Abstract
The Raf kinase inhibitory protein (RKIP) is down-regulated in multiple types of human cancers. Decreased RKIP transcription activity may be one of the major mechanisms responsible for the downregulation of RKIP expression in human diseases. To test this hypothesis, we need to gain basic knowledge of the transcriptional regulation of RKIP. To achieve this objective, we made a systematic effort to identify cis-acting elements and trans-acting factors that control RKIP promoter activity. We found that full RKIP promoter activity requires the region −56 to +261 relative to the transcription start site. Within the full promoter region, there are two motifs rich in G/C that responded to transcription factor Sp1, one cAMP-responsive element that responded to the transcription factor CREB, and one docking site for the histone acetylase p300. In human melanoma A375 cells and human cervical cancer HeLa cells, mutation or deletion of each of these cis-acting elements decreased promoter activity. In A375 cells, knockdown of the corresponding transcription factors Sp1, CREB, or p300 decreased RKIP promoter activity, whereas overexpression of CREB and p300 increased RKIP promoter activity. The results obtained with HeLa cells also supported the idea that Sp1 and CREB play positive roles in the regulation of RKIP transcription. These findings suggest that regulators of the expression or activity of Sp1, CREB, and p300 are involved in regulating RKIP transcription.
Highlights
Activation of receptor tyrosine kinases by growth factors and cytokines promotes cell proliferation, survival, and migration through activation of the Ras-Raf-MEK-ERK cascade [1,2]
From the 39 end, a single deletion of the region +22 to +261 caused a .3-fold reduction in luciferase activity in both cell lines (Fig. 1D). These results indicate that the region of the inserted Raf kinase inhibitory protein (RKIP) sequence from 256 to +261 is sufficient for full RKIP promoter activity and that the region 2813 to 2194 negatively regulates the promoter activity
Three kinds of cis-acting elements and the corresponding transcription factors were found to play positive roles in RKIP promoter activity. These results expand upon the previous finding by Okita et al that the region 297 to +1 in the RKIP gene is important for RKIP transcription and provide a solid basis for further investigation of the regulation of RKIP expression in physiological and pathological processes [14]
Summary
Activation of receptor tyrosine kinases by growth factors and cytokines promotes cell proliferation, survival, and migration through activation of the Ras-Raf-MEK-ERK cascade [1,2] This signaling cascade is hyperactivated in human pathological processes, including cancer and Alzheimer’s disease [3,4]. Raf kinase inhibitory protein (RKIP, known as PEBP1) is a well-characterized inhibitor of Raf kinase [7] It is downregulated in multiple types of human cancers [8,9,10], which results in the overactivation of MEK and ERK. Decreased RKIP transcription activity may be one of the major mechanisms responsible for the downregulation of RKIP expression in human diseases To test this hypothesis, we need to gain basic knowledge of the transcriptional regulation of RKIP. We designed the current study to identify the cis-acting elements and the trans-acting factors that regulate RKIP promoter activity
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