Regulation and Function of the Cytokine-Inducible SH-2 Domain Proteins, CIS and SOCS3, in Mammary Epithelial Cells

Sibylle Tonko-Geymayer, Olivier Goupille, Martin Tonko, Claudia Soratroi, Akihiko Yoshimura, Charles Streuli, Andrew Ziemiecki, Reinhard Kofler and Wolfgang Doppler

Institut für Medizinische Chemie und Biochemie (S.T.-G., O.G., C.S., W.D.) and Institut für Pathophysiologie (M.T., R.K.), Abteilung Molekulare Pathophysiologie, Universität Innsbruck, and Tyrolean Cancer Research Institute, A-6020 Innsbruck, Austria; Molecular and Cellar Immunology (A.Y.), Medical Institute of Bioregulation, Kyushu University, Higashi-ku, Fukuoka, 812-8582, Japan; Department of Cell and Structural Biology (C.S.), University of Manchester, Manchester M13 9PT, United Kingdom; and Department of Clinical Research (A.Z.), University of Berne, CH-3004 Berne, Switzerland

Address all correspondence and requests for reprints to: Wolfgang Doppler, Institut für Medizinische Chemie und Biochemie, Universität Innsbruck, Fritz Pregl-Strasse 3, A-6020 Innsbruck, Austria. E-mail: Wolfgang.Doppler{at}uibk.ac.at.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The cytokine-inducible src homology 2 (SH-2) proteins, CIS (cytokine inducible SH-2 domain protein) and SOCS3 (suppressor of cytokine signaling 3), are implicated in the negative regulation of prolactin (PRL) receptor-mediated activation of signal transducer and activator of transcription 5 (STAT5). We have studied the expression and function of CIS and SOCS3 proteins in the mouse mammary gland and in HC11 mammary epithelial cells. CIS and SOCS3 were differentially regulated: high expression levels of CIS mRNA were measured during the second half of pregnancy, whereas SOCS3 expression was high during the first 12 d post conceptum. SOCS3 levels increased, whereas CIS levels decreased, in the initial phase of involution. At the beginning of the lactation period both CIS and SOCS3 were high. PRL and epidermal growth factor (EGF) were able to induce CIS and SOCS3, whereas glucocorticoids inhibited their expression in mammary epithelial cells. The effect of EGF was much stronger on SOCS3 than on CIS. Ectopic expression of both SOCS3 and CIS inhibited STAT5 activation. Our data indicate that in the mammary gland CIS and SOCS3 are involved in regulating STAT5 signaling at three different instances: 1) SOCS3 serves as a mediator of the inhibitory EGF effect on PRL-induced STAT5 activation; 2) CIS and SOCS3 play a role as negative feedback inhibitors of PRL action; 3) Inhibition of CIS and SOCS3 expression by glucocorticoids contributes to the positive effect of glucocorticoids on PRL-induced STAT5 activation.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
THE USE OF transgenic mice has revealed the importance of the signal transducer and activator of transcription 5 (STAT5) in mammary gland development. A homozygous deletion of the gene encoding the predominant mammary gland isoform STAT5A leads to decreased lobulo-alveolar development and reduced production of milk (1). STAT5A function depends on phosphorylation of tyrosine 694 (2), mediated by either receptor-associated tyrosine kinases belonging to the Janus kinase (JAK) family (3), or by receptor tyrosine kinases, e.g. the epidermal growth factor (EGF) receptor (4), platelet-derived growth factor ß-receptor (5), erbB4 (6), or the IGF-I receptor (7). The plethora of extracellular signals capable of activating STAT5 in various cell types is complicating the evaluation of the mechanisms relevant for STAT5 activation in the mammary gland. Activation of the PRL receptor (PRL-R) and its associated Janus kinase 2 (JAK2) by prolactin (PRL) and/or a placental lactogenic hormone are considered to be major triggers responsible for the high levels of tyrosine-phosphorylated STAT5 detectable in the mammary gland during the second half of pregnancy and during the lactation period (8). However, other mechanisms of activation such as signals mediated by the erbB4 receptor must also be taken into account (6). Glucocorticoids have been shown to enhance STAT5-mediated gene expression either by direct synergistic interactions of the glucocorticoid receptor and STAT5 (9) and by an increase of STAT5 tyrosine phosphorylation and nuclear retention (10). Inhibitory pathways, preventing STAT5 activation in mammary epithelial cells, have been described, e.g. long-term activation of the EGF receptor (11) or cultivating cells attached to laminin-containing extracellular matrix (12) impair PRL-mediated STAT5 activation. The matrix dependence in the response to PRL appears to be relevant for confining activation to epithelial cells attached to the extracellular matrix with the correct composition. In addition, PRL-induced STAT5 tyrosine phosphorylation and DNA binding activity is transient (11, 13).

The search for mechanisms responsible for the restricted activation of STAT5 has focused so far on two different types of proteins, namely, tyrosine phosphatases (14, 15) and the family of cytokine-inducible src homology 2 (SH-2) domain proteins [CIS (cytokine-inducible SH-2 domain) proteins; also termed suppressors of cytokine signaling or SOCS proteins] (16). Tyrosine phosphatases appear to be relevant for inhibiting STAT5 activation in cells not attached to the proper extracellular matrix (15, 17) and have been implicated in the negative effect of EGF on PRL signaling (18). SOCS/CIS family members have been shown to function as feedback inhibitors attenuating the response of cytokines. SOCS/CIS proteins are capable of binding to activated cytokine receptor complexes via their SH-2 domain and to exert their function in multiple ways (see Ref. 16 for a review). These include 1) interference with the binding of cytoplasmic effector molecules to the receptor; 2) inhibition of the catalytic activity of JAK tyrosine kinases; and 3) targeting the receptor complexes to the proteasome via an interaction of the conserved carboxy-terminal region of the SOCS/CIS protein with the E3-ubiquitin-ligase of the proteasome. The inhibition of JAK tyrosine kinase activity is specific for the SOCS/CIS proteins SOCS1/JAB and SOCS3/CIS3 and explains the strong inhibitory effect of these two SOCS family members on JAK-dependent signaling pathways.

