(Received for publication, November 2, 1995)
From the
The serine proteinase inhibitor (SPI-3) gene expression is
transcriptionally regulated by interleukin (IL)-6 and glucocorticoids
in hepatic cells. To identify the transcription factors involved in
regulation of the SPI-3 promoter-chloramphenicol acetyltransferase
constructs we overexpressed Signal Transducer and Activator of
Transcription (STAT) proteins (STAT1, STAT3, STAT5B, and STAT6) and
CAAT enhancer-binding protein . Specific signaling pathways were
activated by cointroduced receptors for growth hormone, IL-3, IL-4, or
chimeric receptors containing the cytoplasmic domain of gp130. STAT3
and STAT5B induced transcription via the SPI-3 promoter. The STAT5B
response was substantially enhanced by truncation of the 5`-flanking
region from -1021 to -148. The responsiveness to STAT3 and
STAT5B required the STAT binding element at -132 to -124.
This element was sufficient to confer regulation onto a heterologous
promoter gene construct. In contrast, overexpression of CAAT
enhancer-binding protein
reduced the transcriptional activity of
the SPI-3 promoter, presumably by interfering with STAT protein binding
to the promoter element. The SPI-3 gene is the first example of an
acute phase gene that is responsive to both STAT3 and STAT5B.
At least three members of serine proteinase inhibitor gene
family (SPI) ()are synthesized in rat liver (Hill and
Hastie, 1987; Pages et al., 1990). These genes code for highly
homologous proteins containing different reactive centers resulting in
specific inhibitory spectra. In healthy rats only the SPI-1 and SPI-2
are synthesized, and their expression is controlled by growth hormone
(Le Cam et al., 1987; Paquereau et al., 1992). The
growth hormone-responsive element within promoter regions of both genes
has been identified; however, the transactivating proteins have yet to
be defined (Yoon et al., 1990; Thomas et al., 1995).
The SPI-3 mRNA is barely detectable in healthy rats and is not affected
by growth hormone (Paquereau et al., 1992).
The pattern of expression of the SPI genes is profoundly modified during an inflammatory response, with transcription of the SPI-3 gene being greatly stimulated, whereas that of the SPI-1 and SPI-2 genes is suppressed (Hill and Hastie, 1987; Le Cam and Le Cam, 1987). IL-6 has been shown to be the major activator of SPI-3 gene expression in primary hepatocytes and rat hepatoma H-35 cells, and its activity was further enhanced by dexamethasone (Kordula et al., 1994; Kordula and Travis, 1996). The induction of SPI-3 gene expression by IL-6 occurs at the level of transcription. The SPI-3 promoter containing the region from -148 to -3 is sufficient for mediating the regulation by IL-6 and dexamethasone (Kordula and Travis, 1996). Two functional elements located at -132 to -124 and at -58 to -66 are necessary for maximal IL-6 responsiveness. The distal element serves as a binding site for STAT proteins (SBE), whereas the proximal element is recognized by C/EBP isoforms (Kordula and Travis, 1995, 1996). However, binding of C/EBP to the distal element was also detected (Rossi et al., 1992; Kordula and Travis, 1996). The relevance of the SBE element was demonstrated by mutations and in combination with a heterologous promoter. The C/EBP binding site contributes to the magnitude of IL-6 regulation because mutation of this element decreased the response to IL-6.
To define which one of the STAT proteins known to be activated
by IL-6 and inflammation in the liver cells is acting on the SPI-3
gene, we reconstituted the regulation pathways in hepatoma cells. The
cells were transiently transfected with vectors expressing C/EBP,
STAT proteins (STAT1
, STAT3, STAT5B and STAT6), and different
hematopoietin receptors for the specific activation of the
overexpressed STAT isoforms. The data indicate a novel response pattern
for the SPI-3 gene consisting of an inducing action of both STAT3 and
STAT5B.
