effects of JSH-23 and L-NAME on cell death and recovery after UVB irradiation. Our data indicated that inhibition of NF-B activity had an opposite effect than the inhibition of cNOS on UVB-induced cell death (Fig. 8A versus Fig. 8B), although L-NAME could inhibit the activation of NF-B (Figs. 2 and 3). Similar results were observed from clonogenic assays (Fig. 9, A versus B). One possible reason is that L-NAME inhibits the activity of NF-B via inhibiting cNOS, which contributes to the production of ONOO Ϫ after UVB irradiation. Thus when L-NAME inhibited NF-B, it also reduced the production of ONOO Ϫ , which promotes cell death (33,40,41). An elevation of ONOO Ϫ can lead to the oxidation of cholesterol (42), which plays a critical role in regulating UVB-induced apoptosis via induction of lipid rafts clustering and Fas aggregation (43,44); NF-B activation can induce iNOS expression (45), and an elevated NO⅐ production inhibits caspase 3 activation in late stage of UVB irradiation (14). Based on these studies and our findings, we propose that early activation of cNOS promotes apoptosis via induction of ONOO Ϫ elevation and inhibits apoptosis via activation of NF-B followed by induced expression of iNOS and escalated NO⅐ pro- FIGURE 9. Clonogenic assay of UVB-irradiated HaCaT cells. Cells with or without drug treatment were exposed to 8 mJ/cm 2 UVB irradiation, and 5 ϫ 10 3 cells were plated and cultured in 6-well plates for 6 days. Cells were then fixed with cold methanol and stained by 1% crystal violet in 25% methanol. Colonies with a size greater than 0.4 mm were counted by a Kodak IS in vivo F system equipped with Kodak Molecular Imaging software (Eastman Kodak). The error bars represent S.D. of three sets of independent experiments. A, cells were treated with L-NAME for 1 h before UVB irradiation, and L-NAME was removed at 6, 24, and 144 h after UVB irradiation. B, JSH-23 was added immediately after UVB irradiation and removed at 6, 24, and 144 h after UVB irradiation. C, L-NAME and JSH-23 were added together for 6 h treatment after UVB irradiation. *, p Ͻ 0.05 versus corresponding control; **, p Ͻ 0.05 versus corresponding UVB alone; #, p Ͻ 0.05 versus UVB plus L-NAME; ∧, p Ͻ 0.05 versus UVB plus JSH- 23. duction. This hypothesis is further supported by our data that the acute treatment of L-NAME (not inhibited NF-B) was slightly better than the continuous treatment of L-NAME (inhibited NF-B) in protection of UVB-induced cell death (Fig.  8, lanes 4 and 7 versus lanes 5 and 8) and that JSH-23 and L-NAME could cancel each other's effect on UVB-reduced colony formation (Fig. 9C, lane 5 versus lane 1).
In summary, we propose a signaling pathway as shown in Fig.  10. UVB irradiation activates cNOS, which leads to the phosphorylation of eIF2␣ and translational inhibition of IB synthesis. With the intact degradation pathway of IB, the translation inhibition of IB reduced IB protein level and thus activated NF-B, which protects IKK␣ from UVB-induced reduction. The activated NF-B also protects cells from UVB-induced apoptosis; however, this anti-apoptotic function can be neutralized by the pro-apoptotic effect of ONOO Ϫ .