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Endocrinology Vol. 148, No. 6 2894-2903
Copyright © 2007 by The Endocrine Society

A Molecular Basis for the Sexually Dimorphic Response to Growth Hormone

Chellappagounder Thangavel and Bernard H. Shapiro

Laboratories of Biochemistry, University of Pennsylvania, School of Veterinary Medicine, Philadelphia, Pennsylvania 19104

Address all correspondence and requests for reprints to: Bernard H. Shapiro, Laboratories of Biochemistry, University of Pennsylvania, School of Veterinary Medicine, 3800 Spruce Street, Philadelphia, Pennsylvania 19104-6048. E-mail: shapirob{at}vet.upenn.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Once reserved solely for the treatment of short stature, the now readily available recombinant GH has expanded the use of the hormone to include the treatment of cardiovascular, renal, muscular, skeletal, immunological, psychosocial, and metabolic abnormalities associated with GH deficiency. There are also proposals for the widespread use of the hormone to ameliorate or reverse aging. However, this extensive use of GH has revealed intrinsic sexual dimorphisms in which females are considerably less responsive to the therapeutic regimen than are males. Dynamic changes in the Janus kinase-2 (Jak2)/signal transducers and activators of transcription (Stat5B) signaling pathway [as determined by transducer activation, Stat5B binding to the GH-responsive promoter of the CYP2C11 gene, and expression levels of the suppressors of cytokine signaling family (Socs2, Socs3, and Cis)] were examined in male and female rat-derived primary hepatocyte cultures exposed to the masculine-like episodic GH profile. We report that the cellular actions of GH normally mediated by activation of the Jak2/Stat5B pathway are suppressed in female cells possibly due to an inherent overexpression of Cis, a member of the suppressors of cytokine signaling family that normally down-regulates the Jak2/Stat5B pathway.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
GH PLAYS AN IMPORTANT role in the development, growth, and maintenance of nearly every organ and tissue in the body by regulating protein, lipid, and carbohydrate metabolism in addition to synergistically enhancing the actions of other hormones (1). Whereas males and females secrete the same daily amount of GH, the secretory patterns are sexually dimorphic. In various species examined, including mice, rats, and humans (2), females secrete a so-called "continuous" GH profile comprised of numerous daily pulses interrupted by short-lived interpulses of usually low or undetectable hormone concentrations. In contrast, the "episodic" masculine GH profile is characterized by significantly fewer secretory bursts of the hormone separated by lengthy interpulses that are invariably devoid of GH. In fact, it is the difference between the continuous (female) and episodic (male) circulating GH profiles, and not plasma hormone levels per se that are responsible for phenotypic sexual dimorphisms ranging from growth patterns to expression levels of hepatic enzymes (2, 3, 4). In this regard, GH deficiency causes numerous abnormalities in growth rates, lean body mass; cardiovascular, bone, adipose, and muscle function; protein, carbohydrate, lipid, and electrolyte metabolism; (1, 5, 6, 7, 8, 9); and expression levels of hepatic IGF-1, IGF binding protein, GH-binding protein (3, 5, 6, 10, 11, 12), and cytochrome P450-dependent drug metabolizing enzymes (13, 14). However, hormone replacement therapy has clearly demonstrated an intrinsic, irreversible, sexually dimorphic response. Daily injections, evoking the masculine-like episodic GH profile, the most feasible, yet efficacious therapeutic approach, are significantly more effective correcting these GH deficiency abnormalities in men (or boys) than women (or girls) (6, 7, 8, 9, 10, 11, 12, 13, 14, 15). Subsequent investigations in rats have reported similar intrinsic sex-dependent responses to GH replacement. For example, renaturalization of the masculine episodic GH profile cannot stimulate female hepatocytes, either in vivo or in vitro (16, 17) to express male-like levels of cytochrome P450 isoforms, indicating an irreversible sexually dimorphic responsiveness to GH regulation. Why then would bone, fat, muscle, and liver cells be less responsive to GH replacement in females?

