©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Intermittent Plasma Growth Hormone Triggers Tyrosine Phosphorylation and Nuclear Translocation of a Liver-Expressed, Stat 5-related DNA Binding Protein
PROPOSED ROLE AS AN INTRACELLULAR REGULATOR OF MALE-SPECIFIC LIVER GENE TRANSCRIPTION (*)

David J. Waxman (§) , Prabha A. Ram , Soo-Hee Park , Hee K. Choi

From the (1) Division of Cell and Molecular Biology, Department of Biology, Boston University, Boston, Massachusetts 02215

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Growth hormone (GH) exerts sexually dimorphic effects on liver gene transcription that are regulated by the temporal pattern of pituitary GH release, which is intermittent in male rats and nearly continuous in females. To investigate the influence of these GH secretory patterns on intracellular hepatocyte signaling, we compared the pattern of liver nuclear protein tyrosine phosphorylation in male and female rats. An M 93,000 polypeptide, p93, was found to be tyrosine phosphorylated to a high level in male but not female rats. GH, but not prolactin, rapidly stimulated p93 tyrosine phosphorylation in hypophysectomized rats. Intermittent plasma GH pulses triggered repeated p93 phosphorylation, while continuous GH exposure led to desensitization and a dramatic decline in liver nuclear p93. p93 was cross-reactive with two monoclonal antibodies raised to mammary Stat 5, whose tyrosine phosphorylation is stimulated by prolactin. Intermittent GH pulsation translocated liver Stat 5/p93 protein from the cytosol to the nucleus and also activated its DNA binding activity, as demonstrated using a mammary Stat 5-binding DNA element derived from the -casein gene. p93 is thus a liver-expressed, Stat 5-related DNA binding protein that undergoes tyrosine phosphorylation and nuclear translocation in response to intermittent plasma GH stimulation and is proposed to be an intracellular mediator of the stimulatory effects of GH pulses on male-specific liver gene expression.


INTRODUCTION

Growth hormone (GH)() regulates a broad range of physiological processes, including somatic growth and development, carbohydrate and lipid metabolism, and liver metabolic function (1, 2) . Some of these effects of GH are indirect and are mediated by insulin-like growth factor 1 produced in the liver in response to GH stimulation, while others result from the direct action of GH on target tissues. The effects of GH on responsive cells are transduced by GH receptor (3, 4) , a transmembrane protein expressed on the surface of liver, adipose, kidney, heart, intestine, lung, and muscle cells (5, 6) . GH receptor is a member of the cytokine receptor superfamily (7) and is comprised of three domains: a 246-amino acid extracellular domain that binds to and is dimerized by a single molecule of GH, a short transmembrane segment, and a 350-amino acid intracellular domain that is required for transduction of intracellular signaling events stimulated by GH (3, 8, 9) .

GH stimulates tyrosine phosphorylation of multiple cellular polypeptides, including GH receptor itself, as an early hormonal response (10, 11) . Some of these phosphorylations are catalyzed by a GH receptor-associated kinase identified as Jak2 (12, 13) , a member of the Janus family of tyrosine kinases (14, 15) , while others are catalyzed by downstream kinases (11) . One of these kinases, mitogen-activated protein kinase, triggers a cascade of kinases that ultimately activate ribosomal protein S6, which mediates the stimulatory effects of GH on protein synthesis (16, 17) . Studies in mouse 3T3-F442A preadipocytes (13, 18) and in rat liver in vivo(19) have shown that GH stimulates tyrosine phosphorylation of a latent cytoplasmic transcription factor of M 91,000, designated Stat 1. In human lymphoblastoid IM9 cells, GH can activate, via tyrosine phosphorylation, a 93,000 dalton protein that may be antigenically related to Stat 1 (13, 20) . Stat 1 belongs to a family of Stat proteins (signal transducer/activator of transcription) that mediate the transcriptional responses stimulated by multiple growth factors and cytokines, including interferon-, epidermal growth factor, and various interleukins (21, 22) . Tyrosine phosphorylation of Stat proteins is associated with nuclear translocation and activation of a latent DNA binding activity, leading to transcriptional activation of target genes (21, 22) .

A unique feature of GH action is that many, though not all, of the effects of this polypeptide hormone are dependent on the temporal pattern of hormone release by the pituitary gland (2) . This pattern of pituitary GH secretion is ultimately regulated by gonadal steroids and hence is markedly different between the sexes, particularly in rodent models (23, 24, 25) . The resultant sex-dependent plasma GH profiles in turn regulate the sex-dependent effects of GH on longitudinal bone growth and body weight gain (26, 27, 28) , as well as the sex-dependent expression of a large number of liver gene products, including polypeptide hormone and other receptors (29-31), cytochrome P450 (32, 33) , and other liver enzymes (34, 35, 36) . Studies in the rat model have established that intermittent plasma GH pulsation, a characteristic of adult male rats, activates liver transcription of several genes whose expression is limited to adult males, such as the gene that encodes the steroid 2- and 16-hydroxylase enzyme CYP2C11, while exposure of hepatocytes to GH continuously, which occurs in adult female rats, stimulates the expression in liver of the steroid sulfate 15-hydroxylase CYP2C12 (37, 38). GH replacement studies in hypophysectomized rats have revealed a requirement for a minimum GH ``off-time'' for effective stimulation of CYP2C11 gene expression by intermittent GH pulsation (28). This, in turn, suggests that hepatocytes exposed to GH continuously (as in adult female rats) become desensitized with respect to activation of the signaling pathway that normally induces CYP2C11 gene expression in males (28) .

Although some progress has been made toward the identification of cis-acting elements that may contribute to the GH regulation of these sex-specific liver genes (37, 39, 40) , little is known about the intracellular signaling events that are stimulated by GH and the pathways that lead to the differential activation in liver of male-expressed as compared to female-expressed genes under the influence of pituitary GH secretory patterns. Of particular interest is the possibility that GH may stimulate alternate signaling pathways in liver, depending on whether hepatocytes are exposed to the hormone intermittently or continuously. The present study examines the effects of physiological levels of GH administered to hypophysectomized rats on nuclear protein tyrosine phosphorylation in the liver. We report that intermittent plasma GH, but not continuous plasma GH, stimulates a high steady-state level of tyrosine phosphorylation and nuclear translocation of a 93,000 dalton polypeptide, p93, which we identify as a novel DNA binding protein related to the prolactin-activable mammary gland factor Stat 5 (41, 42) . These findings are discussed in the context of the cellular and molecular mechanisms through which GH secretory patterns regulate the sexual dimorphism of liver gene expression.


