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J. Biol. Chem., Vol. 277, Issue 21, 19042-19048, May 24, 2002

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RANTES-mediated Chemokine Transcription in Astrocytes Involves Activation and Translocation of p90 Ribosomal S6 Protein Kinase (RSK)^*

Ye Zhang, Qiwei Zhai, Yi Luo, and Martin E. Dorf

From the Department of Pathology, Harvard Medical School, Boston, Massachusetts 02115

Received for publication, December 28, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

RANTES (regulated on activation normal T cell expressed and secreted) (10 ng/ml) stimulates the induction of KC and other chemokines in astrocytes. Elements of the signal transduction pathway controlling this response were identified. RANTES induced phosphorylation of MEK, ERK1/2, p90 ribosomal S6 kinases (RSK), and cAMP-response element-binding protein (CREB) in astrocytes. U0126, a pharmacological inhibitor of MEK, blocked the phosphorylation of the downstream elements ERK, RSK, and CREB, inhibited chemokine synthesis, and reduced transcription from a KC promoter construct. Dominant negative mutants of RSK or CREB blocked the transcription driven by the KC promoter. Finally, RANTES treatment induces nuclear translocation of phosphorylated RSK in astrocytes. This novel role for RSK in signaling chemokine responses and synthesis in astrocytes may contribute to the amplification mechanisms responsible for prolonging inflammatory responses in the central nervous system.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Astrocytes are the most abundant cell type within the human central nervous system (CNS).¹ These nonneuronal cells are capable of bidirectional communication with neurons and are thought to process information (1-3). Astrocytes also help maintain the homeostatic climate of the CNS (4). They express receptors for proinflammatory cytokines, bacterial products, complement components, and constituents of the coagulation system (5-9). Thus, astrocytes are highly sensitive to changes in their local environment. These receptors become activated during autoimmune, infectious, inflammatory, or cerebrovascular diseases resulting in the release of cytokines and effector molecules (6, 10, 11).

Chemokines are a family of proinflammatory cytokines that stimulate directional migration of leukocytes. Chemokines are produced by a spectrum of cell types including T-lymphocytes, macrophages, endothelial cells, microglia, and astrocytes (6, 12, 13). Inflammatory responses in the CNS rapidly induce activation of astrocytes. Activated astrocytes are associated with the production of multiple chemokines and cytokines (6).

It has become clear that the chemokine system is also involved in other physiological and pathological processes including embryogenesis, HIV infection, and tumorigenesis (14-17). The importance of the chemokine system to the CNS was first documented with the chemokine SDF-1 (also termed CXCL12) and its receptor CXCR4. In the absence of SDF-1 or its receptor, mice develop serious disorders during the development of CNS tissues (14, 15).

RANTES (also termed CCL5) is another chemokine involved in the ontogenic development of the brain. Astrocytes from 5-week-old fetal human brains release RANTES, which in turn induces proliferation of the astrocyte cultures (18). However, at week 10 of gestation after these cells have expanded, RANTES synergizes with interferon- to inhibit the proliferative response and promote cell survival, facilitating astrocyte differentiation. The signaling mechanisms regulating these RANTES-mediated effects on astrocytes remain poorly defined.

The stimulation of the extracellular signal-regulated kinase (ERK)-mitogen-activated protein kinase (MAPK) pathway can result in cell growth and proliferation, differentiation, and cell survival. Ribosomal S6 kinases (RSK) comprise a family of serine/threonine kinases that lie at the terminus of the mitogen-regulated ERK-MAPK pathway (19). The stimulation of ERK initiates a cascade of activating events including phosphorylation of RSK by ERK and translocation to the nucleus where they phosphorylate nuclear substrates (20). Many RSK substrates have been identified, implicating RSK in a myriad of cellular processes. RSK phosphorylates several transcription factors, among them CREB (21), c-Fos (22), the CAAT enhancer-binding protein (23), and the estrogen receptor (24) as well as interact with the transcriptional coactivator CREB-binding protein (also known as p300) (25). RSK may also have a role in chromatin remodeling through phosphorylation of histone H3 (26). The diversity of these substrates suggests that RSK is involved in the regulation of a wide range of cellular functions. However, the role of RSK in the synthesis of chemokines and chemokine stimulation is unknown to date.

