(Received for publication, August 22, 1995)
From the
Proteins binding at the interleukin-6 response element of the
rat macroglobulin gene were purified by a combination
of chromatographic procedures including binding site-specific
DNA-affinity chromatography as the principal step. Three polypeptides
of 92, 91, and 86 kDa were enriched approximately 6,300-fold from
nuclei of rat livers excised 12 h after the induction of an
experimental acute phase response. Amino acid sequence analysis
identified the 86- and 91-kDa species as two forms of the transcription
factor Stat3 and the 92-kDa species as the factor Stat5b. This
identification was confirmed by gel mobility shift-supershift
experiments using specific antisera for Stat3 and Stat5. Unexpectedly,
activated Stat5 was also detected in the nuclei of untreated control
rats. cDNA clones representing Stat3 and two isoforms of Stat5b were
isolated from a cDNA library prepared with mRNA from rat livers excised
at the peak of an experimental acute phase response. Full-length
Stat5b, predicted from cDNA, consisted of 786 amino acids, while the
variant Stat5b
40C lacked 41 amino acids at the COOH terminus. The
amino acid sequence of rat Stat5b showed 26.7% overall identity with
rat Stat3, 87.3% with sheep Stat5a, 92.5% with murine Stat5a, and 98.7%
with murine Stat5b.
Hepatic acute phase genes have been divided into two classes
according to the cytokines that are their main inducers. Type 1 genes
are activated by interleukin-1 (IL-1), ()interleukin-6
(IL-6), and glucocorticoids; type 2 genes by IL-6 and glucocorticoids,
but not by IL-1. Other IL-6-like cytokines such as leukemia inhibitory
factor, IL-11, and oncostatin M have similar effects as IL-6 on the
induction of these genes in cultured hepatic
cells(1, 2, 3) . Representative type 1 genes
are the C-reactive protein, the complement C3, and the serum amyloid A
genes; representative type 2 genes are the rat fibrinogen, thiostatin
and
macroglobulin (
M)
genes(1, 2, 3) .
Transcription factors
from the C/EBP and NFB-families play a major role in the basal and
cytokine-induced expression of type 1 genes (4, 5, 6) , and members of the family of
signal transducers and activators of transcription (Stat) are important
in the cytokine-induced expression of type 2 genes. Until now, only the
factors Stat1 and Stat3 had been implicated in the IL-6-mediated
induction of target genes (7, 8, 9, 10, 11, 12, 13) .
Stat factors were first discovered as transcription factors that mediate the induction of responsive genes by interferons. Subsequently they were found to be involved in the induction of a broad array of other target genes by various peptide hormones, including growth- and differentiation-promoting hormones and cytokines(14, 15, 16, 17) . The name Stat factors (signal transducers and activators of transcription) points to a characteristic dual function: the transport of the hormonal signal from the cell-surface receptor to the nucleus, and the transcriptional induction of a specific set of target genes. In the absence of an activating signal, Stat factors are present in a functionally latent, monomeric form in the cytoplasm. After ligand binding to a cell-surface receptor, the factors are activated by specific tyrosine phosphorylation through receptor-associated kinases from the JAK/TYK family(14, 15, 16, 17) . They dimerize and translocate to the nucleus, where they bind specific hormone response elements (REs) in the control regions of their target genes, and thus induce the transcription of these genes. Development of the full transcription-inducing potential of Stat factors was reported to require additional serine/threonine phosphorylation(18, 19) .
The factor Stat3
dimerizes and binds at the IL-6 response element (IL-6 RE) of the rat
M gene in response of hepatic cells to IL-6 or
IL-6-like cytokines(7, 12, 13) . In rats and
mice, Stat3 was present in liver nuclei in an active DNA-binding state
as early as 15-60 min after intraveneous injections of IL-6 or
lipopolysaccharides(7, 12, 13) . It was
activated equally fast in cultured hepatoma cells by treatment with
IL-6 or IL-6-like cytokines (7, 20) . Stat3 was
purified from mouse livers 15 min after intraveneous injection of IL-6,
and the resulting sequence information led to the cloning of murine
Stat3 cDNA(12) .
