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Endocrinology Vol. 140, No. 10 4725-4731
Copyright © 1999 by The Endocrine Society

ARTICLES

Growth Hormone (GH) and 17ß-Estradiol Regulation of the Expression of Mouse GH Receptor and GH-Binding Protein in Cultured Mouse Hepatocytes1

Bernardo Contreras and Frank Talamantes

Department of Biology, Sinsheimer Laboratories, University of California, Santa Cruz, California 95064

Address all correspondence and requests for reprints to: Dr. Frank Talamantes, Department of Biology, Sinsheimer Laboratories, University of California, Santa Cruz, California 95064. E-mail: prolactin{at}aol.com


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In the present study, primary mouse hepatocytes from 8- to 10-week-old virgin female Swiss-Webster mice were perfused with collagenase (100 U/ml) using the two-step method. Isolated hepatocytes were plated in a rat tail type I collagen sandwich configuration to examine the regulation of GH receptor (GHR) and GH-binding protein (GHBP) expression by GH and 17ß-estradiol (E2). After 48 h of initial plating, hepatocytes were divided into groups of five replicates and treated for 24 h with medium containing no hormones (controls), GH (100 ng/ml), E2 (10-9 M), E2 (10-9 M) plus GH (100 ng/ml), or E2 plus GH and ICI 182–780 at different concentrations.

Treatment of hepatocytes with GH or E2 alone did not have any effect on the cellular concentrations of GHBP and GHR. However, the combination of E2 and GH up-regulated the cellular concentrations of GHBP and GHR 2- to 3-fold. GHBP and GHR messenger RNA concentrations were also up-regulated 2- to 3-fold. ICI 182–780, a competitive inhibitor of E2 for the estrogen receptor (ER), at different concentrations inhibited the E2 and GH-induced stimulation of GHBP and GHR. Furthermore, ER concentrations increased 5- to 7-fold in hepatocytes treated with E2 and GH compared with those in untreated cells or cells treated with either E2 or GH alone. In the present study we have shown that in cultured hepatocytes from virgin female mice, GH or E2 alone did not affect the concentrations of GHBP and GHR. However, E2 and GH together significantly up-regulated GHR and GHBP expression.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
LIKE OTHER PEPTIDE hormones, GH exerts its effects by binding to a specific, high affinity cell surface transmembrane protein known as the GH receptor (GHR) (1). In addition, a specific GH-binding protein (GHBP) with high affinity and low capacity for GH has been identified in humans, rabbits, sheep, rats, and mice (1). In humans and rabbits, the extracellular binding domain (EBD) of the GHR is proteolytically cleaved to generate the GHBP (1). Therefore, GHBP is identical to the EBD of GHR. However, in mice and rats, GHR and GHBP are generated by alternative splicing from a single primary transcript. Two messenger RNAs (mRNAs) of 3.9–4.5 and 1.2 kb are generated, encoding the GHR and GHBP, respectively (1). GHBP lacks the transmembrane and cytoplasmic domains of the GHR. Instead, these domains are replaced by a 17- and 27-amino acid hydrophilic tail in rats and mice, respectively (1). The primary source of serum GHBP is believed to be the liver.

In humans, there are conflicting data regarding the role of GH as a regulator of serum GHBP. GH deficiency has been associated with low (2) or normal serum GHBP concentrations (2). In cases of acromegaly, serum GHBP concentrations were either normal (2) or lower than normal (3). In children with idiopathic short stature (4) and in GH-deficient children (2), serum GHBP concentrations were significantly increased after GH treatment. In cultures of the human hepatoma cell line HuH7, transcription of hepatic GHR (5) and GHBP (6) were GH dependent.