Among the seven genes of the SOCS/CIS family (16), only SOCS1 has been described so far to have a nonredundant function in mammary gland development, as indicated by the accelerated mammary gland development of mice with a homocygotic deletion of SOCS1 (19). Forced expression of CIS, the founding member of the SOCS family, in transgenic mice harboring a CIS transgene under the control of the ß-actin promoter leads to impaired terminal differentiation of the mammary gland, failure to lactate, and decreased levels of tyrosine-phosphorylated STAT5 (20), indicating also a function of this SOCS protein in mammary gland development. The CIS promoter contains functional STAT5 binding sites (21). In accordance with that, CIS expression in the mammary gland is completely dependent on STAT5, as evidenced by the lack of CIS expression in the mammary gland of mice with homozygous deletion of STAT5A and STAT5B (22). Experiments with transfected 293 cells revealed an inhibitory function of CIS on STAT5 activation by the PRL-R (20). Thus, a model emerges in which CIS acts in a negative feedback loop to block the long-term activation of STAT5. However, mice with targeted deletion of CIS failed to exhibit a mammary gland phenotype, indicating that other SOCS proteins can compensate for the action of CIS. One candidate for such a SOCS protein is SOCS3, the member of the SOCS family most closely related to CIS. SOCS3 has been shown previously to inhibit activation of STAT5 in transfected 293 cells (23) and to block ß-casein gene expression in response to PRL in mammary epithelial cells (19).

Here we show that expression of either CIS or SOCS3 inhibits STAT5 tyrosine phosphorylation in mammary epithelial cells. We have further investigated the expression of these genes during mammary gland development and tested the effect of PRL, EGF, and glucocorticoid hormone treatment. Interestingly, expression of CIS and SOCS3 was not coordinately regulated in the mammary gland and in mammary epithelial cells, one major difference being their differential induction by PRL or EGF. Based on our results, we postulate the involvement of CIS and SOCS3 proteins in the cross-talk between EGF, PRL, and glucocorticoid hormone-dependent signaling cascades.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Developmental Regulation of CIS and SOCS3 Transcripts in the Mouse Mammary Gland
Expression of CIS and SOCS3 transcripts in the mammary gland was demonstrated previously (23), but a detailed analysis during development has not yet been performed. We investigated the abundance of the transcripts in the mammary gland during different stages of pregnancy, lactation, involution, and in virgin animals by using real-time PCR employing CIS- and SOCS3-specific primers (Fig. 1Go). The obtained values were normalized to the abundance of S18 ribosomal RNA transcripts in the same samples. For CIS (Fig. 1AGo) the analysis revealed high expression during pregnancy with the highest levels during the second half (d 12 to d 19 post conceptum), very high levels at the beginning of the lactation period, and a down-regulation of CIS transcripts during involution, with minimum levels detected at d 3 of involution. At later stages of involution (d 6 to d 9), the expression levels approached the levels of virgin and early pregnant mice. Essentially the same expression pattern was observed by Northern blotting with a CIS-specific probe (not shown). Regulation of SOCS3 transcripts (Fig. 1BGo) during mammary gland development was distinct from CIS during pregnancy and involution. The SOCS3 mRNA was most abundant in virgin mice, during the first half of pregnancy (d 5 to d 12) and at the end of the involution period (d 6 to d 9 after pup removal). Similar to CIS, SOCS3 message in the mammary gland was increased at the first day of the lactation period.



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Figure 1. Expression of CIS and SOCS3 mRNA During Mammary Gland Development

The expression levels of CIS (panel A) and SOCS3 (panel B) transcripts relative to S18 ribosomal RNA were determined by real-time PCR in samples derived from mouse mammary gland tissue of different developmental stages. The results are shown as the mean ± SE of three experiments. The developmental stages are indicated at the bottom of each panel. Designation of stages: V, adult nonpregnant females (virgin); P5 to P19, d 5 to d 19 post conceptum (pregnancy); L1 to L14, d 1 to d 14 of the first lactation period; I8 h to I9, involution period from 8 h to 9 d after withdrawal of pups.

 
Real-time PCR analysis of CIS transcripts required lower numbers of PCR cycles than of SOCS3 transcripts in the mammary gland (see differences in relative levels in Fig. 1Go, A and B). This indicates either lower levels or lower efficiency in amplification of SOCS3 transcripts in comparison to CIS. The former possibility was supported by Northern blot experiments, where with RNA extracts from mammary gland tissue and a SOCS3-specific probe only a weak hybridization signal was obtained in comparison to a CIS probe (results not shown). The lower levels of SOCS3 transcripts in comparison to CIS might be due to a weak induction and/or a rapid turnover of SOCS3 transcripts. Alternatively or in addition, it might be due to the fact that, in the mammary epithelium, only a subpopulation of the cells expresses SOCS3.

Regulation of CIS and SOCS3 Expression by PRL in Mammary Epithelial Cells
Because the issue of rapid induction and down-regulation kinetics of SOCS transcripts is difficult to study in vivo, we further investigated expression of these transcripts in the mouse mammary epithelial cell line HC11. The lactogenic hormone PRL was employed as an induction signal, because CIS is induced by STAT5 (21), and PRL is the major known activator of STAT5 in this cell line. To ensure maximum terminal differentiation at the beginning of PRL induction, cells were kept confluent in a medium lacking EGF. Expression of CIS and SOCS3 in response to PRL treatment was analyzed by real-time PCR (Fig. 2Go). Induction of CIS by PRL was more sustained than of SOCS3, and higher maximal inductions levels (7-fold for CIS vs. 2-fold for SOCS3) were reached. Maximum levels of CIS were achieved after 2–4 h and elevated levels were maintained for 24 h (Fig. 2AGo, open squares). SOCS3 induction was more rapid and transient with maximum levels at 2 h and a loss of induction already after 4 h (Fig. 2BGo, open squares). The data with the mouse cell line support the notion that the low steady state levels of SOCS3 transcripts in the lactating mammary glands are the result of the weak and transient induction in response to PRL. The effects of dexamethasone will be discussed below.



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Figure 2. Effect of PRL and Dexamethasone on CIS and SOCS3 mRNA Expression in HC11 Mammary Epithelial Cells

RNA samples were prepared from confluent cultures of HC11 cells treated with PRL and dexamethasone (Dex) and analyzed by real-time PCR. The relative levels of CIS (panel A) and SOCS3 (panel B) transcripts were determined as described in Fig. 1Go. RT-PCR data are shown as the mean ± SE of three independent experiments for 5-d dexamethasone-treated cells (solid squares) or untreated cells (open squares). Treatment time with PRL is indicated on the abscissa.

