Cell surface heparan sulfate participates in CXCL1-induced signaling

Abstract

The CXC subfamily of chemokines plays an important role in diverse processes, including inflammation, wound healing, growth regulation, angiogenesis, and tumorigenesis. The ELR-CXC chemokine, CXCL1 or MGSA/GROalpha, is traditionally considered to attract neutrophils to sites of inflammation. The non-ELR-CXC chemokine, CXCL10 or IP-10, is chemotactic for monocytes, B cells, and activated T lymphocytes. In addition to its role in leukocyte migration, CXCL10 inhibits the angiogenic functions of the ELR-CXC chemokines as well as bFGF and VEGF. Heparan sulfate proteoglycans (HSPGs) are required for the interaction of bFGF and vEGF ligands and their receptors. However, the role of HSPGs in regulating the ELR-chemokines signaling and biological functions is poorly understood. We show here that the CXCL1 maximal binding to CXCR2 expressed on HEK293 and CHO-K1 cells is dependent on the presence of cell surface HSPGs. The cell surface HSPGs on cells are required for CXCL1-induced PAK1 activation. Moreover, CXCL10 can inhibit CXCL1-induced PAK1 and ERK activation as well as the CXCL1-induced chemotaxis through decreasing CXCL1 binding to cell surface heparan sulfate. These data indicate that HSPGs are involved in modulating CXCL1-induced PAK1 activation and chemotaxis through regulating CXCL1 binding activity to CXCR2 receptor. CXCL10 inhibits CXCL1-induced PAK1 activation and chemotaxis by interfering with appropriate binding of CXCL1 to CXCR2 receptor.

Figure 1

CXCL1 binds to soluble heparan sulfate. CXCL1 binding is specific and competed by heparan sulfate. Binding assays were performed as described in Experimental Procedures. CXCL1 (panel A) or soluble heparan sulfate (panel B) was tested for their ability to compete for ¹²⁵I-labeled CXCL1 binding to CXCR2-expressing HEK293 cells and parental HEK293 cells. The starting point was taken in the presence of 2.5 ng/mL ¹²⁵I-CXCL1, without any cold CXCL1. Data are presented as the mean of the absolute binding activity from three independent experiments. The data were analyzed using the Student’s paired t test (p < 0.05).

Figure 2

(A) The role of cell surface HSPGs in facilitating CXCL1 binding to its receptors. The CXCL1 binding sites are heparinase-sensitive. The CXCR2-expressing HEK293 cells were treated with indicated concentration of heparinase in serum free medium for 2 h at 37 °C and then washed with binding buffer two times before the ligand binding assay. Nonspecific binding was determined by performing the binding assay with ¹²⁵I-labeled CXCL1 in the presence of excess 250ng/mL unlabeled CXCL1. Data are presented as the mean of the percentage of binding where specific binding in the presence of heparinase is divided by specific binding in the absence of heparinase from three independent experiments. The data were analyzed using Student’s paired t test (p < 0.05). (B) The comparison of ¹²⁵I-labeled CXCL1 binding to wild-type and HSPG-deficient CHO cells. After wild-type and HSPG-deficient CHO cells were transient transfected with hCXCR2, binding assays were performed as in Figure 1. The CXCL1 specific binding to wild-type CXCR2-expressing CHO cells was set as 100%. The results represent mean of the percentage of CXCL1 binding to HSPG-deficient CXCR2-expressing CHO cells, as compared to wild-type CXCR2-expressing CHO cells from three independent experiments. The data were analyzed using Student’s paired t test (p < 0.05). (C) The expression of hCXCR2 in wild-type and HSPG-deficient CHO cells. After wild-type and HSPG-deficient CHO cells were transient transfected with hCXCR2, the cells were stained with anti-PE-coupled CXCR2 antibody and analyzed by FACS. The data are presented as the mean of the intensity of PE fluorescence from three independent experiments. The data were analyzed using the Student’s paired t test (p < 0.05).

Figure 3

Cell surface heparan sulfate is required for CXCL1-induced PAK1 activation. (A) Depletion of heparan sulfate by heparinase blocks CXCL-induced PAK1 activation in CXCR2-expressing HEK293 cells. The CXCR2-expressing HEK293 cells were treated with heparinase as in Figure 2A before the stimulation with CXCL1 for 10 min. PAK1 kinase assays were performed as described in Experimental Procedures. Endogenous PAK1 activity was determined by an amount of MBP phosphorylation (top panel). The blot was reprobed with PAK1 antibody to monitor equal loading of PAK1 (lower panel). This figure is representative of three different experiments with similar results. (B) Absence of heparan sulfate in HSPG-deficient CHO cells inhibits CXCL1-induced PAK1 activation. CXCR2-expressing wild-type and HSPG-deficient CHO cells were either untreated or treated with 50 ng/mL CXCL1 for the 10 min after serum starvation for 14 h. PAK1 kinase assays were performed as in panel A. This figure is representative of three different experiments with similar results.

Figure 4

CXCL10 inhibits CXCL1-induced signaling, which is required for the chemotaxis. (A) CXCL10 inhibits CXCL1-induced PAK1 and ERK activation. CXCR2-expressing cells were either untreated or treated with CXCL10 at indicated concentrations for 10 min before stimulation with 50 ng/mL CXCL1 for 10 min as in Figure 3A. Endogenous PAK1 activity was determined by an immunocomplex kinase assay as in Figure 1A (left panel). The blot was probed with PAK1 antibody to monitor equal loading of PAK1 (left-lower panel). Phosphorylated ERK1/2 was detected by Western Blot (right-upper panel). The blot was reprobed with ERK antibody monitor equal loading of PAK1 (right-lower panel). These figures are representative of three different experiments with similar results. (B) The effect of the CXCL10 on CXCL1-stimulated CXCR2-mediated chemotaxis in HEK293 cells. CXCR2-expressing HEK293 cells were treated with the carrier buffer for control (empty bars), CXCL10 at 100 ng/mL (striped bars) or CXCL10 at 500 ng/mL (solid bars), and then the cells were loaded into the upper chamber. For the CXCL10 treated cells, CXCL10 was added to the lower chambers at same concentration as the upper chamber. Chemotactic response to CXCL1 stimulation was compared as described under Experimental Procedures. Values represent the means ± S.E. of three independent experiments. The data were analyzed using the Student’s paired t test (p < 0.05).

Figure 5

The effects of CXCL10 on the CXCL1 binding to CXCR2-expressing cells. CXCR2-expressing HEK293 (panel A) and CXCR2-expressing CHO cells (panel B) were incubated for 1 h at 4 °C with ¹²⁵I-CXCL1 in the presence of varying concentrations of CXCL10. Data are presented as the percentage of binding where the specific binding in the presence of competitor is divided by specific binding in the absence of competitor × 100 in three independent experiments. Values represent the mean ± S.E. of three independent experiments performed in duplicate. The data were analyzed using the Student’s paired t test (p < 0.05).