GH signaling in liver by the episodic profile (in contrast to the continuous profile) is initiated by hormone binding and the resulting activation of GH receptors (GHR) on the surface of the target cells. This allows for the recruitment and/or activation of two molecules of Janus kinase-2 (Jak2), which then cross-phosphorylate each other as well as phosphorylating the receptor on key tyrosine residues. Signal transducers and activators of transcription (i.e. Stat5B), latent transcription factors, bind to these phosphorylated receptor docking sites, are in turn phosphorylated, homodimerize, and translocate to the nucleus where they bind to promoter sites initiating transcription of GH-regulated genes. An important negative regulatory mechanism of GH signaling, the suppressors of cytokine signaling (Socs/Cis) family of inhibitory proteins, which depending upon the protein, inhibit GH signaling by inactivating Jak2, by competing with Stat5B for common tyrosine-binding sites on the intracellular tail of the GHR, and/or by a proteasome-dependent mechanism that results in the degradation of the (GH-GHR-Jak2)·Cis complex (18, 19, 20, 21, 22). In this regard, Cis appears to be the dominant, if not sole, factor down-regulating the GH initiated Jak2/Stat5B signaling pathway in cells continuously exposed to the hormone (18, 21). In part, Cis exerts its inhibitory effect by competing with Stat5B for common tyrosine-binding sites in the cytoplasmic domain of the GHR (20, 21). The Cis gene is activated by a feedback loop regulated by Stat5B (19, 22). Accordingly, we have identified sex differences in this signaling pathway that could explain the sexually dimorphic responsiveness of target cells to the masculine episodic GH profile.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Rats, which uniquely express high levels of several sex-specific, GH-dependent hepatic isoforms of cytochrome P450 (2), were housed in the University of Pennsylvania Laboratory Animal Resources facility under the supervision of certified laboratory animal medicine veterinarians and were treated according to a research protocol approved by the Institutional Animal Care and Use Committee of the University. Male and female rats [Crl: CD (SD)BR] were hypophysectomized by the vendor (Charles River Laboratories, Wilmington, MA) at 8 wk of age [by which time adult, sex-dependent patterns of plasma GH and cytochrome P450s are clearly established (3)] and observed in our facility for 5–6 wk (12 h light/12 h dark; lights on 0800 h) to allow sufficient time for any residual pituitary tissue to regenerate. The effectiveness of the surgery was verified by the lack of weight gain over this period and the absence of pituitaries or fragments at necropsy shortly after euthanizing the rats (~1500 h).

Hepatocyte isolation and culture
Preparation of hepatocytes from long-term hypophysectomized rats free of any confounding effects of endogenous GH (23) was performed with minor modifications (17) by in situ perfusion of collagenase through the portal vein of anesthetized rats following standard protocol (24). The viability of the initial cell suspension of hepatocytes was typically between 80–90% (with trypan blue). The hepatocytes were plated at a density of 5 x 106 viable cells per T-25 flask previously coated with matrigel (274 µg/cm2). After allowing 2–3 h for cell attachment, serum-containing medium was removed and replaced by serum-free DMEM/F-12 media supplemented with streptomycin (100 µg/ml), penicillin (100 U/ml), glutamine (2 mM), HEPES (15 mM), insulin (10 µg/ml), bovine transferrine (10 µg/ml), Na2SeO3 (10 ng/ml), aminolevulinic acid (2 µg/ml), glucose (25 mM), linoleic acid-albumin (0.5 mg/ml), and sodium pyruvate (5 mM). The cultures were also supplemented with fungizone (0.25 µg/ml) for the initial 48 h only. Cultures were maintained in a humidified incubator at 37 C under an atmosphere of 5% CO2/95% air.

Hormonal conditions
To replicate the episodic GH profile, about 4 h after isolation, hepatocytes were exposed to recombinant rat GH (rGH) (0.2 ng/ml) (NHPP, Torrance, CA) for 30 min followed by two careful washings with GH-free media that remained in the wells for 11.5 h, at which time the cells were again washed and exposed to the next 30-min pulse of GH (17). On the 5th d, at which time CYP2C11 levels should have returned to normal (17), cells were harvested after 0, 7, 15, 30, 45, 75, 120, 180, and 240 min after the addition of the last GH pulse at 0700 h. To replicate the continuous GH profile, hepatocytes were constantly exposed to a 2-ng/ml concentration of rGH for 12 h, after which time the cells were washed and exposed for another continuous 12 h to the hormone (23). Cells were harvested on the 5th d after the final media change at the same time intervals as cells treated with the episodic profile.