In this report, we examine the effects of RANTES on cultured neonatal murine astrocytes. RANTES stimulation induces TNF- and KC proteins plus a variety of chemokine transcripts. We now demonstrate that the signaling pathway regulating these responses involves the activation of MEK, ERK, and RSK.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Mice-- BALB/cJ mice were purchased from Jackson Laboratories (Bar Harbor, ME) and bred in our animal facilities. Mice were maintained in accordance with the guidelines of the Committee on Animals of the Harvard Medical School.

Reagents-- Recombinant mouse RANTES was purchased from R&D System (Minneapolis, MN). Recombinant murine IL-1 was purchased from Invitrogen. U0126 was purchased from Cell Signaling Technology (Beverly, MA), whereas SB203580 was purchased from Calbiochem. Rabbit antibodies directed to p44/p42 MAPK (ERK1/2), phospho-p44/p42 MAPK (Thr-202/Tyr-204) (P-ERK1/2), phospho-MEK1/2 (Ser-217/Ser-221), phospho-SAPK/JNK (Thr-183/Tyr-185), phospho-p38 MAPK (Thr-180/Tyr-182), p90RSK, phospho-p90RSK (Ser-381), phospho-p90RSK (Thr-360/Ser-364), phospho-p90RSK (Thr-574), and phospho-CREB (Ser-133) were all purchased from Cell Signaling Technology (Beverly, MA). Hoechst 33258 was purchased from Sigma.

Astrocyte Isolation and Culture-- Astrocytes were prepared from neonatal (<24 h) mouse brains as described previously (27). The purity of the astrocyte cultures was >95% as determined by indirect immunofluorescence with anti-glial fibrillary acidic protein antibodies (Dako Corp., Carpinteria, CA).

ELISA-- For the production of supernatants, 2 × 10⁴ astrocytes were cultured in 96-well plates with medium or 100 ng/ml RANTES. Supernatants were collected after the indicated times. ELISA assays for KC (28) and TNF- (29) were performed as detailed previously. Protein levels were determined using recombinant KC or TNF- (R & D Systems) as standards.

SDS-PAGE and Western Blotting-- Astrocytes were treated for the indicated time with medium or 100 ng/ml RANTES. 3 × 10⁵ cells were resuspended in 100 µl of buffer (50 mM HEPES, pH 7.5, 150 mM NaCl, 2 mM EDTA, 2 mM EGTA, 1% Triton X-100, 50 mM sodium fluoride, 5 mM sodium pyrophosphate, 50 mM sodium -glycerophosphate, 1 mM sodium orthovanadate, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 10 µg/ml aprotinin). Protein concentration of the whole cell extract was determined by BCA protein assay kit (Pierce). 10-µg samples were loaded and separated on a 10% SDS-polyacrylamide gel. After transfer to Hybond ECL nitrocellulose membrane (Amersham Biosciences), blots were blocked overnight with 5% bovine serum albumin at 4 °C and then probed with the indicated antibody. Appropriate anti-immunoglobulin reagents were used to develop the blots by enhanced chemiluminescence (Amersham Biosciences).

RNA Isolation and RNase Protection Assay-- RNA was prepared as detailed previously (28). RNase protection assays (RPA) for chemokine message were conducted with multiprobe templates according to the manufacturer's protocol (RiboQuant assay kit, BD-PharMingen, San Diego, CA). Gels were scanned, and radioactive bands were quantitated using a PhosphorImager (Molecular Dynamics, Sunnyvale, CA). Uniformly expressed housekeeping genes, large ribosomal subunit protein 32-3A (L32), or GAPDH were used for normalization.

