Possible Involvement of Truncated Signal Transducer and Activator of Transcription-5 in the GH Pattern-Dependent Regulation of CYP2C12 Gene Expression in Rat Liver
Hanna Helander,
Jan-Åke Gustafsson and
Agneta Mode
Department of Medical Nutrition, Karolinska Institutet, NOVUM, S-141 86 Huddinge, Sweden
Address all correspondence and requests for reprints to: Hanna Helander, Department of Medical Nutrition, Karolinska Institutet, NOVUM, S-141 86 Huddinge, Sweden. E-mail: Hanna.Helander{at}mednut.ki.se.
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ABSTRACT
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The transcription factors signal transducer and activator of transcription (Stat)5a and Stat5b have been implicated in the GH regulation of CYP2C genes in rodent liver. In addition to full-length Stat5 isoforms, truncated Stat5 proteins (Stat5ß), lacking the transactivating domain, have been demonstrated. In this study we found that Stat5ß can be formed by proteolytic cleavage in rat liver nuclei and that the activity of the protease is independent of GH. The GH regulation of the female-specific CYP2C12 gene has recently been shown to be conveyed by two adjacent Stat5-binding elements in the 5'-upstream region. We found that binding of Stat5 in liver nuclear extracts to this site involved simultaneous binding of two Stat5 dimers, most likely both Stat5b homodimers and Stat5a/Stat5b heterodimers. We also investigated Stat5 binding to a potential composite Stat5 element in the 3'-untranslated region (UTR) of CYP2C12. Several Stat5 complexes were formed on this element including Stat5ß-containing complexes. In transient transfection experiments we could demonstrate that the 3'-UTR element reduced GH activation of a CYP2C12-luciferase reporter construct harboring the 5'-Stat5 elements. We speculate that binding of Stat5ß to the 3'-UTR element could be of relevance for the GH-dependent and sex-specific expression of CYP2C12.
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INTRODUCTION
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PITUITARY GH secretion is sexually dimorphic in the rat. The male type of secretion is characterized by an intermittent pulsation whereas the female secretion results in a continuous presence of GH in the plasma (1). A number of GH actions have been shown to be differentially regulated by the sex-specific GH profiles. Prototypic examples of this sex-dependent GH regulation in the rat liver are the cytochrome P450 genes CYP2C11 and CYP2C12, which are transcribed in a male- and female-specific manner, respectively, directly dependent on the GH plasma profile (2). Upon binding to its cognate membrane-bound receptor, GH activates a number of different signaling molecules including proteins in the signal transducer and activator of transcription (Stat) family via a receptor-associated Janus kinase. Activated Stat proteins dimerize and translocate to the nucleus where they bind to specific DNA target sequences to regulate transcription (3). GH has been shown to specifically activate Stat1, Stat3, and Stat5; in particular, Stat5 is implicated in GH signaling to cytochrome P450 (CYP) genes in rodent liver (4, 5). Two different Stat5 forms exist, Stat5a and Stat5b, which are encoded by distinct genes. The two forms have high sequence homology (>95%) at the amino acid level, with the major differences in the carboxy-terminal part (6, 7).
In addition to full-length Stat5, naturally occurring carboxy-truncated forms of both Stat5a and Stat5b have been found in myeloid cell lineages as well as in liver cells. Truncated Stat5 forms are produced either by alternative splicing (8, 9, 10) or by proteolytic cleavage (11, 12, 13, 14), and it is possible that these mechanisms occur in different cell types or, alternatively, coexist in the same cell. A specific Stat5 protease has been identified and characterized as a nucleus-associated protein, able to cleave both active and inactive Stat5 (11). In a recent study, activity of a Stat3- and Stat5-specific protease was demonstrated both in cytosolic and nuclear extracts from acute myeloid leukemia blasts (14). A physiological significance of truncated Stat5 is indicated by phenotypic changes of myeloid progenitor cells upon introduction of noncleavable Stat5 (11). Furthermore, previous data show that carboxy-truncated Stat5 forms, lacking the transactivation domain, can inhibit transcription of Stat5-regulated genes either by heterodimerization with full-length Stat5 proteins or by binding as homodimers (8). It has been shown that, in addition to binding as dimers, Stat5 as well as Stat1 and Stat4 can form tetrameric complexes on two adjacent Stat-binding motifs and that tetramer formation is mediated by the highly conserved N-terminal regions of these proteins (15, 16, 17, 18, 19). Such cooperative DNA binding can serve to selectively bind different Stat proteins on a promoter that contains multiple Stat-binding sites. This kind of DNA configuration with several Stat5-binding motifs seems to be common among several Stat5-responsive genes (15, 16, 20).
The CYP2C12 gene, expressed in the liver of female rats and dependent on continuous GH, harbors potential Stat-binding elements both in the 5'-flanking region and in the 3'-untranslated region (UTR) of the gene. The 5'-flank of the CYP2C12 gene harbors two neighboring identical consensus Stat5-binding elements, whereas the 3'-UTR harbors one consensus Stat5-binding site in close proximity to a half-site. Previous studies on the transcriptional regulation of the CYP2C12 gene have revealed hepatocyte nuclear factor 6 (HNF-6) as a GH-dependent transcription factor (21), and recent data demonstrate that both HNF-4 and HNF-6 together with Stat5 are required for the transcriptional activation of the CYP2C12 promoter by GH (5). In this report we have investigated the binding of full-length and carboxy-truncated Stat5 protein to the elements in the 5'-flank and the 3'-UTR of the CYP2C12 gene and addressed the functional role of Stat5 binding to these elements.
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RESULTS
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Administration of GH to hypophysectomized (Hx) rats mimicking the female or the male characteristic pattern of GH secretion leads to induction in the liver of the female-specific CYP2C12 and the male-specific CYP2C11 gene, respectively (2). In this study, administration of continuous GH for 5 d induced the female-specific P4502C12 mRNA 10-fold whereas the same daily dose of GH administered as daily injections of GH for 5 d induced P4502C11 mRNA 8-fold (data not shown).
Full-Length and Carboxy-Truncated Stat5 Proteins in Rat Liver
Liver nuclear extracts, prepared from normal female rats and Hx female rats untreated or treated with GH, were analyzed by Western blotting. In line with data from other groups (4), we observed a transient nuclear localization of Stat5 in response to single or repeated GH injections. High levels of Stat5 were present in the nuclei 45 min and 4 h after the last injection but not after 12 h (Fig. 1A
, lanes 57). In response to continuous GH, Stat5 was also detected in nuclear extracts but at considerably lower levels, particularly after 5 d of GH exposure (lanes 34). In the cytosol, several immunoreactive Stat5 bands were apparent, corresponding to different phosphorylation degrees of the protein (22).

