©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Separate Signaling Mechanisms Are Involved in the Control of STAT Protein Activation and Gene Regulation via the Interleukin 6 Response Element by the Box 3 Motif of gp130 (*)

Chun-Fai Lai (1), Juergen Ripperger (2), Karen K. Morella (1), Yanping Wang (1) (3), David P. Gearing (4), Georg H. Fey (2), Heinz Baumann (1) (5)

From the (1)Roswell Park Cancer Institute, Department of Molecular and Cellular Biology, Buffalo, New York 14263, the (2)Chair of Genetics, Universität Erlangen-Nürnberg, D-91058 Erlangen, Federal Republic of Germany, (3)Division of Endocrinology, Children's Hospital, Buffalo, New York 14222, and (4)SyStemix, Palo Alto, California 94304

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The cytoplasmic receptor sequences required for the transcriptional control via the IL-6 response element (IL-6RE) and the hematopoietin receptor response element (HRRE) in hepatoma cells were defined by transient expression of wild-type and mutant granulocyte-colony stimulating factor receptor-gp130 chimeric receptors. gp130 generated two separate transcriptional signals, one of which was directed to IL-6RE and required an intact box 3 motif, and another, which was directed to HRRE and was box 3-independent. The activation of DNA-binding of STAT3 required the same gp130 domains as the IL-6RE response. A box 3-independent activation of STAT proteins was achieved by overexpression of the kinases JAK2 or TYK2. The increase in the DNA-binding activity of STAT proteins, however, did not result in a corresponding increase in transcription via either IL-6RE or HRRE. The data indicate that activation of the DNA-binding potential of STAT proteins via gp130 is not sufficient to achieve transcriptional up-regulation of specific target genes.


INTRODUCTION

The receptor for interleukin-6 (IL-6R)()is composed of the 80 kDa ligand-binding subunit (IL-6R) (1) and the signal-transducing subunit, gp130(2) . After ligand binding, the IL-6R subunits are believed to aggregate into a hexameric complex consisting of two molecules each of IL-6, IL-6R, and gp130(3) . Signal transduction has been shown, or predicted, to involve activation of receptor-associated protein tyrosine kinases belonging either to the JAK (4, 5) or to the Bruton's tyrosine kinase (BTK) families(6, 7) . These kinases phosphorylated both themselves and the cytoplasmic portion of the receptor. The phosphotyrosine residues serve in part as recognition sites for SH2-domain-containing signaling proteins, in particular STAT3 (or ARPF) (8) and to a lesser extent, STAT1(9) . STAT proteins are also phosphorylated that then dimerize, translocate to the nucleus, and bind to specific DNA sequences(10) . Binding to the high affinity form of SIE of the c-fos gene and related interferon- activating site sequences provide the identifying feature of the IL-6-regulated STATs(9, 11) .

gp130 contains in its cytoplasmic domain several functional elements including box 1, 2, and 3 motifs (12, 13) and binding sites for Syp (14) and Shc(15) . These elements are necessary for the activation of JAKs, stimulation of proliferation, and/or induction of several immediate response genes(4, 5, 12) . Although a causal relationship between box 3 function, STAT3 activation, and IL-6-responsive gene regulation was expected(8, 14) , we provide evidence that the activation of STAT proteins was facilitated but not absolutely dependent on the presence of a box 3 motif, and that elevated DNA-binding activity of STAT proteins is not sufficient to account for the transcription of target genes via IL-6RE.


EXPERIMENTAL PROCEDURES

Cells

Rat hepatoma H-35, L-, and COS-1 cells were cultured as described(13) . A clonal line of L-cells stably transfected with an expression vector for human G-CSFR-gp130(277)(13) , and pSV2neo was selected and maintained in medium containing 0.4 mg/ml G418. Hormonal treatments were carried out in serum-free medium containing 1 µM dexamethasone alone or in combination with 100 ng/ml human IL-6 (Genetics Institute) or human G-CSF (Immunex Corp.).