 
Regulation of CIS and SOCS3 Expression by EGF
One explanation for the distinct expression pattern of CIS and SOCS3 transcripts during pregnancy and involution (Fig. 1Go) is the differential utilization of separate induction pathways for the two genes. In this respect it has been demonstrated that SOCS3 transcripts can be induced by STAT5-independent signaling pathways (33). This hypothesis was tested by stimulating HC11 cells with the growth factor EGF and measuring steady state levels of CIS and SOCS3 transcripts by real-time PCR at different times after induction (Fig. 3Go). EGF does not stimulate STAT5 induction in these cells (our unpublished observations) and inhibits PRL-induced STAT5 activation by a mechanism that is most likely indirect, because it requires long-term incubation of cells with EGF (11). Induction of CIS transcripts by EGF (Fig. 3AGo, open squares) was much weaker when compared with the effect of PRL (compare Fig. 2AGo with Fig. 3AGo) and reached maximum levels after 30 min (2-fold induction). By contrast, SOCS3 was induced more strongly by EGF (open squares of Fig. 3BGo) than by PRL (compare Fig. 3BGo to Fig. 2BGo). Maximum induction levels were observed after 30 min (4-fold induction). This differential sensitivity of CIS and SOCS3 to PRL and EGF support the concept that the differential expression pattern of CIS and SOCS3 during pregnancy and lactation is due to a difference in sensitivity to induction pathways.



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Figure 3. Effect of EGF and Dexamethasone on CIS and SOCS3 mRNA Expression in HC11 Mammary Epithelial Cells

Expression of CIS and SOCS3 transcripts was determined as described in Fig. 2Go. In panel A, EGF induction of CIS transcripts, and in panel B, EGF induction of SOCS3 transcripts were determined by real-time PCR and are shown as mean ± SE of three different experiments for 5-d dexamethasone-treated cells (solid squares) or untreated cells (open squares). Treatment time with EGF is indicated on the abscissa.

 
Effect of Glucocorticoids on PRL and EGF Induction of CIS and SOCS3
Because the effect of PRL on CIS and SOCS3 induction is probably mediated by STAT5 binding sites in the promoter of these genes, and glucocorticoids have been described to enhance the transcription from the STAT5 target genes ß-casein and whey acidic protein (34), we wished to determine the effect of glucocorticoids on the expression of CIS and SOCS3. We thus compared the effect of PRL on CIS and SOCS3 transcripts levels in cells treated for 5 d with the synthetic glucocorticoid dexamethasone (solid squares of Figs. 2Go and 3Go) with the levels determined in nondexamethasone-treated cells (open squares of Figs. 2Go and 3Go). Surprisingly, treatment with dexamethasone did not increase but actually decreased the abundance of CIS and SOCS3 transcripts. The inhibitory effect of dexamethasone was already observable before PRL or EGF stimulation. This indicates that glucocorticoids inhibit CIS and SOCS3 expression by acting on hormone-independent basal expression and/or steady state levels of these transcripts. For CIS, the effect of dexamethasone was evident during the whole PRL or EGF induction period investigated. Interestingly, in the case of SOCS3, the inhibitory effect of dexamethasone was observable only during the first hour of treatment with PRL or EGF and, in the case of EGF, an increased rather than decreased expression of SOCS3 was observed after 2 h. Maximal induction by EGF shifted from 30 min in the absence of dexamethasone to 2 h in the presence of dexamethasone (Fig. 3BGo). A possible explanation for this shift is that SOCS3 acts as a feedback inhibitor of its own expression: In the absence of dexamethasone, SOCS3 is strongly induced and thus can rapidly act as a feedback inhibitor, whereas in the presence of dexamethasone, the less efficient induction of SOCS3 requires a longer induction time before enough SOCS3 protein has accumulated to exert its inhibitory action.

To verify the results of the real-time PCR analysis by a different approach, we used Northern blotting to analyze expression of CIS and SOCS3 transcripts. The results obtained are in accordance with the real-time PCR data. In Fig. 4AGo, expression of CIS in response to PRL treatment, and in Fig. 4BGo, expression of SOCS3 transcripts in response to EGF treatment are shown as examples. We further controlled the capability of the mammary epithelial cell line to synthesize ß-casein gene transcripts by Northern blotting (Fig. 4AGo, upper blot). The detection of ß-casein transcripts as early as 8 h after PRL administration in dexamethasone-treated cells (lane 6 of Fig. 4AGo) is in agreement with previous results on the regulation of ß-casein gene expression by glucocorticoids and PRL (34).



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Figure 4. Northern Blot Analysis of CIS, SOCS3, and ß-Casein Gene Expression in HC11 Mammary Epithelial Cells

RNA was isolated from confluent cultures of HC11 treated for different times with EGF, PRL, or dexamethasone as indicated at the top of each panel. The amount of RNA loaded on each lane was controlled by ethidium bromide staining of the 28S rRNA (last row of each panel). In panel A, the blot was hybridized with probes specific for ß-casein and CIS transcripts as indicated at the right margin of each row. In panel B, the blot was hybridized with a probe specific to SOCS3.

 
Expression of CIS and SOCS3 Proteins in HC11 Cells
For demonstration of protein expression of CIS and SOCS3, we analyzed CIS or SOCS3 immunoprecipitates by immunoblotting with a second antibody directed against a different epitope (Fig. 5Go). Expression of the CIS protein with an apparent molecular mass of 32 kDa was demonstrable in extracts induced with PRL for 1 and 2 h (Fig. 5AGo, lanes 3 and 4). The HC11 CIS protein migrated at the same position as the CIS protein induced by IL-2 in HT-2 cells (Fig. 5AGo, lane 7). In the presence of 50 µM of the proteasome inhibitor MG132, about 2 times more CIS protein accumulated after a 2-h induction with PRL (compare lanes 4 and 5 of Fig. 5AGo), suggesting that the CIS protein is degraded by the proteasomal pathway, as is the case in other cell types (35, 36).



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Figure 5. Induction of CIS and SOCS3 Protein in HC11 Mammary Epithelial Cells

HC11 mammary epithelial cells were stimulated for the indicated times with either EGF or PRL as described in Materials and Methods and analyzed for expression of CIS and SOCS3 by immunoprecipitation followed by Western blotting. For CIS, a rabbit polyclonal antibody directed against a CIS fragment with the SH-2 domain and the carboxy terminus (31 ) was used for immunoprecipitation and the goat polyclonal antibody N-19 (Santa Cruz Biotechnology, Inc.) was employed for Western blotting. For SOCS3, immunoprecipitates were obtained with a rabbit polyclonal antibody directed against the complete coding sequence of SOCS3 and analyzed by Western blotting using a SOCS3 antibody directed against the amino terminus of SOCS3. The positions of the molecular mass marker are shown on the left margin on each gel. Sizes are indicated in kilodaltons. In panel A, CIS protein expression in response to PRL treatment was analyzed (lanes 1–4). The effect of a 2-h incubation with the proteasome inhibitor MG132 on PRL-induced CIS expression levels was investigated in lane 5. As a control for CIS expression, IL-2-dependent induction of CIS in HT-2 cells was investigated (lanes 6 and 7). These cells have been described to express CIS and SOCS3 protein under these conditions (28 ). In Panel B, SOCS3 protein expression in response to PRL and EGF was analyzed. In lane 5, HC11 cells were treated for 2 h with 50 µM MG 132 before the immunoprecipitates were prepared. In lanes 6 and 7, control HT-2 cells, or HT-2 cells induced with IL-2 for 1 h, were investigated.