EXPERIMENTAL PROCEDURES

Animal Treatments

Adult male and female Fischer 344 rats (8-10 weeks of age) were untreated or were hypophysectomized by the supplier (Taconic, Inc., Germantown, NY), with follow-up care provided as previously described (43) . Animals were maintained at the Laboratory for Animal Care Facility at Boston University (lights on 8 a.m.-8 p.m.) for at least three weeks following surgery to ensure completeness of hypophysectomy (absence of body weight gain). Intact, untreated male and female rats were killed between 7:30 and 7:45 a.m.

Rat GH was administered to hypophysectomized rats by one of three protocols. Protocol A: intraperitoneal injection of GH was at 12.5 µg/100 g body weight, except where a lower dose (3 µg/100 g body weight) is specified. Rats were killed at time intervals ranging from 5 min to 4 h following a single intraperitoneal injection of GH, as specified in individual experiments. Where indicated, a second intraperitoneal injection of GH was administered 4 h after the first injection, and the animals were killed 45 min later. Protocol B: repeat subcutaneous injection of GH was at 12.5 µg/100 g body weight/injection, given at t = 0, 3, and 6 h to maintain a circulating level of GH over time, followed by sacrifice at t = 8 h. GH administered by the subcutaneous route produces periods of continuous circulating plasma hormone lasting 5-6 h (28, 44) and, when given repeatedly, confers a female profile of liver gene expression (45) . Protocol C: longer term continuous GH treatment was by hormone infusion at 2 µg/100 g body weight/h for 1, 3, or 7 days using an Alzet osmotic minipump (model 2001; Alza Corp., Palo Alto, CA). Minipumps were implanted subcutaneously on the backs of the animals under Ketamine anesthesia using protocols provided by the manufacturer and approved by the Boston University Institutional Animal Care and Use Committee. Control animals received vehicle injections or vehicle-filled osmotic minipumps. These treatments correspond to biologically effective GH replacement doses, which mimic the intermittent plasma GH profile of male rats (periodic intraperitoneal GH injection) and the continuous plasma GH profile of females (repeat subcutaneous injection or GH infusion by osmotic minipump) (28) .

Rat GH used in these experiments was a hormonally pure grade obtained from the National Hormone and Pituitary Program, NIDDK (NIDDK-rGH-B-14-SIAFP, biopotency 1.8 I.U./mg). This material was prepared by subtractive immunoaffinity purification and was shown to be devoid of other pituitary hormones, including prolactin, TSH, FSH, and LH. Rat GH was dissolved in 30 mM NaHCO, pH 10.3, containing 0.15 M NaCl, and the pH was immediately lowered to 9.5 by addition of 0.5 M sodium bicarbonate, pH 8.3. Rat albumin (Sigma) was added to 100 µg/ml to stabilize the hormone (27) . In other experiments, hypophysectomized rats were treated with human GH (NIDDK-hGH-B-1, BIO) or with rat prolactin (hormonally pure grade, NIDDK-rPRL-B-8-SIAFP) at 12.5 µg/100 g body weight, intraperitoneally, and the ani-mals were killed 45 min later. Escherichia coli lipopolysaccharide (LPS) (serotype 026:B6; Sigma L-2762) was administered to hypophysectomized rats by intraperitoneal injection at 1 mg/150 g body weight to promote cytokine release (46) . Animals were killed 75 min later.

Preparation of Liver Nuclear Extract and Cytosol

Nuclear extracts were prepared from individual, freshly excised rat livers using established methods (47) , with the addition of the phosphatase inhibitors sodium fluoride (10 mM) and sodium orthovanadate (1 mM) in the homogenization and nuclear lysis buffers and with the inclusion of the following protease inhibitors (Sigma) in the initial homogenization buffer: antipain, chymostatin, and pepstatin (2 µg/ml each), aprotinin, leupeptin (5 µg/ml each), trypsin inhibitor (10 µg/ml), and phenylmethanesulfonyl fluoride (0.1 mM). The final nuclear preparation was dialyzed against NED buffer [25 mM Hepes (pH 7.6 at 4 °C), 40 mM KCl, 0.5 mM phenylmethanesulfonyl fluoride, 0.1 mM EDTA, 1 mM dithiothreitol containing 0.5 mM sodium fluoride, 1 mM sodium orthovanadate, and 10% glycerol] and then snap-frozen and stored in liquid nitrogen. Cytosolic fractions were prepared by dilution of the supernatant obtained after the first centrifugation step (pelleting of nuclei through a 2 M sucrose cushion) with 2 volumes of NED buffer, followed by centrifugation at 100,000 g for 2 h at 4 °C to pellet a microsomal fraction.

Antibodies

Anti-phosphotyrosine antibodies were mouse monoclonal antibody 4G10 (Upstate Biotechnology, Inc.), mouse monoclonal antibody PY20 (Transduction Laboratories, Lexington, KY), and rabbit polyclonal Shafer anti-phosphotyrosine (11, 48) ; the latter antibody was provided by Dr. L. Argetsinger (Univ. of Michigan). Monoclonal anti-Stat 1 antibody, raised to a peptide fragment comprised of Stat 1 amino acids 591-731 (S21120), monoclonal anti-Stat 3, raised to Stat 3 amino acids 1-178 (S21320), and two independent monoclonal anti-Stat 5 antibodies, both raised to amino acids 451-649 of sheep Stat 5 (S21520, lot 2 (clone 85) and lot 3 (clone 89)), were purchased from Transduction Laboratories. Anti-Stat 1 and anti-Stat 3c were provided by Drs. Z. Zhong and J. Darnell (Rockefeller University) and were used for gel supershift experiments carried out as described (49) . Anti-Stat 3c was raised against Stat 3 amino acids 688-727. Antiserum AbN, raised against amino acids 1-67 of mouse APRF (i.e. mouse Stat 3) (50) , was provided by Dr. D. Levy (New York University School of Medicine).

