©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Activation of Acute Phase Response Factor (APRF)/Stat3 Transcription Factor by Growth Hormone (*)

(Received for publication, June 29, 1994; and in revised form, December 2, 1994)

George S. Campbell (1) Debra J. Meyer (1)(§) Regina Raz (2)(¶) David E. Levy (2)(**) Jessica Schwartz (1) Christin Carter-Su (1)(§§)

From the  (1)Department of Physiology, University of Michigan Medical School, Ann Arbor, Michigan 48109-0622 and the (2)Department of Pathology and Kaplan Comprehensive Cancer Center, New York University School of Medicine, New York, New York 10016

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The mechanisms by which the binding of growth hormone (GH) to its cell surface receptor elicits changes in gene transcription are largely unknown. The transcription factor Stat1/p91 has been shown to be activated by GH. Here we show that acute phase response factor or Stat3 (or an antigenically related protein), is also activated by GH. Stat3 has been implicated in the interleukin-6-dependent induction of acute phase response genes. GH promotes in 3T3-F442A fibroblasts the tyrosyl phosphorylation of a protein immunoprecipitated by antibodies to Stat3. This protein co-migrates with a tyrosyl phosphorylated protein from cells treated with leukemia inhibitory factor, a cytokine known to activate Stat3. Tyrosyl phosphorylated Stat3 is also observed in response to interferon-. Stat3 is present in GH-inducible DNA-binding complexes that bind the sis-inducible element in the c-fos promoter and the acute phase response element in the alpha(2)-macroglobulin promoter. The ability of GH to activate both Stat1 and Stat3 (i.e. increase their tyrosyl phosphorylation and ability to bind to DNA) suggests that gene regulation by GH involves multiple Stat proteins. Shared transcription factors among hormones and cytokines that activate JAK kinases provide an explanation for shared responses, while the ability of the different ligands to differentially recruit various Stat family members suggests mechanisms by which specificity in gene regulation could be achieved.


INTRODUCTION

GH (^1)is an important mediator of body growth and regulates both cellular differentiation and proliferation (reviewed in (1) ), functions that necessitate the coordinated regulation of gene expression. The GH receptor, a member of the cytokine receptor superfamily(2) , transmits an intracellular signal through activation of the cytoplasmic tyrosine kinase JAK2(3) . However, molecular mechanisms linking the activated GH receptor-JAK2 complex to gene transcription are largely unknown.

The Stat (Signal Transducers and Activators of Transcription) family of transcription factors provides a signaling pathway linking activated receptor-tyrosine kinase complexes to the nuclear apparatus for transcriptional regulation. Stat proteins were first identified as signaling molecules for the Tyk2/JAK1-coupled receptor for IFN-alpha(4, 5) . In response to IFN-alpha, p91/Stat1alpha, p84/Stat1beta (an alternatively spliced form of Stat1alpha), p113/Stat2, and a 48-kDa non-Stat protein form the heteromeric IFN-stimulated gene factor-3 complex which binds to an IFN-stimulated response element in IFN-alpha induced genes(6, 7) . Stat1alpha (henceforth referred to as Stat1) has been identified as a component of DNA-binding complexes formed in response to multiple growth and differentiation factors which signal via JAK kinases (e.g. IFN-, IL-6, LIF, oncostatin M) (8, 9, 10, 11, 12, 13, 14) . Stat3/APRF (15, 16, 17, 18, 19) was originally identified as a regulator of transcription of acute phase response genes activated by IL-6 (20) and Stat5/mammary gland factor is a transcription factor for prolactin (21) . Stat4 is a newly cloned protein whose function is not yet known (17) .

Based primarily upon studies of Stats1-3, the following model of Stat protein signaling has been proposed (reviewed in (22) ). Unstimulated Stat proteins are monomeric cytoplasmic proteins. When cells are stimulated with ligand, Stat proteins are thought to associate with phosphorylated tyrosyl residues of an activated receptor-tyrosine kinase complex through Stat Src homology (SH)-2 domains and to become tyrosyl phosphorylated. The tyrosyl phosphorylated Stat proteins form homo- and heteromeric complexes that translocate to the nucleus, where they bind specific DNA sequences and presumably regulate the transcription of appropriate target genes.