Plasmids, Transient Transfection, and Luciferase Activity Assay-- The KC reporter plasmid was constructed by using a luciferase reporter gene pGL-3 basic vector (Promega, Madison, WI) driven by mouse KC promoter (2878/+43). The mutation of the CRE site (ACGTCA to ACGAAT) on the KC promoter (193/188) was made using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). Wild-type p90RSK expression (WT-pKH3) and dominant negative p90RSK expression (RSK-pKH3) plasmids were kindly provided by Dr. John Blenis (Harvard Medical School). Wild-type CREB and dominant negative CREB expression plasmids (30) were kindly provided by Dr. M. R. Montminy (San Diego, CA). Astrocytes were transiently transfected with LipofectAMINE 2000 reagent (Invitrogen) according to the manufacturer's protocol. 24 h later, the cells were stimulated with RANTES in absence or presence of the indicated inhibitor, and after an additional 24 h, luciferase activity was determined as recommend by the manufacturer (Promega). Relative luciferase activity was normalized for cell lysate protein concentration as detected by BCA protein assay kit. The relative-fold induction represents the relative intensity of the experimental sample divided by the relative intensity of the medium control.

Immunofluorescence-- Astrocytes were grown on glass coverslips for one day; the cells were then serum-starved for three hours. Astrocytes were treated with RANTES for 5 or 20 min. Following treatment, cells were fixed in 20 °C methanol, permeabilized in ice-cold 0.2% Triton X-100 in phosphate-buffered saline, and incubated with phospho-p90RSK (Thr-360/Ser-364) antibody overnight at 4 °C followed by incubation with Alexa Fluor 555 goat anti-rabbit IgG (Molecular Probes, Eugene, OR). Nuclei were stained with Hoechst 33258 (1:2000) for 5 min. Coverslips were mounted on slides using the ProLong Antifade Kit (Molecular Probes). Slides were stored at room temperature in the dark until observation.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

RANTES Stimulation of Astrocytes Induces KC and TNF- Synthesis-- The ability of RANTES to stimulate chemokine/cytokine transcripts in mouse astrocytes was examined by RPA. Primary neonatal mouse astrocyte cultures were incubated with the indicated dose of RANTES, boiled RANTES, or TCA-4 for 3 h and then harvested for RNA isolation. RANTES (10 ng/mg) stimulated chemokine/cytokine transcription. Following RANTES treatment, the expression of TNF-, RANTES, KC, MIP-1, MIP-2, and MCP-1 transcripts was up-regulated (Fig. 1A). Boiled RANTES (30 min) and a control chemokine TCA-4 failed to induce any chemokine or cytokine transcripts. Kinetic analyses demonstrated that TNF-, KC, and MIP-2 transcripts were detected as early as 60-90 min after RANTES stimulation (Fig. 1B). At 2 h, distinct bands for MIP-1 and MCP-1 became visible, and a faint RANTES band appeared. IL-6 transcripts were the last to appear (4 h). Unstimulated astrocytes expressed message for the housekeeping genes L32 and GAPDH and occasionally traces of RANTES or MCP-1.

View larger version (17K): [in this window] [in a new window]   Fig. 1.   RANTES-induced cytokine and chemokine gene expression in astrocytes. A, astrocytes were stimulated with the indicated dose of RANTES, boiled RANTES (b100), or TCA-4 for 3 h. Total RNA was prepared and assayed by RPA for the expression of TNF-, RANTES, KC, IL-6, MIP-1, MIP-2, MCP-1, L32, and GAPDH message. Representative data from one of three similar experiments are presented. B, kinetics of cytokine and chemokine gene expression. Astrocytes were stimulated with 100 ng/ml RANTES for the indicated times. C, kinetics of KC protein expression were determined by ELISA using culture supernatants collected at the indicated times. D, kinetics of TNF- protein expression were determined by ELISA using culture supernatants collected at the indicated times.

The ability of RANTES to stimulate KC and TNF- protein synthesis was next examined. Primary astrocyte cultures were treated with 100 ng/ml RANTES for the indicated times, and then supernatants were harvested for assay. The viability of the cultures was >95% throughout the 16-h observation period. Kinetic examination indicated that KC proteins were detectable within 8 h and the levels rose rapidly (Fig. 1C). TNF- proteins were induced with similar kinetics. Low levels of TNF- (<120 pg/ml) were detectable after 16 h compared with the 20-30 ng/ml concentrations of KC (Fig. 1, C and D).

