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J Biol Chem, Vol. 274, Issue 7, 4300-4308, February 12, 1999
From the Department of Veterinary Pathobiology, University of
Missouri, Columbia, Missouri 65211
The serum amyloid A (SAA) protein has been
implicated in the progression and pathogenesis of rheumatoid arthritis
through induction of collagenase activity in synovial fibroblast cells that line the joint tissues. We demonstrate that SAA is synergistically induced in synovial cells by interleukin (IL)-1 and IL-6 that are
present at significantly high level in the synovial fluid of arthritis
patients. These cytokines induced phenotypic changes in synovial cells,
promoting protrusion and increased cellular contact. Induction of SAA
under this condition is mediated by promoter elements located between
Serum amyloid A (SAA),1
a member of the acute-phase protein group, is implicated in the
pathophysiology of several chronic inflammatory diseases including
rheumatoid arthritis, amyloidosis, and atherosclerosis. Its level
increases up to 1,000-fold in response to both acute and chronic
inflammatory conditions. Consistent with this observation, the plasma
concentration of SAA in arthritis patients may range from 1 to 1,000 µg/ml or more (1, 2), and SAA has been found in abundance in the
synovial fluid recovered from inflamed joints of rheumatoid arthritis
patients (3, 4). Current hypothesis suggests that locally synthesized
SAA by synovial fibroblast cells in the inflamed joints acts as an
autocrine inducer of matrix metalloproteinase-1 (collagenase), the only
enzyme that degrades interstitial collagen I, II, and III at neutral pH
and causes extensive joint erosion (5, 6). Synovial fluid samples obtained from the knee joints of rheumatoid arthritis patients have
been found to contain high levels of IL-1, IL-6, and a few other
proinflammatory cytokines that may play a critical role in the
manifestation of chronic inflammation and articular destruction (7).
Presence of high levels of proinflammatory cytokines could possibly
trigger local production of SAA in the synovial fibroblast cells.
Indeed, it has been shown that in synovial fibroblast cells, IL-1 can
induce SAA expression (6). However, the molecular mechanism by which
IL-1-mediated SAA gene induction occurs is not well understood.
Moreover, the combinatorial effect of IL-1 and IL-6, the two cytokines
found in abundance in synovial fluid of rheumatoid arthritis patients,
in SAA gene expression in synovial fibroblast cells is also unknown.
The present study was undertaken to elucidate the mechanism by which
IL-1 and IL-6 stimulate SAA in synovial cells.
Because of its link with rheumatoid arthritis, amyloidosis, and
atherosclerosis, the mechanism of SAA gene induction for the past few
years has been a subject of intense investigation by many laboratories,
including ours. SAA biosynthesis during acute-phase response is highly
induced in the liver, and its concentration drops rapidly to a low
background level within a few days. The increase of SAA biosynthesis
during inflammation is due largely to its increased transcription (8).
Stability of mRNA also contributes to the enhanced expression of
SAA in mouse (9) and human (10, 11). Studies on SAA gene transcription
indicated involvement of C/EBP (12-17) and NF- Cell Culture and Transfection--
Rabbit synoviocyte (HIG82)
cells were obtained from American Type Culture Collection. HIG82 cells
were cultured in Dulbecco's modified Eagle's medium containing high
glucose (4.5 g/liter) supplemented with 7% fetal calf serum. HIG82
synoviocytes were derived from the interarticular soft tissue of the
knee joint of a normal female New Zealand White rabbit. These cells
have retained many of the features and, similar to the tissue of
origin, are activated by phorbol myristic acid and express genes coding for enzymes such as collagenase, gelatinase, and caseinase. For induction, HIG82 cells were stimulated with IL-1
Transient transfections of HIG82 cells were carried out by the calcium
phosphate method (30). Transfections were carried out using a mixture
of DNAs containing 10 µg of chloramphenicol acetyltransferase (CAT)
reporter plasmid, 2 µg of plasmid pSV- Plasmid Constructs--
The progressively deleted CAT
reporter plasmids, pSAA( Scanning Electron Microscopy--
HIG82 cells were fixed in 1%
glutaraldehyde in 0.08 M sodium cacodylate buffer (pH 7.4)
at room temperature and washed in the same buffer. The samples were
post-fixed with 1% osmium tetroxide in distilled water for 1 h,
dehydrated in a graded series of ethanol, critical point dried,
sputter coated with gold, and viewed with a Jeol JSM 35 scanning microscope.
Rhodamine-Phalloidin Fluorescence Microscopy--
HIG82 cells
grown on a glass coverslip were rinsed in phosphate-buffered saline
(PBS), pH 7.4, and fixed for 10 min at room temperature in 3.7%
formaldehyde solution. After three washes in PBS, the cells were
incubated by placing the coverslips in 0.1% Triton X-100 in PBS for 5 min, followed by three washes in PBS. The cells were then stained with
rhodamine-conjugated phalloidin for 30 min, washed in PBS, and the
coverslips were mounted on slides for viewing with fluorescence microscope.
RNA Isolation and Northern Blot Analysis--
Total RNA was
isolated from uninduced and various cytokine-induced HIG82 cells by
using guanidinium thiocyanate method (33). 50 µg of each sample of
RNA was fractionated in a 1.1% agarose gel containing 2.2 M formaldehyde and transferred onto nylon membrane, and the
blot was hybridized to 32P-labeled specific cDNA probes
described in the figure legends. The same membrane was subsequently
hybridized to an actin cDNA probe to evaluate the relative amount
of each RNA sample on the membrane.