The transcription of the -1020 and -148 SPI-3
promoter-CAT constructs was not affected by IL-6 or dexamethasone
alone; however, both factors together led to a synergistic activation (Fig. 1). In contrast, the SPI-3 promoter construct containing
the mutated C/EBP binding site at -58 to -66 was responsive
to IL-6 alone, and this response was further enhanced by dexamethasone.
The transcription of the SPI-1 promoter-CAT construct was induced by
IL-6 together with dexamethasone, whereas these factors were separately
ineffective. Overexpression of C/EBP inhibited transcriptional
regulation of the SPI-3 promoter-CAT constructs. The activity of the
SPI-1 promoter-CAT construct was, however, enhanced (Fig. 1).
Both SPI-promoter CAT constructs were not stimulated by IL-1 treatment;
in fact their expression was reduced when IL-1 was combined with IL-6.
These results suggest that the IL-6 stimulation of SPI-3 gene
expression is not likely be mediated by C/EBP
and thus, this gene
falls into the category of the type II acute phase protein genes that
are regulated by mechanisms independent of IL-1 and C/EBP
.
Figure 1:
Effect of C/EBP on the SPI-3 and
SPI-1 promoter-CAT constructs expression. H-35 cells were transfected
with the pSPI-3(-1020)CAT, pSPI-3(-148)CAT,
pSPI-3(-148)mutC/EBP
CAT, and an expression vector
pMSV-C/EBP
as indicated. Six subcultures of each transfection were
treated with medium alone or medium containing Dex, IL-6, and/or IL-1.
The CAT activities were quantified and expressed relative to the
control in each experimental series (fold induction indicated by numbers above the autoradiograms).
The
observed inhibition of the SPI-3 gene expression by C/EBP is
interpreted to represent binding of C/EBP
to the SBE at -132
to -124, thereby preventing interaction of STAT with the adjacent
SBE. This site contains only two mismatches to the C/EBP consensus, and
binding of a C/EBP proteins to this element was demonstrated by DNA
footprinting (Rossi et al., 1992; Le Cam et al.,
1994) and gel shift analysis (Kordula and Travis, 1996). The second
C/EBP binding site at -58 to -66, which represents a C/EBP
consensus sequence and is also present at an identical site in the
SPI-1 gene promoter, appears to be insufficient to mediate suppression
by overexpressed C/EBP
. The promoter construct containing the
mutated C/EBP binding site at -58 to -66 (mut C in Fig. 1) was responsive to IL-6 and still suppressed by
C/EBP
. The opposite effect of C/EBP
on the SPI-1 promoter-CAT
construct likely results from the presence of the additional C/EBP site
at -107 to -99 identified by Le Cam et al.(1994).
We conclude from these data that STATs rather than C/EBPs are critical
for IL-6 regulation of the SPI-3 gene.
Figure 2: Effect of box 3 motif of gp130 on the SPI-3 gene expression. The chimeric receptor forms G-CSFR-gp130(133) and G-CSFR-gp130(133 m3) were cotransfected with pSPI-3(-1020)CAT or pSPI-3(-148)CAT into H-35 cells. Subcultures were treated with G-CSF and/or IL-6 for 24 h in the presence of Dex. The CAT activities were quantified and expressed relative to the control in each experimental series (fold induction indicated by numbers above the bars).
Figure 3:
Comparison of the effects of STAT3 and
STAT5B on the expression of the SPI-3 promoter-CAT constructs. HepG2
cells were cotransfected with pSPI-3(-1020)CAT or
pSPI-3(-148)CAT, IL-3R, IL-3R
, and expression vector
pSV-STAT3 or pSV-STAT5B. Cultures were treated with IL-3 (3)
or IL-6 (6) in the presence of Dex. The CAT activities were
normalized to the internal transfection marker pIE-MUP and expressed
relative to the control culture (fold induction indicated by numbers above the autoradiogram).
