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, 99 (1), 33-40

HGF Upregulates CXCR4 Expression in Gliomas via NF-kappaB: Implications for Glioma Cell Migration

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HGF Upregulates CXCR4 Expression in Gliomas via NF-kappaB: Implications for Glioma Cell Migration

Mine Esencay et al. J Neurooncol.

Abstract

Invasion is a hallmark of malignant gliomas and is the main reason for therapeutic failure and recurrence of the tumor. CXCR4 is a key chemokine receptor implicated in glioma cell migration whose expression is regulated by hypoxia. Here, we report that hepatocyte growth factor (HGF) upregulated CXCR4 protein expression in glioma cells. HGF pre-treatment increased migration of U87MG and LN229 glioma cells towards the CXCR4 ligand, stromal cell-derived factor-1alpha (SDF-1alpha). AMD3100, a CXCR4 inhibitor, inhibited the increased migration of HGF pre-treated LN229 glioma cells towards SDF-1alpha. Following exposure to HGF and hypoxia, both cell lines showed nuclear translocation of NF-kappaB (p65). The HGF- and hypoxia-induced nuclear translocation of NF-kappaB (p65) involved phosphorylation and degradation of IkappaB-alpha. Knock-down of NF-kappaB expression inhibited the induction of CXCR4 expression in response to HGF, but not to hypoxia. However, knock-down of NF-kappaB expression inhibited the induction of CXCR4 expression in response to hypoxia in the presence of HGF. NF-kappaB mediated migration towards SDF-1alpha in response to HGF. Knock-down of NF-kappaB expression resulted in decreased migration of HGF pre-treated glioma cells towards SDF-1alpha. Therefore, HGF upregulates CXCR4 expression via NF-kappaB and leads to enhanced migration. To our knowledge, this is the first report to show that a crosstalk mediated by NF-kappaB exists between the SDF-1alpha/CXCR4 and HGF/c-Met axes relevant to glioma cell migration. These findings imply that effective inhibition of glioma invasion should be directed against several ligand/receptor signaling pathways.

Figures

Fig. 1
Fig. 1
HGF and hypoxia upregulate CXCR4 protein expression U87MG and LN229 glioma cells were cultured in normoxic or hypoxic conditions in the presence or absence of 20 ng/ml of HGF for 16 h. Lysates were collected and analyzed by Western blot for CXCR4 protein expression. β-actin was used as loading control. Data are representative of three independent experiments with similar results. HGF, hepatocyte growth factor; Hyp, hypoxia.
Fig. 2
Fig. 2
HGF pre-treatment increases migration towards SDF-1α U87MG (a) and LN229 (b and c) glioma cells untreated or pre-treated with 20 ng/ml of HGF for 16 h were seeded in migration chambers in the presence or absence of 100 nM of AMD3100 and placed into wells in the presence or absence of 100 ng/ml of SDF-1α. They were allowed to migrate for 24 h in normoxic or hypoxic conditions. Bar graphs indicate the average number of migrated cells per field. Error bars denote mean ± standard deviation. *P<0.001 versus normoxic control; **P<0.001 versus non-SDF-1α exposed cells; ***P<0.001 versus SDF-1α exposed cells; ΔP<0.001 versus HGF pre-treated, SDF-1α exposed cells. Bar graphs represent pooled data from two independent experiments. N & white bars, normoxic conditions; H & grey bars, hypoxic conditions; hatched bars, AMD3100-treated cells.
Fig. 3
Fig. 3
NF-κB contributes to HGF-mediated CXCR4 upregulation U87MG and LN229 glioma cells were cultured in normoxic or hypoxic conditions in the presence or absence of 20 ng/ml of HGF for 30 min and then subjected to (a) immunostaining for NF-κB (p65) and (b) Western blot analysis for p-IκB-α and IκB-α protein expression. (c) Cells were transfected with Scr siRNA or siRNA directed against NF-κB. After 48 h, cells were cultured in normoxic or hypoxic conditions in the presence or absence of 20 ng/ml of HGF for 16 h. Lysates were collected and analyzed by Western blot for NF-κB and CXCR4 protein expression. β-actin was used as loading control. The experiments were repeated twice. HGF, hepatocyte growth factor; Hyp, hypoxia. si NF-κB (−) indicates cells transfected with Scr siRNA and si NF-κB (+) indicates cells transfected with siRNA directed against NF-κB. Original magnifications: x63.
Fig. 4
Fig. 4
NF-κB mediates migration towards SDF-1α in response to HGF LN229 glioma cells were transfected with Scr siRNA or siRNA directed against NF-κB. After 48 h, cells were cultured in normoxic or hypoxic conditions in the presence or absence of 20 ng/ml of HGF for 16 h. Cells untreated or pre-treated with HGF were seeded in migration chambers and placed into wells in the presence or absence of 100 ng/ml of SDF-1α. They were allowed to migrate for 24 h in normoxic or hypoxic conditions. Bar graphs indicate the average number of migrated cells per field. Error bars denote mean ± standard deviation. *P<0.001 versus normoxic control; ***P<0.001 versus SDF-1α exposed cells; P<0.001 versus hypoxic control; □□P<0.001 versus HGF pre-treated cells exposed to SDF-1α. Bar graphs represent pooled data from two independent experiments. N & white bars, normoxic conditions; H & grey bars, hypoxic conditions. si NF-κB (−) indicates cells transfected with Scr siRNA and si NF-κB (+) indicates cells transfected with siRNA directed against NF-κB.
Fig. 5
Fig. 5
Molecular pathways leading to CXCR4 upregulation in glioma This schema diagrams two potential factors, HGF and hypoxia, that may play a role in the upregulation of CXCR4 in glioma. HGF-mediated CXCR4 induction appears to be via NF-κB. Our results indicate a crosstalk between the SDF-1α/CXCR4 and HGF/c-Met axes relevant to glioma cell migration.

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