Two Tandemly Linked Interferon-
-Activated Sequence Elements in the Promoter of Glycosylation-Dependent Cell Adhesion Molecule 1 Gene Synergistically Respond to Prolactin in Mouse Mammary Epithelial Cells
Zhaoyuan Hou,
Sunil Srivastava,
Meenakshi J. Mistry,
Matthew P. Herbst,
Jason P. Bailey and
Nelson D. Horseman
Department of Molecular and Cellular Physiology, University of Cincinnati, Cincinnati, Ohio 45267
Address all correspondence and requests for reprints to: Nelson D. Horseman, Department of Molecular and Cellular Physiology, University of Cincinnati, 231 Albert Sabin Way, Cincinnati, Ohio 45267-0576. E-mail: nelson.horseman{at}uc.edu.
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ABSTRACT
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Previously, we reported that glycosylation-dependent cell adhesion molecule 1 (GlyCAM 1) was a novel target for prolactin (PRL) in the mouse mammary gland. However, the signaling pathway by which PRL regulates GlyCAM 1 expression has not been specified. In the present study, we showed that PRL induced GlyCAM 1 expression in primary mammary epithelial cells of mice through the Janus kinase 2/signal transducer and activator of transcription 5 (Stat5) pathway. Deletion and site-directed mutagenesis analyses of the GlyCAM 1 promoter demonstrated that the two tandemly linked Stat5 binding sites [interferon-
-activated sequence 1 and -2 (GAS1 and GAS2)] in the proximal promoter region were crucial and synergistically responded to PRL. GAS2, a consensus GAS site, was essential and, by itself, weakly responded to PRL, whereas GAS1, a nonconsensus site, failed to respond to PRL but was indispensable for the maximal activity of the GlyCAM 1 promoter. Gel shift assays showed that probe containing GAS1 and GAS2 bound two Stat5 complexes, which represent Stat5 dimer and tetramer, respectively, while GAS2, by itself, bound Stat5 as a dimer only, and GAS1 showed no apparent binding activity. Interruption of tetramer formation by mutation of a tryptophan to alanine (W37A), and a leucine to serine (L83S) in the N terminus of Stat5A attenuated the synergistic effect between the two tandemly linked GAS sites. Overexpression of W37A and L83S mutants in primary mammary epithelial cells suppressed endogenous GlyCAM 1 expression.
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INTRODUCTION
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THE JANUS KINASE 2 (Jak2)/signal transducer and activator of transcription 5 (Stat5) signaling pathway is the best defined cellular signal transduction pathway mediating prolactin (PRL) function (1, 2). Jaks and Stats were discovered by studying the actions of interferons (IFNs) (3). In mammals, four family members of Jak kinases, Jak1, 2, 3 and Tyk2, have been identified. Jak2 is the major kinase associated with PRL receptor and mediates the downstream signal to the target genes (4, 5). Stat proteins currently have seven members identified from mammals, Stat14, Stat5A, Stat5B, and Stat6 (3, 6). Stat5A and B are the primary transcription factors for PRL signaling in the mammary gland and other tissues (7, 8, 9). Specifically, upon PRL binding to its cognate receptors, Jak2, which is associated with the PRL receptor in the absence of ligand, is activated by phosphorylation. Activated Jak2 phosphorylates PRL receptors and creates docking sites for proteins containing SH2 domains, including Stat5 proteins, which are phosphorylated by activated Jak2 after binding to the C termini of PRL receptors. The activated Stat5 proteins dimerize, dissociate from the PRL receptor, translocate to the nucleus, and bind to IFN
-activated sequence (GAS, TTCNNNGAA) to modulate the target gene transcription (2). Additionally, it has been reported that Stat proteins can bind to tandemly linked GAS elements as tetrameric complexes and synergistically enhance target gene transcription (10, 11). Tetramer formation can stabilize the binding of Stat dimers to low-affinity, tandem GAS sites by decreasing the off-rate of the DNA-protein complexes (11, 12, 13).
It has been well documented that PRL stimulates most of its target genes through Jak2 and Stat5 molecules, although other signaling molecules activated by PRL such as MAPK, phosphatidylinositol 3-kinase, or Src kinase may play certain roles in modulating transcription of these genes (14, 15, 16, 17). Genes encoding milk proteins such as ß-casein, whey acidic protein, and ß-lactoglobulin are well characterized PRL targets in the mammary gland. All milk protein genes identified to date contain at least one active Stat5 binding site (GAS) in their promoter regions (14, 18). In tissue culture, Stat5 binding to the GAS site is indispensable for hormonal induction of ß-casein gene expression (19, 20). Experiments in transgenic mice have demonstrated that Stat5 binding sites in whey acidic protein and lactoglobulin gene promoters are critical for maximal gene activity and PRL responsiveness in vivo (21, 22, 23).
We previously demonstrated that PRL was a potent stimulator of glycosylation-dependent cell adhesion molecule 1 (GlyCAM 1) expression in mouse mammary gland and in HC11 mammary epithelial cells (24). The signaling pathway by which PRL induces GlyCAM 1 expression has not been specified. GlyCAM 1 was originally identified from murine high-endothelial venules as a physiological ligand for L selectin (25). Later studies revealed that GlyCAM 1 mRNA was detectable in the virgin mammary gland and highly induced during pregnancy and lactation, and the protein was secreted into milk. The mRNA expression pattern of GlyCAM 1 was similar to that of ß-casein, being induced during pregnancy and lactation and suppressed by pup removal (26). All these features define GlyCAM 1 molecule as a milk-like protein. However, unlike typical milk protein genes, GlyCAM 1 expression is not restricted to the mammary tissues. In addition to high-endothelial venules and mammary gland, GlyCAM 1 mRNA is abundantly expressed in adrenal and salivary glands and is detectable in lung, uterus, and ovary (our unpublished data). In PRL knockout mice, GlyCAM 1 expression in the mammary gland was substantially reduced and was restored by pituitary graft (24).