In other well studied animal models of the
acute phase response, which avoid intraveneous injection of
proinflammatory agents and thus more closely reflect naturally
occurring acute phase responses, the response developed much more
slowly. When turpentine or complete Freund's adjuvant (CFA) were
injected intramuscularly or intraperitoneally, then the transcription
rates of the rat M gene reached maximum values in
vivo at 12-15 h,
M mRNA concentrations at
18 h, and
M plasma protein concentrations at
24-48 h after the
injection(21, 22, 23, 24) . Under
these conditions, the response of the
M gene was
characteristically slower than that of other type 2 acute phase genes,
such as thiostatin and
-fibrinogen(22) . Similarly, the
M gene also behaved as a characteristically slow
responding gene in cell culture experiments using rat and human
hepatoma-derived cell lines (25) . Therefore it had been
suspected that a particular principle may be responsible for the slow
response of the
M gene, which may not be applicable
for the faster responding type 2 acute phase genes.
Characteristic
early- and late-appearing protein-DNA complexes were assembled between
the IL-6 RE and nuclear proteins from hepatoma cells after exposure to
IL-6 or IL-6-like cytokines(25, 26) . The
early-appearing complexes contained tyrosine-phosphorylated
Stat3(7, 12, 13) , and their appearance was
not prevented by treatment with inhibitors of protein synthesis
(cycloheximide; (7) and (20) ). However, assembly of
the later-appearing complexes was inhibited by cycloheximide and thus
was dependent on intermediate protein synthesis (25) . The
components of the late-appearing complexes that require intermediate
protein synthesis have not yet been identified. We hypothesized that
the late-appearing complexes may differ in their composition or their
posttranslational modifications from the early-appearing complexes,
which may provide an explanation for the slow kinetics of induction of
the M gene.
To test this hypothesis, nuclear
proteins specifically binding at the IL-6 RE of the M
gene, which were present in rat livers 12 h after intraperitoneal
injection of CFA, were studied. At this time the transcription rate of
the
M gene was known to reach maximum values in
vivo(21) . As anticipated, activated, DNA-binding Stat3
was found at this late time, but in addition, DNA-binding Stat5b was
also discovered. This finding raised the new hypothesis that Stat5b may
also act as a transcription factor in mediating IL-6 effects for some
of the late-responding type 2 acute phase genes, including the rat
M gene.
Figure 1: Kinetics of induction of specific protein-DNA complexes with the IL-6 RE during an acute phase response in rats. Four to six rats were injected with CFA for each time point, and livers were excised at the indicated times. Nuclear extracts were prepared and pooled, and gel mobility shift experiments were performed with 10 µg of crude nuclear extract/lane and constant quantities (approximately 20,000 cpm) of the radiolabeled TB2 probe. I, weak complex I, routinely observed between the IL-6 RE and nuclear proteins from untreated rat livers; II, complex II, sequence-specific for the IL-6 RE(25, 26) ; F, free TB2 probe; Free, no protein added; unind.: extracts from control livers; IL-6 REBP, complex II with highly purified proteins from acute phase livers, purified as described in this study.
Figure 2: Purification scheme. Sequence of chromatographic steps in our purification scheme as described under ``Experimental Procedures.''
Figure 3: Purification of Stat factors from acute phase rat livers. A, gel mobility shift experiment monitoring the progressive enrichment of complex-II forming proteins at different stages of purification. Nuclear extracts were prepared 10-12 h after injection of CFA and purified. Crude 1-4, 20, 10, 5, and 2.5 µg of crude extract; dilution series to calibrate the assay. Eluates Q, Ph, eluates from Sepharose Q and phenyl-Sepharose columns; FT DNA, Wash, and Eluate D, flow-through, wash, and eluate from specific DNA-affinity column. B, silver-stained SDS-polyacrylamide gel with proteins from various stages of the purification. Molecular mass is shown in kDa. FT SP, flow-through from Sepharose SP; Eluate Q, Ph, D, eluates from Sepharose Q, phenyl-Sepharose, and specific DNA-affinity column.
Figure 4:
Gel
mobility shift-supershift analysis of IL-6 RE-binding proteins with
antisera specific for Stat1, Stat3, and Stat5. Supershift experiments
were performed with crude nuclear extracts from the livers of untreated
control rats (left six lanes), or with extracts prepared 12 h
after the induction of an acute phase response (right six
lanes) with the following antibodies: none, no antibody; Stat5C°, anti-Stat5 antibody from B. Raught and J.
Rosen;
Stat5C, anti-Stat5 C-17 from Santa
Cruz.