Oligonucleotides--
The sequences of oligonucleotides used in
this study are: wild-type Sp1, 5'-TCGACTGGGCGGAGTCTGGA-3'; mutant Sp1,
5'-TCGATCACCATAGTCTGGA-3'; wild-type SAA
( Nuclear Extract Preparation and Electromobility Shift
Assays--
Nuclear extract was prepared from uninduced and
cytokine-induced cells and protein content was measured (34). In
electromobility shift assays for the detection of DNA-protein
interaction, equal protein amounts of nuclear extract were incubated
with a radiolabeled DNA probe as described (29).
32P-Labeled double-stranded DNA probes were labeled by
filling in the overhangs at the termini with a Klenow fragment of DNA
polymerase, incorporating [ Western Blot Assay--
Nuclear extracts (30 µg of protein)
were fractionated in a sodium dodecyl sulfate-12% polyacrylamide gel
and transferred onto nitrocellulose membrane using an electroblotter
(Research Products International Corp.). For the evaluation of relative
amounts of proteins in each lane, proteins were stained with Coomassie
Blue. Immunoblotting was performed using the abovementioned primary antibodies and horseradish peroxidase-conjugated secondary antibody. Chemiluminescence reaction was performed with ECL detection kit using
the manufacturer's protocol (Amersham Life Science Ltd).
Dephosphorylation of Nuclear Extracts--
Dephosphorylation of
nuclear extracts was carried out by using calf intestinal alkaline
phosphatase as described (29). As a control, some reaction mixtures
contained, in addition to calf intestinal alkaline phosphatase, a
combination of phosphatase inhibitors (50 mM NaF, 5 µM okadaic acid, and 1 mM sodium
orthovanadate). These treated nuclear extracts were used in the
electromobility shift assays as described.
Synergistic Induction of SAA by IL-1 and IL-6--
To determine
the pattern by which SAA is expressed in synovium exposed to cytokines,
rabbit synovial cells (HIG82) were incubated in the presence of IL-1,
IL-6, and IL-1 plus IL-6, and the level of SAA mRNA was measured by
Northern blot analysis. As shown in Fig.
1, IL-1 alone had a higher stimulatory
effect than IL-6, whereas the combination of IL-1 and IL-6
synergistically activated SAA mRNA expression.
Cytokine Treatment Triggers Phenotypic Changes in Synovial
Fibroblast Cells--
Cytokine-treated cells exhibited striking
morphological changes when visualized using a phase contrast microscope
(data not shown). To further verify the cytokine-induced cytoskeletal
structural modification, we have analyzed the cells by scanning
electron microscopy (Fig. 2). This
analysis revealed long protrusions in cytokine-treated cells that
extend from one cell and interact with surrounding cells in culture.
Such protrusions are characteristic of cells attempting to establish a
connection with other cells to form an association that are seen in
cells undergoing differentiation. This process is facilitated by the
polymerization of actin that forms filamentous or F-actin giving rise
to polymorphic shapes of the cells with pseudopod-like protrusions
(35). Formation of such structural configuration was evaluated by
rhodamine-conjugated phalloidin staining for F-actin and visualized by
fluorescence microscopy as described (36). Results of this experiment,
shown in Fig. 3, revealed significant
formation of F-actin bundles, a thread-like extensions (Fig. 3,
B-D). Normally slender and elongated protrusions
of F-actin are seen in control HIG82 cells (Fig. 3A). Upon
exposure to cytokines, these become bulky and readily fuse with those
from the adjacent cells creating a connection between the cells. These
results demonstrated a distinct change in the morphology of synovial
cells in response to cytokines like IL-1 and IL-6.
The Promoter Region between Characterization of Nuclear Factors Induced by IL-1 and
IL-6--
To investigate the nuclear factors in cytokine-activated
synoviocyte that might interact with the above cytokine-responsive element, we performed DNA binding assays using SAA promoter
( Increased Level of DNA Binding Activity in Cytokine-induced
Synovial Cells--
A high level of complex a, which is
formed by both SAF and Sp1, potentially could arise because of an
increase of DNA binding activities of these two transcription factors
or it could be because of only SAF activation. To determine the
contribution of Sp1 in the formation of complex a, we
assayed the level of Sp1 DNA binding activity in various cell nuclear
extracts (Fig. 6). Because the SAA
promoter ( Western Blot Analyses of SAF and Sp1--
The increase in SAF and
Sp1 DNA binding activities led us to examine their protein levels.
Western blot analysis of untreated and various cytokine-treated nuclear
extracts with anti-SAF antibody revealed that the level of SAF protein
is essentially the same in these four cell extracts (Fig.
7A). However, the protein
levels of Sp1 was seen to be appreciably higher in cytokine-treated
cells (Fig. 7B). To verify that this is not because of any
difference in total protein content loaded in the gel, we stained the
gel containing fractionated nuclear proteins with Coomassie Blue. As
seen in Fig. 7C, in all cases, the same amounts of nuclear proteins of comparable quality were used. Also, SAF content in Fig.
