Chemokines induce axon outgrowth downstream of Hepatocyte Growth Factor and TCF/β-catenin signaling

Abstract

Axon morphogenesis is a complex process regulated by a variety of secreted molecules, including morphogens and growth factors, resulting in the establishment of the neuronal circuitry. Our previous work demonstrated that growth factors [Neurotrophins (NT) and Hepatocyte Growth Factor (HGF)] signal through β-catenin during axon morphogenesis. HGF signaling promotes axon outgrowth and branching by inducing β-catenin phosphorylation at Y142 and transcriptional regulation of T-Cell Factor (TCF) target genes. Here, we asked which genes are regulated by HGF signaling during axon morphogenesis. An array screening indicated that HGF signaling elevates the expression of chemokines of the CC and CXC families. In line with this, CCL7, CCL20, and CXCL2 significantly increase axon outgrowth in hippocampal neurons. Experiments using blocking antibodies and chemokine receptor antagonists demonstrate that chemokines act downstream of HGF signaling during axon morphogenesis. In addition, qPCR data demonstrates that CXCL2 and CCL5 expression is stimulated by HGF through Met/b-catenin/TCF pathway. These results identify CC family members and CXCL2 chemokines as novel regulators of axon morphogenesis downstream of HGF signaling.

Keywords: axon; beta-catenin; chemokine; hepatocyte growth factor; hippocampal neurons; neurite outgrowth.

Figure 1

Chemokine genes are upregulated by HGF signaling in 2DIV hippocampal neurons. (A) Summarized array data (left) indicating the chemokine genes that are upregulated in HGF-treated (50 ng/ml, 24 h) compared to untreated hippocampal neurons. (Right) Summary of the quantification of sqPCR experiments. Values indicate fold change of the chemokine expression in HGF-treated vs. untreated samples ± s.e.m. (≥3 experiments). (B) Representative sqPCR of samples taken at the indicated PCR cycle to compare the expression of chemokines in untreated and HGF-treated hippocampal neurons. GAPDH was used as a housekeeping gene (image corresponds to 30 PCR cycles). RT-indicates samples in which reaction was run without RT enzyme.

Figure 2

Recombinant chemokines increase axon morphogenesis. (A–H) Hippocampal neurons (2 DIV) control or treated with CXCL2, CCL5, CCL20, and CCL7 (1000 ng/ml) and immunostained for βIII-tubulin to reveal the axon morphology. Images (A–E) were taken at 10× and (F–H) at 20×. Bars = 30 μm. Average axon length compared to control (I) and axon branching shown as an increase vs. control (J) for chemokine treatments at the indicated dose or HGF (50 ng/ml). All refers to a cocktail of the four chemokines (10 ng/ml). ^*p ≤ 0.05, ^**p ≤ 0.01, and ^***p ≤ 0.001.

Figure 3

Chemokine signaling is involved in the axon morphogenesis promoted by HGF. (A) Hippocampal neurons treated with HGF (50 ng/ml) together with anti-CCL20 (40 ug/ml), anti-CXCL2 (40 μg/ml), SB225502 (1.25 nM) or SB324837 (20 nM), and immunostained for βIII-tubulin. Top pictures were taken at 10× and bottom at 20×. Bars = 30 μm. (B and C) Average axon length measurements (B) and axon branching shown as an increase vs. control (C). All refers to a cocktail of the four chemokines (10 ng/ml). ^**p ≤ 0.01 and ^***p ≤ 0.001 when comparing to control neurons. #p ≤ 0.05, ##p ≤ 0.01, and ###p ≤ 0.001 when comparing to HGF-treated neurons.

Figure 4

Met and TCF inhibition reduce the axon outgrowth induced by HGF. (A) Hippocampal neurons treated with HGF (50 ng/ml) or Wnt-3a (100 ng/ml) alone or together with FH535 (10 μ M) or SU11274 (2 μ M) were immunostained for βIII-tubulin to reveal the axon morphology. Top pictures were taken at 10× and bottom at 20×. Bars = 30 μm. (B) β-catenin transcriptional activity determined using the TOP reported plasmid in transfected Hek293 cells. Relative luciferase activity was determined at 48 h after transfection. Treatments were applied for the last 24 h (see Materials and Methods for details). ^*p ≤ 0.05, ^**p ≤ 0.01.

Figure 5

CXCL2 and CCL5 expression is regulated by HGF signaling through TCF/β-catenin. (A) qPCR analysis indicates that CXCL2 relative levels in 2 DIV hippocampal neurons increase upon HGF stimulation (50 ng/ml, 24 h), decrease below control levels in neurons co-treated with SU11274 (2 μM) plus HGF and remain similar to control levels in neurons co-treated with FH535 (10 μM) plus HGF. (B) CXCL2 relative levels upon HGF stimulation in pervanadate (perv)-treated (last 2 h) neurons. (C) CXCL2 relative levels in 4 DIV hippocampal neurons transduced with lentiviral vectors driving the expression of scrambled or β-catenin shRNAs, untreated or treated with HGF stimulation, indicate that HGF does not increase CXCL2 levels in neurons expressing β-catenin shRNA. Lower panel: Western-blot for β-catenin shows a reduction in β-catenin levels in hippocampal neurons expressing scrambled (scr) or β-catenin (β-cat) shRNAs. β-actin was used as a loading control. (D) CCL5 relative levels (4 DIV) increase by HGF and significantly decrease when neurons were co-treated with HGF and FH535. ^*p ≤ 0.05, ^**p ≤ 0.01 when comparing to control neurons. #p ≤ 0.05, ##p ≤ 0.01 when comparing to HGF-treated neurons.