(Received for publication, June 27, 1995; and in revised form, August 3, 1995)
From the
Transient transfection of expression vectors for various members of the hematopoietin receptor family and STAT proteins into COS-1 cells indicated that each receptor was capable of stimulating the DNA binding activity of STAT1, STAT3, and STAT5B. However, gp130 preferentially activated STAT1 and STAT3. Activation of STAT5B differed from that of the other two in that the box 3 sequence motif in the cytoplasmic domain of gp130 was not required. Moreover, STAT5B and STAT3 enhanced gene transcription via separate regulatory elements. This study has identified two potential signal transduction pathways by which hematopoietin receptors, including the interleukin-6 receptor, control transcription of acute phase plasma protein genes in hepatic cells.
The transcriptional regulation of acute phase plasma protein
genes in hepatic cells by IL-6 ()has been correlated with
the activation of DNA binding properties of STAT3 by the signaling
activity of
gp130(1, 2, 3, 4, 5) . The
suggested function of STAT3 as a transcription factor was supported by
the finding that overexpression of STAT3 and its activation by cytokine
receptor and Janus kinases resulted in enhanced transcription via
IL-6-responsive gene elements(6) . However, the model proposing
a principal role for STAT3 as a mediator of acute phase response (3, 7) needed to be refined because: 1)
gp130-dependent transcription via certain elements, such as the HRRE,
was found to be independent of STAT3(8) ; and 2) the acute
phase response of the liver included activation of not only STAT3 but
also of other members of the STAT protein family, such as
STAT5B(9) . (
)The study of the transcription control
mechanisms by specific STAT isoforms has been difficult primarily due
to the lack of adequate experimental assay systems. Recently, we have
developed techniques to reconstitute the function of hepatic and
non-hepatic hematopoietin receptors in transiently transfected hepatoma
cells(10) . We could define the cytoplasmic domains of the
signal transducing receptor subunits required for the induction of
transcription through specific regulatory
elements(8, 11) . This cell assay system was used to
characterize the specificity of STAT protein activation by
hematopoietin receptors. Two distinct signaling pathways were
identified: one specified by the box 3-dependent activation of STAT3
and transcriptional stimulation via an IL-6RE, and the other specified
by the box 3-independent activation of STAT5B and transcriptional
stimulation via HRRE.
Figure 1: Expression and analysis of STAT proteins. Triplicate aliquots of whole cell extracts from H-35 cells after 15 min treatment with IL-6 containing 5 µg (H-35-Std) of protein or from COS-1 cells transfected with expression vectors for STAT5B (2.5 µg/ml) and PRLR and treated for 15 min with PRL containing 1 µg of protein (STAT5B) were reacted with the oligonucleotides listed at the top (30,000 cpm; 300,000 cpm/ng). The complexes were separated by polyacrylamide gel electrophoresis, and the autoradiogram was exposed for 16 h. Note qualitative and quantitative differences in affinity. The positions of SIF-A, -B, and -C and the STAT5B oligomeric complex (*) are indicated on the left.
Differences in binding specificity for the probes emerged when
tested with STAT5B (Fig. 1). STAT5B did not detectably bind to
the SIE. The -casein element yielded two complexes: one
co-migrating with SIF-B and likely containing the predicted STAT5
dimer, and the other showing a much slower mobility and co-migrating
with the single complex observed with the dimeric TB-2. Since both
complexes reacted with anti-STAT5 detectable by supershift EMSA (data
not shown), the slow mobility complexes were interpreted to be
consistent with a STAT5B tetramer. The comparison of the patterns in Fig. 1also indicated that in extracts of IL-6-treated H-35
cells, STAT1 and STAT3 predominate, obscuring detection of
STAT5-containing complexes. We could, however, detect STAT5 in nuclear
extract of IL-6-treated cells by Western blot analysis (data not
shown).
To define the activation of specific STAT proteins by gp130, we transiently over expressed rat STAT1, STAT3, and STAT5B together with the chimeric receptor G-CSFR-gp130 in COS-1 cells. The chimeric construct permitted analysis of its signaling independent of the endogenous gp130(11) . We verified that the STAT expression vectors yielded abundant and approximately equal amounts of STAT proteins (Fig. 2A; (6) ). To grade the preference of gp130 for specific STAT proteins, we compared its action with that of co-introduced IL-3R (Fig. 2B). IL-3R was chosen because of its strong stimulation of transcription via HRRE in hepatoma cells (12) and its difference from gp130 in lacking a box 3 motif.