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Figure 1. GH-Dependent Nuclear Translocation of Full-Length and Truncated Stat5
Nuclear extracts (NE) (30 µg) and cytosolic proteins (100 µg) from livers of normal female (F) (lane 1) or Hx female rats untreated (lane 2) or treated with GH (0.5 mg/kg/d) were separated by SDS-PAGE (7.5%) and analyzed by Western blotting. GH was administered to Hx rats either by minipump infusion (mp) for 1 d (lane 3) or 5 d (lane 4) or by daily injections for 5 d (lanes 57). The animals injected with GH were killed at the indicated time points after the last injection (45 min, 4 h, or 12 h). A, An antibody recognizing a sequence of Stat5 present in both full-length and truncated Stat5 proteins was used (S21520). B, Specific antibodies directed against the carboxy terminus of Stat5a (sc-1081) or Stat5b (06-554) were used.
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In addition to full-length Stat5, truncated Stat5 (Stat5ß) proteins were detected in all nuclear extracts where full-length Stat5 was present (Fig. 1A
). When specific antibodies directed against the carboxy terminus of Stat5a or Stat5b were used, only full-length and no Stat5ß proteins were detected (Fig. 1B
). Stat5ßs generated by differential splicing are described as 84-kDa (Stat5a) or 86-kDa (Stat5b) proteins, whereas proteolytic cleavage of Stat5a and Stat5b results in proteins of 77 kDa and 80 kDa, respectively (8, 9, 10, 13). In the Western blot experiments with antisera against Stat5, we detected three shorter Stat5 forms of 80, 77, and 74 kDa in apparent molecular mass in rat liver nuclear extracts (Fig. 1A
).
We could not detect any Stat5ß in the cytosolic extracts (Fig. 1A
), indicating that the Stat5ß is generated in the nucleus, e.g. by proteolytic processing. Consistent with proteolytic cleavage, a band of approximately 16 kDa was detected in the nuclear extracts, separated on a dense gel, when Western blotting was performed with antiserum against the carboxy terminus of Stat5a (Fig. 2
). However, no such band was detected using the antiserum against the carboxy terminus of Stat5b, despite the fact that Stat5b is more abundant than Stat5a in rat liver (9). To further investigate whether the liver nuclear extracts possessed proteolytic activity, we performed an experiment in which nuclear extract from COS7 cells, transiently transfected with a Stat5a expression plasmid, was incubated with nuclear extracts from Hx rats. The nuclear extracts were not treated with phenylmethylsulfonyl fluoride (PMSF) during the extract preparation, because PMSF has previously been shown to inhibit the activity of the Stat5-specific protease (11, 12). With Western blot analysis, formation of a truncated, Stat5 immunoreactive band was observed after incubation of recombinant Stat5a with liver nuclear extracts from Hx rats (Fig. 3
, compare lanes 1 and 5). This band, formed by proteolysis of recombinant Stat5a, had a higher mobility than the Stat5ß endogenously present in rat liver. However, the difference may be explained by the size difference between Stat5aß and Stat5bß previously described (7), or by differences in modifications between Stat5 expressed in COS7 cells vs. rat liver. Formation of the band was not affected by addition of the protease inhibitors aprotinin, leupeptin, or pepstatin, but reduced by addition of PMSF (Fig. 3
, lanes 69). Furthermore, the proteolytic activity was detected to a similar extent in nuclear extracts from Hx rats either untreated or treated intermittently with GH (data not shown), indicating that the Stat5 protease activity is independent of GH.

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Figure 2. Detection of a Carboxy-Terminal Stat5 Fragment
Rat liver nuclear extracts from normal female (F) (lane 1), or Hx rats treated with five daily injections and killed 45 min (lane 2), 4 h (lane 3), or 12 h (lane 4) after the last injection, were separated by SDS-PAGE (15%) and analyzed by Western blotting. An antibody directed against the carboxy-terminal part of Stat5a was used (sc-1081).
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Figure 3. Stat5 Protease Activity in Rat Liver Nuclear Extracts
Nuclear extracts from GH-stimulated COS7 cells expressing ovine Stat5a incubated with (lanes 59) or without (lanes 3 and 4) liver nuclear extracts (LNE) from Hx rats were separated by SDS-PAGE (7.5%) and analyzed by Western blotting using a common Stat5 antibody (S21520). The protease inhibitors aprotinin (A), leupeptin (L), pepstatin (P), and PMSF were added at the start of the incubations (lanes 69). Liver nuclear extracts from Hx rats untreated (lane 2) or treated with daily injections of GH and killed 45 min after the last injection (45') (lane 1) were included as references. The putative Stat5aß is indicated by an arrow.
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Stat5 Binding to Tandem Elements in the CYP2C12 Gene
In the 5'-flanking region of CYP2C12, two identical consensus Stat5-binding sites, TTCCTAGAA, spaced by 16 bp, are present just upstream of -4 kb (5), whereas the 3'-UTR harbors one consensus Stat5-binding site, TTCTGAGAA, and 11 bp downstream, a similar imperfect palindrome sequence TTCAAATAA (Fig. 4
) (23). We termed these regions 5'-2C12GLE1, 5'-2C12GLE2, 3'-2C12GLE1, and 3'-2C12GLE2 because of their resemblance with
-interferon-activated sequence (GAS)-like elements (GLEs) (24). To investigate the binding of full-length and carboxy-truncated Stat5 proteins to the GLE sequences of CYP2C12, we used liver nuclear extracts from rats with different GH status, apparently containing different amounts of truncated Stat5 (cf. Fig. 1A
), in EMSAs. In EMSAs carried out with a probe harboring a consensus sequence for binding of Oct factors, we detected similar binding activities in all extracts, indicating an equal quality of the extracts (Fig. 5
). Slightly higher binding activities were observed in extracts from normal animals compared with Hx rats, untreated or treated with GH. This probably reflects some influence of pituitary hormones other than GH.

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Figure 4. Nucleotide Sequences of Potential Stat5-Binding Areas in the 5'-Flank (5') and 3'-UTR (3') of the CYP2C12 Gene
The sequences shown represent the probes used in EMSAs, with the positions of the upper strand oligonucleotides indicated. Boxed sequences indicate potential Stat5-binding sites.
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Figure 5. EMSA of an Oct-Binding Probe with Rat Liver Nuclear Extracts
Protein binding (indicated by an arrow) of liver nuclear extracts (1 µg) from rats with different GH status (see Fig. 1 ) to the Oct probe. Lane 13 contains free probe only.
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The 9-bp core of the 3'-2C12GLE1 sequence is identical with the SPIGLE1 sequence identified as the minimal region of a 45-bp GH-response element in the 5'-flank of the rat serine protease inhibitor gene (SPI) 2.1 and shown to preferentially bind Stat5 (25). Protein binding activity to the 3'-2C12GLE1 probe was absent in liver nuclear extracts from Hx female rats (Fig. 6A
, lane 2). Binding activity was transiently induced after GH injections where binding was observed 45 min but not 12 h after the last injection (lanes 5 and 6). Also in nuclear extracts from normal female rats and from Hx rats treated continuously with GH for 1 or 5 d binding activities were detected (lanes 3 and 4). However, 5 d of continuous GH treatment, compared with 1 d, resulted in reduced complex formation.