Recent findings suggest that Stat family members play a role in the regulation of genes whose transcription is modulated by GH, providing the first insight into how GH might regulate gene transcription. GH has been found to promote the tyrosyl phosphorylation of Stat1 in 3T3-F442A preadipocytes and rat liver. In these systems, Stat1 was also present in a GH-inducible factor (GHIF) complex that binds the high affinity sis-inducible element (SIE) of the c-fos promoter(23, 24) . In IM-9 cells, a 93-kDa tyrosyl phosphorylated protein antigenically related to, but distinct from, Stat1 was present in GH-induced DNA-binding complexes capable of binding to a response region from IFN- induced genes (25) . Its identity is not yet known.

Additional insight into how GH might regulate gene transcription is provided by the evidence presented here that GH activates Stat3/APRF (or an antigenically related protein) in addition to Stat1. The finding that GH stimulates tyrosyl phosphorylation of Stat3 and Stat3 binding to DNA suggests that Stat3 serves as a signaling molecule for GH that links GH receptor binding and subsequent JAK kinase activation at the membrane to regulation of gene transcription in the nucleus. Shared transcription factors among hormones and cytokines that activate JAK kinases provide an explanation for shared responses, while the ability of the different ligands to differentially recruit various Stat family members suggests mechanisms by which specificity in gene regulation could be achieved.


EXPERIMENTAL PROCEDURES

Materials

The stock of 3T3-F442A fibroblasts was kindly provided by H. Green (Harvard University) and maintained as described previously(26) . Recombinant 22-kDa human GH was a gift from Eli Lilly and recombinant murine IFN- a gift of D. Finbloom (Center for Biologics Evaluation, Bethesda, MD). Recombinant human LIF was from Promega. Mouse monoclonal (clone 4G10) antiphosphotyrosine antibody (alphaPY) was purchased from Upstate Biotechnology Inc. Anti-Stat3/APRF serum (alphaStat3) was raised against a glutathione S-transferase fusion protein containing the COOH-terminal amino acids 718-770 of Stat3(19) . Anti-Stat1 serum (alphaStat1), raised against the 39 COOH-terminal amino acids of Stat1(27) , was provided by A. Larner (Center for Biologics Evaluation, Bethesda, MD). ECL detection system was purchased from Amersham.

Methods

Immunoprecipitation and immunoblotting were done essentially as described(23) . Dilutions of 1:500 and 1:5000 were used for alphaStat1 immunoprecipitations and immunoblots, respectively, unless noted otherwise. alphaStat3 was used at a dilution of 1:100 for immunoprecipitation and 1:5000 for immunoblotting. The alphaPY was used at a concentration of 0.15 µg/ml. Electrophoretic mobility shift assays were performed as described (23) using the high affinity SIE probe (m67) of the c-fos promoter(28) , wild-type SIE probe (28) , or APRE probe from the promoter of rat alpha(2)-macroglobulin(20) . The sequence of the m67 probe differs from that of the wild-type c-fos SIE at two positions. The sequence of the wild-type SIE probe is indicated with the two changes present in the m67 probe noted in parentheses: 5`-AGCTTCAG(T)TTCCCGTC(A)AATCCCTAAAGCT-3`. The sequence of the APRE probe is as follows: 5`-GATCCTTCTGGGAATTCCTAGATC-3`.