7A served as an internal normalization control. These
results indicated that increased SAF activity in response to cytokine treatment of HIG82 cells most likely involve a post-translational event. On the other hand, a higher level of Sp1 activity correlates with the Sp1 protein content.
Northern Analysis for SAF and Sp1 mRNAs--
To determine
whether the observed changes in protein content of SAF and Sp1
correlated with the mRNA expression, we performed a Northern blot
analysis (Fig. 8). Consistent with the
Western immunoblot analysis, SAF transcript level remained essentially same in all four cell preparations. This indicated that a
post-translational event is probably responsible for the enhanced SAF
activity in response to cytokine treatment of synoviocyte cells. With
regard to Sp1, an increased expression in response to cytokine was
evident.
Dephosphorylation Reduces the DNA Binding Activities of SAF and
Sp1--
Phosphorylation of transcription factors plays an important
role in the cytokine response. We therefore assessed the role of
phosphorylation in mediating increased DNA binding activity of SAF
during IL-1 plus IL-6 induction of cells. Nuclear extracts were first
dephosphorylated using increasing concentrations of calf intestinal
alkaline phosphatase and then used for DNA-protein interaction studies.
As seen in Fig. 9A,
dephosphorylation severely inhibited formation of the two
cytokine-inducible complexes, a and b. Formation
of the two constitutive complexes, d and e, was not affected by dephosphorylation, but a change in their relative migration was noticed. There was no change in the level or migration pattern of complex c. To rule out the possibility of
degradation of nuclear proteins by any contaminating proteases present
in the phosphatase enzyme, we dephosphorylated IL-1 plus IL-6-treated nuclear extract in the presence of two concentrations of phosphatase inhibitors. Inclusion of phosphatase inhibitors restored the formation of complexes a and b (Fig. 9A,
lanes 4 and 5), which indicated that indeed
phosphorylation is required for their formation. Based on the DNA
binding ability, we concluded that at least two types of SAF proteins
are present in synovial cells. One type is sensitive to phosphatase
treatment, whereas the second type can interact with SAA promoter even
as a dephosphorylated form. Interestingly, the DNA binding ability of
the inducible SAF proteins that form complexes a and
b is affected by dephosphorylation. In contrast, dephosphorylation had no effect on the DNA binding activities of the
constitutively present SAF proteins. Noticeably, some additional faster
migrating bands appeared when interaction of complexes a
and b was prevented (Fig. 9A, lanes 2 and 3).
To analyze the effect of phosphorylation on Sp1 DNA binding activity,
the same dephosphorylated nuclear extracts were incubated with
radiolabeled high-affinity and specific Sp1 oligonucleotide and assayed
for DNA-protein interaction. As shown in Fig. 9B, dephosphorylation severely diminished the formation of Sp1-specific complex a in all cytokine-treated nuclear extracts. In a
control experiment, inhibitors of phosphatase prevented reduction of
complex a formation (Fig. 9B, lanes 4 and 5). These results suggested that Sp1 is phosphorylated
in IL-1 plus IL-6-induced cells.
Phosphatase Inhibitor, Okadaic Acid, Potentiates the DNA Binding
Activities of SAF and Sp1--
To further verify the role of
phosphorylation, we treated HIG82 cells with okadaic acid (OA), a
potent protein phosphatase inhibitor. Nuclear extracts prepared from
untreated and OA-treated cells (10 nM final concentration,
for 24 h) were used in DNA binding assays for SAF and Sp1 (Fig.
10, A and B).
Augmentation of DNA binding of both transcription factors by OA
indicated that a phosphorylation event potentiates these activities.
Consistent with this finding, OA was found to enhance transactivation
potential of both SAF (Fig. 10C) and Sp1 (Fig.
10D) in transient transfection assays.
The results presented here focus on understanding the molecular
basis of SAA gene induction in synovial fibroblast cells in response to
combined stimulation by IL-1 and IL-6 cytokines. A synergistic increase
of SAA expression was noted when synovial cells were exposed to these
two cytokines. The novel findings obtained during this study are: (i)
involvement of Sp1 in mediating SAA gene induction; (ii) induction of
Sp1 in synovial cells during cytokine exposure; and (iii) association
of Sp1 with SAF in mediating synergistic increase of SAA expression
during IL-1 plus IL-6 stimulation of the cells.
Expression of SAA in the synovial cells is implicated in the
induction of collagenase enzyme that is associated with the joint tissue destruction in rheumatoid arthritis (6). SAA, being a member of
the type 1 acute-phase protein group, is synergistically activated by
IL-1 plus IL-6. However, synergistic activation of SAA by IL-1 and IL-6
is only reported in liver cells (38, 39). Incidentally, the synovial
fluid of arthritic patients contains, among other molecules, high
levels of both IL-1 and IL-6. In such a milieu, a relatively higher
level of SAA expression can increase local collagenase production (6)
and most likely increases the severity of the disease. We have
demonstrated that, indeed, the combined presence of IL-1 and IL-6 has a
much higher stimulatory effect on SAA gene expression in synovial
fibroblast cells. We also noticed some morphological changes during
cytokine exposure of these cells (Figs. 2 and 3), evidenced by the
ability of cytokine-treated cells to form extensive elongated
protrusions composed of filamentous actin. Associated with the changes
in cellular morphology, we provided evidence of activation and
induction of two transcription factors, SAF and Sp1, in synovial cells
that are involved in altered gene expression such as that of SAA.