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Figure 6. Binding of Rat Liver Nuclear Proteins to the 3'-2C12GLE1 Probe
A, Binding of nuclear proteins (5 µg) from livers of rats with different GH status (see Fig. 1 ) to the 3'-2C12GLE1 probe was analyzed by EMSA. B, The complex formed between the 3'-2C12GLE1 probe and liver nuclear extracts from either normal female rats (lanes 14) or female Hx rats treated with daily injections and killed 45 min after the last injection (lanes 58) were analyzed by supershift assay. Antibodies directed against Stat5 (lanes 2 and 6), Stat5a (lanes 3 and 7), or Stat5b (lanes 4 and 8) were incubated with the nuclear extracts overnight at 4C before addition of the probe. The complex formed with the 3'-2C12GLE1 probe is indicated with an S and the supershifted protein-DNA complex is marked SS.
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To investigate Stat5 binding to the 3'-2C12GLE1, we performed supershift analysis. As shown in Fig. 6B
, antiserum recognizing both Stat5a and 5b (lanes 2 and 6) was able to supershift the major binding activity in the extracts whereas anti-Stat5a only marginally attenuated the complex formed with nuclear extracts from female rats (lanes 3). Therefore, although continuous GH exposure leads to a maintained level of Stat5a in liver nuclei (cf. Fig. 1B
), this does not seem to be reflected in the binding to the 3'-2C12GLE1 probe. On the other hand, the level of Stat5b in the nucleus correlated well with the binding to the 3'-2C12GLE1 probe, and antiserum against Stat5b did supershift the complex (Fig. 6B
, lanes 4 and 8). This is consistent with Stat5b homodimers being the dominant complex in liver nuclear extracts binding to GAS-like Stat5-binding elements regardless of the mode of GH exposure (26). This probably reflects the greater abundance of Stat5b compared with that of Stat5a in the rat liver (9) rather than a difference in binding affinity, because recent data show that homodimers of Stat5a and Stat5b have similar optimal binding sites (27).
The presence of two GLE sequences in close proximity, such as in the 5'-flank and in the 3'-UTR of the CYP2C12 gene, is apparently common to Stat5 target sequences. First, we investigated the binding of Stat5 proteins to the tandem GLEs in the CYP2C12 5'-flank. EMSA with the 5'-2C12GLE12 probe revealed formation of a major slow migrating band (band A) and some less prominent faster migrating bands (Fig. 7A
). The bands were all dependent on the presence of GH. Thus, binding activity was absent in nuclear extracts from Hx rats (lane 2) and transiently induced after GH injections, i.e. binding was observed 45 min and 4 h but not 12 h after the last injection (lanes 57). Liver nuclear extracts from rats exposed continuously to GH also presented marked binding activities (lanes 1 and 34). The presence of Stat5 in the major complex (A) formed on the 5'-2C12GLE12 probe was verified by the supershift of the complex by the common Stat5a/Stat5b antibody (Fig. 7B
, lanes 2 and 6). Band A was partially supershifted by specific anti-Stat5a antibodies (Fig. 7B
, lanes 3 and 7) and completely supershifted by anti-Stat5b antibodies (Fig. 7B
, lanes 4 and 8). The ability of antisera against Stat5b to completely supershift a complex bound to a single GAS element together with a partial supershift with anti-Stat5a has previously been interpreted as a mixture of Stat5b homodimers and Stat5a-Stat5b heterodimers binding to the element (28, 29). Another possibility is that complex A constitutes a tetramer of Stat5; however, only Stat5a has so far been shown to form tetrameric complexes (15, 27). It is therefore likely that complex A consists of two Stat5 dimers.

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Figure 7. Binding of Rat Liver Nuclear Proteins to the 5'-2C12GLE12 Probe
A, Nuclear extracts (5 µg) from livers of rats with different GH status (see Fig. 1 ) were analyzed by EMSA. The major complex formed with the 5'-2C12GLE12 probe is indicated with an A, and the less prominent faster migrating bands are indicated with a bracket. B, The complexes formed with liver nuclear extracts (5 µg) from normal female rats (lanes 14) or female Hx rats treated with daily injections and killed 45 min after the last injection (lanes 58) were analyzed by supershift assays. Antibodies directed against Stat5 (lanes 2 and 6), Stat5a (lanes 3 and 7), or Stat5b (lanes 4 and 8) were incubated with the nuclear extracts overnight at 4C before addition of the probe. The supershifted protein-DNA complexes are marked SS.
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The potential tandem Stat5 elements in the 3'-UTR of CYP2C12 differ from the elements in the 5'-flank of the gene both in composition and with regard to the distance between the two elements (Fig. 4
). Although one major Stat5 complex was formed on the 5'-2C12GLE12 and the 3'-2C12GLE1 probe with liver nuclear extracts, several bands were formed in EMSAs on the 3'-2C12GLE12 probe (Fig. 8
) and, moreover, in a manner that correlated with the apparent amount of Stat5ß in the nuclear extracts (cf. Fig. 1A
). Of the complexes formed, those labeled a to e were competed away by the addition of excess unlabeled 3'-2C12GLE12 or 3'-2C12GLE1, whereas excess unlabeled 3'-2C12GLE2 had no effect (data not shown). Unlabeled 3'-2C12GLE12 also competitively inhibited formation of complexes below complex e, whereas neither 3'-2C12GLE1 nor 3'-2C12GLE2 did. We focused the further studies on the formation of complexes ae.

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Figure 8. Binding of Rat Liver Nuclear Proteins to the 3'-2C12GLE12 Probe
EMSA with nuclear extracts (5 µg) from livers of rats with different GH status (see Fig. 1 ) and the 3'-2C12GLE12 probe, performed as described in Materials and Methods. The upper complexes formed, ae, are outlined in the figure. The bands below e do not contain Stat1, Stat3, or Stat5 proteins as judged by supershift analysis (data not shown).
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It was most intriguing to find that the type, in addition to the amount, of complexes, ac and e, formed with the 3'-2C12GLE12 probe markedly changed with the different nuclear extracts. Three patterns of complex formation were identified. The complex patterns are schematically presented in Fig. 9
. Pattern I, with mainly band d, is characteristic of nuclear extracts obtained from rats devoid of GH in the circulation. Pattern II and pattern III were both formed with nuclear extracts from rats with GH present in the plasma; characteristic of pattern II was the presence of bands ae (cf. Fig. 8
, lanes 5 and 6), whereas the characteristic of pattern III was the presence of bands c and d and perhaps some e (cf. Fig. 8
, lanes 1, 3, and 4). Apparently, pattern II was formed with nuclear extracts from rats treated intermittently with GH in a male-like fashion (Fig. 8
, lanes 5 and 6), whereas pattern III was formed with nuclear extracts from rats exposed continuously to GH in a female-like fashion (Fig. 8
, lanes 1, 3, and 4).