RESULTS

In our previous investigation of GH-dependent tyrosyl phosphorylation of Stat1 in 3T3-F442A cells, we observed co-immunoprecipitation of Stat1 and a second, more rapidly migrating, tyrosyl phosphorylated protein (p85)(23) . The appearance of this 85-kDa phosphoprotein was GH-dependent. Because p85 was not recognized by alphaStat1 in Western blots (Fig. 1, lanes G-L, and (23) ), we speculated that p85 was associated with Stat1. However, experiments using serial dilutions of alphaStat1 (Fig. 1) revealed that the amount of p85 immunoprecipitated by alphaStat1 from GH-treated cells diminished throughout the dilution series, while the amount of tyrosyl phosphorylated Stat1 remained constant. This argues against the presence of p85 in the alphaStat1 immunoprecipitates because of its association with Stat1. Rather, it suggests that p85 is antigenically related to Stat1.


Figure 1: Serial dilution of alphaStat1 diminishes precipitation of p85 but not Stat1. Whole cell lysates of control (lanes A, C, E, G, I, and K) or GH-stimulated (500 ng/ml, 15 min, 37 °C) (lanes B, D, F, H, J, and L) 3T3-F442A fibroblasts were immunoprecipitated with the indicated dilutions of alphaStat1 and immunoblotted with alphaPY (lanes A-F). The blot was re-probed with alphaStat1 (lanes G-L).



p85 migrates in SDS-polyacrylamide gels at a position similar to that reported for the recently identified Stat3(15, 16, 18) . To determine if p85 might be Stat3, whole cell lysates were prepared from 3T3-F442A fibroblasts incubated with or without GH for 15 min. Proteins were immunoprecipitated with alphaStat3 and analyzed by alphaPY immunoblot. GH strongly promotes tyrosyl phosphorylation of proteins immunoprecipitable by alphaStat3 (Fig. 2, lane D). The response to GH was compared to that of LIF (a cytokine reported to promote tyrosyl phosphorylation of Stat3, (18) ) and IFN- (a cytokine reported not to affect the phosphorylation of Stat3, (16) and (18) ). As expected, LIF promotes tyrosyl phosphorylation of a protein immunoprecipitable by alphaStat3 (Fig. 2, lane F) which co-migrates with the GH-induced phosphoprotein. Surprisingly, IFN- also promotes tyrosyl phosphorylation of a protein recognized by alphaStat3 that co-migrates with the GH- and LIF-induced 85-kDa phosphoprotein, although IFN- is significantly less effective than GH or LIF. As reported previously, immunoprecipitated Stat3 appears on alphaPY immunoblots as a doublet (p89/p87, (15) ; p89/p92, (16) ). The doublet is clearly evident in the immunoprecipitates from IFN- stimulated cells (lane H); it is also seen in the immunoprecipitates of LIF and GH stimulated cells with shorter exposures of the immunoblot. alphaStat3 immunoprecipitates from LIF-stimulated cells contain a tyrosyl phosphorylated protein that migrates at a position appropriate for LIF receptor component gp130 (lane F, diffuse band most proximate to Stat3). This is consistent with the reported ability of Stat3/APRF to associate with gp130(13) .


Figure 2: GH promotes tyrosyl phosphorylation of Stat3. 3T3-F442A fibroblasts were incubated for 15 min at 37 °C with vehicle (lanes A, B, and I), GH (500 ng/ml, lanes C, D, and J), LIF (65 ng/ml, lanes E, F, and K), or IFN- (10 ng/ml, lanes G, H, and L). Lysates were prepared and proteins immunoprecipitated with alphaStat1 (lanes A, C, E, and G) or alphaStat3 (lanes B, D, F, and H-L) and immunoblotted with alphaPY. Lanes A-H, cells were lysed using a nondenaturing buffer containing 0.1% Triton X-100 as described(23) . Lanes I-L, cells were lysed by boiling in 50 mM Tris, 137 mM NaCl (pH 7.5) containing 0.75% SDS, 100 mM dithiothreitol, 100 µg/ml leupeptin, 100 µg/ml aprotinin. The lysates were diluted 10-fold with lysis buffer (23) prior to immunoprecipitation.



