Regulation Of DKK-1 Expression By p38 MAPK Signaling Via CREB Myeloma Cells
Authors: Yang Jing , Zhong Yuping , He Jin , Liu Zhiqiang , Liu Huan , Lin Pei , Orlowski Robert Z. , Kwak Larry W. 
Published: October 2019

Abstract
Bone destruction is a hallmark of multiple myeloma and severely compromises a patient’s quality of life. Recently, we have demonstrated that p38 mitogen-activated protein kinase (MAPK), which is constitutively activated in myeloma cells, is a master regulator of myeloma-mediated bone destruction. We have observed that myeloma cell p38 MAPK upregulated the production of dickkopf-1 (DKK-1), of which inhibits osteoblast differentiation and bone formation by inhibiting Wnt signaling and enhances osteoclast differentiation and bone resorption by upregulating RANKL production. Our results have shown that treatment of SB203580 (SB20), a p38 MAPK inhibitor, downregulated, while treatment of anisomycin, a p38 MAPK activator, upregulated DKK-1 expression. However, the mechanism underlying the regulation of DKK-1 expression by p38 MAPK in myeloma cells remains poorly elucidated. Previous studies have suggested that CREB is a p38 MAPK-targeted transcriptional factor. We therefore hypothesized that myeloma cell p38 MAPK may upregulate the transcriptional levels of DKK-1 through CREB. To verify whether p38 MAPK regulates CREB phosphorylation in myeloma cells, ARP-1 and U266 cells were treated with SB20 or anisomycin. Western blotting results showed that SB20 treatment significantly reduced, while anisomycin treatment enhanced phosphorylation of CREB in both cell lines. Database analysis of DKK-1 promoter regions predicted a couple of potential CREB-binding sites localized around 1.3 kb upstream from the starting codons. CHIP assay further indicated that CREB specifically bound to the one of the putative binding sites (-1,354 bp). To validate the CHIP assay results, we designed three constructs: construct 1 containing the whole promoter (-2,542 bp to -220 bp), which has previously been shown to have strong transcriptional activity; construct 2, as a truncated portion of the promoter containing the putative CREB-binding sites (-1,392 bp to -220 bp); and construct 3, as a truncated portion of the promoter containing neither CREB-binding sites (-1073 bp to -220 bp). These constructs were amplified by PCR and cloned into a luciferase reporter gene vector (pGL2) respectively. To examine the effect of CREB on the transcription activities of DKK1 in myeloma cells, reporter constructs were transfected into ARP-1 and U266 cells. In line with our findings in CHIP assay, we observed that in both myeloma cells, the construct 2 had similar strong transcriptional activity as the construct 1 did, the construct 3 had little activity. These results demonstrated that there was a CREB-binding site in DKK-1 promoter, and is required for p38 MAPK-upregulated DKK-1 expression in myeloma cells. In conclusion, our results uncover a mechanism of myeloma cell p38 MAPK in osteoblast inhibition and osteoclast activation by which p38 MAPK transcriptionally upregulates DKK-1 expression via CREB.

Disclosures:
Orlowski: Bristol-Myers Squibb: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Celgene: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Millennium: The Takeda Oncology Company: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Onyx: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Resverlogix: Research Funding; Array Biopharma: Honoraria, Membership on an entity’s Board of Directors or advisory committees; Genentech: Honoraria, Membership on an entity’s Board of Directors or advisory committees; Merck: Membership on an entity’s Board of Directors or advisory committees.