Analysis of CYP2C11 mRNA by Northern blotting
Total cellular RNA, isolated as previously reported (17, 23), was resolved on denaturing 1% agarose gels and transferred onto Nytran N filters from Schleicher and Schuell (Keene, NH). The Northern blot was probed and reprobed with {gamma}-32P-labeled oligonucleotide probes, using hybridization and high stringency washing conditions as described previously (25). The sequence of the oligonucleotide probe for CYP2C11 has been reported (26). The consistency of RNA loading between samples was confirmed by ethidium bromide staining of 18S and 28S ribosomal RNAs and was verified using an 18S oligonucleotide probe (27). The hybridized mRNA signals were quantified by scanning the autoradiographs using a FluorChem IS-8800 Imager (Alpha Innotech, San Leandro, CA). The mRNA signals were normalized to the 18S rRNA signals in each lane, which exhibited a mean variation of only ±5%, indicating results were independent of loading errors.

Semiquantitative RT-PCR analysis of Cis, Socs2, and Socs3 mRNAs
Cis, Socs2, and Socs3 mRNAs were analyzed by semiquantitative RT-PCR as previously reported (15, 28). The cDNA was made from total cellular RNA isolated from cultured hepatocytes. PCR primers used for Cis, Socs2, Socs3 (28), and rat ß-actin (29) have been reported. The linear range of product amplification was established for each primer pair in pilot experiments. In all experiments, controls were carried out without cDNA (RT step) and yielded no products after PCR, indicating that there was no detectable amount of DNA in the RNA. The PCR products were analyzed on 2% agarose gel containing ethidium bromide with 1x TAE buffer. The mRNA signals were visualized and quantitated by using a FluorChem IS-8800 Imager (Alpha Innotech). The mRNA signals were normalized to the rat ß-actin internal control.

Preparation of whole cell lysate and Western blot analysis
Whole cell lysate was extracted from cultured primary hepatocytes at various times after the last GH pulse, and the protein concentrations of the cell lysates were measured by using the Bio-Rad protein assay reagent (Bio-Rad Laboratories, Hercules, CA). Fifty micrograms of whole cell lysate protein were electrophoresed on 0.75-mm-thick sodium dodecyl sulfate-polyacrylamide (7.5%) gels and electroblotted onto nitrocellulose membranes for immunoblotting (IB). Accordingly, the blots were probed with antibodies against Jak2, phospho-Stat5 (Upstate, Lake Placid, NY), Stat5B (BD Biosciences, San Jose, CA), Stat5A, or Cis (Santa Cruz Biotechnology, Santa Cruz, CA). Additional fractions of the total cell lysates were immunoprecipitated (IP) with either Jak2 (Upstate) or Stat5B (BD Biosciences) antibodies. Next the immunoprecipitates were probed with anti-phosphotyrosine, anti-pY, (Upstate). This order allowed us first to concentrate the Jak2 and Stat5B proteins and then specifically measure the activated component. Signals of the relative protein levels were quantitated by using a FluorChem IS-8800 Imager (Alpha Innotech). Signals were normalized to a control sample that was repeatedly run on each blot and exhibited a concentration variant between blots of 2.8–6.1% for the different proteins. Last, blots were stripped and reprobed with actin antibody (Santa Cruz Biotechnology), normalized with actin loading control, and the resulting findings were comparable to those obtained with internal controls of the assayed protein were run repeatedly on all blots.