The 5'-flanking region of the GlyCAM 1 genomic sequence in the promoter region has a number of potential Stat5 binding sites. An 800-bp fragment of GlyCAM 1 promoter contains four potential Stat5 binding sites (GAS), two of which are tandemly linked and conserved in all members of GlyCAM 1 family. This fragment was shown to respond to PRL stimulation in Chinese hamster ovary (CHO) cells (24). Therefore, we speculate that PRL induces GlyCAM 1 expression through the Jak2/Stat5 pathway, and that the two conserved GAS sites might respond to PRL. In this study, we report that Jak2 and Stat5 critically mediated PRL induction of GlyCAM 1 expression in the mammary epithelial cells, and that the two tandem GAS sites in the proximal promoter region cooperatively responded to PRL stimulation.
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RESULTS
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PRL Is Sufficient to Induce GlyCAM 1 Expression in Mouse Primary Mammary Epithelial Cells
Previously we reported that GlyCAM 1 expression in the mammary gland was suppressed in PRL knockout mice and was induced by high levels of PRL. PRL combined with insulin and dexamethasone stimulated GlyCAM 1 expression in HC11 cells, an established mouse mammary epithelial cell line (24). These results indicate that PRL is necessary for GlyCAM 1 expression both in vivo and in vitro. However, it is not clear whether PRL is sufficient to induce GlyCAM 1 expression in primary mammary epithelial cells. To examine direct regulatory stimulation of GlyCAM 1 expression by PRL, experiments in mouse primary mammary epithelial cells (PMECs) were performed. PMECs were isolated from the mammary glands of virgin mice (ICR, 8 wk old). For PRL treatment, PMECs were plated on conventional plastic tissue culture plates, or on basement membrane reconstituted in six-well plates to allow formation of three-dimensional epithelial organoids (27). After 24 h of treatment with ovine PRL (oPRL, 5 µg/ml), PMECs were collected and assayed for GlyCAM 1 mRNA expression by RT-PCR. For time course experiments, the medium containing oPRL (1 µg/ml) was changed every 24 h. The amount of total RNA used for RT-PCR was normalized by glyceraldehyde-3-phoshate dehydrogenase (GAPDH) level. GlyCAM 1 mRNA was highly induced in PMECs by PRL, and no apparent difference was observed between conventional and reconstituted basement membrane cultures (Fig. 1A
). PRL stimulated GlyCAM 1 expression in a dose-dependent manner (Fig. 1B
), and GlyCAM 1 expression level remained elevated after 24 h (Fig. 1C
). These results show that PRL is sufficient to induce GlyCAM 1 expression in mouse mammary epithelial cells.

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Fig. 1. PRL Is Sufficient to Induce GlyCAM 1 Expression in Mouse PMECs
A, PMECs (5 x 105/well) were seeded in conventional plastic tissue culture plates or in basement membrane reconstituted in six-well plates. After 24 h oPRL (5 µg/ml) was added into the medium for another 24 h. PMECs were harvested and GlyCAM 1 expression was analyzed by RT-PCR. Experiments were repeated twice with similar results. B, Dose-response relationship for GlyCAM 1 expression was tested in PMECs cultured on plastic tissue culture plates. Various concentrations of oPRL were added for 24 h, and GlyCAM 1 mRNA was quantified by RT-PCR. The graph showed the results from one experiment in triplicate. C, GlyCAM 1 mRNA levels remained elevated after 24 h. PMECs were stimulated with oPRL at a concentration of 1 µg/ml and the medium was changed every 24 h. GlyCAM 1 expression levels were measured by RT-PCR. Quantification of the PCR amplifications was analyzed by QuantityOne software (Bio-Rad Laboratories, Hercules, CA), and data shown in panels B and C were in arbitrary units (ratio of GlyCAM 1 to GAPDH). Experiments were repeated twice in triplicate, and data shown are averages from one experiment.
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Jak2 and Stat5 Are Indispensable for PRL-Dependent GlyCAM 1 Expression
It has been well documented that Jak2 and Stat5 are the major signaling molecules mediating PRL induction of target gene expression in a variety of tissues. Thus, we tested the roles of Jak2 and Stat5 in GlyCAM 1 expression after PRL stimulation. PMECs were cultured in six-well plates and treated with oPRL (1 µg/ml), combined with genistein (a general tyrosine kinase inhibitor, 25 µM), or AG490 (a Jak2 inhibitor, 20 µM) for 24 h. AG490 and genistein have been shown to effectively inhibit PRL-activated Jak2 activity in mammary epithelial cells (28, 29, 30). GlyCAM 1 mRNA levels were tested by RT-PCR. The results showed that either AG490 or genistein blocked PRL stimulation of GlyCAM 1 expression (Fig. 2A
). These observations indicate that a tyrosine kinase related to Jak2 is part of the signal transduction pathway for GlyCAM 1 induction. To test the role of Jak2 in PRL induction of GlyCAM 1 expression, CHO D6 cells were cotransfected with dominant negative Jak2 constructs (Jak KD and Jak829) and mouse GlyCAM 1 promoter reporter Gly800-Luc. GlyCAM 1 promoter activity was induced 5-fold in the presence of control vector, whereas Jak KD and Jak829 effectively attenuated the PRL stimulation of the promoter activity (Fig. 2B
). These studies indicate that Jak2 is crucial for PRL induction of GlyCAM 1 expression in mouse mammary epithelial cells.

