From the Institute of Molecular Biology, Jagiellonian
University, 31-120 Kraków, Poland, § Athena
Neurosciences Inc., South San Francisco, California 94080, the
¶ Institute of Biochemistry, Rhenisch-Westfalische Technische
Hochschule, Aachen, D-5100 Germany, and the
Department of
Biochemistry and Molecular Biology, The University of Georgia,
Athens, Georgia 30602
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ABSTRACT |
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1-Antichymotrypsin (ACT) is
an acute phase protein expressed in the brain which specifically
colocalizes with amyloid-
during Alzheimer's disease. We analyzed
ACT synthesis in cultured human cortical astrocytes in response to
various cytokines and growth factors. Oncostatin M (OSM) and
interleukin (IL)-1
were potent stimulators of ACT mRNA
expression, whereas tumor necrosis factor-
had modest activity, and
IL-6 and leukemia inhibitory factor (LIF) were ineffective. The finding
that OSM, but not LIF or IL-6, stimulated ACT expression suggests that
human astrocytes express a "specific" OSM receptor, but not IL-6 or
LIF receptors. However, cotreatment of human astrocytes with soluble
IL-6 receptor (sIL-6R)·IL-6 complex did result in potent stimulation
of ACT expression. When the human ACT gene was cloned, two elements
binding STAT1 and STAT3 (signal transducer and activator of
transcription) in response to OSM or IL-6·sIL-6R complexes could be
identified and characterized. Taken together, these findings indicate
that OSM or IL-6·sIL-6 complexes may regulate ACT expression in human
astrocytes and thus directly or indirectly contribute to the
pathogenesis of Alzheimer's disease.
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INTRODUCTION |
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1-Antichymotrypsin
(ACT)1 is one of the major
positive human acute phase proteins produced by the liver and secreted
into blood plasma (1, 2). The expression of this proteinase inhibitor in hepatic cells is enhanced by interleukin (IL)-6 and glucocorticoids and to a lesser extent by IL-1 (3, 4). Although ACT is also found in
the brain, the plasma-derived inhibitor is separated from this origin
by a tight blood-brain barrier consisting of endothelial cells. For
this reason it is believed that astrocytes are the likely source of ACT
produced within the central nervous system (5). Significantly, ACT has
been identified as one of the amyloid-associated proteins found in the
brains of patients with Alzheimer's disease (6, 7). The pathological
feature of this disease is cerebral degeneration with neuronal cell
death and deposition of abnormal proteins in the form of amyloid
plaques and neurofibrillary tangles. Because the expression of ACT is enhanced dramatically in affected brain regions in Alzheimer's disease, a state of cerebral "acute phase" in response to neuronal degradation has been proposed. IL-1 and IL-6, which are produced by
cells of CNS, were suggested to induce ACT expression in astrocytes (8). Indeed, the induction of ACT expression by IL-1 has been shown in
human astrocyte cultures (9); however, regulation by IL-6 has not been
confirmed.
To understand the control of ACT expression in the brain we used human astrocyte cultures and analyzed the pattern of its synthesis after stimulation with a variety of factors including IL-1 and cytokines of the IL-6 family. We have also cloned the 5'-flanking region of the ACT gene and performed analysis of its transcriptional activity. Our results suggest that at least one cytokine, oncostatin M (OSM), may play an important role in up-regulating ACT expression in astrocytes, whereas IL-6 requires the presence of soluble IL-6 receptor (sIL-6R).
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MATERIALS AND METHODS |
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Cell CultureHuman cortical astrocyte cultures were
established using dissociated human cerebral tissue at 16-20 weeks
gestation. The protocol for obtaining postmortem fetal neural tissue
complied with federal guidelines for fetal research and with the
Uniformed Anatomical Gift Act. Cortical tissue was washed three times
in Ca2+/Mg2+-free Hanks' balanced salt
solution and then dissociated by repeated pipetting. DNase (Sigma) was
added to a final concentration of 0.05 mg/ml, and the solution was
passed through a 100-µm nylon cell strainer (Falcon). The cells
obtained were then centrifuged for 5 min at 200 × g,
resuspended in a trypsin/EDTA solution (0.05% trypsin and 0.53 mM EDTA in Hanks' balanced salt solution, Life Technologies, Inc.), and incubated for 20 min at 37 °C. Modified Eagle's medium containing 1% glucose, 1 mM sodium
pyruvate, 1 mM glutamine, and 10% fetal bovine serum
(MEM/FBS) and DNase (final concentration of 0.05 mg/ml) were added, and
the cells were again pelleted by centrifugation and resuspended in
MEM/FBS. 1.6 × 108 cells were seeded in a T-150
tissue culture flask coated with polyethyleneimine. Cultures were
maintained in an H2O-saturated incubator with an atmosphere
of 95% air and 5% CO2 at 37 °C. The culture medium was
changed 1 and 4 days after plating, and the cultures were then left
undisturbed for at least 1 week.