Western Blot Analysis

Rat liver nuclear extracts (15 µg protein/lane) were electrophoresed through standard Laemmli sodium dodecyl sulfate-polyacrylamide (10%) gels, electrotransferred to nitrocellulose, and then probed with anti-phosphotyrosine or anti-Stat antibodies. Nitrocellulose sheets were blocked for 1 h at 37 °C with 4% non-fat dry milk (Blotto) (w/v) dissolved in 10 mM potassium P, pH 7.4, 0.9% NaCl, 0.3% Tween 20 (anti-phosphotyrosine antibody) or with 3% bovine serum albumin dissolved in 10 mM Tris-Cl, pH 7.5, 0.1 M NaCl, 0.1% Tween 20 (anti-Stat 5). In the case of the anti-phosphotyrosine antibody probings, a special grade of phosphotyrosine-free non-fat dry milk (Upstate Biotechnology, Inc., 17-1056) was used to block nonspecific binding sites. Nitrocellulose blots were washed and then probed for 1-2 h at 20-25 °C with antibodies diluted 1/3000 in blocking solution. Detection was with horseradish peroxidase-conjugated secondary antibody (1/3000) followed by enhanced chemiluminescence (ECL) imaging on x-ray film using ECL reagent (Amersham Corp.).

Phosphatase Treatment

Nuclear extract protein samples (25 µg) were incubated for 45 min at 37 °C with protein phosphotyrosine phosphatase 1B (20 µl containing 2 µg of glutathione S-transferase-phosphatase 1B fusion protein conjugated to 10 µl of agarose beads (Upstate Biotechnology, 14-109)) in a total volume of 70 µl containing 25 mM Tris-Cl, pH 7.0, 50 µM CaCl, and 25 µg of bovine serum albumin. Phosphatase was activated by preincubation for 15 min at 37 °C as suggested by the supplier. Control samples included the phosphatase inhibitor sodium orthovanadate (0.1 mM). Phosphatase-treated samples were analyzed by phosphotyrosine Western blotting and by gel mobility shift assay using a rat -casein promoter probe (see below).

Immunoprecipitation Analysis

Rat liver nuclear extracts (50 µg) were incubated with 3 µg of anti-phosphotyrosine antibody 4G10 or 2 µl of anti-Stat 3 antibody AbN overnight at 4 °C in IP buffer (1% Triton X-100, 150 mM NaCl, 10 mM Hepes, pH 7.4, 1 mM EDTA, 1 mM EGTA, 0.2 mM sodium orthovanadate, 0.2 mM phenylmethanesulfonyl fluoride, 0.5% Nonidet P-40 containing 1 µg/ml each aprotinin, leupeptin, and pepstatin) in a total volume of 100 µl. Protein A-Sepharose (25 µl of a 50% suspension) was added to each sample followed by incubation for 2 h at 4 °C with gentle shaking. Samples were centrifuged for 4 min at 14,000 g, and the Sepharose pellet was then washed three times with 500 µl of IP buffer. The washed beads were resuspended in 30 µl of of 2 Laemmli sample buffer (3% sodium dodecyl sulfate, 10% 2-mercaptoethanol, 2% glycerol, 200 mM Tris-HCl, pH 6.8, containing pyronin Y dye), boiled for 10 min, and centrifuged for 4 min at 14,000 g; the supernatant was then analyzed on 10% sodium dodecyl sulfate-polyacrylamide gels.

Gel Mobility Shift Analysis

Double-stranded oligonucleotide probes, P-end labeled on one strand using T4 kinase, were incubated for 30 min at room temperature with 5 µg of liver nuclear extract protein dissolved in 5 µl of NED buffer and 10 µl of 10 mM Tris-HCl buffer, pH 7.5, containing 10 fmol of DNA probe, 2 µg of poly(dI-dC), 4% glycerol, 1 mM MgCl, 0.5 mM EDTA, 0.5 mM dithiothreitol, 50 mM NaCl (15 µl, total volume). Samples were electrophoresed at room temperature through non-denaturing polyacrylamide gels (4% acrylamide, 0.05% bisacrylamide) in 0.5 TBE buffer (44.5 mM Tris, 44.5 mM boric acid, 5 mM EDTA, pH 8.0) for 2.5 h at 100 volts using standard methods. DNA probes used in these studies were as follows: (a) rat -casein gene Stat 5/MGF response element, nucleotides -101 to -80 (51) , 5`-GGACTTCTTGGAATTAAGGGA-3` (sense strand, oligonucleotide ON-257) and 5`-gTCCCTTAATTCCAAGAAGTCC-3` (antisense strand, ON-258) and (b)SIE probe, 5`-gtcgaCATTTCCCGTAAATCgtcga-3` (sense strand, ON-242) and 5`-gacGATTTACGGGAAATGtcgac-3` (antisense strand, ON-243) (52) . Nucleotides used to facilitate T4 kinase 5`-labeling with [-P]ATP or other purposes are shown in lower case. Unlabeled double-stranded oligonucleotides used as competitors for the gel shift experiments were obtained from Santa Cruz Biotechnology, Inc. and included SIE mutant oligonucleotide (mutation of TTCCCG to CCACCG; sc-2536), and GAS/ISRE consensus oligonucleotide (sc-2537).