 
SOCS3 protein expressed in HC11 cells had the same molecular mass as IL-2-induced SOCS3 in HT-2 cells (Fig. 5BGo). It was present as a doublet with the apparent molecular mass of 26 and 28 kDa and was present already before stimulation with EGF or PRL (Fig. 5BGo, lanes 1 and 8). PRL treatment did not significantly elevate SOCS3 protein expression levels (Fig. 5BGo, lanes 1–4). Treatment with EGF for 30 min to 2 h increased the abundance of the SOCS3 protein approximately 2-fold (compare lane 8 to 11 of Fig. 5BGo), and in untreated cells expression was increased by treatment of cells with MG132 (compare lanes 1 and 5 of Fig. 5BGo), similar as observed for the CIS protein.

Regulation of CIS and SOCS3 Gene Expression by PRL and EGF in Vivo
To investigate the effects of PRL and EGF on the expression of CIS and SOCS3 in vivo, nonpregnant female mice were injected with either EGF or PRL. One hour after injections, the relative expression levels of CIS and SOCS3 mRNA were determined by RT-PCR in the mammary gland as well as in the liver (Fig. 6Go). CIS steady state levels did not change significantly in response to PRL in mammary gland and liver and were strongly induced by EGF in the liver but not in the mammary gland (Fig. 6AGo). The tissue-specific effect of EGF on CIS expression is in accordance with the dependence of CIS expression on STAT5 activation and the tissue-specific action of EGF on STAT5 tyrosine phosphorylation, which is strongly induced in the liver (37) but weakly induced in the mammary gland (38). The lack of a significant effect of PRL on CIS expression in liver can be attributed to the fact that in the liver PRL does not activate STAT5 (39). However, the failure of PRL to induce CIS expression in the mammary gland was surprising, because PRL has been shown to increase STAT5 tyrosine phosphorylation in the mammary gland of virgin mice (38). One explanation could be that the increase of STAT5 tyrosine phosphorylation in response to PRL, which is relatively small in comparison to the effect of GH (38), is not sufficient to further increase the already relatively high CIS expression levels in the mammary gland of virgin mice (Fig. 1Go, Fig. 6AGo).



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Figure 6. Effect of PRL and EGF on CIS and SOCS3 Expression in Nonpregnant Female Mice

Female 10-wk-old mice were treated for 1 h with either vehicle (control), 5 µg/g recombinant ovine PRL (PRL), or 5 µg/g recombinant murine EGF (EGF). Expression levels of CIS (panel A) and SOCS3 (panel B) transcripts in mammary gland and liver tissue were determined as in Fig. 1Go. Results are shown as the mean ± SE of two to three experiments. Statistically significant differences between control and EGF/PRL-treated mice (P <= 0.05) are marked by asterisks.

 
EGF was a potent inducer of SOCS3 expression in the mammary gland (Fig. 6BGo). This strong effect of EGF on SOCS3, but not on CIS, expression (Fig. 6AGo) in the mammary gland of virgin mice is reminiscent of the differential effects of EGF obtained with mammary epithelial cells in culture, where SOCS3 was more strongly induced than CIS (Fig. 3Go), and the effect of EGF on SOCS3 expression was more pronounced than the effect of PRL (compare Figs. 2Go and 3Go). In the liver, by contrast, PRL had a stronger effect than EGF in inducing SOCS3 (Fig. 6BGo, left part of panel). This difference between mammary gland and liver in the responsiveness of SOCS3 to EGF and PRL is likely due to tissue-specific differences in the intracellular signaling pathways induced by the two cytokines as discussed above for the expression of CIS.

Inhibition of PRL-R-Mediated Activation of STAT5 by CIS in COS-7 Cells
Ectopic expression of CIS and/or SOCS3 has been demonstrated to inhibit a variety of JAK tyrosine kinase-dependent signaling pathways (16). In the case of mammary epithelial cells, the PRL-R/JAK2/STAT5 pathway represents a likely target. A potent inhibitory action of ectopic expression of SOCS3 on PRL-R-dependent signaling pathways in nonmammary cells has been reported and attributed to the inhibition of JAK2 tyrosine kinase activity. However, in the case of CIS, conflicting results have been published (20, 23, 40). To reconcile this issue, we investigated the relative effects of SOCS3 and CIS on three different consequences of STAT5 activation by the PRL-R, namely tyrosine phosphorylation, DNA binding activity, and transcriptional activity. We first investigated STAT5 transiently expressed in COS-7 monkey kidney cells. In the presence of transfected PRL-R, PRL induced a rapid increase in the amount of tyrosine-phosphorylated STAT5 with maximum levels between 30 min and 2 h (Fig. 7AGo, lanes 1–9). In accordance with previous reports on the strong inhibitory action of SOCS3 on PRL-R-mediated STAT5 activation (23, 40), complete inhibition of tyrosine phosphorylation of STAT5 by ectopic expression of SOCS3 was observed (Fig. 7AGo, lanes 19–21). In the presence of CIS, STAT5 phosphorylation was reduced by 30–50% during the first 4 h of PRL treatment (Fig. 7AGo, lanes 10–17, and Fig. 7BGo). Inhibition was evident after 5 min of PRL treatment. In cells treated for 24 h with PRL, CIS did not significantly affect STAT5 tyrosine phosphorylation (compare Fig. 7AGo, lanes 9 and 18). This observation is in contrast to the findings of the CIS effect on the GH receptor-mediated tyrosine phosphorylation of STAT5B, which was much more pronounced after longer hormone stimulation periods (35) and was attributed to CIS-mediated proteosomal degradation of the activated GH-R.