Figure 4:
Effect of STAT proteins on the expression
of the SPI-3 promoter-CAT constructs. HepG2 cells were cotransfected
with pSPI-3(-1020)CAT, pSPI-3(-148)CAT, IL-2R,
IL-4R
, and expression vectors pSV-STAT1, pSV-STAT3, pSV-STAT5B, or
pDC-STAT6 as indicated. Cultures were treated with IL-4 (4) or
IL-6 (6) in the presence of Dex. The CAT activities were
normalized to the internal transfection marker pIE-MUP and expressed
relative to the control culture (fold induction indicated by numbers above the bars).
Figure 5: Effect of GHR-mediated activation of overexpressed STAT5B on the SPI-3 promoter-CAT construct expression. H-35 cells (A) or HepG2 cells (B) were cotransfected with pSPI-3(-1020)CAT, pSPI-3(-148)CAT, or pSPI-3(-148)mutStatCAT, GHR, and STAT5B as indicated. Cells were treated with IL-6 or GH in the presence of Dex for 24 h. The CAT activities were normalized to the internal transfection marker pIE-MUP and expressed relative to the control culture (fold induction indicated by numbers above the bars).
Figure 6: Analysis of the effect of STAT3 and STAT5B on the expression of p4xStatCAT. H-35 cells (A) or HepG2 cells (B and C) were cotransfected with p4xStatCAT and vectors expressing GHR or IL-3R and STAT5B or STAT3. Cells were treated with IL-6, GH, or IL-3 in the presence of Dex for 24 h. The CAT activities were normalized to the internal transfection marker pIE-MUP and expressed relative to the control culture (numbers above the bars).
Figure 7: Analysis of the -1020 to -155 fragment of the SPI-3 gene. HepG2 cells were transfected with p(-1020-155)tkCAT, pINV(-1020-155)tkCAT, p4xStattkCAT, or p(-1020-155)4xStattk CAT and treated with IL-6 in the presence of Dex for 24 h. The CAT activities were normalized to the internal transfection marker pIE-MUP and expressed relative to the control culture (fold induction indicated by numbers above the bars). Light bars, control; dark bars, IL-6.
The control of expression of the SPI gene family in liver by GH and inflammatory cytokines (i.e. IL-6) represents a striking example of fundamentally different regulatory effect for closely related genes. Although we could reproduce a liver-like regulation of SPI-3 gene by IL-6 and glucocorticoids in tissue culture, the same experimental tools proved not to be optimal to define the molecular basis for the differential effect of GH on the expression of the SPI-3 and SPI-1 genes. As already suggested by the studies of Waxman et al.(1995), hepatic gene regulation by GH may require consideration of frequencies and magnitude of signaling, processes that have not yet been approached in reconstituted systems as applied here.
The main focus of our studies was on the induction of SPI genes by IL-6. We analyzed in detail the regulation of the SPI-3 gene because the pattern of expression of the cloned promoter in transfected hepatoma cells was qualitatively similar to that of the endogenous SPI-3 gene. Previous results (Kordula and Travis, 1996) suggested that STAT isoforms, C/EBP isoforms, and GR may control the SPI-3 gene expression. We did not address the specific role of GR in the induction of the SPI-3 gene expression in this study. However, glucocorticoids are necessary for the maximal expression of the SPI-3 gene in H-35 cells (Fig. 1; Kordula and Travis(1996)). Moreover, the enhancing effect could be demonstrated by overexpression GR in HepG2 cells that resulted in a 30-fold higher expression of the SPI-3 promoter-CAT construct (data not shown). Although the results suggest a direct action of GR via a GRE within the SPI-3 promoter, the mode of action still needs to be proven.
