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Endocrinology Vol. 145, No. 12 5704-5713
Copyright © 2004 by The Endocrine Society

Activation of the Glucocorticoid Receptor or Liver X Receptors Interferes with Growth Hormone-Induced akr1b7 Gene Expression in Rat Hepatocytes

Pia Kotokorpi, Cissi Gardmo, C. Staffan Nyström and Agneta Mode

Department of Medical Nutrition, Karolinska Institutet, Novum, S-141 86 Huddinge, Sweden

Address all correspondence and requests for reprints to: Agneta Mode, Department of Medical Nutrition, Karolinska Institutet, Novum, S-141 86 Huddinge, Sweden. E-mail: agneta.mode{at}mednut.ki.se.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The akr1b7 gene encodes an aldo-keto reductase involved in detoxification of isocaproaldehyde, the product from side chain cleavage of cholesterol, and of 4-hydroxynonenal (4-HNE) formed by lipid peroxidation and cleavage. Here we show that the expression of akr1b7 mRNA in rat liver is sexually differentiated, expressed in females but not in males, and regulated by the sexually dimorphic secretion pattern of GH. A GH dose-dependent induction of akr1b7 was demonstrated in cultured primary rat hepatocytes, which was sensitive to cycloheximide. Activation of the glucocorticoid receptor (GR) or liver X receptors (LXR) by dexamethasone (Dex) and T1317, respectively, attenuated the GH-induced expression of akr1b7 and CYP2C12, the prototypical rat hepatic gene dependent on the female-characteristic secretion pattern of GH. In contrast, neither Dex nor T1317 had any repressive effect on the GH induction of IGF-I mRNA. A common mechanism for LXR- and GR-mediated repressive actions on gene transcription is inhibition of nuclear factor (NF)-{kappa}B; however, EMSAs and pharmacological interference with NF-{kappa}B signaling provided no evidence for the involvement of NF-{kappa}B in the repressive action of Dex and T1317 on GH-induced akr1b7 expression.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
MEMBERS OF THE aldo-keto reductase (AKR) gene superfamily encode nicotinamide-dependent oxidoreductases and are involved in detoxification of harmful aldehydes and ketones produced by diverse metabolic reactions (1). AKR1B7 was initially described as a secretory mouse vas deferens protein (2) and later found to be highly expressed in the adrenal cortex in rodents and in man (3, 4). AKR1B7 is suggested to be the major enzyme responsible for elimination of isocaproaldehyde produced from side-chain cleavage of cholesterol during steroidogenesis (4). 4-Hydroxynonenal (4-HNE), a major product of lipid peroxidation with strong electrophilic properties and thereby cytotoxic, has also been shown to be metabolized to a less toxic compound by AKR1B7. Consistent with the enzymatic activity of AKR1B7, significant expression has also been detected in rodent testis, ovaries, and intestine, whereas low levels are expressed in kidney, lung, and liver (3, 5).

The expression of the akr1b7 gene in steroidogenic tissues appears regulated by trophic pituitary hormones: ACTH in the adrenal gland, FSH in ovaries and LH in Leydig cells (4, 6). In the mouse vas deferens, the main regulator is androgens (7). Recently, mouse intestinal expression of akr1b7 was demonstrated to be induced by liver X receptor (LXR) ligands (8) and liver gene expression profiling has indicated LXR agonist induction of akr1b7 also in mouse liver (9). LXR, a member of the nuclear receptor family, has emerged as a key factor in control of cholesterol homeostasis (10). Activation of LXR stimulates reverse cholesterol transport from macrophages to the liver where it can be converted to bile acids or eliminated in the bile (11). However, a less beneficial effect of LXR activation in terms of lipid homeostasis is the stimulatory effect on hepatic lipogenesis (12, 13).

A broad array of liver proteins involved in the metabolism of endogenous compounds as well as xenobiotics is sexually dimorphic because of the regulation by the sex-specific GH secretion patterns (14, 15). GH secretion is sexually differentiated in all mammals, which is particularly pronounced in rodents (16, 17). The secretion pattern in male rats is characterized by high peaks of GH release every 3–4 h with low or undetectable levels in between. In contrast, the female rat secretes GH in more frequent pulses with lower peak amplitude and with higher basal levels, resulting in a continuous presence of GH in plasma. The female GH pattern can be mimicked in male and hypophysectomized (Hx) rats by continuous administration of GH, which then results in feminization of liver target gene expression. Prototypical genes subjected to GH pattern-dependent regulation include members of the cytochrome P450 (CYP) gene family (18, 19). Key signaling pathways for effects of GH are the Janus kinase (Jak)2/signal transducer and activator of transcription 5 (Stat5), the Ras/MAPK and the insulin receptor substrate-phosphatidylinositol 3'-kinase pathways and a vast number of transcription factors are implicated in GH regulation of gene transcription (20). Although transcriptional effects of the male characteristic pattern of GH secretion is conveyed by the Jak2/Stat5 pathway (21), the molecular mechanisms for gene regulation by continuous GH have not yet been resolved. However, it has been shown that GH induction of the female specific CYP2C12 gene includes cooperation of Stat5, hepatic nuclear factor (HNF)-6 and HNF-4 (22).

In search for novel GH target genes expressed in rat liver, dependent on the female-specific GH pattern, we used subtractive suppressive hybridization and identified clones (F15, F18, E20) corresponding to the akr1b7 gene (23). A sexually dimorphic akr1b7 expression in rat liver would be discordant with what has been found in the mouse in which the expression in nonreproductive tissues is similar between the sexes (3). This prompted us to investigate the hormonal regulation of rat liver akr1b7 mRNA expression in more detail.

