TNF-α mediates SDF-1α–induced NF-κB activation and cytotoxic effects in primary astrocytes
Yulong Han, Tao He, DeRen Huang, Carlos A. Pardo, Richard M. Ransohoff
Yulong Han, Tao He, DeRen Huang, Carlos A. Pardo, Richard M. Ransohoff
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TNF-α mediates SDF-1α–induced NF-κB activation and cytotoxic effects in primary astrocytes

Abstract

Stromal-derived cell factor-1α (SDF-1α; CXCL12) and its receptor, CXCR4, are constitutively expressed on neuroepithelial cells and are believed to be involved in both development and pathological processes, such as AIDS-associated neurologic disorders. Here, we demonstrate that SDF-1α activates NF-κB, stimulates production of chemokines and cytokines, and induces cell death in primary astrocytes, effects that depend on ongoing secretion of TNF-α. SDF-1α upregulated TNF-α mRNA and protein secretion, as well as TNF receptor 2 expression. TNF-α treatment mimicked SDF-1α induction of NF-κB, IL-1α/β, and RANTES, as well as cell death; neutralizing antibodies against TNF-α opposed these responses. We also found that SDF-1α activated Erk1 and Erk2 (Erk1/2) MAPK in a biphasic fashion. Early Erk1/2 activation was stimulated directly by SDF-1α and late activation was mediated by TNF-α. PD98059 suppression of early Erk1/2 activation correlated with reduction of SDF-1α–induced TNF-α expression. Late Erk1/2 activation was involved in TNF-α–stimulated NF-κB activation and cytokine induction. SDF-1α was induced in reactive CXCR4-positive astrocytes near axotomized spinal cord motor neurons, consistent with autocrine SDF-1/CXCR4 signaling in these cells. We propose that these novel effects of SDF-1α are relevant to the pathogenic and developmental roles of SDF-1α in the CNS.

Authors

Yulong Han, Tao He, DeRen Huang, Carlos A. Pardo, Richard M. Ransohoff

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Figure 1

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SDF-1α activates NF-κB in primary astrocytes. (a–f) Immunofluorescence s...
SDF-1α activates NF-κB in primary astrocytes. (a–f) Immunofluorescence staining of ventral lumbar spinal cord section: anti-GFAP (a and d, red), anti–SDF-1α (b, green), and anti-CXCR4 (e, green). Colocalization of GFAP and SDF-1α (c) or CXCR4 (f) within astrocytes (arrowheads). (g–i) SDF-1α induces NF-κB DNA binding activity. Primary astrocytes were incubated with medium alone or SDF-1α (2 hours) or IFN-γ (250 U/ml; 15 minutes) or TNF-α (30 minutes). Shown are results of EMSA with Fcγ-GAS (g) and IP-10 κB2 probes (g–h). SDF-1α selectively induces NF-κB DNAbinding complex (g) competed by wild-type but not mutant κB elements (h) or supershifted by anti-p50 and anti-p65 antibodies (i). (j) SDF-1α stimulates NF-κB reporter in luciferase activity assay. Primary astrocytes were cotransfected with p6κB-luc and pRL-TK constructs and treated with SDF-1α for 6 hours in the presence or absence of pertussis toxin (PTX) or PD98059 (PD). Shown are mean ± SD(Student’s t test:*PTX + SDF-1α vs. SDF-1α, P = 0.5689; **PD + SDF-1α vs. SDF-1α , P < 0.001). (k) PTX does not suppress SDF-1α- or TNF-α–stimulated activation. Primary astrocytes were treated with SDF-1α for 2 hours or TNF-α for 30 minutes in the presence or absence of PTX. Shown are results of EMSA with IP-10 κB2 probes. Data represent three or more than three experiments (g–k).