Chromatin immunoprecipitation assays (ChIP)
A ChIP assay was performed on harvested hepatocytes 0, 7, 15, 30, 45, 75, 120, and 240 min after the hormone pulse on the 5th d in culture. The ChIP assay was performed as described (30, 31) with slight modifications. Hepatocytes were treated with 1% formaldehyde for 10 min at room temperature. The cross-linking reaction was stopped by adding glycine to a final concentration of 0.125 M. The cells were harvested and washed three times with ice-cold Dulbecco’s PBS buffer containing 5 mM EDTA. The nuclei were subsequently isolated and lysed. The lysate was sonicated to generate DNA fragments with an average length of 100-1000 bp. After removal of cell debris by centrifugation, the chromatin concentration was measured and about 10% of the chromatin was kept as an input, and the rest of the chromatin was diluted 3-fold. Equal concentrations of chromatin from all time points were precleared with protein A agarose beads in the presence of 1 mg/ml BSA and 2 µg of sonicated salmon sperm DNA to reduce the nonspecific background. After removal of beads by centrifugation, 2 µg of Stat5B-specific antibody (BD Biosciences) was added and kept at 4 C overnight on a rotary platform. The immune complexes were collected by centrifugation after adding protein A agarose beads and kept at 4 C for 1 h. The immunoprecipitates were washed sequentially and eluted as described (30). Elutes were pooled and heated at 65 C for 6 h to reverse the formaldehyde cross-linking and also treated with DNase-free RNAase to remove RNA. The samples were treated with 40 µg/ml of proteinase K at 45 C for 1 h, followed by phenol/chloroform extraction and ethanol precipitation. The same process was also carried out for input chromatin. The IP DNAs and input DNAs were analyzed by semiquantitative PCR using forward 5'-AAG GGG AAG CTT CCT AAG CA-3' (–1280/–1260) and reverse 5'-GCC TCC ATG TAT GTC TGT GTG-3' (–1010/–1031) primers of the CYP2C11 promoter made from the CYP2C11 gene (GenBank X79081) to detect the promoter among the IP DNA. A negative control with a forward primer 5'-TGC ACA CCT TAA ATG TAG GC-3' (–1640/–1621) and reverse 5'-TGG CTC CTC TTC AAG TGG TA-3' (–1421/–1440) primer of a non-Stat5B binding region of the CYP2C11 promoter (GenBank X79081) was used to determine the specificity of Stat5B binding to its binding region. The PCR products were resolved on 2% agarose gel containing ethidium bromide, and the band intensities at each time point were quantitated by using a FluorChem IS-8800 Imager (Alpha Innotech).

Confirmation of the Stat5B binding motif among the ChIP-PCR product by Southern blotting analysis
The PCR products (DNA) from the CHIP assays were denatured and transferred onto Nytran N filters from Schleicher and Schuell. Southern blotting was carried out (32, 33) to confirm the Stat5B-binding motif in the PCR product by using a {gamma}-32P-labeled nucleotide sequence of the Stat5B binding sites on the CYP2C11 promoter (34). The signals were scanned and quantitated by using a FluorChem IS-8800 Imager (Alpha Innotech). The signals were normalized with a positive control that was repeatedly run on each blot.

Statistics
All the data were subjected to ANOVA. Significant differences were determined with t statistics and the Bonferroni procedure for multiple comparisons.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Sexually dimorphic response of Jak2 to episodic GH
For 5 d in culture, cells were exposed to an episodic GH profile shown in vivo and in vitro to be a highly effective inducer of numerous sex-dependent physiologic functions including body weight gain, hepatic IGF, and various isoforms of hepatic cytochrome P450 (3, 17, 35, 36). Cells were harvested at various time points after the final GH pulse. Whereas total Jak2 concentrations were unaffected by GH or sex, activation (i.e. phosphorylation) of Jak2 was influenced by both factors (Fig. 1Go). Fifteen minutes after the GH pulse, activated Jak2 levels peaked and returned to pre-GH induction zero levels between 45–75 min in both sexes. However, concentrations of the activated tyrosine kinase were twice as great in male hepatocytes than in female cells.