In rodents, GH binding studies showed that in adult rats, females have about twice the number of hepatic GHRs compared with male rats (2). Others have evaluated the effects of hypophysectomy on GH binding in the liver with variable results. Hypophysectomy reduced GH binding in liver membranes from female rats (7), whereas the opposite effects have been found in hypophysectomized male rats (8). On the other hand, one study reported that hypophysectomy did not have any effect on concentrations of hepatocyte GHRs in either female or male rats (7), but in another study, hypophysectomy reduced GH binding in liver membranes from female rats, whereas in male rats no effects were found, provided the membranes were stripped of endogenous GH. Subsequent GH treatments increased GH binding of liver membranes from both sexes (7). Furthermore, continuous infusion of GH increased GH binding of liver membranes in both intact female and male rats, and it was concluded that in rats, GH regulated its own receptor independently of the sex or pituitary status of the animal (7). However, in a different study, hypophysectomy and GH treatment were without effect on hepatic GHR mRNA concentrations in male rats (7), although hypophysectomy of pregnant mice lowered and GH treatment partially restored hepatic GHR and serum GHBP levels (9). In GH-deficient dwarf rats, the concentrations of hepatic GHR and serum GHBP were lower than those in normal rats, and continuous GH infusion restored hepatic GHR and serum GHBP concentrations to those found in normal rats (10). The effects of GH on GHR expression in cultured rat hepatocytes are conflicting, with one study showing a stimulatory effect of GH on GH binding (11), whereas in another study, GH did not increase the mRNA level for GHR (12). Studies of cultured porcine hepatocytes confirm a stimulatory effect of GH on the expression of its own receptor (13).

In adult rodents, GH pulsatility is sexually dimorphic (14). Male rodents exhibit GH secretory peaks lasting 1–1.5 h every 3–4 h, followed by distinct trough periods of low GH concentrations. In female rats, GH pulses are more random, the peaks of the GH pulses are substantially lower, and trough GH concentrations between peaks are higher. However, these two distinct GH pulse patterns do not become evident until the onset of puberty, and sex steroids play a major role in these changes. Estrogen treatment of male rats resulted in a female-like GH pulse pattern (14). Estrogen-deficient female rats expressed a partial masculinization pattern of GH secretion (14).

Hepatic GHR and serum GHBP concentrations are also sexually dimorphic. The more continuous female GH pulse pattern is associated with elevated hepatic GHR and serum GHBP (10). The stimulatory effect of estrogen on hepatic GHR and serum GHBP is believed to be mediated by the GH pulse pattern (10). Therefore, any direct effects estrogen may have on the synthesis of hepatic GHR and serum GHBP may be masked by the effect estrogen has on the GH pulse pattern. Consequently, to circumvent the in vivo effect that estrogen has on GH pulses in rodents, an alternate in vitro model was needed to evaluate the effects of estrogen on hepatic GHR and GHBP.

One method of studying how factors such as estrogen and GH influence hepatic GHR and GHBP is to use cultured hepatocytes. The collagen sandwich configuration method provides a system for culturing highly differentiated and viable hepatocytes for long periods of time (15). The simplicity of the system makes it ideally suited for evaluating the effects of individual factors on the hepatic GH-GHR/GHBP axis. In the present study, primary mouse hepatocytes cultured in a collagen sandwich configuration were used to examine the regulation of mouse (m) GHR and mGHBP expression by GH and 17ß-estradiol (E2).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and materials
Eight- to 10-week-old virgin female Swiss-Webster mice were purchased from Simonsen Laboratories (Gilroy, CA), housed in a controlled environment with a 14-h light, 10-h dark lighting cycle, and fed ad libitum. The care and use of these animals was approved by the university animal care committee. The bicinchoninic acid protein assay kit was purchased from Pierce Chemical Co. (Rockford, IL). The estrogen receptor (ER) enzyme immunoassay (EIA) kit was obtained from Abbott Laboratories (North Chicago, IL). The MAXIscript-T7 in vitro transcription kit and the Direct Protect Lysate ribonuclease protection assay (RPA) kit were obtained from Ambion, Inc. (Austin, TX). mGH was purified as previously described (16). Rat tail collagen type I was prepared as previously described (17). Collagenase type II was purchased from Worthington Biochemical Corp. (Freehold, NJ). Percoll was obtained from Pharmacia Biotech (Piscataway, NJ). Mouse albumin and rabbit antimouse albumin antiserum were obtained from Cappel Research Products (Durham, NC). Na125I was purchased from Amersham Pharmacia Biotech (Arlington Heights, IL). Goat antirabbit antiserum was obtained from Antibodies, Inc. (Davis, CA). HBSS, Ca2+,Mg2+-free HBSS, BSA, DMEM-Ham’s F-12, amino acids, E2, rabbit IgG {gamma}, human holo-transferrin, linoleic acid, pyruvic acid, trypan blue, thimerosal, gelatin, polyethylene glycol (Mr = 8000), and Sephadex G-100 were obtained from Sigma Chemical Co. (St. Louis, MO). All other reagents were purchased from Fisher Scientific (Pittsburgh, PA).