RESULTS

Liver Nuclear Factor p93: GH-dependent Tyrosine Phosphorylation in Male but Not Female Rats

GH stimulates the tyrosine phosphorylation of multiple cellular proteins in a reaction catalyzed by Jak2, a tyrosine kinase that is activated following GH binding to the GH receptor. Because many of the effects of GH on liver gene expression are sex dependent and are regulated by the temporal pattern of GH stimulation of hepatocytes, we examined whether there are any differences between male and female rats in their patterns of liver nuclear protein tyrosine phosphorylation. We examined nuclear proteins because the sex-dependent effects of GH on liver gene expression are manifest at the level of transcription initiation (37, 38) , and tyrosine phosphorylation stimulated by cytokines and other growth factors is associated with protein translocation to the nucleus (21, 22) . Fig. 1A shows a Western blot of liver nuclear extracts prepared from adult male and adult female rat liver and probed with anti-phosphotyrosine antibodies. Several immunoreactive protein bands are common to both male and female nuclear extracts. In contrast, an anti-phosphotyrosine immunoreactive protein of 93,000 daltons, designated p93, was prominent in male liver nuclear extracts but was present at low or undetectable levels in female liver nuclei. This same sex-dependent pattern of p93 tyrosine phosphorylation was observed using three distinct anti-phosphotyrosine antibodies: Shafer polyclonal anti-phosphotyrosine (Fig. 1A) and monoclonal antibodies 4G10 (Fig. 2, see below) and PY20 (data not shown). Treatment of the liver nuclear extract with phosphotyrosine phosphatase resulted in a loss of the p93 band, confirming the phosphotyrosine nature of this protein (data not shown). p93 was not detectable with anti-phosphotyrosine antibody in nuclear-free liver cytosol, indicating that this tyrosine-phosphorylated protein is localized in the nucleus (see below).


Figure 1: Tyrosine phosphorylation of nuclear p93 in male rat liver and in GH-treated hypophysectomized rats. Panel A, Western blot of liver nuclear extracts prepared from individual male (M) and female (F) rats probed with Shafer anti-phosphotyrosine antibody. Panel B, Western blot of liver nuclear extracts probed with monoclonal anti-phosphotyrosine antibody 4G10. Shown are hypophysectomized (Hx) rats that were sham-injected (lane2) or were treated with rat GH (3 µg/100 g body weight, intraperitoneally) and then killed 5, 15, or 45 min later (lanes3-5). Treatment of hypophysectomized male rats with prolactin (PRL) (lane6) or with LPS (lane7) was as described under ``Experimental Procedures.'' Lane1, untreated male control.




Figure 2: p93 is electrophoretically distinct from Stat 1 and Stat 3. Shown are Western blots of liver nuclear extracts probed with anti-Stat 3 (lane1), anti-phosphotyrosine antibody 4G10 (lanes2-6), and anti-Stat 1 (lane7). The seven nuclear samples were electrophoresed on a single SDS gel, which was transferred to nitrocellulose and then cut into three strips (lanes1, 2-6, and 7) for parallel detection with the three indicated antibodies. The three strips were aligned prior to exposure to x-ray film for detection of the chemiluminescent signal by ECL. Liver nuclear extracts were prepared from individual male (M) (lanes2 and 7), female (F) (lane3), and hypophysectomized (Hx) rats that were sham-injected (lane4) or were injected with rat GH then killed 45 min later (lanes1, 5, and 6). *, higher molecular weight nuclear protein whose tyrosine phosphorylation is enhanced by GH.



The influence of GH on the tyrosine phosphorylation of p93 was examined by hypophysectomy and GH replacement experiments. p93 was not detectable on anti-phosphotyrosine Western blots of liver nuclear extracts prepared from hypophysectomized male rats but was restored to a level comparable with intact male rats in nuclear extracts prepared from livers of hypophysectomized rats given a physiologic replacement dose of rat GH and then killed either 15 or 45 min later (Fig. 1B, lanes4 and 5). Tyrosine phosphorylation of p93 was not stimulated by prolactin (lane6), demonstrating that this phosphorylation is a GH-specific response. Moreover, bacterial LPS, which promotes the release of multiple cytokines and stimulates the phosphorylation of Stat 3 both in mouse liver (46) and in rat liver,() did not stimulate tyrosine phosphorylation of p93 (lane7). The rapid appearance of tyrosine-phosphorylated p93 in the nucleus following GH treatment in vivo suggests that GH stimulates the phosphorylation of preexisting p93 protein.

p93 Is Distinct from Stat 1 and Stat 3

GH can stimulate the tyrosine phosphorylation of proteins similar or perhaps identical to the Stat proteins that can be activated by various cytokines and growth factors (13, 18, 19, 20) . Therefore, we investigated whether p93 corresponds to Stat 1 or Stat 3, two Stat proteins that are known to be expressed in liver cells. A direct comparison of the electrophoretic mobility of p93, detected by phosphotyrosine Western blotting with that of Stat 1 and Stat 3, which were detected in liver nuclear extracts using their respective Stat form-specific antibodies, revealed that p93 migrated slower than Stat 3 (major band at M 89,000 (p89)) (Fig. 2, lane1) and slower than both bands of the Stat 1/Stat 1 doublet (M 91,000 (p91) and M 84,000 (p84), respectively) (Fig. 2, lane7). No Stat 1 or Stat 3 immunoreactivity was seen on the Western blots at a position corresponding to the migration of p93, indicating that p93 is not a phosphorylated form of Stat 1 or Stat 3. This conclusion was confirmed by immunoprecipitation of p93 with anti-phosphotyrosine antibody, followed by Western blotting with anti-Stat 1 and anti-Stat 3 antibodies (data not shown).

Intermittent Plasma GH Pulsation, but Not Continuous Plasma GH Treatment, Triggers Repeated Phosphorylation of p93 in Vivo

We next investigated the hormonal basis for the marked male specificity of p93 tyrosine phosphorylation seen in intact rats. One possibility, that p93 protein is not expressed in female rat liver, was ruled out by our finding that GH stimulated the tyrosine phosphorylation of p93 within 15 min of a single hormone injection in hypophysectomized female rats (Fig. 3A). Thus, female rats are inherently responsive to a pulse of GH.