RANTES Activates the MAPK Pathway in Astrocytes-- To examine the components of the RANTES signal transduction pathway, we evaluated the phosphorylation of the MEK and MAPK. To minimize basal kinase activity, astrocytes were serum-starved at least 3 h before treatment with chemokine. Whole cell lysates were separated by SDS-PAGE and examined by Western blot. Antibodies that specifically react with phosphorylated MEK or phosphorylated ERK were used to detect the active kinases. RANTES-induced phosphorylation of MEK, ERK1, and ERK2 was noted within 5 min, peaked 20-60 min, and lasted for over 2 h (Fig. 2A). For normalization, total ERK protein levels were determined with an Ab reacting with both the phosphorylated and nonphosphorylated proteins (Fig. 2B).

View larger version (35K): [in this window] [in a new window]   Fig. 2.   RANTES induced phosphorylation of MEK and ERK1/2 but not JNK or p38. A, astrocytes were stimulated with 100 ng/ml RANTES for the indicated times, and cell lysates were prepared for analysis by Western blotting. Blots were stained with anti-phospho-MEK1/2 Ab, anti-phospho-ERK1/2 Ab, and antibodies that detected total ERK1/2 expression. B, Pooled data from 3 to 4 independent experiments. Gels were scanned on a PhosphorImager to quantitate the data. The results were normalized based on the levels of total ERK1/2. Gray-shaded bars represent phospho-MEK, solid bars are phospho-ERK1, and open bars represent phospho-ERK2 ± S.E. C, astrocytes were stimulated for 20 min with medium, 100 ng/ml RANTES, or 20 ng/ml IL-1. Cell lysates were analyzed by Western blotting with anti-phospho-ERK1/2, anti-phospho-SAPK/JNK, and anti-phospho-p38 Ab.

To determine whether RANTES also induced phosphorylation of the p38 and SAPK/JNK MAPKs, the state of these enzymes was examined directly by Western blot. As illustrated in Fig. 2C, RANTES treatment failed to phosphorylate p38 or SAPK/JNK. Kinetic studies indicated that phosphorylation of p38 or JNK was not detectable from 5 to 120 min after RANTES stimulation (data not shown). Furthermore, the treatment with the p38 inhibitor SB203580 failed to modulate RANTES-mediated transcription of chemokine RNA (data not shown). All three MAPKs were activated following treatment with the cytokine IL-1 (Fig. 2C) in accordance with published results (31, 32).

To examine the effects of the MAP kinase pathway on induction of chemokine transcripts, we pretreated astrocyte cultures with the specific MEK1/2 inhibitor, U0126, for 1 h. The treatment with 1 µM U0126 partially inhibited transcription, whereas incubation with 50 µM U0126 completely inhibited TNF- and chemokine transcription (Fig. 3). The data indicate that MEK is involved in the intracellular signal required for RANTES-mediated chemokine induction of astrocytes.

View larger version (35K): [in this window] [in a new window]   Fig. 3.   The effects of the MEK inhibitor U0126 on the induction of TNF- and chemokine transcripts. Astrocytes were pretreated with the indicated amount of U0126 and then stimulated with 100 ng/ml RANTES for 3 h, and total RNA was prepared and assayed by RPA as for Fig. 1.

To establish that RANTES up-regulated chemokine production by the activation of promoter elements, primary astrocyte cultures were transiently transfected with a murine KC promoter-luciferase construct, and reporter activity was monitored after RANTES treatment. RANTES stimulated reporter activity in a dose-dependent fashion (Fig. 4A). The treatment of the transfected cells with U0126 inhibited reporter activity, whereas incubation with SB203580, an inhibitor of p38 MAPK, failed to effect this response (Fig. 4B).

View larger version (16K): [in this window] [in a new window]   Fig. 4.   The effect of U0126 pretreatment on RANTES induced KC reporter gene expression. A, astrocytes were transfected with a luciferase reporter construct driven by the murine KC promoter. 24 h later, cells were stimulated in the presence of the indicated dose of RANTES. Values are presented as arbitrary luciferase units and represent the mean ± S.E. of triplicate experiments. B. astrocytes transfected with the above luciferase reporter construct were stimulated in the absence or presence of the indicated amount of U0126 or 5 µM SB203580, an inhibitor of p38, plus 100 ng/ml RANTES for 24 h before luciferase activity was measured.