Cell lysates were also subjected to immunoprecipitation with alphaStat1 (Fig. 2, lanes A, C, E, and G). 91- and 85-kDa tyrosyl phosphorylated proteins are present in the alphaStat1 immunoprecipitates prepared from GH, IFN-, and LIF-treated cells. Consistent with p85 in the alphaStat1 precipitate being Stat3, the amount of tyrosyl phosphorylated 85-kDa protein immunoprecipitated by alphaStat1 and by alphaStat3 showed the same relationship for GH versus LIF versus IFN--treated cells, and p85 co-migrated with the phosphoprotein immunoprecipitated with alphaStat3. Re-probing the blot in Fig. 2with alphaStat3 revealed the co-migration of Stat3 with the 85-kDa tyrosyl phosphorylated proteins immunoprecipitated by either alphaStat1 or alphaStat3 (data not shown). The latter blot also revealed that ligand treatment affects the level of tyrosyl phosphorylation of Stat3 and not the amount of Stat3.

To confirm that the tyrosyl phosphorylated protein immunoprecipitated by alphaStat3 was precipitated through direct binding to the antibody rather than through association with another protein recognized by the antibody, alphaStat3 immunoprecipitations were performed using cell lysates boiled in the presence of SDS and dithiothreitol to dissociate protein complexes (Fig. 2, lanes I-L). A tyrosyl phosphorylated protein co-migrating with alphaStat3 is evident in the lysates from both GH- and LIF-stimulated cells. A longer exposure of the autoradiogram also revealed the presence of two bands co-migrating with the Stat3 doublet when cells were treated with IFN (data not shown). These proteins co-migrated with proteins recognized by antibody when the Western blot was reprobed with alphaStat3. The amount of Stat3 precipitated under these conditions was less than the amount precipitated under nondenaturing conditions, presumably because of the reduced ability of alphaStat3 to immunoprecipitate SDS-denatured Stat3 (data not shown). The identity of the band migrating just above Stat3 in Fig. 2, lanes I-L, is unknown. It is believed not to be Stat1 because in contrast to Stat1, it appears in alphaPY Western blots in the absence of GH.

Stat3 containing DNA-binding complexes have been shown to interact with the c-fos SIE(16) . Our previous data indicate that GH promotes the formation of SIE-binding complexes containing multiple components(23) . It was therefore logical to ask if Stat3 might be part of GH-inducible SIE-binding complexes. When nuclear extracts from control and GH-stimulated 3T3-F442A fibroblasts are incubated with a high affinity SIE probe in an electrophoretic mobility shift assay, GH promotes the formation of SIE-binding complexes containing three bands (Fig. 3A, lane B), a complex designated GHIF. Preincubation of the nuclear extracts with alphaStat3 (Fig. 3A, lane D) results in a ``supershift'' of the complex such that the most slowly migrating band (A), and to a lesser extent the middle band (B), is diminished. The most rapidly migrating band (C) is not detectably altered by the antibody. These data suggest that bands A and B contain Stat3.


Figure 3: The GH-inducible SIE binding complex contains Stat3. A, 3T3-F442A fibroblasts were incubated for 30 min with vehicle (lane A), GH (500 ng/ml, lanes B-D), LIF (65 ng/ml, lanes E-G), or IFN- (10 ng/ml, lanes H-J). Nuclear extracts from cells were preincubated for 20 min at room temperature with alphaStat1 (1:1000 dilution) (lanes C, F, and I) or alphaStat3 (1:100 dilution) (lanes D, G, and J). Electrophoretic mobility shift assays were performed using a high affinity SIE probe (m67). B, 3T3-F442A fibroblasts were incubated with vehicle (lane A) or GH (500 ng/ml, lanes B-E). Nuclear extracts were incubated with nonimmune rabbit serum (1:40 dilution, lane C), a 100-fold excess of unlabeled high affinity SIE probe (lane D) or a 100-fold excess of unlabeled wild-type SIE probe (lane E).