Figure 2:
Activation of STATs in COS-1 cells and
activation by gp130 and IL-3R. A, expression vectors for
G-CSFR-gp130(277) (1 µg/ml) and either for STAT5B or STAT3
(concentrations indicated) were transfected into COS-1 cells. Whole
cell extracts of duplicate subcultures (control and G-CSF-treated)
containing 5 µg of protein were analyzed by Western blotting for
STAT protein expression. Blots were processed simultaneously for
chemiluminescent reaction. B, COS cells were transfected with
expression vectors for G-CSFR-gp130(277), IL-3R and IL-3R
(1
µg/ml each), and STAT1, STAT3, or STAT5B (2.5 µg/ml) as
indicated at the top. Triplicate subcultures were treated as
control or with IL-3 (3) or G-CSF (G) as shown at the bottom. Identical amounts of whole cell extracts were reacted
with SIE,
-casein element, and TB-2, and processed for
EMSA.
Both receptor types were capable of transducing the signal to each STAT isoform. The comparison also indicated that IL-3R preferred STAT5B, whereas gp130 preferred STAT1 and STAT3, consistent with the action of native IL-6R in H-35 cells (Fig. 1). The EMSA patterns as well as antibody supershift assays (not shown) did not provide any evidence for appreciable formation of heterodimers between STAT1 and STAT5B or between STAT3 and STAT5B. TB-2 contains two IL-6REs in a 20-base pair span and was thus predicted to bind two STAT dimers. Surprisingly, this probe did not produce detectable amounts of mixed tetrameric complexes containing one homodimer each of STAT1 and STAT5B, as judged from the absence of complexes with intermediate mobilities on EMSA (Fig. 2B) or reacting to both anti-STAT1 and anti-STAT5 (not shown). We hypothesized that either two different STAT dimers were incompatible in simultaneous binding to TB-2, or that STAT5B binds as a preformed tetramer. The results obtained with pairwise combinations of STATs indicated a certain degree of competition among STATs for activation by the receptors that was correlated with the preferred usage of the particular STAT by the receptor.
Figure 3: Influence of box 3 motif on STAT activation. COS-1 cells were transfected with expression vector for G-CSFR-gp130(133) wild type (wt) or M3 (1 µg/ml each) and STAT5B or STAT3 (2.5 µg/ml) and JAK2 (0.1 µg/ml). Whole cell extracts were used for EMSA with TB-2 as probe. One reaction with extract containing active STAT3 was incubated with anti-STAT3 to supershift the STAT3-containing complex (wt + Ab).
From these results, we concluded that gp130 utilizes separate mechanisms to activate STAT3 and STAT5B. The two pathways have distinct requirements for cytoplasmic receptor domains, but both are dependent on the action of JAKs. One consequence of JAK action is the tyrosine phosphorylation of STATs, thereby inducing dimerization and manifestation of DNA binding activity(24, 25, 26) . Since direct binding of STATs via their SH2 domain to a phosphotyrosine residue in the receptor molecule is not mandatory for their activation(27) , an alternative mechanism for bringing STATs in contact with receptor-bound kinases must exist. One possibility yet to be tested is that STATs directly interact with the kinase or connector molecules.
One prediction for the proposed mechanism of interaction between receptors and STATs was that other hematopoietin receptors lacking a box 3 sequence but utilizing JAK2 should also activate STAT5B with comparable efficiency to IL-3R in our assay system. As anticipated, PRLR and GHR, which have been associated with the STAT5 pathway(24, 26, 28) , promoted activation of STAT5B (Fig. 4). Nevertheless, these receptors also showed activation of STAT3, albeit at variable and much lower levels and only when STAT3 was provided in sufficiently high concentrations (Fig. 4; (6) ).
Figure 4: Action of GHR and PRLR. COS-1 cells were transfected with expression vectors for GHR and PRLR (1 µg/ml each) and for STAT3 or STAT5B (2.5 µg/ml) as indicated. STAT activities were determined as in Fig. 2.
Functional screening of various regulatory elements
indicated that all elements containing a core sequence related to the
-activating site (GAS) including the IL-4RE/GAS of the Fc
R1
gene,
-casein PRE, the IL-6RE of the
-macroglobulin gene, and, foremost, HRRE, were
responsive to STAT5B. Transfection of GHR and PRLR together with the
HRRE-CAT construct and increasing amounts of STAT5B expression vector
established a dose-dependent increase of CAT gene transcription (Fig. 5). The enhancing effect of STAT5B was noted on both the
basal and receptor-mediated transcription. The change in
transcriptional regulation via HRRE was STAT5B-specific because STAT3 (Fig. 5), STAT1, or STAT6 (data not shown; (6) ) proved
to be ineffective.