Figure 1
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  		FIG. 1. Total Jak2, IB: anti-Jak2 (top), and phospho-Jak2, IP: anti-Jak2, IB: anti-pY (bottom) levels in primary hepatocytes derived from hypophysectomized male and female rats exposed to episodic rGH for 5 d in culture. Cells were harvested and analyzed at different time points between 0 and 240 min after the final hormone pulse. Sufficient viable cells were isolated from a single liver for all determinations at every time point presented in the figure. Each data point is a mean ± SD for cells from five or more rats. *, P < 0.01 compares females with males at the same time point. Absolute values should not be compared between panels. A representative Western blot of IP phospho-Jak2 (p-Jak2) is included in the bottom panel.

 
Sexually dimorphic recruitment of Stat5A and B by episodic GH
After phosphorylation of Jak2, the next step in episodic GH-induced signal transduction is the recruitment of Stat5 molecules. Approximately 30 min after the final GH pulse on the 5th d in culture, we observed a small elevation (P > 0.05) in total Stat5A in both sexes. However, at all measured time points, total Stat5A concentrations were consistently approximately 35% greater in female hepatocytes (Fig. 2Go). Whereas baseline levels of total Stat5B exhibited no sex differences, 30 min after the GH pulse, the increase in the concentration of the transcription factor in female hepatocytes was only about half as much as in similarly treated male hepatocytes (Fig. 2Go). In fact, during the entire response period (7–75 min after GH) total Stat5B levels were always greater in the male cells. (The unexpected increase in Stat5B protein first observed at 7 min of GH exposure can be explained by the possible greater affinity of the antibody for the phosphorylated form of Stat5B than to the nonphosphorylated form.)


Figure 2
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  		FIG. 2. Total Stat5A, IB: anti-Stat5A (top), and total Stat5B, IB: anti-Stat5B (bottom) levels in primary hepatocytes derived from hypophysectomized male and female rats exposed to episodic rGH for 5 d in culture. Cells were harvested and analyzed at different time points between 0 and 240 min after the final hormone pulse. Sufficient viable cells were isolated from a single liver for all determinations at every time point presented in the figure. Each data point is a mean ± SD for cells from five or more rats. *, P < 0.01 compares females with males at the same time point. Absolute values should not be compared between panels. Representative Western blots of Stat5A, Stat5B, and their actin loading controls are included on the right.

 
Sexually dimorphic activation of Stat5B by episodic GH
In agreement with the expected sequence of events, levels of phospho-Stat5 peaked 15 min after phospho-Jak2 peaked in cells from both sexes (Fig. 3Go). However, induction levels of the activated transcription factor were several fold greater in male hepatocytes. Stat5 antibodies recognize both the A and B forms, with the latter being implicated in episodic GH action (18, 19, 21) and the former exhibiting no such responsiveness (Fig. 2Go). By immunoprecipitation, we measured the levels of phospho-Stat5B. Although phospho-Stat5B peaked in both male and female cells 30 min after the GH pulse, more than twice as many molecules of the transcription factor were activated in the male hepatocytes (Fig. 3Go). When phospho-Stat5B levels (Fig. 3Go) were corrected to reflect the different protein concentration used to measure total Stat5B (Fig. 2Go), the normalized percent of the total Stat5B that was actually phosphorylated remained about twice as high in the male than female hepatocytes 15–75 min after the rGH episodic pulse (Fig. 3Go). When we exposed the male and female cells to a continuous GH profile, we observed minimal Stat5B phosphorylation (data not shown) reflecting about 10–15% of the levels induced by the episodic profile and in agreement with findings using the rat liver cell line CWSV-1 (37) as well as intact rats (38).


Figure 3
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  		FIG. 3. Phospho-Stat5, IB: anti-p-Stat5 (top), and phospho-Stat5B, IP: anti-Stat5B, IB: anti-pY (bottom) and percent of the total p-Stat5B that is phosphorylated (bottom inset) in primary hepatocytes derived from hypophysectomized male and female rats exposed to episodic rGH for 5 d in culture. Cells were harvested and analyzed at different time points between 0 and 240 min after the final hormone pulse. Sufficient viable cells were isolated from a single liver for all determinations at every time point presented in the figure. Each data point is a mean ± SD for cells from five or more rats. *, P < 0.01 compares females with males at the same time point. Absolute values should not be compared between panels. Representative Western blots of p-Stat5 and their actin loading controls as well as p-Stat5B are included on the right.