Preincubation of the nuclear extracts with alphaStat1 (used at a dilution that preferentially interacts with Stat1, see Fig. 1) diminishes all three bands. In contrast to results with alphaStat3, alphaStat1 preferentially reduce bands B and C (Fig. 3A, lane C). The partial reduction of band A is thought to reflect cross-reactivity of alphaStat1 with Stat3 since a similar complex was not observed using a different Stat1 antibody (data not shown). LIF and IFN- also induce SIE-binding complexes which migrate as three bands similar to those of the GH-induced complexes (Fig. 3A, lanes E and H, respectively), (^2)although the relative amounts of the three bands differ for each ligand. Whereas GH favors the formation of band B, LIF favors bands A and B, and IFN- favors band C, suggesting that IFN-, GH, and LIF do not induce identical SIE-binding complexes. As with the extracts from GH-treated cells, preincubation of the extracts from either LIF- or IFN--treated cells with alphaStat3 results in a supershift such that bands A and B are reduced, while preincubation with alphaStat1 leads to a shift of the complex with reduction primarily of bands B and C.

The DNA-binding complexes induced by GH are unaffected by preincubation with non-immune serum (Fig. 3B, lane C), indicating that the abilities of alphaStat1 and alphaStat3 to affect the mobility of the SIE complex are specific. GHIF binding is blocked by the addition of unlabeled oligonucleotide (m67) based on the high affinity SIE probe (Fig. 3B, lane D), indicating specificity of binding. The GHIF complex also recognizes the wild-type SIE sequence of the human c-fos gene since unlabeled oligonucleotide based on the wild-type SIE reduces GHIF binding (Fig. 3B, lane E). Further evidence that GH induces the binding of Stat1 and Stat3 to the wild-type SIE is provided by the finding that GH promotes the formation of complexes bound to wild-type SIE which contains three bands (Fig. 4, A and B, lane B) that co-migrate in electrophoretic mobility shift assays with the three GH-dependent bands formed using the high affinity SIE probe. The DNA-binding complexes formed using the wild-type SIE probe are present at a substantially reduced level compared to those formed using the high affinity m67 SIE probe (data not shown), as expected from its lower affinity for the sis-inducible factor. GHIF binding to the wild-type SIE probe is blocked by the addition of either unlabeled oligonucleotide based on the wild-type SIE sequence (Fig. 4A, lane D) or the high affinity SIE sequence (m67) (Fig. 4A, lane C), indicating specificity of binding. Consistent with the presence of Stat3 in the GHIF complex that binds the wild-type SIE probe, incubation of the nuclear extracts from GH-treated cells with alphaStat3 (Fig. 4B, lane D) prior to addition of the wild-type SIE probe results in a supershift of the upper two bands. Consistent with the presence of Stat1 in the GHIF complex that binds the wild-type SIE probe, incubation of the nuclear extracts from GH-treated cells with alphaStat1 (Fig. 4B, lane C) prior to addition of the wild-type SIE probe preferentially supershifted the lower two bands. The three bands present in wild-type SIE-binding complexes induced by GH were also present when the cells were incubated with LIF (Fig. 4B, lane E). In contrast, only the two lower bands were visible when the cells were incubated with IFN (Fig. 4B, lane H). The relative ratios of the different bands for each ligand were the same as those observed using the m67 high affinity SIE probe (Fig. 3A). As expected, incubation of the nuclear extracts from LIF and IFN-treated cells with alphaStat1 prior to addition of the wild-type SIE probe preferentially decreased the intensity of the lower two bands (Fig. 4B, lanes F and I) while incubation of the nuclear extract from LIF-treated cells with alphaStat3 preferentially decreased the intensity of the upper two bands (Fig. 4B, lane G). Incubation of the nuclear extract from IFN-treated cells with alphaStat3 had no detectable effect on the band pattern, consistent with the absence of band A.