Figure 5: cDNA and derived amino acid sequence of rat Stat3. cDNA sequence of the coding region plus a portion of the 3`-untranslated region of the Stat3 mRNA. The four peptide sequences obtained from the purified 91-kDa protein and the peptide obtained from the 86-kDa species by amino acid sequence analysis (Table 2) are underlined.
Figure 6:
cDNA and derived amino acid sequence of
rat Stat5b. A, cDNA sequence for the coding region and
portions of the 5`- and 3`-untranslated regions of the full-length
Stat5b mRNA. The two peptide sequences obtained from the purified
92-kDa protein by amino acid sequence analysis (Table 2) are underlined. N, position, at which a stop
codon is found in the mRNA for the Stat5b
40C protein (B). B, cDNA sequence of the portion of the mRNA for the
Stat5b
40C variant that covers the region containing the stop codon
(*) and part of the sequence of the 3`-untranslated region. This
sequence differs from the corresponding sequence of the mRNA for
full-length Stat5b, indicating that the variant most likely was
generated by alternative splicing of the same initial
transcript.
Figure 7:
Comparison of the amino acid sequences of
the two isoforms of Stat5. Identical amino acid residues are boxed. Open bars, position of Src homology domains
type 2 and type 3 (SH2 and SH3, respectively). Black mark,
single tyrosine that is phosphorylated by JAK/TYK kinases upon
activation of Stat factors. BD1 and BD2, binding
determinants 1 and 2 (sequences within amino acids 400-500 of
Stat1 and Stat3 that determine the specific DNA sequences at which
these factors bind)(42) . Within this overall region, BD1 and
BD2 showed a particularly high degree of conservation among the
different Stat factors, and these subdomains were shown by mutagenesis
to directly affect the DNA binding specificity of Stat1 and
Stat3(42) . -, no amino acid at this position; , no
consensus amino acid at this position. m, murine; r,
rat; sh, sheep.
The main new results and conclusions drawn from this study were as follows.
1) An efficient purification scheme for Stat3 and Stat5b from rat liver nuclei was designed and optimized that allowed the isolation of Stat factors in their natural state in the 10-µg (100 pmol) range, starting from 50 to 100 rats.
2) Activated Stat5 was present in liver nuclei of control rats, prior to the onset of an inflammatory response.
3) Both Stat3 and Stat5b were present in rat liver nuclei late in an acute phase response in vivo, suggesting both may participate in the transcriptional induction of some type 2 acute phase genes mediated by IL-6. Stat1 was not enriched by our purification scheme.
4) cDNAs corresponding to two isoforms
of Stat5b, the full-length form and a 40C variant lacking the
COOH-terminal 41 amino acids, were isolated, suggesting each factor may
play a functional role.
A few comments may be added to each of these points.
Schibler and co-workers (27) established that it is an advantage for the purification of nuclear transcription factors, to start with purified nuclei rather than with whole cell extracts. Consequently, their procedures for the purification of nuclei from rat livers were adapted to our system. To reach the 100 pmol range of pure proteins needed for partial aminoacid sequence analysis, it was necessary to start each preparation with 8-10 livers and to pool nuclear extracts from 50-100 livers, before continuing with the chromatographic steps. To process 8 or more livers in one batch, a custom-designed homogenizer was used(28) . The procedures for the extraction of proteins from purified nuclei (27) were optimized to reach the highest possible yield of complex II-forming proteins, which resulted in the modifications of the published procedures described above.
Several chromatographic matrices were tested to remove nucleases contained in the initial nuclear protein extracts, but no single matrix achieved their complete removal. Reduction of the nuclease activity to acceptable levels that no longer damaged the DNA-affinity column required the combination of three conventional chromatographic steps.