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Fig. 2. Jak2 Is Crucial for PRL-Dependent GlyCAM 1 Expression
A, Either AG490 or genistein blocked PRL stimulation of GlyCAM 1 expression in PMECs. PMECs were cultured in six-well plates and stimulated with oPRL (1 µg/ml), combined with either genistein (a general tyrosine kinase inhibitor, 25 µM), or AG490 (a Jak2 inhibitor, 20 µM) for 24 h. The GlyCAM 1 expression levels were measured by RT-PCR. B, Dominant negative Jak2 constructs Jak KD and Jak829 effectively blocked the PRL stimulation on GlyCAM 1 promoter in CHO D6 cells. Twenty four hours before transfection, CHO D6 cells were seeded at 0.5 x 105 cells per well in 12-well plates. ß-Galactosidase plasmids (50 ng), 250 ng of GlyCAM 1 promoter reporters, and 50 ng of JakKD or Jak829 plasmids or parental vectors together with 1 µl Fugene 6 agent were added into each well. After 4 h incubation with plasmids and Fugene 6 agent, cells were stimulated by oPRL (0.5 µg/ml) for 20 h and harvested in 200 µl 1x lysis buffer. Cell lysis (4 µl and 20 µl) was used for measuring luciferase and ß-galactosidase activities, respectively. Promoter activities were represented by relative units (ratio of luciferase activity to ß-galactosidase number), unless indicated specifically.
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To test the role of Stat5 in GlyCAM 1 expression, C-terminal truncated Stat5A and B [dominant-negative (DN) Stat5A and DN Stat5B] were cloned from mouse mammary epithelial cells and sequenced. These truncated isoforms lack the C-terminal transactivation domains and function in a dominant negative fashion (31). DN Stat5A or DN Stat5B suppressed the PRL stimulation of Gly800-Luc activity in CHO D6 cells (Fig. 3A
). This study indicated that either Stat5A or B can mediate PRL signaling to the GlyCAM 1 promoter, which was further supported by data obtained from COS7 cells. COS7 cells express negligible levels of endogenous Stat proteins and have been successfully used to reconstitute a PRL signaling pathway in the presence of exogenous PRL receptor and Stat5 (7, 32, 33). In the absence of recombinant Stat5, PRL failed to stimulate Gly800-Luc activity in COS7 cells cotransfected with PRL receptor cDNA only. In the presence of PRL receptor and either Stat5A or B, PRL significantly induced Gly800-Luc luciferase activity in COS7 cells (Fig. 3B
). Therefore, Jak2 and Stat5 appear to be indispensable for PRL stimulation of GlyCAM 1 expression.

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Fig. 3. Either Stat5A or B Effectively Transduces PRL Stimulation of Gly800-Luc in Vitro
A, C-terminal truncated Stat5A and B suppressed the PRL stimulation on GlyCAM 1 promoter in CHO D6 cells. Experiments were carried out essentially the same as described in Fig. 2B . B, In the presence of PRL receptor, either Stat5A or B could mediate PRL stimulation of GlyCAM 1 promoter in COS7 cells. One day before transfection, COS7 cells (0.5 x 105 cells per well) were seeded in 12-well plates. Pigeon PRL receptor plasmids (50 ng), 50 ng of ß-galactosidase plasmids, 250 ng of Gly800-Luc plasmids, and 50 ng of wild-type Stat5A or Stat5B or parental vectors were used for transfection per well. Transfection and assays were performed essentially the same as in Fig. 2B .
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The Two Tandem Stat5 Binding Sites in the Proximal Promoter Synergistically Respond to PRL Stimulation
Gly800-Luc contains an 800-bp fragment of the GlyCAM 1 proximal promoter, including four potential Stat5 binding sites (GAS). To identify the active PRL response elements, promoter deletion and site-directed mutagenesis were performed. Promoter-reporter constructs Gly254-Luc (deletion of GAS3 and -4) and Gly104-Luc (deletion of all four GAS sites) were generated by PCR (Fig. 4A
). The luciferase activities of these three constructs stimulated by PRL were measured in CHO D6 cells. Gly254-Luc responded to PRL in a level similar to that of Gly800-Luc, whereas Gly104-Luc did not respond to PRL stimulation (Fig. 4B
). These results suggest that GAS3 and GAS4 do not mediate a response to PRL stimulation, and that positive regulatory elements reside in the fragment between -254 and -104, containing GAS1 and GAS2.

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Fig. 4. The Two Tandem Stat5 Binding Sites in the Proximal Promoter Cooperatively Respond to PRL Stimulation
A, Diagram showing the GlyCAM 1 promoter luciferase reporters and potential GAS elements found in the GlyCAM 1 promoter region. B, PRL response elements resided in the promoter region between -254 and -104 containing GAS1 and GAS2. Promoter activities were represented by fold induction (the ratio of PRL stimulated to the blank control). C, GAS1 and GAS2 synergistically responded to PRL stimulation. Mutations were carried out by site-directed mutagenesis kit, and the mutants were confirmed by sequencing. Mutation of GAS1 (ggCCCAcgA) eliminated about 70% of PRL responsiveness compared with wild-type p254, and mutation of GAS2 (ggCCCAcgA) completely abolished the PRL stimulation of GlyCAM 1 promoter activity.