Figure 4: GH induces Stat3 binding to the wild-type SIE. A, 3T3-F442A fibroblasts were incubated for 30 min with vehicle (lane A) or GH (500 ng/ml, lanes B-D). Nuclear extracts were incubated with a 100-fold excess of unlabeled high affinity SIE probe (lane C) or wild-type (wt) SIE probe (lane D). B, 3T3-F442A fibroblasts were incubated for 30 min with vehicle (lane A), GH (500 ng/ml, lanes B-D), LIF (65 ng/ml, lanes E-G), or IFN- (10 ng/ml, lanes H-J). Nuclear extracts from cells were preincubated for 20 min at room temperature with alphaStat1 (1:1000 dilution) (lanes C, F, and I) or alphaStat3 (1:20 dilution) (lanes D, G, and J). Electrophoretic mobility shift assays were performed using the wild-type SIE probe.



Stat3 was originally identified as an IL-6 dependent transcription factor that binds to an APRE in a variety of promoters of genes encoding acute phase response proteins. If the protein activated by GH is authentic Stat3, one would predict that, like IL-6, GH would promote binding of Stat3-containing nuclear protein complexes to the APRE. Fig. 5illustrates that GH does promote the binding of nuclear proteins to an oligonucleotide probe corresponding to the APRE in the rat alpha(2)-macroglobulin gene. As in the experiment using the SIE probes, multiple DNA-binding complexes were formed, with two (designated bands A and B) being readily visible in Fig. 5(lane B) and three visible in other experiments not shown. The same bands were present in extracts from IL-6-treated cells (Fig. 5, lane E). Consistent with the presence of Stat3 in bands A and B, band A was substantially reduced and band B was partially reduced in intensity when nuclear extracts from GH and IL-6-treated cells were pretreated with alphaStat3 prior to addition of the oligonucleotide probe. Preincubation of nuclear extracts with alphaStat3 caused the appearance of a faint band migrating above band A for both GH and IL-6 treated cells, consistent with alphaStat3 altering the mobility of bands A and/or B. This band, denoted by the arrow, is visible in Fig. 5for IL-6 treated cells and in the original autoradiograph for the GH-treated cells (Fig. 5, lane C). The fact that band B is not diminished by alphaStat3 to the same extent as band A suggests that this band may contain factors not present in band A. A smaller band A signal for the IL-6-treated cells compared to GH-treated cells is consistent with the decreased ability of IL-6 compared to GH to stimulate tyrosyl phosphorylation of Stat3 in these cells (data not shown). Specificity of binding is indicated by the observation that GHIF binding is blocked by the addition of unlabeled oligonucleotide based on the alpha(2)-macroglobulin APRE sequence (Fig. 5, lanes D and G).


Figure 5: GH induces Stat3 binding to the rat alpha-macroglobulin APRE. 3T3-F442A fibroblasts were incubated for 30 min with vehicle (lane A), GH (500 ng/ml, lanes B-D), or IL-6 (25 ng/ml, lanes E-G). Nuclear extracts from cells were preincubated for 20 min at room temperature with alphaStat3 (1:100 dilution) (lanes C and F) or a 100-fold excess of unlabeled APRE probe (lanes D and G). Electrophoretic mobility shift assays were performed using the rat alpha(2)-macroglobulin probe.




DISCUSSION

As currently understood, activation of Stat proteins by ligands requires tyrosyl phosphorylation and results in increased binding to specific DNA sequences. Recognition that both Stat1 (23, 24) and Stat3 (this paper) (or antigenically regulated proteins) are thus activated by GH suggests two interacting pathways linking the GH receptor and the nucleus, since Stat proteins have been shown to bind both membrane receptors and specific DNA sequences and to function as transcription factors (reviewed in (22) ). Stats 1 and 3 are the first identifiable transcription factors shown to be regulated by GH. Like GH, other hormones and cytokines (e.g. IFNalpha, IFN, prolactin, LIF, oncostatin M, IL-6, ciliary neurotrophic factor, and epidermal growth factor) have recently been shown to utilize JAK tyrosine kinases and Stat proteins in their signaling pathways(8, 11, 12, 13, 14, 27, 29) , a finding that provides an explanation for why many of these cytokines/hormones elicit similar responses. However, it also raises the question of how different ligands elicit specific responses. One source of specificity is the cell-specific distribution of the receptors. Specificity could also arise from cell-specific expression of various Stat proteins which would offer a potential explanation for why GH stimulates Stat1 and 3 in 3T3-F442A fibroblasts ((23) , this paper) but apparently not in IM-9 lymphocytes(25) . The finding that IFN- appears to activate Stat3 in 3T3-F442A fibroblasts (this paper) and NIH3T3 cells (19) but not in HepG2 (16) or IM-9 cells (25) is also consistent with cell specific expression of either Stat3 or an accessory protein. Alternatively, assay sensitivity may have been greater in the fibroblast studies, or the protein recognized by alphaStat3 in the 3T3-F442A and/or NIH3T3 cells may be antigenically related to Stat3 rather than authentic Stat3.