 
Sex differences in episodic GH-directed Stat5B binding to the CYP2C11 promoter
Because expression of the CYP2C11 gene is solely regulated by the episodic GH profile (2, 35, 36), we examined the binding kinetics of phospho-Stat5B to the CYP2C11 promoter by ChIP assay. In agreement with the expected sequence of signaling events, maximum binding of activated Stat5B to the promoter occurred between 30 and 45 min after the GH pulse in both sexes of hepatocytes (Fig. 4Go). However, almost twice the amount of the transcription factor was bound to the CYP2C11 promoter in male hepatocytes than in female hepatocytes. PCR amplification of a negative (Neg) control using primers flanking the CYP2C11 promoter at a genomic sequence not including the Stat5B binding site demonstrated no measurable nonspecific binding (Fig. 4Go). In conformation, using Southern blotting, we observed twice as much of the Stat5B-binding motif of the CYP2C11 promoter bound to the activated transcription factor in hepatocytes from male rats (Fig. 4Go).


Figure 4
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  		FIG. 4. Graphic quantitation of Stat5B binding to the CYP2C11 putative promoter (ChIP assay, left) and the occupied Stat5B-binding motif in the CYP2C11 promoter (Southern blot, right) as well as representative ChIP and Southern blots determined in primary hepatocytes, derived from hypophysectomized male and female rats, exposed to episodic rGH for 5 d in culture. Cells were harvested and analyzed at different time points between 0 and 240 min after the final hormone pulse. Sufficient viable cells were isolated from a single liver for both the ChIP assay and Southern blotting determinations for every time point presented in the figure. Each data point is a mean ± SD for cells from five or more rats. *, P < 0.01 compares females with males at the same time point. Absolute values should not be compared between panels.

 
Sex differences in Cis/Socs expression regulated by episodic GH
The Cis/Socs family of proteins function as negative regulators of episodic GH-induced Jak/Stat signal transduction. It would appear that the same GH pulse responsible for initiating the activation of the Jak2/Stat5B pathway subsequently induces expression of the Cis/Socs genes whose transcribed proteins, in turn, terminate the Jak2/Stat5B signal to prepare the target cell to respond to the next pulse of GH (18, 20, 21, 22). According to this proposed course of events in which maximum nuclear Stat5B binding occurs 30–45 min after the GH pulse; termination of the reaction as characterized by an elevated expression of the Cis/Socs genes should then follow, as we observed (Fig. 5Go), within 45–75 min of the hormone pulse. Disregarding Socs3, the expression levels of which were not sexually dimorphic and which is not involved in episodic GH-induced signal transduction (20, 39, 40), only hepatocytes from males exhibited a significant (P < 0.001) elevation in Cis and Socs2 mRNAs after the hormone pulse, with changes in Cis mRNA being far more dramatic (~100%) than Socs2 (~25%) (Fig. 5Go). Despite the poor responsiveness of these two inhibitory genes in female hepatocytes to the GH pulse, Cis and Socs2 levels were consistently higher in female hepatocytes at every measured time point. Cis protein levels in episodic GH-treated male and female hepatocytes were in agreement with transcript concentrations (Fig. 6Go).


Figure 5
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  		FIG. 5. Cis (top), Socs2 (middle), and Socs3 (bottom) mRNA levels in primary hepatocytes, derived from hypophysectomized male and female rats, exposed to episodic rGH for 5 d in culture. Cells were harvested and analyzed at different time points between 0 and 240 min after the final hormone pulse. Sufficient viable cells were isolated from a single liver for Cis, Socs2, and Socs3 mRNA determinations by RT-PCR for every time point presented in the figure. Each data point is a mean ± SD for cells from five or more rats. *, P < 0.01 compares females with males at the same time point. Absolute values should not be compared between panels.