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Figure 9. Schematic Presentation of the Different Binding Patterns Formed on the 3'-2C12GLE12 Probe
Pattern I is characteristic for nuclear extracts obtained from rats devoid of GH in the circulation, whereas patterns II and III were both formed with nuclear extracts from rats with GH present in the plasma. The formation of pattern II and III appeared to correlate with the abundance of Stat5ß in the extracts (cf. Fig. 1 ).
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Interestingly, when the 3'-2C12GLE12 probe was mutated so that the 3'-2C12GLE2 TTC half-site was destroyed, 3'-2C12GLE12mut (Fig. 4
), the binding pattern formed with the extract from normal female rats changed from type III to a type similar to II, i.e. the formation of complex c decreased in favor of the e complex (Fig. 10
, lanes 12). However, no a or b bands were formed. Similarly, in extracts from rats treated with daily GH injections, the a and b bands disappeared when the 3'-2C12GLE2 TTC half-site was mutated (lanes 34). Thus, the presence of an intact 3'-2C12GLE2 favored binding to 3'-2C12GLE12 to form complex c with nuclear extracts with apparently low levels of Stat5ß and allowed complex a and b to form with nuclear extracts with apparently high levels of Stat5ß (cf. Fig. 1A
). Furthermore, the mutation led to a predominance of complex e formation independently of what type of extract was used.

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Figure 10. Comparison of Binding of Rat Liver Nuclear Proteins to the 3'-2C12GLE12 and the Mutated 3'-2C12GLE12 Probes
EMSA with nuclear extracts from livers of normal female rats (lanes 1 and 2) or female Hx rats treated with daily injections and killed 4 h after the last injection (lanes 3 and 4) was performed with the 3'-2C12GLE12 probe (lanes 1 and 3) and the 3'-2C12GLE12mut probe (lanes 2 and 4) where the 3'-2C12GLE2 TTC half-site was destroyed (see Fig. 4 ). Complexes ae are indicated.
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In attempts to unravel the identity of the complexes, we used different Stat antibodies in supershift assays. Antibodies recognizing both Stat5a and Stat5b supershifted or mitigated the bands ac and e, indicating that all four GH-dependent bands are formed by Stat5 proteins interacting with the 3'-2C12GLE12 probe (Fig. 11
, lanes 3 and 7). It should be mentioned that none of the bands below e (see Fig. 8
) were affected by addition of Stat5 antibodies (data not shown). Moreover, addition of antibodies against the Stat5-related proteins, Stat1 and Stat3, did not affect the intensity of any of the complexes (data not shown). Antibodies against the specific carboxy terminus of Stat5a had little effect on the abundance of the formed complexes; only a minor supershift of complex c formed with the nuclear extract from female rats was detected on the original film (lane 4). On the other hand, antisera previously shown to specifically bind Stat5b (22) completely shifted bands a and c (lanes 2 and 6). This indicates that bands a and c are comprised of Stat5b complexes. However, the ability of Stat5a antiserum to affect band c, although modestly, could indicate that band c comprises some heteromeric Stat5a/Stat5b complexes in addition to homomeric Stat5b complexes. The fuzzy appearance of band c is most likely due to the extended gel run by which the different Stat5 dimers tend to be resolved. The Stat5 immunoreactivity of bands b and e, supershifted by the common Stat5a and Stat5b antibody, contrasted by their lack of reactivity with either isoform-specific antibody, indicates that the Stat5 proteins forming bands b and e lack the carboxy terminus to which the specific Stat5a and Stat5b antibodies are raised. This suggests therefore that the 3'-2C12GLE12 element binds not only full-length Stat5 but also carboxy-truncated forms of Stat5.

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Figure 11. Stat5 Binding to the 3'-2C12GLE12 Probe
The complexes formed between the 3'-2C12GLE12 probe and liver nuclear extracts from either normal female rats (lanes 14) or female Hx rats treated with daily injections and killed 45 min after the last injection (lanes 58) were analyzed by supershift assays. Antibodies directed against Stat5 (lanes 3 and 7), Stat5a (lanes 4 and 8), or Stat5b (lanes 2 and 6) were incubated with the nuclear extracts overnight at 4C before addition of the probe. Complexes ae are indicated. The lower band in lane 3 and part of lane 4 is an artifact.
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Stat5 as well as Stat1 and Stat4 have been shown to form tetrameric complexes on tandemly linked Stat-binding motifs. Tetramer complex formation is mediated by the highly conserved N-terminal region of the Stat proteins. As a consequence, both full-length and carboxy-truncated Stat5 isoforms would theoretically be able to form tetrameric complexes on duplicated response elements. Due to the short migration in the EMSA experiments of the major complex bound to the 5'-2C12GLE12 probe and of the complexes a and b bound to the 3'-2C12GLE12 probe, it is plausible that these complexes represent binding of Stat5 tetramers and/or two dimers. Moreover the inability of complexes a and b to bind the 3'-2C12GLE12mut probe, harboring only one GLE, further supports the idea that these complexes are formed by binding of either tetramers or duplicated Stat5 dimers to the 3'-2C12GLE12 probe. So far, Stat5b has not been shown to form tetrameric complexes on either single or tandem response elements (15, 27), and therefore it is likely that complex a represents binding of two dimers to the 3'-2C12GLE12 probe. The tentative identities of the Stat5 complexes in bands ac and e are indicated in Table 1
.
To further investigate the binding of full-length and carboxy-truncated Stat5 to the 3'-2C12GLE12 and the 5'2C12GLE12 probe, EMSAs were performed with nuclear extracts from GH-stimulated COS7 cells transfected with expression vectors for either Stat5, Stat5ß, or the empty vector (pXM). As demonstrated by Western blotting of cytosolic extracts, the expression level of Stat5 and Stat5ß was similar in these cells (Fig. 12A
, lanes 3 and 4), whereas cells untransfected or transfected with pXM expressed no Stat5 (lanes 1 and 2). However, the amount of recombinant Stat5ß in nuclear extracts from cells stimulated with GH was approximately 5-fold higher than the corresponding amount of full-length Stat5, as judged by the Western blot experiments (Fig. 12A
, lanes 510). This is in line with previous data showing that Stat5ß has higher resistance toward dephosphorylation compared with full-length Stat5 and therefore is less rapidly translocated out of the nucleus (8, 30). Based on the estimated difference in amount of nuclear full-length and truncated recombinant Stat5 in GH-stimulated cells (Fig. 12A
), 5 times less Stat5ß-harboring nuclear extract than full-length Stat5-harboring extracts were used when the following EMSAs were performed.