Figure 3: Influence of intermittent versus continuous GH treatment on p93 tyrosine phosphorylation. Shown are Western blots of rat liver nuclear extracts probed with anti-phosphotyrosine antibody 4G10. Panel A, liver nuclear extracts prepared from intact female rats (F, lane2) or from hypophysectomized female rats (Hx) that were untreated (lane3) or were treated with rat GH (3 µg/100 g body weight) and then killed 5, 15, or 45 min later (lanes4-6). Nuclear extract from a hypophysectomized male rat treated with rat GH and killed 45 min later is shown for comparison in lane1. Tyrosine phosphorylation of the band marked * was activated by GH within 5 min. Panel B, nuclear extracts prepared from hypophysectomized male rats that were untreated (lane2) or were killed 45 min (lanes3 and 4) or 4 h (lanes5 and 6) following a single intraperitoneal injection of rat GH. Lanes7 and 8, nuclear extracts prepared from hypophysectomized male rats injected with rat GH at t = 0 and again at t = 4 h, then killed 45 min after the second injection (GH pulses = 2) to test for rephosphorylation of p93. Lane1, untreated male nuclear extract. Panel C, nuclear extracts prepared from hypophysectomized female rats exposed to GH continuously for 8 h by repeat subcutaneous injection at t = 0, 3, and 6 h (lanes4 and 5) or for 1 day (lanes6 and 7), 3 days (lanes8 and 9), or 7 days (lanes10 and 11) using an osmotic minipump. Lane1, untreated male control; lane2, hypophysectomized female rat given multiple subcutaneous vehicle injections (control for lanes4 and 5); lane3, hypophysectomized female rat killed 45 min after a single intraperitoneal injection of rat GH. Several nonspecific bands are prominent in the blot shown in panelC.



We next considered whether the preferential tyrosine phosphorylation of p93 seen in male rat liver may be a consequence of the differential stimulatory effect of intermittent GH (male plasma GH profile) versus continuous GH (female plasma GH profile). To test this hypothesis, biologically effective replacement doses of GH were given to hypophysectomized male rats by intraperitoneal injection at 4 h intervals to mimic the frequency of plasma GH pulsation that occurs naturally in intact male rats (2, 23) and is required to stimulate male-specific, GH-dependent liver gene expression (28) . As shown in Fig. 3B, GH stimulated tyrosine phosphorylation of p93 at 45 min, as seen in our earlier experiments, and this was followed by a significant, albeit partial decline in tyrosine-phosphorylated p93 levels after 4 h. A second injection of GH at 4 h restimulated p93 tyrosine phosphorylation to a level comparable with that achieved following the initial GH pulse. Thus, p93 can undergo repeated tyrosine phosphorylation in response to an intermittent, adult male plasma GH profile. By contrast, although p93 phosphorylation was initially induced when GH was delivered to hypophysectomized rats continuously, sustained levels of tyrosine-phosphorylated p93 were only maintained for a limited time period. Tyrosine-phosphorylated p93 was present at a reduced level after 8-24 h of continuous GH exposure and was very low or undetectable after 3 or 7 days of continuous GH treatment (Fig. 3C). Thus, the continuous plasma GH profile in adult female rats is ineffective in supporting high steady-state levels of tyrosine-phosphorylated p93. This indicates that hepatocytes exposed to GH continuously become desensitized with respect to GH-induced p93 tyrosine phosphorylation.

p93 Corresponds to a GH-regulated, Mammary Stat 5-related Liver Nuclear Protein

Mammary gland factor has recently been identified as a Stat family member, designated Stat 5, that can be activated by prolactin (41, 42) . The polypeptide hormones GH and prolactin belong to the same gene family, bind to receptors that are structurally homologous (7) , and both activate the tyrosine kinase Jak2 (12, 53). We therefore investigated whether p93 might correspond to a liver-expressed, mammary Stat 5-like protein. Western blot analysis of adult male rat liver nuclear extracts probed with an anti-Stat 5 monoclonal antibody (clone 85) revealed a closely migrating protein doublet of M 92,500-93,000 (Fig. 4A). The upper band of this doublet corresponds to p93 detected with anti-phosphotyrosine antibody, as established by the precise overlay of the upper Stat 5 band with the p93 band when the two are visualized on separate x-ray films prepared from a single nitrocellulose immunoblot probed sequentially with the two antibodies. In agreement with this conclusion, treatment of the nuclear extracts with phosphotyrosine phosphatase resulted in an apparent conversion of the upper Stat 5-immunoreactive band to the lower band (data not shown). As was observed for p93, the Stat 5-immunoreactive protein bands were absent or were present at much lower levels in female liver nuclear extracts (Fig. 4A). Moreover, the liver Stat 5 protein was increased in the nuclear fraction following GH treatment with the same kinetics as p93 (Fig. 4B, lanes3-6; cf.Fig. 1B). A second, independent anti-mammary Stat 5 monoclonal antibody (clone 89) gave the same pattern of protein bands and the same pattern of liver Stat 5 regulation, further supporting the relatedness of liver Stat 5/p93 and mammary Stat 5. Neither anti-Stat-5 antibody was cross-reactive with Stat 1 or Stat 3, as evidenced by the absence of detectable bands at the migration positions of the latter two Stats.


Figure 4: Liver Stat 5/p93 detected by anti-mammary Stat 5 monoclonal antibody in liver nuclear extracts. Shown are Western blots (WB) of rat liver nuclear extracts (panelsA and B) or immunoprecipitates of nuclear extracts (panelC) probed with anti-Stat 5 monoclonal antibody (clone 85); similar results were obtained with a second anti-Stat 5 monoclonal antibody (clone 89; data not shown). Panel A, male-dominant nuclear expression of liver Stat 5 protein, evident from nuclear extracts prepared from four independent pairs of male (M) and female (F) rats. Panel B, rapid increase in liver Stat 5 in nucleus of hypophysectomized (Hx) male rats treated with GH (3 µg/100 g body weight, intraperitoneally), but not prolactin (PRL) or LPS, as indicated. Panel C, nuclear extracts shown in panelB were immunoprecipitated with anti-phosphotyrosine antibody 4G10 (anti-phosphotyrosine) and analyzed by sequential Western blotting with anti-phosphotyrosine then anti-Stat 5, as indicated in lanes1-7. Lane8, liver nuclear extract from untreated male rat included as a standard.