 

Figure 6
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  		FIG. 6. Cis protein, IB: anti-Cis, levels in primary hepatocytes, derived from hypophysectomized male and female rats, exposed to episodic (top) or continuous (bottom) rGH, for 5 d in culture. Cells were harvested and analyzed at different time points between 0 and 240 min after either the final hormone pulse (episodic) or the last media change (continuous). Sufficient viable cells were isolated from a single liver for Cis determinations at every time point presented in the figure. Each data point is a mean ± SD for cells from five or more rats. *, P < 0.01 compares females with males at the same time point. Absolute values should not be compared between panels. Representative Western blots of Cis and their actin loading controls are included on the right.

 
Sex differences in Cis/Socs expression regulated by continuous GH
Female liver is normally exposed to a continuous GH profile that can only minimally activate the Jak2/Stat5B pathway (37, 38, 41). Accordingly, this feminine GH profile may result in a continuous overexpression of the Cis/Socs family of inhibitory proteins responsible for blocking the activation of the Jak/Stat signal transduction pathway in females. We tested this hypothesis by exposing hepatocytes from both sexes to constant GH at a highly effective concentration (2 ng/ml) shown to restore normal levels (mRNA and protein) of Jak/Stat-independent female isoforms of hepatic cytochrome P450 (23). Cells were harvested after 5 d in culture at various times after the last of 10 media changes. Despite the fact that the cells were continuously exposed to GH, they responded with an increased transcription of Cis/Socs in both sexes (male ~50%; females ~18%, P < 0.01 for all comparisons). 1 Similar to the effects of episodic GH (Fig. 5Go), female hepatocytes exposed to continuous GH expressed considerably greater concentrations of Cis and Socs2 mRNAs than male cells at every time point measured (Fig. 7Go). In fact, when the cells were first harvested from the hypophysectomized rats, Cis mRNA was nine times greater (5919 ± 206/645 ± 66; female/male) and Socs2 mRNA was 25% higher (8297 ± 206/6601 ± 244; female/male) in female hepatocytes; this despite an absence of GH for at least 5 wk and suggesting sex-dependent, irreversible imprinting. Cis protein levels in GH-continuously treated hepatocytes were in agreement with transcript concentrations, with Cis protein remaining at considerably higher levels in females at all time points, but male cells exhibiting a greater GH-induced increase in Cis (~50%, P < 0.01) than female cells (~25%, P < 0.01) (Fig. 6Go).


Figure 7
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  		FIG. 7. Cis (top), Socs2 (middle), and Socs3 (bottom) mRNA levels in primary hepatocytes, derived from hypophysectomized male and female rats, exposed to continuous rGH for 5 d in culture. Cells were harvested and analyzed at different time points between 0 and 240 min after the last media change. Sufficient viable cells were isolated from a single liver for Cis, Socs2, and Socs3 mRNA determinations by RT-PCR for every time point presented in the figure. Each data point is a mean ± SD for cells from five or more rats. *, P < 0.01 compares females with males at the same time point. Absolute values should not be compared between panels.

 
Irreversible sex-dependent responsiveness of hepatic CYP2C11 to episodic GH
Although the present results demonstrate a reduced responsiveness of the Jak2/Stat5B signal transduction pathway to episodic GH in female hepatocytes, the significance of this sexual dimorphism depends upon its biological consequences. Accordingly, we measured expression levels of CYP2C11, the male-specific isoform comprising more than 50% of the total cytochrome P450 in male rat liver (42). CYP2C11 expression is solely dependent upon exposure to the episodic GH profile; albeit its male specificity. The feminine profile of continuous GH secretion is completely suppressive. In the absence of GH (e.g. hypophysectomy), CYP2C11 is expressed at 20–30% of intact male-like concentrations in both sexes. In conformation (2, 16, 35), we have observed the expected sexual dimorphic expression and regulation of CYP2C11 by GH (Fig. 8Go). Because induction of CYP2C11 is reported to be mediated by episodic GH-activation of the Jak2/Stat5B pathway (39, 41), subnormal activation of the signaling pathway in female hepatocytes should reduce expression levels of the isoform. Indeed, whereas exposure of male hepatocytes to episodic GH restored CYP2C11 to intact male-like expression levels, female hepatocytes exposed to the identical episodic profile expressed no detectable concentrations of the isoform (Fig. 8Go).