The mutational analysis of the SPI-3 promoter and measurement of function in transfected hepatoma cells implied participation of STAT isoforms (Kordula and Travis, 1996). Both STAT3 and STAT5B are expressed in hepatocytes and hepatoma cells. However, the level of STAT5 protein, detectable by Western blot analysis and EMSA, is severalfold lower than that of STAT3 (Ripperger et al., 1995; Lai et al., 1995b and data not shown). The precise contribution of the individual endogenous STAT isoforms to the stimulation by the IL-6R could not experimentally be determined. By using the alternative approach of ectopically expressed hematopoietin receptors and STATs, the roles of both STAT3 and STAT5B as inducers were demonstrated. The responsiveness to STAT3 and STAT5B was attributed to the STAT element of the SPI-3 gene at -132 to -124 (Fig. 6). This element was recognized by STAT3 and STAT1 (Kordula and Travis, 1996; Kordula and Travis, 1995), but the binding of STAT5B could not be detected (data not shown). This observation suggests that the binding of STAT factors observed in the in vitro experiments may not always correlate with the activation of transcription via STAT elements in cells. Alternatively, the STAT5B-specific induction is achieved by a promoter sequence that is larger than that applied to DNA binding assay. A striking finding is the influence of surrounding promoter sequences on STAT action. The sensitivity to STAT5B is reduced in the presence of a sequence 5` to the STAT site. The (-1020 to +3) fragment from the SPI-3 promoter is strongly STAT3 responsive and only weak to STAT5B. The 5` fragment (-1020 to -155) neither contains any detectable functional STAT3 binding site nor confers responsiveness to IL-6 onto tk promoter (Fig. 7). The deletion of this fragment has been shown to result in the responsiveness of the truncated promoter to GH (Paquereau et al., 1992). Our data are consistent with this observation since a promoter truncated to -148 is significantly stimulated by STAT5B, which is known to be activated by GHR. However, identity of a suppressive element for response to STAT5B of the -1020 promoter is not yet known. The modulator element is likely located between position -304 and -148 because the response to STAT3 and STAT5B of the 304-base pair-long SPI-3 promoter was similar to that of the 1020-base pair-long promoter (data not shown).
STAT3 has been proposed to be a key signaling molecule controlling
expression of the acute phase genes activated by IL-6 (including the
SPI-3 gene) (Wegenka et al., 1993; Akira et al.,
1995; Kordula and Travis, 1996). Our new data show that STAT5B
contributes to the regulation of the SPI-3 gene. Thus, the SPI-3 gene
appears to be the first example of a liver gene that is a target of the
two separate signaling pathways generated by gp130 (Lai et
al., 1995a, Lai et al., 1995b). The box 3
motif-independent pathway, which is associated with the activation of
STAT5B, has also been detected with other hematopoietin receptors
including GHR (Morella et al., 1995b). Because of its
sensitivity to STAT3 and STAT5B, the SPI-3 gene differs from other
acute phase genes that have been studied for STAT isoforms specificity.
The genes coding rat -acid glycoprotein, rat and human
haptoglobin, rat hemopexin, rat
fibrinogen, and human C reactive
protein are strictly STAT3 responsive with minor to nondetectable
activation by STAT5B. (
)
The inhibition of the SPI-3
transcription by overexpressed C/EBP suggests that the C/EBP
isoforms might significantly modulate regulation by STATs. Both STAT3
and C/EBP
are activated during inflammation in liver and by IL-6
in hepatoma cells and bind to sequence similar gene elements (Baumann et al., 1992; Alam et al., 1992; Wegenka et
al., 1993). However, the two factors are not regulated with
similar kinetics. STAT3 is rapidly activated in response to
inflammatory factors, whereas activation of C/EBP is delayed (Baumann et al., 1992). This difference in activation may represent a
mechanism explaining the transition from gene induction to repression,
in other words a switch from STAT-mediated activation to C/EBP-mediated
inhibition. This transition would equal the course of the SPI-3 gene
regulation following inflammation or IL-6 treatment (Hill and Hastie,
1987; Kordula and Travis, 1996). In this report the regulation of the
SPI-3 gene transcription emerges as one of the best models to elucidate
the processes governing activation as well as deactivation of an acute
phase gene in liver cells.