The identification of p93 as the Stat 5-immunoreactive protein seen on these Western blots was confirmed by immunoprecipitation of p93 from rat liver nuclear extracts with anti-phosphotyrosine antibody 4G10, followed by SDS-gel electrophoresis and sequential Western blotting with anti-phosphotyrosine and then anti-Stat 5 antibody (Fig. 4C). This identification is further supported by immunoprecipitation of the tyrosine-phosphorylated and the Stat 5-immunoreactive p93 protein by antibody AbN (data not shown). AbN is a polyclonal antibody that was raised to a conserved region at the amino terminus of Stat 3 and is cross-reactive with several unidentified Stat-like proteins (50) , including the liver Stat 5-related protein p93. Nuclear accumulation of liver Stat 5 was repeatedly stimulated by an intermittent pattern of GH replacement, as revealed by Western blot analysis with anti-Stat 5 antibody (Fig. 5A). This effect was more apparent when tyrosine-phosphorylated liver Stat 5 (i.e. the upper band of the Stat 5 doublet seen in Fig. 5A) was selectively immunoprecipitated with anti-phosphotyrosine antibody (Fig. 5B). The nuclear level of liver Stat 5 was also suppressed by continuous GH treatment in a manner indistinguishable from that of p93 (data not shown). Liver Stat 5/p93 apparently differs from Stat 5 cloned from sheep mammary gland (41) , however, insofar as it does not respond to prolactin (Fig. 4B, lane7, and Fig. 4C, lane6; cf. unresponsiveness of p93, Fig. 1B).


Figure 5: Elevation of nuclear levels of liver Stat 5 by intermittent GH stimulation. Panel A, shown is a Western blot (WB) of liver nuclear extracts prepared from individual hypophysectomized (Hx) male rats treated with GH by intermittent intraperitoneal injection to mimic the pulsatile plasma GH profile of intact males, using the protocol described under Fig. 3B, and then probed with anti-Stat 5 antibody (clone 85). Panel B, samples from the experiment shown in panelA were immunoprecipitated with anti-phosphotyrosine antibody 4G10 and then probed sequentially with anti-phosphotyrosine 4G10 and then anti-Stat 5 antibodies, as indicated. Samples are from the same experiment shown in Fig. 3B. Samples shown in panelB, lanes5 and 6, correspond to the individual liver nuclear extracts shown in panelA, lanes6 and 7.



GH Stimulates Tyrosine Phosphorylation of Cytosolic Liver Stat 5/p93

Liver cytosolic extracts were analyzed by Western blotting to determine whether hepatocytes contain a cytoplasmic pool of the mammary Stat 5-related protein that could serve as a precursor of the tyrosine-phosphorylated p93, which accumulates in the nucleus following GH stimulation. Western blot analysis of these samples revealed a Stat 5-immunoreactive protein that is expressed at a low level in liver cytosol in hypophysectomized rats (Fig. 6A, lane4), as well as in intact male and female rats (data not shown). The cytosolic Stat 5-immunoreactive protein seen in these samples (designated Stat 5 band b, Fig. 6A) migrated distinctly faster than the liver Stat 5 protein seen in the nucleus (lane4versus3). Moreover, the cytosolic protein was not tyrosine phosphorylated, as determined by immunoblotting with anti-phosphotyrosine antibody (Fig. 6B, lane4). GH treatment led to a noticeable decrease in the mobility of the liver Stat 5 protein detected in the cytosol (i.e. conversion of band b to band a). This mobility decrease was first detectable at 5 min, as revealed by a longer exposure of the blot shown in Fig. 6A, and was maximal at 15 and 45 min after hormone injection (lanes5-7versus4). The lower mobility liver Stat 5 protein present in the cytosol (band a, Fig. 6A) corresponds to the tyrosine-phosphorylated form, as determined by anti-phosphotyrosine Western blotting (Fig. 6B). Moreover, the time course for appearance of this upper band (lanes5-7) paralleled the appearance of the tyrosine-phosphorylated p93 in the nucleus (cf.lanes2 and 3). Thus, GH induces a rapid tyrosine phosphorylation of liver Stat 5 present in a cytosolic precursor pool, and this is closely followed by translocation of the phosphorylated protein to the nucleus.


Figure 6: GH-stimulated phosphorylation of liver cytosolic Stat 5. Shown are Western blots of liver nuclear extracts (15 µg/lane; lanes1-3) and the corresponding cytosols (70 µg/lane; lanes4-9) probed sequentially with anti-Stat 5 (clone 85) (panel A) and anti-phosphotyrosine antibody 4G10 (panel B). Hypophysectomized (Hx) male rats were untreated (lanes1 and 4) or were treated with rat GH (3 µg/100 g body weight, intraperitoneally) and then killed 5 min (lanes2 and 5), 15 min (lanes3 and 6), or 45 min later (lane7). Hypophysectomized rats treated with prolactin (PRL, lane8) or LPS (lane9) are included as controls. Two bands were detected with anti-Stat 5 in hypophysectomized liver cytosol (bands marked a and b); bandb predominates in the hypophysectomized rat liver sample (lane4) and is converted to the more slowly migrating band a following treatment with GH but not prolactin or LPS. The tyrosine-phosphorylated p93 band seen in panelB coincides with Stat 5, band a, as determined by direct overlay of the x-ray films of the two ECL probings. The changes in cytosolic Stat 5 banding pattern shown in lanes4-7 for hypophysectomized male rats were also observed following GH treatment of hypophysectomized female rats (data not shown).



GH Activates DNA Binding Activity of Liver Stat 5/p93

In view of the immunochemical similarity between liver Stat 5/p93 and mammary gland Stat 5, we examined the effects of GH treatment on liver nuclear protein binding to a mammary Stat 5-binding DNA response element found upstream of the rat -casein gene (51) . As shown in Fig. 7A, a discrete gel mobility shift complex was formed upon incubation of the -casein promoter DNA probe with nuclear extracts prepared from male but not female or hypophysectomized male rat liver. This nuclear DNA binding activity was rapidly increased by a pulse of GH given to either male or female hypophysectomized rats (Fig. 7B), with kinetics similar to the tyrosine phosphorylation of p93 (cf.Fig. 1B and 3A). By contrast, neither prolactin nor LPS induced this gel mobility shift complex. Continuous GH treatment suppressed this nuclear protein binding activity in parallel to p93 and liver Stat 5 protein (e.g.Fig. 7CversusFig. 3C). A low level of DNA binding activity was detected in cytosolic extracts of GH-treated hypophysectomized rats (Fig. 7D, lanes4 and 5), in agreement with the low level of tyrosine-phosphorylated liver Stat 5/p93 protein in liver cytosol. Little or no gel shift complex was formed by cytosolic extracts of hypophysectomized male rat liver (Fig. 7D, lane2), despite the presence of liver Stat 5-immunoreactive protein (albeit not tyrosine-phosphorylated protein) (cf.Fig. 6 , lane 4). This suggests that tyrosine phosphorylation is required to activate the DNA binding activity of liver Stat 5. This conclusion is supported by the loss of DNA binding activity upon dephosphorylation of liver Stat 5/p93 by treatment with phosphotyrosine-specific phosphatase (Fig. 7E). Thus, GH-stimulated tyrosine phosphorylation and nuclear translocation of liver Stat 5/p93 is associated with a specific activation of this factor's latent binding activity toward a Stat 5 DNA response element.