Liver perfusions
Hepatocytes were isolated using collagenase (100 U/ml) and the two-step liver perfusion method via the portal vein as described by Seglen et al. (18). To isolate viable parenchymal cells, the protocol of Kreamer et al. (19) was used. Nine volumes of stock Percoll were added to 1 vol 10 x HBSS. A suspension of isolated hepatocytes was adjusted to 5.0–7.0 x 106 cells/ml culture medium, which consisted of DMEM-Ham’s F-12 supplemented as described by Hutson et al. (20), except that insulin and corticosterone were not included. Twenty milliliters of the diluted cell suspension were mixed with 20 ml diluted Percoll. The solution was centrifuged at 50 x g for 10 min at 4 C. The viable parenchymal cells, which pelleted at the bottom of the tube, were resuspended and washed twice with culture medium and centrifuged at 50 x g for 2 min at 4 C. Viability and cell number were assessed using 0.4% trypan blue dye. This method resulted in cell viability in the 95–98% range. Cell yield usually resulted in 4.5–5.0 x 107 cells/liver.

Cell culture
Hepatocytes were plated in 60 x 15-mm dishes that were precoated with 1 ml rat tail type I collagen at a density of 2.2 x 106 cells/dish. The cells were incubated in 4 ml culture medium at 37 C in 5% CO2-95% air. After 5 h, the medium was removed, and the cells were overlaid with a second layer of collagen. The second collagen layer was allowed to gel for 30 min before 2 ml culture medium were added (15). Thereafter, media were changed every 24 h. After a 48-h cell recovery period, media were changed to 2 ml culture medium supplemented with different hormones. E2, diluted in ethanol to a stock concentration of 10-2 M, was used at final concentrations ranging from 10-11-10-6 M. mGH, diluted in 10 mM NaOH to a stock concentration of 1 mg/ml, was used at concentrations of 10, 100, and 1000 ng/ml. When tested in combination, mGH was used at a concentration of 100 ng/ml, and E2 at 10-9 M. After 24 h of hormone treatment, the collagen-sandwiched cells were transferred into borosilicate glass tubes, and 2 ml 4.3 M acetic acid were added to dissolve the collagen. Samples were incubated for 30 min at 37 C, followed by a brief centrifugation to collect the cells. Cells were used for DNA assays, protein assays, RIAs, ER assays, or RPAs. DNA was measured from pelleted cells from each group as previously described (21). Cells used for protein assays and RIAs were lysed in 1 ml RIA-Triton buffer [1% (wt/vol) Triton X-100, 10 mM Na2HPO4, 10 mM Na2EDTA, 150 mM NaCl, 0.1% (wt/vol) RIA grade BSA, and 0.01% (wt/vol) thimerosal, pH 7.5] and centrifuged for 1 min at 10,000 x g, and the supernatants were stored at -20 C. Cells used to measure ER concentrations were lysed in 500 µl homogenization buffer as described in Abbott’s EIA instruction manual. Samples were assayed immediately. Cells used for RPAs were lysed in 100 µl RPA Direct Lysis Buffer (Ambion, Inc.) and centrifuged for 15 min at 10,000 x g at 4 C, and the supernatants were stored at -20 C. All hormone treatments were replicated five times in each culture. Each experiment was repeated at least three times, unless otherwise noted.