RANTES Stimulates Activation of RSK in Astrocytes-- The p90 ribosomal S6 protein kinases (RSKs) are a family of Ser/Thr protein kinases that are stimulated through the ERK pathway (19). RSKs participate in the regulation of transcription factors such as CREB, CREB-binding protein and p300, and c-Fos (19, 21, 25, 33). In addition, RSK participates in the phosphorylation of IB, leading to the activation and nuclear translocation of NF-B (34, 35). The activation of RSK is a stepwise process involving the phosphorylation of multiple residues (19). To determine whether RANTES can mediate RSK activation in astrocytes, cells were treated with 100 ng/ml RANTES, and the phosphorylation of multiple RSK residues was examined by Western blot (Fig. 5A). Increased phosphorylation was observed with all phospho-RSK Ab examined including reagents specific for phosphorylation at residues Ser-381, Thr-360/Ser-364, and Thr-574. The phosphorylation of RSK was noted within 5 min, peaked at 60 min, and was sustained for over 2 h.

View larger version (41K): [in this window] [in a new window]   Fig. 5.   RANTES induced phosphorylation of p90RSK via the MAPK pathway. A, astrocytes were stimulated with 100 ng/ml RANTES for the indicated time. Cell lysates were assayed by Western blotting with anti-phospho-p90RSK (Ser-381) (P-RSK (Ser381)), anti-phospho-p90RSK (Thr-360/Ser-364) (p-RSK (Thr360/Ser364)), and anti-phospho-p90RSK (Thr-574) (p-RSK (Thr574)) Ab. The same lysates were also analyzed for the total expression of the kinase using anti-p90RSK antibody to ensure equal protein loading. B, dose response of RANTES-mediated phosphorylation of ERK and RSK. Blots were probed with anti-phospho-RSK (Ser-381), anti-phospho-ERK, and anti-total ERK. Representative data are from one of three experiments. C, astrocytes were pretreated with the indicated concentrations of U0126 and stimulated with 100 ng/ml RANTES for 20 min. Western blots were performed as indicated above.

There is a correlation between the dose of RANTES required to stimulate chemokine transcription (Fig. 1A) and the dose sufficient to phosphorylate ERK and RSK (Fig. 5B). Thus, 10 ng/ml RANTES (~1 nM) triggers the phosphorylation of these kinases and increases transcription.

To establish the interrelationship between MEK and RSK, astrocytes were treated with graded doses of U0126 before stimulation with RANTES. After 20 min, lysates were prepared and examined for ERK and RSK phosphorylation. Treatment with the MEK inhibitor blocked RSK phosphorylation in a dose dependent fashion (Fig. 5.C). Therefore, MEK is a critical upstream kinase responsible for activation of RSK in the RANTES signal transduction pathway.

To examine the RSK dependence of RANTES-stimulated activation of the KC promoter, we employed a dominant negative mutant of RSK. The two phosphorylation sites (K112R/K464R) required for kinase activity were mutated, resulting in a kinase-defective protein (33, 36). Astrocytes were cotransfected with the luciferase-KC promoter construct along with wild-type RSK, mutant RSK, or vector controls. The co-transfected cells were stimulated with RANTES and monitored for luciferase reporter activity. Dominant negative RSK specifically suppressed reporter activity (Fig. 6), demonstrating the importance of this enzyme in regulating the transcription of the chemokine KC.

View larger version (12K): [in this window] [in a new window]   Fig. 6.   The effect of dominant negative p90RSK plasmid on the KC reporter gene. Astrocytes were co-transfected with the luciferase reporter construct driven by murine KC promoter and expression plasmids for the wild-type p90RSK or the kinase-defective mutant of p90RSK. Transfected astrocytes were stimulated with medium (open bars) or 100 ng/ml RANTES (solid bars) for 24 h before the cells were harvested to detect luciferase activity. Values are given in arbitrary luciferase units and represent the mean ± S.E. of triplicate experiments.

