Regulation of MSK1-Mediated NF-κB Activation Upon UVB Irradiation

Abstract

Nuclear Factor Kappa-B (NF-κB) is a transcription factor that controls expression of genes involved in the immune and inflammatory responses as well as being a key component in the onset of cancers. In this study, we provided evidence that mitogen- and stress-activated protein kinase (MSK1) is responsible for a noncanonical late-phase activation of NF-κB upon UVB irradiation. Our data demonstrated that following UVB irradiation, MSK1 is activated via phosphorylation at the 24 h time point coinciding with translocation of NF-κB into the nucleus. Investigations into the signaling pathways upstream of MSK1 through the use of specific inhibitors for mitogen-activated protein kinase and p38 revealed that both kinases are required for full phosphorylation during the late phase (24 h), while p38 is paramount for phosphorylation during the early phase (6 h). Electromobility shift assays (EMSA) showed that inhibition of MSK1 resulted in a marked reduction in NF-κB binding affinity without altering the nuclear translocation of NF-κB. Supershift EMSA implicate that the p65, but not p50, isoform of NF-κB is involved in late-phase activation in response to UVB irradiation. Together, the results of these studies shed light onto a novel pathway of MSK1-mediated late-phase activation of NF-κB in response to UVB irradiation.

Figure 1

Expression levels of MSK and phosphorylated MSK following treatment with UV and specific inhibitors. A) JB6 CL41 cells were pretreated for 1 h with or without the inhibitors PD98050 (50 μM) or SB203580 (10 μM) and irradiated with 50 mJ/cm² UVB. Whole cells lysates were collected after 6 h and analyzed via western blot. B) Identical to A with the exception of lysates being collected at 24 h. C) Cells were pretreated with both PD98050 (50 μM) and SB203580 (10 μM) and lysates collected at 6 and 24 h post irradiation.

Figure 2

Electromobility shift assay for NF-κB binding activity. A) JB6 CL41 cells were pretreated for 1 h with the inhibitors PD98059 (50 μM) or SB203580 (10 μM) and irradiated with 50 mJ/cm² UVB. Nuclear lysates were collected after 6 or 24 h and incubated with radiolabeled NF-κB consensus sequence. Samples were then ran on a non-denaturing gel and visualized with exposure to radiosensitive film. B) Identical to A with the exception of samples being pretreated with a combination of PD98059 (50 μM) and SB203580 (10 μM) or H89 (10 μM).

Figure 3

EMSA Supershift assay for NF-κB binding activity. JB6 CL41 cells were irradiated with 50 mJ/cm² UVB. Nuclear lysates were collected after 6 or 24 h. Samples for supershift analysis were incubated with p50 or p65 antibody before being incubated with radiolabeled NF-κB consensus sequence. A competition test was run on two samples by adding 20 ng unlabeled probe to the incubation mixture. Samples were then ran on a non-denaturing gel and visualized with exposure to radiosensitive film

Figure 4

Electromobility shift assay for NF-κB binding activity. A) Stably transfected JB6 CL41 cells containing a pCMV5-Flag-MSK1-A195/NH₂ vector for a dominant-negative N-terminal kinase dead MSK1 mutant were pretreated for 1 h with the inhibitors PD98059 (50 μM) or SB203580 (10 μM) and irradiated with 50 mJ/cm² UVB. Nuclear lysates were collected after 6 or 24 h and incubated with radiolabeled NF-κB consensus sequence. Samples were then ran on a non-denaturing gel and visualized with exposure to radiosensitive film. B) Identical to A with the exception of samples being pretreated with a combination of PD98059 (50 μM) and SB203580 (10 μM) or H89 (10 μM).

Figure 5

Immunohistochemical staining of NF-κB translocation. Cells were grown on glass cover slips and pretreated for 1 h with the inhibitors PD98059 (50 μM), SB203580 (10 μM), H89 (10 μM) or a combination and irradiated with 50 mJ/cm² UVB then fixed after 6 and 24 h. Fixed cells were incubated with primary antibody for p65 then FITC-conjugated secondary antibody and visualized via fluorescence microscopy.

Figure 6

Proposed model of MSK-1mediated NF-κB activation following UVB irradiation.