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Figure 7. Effect of CIS and SOCS3 Expression on STAT5 Tyrosine Phosphorylation in COS-7 Cells

A, Western blot analysis of tyrosine-phosphorylated STAT5 in transiently transfected COS-7 cells either coexpressing STAT5A and the PRL-R (-CIS/-SOCS3, lanes 1–9) or STAT5, the PRL-R and CIS (+CIS, lanes 10- 18) or SOCS3 (+SOCS3, lanes 19–21). STAT5 (5 µg), 5 µg PRL-R, and 5 µg CIS expression vector per 20 µg transfected DNA were used. Cells were stimulated with PRL (5 µg/ml) 48 h after transfection for the time period indicated at the top of each panel. WCEs were prepared and equal amounts of protein were analyzed by using an antibody against phosphotyrosine (band corresponding to phosphorylated STAT5 is marked by STAT5-PY). The membrane was stripped and reincubated with polyclonal STAT5 antibody to visualize the total protein level of STAT5 (marked by STAT5). The position of the 90-kDa molecular mass marker is indicated at the left margin. B, The relative abundance of tyrosine-phosphorylated STAT5 normalized to total STAT5 protein levels was analyzed by densitometric analysis of the experiment shown in panel A. Results are presented as percentage of maximum phosphorylation observed in COS-7 cells induced by PRL for 30 min.

 
The effect of CIS on STAT5 DNA binding activity was investigated by EMSA, using the STAT5 binding site of the lactogenic hormone response element of the ß-casein gene promoter [lactogenic hormone response region (LHRR) (11)] as a probe. A representative experiment is shown in Fig. 8Go. CIS inhibited the STAT5 DNA binding activity observed after 5 and 60 min of PRL stimulation by 70% and by 46%, respectively (Fig. 8Go, A and B). This effect is comparable to its effect on tyrosine phosphorylation shown in Fig. 7Go. After 5 h PRL stimulation, inhibition of DNA binding by CIS was less pronounced than after 5 min (32%, compare lanes 5 and 10 of Fig. 8AGo). Experiments with SOCS3 revealed a complete inhibition as early as after 15 min (Fig. 8AGo, lane 11). To determine whether the reduced STAT5 DNA binding activity observed in vitro results in decreased STAT5 transcriptional activity, a ß-casein gene promoter-driven luciferase construct was cotransfected with the STAT5A and PRL-R expression vectors. The effect of a 24-h PRL induction on luciferase expression was determined in the presence or absence of CIS and SOCS3. PRL-R/JAK2/STAT5-mediated promoter activity was inhibited 48% by CIS and 97% by SOCS3 (Fig. 8CGo). The combined results of Figs. 7Go and 8Go lead to the conclusion that both CIS and SOCS3 inhibit STAT5 in cells overexpressing the PRL-R and STAT5. SOCS3 completely inhibited STAT5 activation, whereas the effect of CIS was partial. No significant difference in the relative effects of CIS on tyrosine phosphorylation, DNA binding activity, and transactivation by STAT5 was detectable. This implies that the primary inhibitory action is on tyrosine phosphorylation, and that the other effects are the consequence of this inhibition.



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Figure 8. Inhibitory Effect of CIS and SOCS3 on STAT5 DNA Binding and ß-Casein Gene Promoter-Dependent Transcription in COS-7 Cells

A, STAT5 DNA binding activity was analyzed with the same WCE as used for the experiment of Fig. 7Go and by EMSA and with a probe containing the LHRR of the rat ß-casein gene promoter. Equal amount of protein (8 µg) was loaded. Arrows at the right margin mark the position of STAT5A-specific (STAT5A) and nonspecific (ns) complexes. B, PhosphorImager analysis of the relative levels of the STAT5A DNA binding complex shown in panel A. STAT5 DNA complex formation is shown as a percentage of maximum complex formation observed in cells treated for 1 h with PRL in the absence of CIS. C, Effect of CIS and SOCS3 expression on PRL-mediated induction of the ß-casein gene promoter activity. CIS or SOCS3 expression vector (2.6 µg per 20 µg transfected DNA) was used, together with expression vectors for STAT5A (4 µg), PRL-R (4 µg), 0.15 µg SV40 Renilla, and 8 µg ß-casein gene luciferase constructs. In controls, the CIS or SOCS3 expression vector was replaced with the equivalent amount of bluescript vector. Twenty-four hours after transfection, cells were treated with PRL for 24 h (+PRL), or remained untreated (-PRL). Luciferase and Renilla activities of extracts were measured, and normalized luciferase activity was calculated. Induction ratios are shown as mean ± SE of three experiments and are presented relative to the activity of untreated controls.

 
Inhibition of PRL-R-Mediated Activation of STAT5 by CIS and SOCS3 in HC11 Cells
To determine whether CIS and SOCS3 inhibit endogenous STAT5 expressed in a mammary epithelial cell, we evaluated the effect of CIS and SOCS3 expression on the activation of STAT5 by PRL in HC11 cells. Cells stably transfected with doxycycline-inducible CIS or SOCS3 expression vectors were employed. Two different clones of CIS and SOCS3 transfectants were investigated for the effect of a 15-min PRL stimulation on STAT5 tyrosine phosphorylation in the presence or absence of doxycycline treatment. As controls, we used the parental HC11 cell line or clonal derivatives stably transfected with a doxycycline-inducible luciferase expression vector. Tyrosine-phosphorylated STAT5A relative to total STAT5A levels was determined by immunoblotting of STAT5A immunoprecipitates with a phosphotyrosine-specific antibody (upper blot of Fig. 9AGo) or a STAT5-specific antibody (second blot of Fig. 9AGo). Doxycycline-inducible expression of CIS and SOCS3 proteins was determined in immunoprecipitation experiments with an antibody against the common myc tag of the CIS and SOCS3 constructs (last row of Fig. 9AGo). The results revealed an inhibitory action of both CIS and SOCS3 on STAT5 tyrosine phosphorylation. The percentage of inhibition was quantified by densitometric analysis of the experiment shown in Fig. 9AGo and of additional experiments not shown and was 48 ± 10% for the CIS clones and 59 ± 21% for the SOCS3 clones. Control cells not expressing CIS or SOCS3 in response to doxycycline were not inhibited by doxycycline (Fig. 9AGo), indicating that the inhibition observed with the CIS and SOCS3 clones was not an unspecific effect of the doxycycline treatment. The incomplete inhibition of STAT5 activation by CIS and SOCS3 can be attributed to the fact that only between 50 and 75% of our transfectants expressed CIS and SOCS3, as determined by immunofluorescence with an antibody against the myc tag of the transfected genes (data not shown). When correcting for the fraction of cells not expressing the SOCS proteins, both CIS and SOCS3 have a strong inhibitory effect on the activation of the endogenous STAT5 in mammary epithelial cells. In contrast to HC11 cells, in COS-7 cells only SOCS3 had a strong inhibitory effect on STAT5 activation (Figs. 7Go and 8Go). A possible explanation for the partial effect of CIS on STAT5 activation in COS-7 cells is the substantially higher levels of exogenously expressed STAT5 in comparison to the endogenous levels of STAT5 in HC11, as will be discussed in the next section.