Assays for protein, albumin, GHR, GHBP, and ER
The protein concentrations of cell lysates were measured using a bicinchoninic acid protein assay kit. RIAs to measure GHR (22) and GHBP (23) concentrations were used as previously described. Cellular ER contents were measured using an EIA kit.

An RIA for mouse albumin was developed. Conditioned medium was diluted 1000-fold in RIA-G buffer [10 mM Na2HPO4, 10 mM Na2EDTA, 150 mM NaCl, 0.1% (wt/vol) gelatin, 0.01% (wt/vol) thimerasol, and 0.02% (wt/vol) rabbit IgG {gamma}, pH = 7.5). Rabbit antimouse albumin antiserum was diluted 1:25,000 (vol/vol) in RIA-G buffer. Mouse albumin was iodinated with Na125I by the Iodogen method (24). Radiolabeled albumin was separated from free 125I on a Sephadex G-100 column. Mouse albumin standards were diluted in RIA-G buffer in the range of 0.1–100 ng/ml. One hundred microliters of standard or diluted conditioned medium were incubated with 100 µl diluted antiserum and 100 µl diluted tracer (10,000 cpm). Nonspecific binding was determined by substituting RIA-G buffer in place of primary antiserum. All samples were assayed in triplicate. The mixture was incubated for 24 h at room temperature before 100 µl goat antirabbit antiserum, diluted 1:16 (vol/vol) in RIA-G buffer, were added to the samples. The samples were incubated for an additional 30 min at room temperature, before 100 µl 30% polyethylene glycol were added. The samples were vortexed and centrifuged at 9,000 x g for 20 min at 4 C. The supernatants were aspirated, and the pellets were counted in a {gamma}-counter (Packard Instrument Co., Meriden, CT). A 1:25,000 dilution of the primary antiserum provided 45–55% total binding, and nonspecific binding ranged between 4–8% (n = 5 assays). The intraassay coefficient of variation was 9.2% (n = 10), and the interassay coefficient of variation was 10.1% (n = 5 assays) from pooled conditioned medium.

Synthesis of complementary RNA probes
Mouse GHR/GHBP and cyclophilin riboprobes were transcribed and [{alpha}-32P]UTP labeled as previously described (25). In the RPAs, the 256-nucleotide (nt) mGHR/GHBP riboprobe yielded two protected fragments, a 256-nt fragment corresponding to mGHBP and a 150-nt fragment corresponding to mGHR (Fig. 1Go). The cyclophilin riboprobe resulted in a 103-nt protected fragment.



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  		Figure 1. A mouse GHR/GHBP riboprobe of 256 nt was transcribed and [{alpha}-32P]UTP labeled as described in Materials and Methods. In RPAs, this riboprobe yielded two protected fragments, a 256-nt fragment for mGHBP and a 150-nt fragment corresponding to mGHR. The protected fragments are complimentary to 150 nt of the mRNA that encodes portion of the EBD shared by both mGHR and mGHBP. The remaining 106 nt that are complimentary to the mRNA encoding the hydrophilic tail (TP) are unique to the mGHBP mRNA.

 
Direct RPAs
Ribonuclease protection assays were carried out overnight as described in the Direct Protect Lysate RPA instruction manual (Ambion, Inc.). The bands corresponding to probe fragments protected by GHR, GHBP, and cyclophilin mRNAs were quantitated by phosphorimaging (PhosphorImager, Molecular Dynamics, Inc., Sunnyvale, CA).

