CpG DNA induces macrophage cyclooxgenase-2 (Cox-2) production. In this study, we have investigated a biochemical signaling pathway and transcription factors responsible for transcriptional regulation of the Cox-2 gene expression induced by CpG DNA. CpG DNAinduced Cox-2 promoter activity was completely inhibited by an endosomal acidification inhibitor (chloroquine), a TLR9 antagonist inhibitory CpG DNA, or overexpression of a dominant negative (DN) form of MyD88. In contrast, overexpression of DN-IRAK1 or DN-TRAF6 only partially inhibited CpG DNA-induced Cox-2 promoter activity and NF-B activation, indicating the presence of additional signaling modulators downstream of MyD88. CpG DNA-induced Cox-2 promoter activity was substantially suppressed in cells overexpressing super-suppressive IB (IB-arachidonic acid), DNp38, or DN-CREB. In addition, Cox-2 promoter-luciferase reporters with alterations in predicted cis-acting transcriptional regulatory elements revealed that C/EBP, Ets-1, NF-B, and CREB-binding sites are essential for optimal Cox-2 expression in response to CpG DNA. Conclusively, these results demonstrate that endosomal DNA processing and TLR9/MyD88-dependent activation of NF-B and p38 are required for transcriptional regulation of Cox-2 expression induced by CpG DNA, and suggest that interleukin-1 receptor-associated kinase and/or TRAF6 may be a diverging point for NF-B activation in response to CpG DNA in RAW264.7 cells.

cAMP Responsive Element Binding protein (CREB) is a transcription factor that promotes expression of genes involved in cell growth and survival. Signaling via reactive oxygen species (ROS) has been reported to attenuate CREB activity by phosphorylation of CREB at the inhibitory site serine‐121. However, ROS signaling has also been reported to enhance CREB activity via phosphorylation at the positive regulatory site serine‐133. We investigated the effects of ROS on CREB activity in murine B lymphocyte cells and observed that hydrogen peroxide induced CREB phosphorylation at ser‐133. Menadione, a superoxide inducer, also promoted CREB phosphorylation at ser‐133. In contrast to our prediction, we observed that peroxide did not induce ser‐121 phosphorylation but rather suppressed ser‐121 phosphorylation. Menadione treatment, however, did not exhibit this marked suppression of ser‐121 phosphorylation. ATM, a kinase activated by DNA damage and ROS, has been previously reported to mediate CREB phosphorylation at ser‐121 in other cell types, and we observed a similar role for ATM in B cells. Intriguingly, we also observed that U0126, an inhibitor of the MEK‐ERK pathway, also suppressed ser‐121 phosphorylation of CREB. Our data therefore illustrate novel mechanisms of CREB regulation in B lymphocytes and suggest that different species of ROS can mediate distinct effects on CREB activation.

The cyclic AMP response element binding protein (CREB) is a bZIP transcription factor activated by phosphorylation within its kinase‐inducible domain (KID), which is mediated by PKA and other kinases. CREB activation promotes the expression of genes regulating cell proliferation and cell survival, and CREB activation in neurons plays an essential role in memory formation. Herein, we have investigated the evolution of CREB in metazoan and unicellular opisthokont lineages. We identified CREB homologs with conserved bZIP domains and KID‐homologous phosphoacceptor domains in basal metazoans including cnidarians, ctenophores, placozoans, and sponges as well as choanoflagellates, the closest unicellular relatives of metazoans. The metazoan and choanoflagellate CREB homologs also possessed conserved residues known to be required for the formation of CREB‐coactivator complexes. This report therefore establishes the pre‐metazoan ancestry of CREB as a kinase‐inducible transcriptional regulator.

Cyclic AMP responsive‐element binding protein (CREB) is a transcription factor linked to proliferation and survival of many cell types, including B lymphocytes and B cell lymphomas. Cancer cells can increase resistance to chemotherapy via activating survival signaling pathways, and we investigated whether CREB activating pathways are associated with chemoresistance signaling in B lymphomas. B lymphoma cell lines were treated with anti‐mitotic drugs including etoposide, which induces DNA damage, and colchicine, which promotes microtubule depolymerization. We observed that etoposide and colchicine promoted CREB phosphorylation via signaling pathways involving protein kinase C (PKC) and mitogen activated protein kinases (MAPK). We next tested whether the CREB activating PKC and MAPK pathways influenced chemosensitivity. PKC and MAPK inhibitors, used either singly or in combination with colchicine or etoposide, enhanced B lymphoma growth arrest and cell death. Our results support the continuing study of combination therapies pairing traditional anti‐neoplastic drugs with selective signal transduction inhibitors to combat chemoresistance and increase chemotherapeutic efficacy.