The synthetic oligonucleotide
TB2, representing a tandem repeat of the core IL-6 RE of the rat
M gene, preferentially enriched proteins with high
binding affinity for this particular version of the IL-6 RE. This
version differs in one nucleotide from the consensus IL-6 RE (42) and consequently shows a characteristically skewed
spectrum of binding properties for DNA-binding factors. At the time,
when this procedure was established, it was unknown which factors were
to be expected in complex II. The procedure was designed to purify any
factors binding at this IL-6 RE. It is now clear that the TB2 sequence
introduced a bias in favor of Stat5b, which binds better at this
sequence than at other known binding sites for Stat factors such as the
gamma-activated site and serum-inducible element(11) . However,
this bias is not an artifact of our procedure, but rather reflects a
natural preference of the IL-6 RE of the rat
M gene
for Stat5b. The fact that Stat5b was isolated with our procedure from
rodent livers, while it had escaped detection by other
authors(7, 12, 13) , is probably due to the
choice of the TB2 oligonucleotide as the DNA-affinity reagent. In two
cases an oligonucleotide was used as the affinity reagent that
contained a mutated IL-6 RE sequence(7, 12) . The
sequence had been mutated to convert it to a palindrome, which is now
known to bind Stat1 and Stat3 more strongly than Stat5b. This may
explain why those authors only enriched Stat3 but not Stat5b. A second
group of authors used the sequence corresponding to the IL-6 RE from
the human
M gene as the affinity reagent, which
differs from that of the rat
M gene(13) . The
human
M gene is not a strongly responding acute phase
gene. These authors enriched Stat1 and Stat3 from rat livers but not
Stat5b, probably again because their affinity reagent was not binding
Stat5b as strongly as TB2. The particular binding properties of the TB2
oligonucleotide also provide a partial explanation for the absence of
Stat1 among the proteins enriched by our procedure.
This purification scheme can now be used to purify Stat5b in large quantities in its natural state, for example to study naturally occurring posttranslational modifications. In view of the less severe problems with nucleases encountered by other authors in the mouse system(12) , the procedure presented here can probably be further improved by using magnetic beads coated with the affinity oligonucleotide to accelerate the purification. Overall, it is remarkable that in this procedure Stat3 and Stat5b co-purified over four chromatographic steps, demonstrating a great similarity of their chromatographic properties. It is further remarkable that after a 6,300-fold purification the yield was still as high as 27% (Table 1). This estimation of the final yield may be an overestimate due to the difficulty of correctly assessing the content of these factors in the initial crude nuclear lysates.
Assuming the
27% figure for the yield to be correct, 10 µg of Stat5b purified
from 100 livers would correspond to approximately 2,700 molecules of
Stat5b/cell. If the yield was overestimated by 1 order of magnitude,
then the content would be 27,000 molecules of Stat5b/cell. Thus a rat
liver hepatocyte at the peak of an acute phase response contains at
least 2,000-3,000 molecules of Stat5b/cell and possibly up to
10-fold more. This is a normal copy number for nuclear transcription
factors. Copy numbers vary from 600 molecules/cell for hepatocyte
nuclear factor 1 (HNF1) ()to several 100,000 molecules/cell
for the general factor SP1. (
)
We cannot yet definitively
state that Stat5 was present in hepatocytes as opposed to other cell
types of the liver. However, additional experiments ()demonstrated the presence of Stat5 in hepatoma cells by
immunoreactivity with Stat5-specific antibodies. While we cannot
exclude the presence of Stat5 in other cell types of the liver, the
finding that it is present in hepatoma cells makes it very plausible
that it should also be present in hepatocytes in vivo.
The
purified new proteins were identified as Stat3 and Stat5b by sequence
comparison. The cDNA sequence of our Stat5b clones (Fig. 6)
showed a 98.7% match with the published mouse Stat5b sequence, but only
a 92.5% and an 87.3% match with the mouse and ovine Stat5a sequences,
respectively. The peptide sequences obtained for the purified proteins (Table 2) showed a complete match with our cDNA-derived amino
acid sequences for Stat3 and Stat5b ( Fig. 5and 6). It was
possible to obtain distinct PCR products for both Stat5a and Stat5b by
using PCR primers derived from the published murine Stat5a sequence in
combined reverse-transcription PCR experiments with rat liver
mRNA. Thus, rat livers contain mRNAs for both Stat5a and
Stat5b, but so far peptide sequences and cDNA clones have been obtained
only for Stat5b. Stat5a may have escaped detection or may have been
less strongly enriched by affinity chromatography with the TB2
oligonucleotide than Stat5b. Most likely, Stat5a was present in lower
copy numbers per cell than Stat5b, and therefore may not have been
detected among the purified factors. The 86- and 91-kDa forms of Stat3,
which were purified in this study, probably represented the same
polypeptide in different secondarily modified states, most likely
differing in their patterns of serine-threonine
phosphorylation(18, 19) . Until now, only one type of
Stat3 mRNA has been found in mice, rats, and humans, and it is
therefore unlikely that these two different proteins were derived from
different mRNA species.