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GAS1 and GAS2, spaced by seven nucleotides, are located between -110 and -135 of the mouse GlyCAM 1 gene promoter. GAS2 is a consensus site (TTCCCAGAA), whereas GAS1 has one mismatch (TTCCCAaAA). To determine whether either GAS1 or GAS2, or both, are required for PRL responsiveness, site-directed mutagenesis was performed. Mutation of GAS1 (ggCCCAcgA) eliminated about 70% of PRL responsiveness compared with wild-type Gly800-Luc (Fig. 4C
). This indicates that GAS1, the nonconsensus site, is needed for maximal response to PRL. In contrast, mutation of GAS2 (ggCCCAcgA) completely eliminated the PRL stimulation of Gly800-Luc activity. These results reveal that a synergy exists between the two tandem GAS sites in responsiveness to PRL; GAS2 is essential and weakly responded to PRL; GAS1, by itself, is unable to respond to PRL but is indispensable for the maximal responsiveness to PRL.
Stat5A Mutants W37A and L83S Inhibit the Synergy Between GAS1 and GAS2 in Response to PRL Stimulation
We speculated that formation of Stat5 tetrameric complexes may mediate the synergy between GAS1 and GAS2 in response to PRL. It has been reported that Stat proteins bind to tandemly linked GAS motifs as tetrameric complexes and synergistically enhance target gene transcription (10, 11). Tetramer formation can stabilize the binding of Stat dimers to low-affinity tandem GAS sites by decreasing the off rate of the DNA-protein complexes (11, 12, 13). Two GAS sites spaced 11 nucleotides apart in the PRRIII region of the IL-2 receptor
-gene promoter were shown to be required simultaneously for the responsiveness to IL-2 stimulation by binding Stat5 tetrameric complex (12). The two tandem GAS sites, spaced by seven nucleotides in the GlyCAM 1 promoter region, are comparable to that of IL-2 receptor
-gene. The presence of both sites is necessary for the full functional activity of Gly800-Luc activity (Fig. 4C
). To test whether Stat5 tetramer mediates the synergy between GAS1 and GAS2, two Stat5A mutants, W37A and L83S, were made. The invariant tryptophan in the N terminus of Stat proteins (W37) was shown to be a crucial residue involved in Stat dimer-dimer interaction (34). It was shown that mutation of the conserved tryptophan to alanine (W37A) interrupted Stat5 tetramer formation and suppressed the IL-2 stimulation on IL-2 receptor
-chain expression (12). The leucine residue L83, conserved in all Stat proteins, is one of the key residues in the coiled-coil region. Mutation of the leucine L83 to serine is expected to interfere with the proper folding of the N terminus of Stat5 and to deform its hook-like structure (34). Therefore, we predicted that either W37A or L83S mutants could suppress PRL stimulation on Gly800-Luc activity containing the two tandem GAS sites by disrupting Stat5 tetramer binding. Luciferase assays showed that either W37A or L83S effectively suppressed Gly800-Luc activity in responding to PRL (Fig. 5A
); moreover, W37A and L83S effectively suppressed the responsiveness of the PRE-Luc reporter to PRL. The PRE-Luc reporter contains six tandem copies of GAS sequence identified from the rat ß-casein promoter, and adjacent GAS elements are spaced by 11 nucleotides (Fig. 5B
). These studies indicate that interference with the N-terminal structure of Stat5 can block the synergy between two tandem GAS elements in responding to PRL. Furthermore, Stat5B W37A displayed similar inhibition on GlyCAM 1 promoter reporter activity (data not shown). Because Stat5A and Stat5B behave similarly, the following experiments were done with Stat5A only.

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Fig. 5. Stat5 Mutants W37A and L83S Inhibit the Synergy between GAS1 and GAS2 in Response to PRL Stimulation
A, Stat5A mutants W37A and L83S attenuated PRL stimulation of GlyCAM 1 promoter containing the two tandem GAS sites. B, W37A and L83S effectively suppressed the responsiveness of the PRE-Luc reporter to PRL. The PRE-Luc reporter contains six tandem copies of GAS sequence identified from the rat ß-casein promoter, and adjacent GAS sequences are spaced by 11 nucleotides. W37A, L83S, and parental vectors (50 ng) were transected into each well. Luciferase and ß-galactosidase assays were carried out as described in Fig. 2B .
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Probe Containing the Two Tandem GAS Sites Binds Stat5A as Dimer and Tetramer
To confirm the physical association between Stat5 tetrameric complexes and the two tandem GAS elements in the GlyCAM 1 promoter, gel shift assays were performed with probes containing GAS1 or GAS2, or both together. Probes were derived from the wild-type or mutated GlyCAM 1 promoters and the sequences were shown in Fig. 6A
. Probe cas contains a GAS element derived from the rat ß-casein gene promoter and has been shown to bind Stat5 as a dimer (5, 7). PRRIII contains two tandem GAS elements derived from the IL-2 receptor
-gene promoter and was shown to bind Stat5 as a tetramer complex (35). Therefore, probes cas and PRRIII were used as positive controls for Stat5 dimer and tetramer complexes, respectively. Flag-tagged wild-type Stat5A, W37A, and L83S were overexpressed in COS7 cells, isolated by immunoprecipitation with anti-flag M2 affinity gel, and eluted with 3xflag peptides. The recombinant proteins were tested by Western blot using anti-Stat5A antiserum or antiphosphotyrosine antibody. All three proteins were tyrosine phosphorylated in a PRL-dependent manner, and the mutations of the tryptophan to alanine and the leucine to serine did not affect the tyrosine phosphorylation upon PRL stimulation (Fig. 6B
). Gel shift assays showed that G12 containing the tandem GAS sites bound two complexes of wild-type Stat5A (Fig. 6C
, left lane), both of which were supershifted by adding anti-Stat5 antibody (Fig. 6C
, right lane). The slow complex showed a migrating rate similar to that of PRRIII and was likely to represent Stat5 tetramer, whereas the fast complex showed a migrating rate similar to that of cas probe and has been confirmed as a dimer complex (Fig. 7A
). Probe G2 bound the fast migrating complex only, whereas G1 showed no apparent binding (Fig. 7A
). As expected, mutants W37A and L83S formed the fast migrating complexes only, and no tetramer complex was seen (Fig. 7B
).