Figure 8
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  		FIG. 8. CYP2C11 mRNA expression in "zero time" (i.e. immediately after isolation and before plating) hepatocytes derived from intact and hypophysectomized male and female rats as well as primary hepatocytes from hypophysectomized rats exposed to either episodic rGH or its diluent for 5 d (5d) in culture. Sufficient viable hepatocytes were isolated from each hypophysectomized rat for every treatment presented in the figure. Values are presented as a percentage of CYP2C11 mRNA in hepatocytes from intact male rats at zero time arbitrarily designated 100%. Each data point is a mean ± SD for cells from five or more rats. ND, Not detectable. *, P < 0.01 when compared with intact males at zero time.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The episodic masculine-like GH profile produces its effects by initiating a specific series of signaling events at its target cell that sequentially include binding to its transmembrane receptor causing GHR activation, recruitment, and phosphorylation (i.e. activation) of Jak2, phosphorylation of the GHR, phosphorylation, homodimerization, and nuclear translocation of Stat5B, which initiates transcription of GH-responsive genes by binding to their promoters (18, 19, 20, 21, 22). In order for this signal transduction pathway to be effective, threshold levels of Stat5B (and possibly Jak2) have to be activated, translocated into the nucleus, and then bound to the promoter of GH-regulated genes (40, 43, 44). Because all of these signal transduction events were blunted by at least 50% in the female hepatocytes, and considering that as much as 70% of normal phospho-Stat5B translocated nuclear levels are insufficient to initiate episodic GH-transcription of the CYP2C11 gene (44), it seems reasonable to conclude that the sexually dimorphic responses to episodic GH are due to a failure by the hormone to stimulate an optimum cascade of signal transducers in the target cells of females. The reason for this insufficient responsiveness to GH in female cells may be due to the persistent overexpression of Cis, the sole member of the Socs/Cis family of inhibitory proteins responsible for terminating the episodic GH-activated Jak2/Stat5B pathway (18, 21, 39, 40). In this regard, compared with males, Cis levels are dramatically higher in the livers of intact females (18, 45). Moreover, we have found that female hepatocytes that were either GH-deficient (hypophysectomized), continuous, or episodic GH-treated expressed dramatically higher levels of Cis and it is the overexpression of the Cis protein in continuous GH-treated rats that is known to prevent Jak2/Stat5B-directed transcription of GH-dependent genes (18, 39). In contrast, male cells exposed to episodic GH express elevated levels of Cis for only a transient period, down-regulating the Jak2/Stat5B pathway after transcription initiation and allowing a subsequent reactivation of the pathway by the next GH pulse.

Under normal circumstances, the female secretes a continuous profile of GH suppressing the activation of the Jak2/Stat5B signal transduction pathway that otherwise could be responsible for the masculinization of numerous sexual dimorphisms ranging from musculoskeletal development to hepatic enzymes. Clearly, females were not intended to be exposed to the masculinizing episodic GH profile. However, it would seem that as an added precaution, females inherently overexpress Cis, guaranteeing that even independent of GH status, the Jak2/Stat5B pathway and dependent functions will be suppressed. Thus, we see a possible explanation for the subnormal responsiveness of females to the usual intermittent GH therapy.


   Footnotes
 
First Published Online March 1, 2007

Abbreviations: ChIP, Chromatin immunoprecipitation assay; GHR, GH receptor; IB, immunoblotting; IP, immunoprecipitated; Jak2, Janus kinase-2; rGH, rat GH; Stat, signal transducer and activator of transcription.

1 Although it is possible that the concentration of GH declined between every 12 h of media change, it is unlikely that the hormone was ever eliminated (e.g. metabolized) from the culture. The GH regimen used in the experiment induces normal levels (mRNA and protein) of CYP2C12, an isoform whose expression is absolutely dependent upon continuous exposure to the hormone (3 16 ). Back

This work was partially supported by National Institutes of Health Grant GM-45758.

Disclosure Summary: C.T. and B.S. have nothing to declare.

Received September 28, 2006.

Accepted for publication February 21, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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