Astrocyte cultures were prepared by multiple passaging of the established mixed brain cell cultures. The cells from one T-150 flask were replated into three to five T-150 flasks. Just before confluence the cells were repassaged by trypsinization. This process was repeated until the cultures were >98% pure astrocytes as judged by immunocytochemistry analysis for glial fibrillary acidic protein (three to four passages). Human astrocytoma CCF-STTG1 cells were obtained from ATCC (Rockville, MD) and cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, antibiotics, sodium pyruvate, and nonessential amino acids.
Cytokines and Cell Stimulation--
Cells were stimulated with
50 ng/ml recombinant human IL-6 (1.9 × 109 units/mg),
25 ng/ml OSM (4.7 × 107 units/mg), 5 ng/ml
recombinant human IL-1 (1.2 × 108 units/mg) (all a
gift from Immunex Corp., Seattle), 200 units/ml recombinant human
interferon- (2.0 × 107 units/mg) (Boehringer), 25 ng/ml recombinant human epidermal growth factor (Upstate Biotechnology
Inc., Lake Placid, NY), 50 ng/ml sIL-6R (R&D Systems Inc., Minneapolis,
MN), 10 ng/ml TNF-
(a gift of Cutter Laboratories Inc., Berkeley,
CA), 10 units/ml recombinant human LIF (a gift of Dr. Heinz Baumann,
Roswell Park Cancer Institute, Buffalo, NY), 100 ng/ml phorbol
12-myristate 13-acetate, or 1 µM dexamethasone (both from
Sigma). Actinomycin D (Sigma) was used at 5 µg/ml. OSM and sIL-6R in
cerebrospinal fluid were measured using enzyme-linked immunosorbent
assay as recommended by the supplier (R&D Systems Inc., Minneapolis,
MN).
RNA Preparation and Northern Blot Analysis-- Total RNA was prepared using the phenol extraction method (10, 11). 5-µg samples of RNA were subjected to formaldehyde gel electrophoresis using standard procedures (12) and transferred to Hybond-N membranes (Amersham) according to the manufacturer's instructions. The filters were prehybridized at 68 °C for 3 h in 10% dextran sulfate, 1 M sodium chloride, and 1% SDS and hybridized in the same solution with a 1.4-kilobase EcoRI/EcoRI fragment of ACT cDNA (a gift of Dr. H. Rubin, University of Pennsylvania) labeled by random priming (13). After the hybridization, nonspecifically bound radioactivity was removed by washing in 2 × SSC at room temperature followed by two washes in 2 × SSC and 1% SDS at 68 °C for 20 min.
Synthetic Oligonucleotides--
The following
oligonucleotides were used to obtain a 352 to +25 fragment of ACT
promoter: 5'-TAGTCTAGAAATATACCAAAATGTTAG-3' and
5'-GGTGGATCCAAAGCCGTCTGTCTGGAG-3'; primer
5'-ATCATCTAGACATTTCCAGTCCGA-3' was synthesized to obtain a
deletion mutant (
103 to +25). Mutants containing point mutations in
the ACT-A and/or ACT-B elements were generated by polymerase chain
reaction using the PWO polymerase (Boehringer Mannheim) and the
following primers: 5'-GTATGAGCTCAAATTATCATCTGGT-3', 5'-ATTTGAGCTCATACGGGCTTGTTAG-3',
5'-CCAGGGTACCAACAGAACACTTGGT-3', and
5'-TGTTGGTACCCTGGAAATGACCAGA-3'. All oligonucleotides used for gel retardation assays were designed to contain single-stranded 5'-overhangs of 4 bases at both ends after annealing (Table
I).