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Fig. 6. The Two Tandem GAS Sites Specifically Bind Stat5 as Two Complexes
A, Probes G12, G1, and G2 were derived from the wild-type GlyCAM 1 promoter and the mutants. Probe cas contains a GAS element derived from the rat ß-casein gene promoter and has been shown to bind Stat5 as a dimer. PRRIII contains two tandem GAS elements derived from the IL-2 receptor -gene promoter and was shown to bind Stat5 as a tetramer complex. Probes were labeled with [32P]dCTP by klenow fill-in. B, Wild-type Stat5A, W37A, and L83S proteins were tyrosine phosphorylated in a PRL-dependent manner, and the mutations of the tryptophan to alanine and the leucine to serine did not prohibit tyrosine phosphorylation level. Flag-tagged wild-type Stat5A, W37A, and L83S were overexpressed in COS7 cells, and the proteins were isolated by immunoprecipitation with anti-flag M2 affinity gel and eluted with 3xflag peptides. The proteins were tested by Western blot using anti-Stat5A antiserum and antiphosphotyrosine antibody. C, G12 containing the two tandem GAS sites bound two complexes of wild-type Stat5A. Flag-tagged wild-type Stat5A (50 ng) was used for gel shift assays in 20 µl reaction containing 20,000 cpm of probes, 2 µg poly(dIdC), 10 mM Tris HCl (pH 7.5), 10 mM HEPES, 50 mM KCl, 1.25 mM dithiothreitol, 1.1 mM EDTA, and 12% glycerol. Supershift was performed by preincubating the Stat5 proteins with 1 µg of the anti-Stat5 antibody for 15 min before adding G12 probes. The results showed that GAS12 bound two complexes, the slowly migrating and the fast migrating complexes (left lane), and both complexes were supershifted by anti-Stat5 antibody (right lane).
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Fig. 7. Stat5A Mutants W37A and L83S Form Dimer Complex on the Two Tandem GAS Sequences
A, G12 bound Stat5A as dimer and tetramer complexes. The slow complex showed a migrating rate similar to that of PRRIII and was likely to represent Stat5 tetramer. The fast complex showed a migrating rate similar to that of cas probe which binds Stat5 protein as a dimer. Probe GAS2 bound the fast migrating complex only, whereas GAS1 showed no apparent binding. B, As expected, mutants W37A and L83S bound to G12 as the fast migrating complexes and no tetramer complexes were seen.
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Stat5A Mutants W37A and L83S Suppress Endogenous GlyCAM 1 Expression in PMECs
Stat5A mutants W37A and L83S were shown above to suppress GlyCAM 1 promoter activity by interrupting Stat5 tetramer-DNA complex. To test the significance of Stat5 tetramer in mammary epithelial cells, W37A or L83S or control vectors or ß-galactosidase plasmid (2 µg/well) were transfected into PMECs cultured on six-well plates with lipofectamine plus reagent. The transfection efficiency was indicated by X-gal staining (Fig. 8A
). PMECs was stimulated with oPRL as described in Fig. 1
. After 24 h of transfection, cells were stained for ß-galactosidase activity. Transfection efficiency was represented by percentage of positive stained cells. Transfection efficiency was affected by the cell density and batch of preparations. In Fig. 8A
, 30 (±4.1) % of PMECs were positively stained. The GlyCAM 1 mRNA levels were measured by RT-PCR. Consistent with the reporter and gel shift data, both W37A and L83S suppressed the induction of endogenous GlyCAM 1 mRNA expression, whereas wild-type Stat5A induced GlyCAM 1 expression (Fig. 8B
).

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Fig. 8. Stat5A Mutants W37A and L83S Suppress Endogenous GlyCAM 1 Expression in PMECs
A, Transfection efficiency was indicated by X-gal staining and 30 (±4.1) % of PMECs were positively stained. X-gal staining was done using LacZ reporter assay kit following the manufacturers protocol. ß-Galactosidase plasmids (2 µg/well in six-well plates) were transfected into PMECs with lipofectamine plus reagent. After 24 h, cells were washed with PBS and fixed with 1x fixative solution for 10 min. After three washes with PBS, X-gal staining solution was added to each well and incubated at 37 C for 4 h. Positively stained PMECs were counted under a light microscope. Arrow showed a positively stained epithelial cell. B, Wild-type Stat5A, W37A, L83S, and parental vectors (2 µg/well) were transfected into PMECs cultured in six-well plates with lipofectamine plus reagent, and cells were stimulated by PRL (1 µg/ml) for 24 h. GlyCAM 1 mRNA levels were measured by RT-PCR. As expected, either W37A or L83S suppressed the endogenous GlyCAM 1 expression.
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DISCUSSION
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Previously we demonstrated that PRL was a potent stimulator of GlyCAM 1 expression in the mouse mammary gland and in HC11 mammary epithelial cells (24). However, it was not clear whether PRL alone could induce GlyCAM 1 transcription in mammary epithelial cells. Primary mammary epithelial cells are an ideal model with which to study the interplay between lactogenic hormones and growth factors on the mammary epithelial cell proliferation, differentiation, and lactation (27, 36). When cultured in reconstituted basement membrane (Matrigel), PMECs form spherical, cystic structures called mammospheres that closely resemble the in vivo architecture. In this study, we showed that PRL alone induced GlyCAM 1 expression in both conventional cultured PMECs and mammospheres (Fig. 1
). The different responsiveness to PRL stimulation between PMEC and HC11 cells may be indicative of intrinsic property changes in the evolution of HC11 cells in culture. These changes imply limitations on using HC11 cells as a model system to study the effects of hormones and growth factors on mammary epithelial cell function.