Figure 7: Gel mobility shift analysis of liver Stat 5/p93 DNA binding activity using -casein promoter probe. Liver nuclear extracts were analyzed by gel mobility shift assay using a rat -casein gene mammary Stat 5 response element probe. Panel A, liver nuclear extracts prepared from individual male (M) (lanes1, 2), female (F) (lanes3 and 4), and hypophysectomized male rats (lane5). Panel B, time course for activation of liver Stat 5/p93 following intraperitoneal injection of rat GH (3 µg/100 g body weight) in hypophysectomized (Hx) male (lanes2-5) and female rats (lanes6-9). Lane1, intact male rat liver nuclear extract (M). Panel C, influence of continuous GH treatment in hypophysectomized female rats for times ranging from 45 min to 3 days. Samples analyzed are the same ones shown in Fig. 3C. A second, lower mobility protein-DNA complex with the same binding specificity as the major complex is evident in this experiment. Panel D, gel shift activity detected in liver cytosol (20 µg; lanes2-5) of male hypophysectomized rats treated with GH. Samples are the same as those shown in Fig. 6. Gel shift activity of liver nuclear extract from untreated male rat (5 µg) is shown for comparison in lane1. Panel E, effect of phosphotyrosine phosphatase (PY-PTase) treatment on gel shift complex formation. Liver nuclear extracts from two individual untreated male rats were incubated for 45 min at 37 °C either with or without phosphatase (PTase) in the absence or presence of 0.1 mM sodium orthovanadate, as indicated. Panel F, effect on gel shift complex formation of unlabeled DNA competitors present at a 20-fold (lane2) or 50-fold (lanes3-6) molar excess over P-labeled -casein probe. Competitors used were -casein probe (lanes2 and 3), SIE (lane4), SIE mutant (lane5), and GAS/ISRE (lane6).



Competition experiments were carried out to examine the relationship of the GH-inducible, liver Stat 5 protein/-casein DNA gel shift complex to protein-DNA complexes formed by other Stat proteins (Fig. 7F). Formation of the -casein DNA complex was fully inhibited by unlabeled -casein DNA probe (lanes2 and 3) but was not inhibited by an oligonucleotide containing a consensus binding site (21) for the interferon--activated GAS/ISRE sequence of the Ly6 gene (lane6); thus, liver Stat 5/p93 does not bind to the GAS site. The high affinity c-sis-inducible element of the human c-fos gene (SIE site m67 (54) ), which binds tightly to a broad range of activated Stat proteins (49, 50, 52, 55) , inhibited -casein DNA complex formation only partially (lane4), suggesting that liver Stat 5/p93 binds to the -casein promoter site with a substantially higher affinity than to the SIE site. The partial inhibition by SIE is specific, however, since it was not observed when using an SIE probe containing a mutated binding site (lane5versus4). The distinct nature of the GH-inducible liver Stat 5--casein gel shift complex was further highlighted by the inability of antibodies to Stat 3 (or to Stat 1) to supershift this complex, despite the strong supershift conferred by this antibody on a complex formed between the SIE probe and LPS-activated rat liver nuclear extracts (data not shown).


DISCUSSION

The present study identifies a 93,000 dalton latent cytoplasmic DNA binding protein, designated liver Stat 5/p93, as a likely intracellular mediator of the stimulatory effects of intermittent plasma GH pulses on male-specific liver gene transcription. Pituitary GH secretory profiles, which are sexually differentiated in many species (23, 25, 56) , including humans (57, 58) , are responsible for establishing and for maintaining the sex-dependent patterns that characterize liver gene expression. These effects are best understood in the rat liver model, where in males intermittent pituitary GH secretion, and the resultant pulsatile plasma GH pattern, activates transcription of male-expressed genes, while in females the more frequent occurrence of pituitary GH secretory events, and the resultant continuous plasma GH pattern, activates an adult female-specific pattern of gene transcription (32, 33) . Liver Stat 5/p93 is shown to be activated by GH via tyrosine phosphorylation, which uncovers this protein's latent DNA binding potential and stimulates its translocation from the cytosol to the nucleus. This phosphorylation event, which is likely catalyzed by the GH receptor-associated tyrosine kinase Jak2 (12, 13) , is an early, probably primary response of the hepatocyte to GH stimulation. Liver Stat 5 thus exhibits each of the general characteristics associated with other Stat family members, including those activated by interferons and , epidermal growth factor, interleukin 6, and various other cytokines and growth factors (21, 49, 59, 60) .

In contrast to other Stats, liver Stat 5 appears to be uniquely responsive to the temporal pattern of hormone stimulation, which in the case of GH is crucial for discriminating between the effects of male versus female plasma hormone profiles on liver gene expression. Tyrosine phosphorylation, nuclear translocation, and activation of liver Stat 5's DNA binding potential were supported at a high steady-state level in an intact rat model by the intermittent plasma GH pattern associated with adult males but not by the continuous plasma GH profile that is characteristic of adult females. Although GH-naive hypophysectomized female rats initially responded to GH treatment by tyrosine phosphorylation of p93 as in hypophysectomized males, continuous GH exposure desensitized the hepatocytes and led to a dramatic decline in the steady-state level of tyrosine-phosphorylated p93. The relatively long time period required for complete desensitization of liver Stat 5/p93 with respect to GH-induced tyrosine phosphorylation (>8-24 h) suggests that this process is mechanistically distinct from the more rapid desensitization of Jak2 kinase with respect to further GH activation (but not to subsequent interferon- activation), which is seen in cultured IM-9 cells exposed to GH continuously for 1 h (13) . Additional studies are required to establish the precise mechanism for the desensitization observed in our in vivo studies. Conceivably, this desensitization may involve a feedback inhibitory effect of continuous GH that is manifest (a)at the level of GH receptor activation via dimerization (8) , (b)at the level of Jak2 kinase recruitment and activation (12, 13) , or (c)by the availability of functionally active cytoplasmic liver Stat 5 protein for tyrosine phosphorylation. This desensitization is unlikely to reflect plasma membrane GH receptor down-regulation in response to prolonged GH stimulation, since GH receptors are, in fact, up-regulated by continuous GH treatment and, consequently, are abundant on the hepatocyte surface in female rats (61, 62).