Figure 5: Stimulation of transcription by STAT5B. HepG2 cells were transfected with pHRRE-CAT (10 µg/ml) and expression vectors for GHR and PRLR (1 µg/ml each) and increasing amounts of STAT5B or STAT3 as noted. Subcultures were treated without cytokines (control) or with PRL, GH, or IL-6. The change in CAT activity was calculated relative to control cells without STAT supplementation. Values represent means of two separate experimental series.
In HepG2 cells, both GHR and PRLR were able to
control transcription, in part via STAT5B. This finding differs from
similar experiments with STAT5A in COS cells reported by Gouilleux et al.(29) , who observed that PRLR but not GHR or
erythropoietin receptor mediated enhanced gene transcription via the
-casein element. Both studies concur in that the magnitude of
STAT5 activation by the receptor did not correlate well with the
magnitude of transcriptional induction. For instance, in Fig. 5,
neither GHR nor PRLR reconstituted an increase in transcription of
comparable magnitude with that by the endogenous IL-6R.
To confirm the role of STAT5B in hematopoietin receptor signaling leading to gene transcription, we extended the analyses to other receptor combinations. We focused on those hematopoietin receptors that had shown low action in HepG2 cells and for which no autocrine pathway or response to serum factors was detectable. Moreover, for some of these receptors, the activation of STAT5 had been already reported (30, 31, 32) . We achieved transcription-enhancing effects via HRRE and related GAS sequences with c-Mpl, and box 3-deleted constructs of G-CSFR-LIFR(140), G-CSFR-gp130(40), and G-CSFR(27). The most prominent effect of STAT5B on transcription was, however, obtained with IL-2R and IL-4R (Fig. 6). STAT5B enhanced the signal of the transfected receptor, resulting in a transcription of the reporter gene that equaled or even exceeded the effect of the endogenous IL-6R. Moreover, when this optimal experimental receptor system was used in combination with the IL-6RE-CAT construct, the results illustrated the clear difference in the regulatory actions of STAT5B and STAT3. STAT5B enhanced transcription via HRRE but not via IL-6RE, and STAT3 was ineffective via HRRE (Fig. 5) but enhanced transcription via IL-6RE (Fig. 6). What still needs to be determined is the extent to which STAT3 and STAT5, or functionally redundant factors, contribute to the regulation of genes by IL-6 in hepatoma cells.
Figure 6:
Transcription control by IL-2R and IL-4R
via STAT5B. HepG2 cells were transfected with either pHRRE CAT or
pIL-6RE-CAT (10 µg/ml), expression vectors for IL-2R,
IL-2R
and IL-4R (1 µg/ml each) and either increasing amounts
of STAT5B (noted in leftpanel) or STAT3 or STAT5B (5
µg/ml) (rightpanel). Subcultures were treated
with IL-2, IL-4, or IL-6, and the change in CAT activity was calculated
relative to control culture. Similar regulatory effects were measured
in three additional experiments.
This study demonstrates that hematopoietin receptors use two distinguishable pathways to regulate gene transcription. The following model is proposed. The basic signaling pathway probably exerted by most if not all hematopoietin receptors requires minimally the box 1 motif. Box 1 engages one of the JAK family members(33) , which recruits primarily STAT5 and much less the other STATs. Upon phosphorylation, the dimeric or oligomeric STAT5 interacts with GAS/PRE/TB-2-related sequences. If the receptor bears a box 3 or related sequence, STAT 1, STAT3(8) , or STAT6 (34) are also recruited to receptors by using box 3 as docking site. The subsequent activation process by the receptor-bound JAK may be similar to that affecting STAT5. The genetic targets of STAT3 differ from that of STAT5; thus, separate sets of genes may be controlled by specific members of the STAT family. Although the precise action of STAT proteins as transcription factors has yet to be defined, it is commonly assumed that the binding of STATs to the DNA element enhances transcription of the genes in cis. Although STAT1 and STAT6 are also regulated by some of the hematopoietin receptors used in our study, no transcription- controlling action of these STATs could be detected by the approach of overexpression(6) . It is conceivable that under physiological conditions STAT1 or STAT6 can interfere with or modulate the action of the other STAT proteins by competition for binding to the same DNA sequences.
Unquestionably, the transfection experiments are crude reconstitutions of the normal regulatory systems operative in cells and may yield artificially exaggerated responses. However, these experiments have emphasized the importance of (a) the relative concentrations of receptors, kinases, and STATs and (b) the sequence specificity of the binding of STATs to DNA response elements. A most notable effect also is that excess amounts of JAKs lead to ligand-independent activation of STATs and transcription, suggesting that under such conditions alternative pathways that are generally unnoticed have become predominant. Moreover, every transcription assay is performed in cells that contribute a complex combination of endogenous factors, some of which may significantly influence the observed regulatory events.