1B). 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 around the CXCR4 promoter corresponds to increased CXCR4 expression in HeyA8 ovarian DPA-714 cell DPA-714 collection. Taken together, our results suggest that high phospho-ERK levels and NFAT3 expression plays a novel role in regulating CXCR4 expression. Introduction CXCR4 belongs to a large family of G protein-coupled receptors that specifically binds to CXCL12, a chemokine also known as stromal derived factor-1 alpha (SDF-1). Among numerous biological processes, CXCR4 plays a critical role in WHIM syndrome, HIV entry, malignancy progression and metastasis [1]-[3]. While other GPCR family members are overexpressed in few specific cancers, CXCR4 is usually CALML5 overexpressed in more than 23 different types of malignancy [4]. Since the CXCR4 receptor is critical in the process of hematopoiesis, development, and vascularization, the deregulation of the CXCR4 signaling pathways may contribute to tumorigenesis [1]. The activation of CXCR4 by the ligand SDF-1 prospects to activation of various signaling pathways including Janus kinase/Transmission Transducer and Activator of Transcription 3 (Jak/STAT3), Nuclear factor kappa-light-chain-enhancer of activated B cells (NFB), Mitogen-activated protein kinase kinase (MEK1/2), and Extracellular signal regulated kinase (ERK) [5]C[8]. In hematopoietic cells, activation of CXCR4 through the Jak/STAT3 signaling pathways prospects to cytoskeletal reorganization and cell migration [9]. In many tumor types, STAT3 is usually constitutively activated and deregulated STAT3 signaling may contribute to the process of tumorigenesis [10]. More recently, small cell lung carcinoma (SCLC) cells lines and main SCLC tumors show increased phosphorylation of STAT3, and treatment of SCLC cell lines with SDF-1 further increased STAT3 phosphorylation [7]. Additional investigation showed that upon SDF-1 treatment, JAK2 co-immunoprecipitated with CXCR4 supporting the link between the Jak/STAT3 signaling pathway and CXCR4 [7]. CXCR4 mediated cell migration in a human osteosarcoma cell collection entails the DPA-714 MEK1/2, ERK, and NFkb signaling pathways [6]. The activation of CXCR4 upon SDF-1 binding also prospects to the dissociation of the trimeric G-proteins into G monomer and G dimer. Downstream signaling events triggered by the G 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]. NFAT is usually a ubiquitous transcriptional factor that transactivates many cytokines including Interleukin-2, 3, 4, 12, inflammatory cytokines, and growth factors [13]C[16]. In human peripheral blood lymphocytes, CXCR4 expression is usually mediated by calcium and calcineurin activity, thus showing the relationship of CXCR4 regulation and the calcineurin-NFAT pathway [12]. The promoter region of CXCR4 is usually well characterized and the basal CXCR4 transcription is usually shown to be controlled mainly by two transcriptional factors, a positive regulating Nuclear Respiratory Factor-1 (NRF-1) and a negative regulating Ying Yang 1 (YY1) [17], [18]. Additionally, CXCR4 expression can be upregulated by calcium and cyclic adenosine monophosphate (cAMP) and by numerous cytokines including IL-2, IL-4, IL-7, IL-10, IL-15, and DPA-714 TGF-1 [18]C[21]. In contrast, inflammatory cytokines such as TNF-, INF-, and IL-1 all have been shown to suppress CXCR4 expression [22]C[24]. Regulation of CXCR4 expression is usually important in cell migration, transcription, and cellular trafficking. A better understanding of the signaling pathways and transcriptional factors involved in regulating CXCR4 expression is essential in elucidating the role of CXCR4 in malignancy. Although reports of various cancer types showing high levels of CXCR4 expression, we have experimentally observed that cell lines of various solid tumors exhibit weak cell surface CXCR4 expression MDA-MB-231 and MCF-7 of whole cell extracts and tumor xenograft showed CXCR4 protein expression with tumor xenograft having higher CXCR4 expression (Fig. 1C). Using additional adherent cell lines with low CXCR4 surface expression, we investigated whether 3D culturing could alter the levels of CXCR4 expression. Open in a separate window Physique 1 HeyA8 Ovarian cell collection expresses low levels of cell surface CXCR4.Jurkat and HeyA8.