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Figure 12. Binding of Full-Length and Carboxy-Truncated Stat5a to the 5'-2C12GLE12 and the 3'-2C12GLE12 Probes
A, Cytosolic proteins (lanes 14) and nuclear extracts (lanes 510) (NE) from GH-stimulated COS7 cells untransfected (lane 1) or transfected with either the empty vector pXM (lane 2), pXM-MGF expressing full-length Stat5a (lanes 3 and 57), or pXM-MGFß expressing Stat5aß (lanes 4 and 810) were separated by SDS-PAGE (7.5%) and analyzed by Western blotting. In lanes 510, different amounts of the nuclear extracts were loaded onto the SDS-PAGE. An antibody recognizing a sequence of Stat5 present both in full-length and carboxy-truncated Stat5 proteins was used (S21520). B and C, Binding of nuclear proteins from GH-stimulated COS7 cells transfected with pXM-MGF (Stat5a) or pXM-MGFß (Stat5aß) to the 5'-2C12GLE12 (B) or the 3'-2C12GLE12 (C) probe, analyzed by EMSA. The following relative ratios of Stat5a to Stat5aß (in percent) were used; 100:0 (lane 1), 95:5 (lane 2), 90:10 (lane 3), 80:20 (lane 4), 60:40 (lane 5), 50:50 (lane 6), 40:60 (lane 7), 20:80 (lane 8), and 0:100 (lane 9). Binding of nuclear extracts from normal female rats (F) (lane 10) and Hx female rats treated with daily injections and killed 45 min after the last injection (45') (lane 11) were included as references. The complexes A and ae, formed with liver nuclear extracts, and the tentative identities of the complexes formed with recombinant Stat5a and/or Stat5aß are indicated in the figure: homomeric Stat5a complexes (5/5), heteromeric Stat5a/Stat5aß complexes (5/5ß), and homomeric Stat5aß complexes (5ß/5ß).
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When recombinant full-length Stat5 was incubated with the 5'-2C12GLE12 probe, a faster migrating band was formed in addition to complex A (Fig. 12B
, lane 1). The faster migrating band is likely to represent binding of single Stat5 dimers to the probe. By increasing the ratio of carboxy-truncated Stat5 in the reactions (lanes 28) additional bands were formed with the 5'-2C12GLE12 probe, indicating that Stat5ß also can bind to this element. These bands are likely to represent heteromeric Stat5-Stat5ß and homomeric Stat5ß complexes, with the upper bands corresponding to binding of tetrameric or two-dimeric complexes and the lower bands corresponding to binding of single dimers (see Fig. 12B
). When nuclear extracts, either mixed to a low Stat5/Stat5ß ratio or expressing only Stat5ß, were incubated with the 5'-2C12GLE12 probe, mainly a dimer complex was formed (lanes 8 and 9), indicating that Stat5ß tetramers or two-dimer complexes have a low affinity for this element. Furthermore, comparison of lanes 1 and 9 indicates that Stat5ß could have a lower affinity for the 5'-2C12GLE12 probe compared with full-length Stat5.
When recombinant full-length Stat5 was incubated with the 3'-2C12GLE12 probe, two bands were formed, likely to correspond to a tetramer/two-dimer complex and a one-dimer complex (Fig. 12C
, lane 1) similar to the binding to the 5'-2C12GLE12 probe. Furthermore, full-length Stat5 seemed to be more effective in binding as a tetramer/two-dimer complex than binding as a single dimer to this probe. With increasing amounts of Stat5ß, additional bands were formed (lanes 28) in a similar manner as observed with the 5'-2C12GLE12 probe (cf. Fig. 12B
). However, binding of heteromeric Stat5-Stat5ß and homomeric Stat5ß complexes to the 3'-2C12GLE12 was evident at higher Stat5-Stat5ß ratios compared with binding to the 5'-2C12GLE12 probe. Moreover, Stat5ß alone was able to form both tetramer/two-dimer and one-dimer complexes to the 3'-2C12GLE12, with a preference for the tetramer/two-dimer complex (lane 9).
GH-Dependent Regulation of CYP2C12 Promoter Constructs via the 5'- and 3'-2C12GLE12 Elements
To address the functional role of the tandem GLEs in the 5'-flanking region and in the 3'-UTR of the CYP2C12 gene, transient transfections with reporter plasmids harboring the GLEs were performed in primary rat hepatocytes (Fig. 13
). No GH-dependent activation was detected using either a construct harboring only the proximal promoter of CYP2C12 in front of the luciferase gene (-549Luc), or a construct where the 32C12GLE12 element was inserted downstream of the luciferase gene (-549Luc/3'-GLE12). However, when the 5'-2C12GLE12 sequence was put in front of -549Luc (5'-GLE12/-549Luc), GH induced the reporter gene activity 3.3-fold (P < 0.001). This is consistent with results from Sasaki et al. showing that the 5'-2C12GLE12 sequence confers GH responsiveness in rat liver (5). The relatively low-fold of induction upon GH treatment is in consonance with previous reports of transfection experiments with single or duplicated GLEs (5, 31). Interestingly, the GH inducibility was reduced by 37% when the construct harbored both the 5'-2C12GLE12 upstream of the CYP2C12 promoter and the 3'-2C12GLE12 downstream of the luciferase gene (5'-GLE12/-549Luc/3'-GLE12). The reduced GH inducibility of 5'-GLE12/-549Luc/3'-GLE12 compared with 5'-GLE12/-549Luc was statistically significant (P < 0.01). In the hepatocyte culture system, cotransfection of a reporter plasmid for normalization leads to squelching of the hormone response, and therefore protein normalization was used. In addition to primary hepatocytes, we have used the GH receptor stably transfected rat hepatoma cell line BRL-4 for transient transfection experiments. In these cells, normalization against a cotransfected reporter plasmid is possible and we obtained concordant results (data not shown).

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Figure 13. GH Induction of CYP2C12 Reporter Gene Constructs via the 5'- and 3'-2C12GLE12 Elements
Primary rat hepatocytes were transiently cotransfected with different CYP2C12 firefly luciferase constructs and pXM-MGF. After transfection, the cells were cultured in the absence (white bars) or presence (gray bars) of bGH (100 ng/ml) for 12 h and then harvested. The basal activity (luciferase units/µg protein) of -549Luc, in the absence of GH, was given the value 1. All values represent the mean ± SEM of 8 independent transfections from one representative experiment. Significant difference between two groups at P < 0.01 (**) or P < 0.001 (***) (ANOVA, followed by Students Newman-Keuls test).
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DISCUSSION
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In this study with rat liver extracts, we found that Stat5ß colocalized with full-length Stat5 in the nuclei after GH stimulation and that Stat5ß was absent in the cytosol. This indicates that Stat5ß can be formed by a proteolytic cleavage mechanism in the liver, as has been demonstrated in myeloid cells (8, 11, 12, 13), in addition to the alternative splicing mechanism previously demonstrated (9). Furthermore, a cleavage product of 16 kDa was detected in liver nuclear extracts using a Stat5a-specific antibody, directed against the carboxy terminus of the Stat5a protein. The absence of a low molecular weight band using an antibody against the carboxy terminus of Stat5b was surprising because Stat5b is more abundant in rat liver than Stat5a (9). This could be due, however, to the lower sensitivity of the Stat5b antibody than of the Stat5a antibody that we have observed. It could also indicate that Stat5a is more susceptible to proteolytic cleavage than Stat5b.