Cell specificity of receptor and/or Stat expression cannot account entirely for the specificity of cytokine responses. Although GH, IFN-, and LIF all activate JAK2 tyrosine kinase in 3T3-F442A fibroblasts, they clearly differ in their ability to activate Stat1 and 3, as judged by both extent of tyrosyl phosphorylation and relative amounts of the three bands in the high affinity SIE-binding complexes. Among the three ligands, GH is by far the most potent in activating JAK2 in 3T3-F442A cells, while LIF and IFN- are significantly less potent. (^3)In contrast, LIF is the most potent activator of Stat3, while IFN- is the least potent. The lack of correlation between the activation of JAK2 and Stat3 by GH, LIF, and IFN- indicates that JAK2 activation is unlikely to be sufficient for ligand-dependent stimulation of Stat3 in 3T3-F442A fibroblasts, even though co-expression of JAK2 and Stat3 in COS cells appears to be sufficient for binding of Stat3 to the c-fos SIE(19) . JAK2 could be the activating Stat3 kinase for GH, LIF and IFN- if Stat3 activation was facilitated by binding of Stat3 to phosphorylated tyrosines in the different receptors. Phosphorylated tyrosines in the receptors for GH, LIF and IFN- are likely to have different affinities for the SH2 domain of Stat3. In support of this idea, a tyrosyl phosphorylated protein corresponding in size to LIF receptor component gp130 is precipitated by Stat3 antibodies (this paper and (13) ) and Stat1 has been shown to bind to the epidermal growth factor receptor by a mechanism involving the SH2 domain of Stat1(30) . However, we do not detect tyrosyl phosphorylated bands corresponding in size to the GH receptor or to JAK2 in the alphaStat3 immunoprecipitates from GH-treated cells, suggesting a lower affinity of Stat3 for these proteins compared to LIF receptor components, fewer receptors for GH compared to LIF, or fewer Stat3 binding sites per receptor. Alternatively, a protein other than or in addition to JAK2 may be required for activation of Stat3 by GH.

Signalling specificity in response to various cytokines/hormones that activate JAK kinases and Stat proteins is most likely related to, at least in part, the ability of Stat proteins to form homo and heterodimers(4, 31) . Different combinations of Stat proteins could provide specificity for these different ligands, as has been shown for other transcription factors(32) . Supporting this is the finding that, relative to each other, the intensities of the three ligand-induced bands observed in the SIE electrophoretic mobility shift assay were different for LIF, IFN-, and GH-treated cells. These three ligand-induced bands are hypothesized by us and others (33, 16) to contain Stat3 homodimers (band A), Stat1/Stat3 heterodimers (band B), and Stat1 homodimers (band C). However, it should be noted that in contrast to what one might expect if Stat1 and Stat3 formed heterodimers, little or no Stat1 was precipitated by alphaStat3 (Fig. 2) and at a concentration of alphaStat3 that preferentially precipitates Stat1, only a small amount of Stat3 was detected in the alphaStat1 precipitate (Fig. 1, lane B). Preliminary evidence indicates that this inability to detect Stat1/Stat3 heterodimers by immunoprecipitation may be due at least in part to dissociation of Stat complexes during the solubilization and subsequent immunoprecipitation steps. An inability to detect Stat1/Stat3 heterodimers, assuming they exist, could also be explained if the ratio of heterodimers to homodimers and monomers is low or if the Stat antibodies immunoprecipitate Stat monomers and/or homodimers more effectively than heterodimers.