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Library Screening and Plasmid Construction--
A human genomic
library (from a HT1080 fibrosarcoma cell line) was obtained from
Stratagene (La Jolla, CA). 5 × 105 phages were
screened using a (352 to +25) polymerase chain reaction fragment of
the ACT promoter. A 4517-base pair EcoRI/EcoRI
fragment containing the 5'-flanking region of the ACT gene was cloned
into pUC19 (pUCACT). Plasmid ptkCAT
EH is a derivative of pBLCAT2
(15). Plasmids pACT-352CAT and pACT-103CAT were obtained by insertion of the XbaI/BamHI- or
XbaI/XhoI-digested polymerase chain reaction products into the XbaI/BglII or
XbaI/XhoI sites of ptkCAT
EH, respectively.
Plasmids pACT-247CAT, pACT-156CAT, and pACT-68CAT were generated by
insertion of MseI(blunt)/XhoI,
BstNI(blunt)/XhoI, and
BstNI(blunt)/XhoI fragments into the
BamHI(blunt)/XhoI sites of ptkCAT
EH. Plasmid
pACT-2431CAT was produced by inserting a PstI/PstI fragment from pUCACT into the
PstI/PstI sites of pACT-352CAT, and plasmid
pACT-1505CAT was formed by inserting a XbaI/XhoI
fragment from pACT-2431CAT into the XbaI/XhoI
sites of ptkCAT
EH. Plasmid pACT-1514CAT derives from pACT-2431CAT,
from which the SphI/SphI fragment was deleted.
Plasmid pACT-3573CAT was obtained by cloning the
XbaI/XbaI fragment from pUCACT into the
XbaI site of pACT-1505CAT. Plasmids pACTmutA-CAT,
pACTmutB-CAT, and pACTmutAmutB-CAT analogous to pACT-156CAT but with
introduced point mutations in the ACT-A and/or ACT-B elements were
generated by insertion of XbaI/XhoI-digested polymerase chain reaction products into the
XbaI/XhoI sites of ptkCAT
EH. Plasmids
p6x(ACT-A)tkCAT, p6x(ACT-B)tkCAT, p3x(ACT-(A+B))tkCAT, and
p(ACT-(A+B))tkCAT were obtained by insertion of a double-stranded oligonucleotide(s) with BglII ends into the BamHI
site of ptkCAT
EH. All constructs were sequenced on both strands.
Transient Transfections--
Cells were transfected in
Dulbecco's modified Eagle medium supplemented with 10% fetal calf
serum using calcium phosphate precipitates (16), with 5 µg of plasmid
DNA and 3 µg of internal control plasmid pCH110 (Pharmacia Biotech
Inc.). Cells were incubated with precipitate for 6 h, washed
twice, and media changed. One day after transfection, cells were
stimulated and then cultured another 24 h and harvested. Protein
extracts were prepared by freeze-thawing (17), and the protein
concentration was determined by the BCA method (Sigma). Chloramphenicol
acetyltransferase (CAT) and -galactosidase assays were performed as
described (18, 19). CAT activities were normalized to the internal
control
-galactosidase activity and are the means ± S.E. of
three to eight determinations.
Extract Preparation and Gel Retardation Assays--
Nuclear
extracts were prepared as described (20, 21). Double-stranded DNA
fragments were labeled by filling in 5'-protruding ends with Klenow
enzyme using [-32P]dCTP (3,000 Ci/mmol). Gel
retardation assays were carried out according to published procedures
(22, 23). 2-5 µg of nuclear extracts and approximately 10 fmol
(10,000 cpm) of probe were used. The polyclonal anti-STAT3 (C20) and
anti-STAT1 (E23) antisera were from Transduction Laboratories
(Lexington, KY).
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RESULTS |
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OSM, IL-1, and TNF- Regulate the Expression of ACT in Human
Astrocytes--
ACT is produced by human astrocytes with IL-1 having
already been shown to induce its mRNA expression (9). We analyzed the pattern of ACT production in human astrocyte cultures in response to other factors regulating gene expression during inflammation and the
acute phase response. Human astrocytes were stimulated with IL-1,
TNF-
, IL-6, OSM, LIF, phorbol 12-myristate 13-acetate, interferon-
, and epidermal growth factor and analyzed by Northern blotting. An example of the results obtained is shown in Fig. 1A. In addition to IL-1, as
reported previously, TNF-
and OSM were potent stimulators of ACT
mRNA synthesis, and this was enhanced greatly by the synthetic
glucocorticoid dexamethasone. Dexamethasone alone had only a small
effect. In contrast, IL-6, LIF, epidermal growth factor,
interferon-
, and phorbol 12-myristate 13-acetate were ineffective
(Fig. 1A and data not shown). OSM was the most potent
stimulator of ACT expression, not only in human astrocytes but also in
the human astrocytoma cell lines U373 and CCF-STTG1 (data not shown).
