The results presented in this paper show that SP600125 is an efficient inhibitor of histone H3 Ser10 phosphorylation in vivo and demonstrate a novel mechanism by which repression of the LDL receptor expression is mediated bcl-2 by this modification. We report here for the first time, for any cell type, that SP600125 inhibits histone H3 Ser10 phosphorylation and the inhibitory effect appears to be not a direct consequence of JNK inhibition for several reasons. First, SP600125 reduced histone H3 Ser10 phosphorylation with a concentration dependence that does not parallel the inhibition of p46 54JNK, and a close match is expected if SP600125 mediates its effect through this kinase. Second, another selective polypeptide p46 54JNK inhibitor, JNK 1, does not block histone H3 Ser10 phosphorylation at concentrations that completely blocked p46 54JNK.
Third, histone H3 is poorly phosphorylated in vitro by the purified p46 54JNK compared to other MAPKs, and a complete deficiency of the p46 54JNK1 or p46 54JNK2 isoform did not SU11274 affect histone H3 Ser10 phosphorylation. Finally, lack of p46 54JNK involvement is consistent with an earlier study showing that SP600125 is capable of inhibiting a variety of kinases. The potential therapeutic value, together with the commercial availability of this chemical inhibitor, has made this kinase the subject of intensive studies during the past years. Since its first report in 2001 as a selective p46 54JNK inhibitor, SP600125 has become the pharmacological inhibitor of choice for assessing the role of this kinase in a range of biochemical and biological events. A perusal of the existing literature on studies using SP600125 for the inhibition of p46 54JNK isoforms in cultured cells indicates that this compound is typically used at concentrations ranging from 1 to 50 M.
Based on the studies reported here, we anticipate that concentrations of SP600125 sufficient to inhibit p46 54JNK activities in cultured cells and tissues would also suppress histone H3 Ser10 phosphorylation. The extent to which histone H3 Ser10 phosphorylation suppression by SP600125 compromises its usefulness as a JNK inhibitor will depend on the nature of the study. In view of our results, data obtained with SP600125 must be interpreted with great caution. An overwhelming majority of the studies that have led to the present models on the role of histone H3 Ser10 phosphorylation have focused on the mechanisms of gene activation.
Therefore, while these studies have been quite valuable for providing the framework within which to study the events that induce gene expression, the results are limited in their application to transcription initiation events. Two of the central questions are how nuclear factors and associated proteins gain access to the chromatin templates and how histone modifications play a role in the initiation process. The LDL receptor promoter is an ideal model system with which to study the role of histone H3 Ser10 phosphorylation in this process. The LDL receptor promoter contains the initiator region and the binding sites for a limited number of transcription factors, including Sp1 and SREBP 2. The Sp1 site adjacent to SRE 1 is crucial for basal LDL receptor transcription, as a mutation of this region in the human gene leads to a dramatic decrease in promoter activity.