Statistical analysis
GHR, GHBP, and ER concentrations in cell lysates were normalized to total cellular protein concentrations. Albumin from conditioned medium was also normalized to total cellular protein concentrations. The RPA values for GHR and GHBP mRNAs were normalized to values of the mouse cyclophilin mRNA protected fragment. Normalized values were analyzed using one-way ANOVA followed by Scheffe’s test. P < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Albumin concentrations in conditioned media were measured to ensure that hepatocytes remained differentiated and viable for at least 1 week. Albumin in cell lysates was undetectable, but was secreted into the culture medium. Secreted albumin concentrations remained constant between days 2–8 of culture (mean ± SE; picomoles per mg protein of whole cell lysates; P > 0.05; n = 10 wells): day 2, 250 ± 15; day 3, 245 ± 18; day 4, 255 ± 20; day 5, 266 ± 20; day 6, 240 ± 25; day 7, 255 ± 18; and day 8, 262 ± 20.

Treatment of hepatocytes with GH or E2 alone did not have any effect on the concentration of GHBP in culture media (data not shown) or on cellular levels of GHBP (Fig. 2aGo) and GHR (Fig. 2bGo). However, the combination of E2 and GH up-regulated the cellular concentrations of GHBP (Fig. 2aGo) and GHR (Fig. 2bGo) 2- to 3-fold, which was significantly higher than concentrations found in controls. E2 plus GH up-regulation of GHBP was also evident in conditioned medium (data not shown). In cultured hepatocytes, E2 plus GH up-regulated GHBP mRNA (Fig. 3Go, a and b) and GHR mRNA (Fig. 3Go, a and c) 2- to 3-fold, whereas GH and E2 alone were without effect.



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  		Figure 2. GHBP and GHR concentrations from cultured hepatocytes of female virgin mice. Cells were cultured in a collagen sandwich configuration and maintained in hormone-free culture medium for 48 h as described in Materials and Methods. Cells were then divided into groups of five and treated for 24 h with culture media containing different hormones. Cells were lysed, and lysates were used in RIAs to measure concentrations of GHBP and GHR as described in Materials and Methods. A and B show the effects on GHBP and GHR, respectively. Results from the following groups are shown: a, no hormones; b, GH (100 ng/ml); c, E2 (10-9 M); d, E2 (10-9 M) plus GH (100 ng/ml). Each bar represents the mean ± SEM (n = 5). Results are representative of one experiment. Experiments were repeated three times. *, GHR and GHBP concentrations were significantly elevated compared with those in all other groups (P < 0.05).

 


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  		Figure 3. GHBP and GHR mRNA concentrations were quantitated in hepatocytes cultured for 24 h in medium supplemented with different hormones as described in Materials and Methods. A, A representative phosphorimage of GHBP, GHR, and cyclophilin mRNAs. Each lane represents RNA lysate collected from one 60-mm dish plated with 2.2 x 106 hepatocytes as described in Materials and Methods. Messenger RNA from the following groups are shown: no hormones (NH), GH (100 ng/ml), E2 (10-9 M), E2 (10-9 M) plus GH (100 ng/ml), and control cell lysate (C) from the Ambion kit. B and C show the concentrations of mRNAs for GHBP and GHR, respectively. The columns represent the phosphorimage counts for each group that were normalized to the cyclophilin mRNA phosphorimage counts. The results from the following groups are shown: a, no hormones; b, GH (100 ng/ml); c, E2 (10-9 M); and d, E2 (10-9 M) plus GH (100 ng/ml). Each bar represents the mean ± SEM (n = 5). Results are representative of one experiment. Experiments were repeated three times. *, GHR and GHBP mRNA concentrations were significantly elevated compared with those in all other groups (P < 0.05).

 
The stimulation of E2 plus GH on GHR and GHBP did not appear to result from stimulation of general protein synthesis, as the concentrations of albumin in the media remained unchanged. Albumin values from hepatocytes treated with GH or E2 alone also remained constant (Fig. 4Go). Furthermore, the DNA concentrations remained unchanged among the various groups (data not shown).