Comparable phenomenon has been observed in other cell types. For
example, onset of differentiation in preadipocytes induced by exogenous
hormonal agents, triggers activation of C/EBP- The mechanism of SAA induction was investigated by transfection of
synovial cells with various SAA promoter-CAT constructs followed by
induction with IL-1 and IL-6. The region between An important result of this study is the identification of Sp1 as a
mediator of cytokine response for SAA gene induction. Involvement
of Sp1 is documented by DNA-protein interaction (Fig. 5). Following
cytokine addition, the DNA binding activity of Sp1 was increased and
IL-1 plus IL-6 had the most stimulatory effect (Figs. 5 and 6).
Increased Sp1 DNA binding activity correlated very well with increased
SAA mRNA expression and increased SAA promoter activity. Higher DNA
binding activity of Sp1 following cytokine treatment of HIG82 cells is
partly because of an increase in Sp1 protein level (Fig.
7B). Sp1 protein level is known to be increased during
development (41), cellular differentiation (41), and SV40 infection
(42).
The loss of DNA binding activity of Sp1 following dephosphorylation
(Fig. 9B) and potentiation of this activity by okadaic acid
(Fig. 10B) suggest that phosphorylation of Sp1 is helpful for its DNA binding function as well as for its transactivation potential. The significance of phosphorylation of Sp1 has recently been
demonstrated in several observations where strong circumstantial evidences suggested that phosphorylated Sp1 is an active moiety in
transcription. Phosphorylation of Sp1 at the serine 131 position is
found to be important for supporting Tat-activated transcription from
the HIV-1 promoter. A DNA-dependent protein kinase
augmented by Tat protein mediates phosphorylation of Sp1 at serine 131 (43). Also, phosphorylation of Sp1 was found to be necessary for its binding to the Sp1 binding element present in the
Sp1, whose binding element is present in the regulatory regions
of a wide range of vertebrate genes, is traditionally viewed as a
constitutive transcription factor required only for the maintenance of
housekeeping genes. However, its role as a regulatory factor is
recently getting identified and recognized. Sp1 is shown to regulate
phorbol 12-myristate 13-acetate and okadaic acid-mediated induction of
WAF/CIP1 gene, a cyclin-dependent kinase protein (46), to
reverse Erb-B2 protooncogene-mediated down-regulation of
It is interesting to note that Sp1 alone did not interact with
SAA promoter, although the GGGAGG binding element is considered to be a
moderately well interacted site for Sp1-type GC box DNA-binding proteins. Sp1 interacted with SAA promoter as an SAF/Sp1 heteromer and
also only under cytokine-induced conditions. It may be argued that
because Sp1 binding element is embedded in the SAF binding element, a
functional interference prevented Sp1 from interacting to this site.
Second, it is possible that to interact with SAA promoter, Sp1 either
needs to be dissociated from an inhibitor protein (55), or
post-translationally modified via glycosylation and/or phosphorylation
(56). We believe that for SAF/Sp1 interaction, post-translational
modification of SAF is also a requirement, because under normal
condition, constitutively present Sp1 and SAF did not form any
heteromeric complex with SAA promoter. In a previous study (23) using
an in vitro reconstituted system, potential physiological
importance that can arise during combinatorial regulation of these two
factors was suggested. In this report, we documented an in
vivo biological condition triggered by IL-1 and IL-6 in
synoviocyte cells, which allowed activation of both of these
transcription factors for their association to mediate induction of SAA.
*
This work was supported by United States Public Health
Service Grant DK49205 and funds from the College of Veterinary
Medicine, University of Missouri.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.
The abbreviations used are:
SAA, serum amyloid A; SAS, SAA-activating sequence; SAF, SAS binding factor; CAT, chloramphenicol acetyltransferase; C/EBP, CAAT/enhancer-binding
protein; EMSA, electrophoretic mobility shift assay; OA, okadaic acid; PBS, phosphate-buffered saline.
Activation of Sp1 and Its Functional Co-operation with Serum
Amyloid A-activating Sequence Binding Factor in Synoviocyte Cells
Trigger Synergistic Action of Interleukin-1 and Interleukin-6 in Serum
Amyloid A Gene Expression*
ABSTRACT
Top
Abstract
Introduction
References
254 and 226, which contains binding sites for transcription factors
Sp1 and SAA activating sequence binding factor (SAF). Mutation of these
sequences abolishes SAA promoter response to IL-1 and IL-6. The role of
Sp1 in SAA induction was demonstrated by increased DNA binding
activity, phosphorylation, and increased protein content of Sp1 during
cytokine treatment. Sp1 interacts with the SAA promoter in association with SAF as an SAF·Sp1 heteromeric complex. Furthermore, using a
phosphatase inhibitor, we demonstrated increased transactivation potential of both Sp1 and SAF as a consequence of a phosphorylation event. These results provide first evidence for cytokine-mediated activation of Sp1 in synovial fibroblast cells and its participation in
regulating SAA expression by acting in conjunction with SAF.