This finding correlates with the observation that OSM strongly
activates ACT synthesis in bronchial epithelial cells (24). The OSM- or
IL-1-induced ACT mRNA was very stable with a half-life of at least
12 h in human astrocytes (Fig. 1B) as well as in the
CCF-STTG1 cell line.
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sIL-6R Restores Responsiveness of Astrocytes to IL-6-- The family of IL-6 type cytokines is characterized by the use of a common receptor subunit, the signal transducing protein gp130 (26). Signaling through gp130 results in the phosphorylation of tyrosine residues of latent cytoplasmic proteins called STATs, particularly STAT1 and STAT3, followed by their dimerization, translocation to the nucleus, binding to specific elements within the promoters of target genes, and activation of transcription (26-28). Because the synthesis of ACT mRNA in astrocytes was induced by OSM but not by IL-6 (Figs. 1A and 2B), and astrocytes were reported to produce IL-6 (29), we hypothesized that these cells lack IL-6 receptors (IL-6R) on their cell surface. We analyzed the expression of IL-6R (gp80) in these cells by Northern blotting but could not detect any IL-6R mRNA, although it was easily detectable in HepG2 cells (data not shown). Next, to show that functional IL-6 receptors are missing from astrocytes, cells were stimulated with OSM, LIF, IL-6, or IL-6 together with sIL-6R. The IL-6·sIL-6R complexes have been shown previously to bind to gp130 and activate its signaling (26, 30). The induction of ACT expression was measured by Northern blotting, and activation of STAT proteins was examined by following their ability to bind to the high affinity sis-inducible element of human c-fos gene promoter (SIE) (31). OSM was found to activate STAT3 and STAT1 and induce ACT mRNA synthesis (Fig. 3, A and B; see also Fig. 5B). In contrast, IL-6 and LIF were incapable of inducing ACT mRNA synthesis or activating STAT factors. However, activation of STAT proteins and ACT expression was observed when sIL-6R was used together with IL-6, indicating that human astrocytes lack functional IL-6 receptors and most likely do not express LIF receptors. The IL-6·sIL-6 receptor cR complex strongly activated STAT3 but STAT1 activation could also be seen after longer exposure of a gel (data not shown). As a positive control astrocytoma cells were stimulated with IL-6, LIF, and OSM (Fig. 3B, right panel). In these cells all three cytokines activated STAT3 (OSM also activated STAT1); however, a weak action of IL-6 and LIF suggested a small number of the corresponding surface receptors (Fig. 3B, right panel). Furthermore, when astrocytes were transfected with the ACT-CAT construct the sIL-6R restored their responsiveness to IL-6 (Fig. 3C).
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Identification of STAT-binding Elements within the ACT
Promoter--
Because both OSM and IL-6·sIL-6R greatly enhance ACT
expression in astrocytes as well as activate transcription of the
3.6-kilobase ACT-CAT construct, a series of 5'-deletion mutants was
made to identify the elements conferring this responsiveness.
Constructs were transiently transfected into astrocytes, and their
responsiveness to either OSM, dexamethasone, or OSM together with
dexamethasone was assayed. The construct containing the
156 to +25
fragment of the ACT promoter and all of the longer constructs were
fully responsive to OSM (or OSM with dexamethasone), whereas
dexamethasone alone had no effect (Fig.
4). However, the truncation to
103 resulted in a drastic loss of responsiveness to OSM by 80%. The responsiveness to OSM was abolished completely by further truncation to
68. The enhancing effect of dexamethasone (approximately 2-fold) was
observed with all constructs, suggesting an activation of the
transcriptional machinery rather than induction via a specific glucocorticoid receptor-responsive element. Because we could not detect
any glucocorticoid receptor-responsive element within ACT promoter
constructs, and the glucocorticoid receptor was recently shown to
directly interact with the STAT5 protein (32), it is possible that the
enhancing effect of dexamethasone is mediated by an interaction of the
glucocorticoid receptor with STATs.
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Functional Analysis of STAT Binding Elements-- To correlate binding of STAT1 and STAT3 complexes with the transcriptional activity of the ACT promoter, point mutations were introduced into the STAT binding sites. Mutations of both sites A and B had the same drastic effect on promoter activity, reducing the responsiveness by 80%, whereas the promoter with both mutated sites was no longer induced by OSM (Fig. 6). These results indicate that both sites contribute equally to the activation of ACT transcription by OSM.