It has been well documented that Jak2 and Stat5 are the major molecules mediating PRL induction of target gene expression in a variety of tissues, including the mammary gland (37, 38). Consistently, we showed that PRL induced GlyCAM 1 expression in mammary epithelial cells through a Jak2/Stat5 pathway. Stat5 was identified as a PRL-induced mammary gland transcription factor (7). The two genes encoding Stat5A and B are colocalized to murine chromosome 11, and the proteins are approximately 95% identical in amino acid sequences (8, 9). Mice bearing disrupted Stat5A, Stat5B, or both have been generated. Stat5A-/- mice display defective mammary gland development and lactogenesis, whereas Stat5B-/- mice exhibit a loss of sexually dimorphic growth due to defective GH signaling (39, 40). The differential effects of Stat5A and B deletion may be due to protein levels, extent of activation, and/or an isotype-specific function (41). Analysis of milk protein expression levels in Stat5A-/- and Stat5B-/- mice showed that Stat5A and B have overlapping and distinctive functions in the mammary gland. Although no data are available on GlyCAM 1 expression in these mouse models, we showed that in vitro either Stat5A or B efficiently and crucially mediated PRL stimulation on GlyCAM 1 promoter activity.
Both Jak1 and Jak2 were reported to associate with the PRL receptor and are activated upon PRL stimulation; however, the downstream signaling of Jak1 is not clear (42, 43). By using mutant cell lines defective in Jak1 or Jak2, it was shown that Jak2 is absolutely required for PRL-dependent phosphorylation of PRL receptor, activation of Stats, and induction of milk protein gene promoters (4, 5). In addition to Stat5, Stat1 and Stat3 can be activated upon PRL stimulation (44, 45). It has been shown that Stat1 specifically transduces interferon stimulation in vivo (46). Stat3 is specifically activated at the onset of the involution in the mammary gland (47). No PRL-target genes identified to date are regulated through Stat3. Moreover, it is not clear whether other signaling molecules activated by PRL such as MAPKs, Src kinase, or phosphatidylinositol 3-kinase might play roles in inducing GlyCAM 1 expression in vivo (15, 16, 17). Obviously, Jak2 and Stat5 play crucial roles in mediating PRL signaling to GlyCAM 1 promoter.
The simultaneous presence of GAS1 and GAS2 is required for the full functional promoter activity upon PRL stimulation (Fig. 4C
). Although this is not the first example showing a synergy between two tandemly linked GAS elements in natural promoters, our results clearly showed the differential roles of the two GAS sites in the cooperation. GAS2 is a consensus site and responds weakly to PRL, whereas GAS1 is a nonconsensus site containing a one-nucleotide mismatch. By itself, GAS1 cannot respond to PRL. However, GAS1 is indispensable for the full functional promoter activity. These observations imply that formation of Stat5 tetramer-DNA complex on the GlyCAM 1 promoter is a sequential process: GAS2 may bind Stat5 dimer at a relatively high affinity. The GAS2-Stat5 complex would then function like an anchor. GAS1 binds Stat5 dimer only weakly, but the GAS1-Stat5 complex can be stabilized by interacting with the GAS2-Stat5 complex.
Stat tetramer formation is mediated by highly conserved N-terminal regions. Deletion of the N-terminal approximately 100 residues of Stat1 and Stat4 abolishes cooperative binding to DNA as tetramers but does not affect dimer formation and DNA binding (10). Crystallographic analysis revealed that the N terminus of Stat4 is helical, and eight helices in total are packed together to form a hook-like structure (34). Tryptophan 37 (W37 in mouse Stat5A and B) in helix 4, conserved in all Stat proteins, is a central anchor residue at the interface of the hooks and was shown to be a crucial residue in the tetramer formation. Mutation of the tryptophan to alanine (W37A) can interrupt tetramer formation and suppress target gene transcription. The results obtained from the gel shift, reporter assays, and PMECs on the two tandem sites in GlyCAM 1 promoter provide affirmative support for the importance of the W37 residue. W37A and L83S mutants inhibited formation of Stat5 tetramer-DNA complex and suppressed GlyCAM 1 promoter activity when stimulated by PRL. The conserved leucine residue L83 in helix 7 is one of the key residues in the antiparallel coiled-coil region. Mutation of the leucine L83 to serine is supposed to interfere with the proper folding of the N terminus of Stat5 and to deform its hook-like structure. These studies indicate that the proper hook-like structure is essential for Stat tetramer formation. It has been suggested that N termini of Stat proteins are important for protein-protein interaction, nuclear translocation, and deactivation (48). The coiled-coil region in the N terminus was shown to involve protein-protein interactions such as recruiting cofactors (34). However, it was not clear how much these effects contribute to GlyCAM 1 suppression by W37A and L83S mutants.