INTRODUCTION
Top
Abstract
Introduction
References
B (15, 18-20) in
human, mouse, rat, and rabbit. Involvement of common promoter elements
in these species indicates a signaling and response pathway that
presumably remained conserved during evolution. Although liver is a
major site of SAA expression, extrahepatic tissues are also involved in
expressing this gene. Such a local production of SAA is linked to the
pathogenesis of several diseases. For example, in rheumatoid arthritis,
SAA biosynthesis in the synovial cells of joint tissue is highly
induced (3-6). Also SAA expression in macrophage cells (21-23) and in
aortic smooth muscle cells (24) is likely to play a significant role in
atherosclerosis. Recent studies indicate that SAA is also synthesized
in the brain (25). Although it is expressed in both hepatic and
extrahepatic cells, the expression level of SAA has been found to vary
markedly depending on the cell types (26) and on the nature of
inflammatory agents (27). Part of the difference lies with the
complexity of the SAA gene with the presence of multiple isoforms in
many species (reviewed in Ref. 28). In humans, for example, multiple
SAA isoforms, designated as SAA 1-4 have been reported among which
SAA4 is constitutively expressed, SAA3 is not expressed, and SAA1 and
SAA2 isoforms are expressed with varying degrees of induction in
different tissues. In mouse, five SAA isoforms have been reported.
Mouse SAA1 and SAA2 isoforms are expressed and induced predominantly in
the liver, whereas SAA3 isoform is induced in multiple tissues during
inflammation. Similar to human SAA4, mouse SAA5 is constitutively
expressed, although mouse SAA4, which is analogous to human SAA3, is
not expressed. In rabbit so far three isoforms of SAA have been
reported. Nonhepatic induction of mouse SAA3 has been extensively
studied, which revealed that C/EBP-
and SAA enhancing factor are
major regulators (13). In rabbit, besides C/EBP and NF-B, a novel transcription factor termed as SAF is involved in the cytokine-induced expression of rabbit SAA2 gene (29). In this report, we provide the
first evidence of the involvement of Sp1 in inducing rabbit SAA2 gene
expression in response to IL-1 and IL-6 cytokines in synovial cells.
MATERIALS AND METHODS
(200 units/ml), IL-6 (1000 units/ml), or both for 48 h.
-gal (Promega) as a control
for measuring transfection efficiency, and carrier DNA so that the
total amount of DNA in each transfection remained constant at 16 µg.
Cells were harvested 48 h post-transfection, and CAT activity was
determined from cell extracts as described previously (30). For CAT
assays, extracts were heated at 60 °C for 10 min to inactivate
endogenous acetylase and assayed for -galactosidase activity.
Different effectors used in the transfection assay had no effect on
-galactosidase expression. An equivalent amount of each cell extract
was used in the determination of CAT activity. All values reported have
been corrected for background activity, which was determined from
mock-transfected cells. All transfection experiments were performed at
least three times.
401/+63)CAT, pSAA(314/+63)CAT,
pSAA(280/+63)CAT, pSAA(193/+63)CAT, pSAA(135/+63)CAT and
pSAA(35/+63)CAT were constructed by cloning various portions of the
SAA promoter into the basic promoterless plasmid vector pBLCAT3 (31).
Progressive deletions from the 5'-end of the rabbit SAA gene promoter
fragments were constructed by using restriction endonuclease cleavage
sites present in the SAA gene or using the polymerase chain reaction
method to create suitable fragments of the SAA promoter. Plasmids
pSAA(280/193)CAT, pSAA(254/193)CAT and pSAA(254/226)CAT
were constructed using plasmid vector pBLCAT2 (31). pBLCAT2 contains an
human sarcoma virus thymidine kinase promoter. Background effect of
this vector was normalized by separately transfecting cells with the
plasmid vector in an amount equal to that used for SAA
promoter-containing pBLCAT2 derivatives and grown in the absence and in
the presence of IL-1 and IL-6. These cytokines did not have any effect
on pBLCAT2 vector alone. The mutated pmtSAA(254/226)CAT plasmid was
constructed by ligating a mutated sequence to the plasmid vector
pBLCAT2. The sequence of mutated oligonucleotide was
5'-CAATGAGTCGAGACCGTCGACATCCATGG-3'. Underlined bases represent altered sequences. The rationale for selecting the sites for mutation was based upon fine-structure mapping
of SAF-binding site followed by a systematic base substitution analysis
of this region. Full-length functional Sp1 cDNA and a mutated
frameshift Sp1 cDNA were isolated from pSVSp1F and pSVSp1FX plasmids (32) and subcloned into pCMV4 vector. pCMVSAF (23) contained a
cDNA-encoding SAF cloned into pCMV4.
254/226), 5'-CCCTTCCTCTCCACCCACAGCCCCCATGG-3'; mutant SAA
(254/226),
5'-CAATGAGTCGAGACCGTCGACATCCATGG-3'.
Underlined bases represent altered sequences determined by base
substitution analysis of SAF-binding region.
-32P]dCTP. In some binding
assays, competitor oligonucleotides were included in the reaction
mixture. For antibody interaction studies, antibodies were added to the
reaction mixture during a preincubation period of 30 min on ice.
Anti-Sp1 antibody was obtained from Santa Cruz Biotechnology and
anti-SAF antibody was prepared as described (23).
RESULTS
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Fig. 1.