Expression Vectors and CAT Reporter Gene Constructs

Expression vectors encoding the chimeric human receptor G-CSFR-gp130(277) and the truncated forms with cytoplasmic domains of 230, 165, 133, 109, 90, 65, 40, and 1 (cyto) residues, respectively, have been described(13) . Tyrosine 125 was mutated to alanine (Y125A) in box 3a (termed M3) of G-CSFR-gp130(277) and(133) , and the prolines 14 and 16 were mutated to leucines (P14/16L) in box 1 (termed M1) of G-CSFR-gp130(133) by using the in vitro mutagenesis kit from Clontech. Rat STAT1 cDNA (a 2,300-bp EcoRI-XhoI fragment) and rat STAT3 cDNA (a 3,000-bp EcoRI-EcoRV fragment).() were inserted into the NotI site of pSV-Sport 1 (Life Technologies, Inc.). The 4,176-bp human TYK2 cDNA (provided by Dr. J. J. Krolewski) (16) was inserted into the KpnI-NotI sites of pDC302(17) . The expression vector for mouse JAK2 in pEF-BOS has been provided by Dr. D. M. Wojchowski(18) . The CAT reporter gene constructs pHRRE-CAT and pIL-6RE-CAT have been described(19) .

Cell Transfection and Analysis

Plasmid DNA was transfected by the DEAE-dextran method (20) optimized for each application (see figure legends). Cell cultures were subdivided, treated, and analyzed for CAT activity as described(13, 21) . STAT protein activation was determined in whole cell extracts prepared from cells after treatment with cytokines for 15 min at 37 °C by EMSA(9) . The high affinity SIEm67 oligonucleotide served as binding substrates(9) . STAT3-containing complexes were identified by ``supershift'' assay using 1 µg of rabbit antibodies against STAT3 (Santa Cruz Biotechnology).

To determine G-CSFR-gp130 protein expression, 20 µl of whole cell lysate from 5 10 transfected cells was mixed with 25 µl of SDS buffer and electrophoresed in a 6% SDS-polyacrylamide gel. The proteins were transferred to Immobilon membrane (Millipore), incubated with sheep anti-human G-CSFR (provided by Dr. D. J. Tweardy), and processed for chemiluminescent reaction according to the supplier (DuPont NEN).


RESULTS AND DISCUSSION

Cytoplasmic Domain of gp130 Mediates Two Signals

The chimeric G-CSFR-gp130 receptor form was used to define the signaling reactions initiated by the cytoplasmic domain of gp130 independently of the resident receptor subunits for IL-6-type cytokines(13, 21) . Transfection of G-CSFR-gp130 with full-length or progressively truncated cytoplasmic domains into H-35 cells allowed the mapping of the regions required for gene activation via two characteristic elements, IL-6RE and HRRE (Fig. 1). G-CSFR-gp130(133) was the minimal construct generating a significant transcriptional response via IL-6RE. Deletion of the carboxyl-terminal box 3 repeated motifs b, c, and d (Fig. 1) diminished the magnitude of regulation 2-fold. Removal of residues 133-109, containing the box 3a sequence, eliminated all detectable IL-6RE response. However, regulation via HRRE by the same receptors was maintained. The HRRE response decreased to 5-10% only after removal of box 2 and was lost with deletion of box 1.


Figure 1: Differential regulation of IL-6 responsive gene elements by gp130 motifs. The chimeric receptor forms of G-CSFR-gp130 indicated at the top (1 µg/ml) were cotransfected with pIL-6RE-CAT (6 µg/ml) and HRRE-CAT (6 µg/ml) into H-35 cells. Subcultures were treated with G-CSF for 24 h. The CAT activities were normalized to the internal transfection marker pIE-MUP and expressed relative to the control culture in each experimental series.