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  		Figure 4. Albumin concentrations in conditioned medium from cultured hepatocytes. Cells were cultured and treated as described in Fig. 2Go. The results from the following groups are shown: a, no hormones; b, GH (100 ng/ml); c, E2 (10-9 M); and d, E2 (10-9 M) plus GH (100 ng/ml). Each bar represents the mean ± SEM (n = 5). Results are representative of one experiment. Experiments were repeated three times.

 
To assess the involvement of ER in the up-regulation of GHR and GHBP expression, cells were incubated for 24 h with E2 plus GH in the presence or absence of ICI 182–780, a competitive inhibitor of E2 for the ER (26). ICI 182–780 inhibited the E2- plus GH-induced stimulation of GHBP (Fig. 5aGo) and GHR (Fig. 5bGo). The ER values in cultured hepatocytes did not change when cells were treated with either E2 or GH alone. However, the ER concentrations increased 4- to 6-fold in hepatocytes treated with both E2 and GH (Fig. 6Go).



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  		Figure 5. The effects of ICI 182–780 on the E2 plus GH stimulation of GHR and GHBP production. Hepatocytes were plated and cultured as described in Fig. 2Go. Cells were treated with E2 (10-9 M) plus GH (100 ng/ml) and different concentrations of ICI 182–780. A and B show the effects on GHBP and GHR, respectively. The results from the following treatments are shown: a, no hormones; b, E2 (10-9 M) plus GH (100 ng/ml) without ICI 182–780; c, E2 (10-9 M) plus GH (100 ng/ml) with ICI 182–780 (10-10 M); d, E2 (10-9 M) plus GH (100 ng/ml) with ICI 182–780 (10-9 M); e, E2 (10-9 M) plus GH (100 ng/ml) with ICI 182–780 (10-6 M); and f, ICI 182–780 (10-6 M) alone. Results are representative of one experiment. Experiments were repeated three times. *, Significantly different from the NH group (P < 0.05). **, Significantly different from the E2 plus GH group (P < 0.05).

 


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  		Figure 6. ER concentrations in hepatocytes cultured and treated with different hormones as described in Fig. 2Go. The results from the following groups are shown: a, no hormones; b, GH (100 ng/ml); c, E2 (10-9 M); and d, E2 (10-9 M) plus GH (100 ng/ml). Results are representative of one experiment. Experiments were repeated three times. *, Significantly different from all other groups (P < 0.05).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Studies in rodents have shown that the E2 up-regulation of the liver GHR/GHBP is mediated by E2 regulation of the GH pulse pattern. To circumvent these effects, in the present study cultured hepatocytes from female mice were used to determine whether E2 and GH have direct effects on the hepatic GHR/GHBP. These studies provide additional information on how GH and E2 affect the expression of the hepatic GHR and GHBP. GH or E2 alone did not change the cellular concentrations of GHBP and GHR in cultured hepatocytes, nor were secreted concentrations of GHBP affected.

Some in vivo studies have shown that hypophysectomy and GH treatment did not affect the hepatic GHR and serum GHBP concentrations (7), and GH did not affect steady state levels of GHR and GHR mRNA in cultured hepatocytes (12). Other studies in rats have shown that GH regulates its own receptor in the liver and other tissues. For example, GH binding to liver membranes was reduced by hypophysectomy and increased by subsequent GH treatment (7). Also, in both intact male and female rats, continuous GH infusion increased GH binding of liver membranes (7). In pregnant mice, hypophysectomy lowered GHR and GHBP concentrations, and subsequent GH treatments partially restored GHR and GHBP concentrations (9). GH regulation of its own receptor in the liver is supported by studies in the GH-deficient dwarf rat, where continuous GH infusion increased hepatic GHR and serum GHBP concentrations to levels similar to those in normal rats (10). In these in vivo studies, GH could have interacted with other growth factors or steroids to regulate its own receptor. It appears that in vivo, GH is capable of regulating its own receptor in the liver, but in cultured hepatocytes, results have been less definite. Studies on rat cultured hepatocytes have shown an increase (11) and no change (12) in GHR mRNA expression. Studies of porcine cultured hepatocytes have supported a stimulatory effect on GHR mRNA expression (13). In cultured hepatocytes, GH might require the permissive effects of other factors to up-regulate the expression of GHR and GHBP.

