Abstract
The G-protein coupled chemokine (C-X-C motif) receptor CXCR4 is linked to cancer, HIV, and WHIM (Warts, Hypogammaglobulinemia, Infections, and Myelokathexis) syndrome. While CXCR4 is reported to be overexpressed in multiple human cancer types and many hematological cancer cell lines, we have observed poor in vitro cell surface expression of CXCR4 in many solid tumor cell lines. We explore further the possible factors and pathways involved in regulating CXCR4 expression. Here, we showed that MEK-ERK signaling pathway and NFAT3 transcriptional factor plays a novel role in regulating CXCR4 expression. When cultured as 3D spheroids, HeyA8 ovarian tumor cells showed a dramatic increase in surface CXCR4 protein levels as well as mRNA transcripts. Furthermore, HeyA8 3D spheroids showed a decrease in phospho-ERK levels when compared to adherent cells. The treatment of adherent HeyA8 cells with an inhibitor of the MEK-ERK pathway, U0126, resulted in a significant increase in surface CXCR4 expression. Additional investigation using the PCR array assay comparing adherent to 3D spheroid showed a wide range of transcription factors being up-regulated, most notably a> 20 fold increase in NFAT3 transcription factor mRNA. Finally, chromatin immunoprecipitation (ChIP) analysis showed that direct binding of NFAT3 on the CXCR4 promoter corresponds to increased CXCR4 expression in HeyA8 ovarian cell line. Taken together, our results suggest that high phospho-ERK levels and NFAT3 expression plays a novel role in regulating CXCR4 expression.
Highlights
CXCR4 belongs to a large family of G protein-coupled receptors that binds to CXCL12, a chemokine known as stromal derived factor-1 alpha (SDF-1a)
Since SDF-1a mediated activation of NF-kb pathway has been reported to be dependent on the MEK/Extracellular signal regulated kinase (ERK) pathway [8, 28], we set out to determine if HeyA8 adherent cells were responsive to SDF1a
We show that the activation of the ERK pathway inhibits CXCR4 expression
Summary
CXCR4 belongs to a large family of G protein-coupled receptors that binds to CXCL12, a chemokine known as stromal derived factor-1 alpha (SDF-1a). The stimulation of CXCR4 by the ligand SDF-1a leads to activation of various signaling pathways including Janus kinase/Signal Transducer and Activator of Transcription 3 (Jak/STAT3), Nuclear factor kappa-light-chain-enhancer of activated B cells (NFkB), Mitogen-activated protein kinase kinase (MEK1/2), and Extracellular signal regulated kinase (ERK) [5,6,7,8]. STAT3 is constitutively activated and deregulated STAT3 signaling may contribute to the process of tumorigenesis [10]. Downstream signaling events triggered by the Gbc protein result in an increase in intracellular calcium and various protein kinases [11] This activates a serine/threonine phosphatase calcineurin which triggers the activation and translocation of various transcriptional factors including Nuclear Factor activated in T-cells (NFAT) [12]. In human peripheral blood lymphocytes, CXCR4 expression is mediated by calcium and calcineurin activity, showing the relationship of CXCR4 regulation and the calcineurin-NFAT pathway [12]
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