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DISCUSSION |
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The role of inflammatory cytokines, including TNF-, IL-1, and
IL-6, within the CNS has been extensively studied over the last several
years. Specifically, IL-1 and IL-6 have attracted much attention and
have been suggested to mediate a cerebral acute phase response and
induce the expression of cerebral acute phase proteins in astrocytes,
including ACT (8). In addition, IL-6, which is the major regulator of
liver acute phase proteins, has also been suggested to participate in
the pathogenesis of Alzheimer's disease (33, 34). Our results,
however, clearly show that IL-6 failed to activate STAT factors or to
induce ACT expression in cultured human astrocytes (Fig. 3). The lack
of response of these cells to IL-6 results from the absence of the
subunit of the IL-6R (gp80), because sIL-6R restored both activation of STAT proteins and induction of ACT expression (Fig. 3). Significantly, LIF, another IL-6 type cytokine, neither activated STAT proteins nor
induced ACT expression in human astrocytes. We did not address specifically the question as to which of the components involved in LIF
signaling was missing in astrocytes. However, considering that
astrocytes produce both IL-6 and LIF (35, 36) we speculate that LIF-R
is a likely candidate. Contrary to our present observations with human
astrocytes, IL-6 has been reported to up-regulate nerve growth factor
expression in mouse astrocytes (37). We have analyzed the response of
rat astrocytes to IL-6 in terms of STAT activation and regulation of
gene expression. IL-6 activated STAT3 poorly in these cells and weakly
induced the expression of the serine proteinase inhibitor-3 gene, a rat
homolog of the ACT gene. As in human astrocytes, IL-6·sIL-6R
complexes improved significantly both activation of STAT factors and
induction of serine proteinase inhibitor-3 gene transcription in these
cells.2 These results suggest
that both sets of astrocytes express only small amounts of IL-6Rs or
lack the presence of IL-6Rs on their cell surfaces. Also, human
astrocytoma cells CCF-STTG1 and U373 barely respond to IL-6 (Fig. 2 and
data not shown). Thus, IL-6 alone cannot trigger the activation of
specific proteins, including ACT, in human astrocytes.
In contrast to IL-6 and LIF, OSM emerges as a potent stimulator of ACT expression in astrocytes (Figs. 1 and 3). These three cytokines possess overlapping activities because of their signaling through receptors containing the shared signal transducer gp130 (26). Binding of IL-6 to the IL-6R results in the homodimerization of gp130, whereas LIF triggers heterodimerization of gp130 with LIF-R (26). OSM can signal via two receptors: LIF-R·gp130 (also a receptor for LIF) and OSM-R·gp130 (specific OSM receptor) (26, 38-40). Because LIF failed to activate STAT proteins and induce the expression of ACT, we conclude that signaling via the LIF-R·gp130 system is not functional in human astrocytes. The response to OSM on the other hand is triggered through a specific OSM receptor composed of OSM-R and gp130. In contrast to human astrocytes, human astrocytoma cells CCF-STGG1, known to contain mRNA for gp130, LIF-R, and OSM-R (40), can respond to LIF (Fig. 2). Thus, these cells contain functional LIF receptors in addition to functional specific OSM receptors. It should be noted that the other well known example of signaling utilizing the specific OSM receptor but not the LIF-R·gp130 complex is the control of growth of the acquired immunodeficiency syndrome-associated Kaposi's sarcoma cells by OSM (38). Thus, astrocytes, in parallel with Kaposi's sarcoma cells, express a specific OSM receptor that is being utilized to activate STAT proteins and induce expression of specific genes, including ACT. Recently, a specific OSM receptor has been shown to activate STAT5B efficiently (41, 42); however, ACT expression is not up-regulated by STAT5B overexpression.3
The activation of ACT expression by IL-6·sIL-6R and OSM raises the question as to whether these factors can be found in the CNS. The expression of IL-6 by astrocytes, microglia, and also by astrocytoma cells is well documented (36, 43). Furthermore, IL-6 can be detected readily in cerebrospinal fluid of patients during viral and bacterial meningitis (37, 44), HIV (human immunodeficiency virus)-induced encephalopathy (45), Parkinson's disease (46), multiple sclerosis (47), after trauma (48), and in Alzheimer's disease (33, 34, 49). The sIL-6R that is necessary for IL-6 action on human astrocytes was detected in the cerebrospinal fluid of normal patients (1.6 ng/ml) (50), and its level was elevated to 6.6 ng/ml in the cerebrospinal fluid of a