Box 3 Is Necessary for the Activation of STAT3 and Transcription through IL-6RE

To identify whether gene regulation by the G-CSFR-gp130 is correlated with the ability to activate STAT proteins, the receptors were transfected into L-cells. This cell type has a higher transfection efficiency than H-35 cells, thus yielding a receptor protein expression detectable by Western blotting activation of endogenous STAT proteins(19) , low level regulation of cotransfected IL-6RE-containing CAT gene constructs (see Fig. 4A and Ref. 21). Wild-type G-CSFR-gp130(277) mediated a G-CSF-specific activation of STAT proteins producing a SIF complex that comigrated on EMSA with SIF-A induced by the endogenous IL-6R (Fig. 2A). STAT3 contributed essentially to all DNA-binding activity detected by EMSA as shown by an antibody-mediated supershift assay (Fig. 2B).


Figure 4: Effect of ectopically expressed STAT proteins on gene regulation. A, L-cells were transfected with G-CSFR-gp130(133wt) (1.5 µg/ml), Hp(IL-6RE)CAT (2 µg/ml), and, where indicated, with expression vectors for STAT1 (1.5 µg/ml), STAT3 (1.5 µg/ml), or JAK2 (0.1 µg/ml). Subcultures of the transfected cells were used to determine the activation of STAT proteins by EMSA after 15 min of G-CSF treatment and increased expression of CAT activity after 24 h of treatment. All analyses were normalized to the expression of the internal transfection marker pIE-MUP (13, 21). B, gene regulation in H-35 cells. Various receptor constructs (1 µg/ml), pIL-6RE-CAT (6 µg/ml), and expression vectors for STAT1 (3 µg/ml), STAT3 (3 µg/ml), JAK2 (0.1 µg/ml), or TYK2 (0.1 µg/ml) were cotransfected as indicated. The subcultures were treated with the indicated agonists, and the magnitude of stimulation of CAT enzyme activity was calculated relative to the control in each series. Mean and S.D. of three separate experiments are shown. C, activation of HRRE signal by JAK2. H-35 cells were transfected with G-CSFR-gp130(40) (1 µg/ml), HRRE-CAT (5 µg/ml), and expression vectors for STAT3 (3 µg/ml) and JAK2 (0.1 µg/ml) as indicated. The relative changes in CAT activities were quantitated.




Figure 2: Correlation of STAT protein activation and transcriptional regulation through the IL-6RE. A, activation of STAT protein in L-cells. The G-CSFR-gp130 receptor forms indicated at the top (3 µg/ml) were transfected into L-cells. Subcultures were treated for 15 min with or without G-CSF, extracted, and analyzed by EMSA. Extracts from IL-6-treated L-cells and a G-CSF-treated G-CSFR-gp130(277) stable L-cell line served as standards. B, identification of STAT3-containing SIF complex. The extract from G-CSFR-gp130(133wt)-transfected and G-CSF-treated culture from A was analyzed by EMSA with or without inclusion of anti-STAT3 antibodies in the binding reaction. C, equal amounts of whole cell extracts from L-cells transfected with the indicated receptors from A were analyzed by Western blot for the expression of chimeric G-CSFR proteins. The patterns show the characteristic two forms of the mature chimeric G-CSFR proteins. D, gene regulating activities of the G-CSFR-gp130 receptor forms. The receptors indicated at the top were cotransfected into H-35 cells together with either Hp(IL-6RE)-CAT or HRRE-CAT. Subcultures were treated with G-CSF, and the effects on the CAT activities relative to control cells were determined. The relative changes are given above the autoradiograms.



G-CSFR-gp130(133) was 3 times less active than the full-length form (Fig. 2A). Deletion to residue 109 abrogated detectable STAT protein activation. The functionally relevant sequence within the 133-residue form was determined by mutation. The Y125A mutant box 3 (M3) abolished STAT activation (Fig. 2A) but did not affect receptor protein expression as demonstrated by Western blotting (Fig. 2C). By contrast, the M3 mutation in full-length gp130(277) did not influence STAT activation (Fig. 2A). Thus, the repeated box 3 motifs (b, c, and d) were functionally redundant(14) .