Stat3 was originally identified as a protein that binds to an IL-6 response element in the promoter of several IL-6 inducible acute phase response genes in liver(15, 20) . This element can confer IL-6 responsiveness to a heterologous promoter(34) . Thus, Stat3 is thought to play a role in the regulation of these genes by IL-6. The finding here that GH can also induce binding of Stat3 containing complexes to APRE raises the possibility that GH might also regulate the expression of acute phase response genes in liver. Similarly, it seems likely that Stat3 plays a role in the GH-dependent regulation of c-fos, since Stat3 binds to the SIE of the c-fos gene (this paper and (16) and (19) ) and since GH activates c-fos transcription(26) . The SIE has been shown to mediate induction of c-fos transcription in response to platelet-derived growth factor (NIH3T3 fibroblasts, (28) ) and epidermal growth factor (COS cells overexpressing Stat1, (30) ), two growth factors shown recently to activate Stats 1 and 3(16, 19, 33, 35) . Consistent with the SIE and the proteins that bind it playing a critical role in the regulation of c-fos gene expression, Curran and colleagues (^4)have observed that mutation of the SIE obliterates normal expression of Fos in transgenic mice and Fos inducibility in fibroblasts derived from them. Stimulation of Stat3 DNA binding to the SIE alone would not necessarily be expected to be sufficient for full induction of c-fos, since the serum response element of the c-fos promoter can mediate induction of c-fos by GH(36) . Whether proteins that bind to the SIE regulate c-fos expression independently or must act in concert with factors that bind to other response elements (e.g. serum response element) remains to be determined. It seems likely, however, that Stat1 and -3 participate in the regulation of genes other than c-fos. Thus, the finding that multiple Stat proteins are activated by GH not only provides insight into the molecular mechanism by which GH and other members of the cytokine receptor family are likely to regulate gene expression, but also suggests strategies to identify new target genes for GH and additional GH-dependent transcription factors.


FOOTNOTES

*
This work was supported in part by National Institutes of Health Grants R01-DK34171 (to C. C.-S. and J. S.) and RO1-A128900 (to D. E. L.), National Science Foundation Grant IBN9291667 (to J. S.), and Cancer Research Institute Grant (to D. E. L.). G. S. C. and D. J. M. contributed equally to the work. 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.

§
NIH Predoctoral Trainee (GM07315).

Fellow of the Consejo Superior de Investigaciones Cientificas.

**
Pew Scholar.

§§
To whom correspondence should be addressed: Dept. of Physiology, University of Michigan Medical School, Ann Arbor, MI 48109-0622. Fax: 313-936-8813.

(^1)
The abbreviations used are: GH, growth hormone; APRF, acute phase response factor; LIF, leukemia inhibitory factor; IL, interleukin; IFN, interferon; SIE, sis-inducible element; GHIF, GH-inducible factor; APRE, acute phase response element; Stat, signal transducers and activators of transcription; alphaPY, antiphosphotyrosine antibody.

(^2)
While the presence of band A is difficult to detect using extracts from IFN-treated cells (Fig. 3A, lane H) due to the intensity and diffuseness of band B, band A is detectable when the extracts are treated with alphaStat1 (Fig. 3A, lane I) which diminishes the intensity of band B.

(^3)
L. S. Smit, D. J. Meyer, L. S. Argetsinger, G. W. Hsu, M. G. Myers, M. F. White, N. Billestrup, G. Norstedt, and C. Carter-Su, manuscript in preparation.

(^4)
T. Curran, personal communication.


ACKNOWLEDGEMENTS

We thank Drs. L. Argetsinger, L. Smit, J. VanderKuur, and K. Rosenspire for helpful discussions.


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