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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online February 5, 2003 as doi:10.1096/fj.02-0962fje. |
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Department of Pathology, Harvard Medical School, Boston, MA 02115;
* Department of Neurology, Children’s Hospital, Boston, Massachusetts, USA
2Correspondence: Department of Pathology, Harvard Medical School, Armenise Building D-530, 200 Longwood Ave., Boston, MA 02115, USA. E-mail: dorf{at}hms.harvard.edu
SPECIFIC AIMS
Astrocytes release a spectrum of proinflammatory mediators following stimulation with the chemokine RANTES. We examined the signaling elements controlling this response.
PRINCIPAL FINDINGS
1. Decreased intracellular cAMP levels after RANTES stimulation
RANTES stimulation of primary neonatal mouse astrocytes induced chemokine and cytokine transcription, including de novo induction of mRNA for KC, RANTES, MIP-1
, MIP-2, MCP-1, and TNF-. Astrocytes have two high-affinity RANTES receptors: CCR1 (CC chemokine receptor 1) and CCR5. These 7-transmembrane spanning G-protein-coupled receptors often modulate adenylyl cyclase activity. RANTES (100 ng/mL) decreased intracellular cAMP levels by 68% in a dose-dependent fashion. This response is chemokine specific as another CC chemokine, TCA4, failed to significantly reduce cAMP levels. Kinetic analyses demonstrated that intracellular cAMP was dramatically decreased within 1 min and was slowly recovering at 20 min. Forskolin, an activator of adenylyl cyclase, increased intracellular cAMP levels 
fourfold. RANTES treatment inhibited forskolin-induced cAMP accumulation in a dose-dependent manner.
Chemokine receptors are generally associated with pertussis toxin (PTx) -sensitive Gi proteins. PTx inhibited the induction of chemokine and cytokine mRNA. PTx also reversed the marked decrease in intracellular cAMP levels following RANTES stimulation. The data suggest RANTES-mediated modulation of cAMP and induction of most proinflammatory mediators are dependent on Gi proteins.
2. Protein kinase A activity is decreased in RANTES-treated astrocytes
PKA activity was inhibited by 60% after treatment with 100 ng/mL RANTES (Fig. 1
A). Kinase activity was maximally reduced 10 min after RANTES stimulation. In contrast, treatment with forskolin or cAMP analogs (db-cAMP and 8-bromo-cAMP) activated astrocyte PKA activity (Fig. 1B
). To examine the role of PKA in up-regulation of a prototype inflammatory mediator, MIP-1, three PKA inhibitors—H-89, Rp-8-bromo-cAMP, and PKI (protein kinase A inhibitor 14–22 amide)—were used. All three PKA inhibitors induced expression of transcripts for MIP-1 (Fig. 1C
) and other proinflammatory mediators (Fig. 1D
).
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3. RANTES stimulates activation of Raf-1 in astrocytes
RANTES induced Raf-1 kinase activity in 1 to 5 min; Raf-1 kinase activity peaked after 5–10 min (Fig. 2
A). Measurement of Raf-1 activity was based on phosphorylation of MEK, thereby directly demonstrating the role of Raf-1 in initiation of the MAPK pathway in astrocytes. Increased Raf-1 enzyme activity was accompanied by dephosphorylation of an inhibitory phosphate site, Ser 259 (Fig. 2B
). The data demonstrate that RANTES stimulates Raf-1 activation in astrocytes. Treatment with the Raf-1 inhibitor blocked gene expression in a dose-dependent fashion (Fig. 2C
). All concentrations of this inhibitor failed to affect astrocyte viability or expression of the housekeeping genes L32 and GAPDH. The Raf-1 inhibitor also blocked MEK and erk1/2 phosphorylation induced by RANTES linking Raf-1 to the MAPK pathway and to production of proinflammatory mediators in astrocytes.
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To examine the Raf dependence of RANTES-stimulated activation of the MIP-2 promoter, we used dominant negative and constitutively active mutants of Raf. The phosphorylation site (Ser 621) required for kinase activity was mutated, resulting in a kinase defective protein. Dominant negative Raf specifically suppressed reporter activity (Fig. 2D
), demonstrating the importance of this enzyme in regulating the transcription of the chemokine MIP-2. Constitutively active mutant Raf was sufficient to induce transcription from the MIP-2 promoter (Fig. 2D
).
