CXCR7 is induced by hypoxia and mediates glioma cell migration towards SDF-1α

Abstract

Background: Glioblastomas, the most common and malignant brain tumors of the central nervous system, exhibit high invasive capacity, which hinders effective therapy. Therefore, intense efforts aimed at improved therapeutics are ongoing to delineate the molecular mechanisms governing glioma cell migration and invasion.

Methods: In order to perform the studies, we employed optimal cell culture methods and hypoxic conditions, lentivirus-mediated knockdown of protein expression, Western Blot analysis, migration assays and immunoprecipitation. We determined statistical significance by unpaired t-test.

Results: In this report, we show that U87MG, LN229 and LN308 glioma cells express CXCR7 and that exposure to hypoxia upregulates CXCR7 protein expression in these cell lines. CXCR7-expressing U87MG, LN229 and LN308 glioma cells migrated towards stromal-derived factor (SDF)-1α/CXCL12 in hypoxic conditions in the Boyden chamber assays. While shRNA-mediated knockdown of CXCR7 expression did not affect the migration of any of the three cell lines in normoxic conditions, we observed a reduction in the migration of LN229 and LN308, but not U87MG, glioma cells towards SDF-1α in hypoxic conditions. In addition, knockdown of CXCR7 expression in LN229 and LN308 glioma cells decreased levels of SDF-1α-induced phosphorylation of ERK1/2 and Akt. Inhibiting CXCR4 in LN229 and LN308 glioma cells that were knocked down for CXCR7 did not further reduce migration towards SDF-1α in hypoxic conditions and did not affect the levels of phosphorylated ERK1/2 and Akt. Analysis of immunoprecipitated CXCR4 from LN229 and LN308 glioma cells revealed co-precipitated CXCR7.

Conclusions: Taken together, our findings indicate that both CXCR4 and CXCR7 mediate glioma cell migration towards SDF-1α in hypoxic conditions and support the development of therapeutic agents targeting these receptors.

Figure 1

Hypoxia upregulates CXCR7 protein expression. (a) U87MG, (b) LN229 and (c) LN308 glioma cells were cultured in normoxic or hypoxic conditions for 3, 6, 12, 18 and 24 h. Total cell lysates were collected and analyzed by Western blot for HIF-1α and CXCR7 protein expression. β-Actin was used as loading control. Data are representative of two independent experiments with similar results. N, normoxia (20% O₂); H, hypoxia (1% O₂).

Figure 2

CXCR7 mediates the migration of LN229 and LN308 glioma cells towards SDF-1α in hypoxic conditions. shRNA-infected U87MG, LN229 and LN308 glioma cells were seeded in migration chambers in the presence or absence of 100 ng/ml (10 nM) of SDF-1α in the lower well. They were allowed to migrate for 8 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 hypoxic cells. Bar graphs represent pooled data from two independent experiments. N, normoxia (20% O₂); H, hypoxia (1% O₂); white bars, shLacZ; grey bars, shCXCR7 S4; hatched bars, shCXCR7 S5.

Figure 3

Inhibiting CXCR4 in glioma cells that are knocked down for CXCR7 does not further reduce migration towards SDF-1α. shRNA-infected LN229 and LN308 glioma cells were seeded in migration chambers with or without 100 nM of AMD3100 and in the presence or absence of 100 ng/ml (10 nM) of SDF-1α in the lower well. They were allowed to migrate for 8 h in hypoxic conditions (1% O₂). Bar graphs indicate the average number of migrated cells per field. Error bars denote mean ± standard deviation. ^*P<0.001 versus non-SDF-1α exposed cells; ^**P<0.001 versus SDF-1α exposed cells. Bar graphs represent pooled data from two independent experiments.

Figure 4

SDF-1α induces CXCR7-mediated phosphorylation of ERK1/2 and Akt in LN229 and LN308 glioma cells. shRNA-infected LN229 and LN308 glioma cells were exposed to SDF-1α for 15 min and analyzed for total and phosphorylated ERK1/2, Akt and FAK by Western blot analysis. Data represent one of two independent experiments.

Figure 5

Inhibiting CXCR4 in glioma cells that are knocked down for CXCR7 does not further reduce levels of SDF-1α-induced phosphorylation of ERK1/2 and Akt. shRNA-infected LN229 and LN308 glioma cells were exposed to SDF-1α for 15 min in the presence or absence of 100 nM of AMD3100 and analyzed for total and phosphorylated ERK1/2 and Akt by Western blot analysis. Data represent one of two independent experiments.

Figure 6

CXCR4 and CXCR7 bind in glioma cells. LN229 and LN308 glioma cells were transfected with an empty vector (EV) or HA-tagged CXCR4. Whole cell extracts (WCE) were immunoprecipitated (IP) with anti-HA resin and samples were subjected to Western blot analysis using anti-HA and anti-CXCR7 antibodies. Data are representative of two independent experiments with similar results.