Gene-regulatory function of the receptor mutants was tested in H-35 cells (Fig. 2D). The M3 mutation in gp130(133) inactivated the regulation of the IL-6RE-CAT gene but not of the HRRE-CAT gene. The P14/16A mutant box 1 (M1) abolished all signaling function of gp130(133), i.e. activation of STAT proteins in L-cells and transcriptional regulation of IL-6RE- and HRRE-CAT genes in H-35 cells, even though the M1 protein was abundantly expressed in transfected L-cells (data not shown; see also Fig. 4C below).

Box 3-independent STAT Activation by JAK2

If active STAT proteins cause transcriptional regulation, a change in their cellular concentration should entail a corresponding change in the transcriptional activity of IL-6RE-containing genes. To assess whether G-CSFR-gp130 could activate excess amounts of ectopically expressed STAT proteins and thus prominently alter the intracellular concentrations of active STAT proteins, expression vectors for G-CSFR-gp130s forms and for rat STAT1 and STAT3 were cotransfected into COS-1 cells (Fig. 3A). COS-1 cells were used because of high expression of transfected vectors and the low level contribution of endogenous STATs, hence permitting clear demonstration of the specificity of STAT protein activation. Transfected G-CSFR-gp130 alone produced a minor increase of the endogenous SIF-C protein-DNA complex (Fig. 3A). Transfected rat STAT1 and 3 were strongly activated, producing rat SIF-C and SIF-A complexes, respectively, which showed different mobilities from the corresponding endogenous SIF complexes (Fig. 3A). Inclusion of JAK2 caused two notable effects: an increase in basal level of activated STAT3 (Fig. 3A) or STAT1 (not shown), and a further induction following G-CSF treatment. Excess levels of JAK2 were capable of increasing basal levels of activated STATs even in the absence of ligand-receptor interaction. This JAK2-mediated, receptor-dependent activation of STAT3 did not require box 3. In fact, box 1 was sufficient, as demonstrated by the data obtained with G-CSFR-gp130(40) (Fig. 3A). The resulting intensities of SIF complexes exceeded by a factor of 1,000-10,000 those achieved in COS cells not supplemented with STAT proteins.


Figure 3: Modulation of SIF by overexpression of STATs and JAKs. A, the indicated receptors (2 µg/ml) were transfected into COS-1 cells alone or with STAT1 (2 µg/ml), STAT3 (2 µg/ml), or JAK2 (0.15 µg/ml). Subcultures were treated as indicated, extracted, and analyzed by EMSA. B, as in A, COS-1 cells were cotransfected with the indicated receptor forms (2 µg/ml) STAT3 (2 µg/ml) TYK2 (0.15 µg/ml) and analyzed as in A.



TYK2 has been noted to interact with gp130(8) . Therefore, we tested whether TYK2 was also effective in activating STAT proteins (Fig. 3B). In the absence of any transfected kinase, the box 3-specific activation of coexpressed rat STAT3 by G-CSFR-gp130(133) was detectable, probably mediated by the resident COS cell kinases. Overexpressed TYK2, like JAK2, promoted a several hundred-fold increase of STAT3 activation independently of agonist treatment and presence of box 3. Neither TYK2 nor JAK2 was effective with transfected G-CSFR-gp130(133M1), suggesting that the kinase action was linked to box 1 element in agreement with the published observations for other hematopoietin receptors(4, 5, 12, 22) .

STAT3 and JAK2 Contribute to but Are Not Sufficient for Gene Regulation

The prominent activation of ectopically expressed STAT proteins provided the experimental condition to assess the link between activation of STAT proteins and transcriptional induction of genes. G-CSFR-gp130(133) and STAT proteins were introduced into L- and H-35 cells and their effects on cotransfected IL-6RE-CAT gene determined (Fig. 4, A and B). Although a prominent STAT1 and -3 activation was achieved in L-cells (Fig. 4A), the IL-6RE response after 24 h G-CSF treatment was not appreciably elevated above the control culture. JAK2 alone raised basal activity 2-fold and enhanced the G-CSF effect severalfold. Surprisingly, the combination of JAK2 and the STATs did not increase transcription proportionally to the STAT protein activity.