The gel mobility shift-supershift experiments (Fig. 4) confirmed the presence of Stat5 in rat liver nuclei. The surprising finding was that active Stat5, capable of binding at the IL-6 RE, was present also in the nuclei of untreated control rat livers. The signal causing its activation and the functional significance of activated Stat5 in the nuclei of normal liver cells are unknown. During an acute phase response, the major factor binding at the IL-6 RE clearly was Stat3. However, small amounts of Stat5 were present, and these probably represent the proteins purified in our procedure. The fact that complex II was shifted completely with anti-Stat3 serum and that every complex thus contained at least some Stat3 (Fig. 4) does not imply the formation of heterodimers between Stat3 and Stat5b. No evidence for Stat3/Stat5b heterodimer formation was obtained, when monomeric probes were used instead of the dimeric TB2(43) . Thus, the majority of complexes II probably consisted exclusively of Stat3, and the minority of mixed Stat3/Stat5b complexes most likely contained one homodimer each of Stat3 and 5b bound at each of the two tandemly repeated IL-6 REs of TB2.
The presence of Stat5b in liver cells was unknown until
these data were prepared for publication. Recently, factors
immunologically reactive with anti-Stat5 sera were found in liver cells
and were reported to be activated by growth hormone or epidermal growth
factor(44, 45) . Our observation that Stat5b was
present in an active, DNA-binding form in rat liver nuclei late in an
acute phase response and that it preferentially bound at TB2 suggests
that it may play a functional role in mediating the IL-6 induced
transcription of certain target genes, including the M
gene. Indeed, initial functional evidence has recently been obtained in
transfection studies to support this view. The cDNA coding for rat
Stat5b was placed into an expression vector and transfected into human
hepatoma cells. A reporter construct carrying a chloramphenicol
acetyltransferase reporter under the control of multiple copies of TB2
was co-transfected. After activation of the JAK/Stat signal cascade by
treatment of the cells with suitable cytokines, a cytokine-induced
increase of chloramphenicol acetyltransferase reporter activity was
obtained, which was proportional within a certain range to the dose of
the transfected expression construct. (
)These data were
interpreted to show that Stat5b indeed can function as a transcription
factor in rat liver cells, capable of mediating cytokine-induced
transcription of particular target genes. These data are not proposed
to constitute definitive proof of this hypothesis, but they provide
preliminary evidence in its favor. Similarly, transfection studies with
Stat3 expression constructs into human hepatoma cells were performed
and provided evidence in support of the notion that our rat Stat3 cDNA
clones code for a functionally active transcription factor capable of
mediating cytokine-induced transcription of specific target
constructs(46, 47) .
A carboxyl-terminally
truncated isoform of p91/Stat1, referred to as p84/Stat1
, had
previously been discovered and shown to be generated by alternative
splicing(19, 48) . This shorter variant has been
discussed as a dominant negative inhibitor of Stat1, capable of
competing with the full-length form for the same DNA-binding site, but
no longer capable of transcriptional transactivation(48) .
Consequently, it was proposed that the COOH-terminal 38 amino acids of
Stat1 are essential for its transactivator function(19) . A
carboxyl-terminally deleted version is now also known for Stat5b. Thus,
the COOH-terminal 40 amino acids of both factors may share a common
function. However, the XPXSP-motif conserved in the C
termini of Stat1, Stat3, Stat4, and ovine Stat5a, which has been
discussed as a potential target for mitogen-activated protein
kinase-like activities(19) , is absent in mouse and rat Stat5b.
Potential specific functions for each isoform, their relative
importance, and the mechanism of generation of the short isoform are
currently unknown.
With the availability of these cDNA clones the
initial question, whether Stat5b plays a specific role in the delayed
transcriptional activation of the M gene in comparison
with other type 2 acute phase genes, can now be addressed
experimentally.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) X91810 [GenBank]and X91988[GenBank].
This paper is dedicated to the memory of Dr. Wolfgang Northemann.
Note Added in
Proof-Since submission of this manuscript, the existence of
Stat3, a carboxyl-terminally truncated isoform of Stat3, was
reported. Schaefer, T. S., Sanders, L. K., and Nathans, D. (1995) Proc. Natl. Acad. Sci. U. S. A.92, 9097-9101. A
corresponding shorter mRNA species that was resolved in Northern blot
experiments has not yet been reported.