In the present study, GHR and GHBP were significantly up-regulated when GH and E2 were used in combination. This demonstrates a synergism between E2 and GH to up-regulate the hepatic GHR and GHBP in cultured female mouse hepatocytes. This was not caused by an increase in cell number, because the DNA content of the cultured cells did not differ between experimental groups. Our results are consistent with in vivo studies, where E2 required the presence of GH to up-regulate hepatic GHR and serum GHBP. There it was concluded that such E2 up-regulation of GHR and GHBP was mediated by the effects of E2 on the GH pulse pattern (10). The synergism between E2 and GH seen in our studies is similar to that found in cultured osteoblasts, where the presence of both E2 and GH was required for cell proliferation, whereas either GH or E2 alone was without effect. However, in that study, E2 alone did increased GH binding in cultured osteoblasts (27).

In other tissues, GH has been shown to regulate its own receptor. In rat adipocytes (28) and in cultured rat epiphyseal chondrocytes (29), GH increased GHR mRNA concentrations. In contrast, GH down-regulated its own receptor in cultured fibroblasts (7) and IM-9 lymphocytes (7). Therefore, the effects of GH on its own receptor are probably tissue specific and vary among species, physiological state, and the sex of the animal. Results may also vary when in vivo vs. in vitro studies are compared. GH effects in vivo are not always identical in vitro, probably because other hormones are required that were not identified in the in vivo studies. The results presented here suggest that the mechanism by which GH and E2 regulate the hepatic GHR and serum GHBP is more complex than what in vivo studies suggest. The results in our studies point to an additional mechanism of E2 and GH up-regulation of hepatic GHR and GHBP at the cellular level.

The involvement of hepatic ER in the stimulatory effect of E2 and GH was suggested by the inhibitory effect of ICI 182–780 on the up-regulation of E2 and GH on GHR and GHBP. ICI 182–780 is an antiestrogen that competes with E2 for the binding site on the ER, thus acting as a competitive inhibitor of ligand-dependent ER-mediated effects (30). It is possible that these inhibitory effects were accomplished by reducing the intracellular concentrations of the ER, by blocking the transport of ERs into the nucleus, or by blocking ER dimerization, thus inhibiting DNA binding (30). The inhibitory effects of ICI 182–780 coupled with the increase in ER concentrations in cells treated with E2 and GH suggest that the ER is involved in the E2 and GH up-regulation of GHR and GHBP in cultured hepatocytes. How the ER is involved is unknown. The present study indicates that E2 and GH up-regulates hepatic GHR and GHBP by acting directly on the liver or perhaps by E2 serving a permissive role for the effects of GH regulating its own receptor. The fact that GHR and GHBP were increased only when the ER was also present could point to a variety of possibilities. ER concentrations were probably too low in cultures when GH and E2 were used alone, and this could be the reason why E2 was without effect alone. On the other hand, increased concentrations of ER probably allowed E2 to provide a permissive role for the effects of GH. However, the possibility that ER could be involved in a direct manner, at the transcriptional level, cannot be omitted. Nuclear proteins such as ER, can also regulate transcription of genes lacking estrogen response elements by modulating the activity of other transcription factors, such as activating protein-1 and nuclear factor-{kappa}B (31), or by interacting with other general transcription factors, such as transcription factor IIB and transcription factor IID (31).