Finally, we examined the ability of RANTES to induce nuclear translocation of RSK. Untreated astrocytes display a diffuse distribution of phosphorylated RSK, nuclear translocation was noted 5 min after RANTES treatment, and after 20 min, high levels of phosphorylated RSK were concentrated within the nucleus (Fig. 7).

View larger version (52K): [in this window] [in a new window]   Fig. 7.   RANTES mediated the translocation of phosphorylated p90RSK. Astrocytes stimulated with 100 ng/ml RANTES for 5 or 20 min were stained by anti-phospho-p90RSK (Thr-360/Ser-364) Ab and Hoechst 33258 to detect nuclei.

RSK is known to phosphorylate nuclear proteins including CREB. Phosphorylation of CREB stimulates the recruitment of components of the basal transcription machinery permitting new mRNA synthesis. The activation of gene expression by CREB requires phosphorylation of the Ser-133 residue. We examined the kinetics of CREB phosphorylation in RANTES-stimulated astrocytes by Western blot with a CREB Ser-133-specific Ab. The phosphorylation of CREB was noted in a time-dependent fashion, and this phosphorylation was dependent on the activation of the MAPK pathway as demonstrated by inhibition with U0126 (Fig. 8A). Furthermore, co-transfection of wild type or dominant negative CREB along with the KC promoter-luciferase construct established that CREB was involved in transcription from the KC promoter. Thus, a dominant negative CREB inhibited transcription by the KC promoter (Fig. 8B).

View larger version (14K): [in this window] [in a new window]   Fig. 8.   CREB participates in KC transcription. A, kinetics of CREB phosphorylation at Ser-133 in astrocytes. After stimulation with 100 ng/ml RANTES, cell lysates were harvested at the indicated times. One group was pretreated with 50 µM U0126 to block MEK1 activation. Western blot with anti-phospho-CREB (Ser-133) Ab or anti-ERK Ab. B, co-transfection of the dominant negative CREB mutant blocks luciferase production through the KC promoter in BALB astrocytes. Values are given in arbitrary luciferase units and represent the mean ± S.E. of triplicate experiments. C, astrocytes were transfected with the luciferase reporter plasmid driven by the KC promoter with or without a CREB site (193/188). 24 h later, astrocytes were treated with 100 ng/ml RANTES for 8 h. Luciferase activities were detected and presented in arbitrary units. The mean ± S.E. is presented from three independent experiments.

CREB functions by binding to CRE. We identified at least two potential CRE sites within the 2.9-kb KC promoter (data not shown). The proximal CRE site in the KC promoter is highly conserved, and identical sequences are present in other CXC chemokine promoters. To evaluate the role of the proximal CRE site in KC expression, the site was mutated. The activity of the wild type and mutant promoter constructs were examined following RANTES stimulation in the luciferase reporter assay. The mutation of the proximal CRE site disrupted transcriptional activity (Fig. 8C).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

In this communication, we demonstrate that RANTES stimulates murine astrocytes to synthesize RNA for multiple chemokines including KC, MIP-2, MIP-1, MCP-1, and RANTES plus the cytokines TNF- and IL-6. This in vitro model reflects the complex pattern of chemokines and cytokines produced by astrocytes during chronic infectious and inflammatory diseases (6, 27, 37, 38). The ability of one chemokine to induce a cascade of proinflammatory mediators represents an amplification mechanism that may prolong inflammatory responses in the CNS.

Previous reports (27, 39, 40) indicate that other chemokines including the CXC-chemokines KC, MIP-2, and SDF-1, and the CC-chemokines MIP-1 and MIP-1 also induce chemokine/cytokine amplification, but those reports failed to examine RANTES. The ability of RANTES to stimulate astrocytes is consistent with reports demonstrating that astrocytes express two independent high affinity RANTES receptors, CCR1 (41, 42) and CCR5 (18, 43-45).

