Transcriptional regulation of human osteopontin promoter by C/EBPα and AML-1 in metastatic cancer cells

Abstract

Osteopontin (OPN) is a secreted glycoprotein produced by osteoclasts, macrophages, T cells, hematopoietic cells, and vascular smooth muscle cells. It contributes to macrophage homing and cellular immunity. It also mediates neovascularization, inhibits apoptosis, and plays important roles in extracellular matrix remodeling and angiogenesis. These properties are also characteristics of metastatic cancer cells. Consequently, the OPN gene was found to be upregulated among various metastatic cancer cells. This suggests that OPN is involved in tumor metastasis. How the OPN gene is upregulated in metastatic cancer cells remains to be illustrated. Thus, we investigated the transcriptional activation of the OPN promoter in the human metastatic cancer cell line A2058. We cloned the OPN promoter, and serial deletion analysis of the OPN promoter showed that the region between -170 and -127 may act as an enhancer to control the OPN gene in metastatic tumor cells. This region was found to contain overlapped AML-1 and C/EBP binding site motifs. Gel-mobility-shift assays using the A2058 nuclear extract and AML-1a or C/EBPa (CCAAT/ enhancer binding protein alpha) recombinant protein indicated that these two transcription factors can bind to the overlapped AML-1 /C/EBP binding site motifs on the OPN regulatory sequence from -147 to -127. Surprisingly, the gel-shift experiments did not show supershift complex formation between AML-1 and C/EBPa. Functional analysis showed that the C/EBPa was more potent than the complex of AML-1 and its cofactor CBFb to upregulate the OPN promoter. In addition, AML-1 and C/EBPa did not exhibit transactivation additively or synergistically. Our results suggest that AML-1 and C/ EBPa play an important role in the upregulation of the OPN gene in metastatic tumor cells.

Human myeloma cells express the bone regulating gene Runx2/Cbfa1 and produce osteopontin that is involved in angiogenesis in multiple myeloma patients

“…Other transcription factors could regulate OPN expression in myeloma cells given that a transcriptional regulation of OPN promoter by C/EBPa and to a minor extent by AML-1 in metastatic cancer cells has recently been demonstrated. 12 In this study, we have tested C/EBPa mRNA expression in several HMCLs but we failed to detect it, demonstrating that C/EBPa is not involved in OPN production by myeloma cells. On the other hand, we found that myeloma cells express the two major isoforms of the transcription factor AML-1, AML-1A and AML-1B, as previously described, 38 even if any correlation between OPN expression and AML-1 expression or AML-1A/AML-1B ratio has not been found.…”

“…11 However, other transcription factors may also be involved in the production of OPN by cancer cells as the CCAAT/enhancerbinding protein alpha (C/EBPa) and AML-1, because it has been demonstrated that OPN promoter contains their DNA binding consensus regions. 12 OPN interacts with CD44 as a major receptor promoting cell adhesion, proliferation and survival. 7,13 Recent data indicate that OPN also stimulates endothelial cell survival and migration [13][14][15] and acts as a potent proangiogenic factor in vivo.…”

WS5, a direct target of oncogenic transcription factor Myc, is related to human melanoma glycoprotein genes and has oncogenic potential

“…Furthermore, co-immunoprecipitation revealed direct association of Myc and C/EBP proteins in MC29-transformed cells. C/EBPa is a multifunctional transcription factor that is involved in the specification of the differentiated phenotype through gene activation, but was recently also implicated in the acceleration of cell proliferation and activation of metastasis related genes (Heckman et al, 2003;Liu et al, 2004;Wang and Timchenko, 2005). Notably, functional interactions of Myc with the C/EBPb protein have been reported previously (Mink et al, 1996;Hartl et al, 2003).…”

Abnormal Expression of the ERG Transcription Factor in Prostate Cancer Cells Activates Osteopontin

“…Several known cis-acting transcription factors have been described and most of them have been localized to a conserved region at 250 bp upstream of the proximal promoter. Potential binding sites for transcriptional regulators, such as AP-1, Myc, Oct-1, USF, v-Src, TGF-b/BMPs/Smad/Hox, Wnt/ b-catenin/APC/GSK-3b/Tcf-4, Ras/RRF, TF53, Runx2, and ETS family members, have been identified (14)(15)(16)(17)(18)(19)(20)(21).…”