The GH-activated, tyrosine-phosphorylated liver nuclear protein p93 characterized in this study was identified as a mammary gland factor/Stat 5-related protein on the basis of its immunochemical cross-reactivity with two individual anti-mammary Stat 5 monoclonal antibodies but not with several monoclonal and polyclonal antibodies reactive with Stat 1 and Stat 3. Both of the anti-Stat 5 antibodies consistently yielded two and sometimes three bands on Western blots of GH-stimulated rat liver nuclear protein extracts, suggesting that multiple Stat 5-related proteins, or perhaps a single, multiply phosphorylated Stat 5 protein, may be expressed in rat liver. With both antibodies, the uppermost Stat 5-immunoreactive band coincided with p93 detected with anti-phosphotyrosine antibody, suggesting that it corresponds to the tyrosine-phosphorylated form of liver Stat 5. This identification of p93 as a liver-expressed, mammary Stat 5-related protein is strongly supported by our finding that the GH-activated liver nuclear factor binds specifically to a mammary Stat 5-binding DNA response element derived from the 5`-flank of the rat -casein gene. The inability of a GAS oligonucleotide or a high affinity SIE oligonucleotide, which contain binding sites for Stat proteins 1 and 3, to significantly compete with this DNA binding site lends further support to the distinct nature of liver Stat 5. Although liver Stat 5 is thus related to Stat 5 cloned from sheep mammary gland, liver Stat 5 and mammary Stat 5 seem to be different, since the latter Stat is not detectably expressed at the mRNA level in liver tissue (41) . Moreover, prolactin, which activates mammary Stat 5 (41) , did not activate liver Stat 5 in our experiments, as judged by the lack of an increase in p93 tyrosine phosphorylation or liver Stat 5 protein in liver nuclei and by the absence of an effect on liver nuclear DNA binding activity assayed with the -casein Stat 5 binding site probe. This lack of an effect of prolactin may reflect the absence of the prolactin-activable mammary Stat 5 in liver, since the two other cellular components required for prolactin-induced Stat 5 activation, i.e. the long form of prolactin receptor (7, 63) and Jak2 kinase (53, 64, 65) , are reportedly present in hepatocytes. Liver Stat 5 may not be restricted to hepatocytes, as suggested by the presence of GH-activated, tyrosine-phosphorylated protein(s) of similar size in other GH-responsive cell types (13, 20, 66) . Further studies, including cloning of liver Stat 5 and direct study of its responsiveness to GH and prolactin, are needed to establish the precise relationship between liver Stat 5 and mammary Stat 5.

The broad range of physiological and metabolic effects that GH has on target tissues (1, 2) and in particular the occurrence of sex-dependent effects of GH secretory profiles on long bone growth and liver gene expression suggest that GH may activate several independent or, perhaps, parallel signaling pathways, even within a single cell type. This proposal is supported by the finding that GH can activate multiple Stat proteins in hepatocytes; these include liver Stat 5, as shown in the present study, as well as Stat 3 and, under certain conditions, Stat 1 (19) . It seems likely that these three GH-responsive Stat proteins will contribute to the activation of different subsets of GH-responsive genes and conceivably may each respond in a distinct manner to the temporal pattern of circulating plasma GH levels. This would help explain the broad diversity of the effects of GH, as well as the restriction of sex- and plasma hormone profile-dependent effects of GH to a subset of GH-activable genes. For instance, the rapid activation of the c-fos gene by GH (67, 68) could in part be mediated by the binding of GH-activated Stat 3 and/or Stat 1 to the SIE sequence found within a regulatory element upstream of that gene (54) , while activation of the liver cytochrome P450 gene CYP2C11 by intermittent GH pulsation (37, 38) could be mediated by the binding of GH-activated liver Stat 5 to cognate regulatory elements within or adjacent to CYP2C11. However, since the stimulatory effects of GH pulses on CYP2C11 gene expression take at least 1-2 days to be manifest (69) , as compared with the activation of liver Stat 5/p93 within 15 min of GH treatment (this study), the induction of CYP2C11 gene transcription by GH pulsation may be an indirect response to liver Stat 5 activation. Further studies will be required to determine the molecular mechanisms through which liver Stat 5 activates target genes in response to GH pulses, as well as the cellular mechanism through which hepatocytes exposed to continuous GH become desensitized with respect to liver Stat 5 activation.


FOOTNOTES

*
This work was supported in part by National Institutes of Health Grant DK-33765 (to D. J. W.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Biology, Boston University, 5 Cummington St., Boston, MA 02215. Fax: 617-353-7404; E-mail: djw@bio.bu.edu.

The abbreviations used are: GH, growth hormone; Stat, signal transducer/activator of transcription; LPS, bacterial lipopolysaccharide.

P. A. Ram, S.-H. Park, H. K. Choi, and D. J. Waxman, manuscript in preparation.


ACKNOWLEDGEMENTS

We thank Drs. L. Argetsinger, Z. Zhong, J. Darnell, and D. Levy for providing anti-phosphotyrosine and anti-Stat antibodies.

Note Added in Proof-Western blot analysis of liver Stat 5 levels in a series of intact male rats killed at different times of the day revealed a striking individual variability in liver nuclear Stat 5 levels, with a strong positive correlation between the occurrence of liver Stat 5 protein in the nucleus and the presence of GH in the plasma at the time of death. This finding provides strong additional support for our conclusion that liver Stat 5 undergoes repeated cycles of tyrosine phosphorylation and nuclear translocation in response to naturally occurring plasma GH pulses in intact adult male rats.


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