E2 and GH induction of the hepatic ER is consistent with other studies of cultured rat hepatocytes (32). Although in cultured rat hepatocytes, GH required the presence of other hormones to up-regulate the ER (32), results from in vivo studies indicated GH alone up-regulated the hepatic ER (33), another example of an effect of GH in vivo that is not found in vitro unless other hormones are present.

The GHR/GHBP gene has not been fully characterized in any species, making it difficult to determine how E2, GH, and ER are involved in the regulation of GHR/GHBP. In the liver, alternative splicing of the 5'-untranslated region (UTR) of the GHR/GHBP gene results in heterogeneity in primary transcripts. Five 5'-UTRs have been reported in the rat, and four in the mouse (L1–L4) (1, 34). In the mouse, L1 and L2 are homologs of GHR1 and GHR2 of the rat GHR/GHBP gene, respectively. The transcription of L1 and L2 is regulated by their own promoters. In vivo studies showed that although GHR1 transcription is regulated by GH, transcripts with the other GHR 5'-UTRs are not affected (35). In hepatocytes cultured with a combination of GH, E2 and dexamethasone, a 1.5- to 2-fold induction of mRNA for GHR1 was reported (36). In pregnant mice, L1 is responsible for the increased hepatic GHR and GHBP expression (37). It is possible that the E2 plus GH regulation of GHR/GHBP expression we found in cultured liver cells from adult female mice involves the L1 promoter.

In the liver, GH elevates Jun and Fos concentrations (38). GH also regulates the concentrations of hepatocyte nuclear factor (HNF-1) (39), HNF-3 (39), HNF-6 (40), the CAAT enhancer binding proteins (C/EBPs) (41), and the GH-regulated nuclear factor. GH-regulated nuclear factor is enriched in adult female rats (42). GH also activates the translocation of Stat3 (signal transducer and activator of transcription) and and Stat5 into the nucleus, which, in turn, bind to interferon-{gamma} activation site-like cis elements to activate transcription (43). To date, no interferon-{gamma} activation site-like cis elements have been identified in the L1 and L2 promoters (44), whereas a C/EBP cis element has been identified in the L1 promoter (44). However, a computer analysis of the promoter and upstream regions of mouse L1 and L2 point to several potential activating protein-1- and C/EBP-binding sites, whereas no estrogen response elements have been identified (1).

The requirement for E2 and GH in the induction of GHR/GHBP in cultured hepatocytes could be related to the interaction of the ER and other transcription factors regulated by GH. It is likely that cells depleted of GH have lower levels of GH-dependent transcription factors, which, in turn, might prevent the interaction of any of these transcription factors with ER or other proteins involved in regulating the liver GHR/GHBP expression. The fact that elevated concentrations of ER are correlated with the E2 and GH induction of GHR and GHBP, and the possibility that GH is associated with elevated levels of several transcription factors may account for the need for both E2 and GH in the induction of the expression of GHR and GHBP in cultured mouse hepatocytes. On the other hand, the elevated concentrations of ER could merely be required for the permissive effects of E2 on GH stimulation of its own receptor, without the need for ER interacting with the transcriptional machinery involved in the expression of GHR/GHBP. These studies identified an additional synergism between E2 and GH at the cellular level. The culture system used in these studies provides an ideal system to further study the mechanism involved in E2 and GH regulation of the hepatic GHR/GHBP.


   Acknowledgments
 
The authors appreciate the excellent advice provided by Drs. Ogren and Thordarson throughout the completion of the studies and in the writing of this manuscript. We are also in debt to Dr. Camarillo for providing the GHR RIA, to Dr. Ilkbahar for providing the GHR/BP riboprobe, and to Dr. Kensinger for the assistance in developing the albumin RIA. We also thank Dr. John Ruffin from the Office of Minority Health at the NIH.


   Footnotes
 
1 This work was supported by NIH Grants CA-71590, HD-14966, and GM-08132 (to F.T.). Back

Received January 13, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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