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Figure 9. Effect of Forced Expression of CIS and SOCS3 on STAT5 Activation in HC11 Cells

A, Confluent parental HC11 mammary epithelial cells (HC11) or clonal derivatives of HC11 cells stably transfected with doxycycline-inducible luciferase expression vector (LUC clones), doxycycline-inducible CIS expression vector (CIS clones nos. 8 and 12), or SOCS3 expression vector (SOCS3 clones nos. 7 and 12) were treated with 2 µg/ml doxycycline for 5 d (Doxy +) or were cultivated in a doxycyline-free medium (Doxy -) as indicated at the top of the panel. All cells were stimulated with PRL for 15 min before harvesting the cells. STAT5A tyrosine phosphorylation was assayed in STAT5A immunoprecipitates by Western blotting with a phosphotyrosine-specific antibody (first row, band marked with STAT5-PY). As a loading control, the membrane was stripped and reincubated with polyclonal STAT5 antibody (band marked with STAT5). Expression of CIS and SOCS3 was analyzed in myc immunoprecipitates (last row) by Western blotting with an antibody recognizing the common myc tag of CIS and SOCS3 (position of band marked with CIS and SOCS3). B, The inhibitory effect of CIS and SOCS3 expression on PRL-induced STAT5 tyrosine phosphorylation was quantified by densitometric analysis of autoradiographs and by normalization of the levels of tyrosine-phosphorylated STAT5 to the levels of total STAT5 protein. Results are shown as the percentage of tyrosine phosphorylation observed in cells treated with doxycycline in comparison to not-doxycycline-treated cells. Data were obtained from independent experiments (n in parentheses) including those shown in panel A and are expressed as mean ± SE. Control, untransfected HC11 cells (n = 7); CIS, cells transfected with doxycycline-inducible CIS expression vector (n = 9); SOCS3, cells transfected with doxycycline-inducible SOCS3 expression vector (n = 4). The significance level of difference between control cells and CIS/SOCS3-expressing cells is shown by asterisks (*, P < 0,05; **, P < 0.01).

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Our study demonstrates a noncoordinate regulation of the two cytokine-inducible SH-2 domain proteins CIS and SOCS3 in the mammary gland and in mammary epithelial cells. Both were induced at the onset of the lactation period in the mammary gland. However, we observed a strikingly distinct developmental expression of CIS and SOCS3 genes during pregnancy and involution, and different relative effects of EGF, PRL, and glucocorticoid hormones, on their expression in mammary epithelial cells.

Differential Induction of CIS and SOCS3 in the Mammary Gland and by EGF and PRL in Mammary Epithelial Cells
CIS and, to a lesser extent, SOCS3 were induced at the beginning of the lactation period. PRL-induced STAT5 activation appears to be the major trigger for this effect. This notion is supported by the finding of high tyrosine-phosphorylated STAT5 levels and of STAT5 DNA binding at this stage of mammary gland development (8, 11) and by the effect of PRL on CIS and SOCS3 expression in mammary epithelial cells (Fig. 2Go). STAT5 might also be relevant for mediating the elevated CIS expression during the second half of pregnancy (Fig. 1AGo), because it is preferentially activated during this time period (8). However, in the case of SOCS3, the lower STAT5 activation during late pregnancy in comparison to lactation (8, 11) might not allow accumulation of high SOCS3 levels at this stage of development. This notion is derived from the lower and more transient SOCS3 induction in response to PRL in comparison to CIS in mammary epithelial cells (Fig. 2Go).

STAT5-independent induction pathways appear to be relevant for the elevated levels of SOCS3 during the first half of pregnancy and during the initial phase of the involution period, because STAT5 activation is low or absent during these developmental stages (8). Induction of SOCS3 by EGF in mammary epithelial cells represents such a STAT5-independent pathway, because EGF does not activate STAT5 in HC11 cells (our unpublished observations). In the mammary gland, EGF might be important for contributing to the basal levels of SOCS3 throughout. In mammary epithelial cells, induction of SOCS3 by EGF is a prime candidate for explaining the slow and probably indirect inhibitory effect of EGF on activation of STAT5 DNA binding (11) and tyrosine phosphorylation (Geymayer, S., unpublished results) and thus appears to be an important mediator for the negative cross-talk between PRL-R- and EGF-receptor-mediated signaling cascades. Alternatively or in addition, the tyrosine phosphatase PEST is potentially involved in this negative cross-talk, as suggested recently (18). The signals leading to an induction of SOCS3 during the early stage of pregnancy remain unclear. At the onset of involution, SOCS3 induction might be triggered by STAT3, a known inducer of SOCS3 in tissues other than the mammary gland (23), because STAT3 is strongly activated during this time period by an unknown signaling pathway (41).

Action of Glucocorticoids on CIS and SOCS3 Expression
The effect of dexamethasone in inhibiting SOCS3 and CIS induction was primary on basal expression levels, as revealed by real-time PCR analysis. This negative effect of glucocorticoids does not appear to be mammary gland specific because it has been described recently also in hepatocytes (42). In mammary epithelial cells, the effect on SOCS3 expression was confined to the early time points of induction by PRL and EGF. An explanation for this restricted action is provided by the putative role of SOCS3 as feedback inhibitors of its own expression. Based on this assumption, dexamethasone would curtail the function of SOCS3 as a feedback inhibitor by inhibiting accumulation of SOCS3 transcripts. As a consequence, the induction pathway initiated by PRL and EGF would not be shut down as efficiently and at later time points after PRL or EGF stimulation (beginning with 2 h, Figs. 2Go and 3Go), a situation would be reached in which the diminished feedback inhibition completely balances the inhibitory effect of dexamethasone on SOCS3 expression.

We think that the observed effect of dexamethasone on CIS and SOCS3 might be relevant for some aspects of the well described synergisms between GR and STAT5, in particular for the indirect effect of GR on PRL action (34), which in addition to the direct synergisms between GR and STAT5 (9) contributes to enhancement of milk-protein gene expression by glucocorticoids. In this respect, decreased expression of SOCS3 and CIS is expected to lead to a more sustained activation of STAT5. In accordance, the presence of activated GR led to prolonged STAT5 tyrosine phosphorylation in transfected cells (10), and treatment of HC11 cells with dexamethasone and PRL resulted in 1.5-fold higher STAT5 levels in the nucleus as compared with cells treated with PRL only (13).