A number of genes containing tandemly linked GAS elements in their promoter regions have been identified to form Stat tetramer-DNA complexes. Two tandem GAS sites, spaced by 10 nucleotides in the first intron of the IFN
gene, stably bind Stat4 complexes (10). Similarly, Stat1 can bind two weak tandem GAS sites in the promoter region of the mig chemokine gene and effectively activates transcription by forming a tetrameric complex (49, 50). Stat5 was observed to form tetramer complexes in the IL-2 receptor
-gene promoter and CIS gene promoter (13, 35). Moreover, a few other genes contain putative tandemly linked GAS elements in the promoters such as rat ß-casein, pim-1, cp35, and mouse Bcl-x (51, 52). Therefore, Stat tetramerization on tandemly linked GAS sites may have significant biological impacts. Stat tetramers have longer half-life time and stronger transactivation activity than that of Stat dimers (35). Consensus GAS elements (TTCNNNGAA) are not required for Stat5 tetramerization (51). There are GAS motifs that cannot bind Stat5 dimers in vitro but can bind Stat5 tetramer when interacting with a second neighboring GAS motif. In vitro binding selection showed that Stat5 tetramer can bind a broader range of sequences than can Stat5 dimers (51). The less-stringent nucleotide preferences by Stat tetramers can tolerate some mutation in the GAS elements during evolution and may reflect adaptability of the Jak/Stat pathway to natural selection pressures. Second, Stat tetramer/DNA binding has a higher intrinsic potential of distinguishing individual Stats (10, 13). In the IFN
gene promoter, different Stat proteins bind distinct tandem GAS pairs (10). In vitro selection experiments demonstrated that Stat5A and Stat5B homodimers have no apparent DNA binding preference and have similar DNA binding specificities. However, Stat5A favors tetramer formation and binds to tandemly linked GAS elements, whereas Stat5B prefers binding DNA as dimers (51). The difference in forming tetrameric complexes between Stat5A and Stat5B may explain, to some extent, the different phenotypes observed in the mammary gland of Stat5A-/- and StatB-/- mice (41). Third, Stat tetramerization may create new surfaces, which may interact with different coregulators than Stat dimers (35). Although new factors associated with Stat tetramer have not yet been identified, it would be an interesting question to probe in the future.
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MATERIALS AND METHODS
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Cell Cultures
Primary mouse mammary epithelial cells (PMECs) were prepared from virgin mice (ICR, 8 wk of age) by enzymatic digestion and differential trypsinization, according to the methods of Imagawa with modifications (27). In brief, excised mammary glands were minced with razor blades and then transferred into 50-ml conical tubes and digested in M199 media containing BSA (2.5 mg/ml, fraction V, Sigma Chemical Co., St. Louis, MO), 0.1% collagenase type III (Worthington Biochemical Corp., Freehold, NJ), 2 mM L-glutamine, penicillin (50 U/ml)/streptomycin (50 µg/ml), and amphotericin B (2 µg/ml, Sigma) for 3 h at 37 C with shaking. Organoids were collected by centrifugation and washed twice in PBS. Organoids were allowed to settle in collagen-coated dishes (BD Biosciences, Palo Alto, CA) containing culture medium DMEM/F12 (1:1), 1% FBS, insulin (5 µg/ml), epidermal growth factor (10 ng/ml), and penicillin (50 U/ml)/streptomycin (50 µg/ml) at 37 C under 5% CO2 for 3 d. Fibroblasts were removed by partial trypsin digestion. Resulting epithelial cells (5 x 105 per well) were plated on conventional plastic tissue culture plates, or on basement membrane reconstituted in six-well pates (Matrigel at 1 ml/well, BD Biosciences). Cells were preincubated for 1 d and then treated with oPRL at different doses and time courses. For matrigel culture, the gels were transferred to tubes and digested with dispase, and cells were collected by centrifugation.
Chinese hamster ovary (CHO) D6 cells, which were stably transfected with constitutively active pigeon PRL receptor, were maintained in Hams F12 medium supplemented with 10% FBS, 0.5 mg/ml geneticin (Sigma), 2 mM L-glutamine, and 1x antibiotic/antimycotic (Life Technologies, Gaithersburg, MD). COS7 cells were purchased from ATCC (Manassas, VA) and routinely maintained in DMEM containing 10% FBS, 2 mM L-glutamine, and penicillin (50 U/ml)/streptomycin (50 µg/ml).
RNA Isolation and RT-PCR
Total RNA from PMECs was isolated with Tri Reagent (Molecular Research Center, Cincinnati, OH). RNA was treated with RQ DNase I to remove any genomic DNA contamination according to the manufacturers protocol (Promega Corp., Madison, WI). Two micrograms of the treated total RNA were used for cDNA synthesis in a 20 µl reaction. AMV reverse transcriptase was purchased from Life Technologies. The primer pairs used for GlyCAM 1 and GAPDH amplification were described previously (24). PCR amplification was completed using Taq DNA Polymerase (Promega Corp.) at 94 C for 15 sec, at 60 C for 15 sec, and at 72 C for 60 sec in PCR Express Thermal Cycler (HYBAID, Middlesex, UK).
Site-Directed Mutagenesis and Plasmid Constructs
The GlyCAM 1 promoter-reporter constructs, Gly800-Luc, Gly254-Luc, and Gly104-Luc, were made by cloning the promoter fragments into pGL3 basic vector between SacI and Kpn sites. Four potential Stat5 binding sites are found in Gly800-Luc, two GAS sites are in Gly254-Luc, and no GAS site is in Gly104-Luc (Fig. 2A
). Mutation of GAS1 and GAS2 was done by QuikChange Site-Directed Mutagenesis kit following the manufacturers protocol (Stratagene, La Jolla, CA), and all mutants were confirmed by sequencing. Dominant negative Jak2 plasmids, Jak KD (a double mutation in C terminus kinase domain) and Jak829 (deletion of kinase domain after amino acid 829), were described previously (5). Full lengths of the coding regions of both Stat5A and B were cloned from mouse PMECs by RT-PCR with Pfu DNA polymerase and inserted into pCMV expression vector between SalI and NotI sites (BD Biosciences). Deletion of C-terminal transactivation domains of both Stat5A and B was carried out by PCR to generate dominant negative Stat5 (DN Stat5A and B) plasmids according to Wang et al. (31). Mutation of the tryptophan W37 to alanine (W37A) and the leucine residue L83 to serine (L83S) of Stat5A were made by QuikChange Site-Directed Mutagenesis Kit and confirmed by sequencing. Full lengths of cDNA encoding wild-type Stat5A, W37A, and L83S were cloned into pFLAG-CMV-2 expression vector (Sigma) between EcoRI and XbaI sites. All plasmid were prepared with EndoFree plasmid maxi kit (QIAGEN, Valencia, CA).