patient with Crow-Fukase syndrome (51). We measured the levels of sIL-6R in the cerebrospinal fluid of both normal individuals and patients with Alzheimer's disease using a sensitive enzyme-linked immunosorbent assay. The amount of sIL-6R was not changed significantly in patients with Alzheimer's disease (0.8 ng/ml for both groups, n = 8). However, it is worth mentioning that the concentration of sIL-6R in the cerebrospinal fluid is approximately 1,000-fold higher than the concentration of IL-6. These results suggest that the level of sIL-6R in cerebrospinal fluid probably does not change during Alzheimer's disease; however, the amount of sIL-6R might still be elevated in certain regions of the brain (i.e. plaques). Clearly, the source of sIL-6R within the CNS needs to be defined. The amount of this receptor protein in plasma is much higher than in cerebrospinal fluid (77 ng/ml) (50) because blood-brain barriers prevent plasma proteins from entering the CNS. However, a blood-brain barrier breakdown has been shown to occur in response to overexpression of IL-6 in astrocytes (52, 53). Thus, the expression of IL-6 by reactive astrocytes might potentially result in diffusion of plasma proteins into the CNS (including sIL-6R and ACT). In the light of these findings the up-regulation of IL-6 could conceivably contribute indirectly to a cerebral acute phase response by enhancing the levels of sIL-6R in the CNS and the formation of functional IL-6·sIL-6R complexes.
We could not detect OSM in the cerebrospinal fluid of either controls or patients with Alzheimer's disease using a sensitive enzyme-linked immunosorbent assay (detection limit 2 pg/ml). However, plasma levels of OSM are also very low (below 20 pg/ml) (54). This cytokine is produced by activated T-cells, and these cells readily migrate into the CNS (for review, see Ref. 55). In addition, human microglia can produce OSM and are also a likely source of this cytokine within the CNS.4
The activation of ACT transcription by OSM (or IL-6·sIL-6R) could be correlated with the binding of STAT3 and STAT1 complexes to two elements within the ACT promoter (Fig. 5). These elements bind STAT complexes with different affinities, although both contribute equally to the transcriptional activity of the ACT promoter (Fig. 6). In vitro, the distal site is bound with high affinity by STAT3 and STAT1, whereas the proximal site requires the presence of the distal element. Moreover, the distal site is a true enhancer capable of conferring responsiveness onto a heterologous promoter. The element containing both distal and proximal sites is qualitatively as good as the enhancer element containing only the high affinity distal site (Fig. 7). This observation indicates that the binding of STATs to promoter elements in vitro does not always correlate with the activation of transcription via STAT elements in vivo.
Taken together these studies indicate that the increase of IL-6 alone cannot account for the enhanced ACT expression and that astrocytic responses to IL-6 require the formation of functional IL-6·sIL-6R complexes. Indeed, a role for OSM must now be considered because it is a potent stimulator of ACT expression in cultured human astrocytes.
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ACKNOWLEDGEMENTS |
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We thank Dr. Dale Schenk for a critical reading of the manuscript and Ceci Land for cell culture preparations.
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FOOTNOTES |
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* This work was supported by Research Grants HL26148 and HL37090 from the National Institutes of Health (to J. T.) and by Grant 6 Po4A 010 12 from the Committee of Scientific Research (KBN, Warsaw, Poland) (to T. K.).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.
jwere
** To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, Life Sciences Bldg., University of Georgia, Athens, GA 30602. Tel.: 706-542-1711 or 1334; Fax: 706-542-3719; E-mail, JTravis{at}uga.cc.uga.edu.
1
The abbreviations used are: ACT,
1-antichymotrypsin; IL, interleukin; CNS, central
nervous system; OSM, oncostatin M; -R, receptor; sIL-6R, soluble
interleukin-6 receptor; TNF-
, tumor necrosis factor-
; tk,
thymidine kinase; LIF, leukemia inhibitory factor; CAT, chloramphenicol
acetyltransferase; STAT, signal transducer and activator of
transcription; SIE, sis-inducible element of c-fos
gene.
2 T. Kordula, M. Bugno, D. Bagarozzi, Jr., and J. Travis, unpublished observations.
3 H. Baumann, unpublished observations.
4 R. Rydel and E. F. Brigham, unpublished data.
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REFERENCES |
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