It is now established that stimulation of astrocytes with RANTES induces a storm of proinflammatory chemokines and cytokines. This self-limiting inflammatory cascade may be responsible for prolonging production of inflammatory mediators within the central nervous system. We propose to examine the molecular basis for glial cell responses to the prototype chemokine, RANTES. Specifically, we will define the major signaling requirements for RANTES-mediated astrocyte activation. The proposal lists three specific Aims. The first is to identify the immediate early genes transcribed in this system. Preliminary data suggest that RANTES stimulated astrocytes produce TNF-a and that this cytokine in turn stimulates production of MCP-1 and perhaps other inflammatory mediators. Thus treatment of astrocytes with neutralizing anti-TNF-a Ab blocked induction of MCP-1 transcripts while expression of other chemokines and cytokines remained unaffected. Additional Ab blocking experiments are designed to identify other intermediary factors. Astrocytes derived from TNF-a and IL-1 R knockout mice alone or in combination with neutralizing antibodies will also be used to examine this issue. The role of membrane TNF and its two receptors will also be examined.
Aim #2 focuses on the role of p90RSK in signaling. Astrocytes transfected with a dominant negative RSK mutant failed to transcribe a chemokine promoter-luciferase construct indicating a major role for the RSK kinase in regulating the RANTES signal transduction pathway. Our working hypothesis is that activation of a MAP kinase results in RSK phosphorylation, activation, and nuclear translocation. Preliminary data support this hypothesis but leave several questions open including identifying additional upstream signaling components. The tissue specificity of the RANTES-induced signaling pathway will be compared between astrocytes and microglia.
The third Aim focuses on defining the factors controlling transcription of chemokines in astrocytes. Using mutagenesis of the chemokine promoter used to drive a luciferase reporter we demonstrated one NF-kB site was critical for inducing transcriptional activity. Additional mutagenesis experiments are designed to identify other promoter elements especially those activated by RSK. In summary, the proposed experiments should provide insights into the role of chemokines in glial biology and expand our vision of the broadening field of chemokine biology.

National Institute of Health (NIH)
National Institute of Neurological Disorders and Stroke (NINDS)
Research Project (R01)
Project #
Application #
Study Section
Molecular, Cellular and Developmental Neurosciences 2 (MDCN)
Program Officer
Jacobs, Tom P
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
Harvard University
Schools of Medicine
United States
Zip Code
Di, Yuwei; Li, Shitao; Wang, Lingyan et al. (2008) Homeostatic interactions between MEKK3 and TAK1 involved in NF-kappaB signaling. Cell Signal 20:705-13
Abromson-Leeman, Sara; Ladell, Daniel S; Bronson, Roderick T et al. (2007) Heterogeneity of EAE mediated by multiple distinct T-effector subsets. J Neuroimmunol 192:3-12
Li, Shitao; Wang, Lingyan; Berman, Michael A et al. (2006) RNAi screen in mouse astrocytes identifies phosphatases that regulate NF-kappaB signaling. Mol Cell 24:497-509
Zhai, Qiwei; Luo, Yi; Zhang, Ye et al. (2004) Low nuclear levels of nuclear factor-kappa B are essential for KC self-induction in astrocytes: requirements for shuttling and phosphorylation. Glia 48:327-36
Luo, Yi; Berman, Michael A; Abromson-Leeman, Sara R et al. (2003) Tumor necrosis factor is required for RANTES-induced astrocyte monocyte chemoattractant protein-1 production. Glia 43:119-27