Proteolytically cleaved Stat5 forms are generated through a unique protein-processing event. A Stat5-specific nucleus-associated protease, able to cleave Stat5 at a specific site, has been characterized in myeloid cell lineages (11). The sequence required for cleavage has been delimited to ATYMDQAP, present in both Stat5a and Stat5b, and with the cleavage taking place between the tyrosine and the methionine (11). By cleavage at this site, the protein becomes transcriptionally inactive (12). The Stat5-specific protease has been shown to be inhibited by PMSF but not by other serine protease inhibitors (11, 12). In line with this, the inclusion of PMSF during the liver nuclear extract preparation resulted in apparently less Stat5ß. However, neither in this type of extract nor in extracts prepared by direct boiling of the minced tissue in sodium dodecyl sulfate-containing buffer the formation of Stat5ß was completely abolished (data not shown).
A study performed with mammary gland tissue in which Stat5 signaling is activated by prolactin suggests that truncated forms of Stat5 are exclusively generated in vitro during cell extract preparation (32). This does not, however, rule out the possibility of an in vivo function of the protease in other cells. The demonstration that Stat5ß protein is formed by proteolytic cleavage in myeloid but not in lymphoid cell lineages suggests that the protease is active in vivo in certain cell types (11). Furthermore, several groups have shown that loss of expression and activation of Stat5ß isoforms correlates with maturation of cells indicating a distinct functional role of Stat5ß (13, 33). Moreover, introduction of a noncleavable mutant of Stat5 into a myeloid cell line has been shown to lead to significant phenotypic changes (11). It is conceivable that the Stat5 protease activity is regulated under certain conditions such as differentiation, disease, stress, or by hormones. Our finding that Stat5ß is formed from recombinant Stat5a subjected to incubation with nuclear extracts from Hx rats, either untreated or treated intermittently with GH injections, indicates that the Stat5 protease activity in rat liver is not regulated by GH. However, with all liver extracts analyzed we have noticed a tendency toward a higher apparent ratio of Stat5 to Stat5ß in extracts prepared from rats continuously exposed to GH compared with extracts from rats intermittently exposed to the hormone, but we have not been able to confirm this trend statistically.
If the Stat5 protease is not regulated by the pattern of GH, one could anticipate that when low levels of Stat5 are continuously translocated to the nucleus, as occurs at continuous GH exposure, an equilibrium of Stat5 and Stat5ß prevails. After a GH pulse as in normal male rats, a rapid activation and nuclear translocation of Stat5 ensues. Immediately after the onset of nuclear translocation, it is likely that all of the Stat5 proteins exist in the full-length form. With time, the Stat5 may be cleaved by the specific protease. The ratio of Stat5 to Stat5ß in the nucleus would then be lower in between the GH pulses than immediately after the pulse or during continuous GH, because there is no further nuclear translocation of Stat5 during the GH interpulse period. The higher resistance of Stat5ß to dephosphorylation (8, 30) would lead to prolonged DNA binding of Stat5ß complexes compared with complexes of full-length Stat5. Thus, the pattern of GH exposure of the liver could affect indirectly the amount of Stat5ß in relation to the amount of full-length Stat5.
Characterization of DNA-protein interactions in vitro using single and minimal response elements has certainly contributed to our understanding of GH signaling pathways. However, the presence of two GLEs in close proximity is apparently common to Stat5 target sequences (15, 16, 20). Furthermore, two clustered Stat5 consensus sequences or one consensus and one half-site (TTC or GAA) have previously been shown to allow cooperative binding and formation of Stat5 tetramers (15, 16, 27), and as shown in this study, by using natural tandem elements as probes in EMSAs, additional information may be obtained. The most interesting finding was that binding to the 3'-2C12GLE12 probe resulted in several different complexes whose appearance correlated with the presence of Stat5ß protein in the liver nuclear extract. This is in contrast to the binding to the 5'-2C12GLE12 probe that gave rise to one major complex with the same migration regardless of the amount of Stat5ß in the liver nuclear extract. However, in gel shift experiments with recombinant Stat5a, carboxy-truncated Stat5 also was able to bind to the 5'-2C12GLE12 probe. This difference in Stat5 binding to the 5'-2C12GLE12 probe could be a result of only Stat5a being used in the experiment with recombinant protein, whereas in experiments with endogenous Stat5 from rat liver nuclear extracts Stat5b dominates over Stat5a (9). There could also be differences between rat hepatocytes and COS7 cells in phosphorylation of the protein. It has been shown recently that Stat5a and Stat5b are serine phosphorylated differently (34). Furthermore, serine phosphorylation of Stat5 proteins was shown to be of importance for both Stat5 binding to GLEs and for the transcriptional activation of different reporter constructs harboring Stat5-binding elements. Although recombinant Stat5aß was shown to bind to the 5'-2C12GLE12 element, it is questionable whether binding of Stat5ß to this element is of functional importance for GH regulation of the CYP2C12 gene in rat liver. Heteromeric Stat5-Stat5ß and homomeric Stat5ß complexes were formed more readily with the 3'-2C12GLE12 probe than with the 5'-2C12GLE12 probe, and this was evident both with recombinant Stat5a and with endogenous Stat5 in rat liver nuclear extracts. In fact, with endogenous Stat5, Stat5ß complexes were formed only with the 3'-2C12GLE12 probe. This, together with the apparently low level of Stat5ß found in rat liver nuclear extracts (cf. Fig. 1A
), compared with the Stat5-Stat5ß ratios used with recombinant Stat5a, indicates that binding of Stat5ß complexes to the 3'-2C12GLE12 is more likely to be of physiological relevance than Stat5ß binding to the 5'-2C12GLE12 element.
Taken together, our results show that on the perfect repeat of two GLEs in the 5'-flanking sequence of the CYP2C12 gene a major complex of full-length Stat5a and Stat5b bound to the probe when liver nuclear extract was used. This tandem GLE has been shown to convey GH regulation of the CYP2C12 gene (5). On the other hand, the imperfect GLE repeat in the 3'-UTR of the gene bound several different complexes, of either full-length or carboxy-truncated Stat5. The full-length Stat5 included in these complexes was predominantly the Stat5b isoform. The origin of the carboxy-truncated Stat5 has not been possible to ascertain, but it is likely that both Stat5a and Stat5b, harboring the ATYMDQAP sequence required by the protease, are subjected to proteolytic cleavage, and it is therefore conceivable that both Stat5ß isoforms may bind to the 3'-2C12GLE12.