Transient Transfection and Luciferase Reporter Assay
ß-Galactosidase and pigeon PRL receptor plasmids were described previously (5). Twenty-four hours before transfection, CHO D6 cells were seeded at 1 x 105 cells per well in 12-well plates. ß-Galactosidase plasmid (50 ng) and GlyCAM 1 promoter reporter (250 ng) along with 1 µl Fugene 6 agent (Roche, Indianapolis, IN) were mixed and added into each well following the manufacturers protocol. In COS7 cells, along with ß-galactosidase plasmid and GlyCAM 1 promoter reporter, 50 ng of pigeon PRL receptor plasmid with 50 ng of Stat5 plasmids or 50 ng parental vectors were cotransfected. Four hours after transfection, cells were washed twice with PBS and then changed to serum-free media either with 0.5 µg/ml or without oPRL. Twenty hours after the addition of PRL, cells were harvested and lysed. Luciferase and ß-galactosidase activities were measured with luciferase reporter gene assay kit (Roche) and Galacto-Light Plus chemiluminescent reporter assay for ß-galactosidase kit (Tropix, Bedford, MA), respectively. The transfection efficiency was normalized by ß-galactosidase activity. All transfections for luciferase assays were repeated three times in triplicate, and data shown were from one experiment in triplicate. PMECs were transfected with lipofectamine Plus reagent (Invitrogen, Carlsbad, CA) following the manufacturers instructions. Twenty-four hours before transfection, PMECs were trypsinized from collagen-coated dishes and plated into six-well plates at 5 x 105 cells per well. Just before transfection, the old media were removed and replaced with fresh plain medium after two washes with 1x PBS. W37A, L83S, or parental vectors (2 µg) were used per well. Four hours after transfection, the medium was replaced with fresh culture medium with/without oPRL (1 µg/ml) and incubated for 24 h. Cells were harvested, and total RNA and proteins were isolated with Tri Reagent (Molecular Research Center). Transfection efficiency of PMECs was reflected by X-gal staining. ß-Galactosidase plasmids (2 µg/well in six-well plates) were transfected into PMEC using the same procedure as Stat5 plasmids, and after 24 h cells were stained by LacZ reporter assay kit following the manufacturers protocol (InvivoGen, San Diego, CA).
Overexpression and Purification of Flag-Tagged Stat5 Proteins and Western Blot
Flag-tagged Stat5 fusion proteins were expressed in COS7 cells cultured in 100-mm Petri dishes by transfection of 5 µg flag-tagged wild-type Stat5A, W37A, or L83S plasmids, together with 2 µg PRL receptor. Twenty-four hours after transfection, cells were treated with or without oPRL (0.5 µg/ml) for 30 min and lysed in buffer containing 50 mM Tris HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, and 1% Triton X-100. The recombinant proteins were immunoprecipitated with anti-flag M2 affinity gel (Sigma) and eluted with 3xflag peptides following the manufacturers instruction. Protein concentration was measured by the method of Lowry. Western blot was described previously (5). Anti-Stat5A monoclonal antibody (Zymed Corp., South San Francisco, CA) and phosphor-tyrosine monoclonal antibody (Cell Signaling, Beverly, MA) were used at 1:1000 ratio of dilution.
EMSA
Probes for gel shift assays were synthesized, annealed in 50 mM Tris HCl (pH 7.9), 150 mM NaCl, and 1 mM EDTA and labeled with [32P]dCTP by klenow fill-in. Binding reaction mixtures (20 µl) contained 50 ng flag-Stat5A proteins, 20,000 cpm of probe, and 2 µg poly (dI-dC) in 10 mM Tris HCl (pH 7.5), 10 mM HEPES, 50 mM KCl, 1.25 mM dithiothreitol, 1.1 mM EDTA, and 12% glycerol. The mixtures were incubated for 30 min on ice. Supershift was performed by preincubating the recombinant proteins with 1 µg of a pan-Stat5 antiserum (Stat5 sc-835, Santa Cruz Biotechnology, Inc., Santa Cruz, CA) for 15 min before adding the probes. Electrophoresis was carried out on 5% nondenaturing polyacrylamide gel in 0.3x TBE buffer at 150 V, room temperature for 2.5 h.
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ACKNOWLEDGMENTS
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The authors are grateful to Dr. George A. Jacob and Kathryn M. Nieport for technical help.
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FOOTNOTES
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This work was supported by NIH Grant DK 52134 (to N.D.H.).
Abbreviations: CHO, Chinese hamster ovary; DN, dominant negative; GAPDH, glyceraldehyde-3-phoshate dehydrogenase; GAS; interferon-
-activated sequence; GlyCAM 1, glycosylation-dependent cell adhesion molecule 1; IFN, interferon; Jak, Janus kinase; oPRL, ovine PRL; PMEC, primary mammary epithelial cells; PRL, prolactin; Stat, signal transducer and activator of transcription.
Received for publication February 7, 2003.
Accepted for publication July 7, 2003.
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