Mechanism of Action of CIS and SOCS3 on STAT5 Signaling
The different inhibitory actions of SOCS3 and CIS on PRL-R-induced STAT5 signaling implicate different mechanisms of action. Efficient inhibition by CIS was restricted to cells expressing either the endogenous STAT5 protein, as shown in this study, or limiting levels of a transfected STAT5 gene (20). CIS also inhibited PRL-dependent expression of the endogenous ß-casein gene in mammary epithelial cells under these conditions (19). This effect is possibly mediated by STAT5; however, STAT5-independent pathways must also be taken into consideration. At high expression levels of PRL-R and/or STAT5, only weak (23) or no (40) inhibition by CIS was observed. By contrast, expression of SOCS3 led to an efficient block of STAT5 expression at high and low levels of STAT5 and the PRL-R (Refs. 19 and 23 and this report), and was not restricted to the mammary epithelium. The more potent effect of SOCS3 is possibly attributed to its described inhibitory action on JAK2 tyrosine kinase activity.

In analogy to the mechanism proposed for the inhibitory action of CIS on erythropoietin (31)-, thrombopoietin (3)-, IL-3 (31)-, IL-2 (21)-, and GH (43)-mediated STAT5 activation, the action of CIS on PRL-induced STAT5 activation probably involves binding to the phosphorylated domains of the receptor complex via its SH-2 domain, thereby at least partially impeding the recruitment and activation of STAT5 molecules and its subsequent tyrosine phosphorylation by the PRL-R/JAK2 complex. Similar, SOCS3 might exert part of its inhibitory action by binding to specific sites in PRL-R. However, in contrast to the findings with the other receptors, we have not yet been able to demonstrate binding of CIS or SOCS3 to the PRL-R by coimmunoprecipitation experiments (our unpublished results). The only SOCS protein that we found in immunoprecipitates was SOCS2, as demonstrated previously (40). Thus, if an interaction between CIS or SOCS3 and the PRL-R occurs in vivo, it appears to be not strong enough to allow recovery of PRL-R protein from immunoprecipitates after washing. For a further evaluation of a potential interaction between CIS and/or SOCS3 and specific domains of the PRL-R, functional assays are mandatory. Using PRL-R mutants, it should be possible to determine the role of PRL-R domains, which become phosphorylated on tyrosine upon receptor stimulation. For that purpose, and to overcome the problem of endogenously expressed wild-type PRL-R, we are presently engineering mammary epithelial cell lines expressing chimeric erythropoietin receptor/PRL-R molecules with such mutations.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Plasmid Constructions
For transient expression of N-terminal myc-tagged SOCS/CIS-proteins in 293 and COS-7 cells, pcDNA3-Myc-CIS plasmids (24) were used. For inducible expression in HC11 cells, fragments comprising the coding region of SOCS3/CIS including the myc tag were inserted into the tetracycline-inducible expression vector pBIG2i (25). For CIS, this was done by excision of a HindIII/XbaI fragment, filling up of recessive ends by Klenow polymerase, and insertion of the fragment into EcoRV site of pBIG2i. For SOCS3, the SpeI/XbaI fragment from pcDNA3-Myc SOCS3 was inserted into the SpeI site of pBIG2i. From the resulting construct with the proper orientation of the SOCS3 coding region, a BamHI/BamHI fragment encompassing noncoding regions between the promoter and SOCS3 was deleted to obtain the final construct. The tetracycline-inducible luciferase gene expression vector was constructed by inserting the NheI/XbaI fragment of pGL3basic (Promega Corp., Madison, WI) with the coding region of the luciferase gene into the SpeI site of pBIG2i. The PRL-R expression vector pECEPRLR (26) with the complete coding region of the mouse gene was used. The structures of the expression vector for STAT5 (27), and of the ß-casein gene luciferase (26), have been described. The Renilla luciferase expression vector pRL-SV40 was obtained from Promega Corp.

PRL and EGF Injections
All animal studies were conducted in accordance with the principles and procedures outlined in the NIH Guide for the Care and Use of Laboratory Animals and were approved by the committee on animal care of the Austrian Federal Ministry for Education, Science and Culture. Female Swiss mice (10 wk old) were injected ip with 5 µg/g body weight of recombinant ovine PRL (provided by Dr. Arieh Gertler), 5 µg/g body weight of recombinant murine EGF (Strathmann Biotec AG, Hamburg, Germany), or vehicle and killed 1 h after injections. The livers and mammary glands were snap frozen in liquid nitrogen and stored at -80 C.

Cell Culture and Transfections
COS-7 monkey kidney cells and 293 human embryonic kidney cells were grown in DMEM containing 10% fetal calf serum (FCS); HC11 mammary epithelial cells in RPMI 1640 containing 10% FCS, 5 ng/ml EGF, and 1 µg/ml insulin. The IL-2-dependent T-cell line HT-2 was maintained in RPMI 1640 with 10% FCS, 2 mM glutamine, and 50 µM mercaptoethanol. Before treatment with PRL or EGF, HC11 cells were kept for 2 d in EGF-free medium containing 2% FCS as described previously (11). HT-2 cells were stimulated with IL-2 after arresting cells in RPMI 1640 with 2% FCS for 14 h, similar to a procedure described previously (28). Inductions were carried out with 5 µg/ml ovine PRL (31 U/mg, Sigma, St. Louis, MO), 0.1 µM dexamethasone (Sigma), 5 ng/ml EGF (Sigma), or 50 ng/ml recombinant mouse IL-2 (Insight Biotechnology, Middlesex, UK).

Transient transfections were carried out with the calcium phosphate coprecipitation technique as described (29). A total of 20 µg DNA per six wells of a six-well plate were transfected. For establishment of stable CIS/SOCS3 clones under the control of a tetracycline-inducible promoter, HC11 cells were transfected with 3 µg of the CIS/SOCS3 expression vector with the Geneporter transfection kit, and clones resistant to hygromycin were selected in a medium containing 150 µg/ml hygromycin.

