We previously reported that the stem cell factor (SCF) gene activation is regulated through a functional κB enhancer site originally located in the first intron (+215-GGGAGCTCCC-+225
) of the SCF gene 
. The first important finding of the present study is that this κB intronic enhancer site is occupied by NF-κB and its coactivator CBP upon IL-1β-treatment of primary human lung fibroblasts, and that MSK1 also co-immunoprecipitates at this site. This suggests formation of a transcription complex involving MSK1 at the SCF κB enhancer site in inflammatory conditions. We additionally clearly show that this is dependent upon phosphorylation of the Ser276 residue of the p65 subunit of NF-κB, and that MSK1 is responsible for this phosphorylation. We demonstrate for the first time that this is a prerequisite for upregulated SCF expression in inflammation.
Another important finding of this paper is the suppression of binding of NF-κB, CBP and MSK1 to the SCF intronic enhancer after inhibition of the MAPKs p38 and ERK. These two MAPKs are reported to activate MSK1 in inflammatory conditions in cell lines like HEK293 and L929sA 
. We additionally show that this colocalization is suppressed by the non-selective MSK1-PKA inhibitor H89, indicating that the in vivo occupancy of the SCF intronic enhancer by NF-κB and CBP is dependent upon MSK1 activation.
Transcriptional activity of NF-κB is controlled by phosphorylation of p65 at multiple serine residues. Here we show that p65 is phosphorylated at Ser276 and Ser536 upon IL-1β-treatment in human lung fibroblasts. However, inhibition of p38 and ERK MAP kinases had no effect on p65 Ser536 phosphorylation. This contrasts with p65 Ser536 phosphorylation by the p38 pathway in human bronchial epithelial BEAS-2B cells after pneumococci stimulation 
, or by the MEK-ERK pathway in rat vascular smooth muscle cells in primary culture after angiotensin II treatment 
. We can deduce that different pathways may be involved in Ser536 phosphorylation, depending on the stimulus or cell type studied, which is a new and potently important information.
By contrast, we importantly report that inhibition of the MAPKs p38 and ERK or of the downstream kinase MSK1 blocks the phosphorylation of p65 Ser276 induced by IL-1β. This is consistent with direct p65 phosphorylation by MSK1 at Ser276 by IL-1β, as was also the case for TNFα, another inflammatory cytokine, in human HEK293 and murine L929sA cells 
. This is also in accordance with the phosphorylation of the equivalent sites at p50 (Ser337) and c-Rel (Ser267) of the NF-κB protein family 
during LPS treatment 
, although phosphorylation was dependent on a PKAc subunit. Interestingly, PKAc also leads to p65 Ser276 phosphorylation in response to LPS in murine 70Z/3 pre-B cells 
. The possibility that PKAc might phosphorylate p65 at Ser276 cannot totally be ruled out by our studies, since H89 also inhibits PKA 
. For this reason, we ascertained the role of MSK1 by transfection of a kinase-dead mutant of MSK1 (MSK1 KD), inducing inhibition of Ser276 phosphorylation in response to IL-1β, showing an important reduction (63%) taking into account the transfection efficiency (70%, data not shown) and the competition obtained (MSK1 KD vs
Phosphorylation of p65 at Ser276 (as well as the analogous site in c-Rel) is reported to disrupt the interaction between its C-terminal and N-terminal regions, thus unmasking an interaction site with the histone acetyl transferase CBP 
. In the present study, co-immunoprecipitation experiments demonstrate that p65-CBP interaction is directly dependent upon prior p65 phosphorylation at Ser276 by MSK1. This result conforts the finding that acetylation of p65 at Lys310 by CBP is secondary to Ser276 phosphorylation 
. This acetylation of p65 enhances its DNA-binding activity 
. Similarly, acetylation of p50 and p52 by CBP is linked to DNA-binding at the iNOS and COX-2 promoter 
. Thus we may postulate that NF-κB and CBP interaction with the SCF intronic enhancer is a direct consequence of Ser276 phosphorylation. This is parallel to reports that phosphorylation at Ser337 of the p50 subunit also controls DNA binding of p50 
. Interestingly, the sequence surrounding p50 Ser337 (LRRKS337
DLE) is highly similar to that around p65 Ser276 (LRRPS276
. Moreover, site-specific phosphorylation is reported to target NF-κB to a particular subset of genes 
. Indeed, S276A mutation of p65 inhibits a reporter gene harboring an IL-6-derived κB site (GGGATTTTCC
), while it has no effect when a palindromic κB site derived from the p52/p100 promoter (GGGAATTCCC
) is concerned. We here report that the GGGAGCTCCC
κB site of the SCF gene is also dependent upon Ser276 p65 phosphorylation, and that this determines NF-κB binding.
MSK1 also promotes phosphorylation of histone H3 at Ser10 
, depending on the gene, cell type and stimulus. We asked the question of the role of such MSK1-mediated chromatin modification in p65 DNA binding to the SCF gene, that would therefore affect the accessibility of the κB site. However, ChIP experiments show the absence of modification of H3 Ser10 phosphorylation when MSK1 or its upstream MAPK are inhibited. Since Duncan and collaborators also reported MSK1 not to phosphorylate histone H3 after TNFα treatment 
, we may submit that histone H3 phosphorylation is independent of the MSK1 pathway in inflammation. MSK1-mediated control of p65 binding to the SCF intronic enhancer is thus more likely to be due to Ser276 phosphorylation rather than chromatin remodelling.
Confirming this hypothesis, we finally demonstrate the direct involvement of MSK1-mediated p65 Ser276 phosphorylation in SCF expression. First, blocking the action of MSK1 by either a kinase-dead MSK1 mutant plasmid or anti-MSK1 siRNA leads to inhibition of IL-1ß-induced SCF expression. Second, SCF production is inhibited by mutation of the Ser276 to Cys. Both serve as the final demonstration that this Ser276 phosphorylation by MSK1 is a necessary requisite for SCF expression in inflammation.
In conclusion, we propose a model according to which MSK1 interacts with the enhanceosome and mediates both the binding of NF-κB to the SCF intronic enhancer and its interaction with its coactivator CBP (). MSK1 may therefore be an interesting therapeutic target for inflammatory diseases, in view of its central role in NF-κB activation.
Pathway leading to SCF expression in primary human lung fibroblasts upon IL-1β treatment.