Northern blot analysis of SAA mRNA in
synoviocyte cells. HIG82 cells were grown in control medium
(lane 1) or in the presence of IL-1 (lane 2),
IL-6 (lane 3), or IL-1 plus Il-6 (lane 4) for
48 h. Total RNA (50 µg) prepared from these cells was
fractionated in a formaldehyde-agarose gel, transferred onto nylon
membrane, and hybridized to 32P-labeled SAA cDNA probe.
The same membrane was reprobed with 32P-labeled -actin
cDNA for qualitative and quantitative evaluation of the RNA
samples.
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Fig. 2.
Scanning electron microscopy of synoviocyte
cells following cytokine treatment. HIG82 cells were grown in
culture medium described under "Materials and Methods" in the
absence (panel A), or presence of IL-1 (panel B),
IL-6 (panel C), or IL-1 plus IL-6 (panel D) for
48 h. Cells were processed for scanning electron microscopy as
described under "Materials and Methods." The white bars
in each panel represent 10 nm.
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Fig. 3.
Fluorescence staining of actin filaments of
synoviocyte cells following cytokines treatment. HIG82 cells were
grown in culture medium in the absence (panel A) or in the
presence of IL-1 (panel B), IL-6 (panel C), or
IL-1 plus IL-6 (panel D) for 48 h. Cells on coverslips
were fixed, and actin filaments were stained with rhodamine-conjugated
phalloidin reagent as described under "Materials and Methods."
Magnification × 1,000.
254 to 226 Is Essential for Optimal
IL-1 Plus IL-6 Response--
Cytokine-induced cellular changes
significantly altered SAA transcription causing accumulation of this
transcript. In an effort to identify the cytokine-response element of
SAA promoter, a series of 5'-deletion constructs were prepared (Fig.
4A). These constructs were
transiently transfected in rabbit synovial cells, followed by the
addition of IL-1 plus IL-6. The results from the transfection assay
indicated that deletion of sequences from 314 to 280 has no effect
on cytokine-mediated CAT reporter gene induction. In contrast,
significant reduction in CAT gene induction was noticed when the region
between 280 and 193 was deleted. Further removal of upstream
sequences abolished any measurable effect of cytokines. The results of
functional promoter analysis suggested that sequences between 280 and
193 were most important for IL-1 plus IL-6 mediated inducible
expression of the SAA gene in rabbit synovial cells. Further analysis
of the cytokine-inducible region was done by preparing some additional
deletion constructs. As shown in Fig. 4B, a reporter gene
containing sequences between 254 and 226 retained full
responsiveness to IL-1 plus IL-6 stimulation. Mutation in this region
resulted in a loss of responsiveness to cytokine stimulation. These
results supported the involvement of sequences between 254 and 226
in the cytokine activation of the SAA gene in synovial cells.
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Fig. 4.
Transient transfection assay for SAA
promoter function. HIG82 cells were transfected with CAT reporter
plasmids containing various regions of the SAA promoter. Details of the
CAT constructs, transfection, and CAT assay procedure are described
under "Materials and Methods." These results represent an average
of three independent experiments.
254/226) and various cytokine-treated synovial cell nuclear
extracts. Control, untreated nuclear extract formed three major
complexes, termed as c, d, and e (Fig.
5A, lane 1). The
same protein amount of nuclear extracts from IL-1, IL-6, and IL-1 plus
IL-6-induced cells formed five complexes, a, b,
c, d, and e (Fig. 5A,
lanes 2-4). These results showed that cytokines can induce
some nuclear proteins that form inducible DNA-protein complexes like
a and b with SAA promoter. Noticeably, the levels
of a and b were highest in cells treated with
IL-1 plus IL-6, whereas the levels of constitutively present complexes
c, d, and e remained the same. The
specificity of these DNA-protein complexes was determined by
competition with a molar excess of unlabeled homologous probe (Fig.
5B, lanes 2, 8, 14, and
20) and mutated probe (Fig. 5B, lanes
3, 9, 15, and 21). Because
complex c was not inhibited by the homologous probe, it
indicated that this complex was formed by either a nonspecific or a
very low affinity interaction of a protein with SAA promoter. Polyclonal antibodies were used to determine the identity of these nuclear proteins. This region of the SAA promoter contains an element
where the transcription factor SAF binds. Also a potential Sp1 binding
domain is located in this region of the SAA promoter. Antisera to these
two transcription factors were used. A complete inhibition of complexes
b, d, and e and part inhibition of
complex a were obtained using antibody directed against SAF
protein (Fig. 5B, lanes 4, 10,
16, and 22). At a higher concentration of the anti-SAF antibody, complex a was also completely inhibited
(Fig. 5C). Anti-Sp1 antibody partially supershifted only
complex a (Fig. 5B, lanes 5,
11, 17, and 23) and at a higher
concentration, completely supershifted complex a (Fig.
5C). Complete inhibition or supershifting of complex
a by anti-SAF or anti-Sp1 antibodies indicate that complex
a is formed by a combined interaction of SAF and Sp1 with the SAA probe and not because of a comigration of two independent complexes formed by SAF or Sp1. Also, in a previous study using affinity purified Sp1 protein (23), we noticed that Sp1 itself does not
bind to the SAA promoter. Other three complexes, b,
d, and e, are formed by SAF-like proteins. Taken
together, these results suggested that activation of SAA promoter after
cytokine stimulation is closely related to the induction of both SAF
and Sp1 factors, in which the combination of IL-1 and IL-6 had the most
stimulatory effect. Noticeably, no DNA-protein complex was detected
that was formed by Sp1 alone, indicating that Sp1 cannot directly bind
to the SAA promoter.