RNA Extraction and Northern Blotting
Mouse mammary gland tissue from Swiss mice was frozen by submersion in liquid nitrogen and ground into powder. Total RNA extraction of pulverized organs or HC11 cells was performed by using TRI reagent (Molecular Research Center, Inc., Cincinnati, OH). Integrity of RNA was checked on ethidium bromide-stained agarose gels by staining for rRNA. RNA (8 µg) was resolved by electrophoresis through 1% agarose gel in 3-(N-morpholino)propane sulfonic acid-formaldehyde-buffer, transferred to nylon membranes (Hybond N, Amersham Pharmacia Biotech, Arlington Heights, IL) in 20x standard saline citrate buffer followed by UV cross-linking (125 J). The membrane was prehybridized for 3 h at 65 C in hybridization solution containing 0.5 M sodium phosphate, pH 6.8; 7% sodium dodecyl sulfate; and 10 mg/ml BSA (fraction V, Sigma). DNA probes for Northern blots were generated by excision of mouse CIS from the pcDNA3-Myc-CIS plasmid (24) with EcoRI/XbaI (770 bp), SOCS3 from BSK-mCIS3 with EcoRI/NotI (1000 bp), and of ß-casein cDNA sequence from pCSNß (30) with PstI (440 bp). DNA probes prepared by random priming with [{alpha}-32P]dATP were added and hybridization was continued for 18 h at 65 C. After the membrane had been washed twice at 65 C for 30 min it was exposed to x-ray (Kodak, Rochester, NY). For reprobing, membranes were stripped by incubation for 10 min at 100 C in 0.1% sodium dodecyl sulfate, 0.1x standard saline citrate.

Quantification of RNA Expression Levels by Real-Time PCR
Total RNA was prepared from tissue powder or HC11 cells and reverse transcribed with random primers and the Superscript II Rnase H- reverse transcriptase (Life Technologies, Inc., Vienna, Austria) into cDNA. Analysis was performed by quantitative PCR analysis via real-time PCR using the Abi Prism 7700 sequence detector (PE Applied Biosystems, Vienna, Austria). Sequences for probes [FAM (carboxy-fluorescein modification) label] and primers were selected using the Primer Express software of PE Applied Biosystems and were the following: CIS reverse primer, 5'-ccgggtgtcagctgcac-3'; CIS forward primer, 5'-tcctggccttcccagatgt-3'; CIS probe, 5'-ccttgtgcagcactatgtggcctcc-3'; SOCS3 reverse primer, 5'-agtagaatccgctctcctgcag-3'; SOCS3 forward primer, 5'-gctccaaaagcgagtaccagc-3'; SOCS3 probe, 5'-tggcgcacggcgttcacca-3'; 18S reverse primer, 5'-tcacccgtggtcaccatg-3'; 18S forward primer, 5'-ccatccgaacgtcgtccctat-3'; 18S probe, 5'-actttcgatggtagtcgccgtgcct-3'.

Immunoprecipitation
For immunoprecipitation with HC11 and HT-2 cells, 6 x 106 cells were incubated with 1 ml lysis-buffer [20 mM Tris (pH 8), 137 mM NaCl, 2.7 mM KCl, 1% Brij 96, 10% glycerol, 50 mM orthovanadate, 1 mM dithiothreitol (DTT), 1 mM phenylmethylsulfonylfluoride, 2 µg/ml leupeptin, 5 µg/ml aprotinin] for 1 h at 4 C with shaking. After centrifugation for 10 min at 17,500 x g the supernatant was incubated with the indicated concentrations of specific antibodies overnight. Polyclonal c-Myc antibody (A-14; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), polyclonal STAT5A antiserum (provided by T. Decker), rabbit polyclonal CIS antibody (31), or 2 µl of rabbit polyclonal antibody directed against bacterial expressed SOCS3 were used. After addition of 20 µl protein A agarose for 1 h at 4 C, immunocomplexes were washed three times with washing buffer (20 mM Tris, pH 8; 137 mM NaCl; 2.7 mM KCl; 0.1% Triton X-100; 1 mM orthovanadate; 1 mM DTT) and eluted in sodium dodecyl sulfate sample buffer for analysis by SDS-PAGE.

Western Blotting
Either immunoprecipitates or whole cell extracts (WCEs) were used. WCEs were prepared as described previously (11). Analysis was performed on NuPAGE 4–12% Bis-Tris gels (Novex, San Diego, CA), and the separated proteins were transferred to polyvinylidene difluoride membranes. For immunodetection, membranes were incubated with the appropriate dilutions of either CIS (N-19), SOCS-3 (S-19) goat polyclonal antibodies (Santa Cruz Biotechnology, Inc.), c-Myc (A-14) rabbit polyclonal antibody (Santa Cruz Biotechnology, Inc.), c-Myc (sc-40) monoclonal antibody (Santa Cruz Biotechnology, Inc.), STAT5A monoclonal antibody (Transduction Laboratories, Inc., Lexington, KY), or monoclonal antibody 4G10 (Upstate Biotechnology, Inc. Lake Placid, NY). Antimouse, -rabbit, and -goat secondary antibodies were obtained from Santa Cruz Biotechnology, Inc. The enhanced chemiluminescence protocol of Amersham Pharmacia Biotech was used for detection.

EMSA
A 30-bp double-stranded oligonucleotide probe labeled with [{gamma}-32P]ATP (>6000 Ci/mmol) comprising the proximal STAT5 binding site of the LHRR of the rat ß-casein gene promoter was used (11). Binding reactions were performed with WCEs and analyzed on a 4% polyacrylamide gel as described previously (11).

Luciferase Assay
Transfections were done with the calcium phosphate precipitation technique essentially as described (29). For reporter gene assays, COS-7 or 293 cells were split into six-well dishes at a cell density of 1–2 x 105 cells per well at the day before transfection. Eighteen hours after transfection, precipitates were washed off and cells were treated with 5 µg/ml ovine PRL for 24 h as indicated. For determination of firefly and Renilla luciferase activity, cells were lysed with 250 µl buffer containing 25 mM glycylglycin (pH 7.8), 15 mM MgSO4, 4 mM EGTA, 1 mM DTT, and 0.2% Tween 20, and then were scraped off the dishes, transferred into 1.5-ml centrifuge tubes, incubated under vigorous shaking for 20 min at 4 C, and centrifuged at 10,000 x g for 5 min. Aliquots of the supernatant were taken for determining either firefly luciferase activity (20) or Renilla luciferase activity (32) as described elsewhere.


    ACKNOWLEDGMENTS
 
We thank Dr. Katja Garimorth and Dr. Hermann Dietrich for their help in the animal experiments and Thomas Decker for generously providing us with the polyclonal STAT5 antibody.


    FOOTNOTES
 
This work was supported by the Fonds zur Förderung der Wissenschaftlichen Forschung, Projects F209 and F204.

Abbreviations: CIS, Cytokine-inducible SH-2 domain; DTT, dithiothreitol; EGF, epidermal growth factor; FCS, fetal calf serum; JAK, Janus kinase; LHRR, lactogenic hormone response region; PRL, prolactin; PRL-R, PRL receptor; SH-2, src homology 2; SOCS3, suppressor of cytokine signaling 3; STAT, signal transducer and activator of transcription; WCE, whole-cell extract.

Received for publication September 25, 2001. Accepted for publication February 26, 2002.


    REFERENCES
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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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