Similar signal reconstitution in H-35 cells (Fig. 4B) confirmed the findings made with L-cells and revealed additional important features. 1) Transfected STATs and/or JAKs had no significant effect on action of the endogenous IL-6R or transfected G-CSFR-gp130(133wt); 2) G-CSFR-gp130(133M1) was inactive under all conditions; 3) G-CSFR-gp130(133M3) produced a 2-5-fold transcriptional stimulation through IL-6RE in the presence of overexpressed STAT3, JAK2, or TYK2. However, even under optimal reconstitution conditions, the box 3-deficient G-CSFR-gp130 yielded a maximal IL-6RE response that was only a fraction of that obtained with the wild-type G-CSFR-gp130(133). The data from Fig. 4(A and B) indicate that transcriptional regulation through IL-6RE by gp130 was not strictly correlated with relative amounts of activated STATs. We hypothesize that an additional regulatory factor (or factors) is activated by gp130(133), probably by the action of receptor-associated kinase(s). This factor could not be experimentally identified by binding to SIE or IL-6RE (Fig. 2A; data not shown) and thus was termed ``SIF-independent gene regulator'' or ``SIG.''

Evidence for a functional entity such as SIG in hepatoma cells was provided by the box 3- and STAT3-independent transcriptional regulation through HRRE ( Fig. 1and Fig. 2, A and D). The HRRE signal was, however, dependent on box 1, suggesting that box 1-interacting JAKs may act as potential mediators of that hypothetical signal pathway. The relatively low HRRE regulating activity of G-CSFR-gp130(40) conceivably was due to reduced association with and/or activation of JAKs. Overexpression of JAK2 (Fig. 3, A and B) or TYK2 (data not shown) clearly rectified this inefficient kinase action. Furthermore, cotransfection of the G-CSFR-gp130(40) with JAK2 yielded a 20-fold increase in transcription through HRRE that was independent of cotransfected STAT3 (Fig. 4C).

This study illustrates that gp130, like other hematopoietin receptors (4, 5, 12), can activate various STAT proteins. The prominence of this response and the specificity of STAT-3 for DNA sequences related regulation of genes by IL-6 have led to a model proposing STAT-3 to function as both signal transducers and activators of transcription(4, 5) . However, the data of this study indicate that STAT3 does not act as sole activator of transcription and that other activities controlled by gp130, such as the hypothetical SIG, are likely involved. Contributing activities may include the action of serine kinase noted to enhance DNA binding of STAT proteins(23, 24, 25) . Moreover, the current model of the JAK-STAT pathway from receptor to the regulated gene will need modifications based on the two observations that 1) STAT activation does not strictly depend on phosphotyrosine residues on the receptor molecule(26) , and 2) regulation of the prototypical IL-6-responsive -fibrinogen gene does not appear to involve STAT3(27) .


FOOTNOTES

*
This study was supported by Grant CA26122 from the National Institutes of Health (to H. B.) and Grant DFG Hi291/5-4 from the Deutsche Forschungsgemeinschaft (to G. H. F.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel. 716-845-4587; Fax: 716-845-8389; E-mail: baumann@sc3101.med.buffalo.edu.

The abbreviations used are: IL-6R, interleukin-6 receptor; CAT, chloramphenicol acetyltransferase; EMSA, electrophoretic mobility shift assay; G-CSF(R), granulocyte-colony stimulating factor (receptor); HRRE, hematopoietin receptor response element; IL-6RE, interleukin 6 response element; JAK, Janus kinase; MUP, major urinary protein; SIE, sis-inducible element; SIF, sis-inducible factor; SIG, SIF-independent gene regulator; STAT, signal transducer and activator of transcription; bp, base pair(s).

J. Ripperger, unpublished data.


ACKNOWLEDGEMENTS

We thank Dr. D. M. Wojchowski for providing pEF-BOS-JAK2, Dr. J. J. Krolewski for the TYK2 cDNA, Dr. D. J. Tweardy for sheep anti G-CSFR, and Marcia Held for secretarial assistance.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.