View larger version (45K):
[in a new window]
Fig. 5.
Electrophoretic mobility shift assay for the
detection of DNA binding activity in cytokine-treated synoviocyte
cells. A, 32P-labeled SAA element
( 254/226) was incubated with 10 µg of nuclear extracts prepared
from uninduced (lane 1), IL-1 treated (lane 2),
IL-6 treated (lane 3), and IL-1 plus Il-6 treated
(lane 4) HIG82 cells. DNA-protein complexes were resolved in
a 6% native polyacrylamide gel. Detectable complexes are designated as
a through e. B, the abovementioned DNA
probe and four different nuclear extract preparations, described in
panel A, were incubated in the presence of 50-fold molar
excess of either wild-type (wt) or mutant (mt)
competitor oligonucleotides containing SAA sequences as described under
"Materials and Methods." In some reactions, nuclear extracts were
preincubated with anti-SAF antibody (1 µl of a 10-fold diluted
stock), anti-Sp1 antibody (1 µl of the stock), or nonspecific
(NS) serum. The arrow indicates the migration
position of supershifted complexes in lanes 11,
17, and 23. C, 32P-labeled
SAA element (254/226) was incubated with 10 µg of nuclear extract
prepared from IL-1 plus IL-6-treated HIG82 cells (lanes
1-3). In lane 2, nuclear extract was preincubated with
anti-SAF antibody (3 µl of a 10-fold diluted stock), and in
lane 3, nuclear extract was preincubated with anti-Sp1
antibody (3 µl of the stock) before the addition of the radioactive
probe.
254 to 226) is interacted by both SAF and Sp1 proteins,
we chose a high-affinity and highly specific Sp1 DNA binding element as
a probe for measuring Sp1 activity to avoid any functional interference
from SAF proteins present in the nuclear extracts. Moreover, a previous
study (23) indicated that Sp1 does not bind by itself to the SAA
promoter. With control nuclear extract, three faint DNA-protein
complexes, a, b, and c, in which complex a is major, were detected (lane 1).
Cytokine treatment increased the level of complex a with the
highest activity seen in IL-1 plus IL-6-treated cells (lanes
2-4). The levels of the other two minor complexes, b
and c, were only slightly increased during cytokine
treatment. The formation of these complexes was completely abolished by
the presence of an excess of unlabeled homologous Sp1 oligonucleotide
(Fig. 6B, lanes 2, 7, and
12) but not by the presence of unlabeled mutated Sp1
oligonucleotide (Fig. 6B, lanes 3, 8,
and 13). Anti-Sp1 antibody supershifted only complex a (Fig. 6B, lanes 4, 9, and
14), whereas nonspecific antibody had no effect on these
complexes (Fig. 6B, lanes 5, 10, and
15). These results showed that complex a is
formed by Sp1, and complexes b and c may be
formed by any of the other Sp1-like proteins such as Sp2, Sp3, and Sp4
(37). Thus, Sp1 is highly induced in synoviocyte cells particularly in
response to IL-1, and a synergistic induction is achieved by the
combined action of IL-1 and IL-6.
View larger version (71K):
[in a new window]
Fig. 6.
Cytokine-mediated induction of Sp1 activity
in synoviocyte cells. A, nuclear extracts (10 µg of
protein) prepared from untreated control (lane 1), IL-1
treated (lane 2), IL-6 treated (lane 3), or IL-1
plus Il-6 treated (lane 4) cells were incubated with
32P-labeled high affinity Sp1-binding oligonucleotide. The
resultant DNA-protein complexes were resolved in a 6% native
polyacrylamide gel. Three detectable complexes are designated as
a, b, and c. B, The
DNA-protein complexes formed by nuclear extracts from IL-1 treated
(lanes 1-5), IL-6 treated (lanes 6-10), and
IL-1 plus IL-6 treated (lanes 11-15) HIG82 cells were
further characterized by using competitor oligonucleotides and anti-Sp1
antibody. 50-fold molar excess of either wild-type (wt) or
mutant (mt) Sp1-binding oligonucleotides were added as
competitors. Anti-Sp1 antibody was added at a concentration of 1 µl
of the stock. As a control in antibody inhibition/supershift assay,
nonspecific serum was included in some assays. The arrows
indicate the migration position of the supershifted complex in
lanes 4, 9, and 14.
View larger version (58K):
[in a new window]
Fig. 7.
Western immunoblot analysis for SAF and Sp1
in synoviocyte cells. Nuclear extracts (30 µg of protein)
prepared from untreated (lane 1), IL-1 plus IL-6 treated
(lane 2), IL-6 treated (lane 3) and IL-1 treated
(lane 4) HIG82 cells were fractionated in a 5%/12%
SDS-polyacrylamide gel electrophoresis, electroblotted onto a
nitrocellulose membrane and analyzed for the presence of SAF
(panel A) and Sp1 (panel B) proteins using
specific antisera. Arrows indicate location of SAF- and
Sp1-specific bands. C, Coomassie blue staining of proteins
in the same amounts of nuclear extracts as those in panels A
and B. Molecular masses of standard protein markers are
indicated in kilodalton. Similar patterns of proteins in lanes
1 to 4 indicate that they are of equal quality and suitable for
comparative studies.