Because Stat5ß lacks the trans-activating domain, it can inhibit transcription of genes activated by the full-length Stat5 (8, 12, 30). It is therefore plausible that Stat5ß binding to the 3'-2C12GLE12 motif (bands b and e) could play a role in the low expression of the CYP2C12 gene in male rats. That the 3'-2C12GLE12 motif would be responsible for attenuation of the GH activation of the CYP2C12 gene via the 5'-2C12GLE12 sequence is supported by the transfection experiments carried out in primary rat hepatocytes. In the male rat, a transient transcriptional activation of CYP2C12 could occur in response to each GH pulse that would be terminated by an unfavorable decrease of the Stat5-Stat5ß ratio during the GH interpulse period. Indeed, P4502C12 mRNA is induced in Hx rats by GH injections, but only 1.5- to 3-fold, whereas continuous GH treatment induces the gene at least 10-fold (Ref. 2 and our unpublished observations). As discussed above, a more favorable and constant ratio of Stat5/Stat5ß could prevail in the female rat being continuously exposed to GH, and this ratio would not lead to termination of transciption of the CYP2C12 gene. A recent study by Delesque-Touchard et al. (35) suggests that GH-activated Stat5b suppresses HNF3 and/or HNF6-induced expression of a CYP2C12 promoter construct. However, this construct did not harbor the 5'-2C12GLE12 sequence or any other Stat5-binding elements. The publication by Sasaki et al. (5) clearly demonstrated that the 5'-flanking 2C12GLE12 sequence together with downstream elements binding HNF-4 and HNF-6 convey the GH regulation of the CYP2C12 gene in the female rat. They also found that the GH-responsive CYP2C12 5'-flanking reporter construct was activated in male rats. The authors speculate that the gender difference in expression of CYP2C12 could be the result of an unknown repressor protein(s) present in male rats or due to inactivation at the chromatin level in the male. It is tempting to speculate that the suppressor protein in the male rat could be Stat5ß. Thus, binding of different Stat5 complexes to the 3'-2C12GLE12 element could be involved in the GH-dependent and sex-differentiated expression of the CYP2C12 gene.
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MATERIALS AND METHODS
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Materials
Recombinant bovine GH (bGH) and human GH were provided by American Cyanamid (Wayne, NJ) and Pharmacia \|[amp ]\| Upjohn, Inc. (Stockholm, Sweden), respectively. Complete protease inhibitor cocktail tablets and PMSF were purchased from Roche (Bromma, Sweden), and radionucleotides
-32PdCTP,
-32PdATP, and
-32PdATP were purchased from Amersham Pharmacia Biotech (Uppsala, Sweden). The MGF expression plasmid encoding ovine Stat5a (pXM-MGF) (37, 38) was kindly provided by Dr. B. Groner (Georg Speyer Haus, Institute for Biomedical Research, Frankfurt am Main, Germany). Cell culture reagents were obtained from Life Technologies, Inc. (Täby, Sweden).
Animals
Normal Sprague Dawley rats, 8 wk of age, and female Sprague Dawley rats Hx at 6 wk of age were purchased from Møllegaards Breeding Centre Ltd. (Skensved, Denmark). The animals were maintained under standardized conditions of light and temperature and with free access to animal chow and water. Completeness of hypophysectomy was ascertained by recording the body weight for 1 wk before GH treatments and by inspection of the sella turcica when the animals were decapitated. Bovine GH was administered to Hx animals at a daily dose of 500 µg/kg by either continuous infusion from osmotic minipumps (model 2001; Alza Corp., Palo Alto, CA) or by daily sc injections. The last injection before the animals were decapitated was given ip. The animals were killed at different time points in relation to the GH treatment, as indicated in the figures. All animal procedures were approved by the Stockholm South Ethical Committee of the Swedish National Board for Laboratory Animals.
Analysis of Hepatic mRNA Levels
Levels of rat liver cytochrome P450 2C11 and P450 2C12 mRNA were analyzed in samples of total nucleic acids using specific 35S-UTP-labeled cRNA probes in solution hybridization assays as previously described (38, 39).
Nuclear and Cytosolic Protein Extraction
Rat livers were homogenized in sucrose buffer (0.3 M sucrose; 10 mM HEPES, pH 7.6; 1 mM EDTA; 10 mM KCl) containing 0.35 µg/ml pepstatin, 0.15 mM spermine, 0.5 mM spermidine, 1 mM sodium orthovanadate, 10 mM NaF, 1 mM dithiothreitol, and protease inhibitor cocktail, with or without 1 mM PMSF. Liver nuclei were prepared by centrifugation of the homogenate through buffered 2 M sucrose (2), after which nuclear extracts were prepared as described by Gorski et al. (40). For each extract preparation, pooled nuclei from three rats were used and at least two separate preparations were made. Cytosols were prepared by clearance of pooled homogenates by centrifugation for 30 min at 213,000 x g.
COS7 cells were cultured in DMEM supplemented with 10% fetal calf serum in 10-cm dishes. At 6070% confluency, serum was withdrawn, and the cells were transfected using FuGENE 6 (Roche) according to the manufacturers instructions. Briefly, cells were cotransfected with 5 µg of either pXM-MGF, pXM-MGFß, or the empty vector pXM and 5 µg of PCMV-PrlR. Media were changed 24 h later, and the cells were treated with human GH (400 ng/ml) for 15 min. Cells were rinsed with cold PBS and lysed in solution A (10 mM HEPES, pH 7.9; 1.5 mM MgCl2; 10 mM KCl; 1% Triton X-100). After centrifugation, the supernatant was saved as cytosolic extract, and the pellet was dissolved in 3 vol of buffer C (20 mM HEPES, pH 7.9; 25% glycerol; 420 mM NaCl; 1.5 mM MgCl2; and 0.2 mM EDTA) and incubated for 30 min at 4C. After centrifugation, the supernatant was saved as nuclear extract. All buffers were supplemented with inhibitors, except PMSF, as described above.
Western Blotting
Sodium dodecyl sulfate-solubilizing buffer (10 mM Tris-HCl, pH 6.8; 0.8% sodium dodecyl sulfate; 4.6% glycerol; 0.02% bromophenol blue; and 0.14 M 2-mercaptoethanol) was added to cytosol and nuclear extracts, after which samples were boiled. Proteins were separated on a 7.5% SDS-PAGE gel and transferred to a Hybond ECL nitrocellulose membrane (Amersham Pharmacia Biotech) by semidry blotting. The membrane was blocked overnight in PBS containing 5% fat-free milk protein and 0.1% Tween 20. After washing, the membrane was incubated for 1 h with antibody. Antibodies used were as follows: mouse anti-Stat5 (S21520, Transduction Laboratories, Inc., Lexington, KY, raised against amino acids 451-649 of sheep Stat5) diluted 1:250; rabbit anti-Stat5a (sc-1081, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, raised against amino acids 774-793 of mouse Stat5a) diluted 1:750; and rabbit anti-Stat5b (06-554, Upstate Biotechnology, Inc., Lake Placid, NY, raised against amino acids 774-787 of human Stat5b) diluted 1:1000. All antibodies were diluted in PBS containing 0.1% Tween 20 (TPBS) and 1% milk protein, except for anti-Stat5b in which the dilution was performed according to the supplier. After washing of the membrane in TPBS, the secondary antibody, goat antirabbit IgG, or goat antimouse IgG coupled with horseradish peroxidase, diluted 1:4000 and 1:2000, respectively, in 1% milk protein/TPBS, was applied for 1 h. After secondary antibody incubation, the membrane was further washed in TPBS, after which specific antibody signals were visualized on x-ray film by enhanced chemiluminescence using an ECL kit (Amersham Pharmacia Biotech). For reprobing, membranes were stripped for 30 min at 50 C in a buffer (62.5 mM Tris, pH 6.8; 2% sodium dodecyl sulfate; and 100 mM ß-mercaptoethanol).