4. Cross-talk between PKA and MAPK pathways
To establish the interrelationship between the cAMP/PKA and Raf/MAPK pathways, astrocytes were treated with graded doses of PKA inhibitors (H-89, Rp-8-bromo-cAMP, or PKI) and monitored for Raf-1 kinase activity. The inhibitors of PKA increased Raf-1 kinase activity in a dose-dependent fashion and decreased phosphorylation of Raf-1 on Ser 259. These findings indicate that PKA acts upstream of Raf-1 in the RANTES signaling pathway. H-89 treatment also induced MEK, erk1/2, and RSK phosphorylation in a dose-dependent fashion.
To examine the effects of the MAPK pathway on the induction of proinflammatory mediators, astrocytes were treated with U0126, an inhibitor of MEK, before stimulation with RANTES or H-89. Treatment with 10–50 µM U0126 blocked erk1/2 and RSK phosphorylation. U0126 also inhibited H-89- and RANTES-induced chemokine/cytokine transcription in a dose-dependent manner. Neither U0126 nor Raf-1 inhibitor decreased PKA activity.
CONCLUSIONS AND SIGNIFICANCE
Astrocytes are a major source of inflammatory mediators and are implicated in the pathophysiology of inflammatory and neurodegenerative diseases. These diseases are characterized by scarring lesions containing reactive hypertrophic astrocytes producing chemokines and cytokines that orchestrate migration and activation of leukocytes into the lesions. RANTES is highly expressed in numerous inflammatory or infectious diseases of the central nervous system including multiple sclerosis, Alzheimer’s, neuroAIDS, etc.
The CCR1 and CCR5 RANTES receptors expressed on primary mouse astrocytes belong to the family of G-protein-coupled receptors. The effects of RANTES were sensitive to PTx, and RANTES stimulation resulted in a rapid decrease of cAMP levels indicating association of the RANTES receptors with Gi proteins. Addition of nonhydrolizable cAMP analogs (db-cAMP and 8-bromo-cAMP) generally antagonized the effects of RANTES-induced transcription. These findings support the contention that cAMP plays a central role in RANTES-mediated astrocyte activation.
A key target for cAMP is PKA. Three PKA inhibitors mimicked the effects of RANTES by inducing chemokine and cytokine transcription in astrocytes. These findings indicated that PKA negatively modulates transcription of proinflammatory mediators in astrocytes.
Raf-1 kinase lies at the heart of a signaling network that controls cell proliferation, neoplastic transformation, and differentiation. Many of these effects are transmitted via the MAP kinase (MAPK) pathway. But the role of Raf-1 in RANTES-mediated signaling was unknown. A key step in Raf-1 activation is dephosphorylation of Ser 259 regulating its binding to upstream activators as well as to its substrate, MEK. Recently it was shown that the Ser 259 residue in Raf-1 was a target site for phosphorylation by PKA. In this report, we found RANTES treatment caused dephosphorylation of Ser 259 and activated Raf-1 kinase enzymatic activity. In addition, a Raf-1 inhibitor blocked RANTES-mediated transcription in astrocytes. The combined data indicate that Raf-1 plays a critical role in RANTES-mediated astrocyte activation.
To demonstrate the cross-talk between the PKA and MAPK pathways, the PKA inhibitor H-89 was used. MEK, erk1/2, and RSK were phosphorylated by H-89. Furthermore, a MEK inhibitor, U0126, blocked H-89-induced phosphorylation of erk1/2 and RSK in a dose-dependent fashion, but U0126 had no effect on PKA enzyme activity. The data place PKA upstream of Raf/MEK.
cAMP has divergent effects on MAPK pathway depending on whether signaling is through Raf-1 or B-Raf. B-Raf is present in neuronal cells but not astrocytes. In neurons, cAMP activates the MAPK pathway through Rap 1/B-Raf signaling. However, in astrocytes Raf-1 is the bridge to the MAPK pathway and may serve as a lineage marker.
In astrocytes, chemokines stimulate transcription of proinflammatory mediators, a finding rarely noted in leukocytes. We demonstrate that RANTES down-regulates intracellular cAMP levels with subsequent decreases in PKA activity. Furthermore, PKA cross-talk with Raf-1 links the present results to previous findings indicating MAPK controls transcription through RSK and CREB. This novel signaling cascade is summarized in Fig. 3
. The observations are of direct relevance to diseases in which enhanced levels of RANTES and inflammatory mediators are noted in the CNS.
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FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0962fje; to cite this article, use FASEB J. (February 5, 2003) 10.1096/fj.02-0962fje 
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