View larger version (55K):
[in a new window]
Fig. 8.
Northern blot analysis of SAF and Sp1
mRNAs in synoviocyte cells. Total RNA (50 µg) prepared from
HIG82 cells grown in control medium (lane 1) or in the
presence of IL-1 (lane 2), IL-6 (lane 3), or IL-1
plus IL-6 (lane 4) for 48 h were fractionated as
described in Fig. 1 and under "Materials and Methods." RNA was
hybridized to 32P-labeled SAF cDNA probe. The same
membrane was reprobed with 32P-labeled Sp1 cDNA and
-actin cDNA probes, respectively.
View larger version (75K):
[in a new window]
Fig. 9.
Evidence that phosphorylation is required for
SAF-Sp1 heteromeric complex formation. Nuclear extract (10 µg of
protein) from IL-1 plus IL-6 treated HIG82 cells was incubated for 30 min with increasing amounts of calf intestinal alkaline phosphatase (2 units or 4 units per reaction). The incubation mixture in lane
4 also contained a mixture of phosphatase inhibitors (NaF, 50 mM; okadaic acid, 5 µM; and sodium
orthovanadate, 1 mM) during phosphatase treatment. The
phosphatase inhibitors concentration in lane 5 is half of
that in lane 4. These phosphatase-treated nuclear extracts
were subsequently used in DNA binding assays with either SAF binding
probe (panel A) or Sp1 binding probe (panel
B).
View larger version (39K):
[in a new window]
Fig. 10.
Phosphatase inhibitor, okadaic acid,
potentiates DNA binding and transactivation ability of SAF and
Sp1. Electrophoretic mobility shift assay was performed using SAF
binding probe (panel A) and Sp1 binding probe (panel
B). Nuclear extracts, prepared from HIG82 cells grown in the
absence (lane 1) or in the presence of 10 nM of
okadaic acid for 24 h (lane 2) were used in the binding
assay performed as described under "Materials and Methods." For
transient transfection, HIG82 cells were co-transfected with
pSAA( 254/226)CAT reporter plasmid (10 µg of DNA) and 2 µg of
DNA of either pCMV SAF (panel C) or pCMV Sp1 (panel
D). Following overnight incubation with the plasmids, the cells
were washed and grown for 48 h in fresh medium in the presence or
absence of 10 nM OA. Cell extracts were prepared and CAT
activity was measured as described under "Materials and Methods."
Results represent an average of three separate experiments.
DISCUSSION
(40). C/EBP- in
turn up-regulates expression of 422 adipose P2 and glucose transporter
4 genes.
254 and 226 was
found to be most responsive to IL-1 plus IL-6 stimulation. Another
region between 193 and 135 was seen to contain some cytokine
responsiveness (Fig. 4). Two C/EBP binding sites are located in this
region that earlier were found to be highly active in regulating SAA
gene induction in cultured liver cells and also in liver tissues
(12-18). No effect of NF-B, a known regulator of SAA in liver cells
(15, 18-20), was noticed. For further characterization of the promoter
element between 254 and 226, DNA binding assays were conducted that
detected the formation of specific inducible DNA-protein complexes.
Antibody-mediated ablation/supershift of these complexes suggested the
involvement of Sp1 and SAF transcription factors in the formation of
inducible complexes, which appeared following cytokine stimulation of
the cells (Figs. 5 and 6). It should be noted that some nuclear
factors, which were constitutively present in the cells, belong to the
SAF family of proteins. However, constitutively present SAF isoforms
are distinctly different in terms of their DNA binding abilities from
the cytokine-activated SAF isoforms. Cytokine-activated SAF proteins
are highly sensitive to dephosphorylation (Fig. 9A), which
inhibits their interacting ability to the SAA promoter. These results
demonstrated that cytokine-inducible SAF isoforms may require
phosphorylation to interact with the SAA promoter.
2-integrin core promoter (44). Phosphorylation of Sp1
induced by okadaic acid is shown to facilitate the formation of basal
transcription complex and induce expression of HIV LTR in T lymphocytes
(45). Similar induction of Sp1 activity by okadaic acid was also
observed in U937 leukemic cells (46). Our finding of the potentiation of Sp1 activity by OA along with its increased DNA binding activity indicates that phosphorylation most likely plays a major role in the
augmentation of Sp1 activity for induction of SAA gene in synovial
fibroblast cells during inflammatory conditions.
2-integrin gene (47) and to play a critical role in
cytokine-stimulated expression of the vascular cell adhesion molecule
gene (48). In endometrial epithelium, hormone-induced recruitment of
Sp1 mediates estrogen-responsive activation of the rabbit uteroglobin gene (49). Also, concerted action of Sp1 and sterol response element-binding protein (50), Sp1 and GATA (51), Sp1 and C/EBP (52),
Sp1 and NF-B (53), and Sp1 and Ap1 (54) in promoting respective gene
expression has been demonstrated. This study further extends the role
of Sp1.
FOOTNOTES
To whom correspondence should be addressed: Dept. of Veterinary
Pathobiology, University of Missouri, Columbia, MO 65211. Tel.:
573-882-4461; Fax: 573-884-5414; E-mail: rayb{at}missouri.edu.
REFERENCES
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Abstract
Introduction
References
Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.
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