The strong, rapid, and sustained induction of KC suggests an important role for this chemokine. KC and the structurally related chemokine MIP-2 may contribute to inflammation by recruiting leukocytes to the CNS, and they may also participate in the subsequent repair processes by promoting the growth of oligodendrocytes (46, 47). TNF- is a multipotential cytokine involved in microglial activation, neuronal death, and immune regulation (48, 49). Thus, the release of KC and TNF- during an inflammatory response might be expected to have pathological consequences.

Little is known about the signaling mechanisms involved in astrocyte responses to chemokines. Recent reports noted the involvement of MEK and ERK1/2 in response to SDF-1 (40, 50, 51). We now demonstrate that these enzymes are also involved in astrocyte signal transduction following RANTES treatment. However, RANTES was reported to induce rapid phosphorylation and activation of p38 MAPK as well as the activation of its downstream effector MAPK-activated protein kinase-2 in T cells (52). Pharmacological inhibition of RANTES-dependent p38 MAPK activation blocked MAPK-activated protein kinase-2 activity in T cells (52). However, we failed to detect the activation of p38 or JNK in RANTES-activated astrocytes. The combined data imply that RANTES differentially activates distinct MAPKs in a cell type-specific fashion.

We now demonstrate that RANTES activates the Ser/Thr kinase, RSK, downstream of ERK. In addition, RANTES stimulation causes translocation of phosphorylated RSK to the nucleus. Transfection of a dominant negative RSK mutant that lacks kinase activity specifically inhibited KC promoter-driven transcription. The kinase-defective RSK inhibited but did not completely abolish RANTES-induced transcription, suggesting that other signaling pathways may be involved in the transcriptional activation of KC. To our knowledge, this is the first evidence linking RSK to signaling pathways in astrocytes and the initial data indicating that RSK is involved in chemokine signaling. In the nucleus, RSK apparently phosphorylates CREB. The action of CREB is required for RANTES-mediated induction of chemokine transcription.

In a system that analyzed the growth and differentiation of human first trimester fetal astrocytes, it was shown that RANTES induced nuclear translocation of STAT-1 proteins (18). However, preliminary experiments with mouse neonatal astrocytes suggest that STAT-1 does not play a role in RANTES-mediated chemokine synthesis (data not shown). The combined data suggest that these different RANTES-mediated effector capacities involve separate elements for nuclear translocation and transcriptional activation.

In summary, the phosphorylation and translocation of RSK are novel requirements for RANTES-mediated activation of chemokine synthesis in astrocytes. The ERK-RSK signal transduction pathway used by astrocytes is distinct from the reported mechanisms of chemokine signaling and induction used by leukocytes. Hence, therapeutic strategies to regulate chemokine responses and synthesis may need to be tailored to specific target tissues.

	FOOTNOTES

^* This study was supported in part by National Institutes of Health Grants NS37284 and CA67416 and a grant from the Hoechst Marion Roussel Collaboration.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. of Pathology, Harvard Medical School, Armenise Bldg. D530, 200 Longwood Ave., Boston, MA 02115. Tel.: 617-432-1978; Fax: 617-432-2789; E-mail: dorf@hms.harvard.edu.

Published, JBC Papers in Press, March 13, 2002, DOI 10.1074/jbc.M112442200

	ABBREVIATIONS

The abbreviations used are: CNS, central nervous system; RANTES, regulated on activation normal T cell expressed and secreted; Ab, antibody; CREB, cAMP response element-binding protein; CRE, cAMP response element; ERK, extracellular signal-regulated kinase; L32, large ribosomal subunit protein 32-3A; MAPK, mitogen-activated protein; ELISA, enzyme-linked immunosorbent assay; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; RPA, RNase protection assay; RSK, p90 ribosomal S6 protein kinase; HIV, human immunodeficiency virus; JNK, c-Jun N-terminal kinase; SAPK, stress-activated protein kinase; IL, interleukin; MCP, monocyte chemoattractant protein; TNF, tumor necrosis factor; MIP, macrophage inflammatory protein; STAT, signal transducers and activators of transcription; SDF, stromal cell-derived factor; TCA, thymus-derived chemotactic agent.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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