For detection of the carboxy-terminal part of Stat5 after proteolytic cleavage, proteins were separated on a 15% SDS-PAGE gel and probed with anti-Stat5 (sc-1081 and 06-554) as described above. To analyze Stat5 cleavage activity in rat liver nuclear extracts, 7 µg of nuclear extracts from GH-stimulated COS7 cells expressing ovine Stat5a were incubated with 30 µg of nuclear extracts from Hx rats for 30 min at 37 C. Protease inhibitors were added to a final concentration of 2 µg/ml (aprotinin), 0.5 µg/ml (leupeptin), 0.7 µg/ml (pepstatin), or 0.17 mg/ml (PMSF) to some of the reactions (see Fig. 3
).
EMSA
Liver nuclear extracts (1-10 µg protein) or nuclear extracts from COS7 cells transfected with expression vectors for either full-length (pXM-MGF) or carboxy-truncated (pXM-MGFß) ovine Stat5 were incubated with 2.5 µg poly(dI/dC) for 10 min at room temperature in EMSA buffer (4% Ficoll; 4% glycerol; 12 mM HEPES, pH 7.9; 4 mM Tris-HCl, pH 7.9; 0.14 mM EDTA; and 1 mM dithiothreitol). A total of 30,000 cpm of 32P-labeled probe was added, and the incubation was allowed to proceed for an additional 10 min. For supershift analysis the extracts were incubated with antibody, either overnight at +4 C or for 45 min at room temperature, before addition of the probe. Antibodies used in supershift analysis were anti-Stat5 (raised against amino acids 6132 of sheep Stat5 (41), kindly provided by Dr. L.-A. Haldosén), anti-Stat5a (sc-1081x, Santa Cruz Biotechnology, Inc.), and anti-Stat5b (sc-835x, Santa Cruz Biotechnology, Inc., raised against amino acids 763779 of mouse Stat5). Samples were resolved at room temperature on a prerun native polyacrylamide gel (4.5% for the Oct and 2C12GLE1 probes, and 5.5% for the 5'- and 3'-2C12GLE12 probes) in 0.25x 22.5 mM Tris-borate and 0.5 mM EDTA for 1 h at 250 V. To increase the resolution of the protein complexes bound to the 5'- and 3'-2C12GLE12, the electrophoresis was run for 4 h at 150 V. After electrophoresis, the gel was dried and exposed to an x-ray film. Probes corresponding to CYP2C12 gene sequences (see Fig. 4
) were end labeled with T4 PNK (Amersham Pharmacia Biotech) and gel purified. The Oct probe, 5'-AGC-TTG-ATC-ACC-TCA-GGA-TCC-TTT-GTA-TGC-AAA-TCA-TGT-3' (sense strand), was labeled with Klenow (Amersham Pharmacia Biotech) and purified on a nick-column (Amersham Pharmacia Biotech).
Plasmid Constructs
A luciferase reporter construct driven by the CYP2C12 promoter (-549Luc) was created by PCR amplification of the CYP2C12 promoter (-549 bp to +9 bp) with prolonged primers harboring cloning sites for BglII and HindIII, respectively, followed by subcloning into the multicloning site of the pGL3-Basic vector (Promega Corp., Southampton, UK). Oligonucleotides harboring 5'-2C12GLE12 and 3'-2C12GLE12 (Fig. 4
) flanked by a 5'-overhang of 5'-CTAG-3' were subcloned into the -549Luc vector in front of the CYP2C12 promoter (NheI site) and 3' of the luciferase reporter gene (XbaI site), respectively. The expression plasmid, pXM-MGFß, was made with the Transformer Site-Directed Mutagenesis Kit from CLONTECH Laboratories, Inc. according to the manufacturers instructions. Briefly, a selection primer (5'-GTCCAACTGCAGTTGACGG TAACG-3') and a mutagenic primer (5'-GAAGCAGCGCCACCTACTGAATGTTTGTTC GGGG-3') were used to delete the sequence in pXM-MGF corresponding to amino acids 721794 in the carboxy terminus of ovine Stat5 [MGF (mammary gland factor)/Stat5a]. To obtain the empty vector pXM, pXM-MGF was digested with SalI and NotI to excise the Stat5 (MGF) expressing sequence, and then blunted with Klenow and religated. All plasmids were purified with EndoFree Plasmid Maxi kit from QIAGEN (Merck Eurolab, Stockholm, Sweden), according to the manufacturers instructions.
Transfection Assays
Primary rat hepatocytes were prepared by noncirculating collagenase perfusion through the portal vein of anesthetized female rats according to the method of Bissell and Guzelian (42). Cells were seeded at a density of 2 x 106 per 60-mm dish in 4 ml of Williams E media supplemented with insulin (0.1 µg/ml), penicillin (50 U/ml), streptomycin (50 U/ml), and 2% fetal calf serum. After the initial overnight incubation, the media were changed to serum-free and thereafter renewed daily. The cells were transfected, at 44 h of cell culture age, using FuGENE 6 (Roche) according to the manufacturers instructions. Briefly, cells were cotransfected with 2 µg of plasmid harboring the different CYP2C12 constructs fused to the firefly luciferase reporter gene, and 0.02 µg of pXM-MGF plasmid. After 8 h of transfection, media were changed and the cells were treated or not with bGH (100 ng/ml). After 12 h of bGH treatment, the cells were harvested in PBS and lysed in PLB buffer (SDS Promega Corp.), by aspiration ten times through a 27-gauge needle. Firefly luciferase activity was measured using the GenGlow reagents (B01243963, Thermo Labsystems, Stockholm, Sweden) in a luminometer (Anthos lucy1, Labdesign, Täby, Sweden), and the protein concentration was determined using a Bradford-Coomassie assay (23225ZZ, Pierce Chemical Co., Boule-Nordic AB, Stockholm, Sweden) according to the manufacturers descriptions.
Statistics
Data analysis was performed using one-way ANOVA followed by the Students Newman-Keuls test. Samples were considered significantly different at P < 0.05.
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ACKNOWLEDGMENTS
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We are grateful to Dr. Lars-Arne Haldosén for valuable discussions during the course of this study and for critical reading of the manuscript.
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FOOTNOTES
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This work was supported by grants from the Swedish Medical Research Council (Grants 31X-06807 and 72X-13146) and from Karolinska Institutet funds.
Abbreviations: bGH, Bovine GH; GAS,
-interferon-activated sequence; GLE, GAS-like element; HNF, hepatocyte nuclear factor; Hx, hypophysectomized; MGF, mammary gland factor; PMSF, phenylmethylsulfonyl fluoride; Stat, signal transducer and activator of transcription; TPBS, PBS containing 0.1% Tween 20; UTR, untranslated region.
Received for publication June 13, 2001.
Accepted for publication March 6, 2002.
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