In this study, we have confirmed that akr1b7 mRNA expression is sexually differentiated in rat liver and dependent on continuous GH in vivo. Furthermore, the induction by GH was exerted directly on the hepatocyte and sensitive to cycloheximide (CHX) treatment. We have also demonstrated that the GH induction of akr1b7 is perturbated by activation of the glucocorticoid receptor (GR) or LXR.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and hormonal treatment
The Stockholm South Ethical Committee of the Swedish National Board of Animal Experiments/the Swedish National Animal Welfare Agency approved all animal experiments. Normal and Hx Sprague Dawley rats for in vivo experiments were obtained from Møllegaard (Møllegaard Breeding center Ltd., Ejby, Denmark). The rats underwent Hx at 6 wk of age. Completeness of Hx was ascertained by recording the body weight for 1 wk before hormone treatment and by inspection of the sella turcica at the time the rats were killed. Animals were maintained under standardized conditions of temperature, humidity, and light and with free access to standard laboratory chow and water. To mimic the female GH pattern, animals were given bovine GH (bGH), a generous gift from American Cyanamid Co. (Princeton, NJ), as a continuous infusion by means of Alzet osmotic minipumps (model 2001; Alza, Palo Alto, CA), at a daily dose of 0.5 mg bGH/kg·d for 6 d. Substituted Hx rats were given daily sc injections of cortisol phosphate (C), 400 µg/kg·d (Solu-Cortef, Upjohn, Puurs, Belgium) and T4, 10 µg/kg·d (Nycomed, Oslo, Norway). Animals were killed by decapitation, and liver samples and adrenals were collected for preparation of total nucleic acids (tNA) or total RNA.

Primary rat hepatocyte cultures
Rats for hepatocyte isolation were purchased from BK-Scanbur (Scanbur, Sollentuna, Sweden). Primary hepatocytes were isolated from isoflurane-anesthetized 6- to 8-wk-old rats by nonrecirculating collagenase perfusion as previously described (24). Hepatocytes were seeded onto matrigel-coated 10-cm dishes (10 x 106 cells/dish) in William’s E medium with Glutamax (Invitrogen Life Technologies, Paisley, Scotland, UK) supplemented with 100 U/ml of penicillin and 100 µg/ml of streptomycin (Invitrogen Life Technologies) and insulin (I-5500; Sigma-Aldrich, Stockholm, Sweden) as indicated. Medium was renewed daily. Matrigel was prepared from Engelbreth-Holm-Swarm sarcoma propagated in C57BL/6 female mice and stored at –20 C, as described (25). Hormone and drug treatments were commenced at 48 h of culture age and, unless otherwise indicated, continued for 18 h. Dexamethasone (Dex), T3, T0901317 (T1317), ammonium pyrolidinedithiocarbamate (PDTC), and CHX were purchased from Sigma and pregnenolone 16{alpha}-carbonitrile (PCN) from Upjohn Co. (Kalamazoo, MI). 9-cis RA (9-cis retinoic acid) was a generous gift from Hoffmann-La Roche Inc. (Basel, Switzerland). Stock solutions were prepared in medium or dimethyl sulfoxide (DMSO). When used, the final concentration of DMSO in culture medium was 0.1% and control and/or GH-treated cells received the vehicle. The cells were maintained in a humidified incubator with 5% CO2 at 37 C. Cells were harvested in SET buffer [1% sodium dodecyl sulfate, 10 mM EDTA, 20 mM Tris (pH 7.5)] for subsequent preparation of tNA.

Preparation of RNA and tNA
Total RNA was prepared using the RNeasy Protect Midi kit (QIAGEN, Hilden, Germany). tNA were prepared from tissue samples and hepatocyte culture dishes as previously described (26). The concentration of nucleic acids was measured spectrofotometrically and the DNA was quantified using a fluorometric assay (27).

Whole cellular and nuclear protein extraction
Cultured hepatocytes were harvested and incubated for up to 1 h on ice in PBS containing 5 mM EDTA to dissolve the matrigel. For extraction of whole cellular proteins, cells from two dishes were pooled and pelleted by centrifugation for 5 min at 500 x g, suspended in 400 µl lysis buffer A [10 mM HEPES (pH 7.9), 1.5 mM MgCl2, 10 mM KCl, 1% Triton X-100, 1 mM dithiothreitol, 100 µM Na3VO4, 10 mM NaF, 1 µg/ml pepstatin, 67 µg/ml phenylmethylsulfonyl fluoride] and one tablet Complete Mini protease inhibitor cocktail per 10 ml (Roche Diagnostics, Mannheim, Germany), incubated on ice for 15 min and passed eight times through a 27-gauge needle. The cell lysate was cleared by centrifugation for 20 min at 13,000 x g and aliquots stored at –135 C. For nuclear protein, extraction pelleted cells from three dishes were resuspended in 5 vol buffer A1 (buffer A without Triton X-100) and incubated on ice for 10 min. After centrifugation, the pellet was resuspended in 3 vol buffer A2 (A1 with 0.05% Nonidet P-40) and passed five times through a 27-gauge needle and centrifuged. The pellet was resuspended in 3 vol buffer C [10 mM HEPES (pH 7.9), 26% glycerol, 1.5 mM MgCl2, 0.2 mM EDTA and 420 mM NaCl] and incubated on ice for 30 min. After centrifugation at 24,000 x g for 30 min, the supernatant was collected as nuclear extract and aliquots were stored at –135 C. Liver nuclei were prepared by centrifugation of liver homogenates through buffered 2 M sucrose essentially as previously described (19), after which nuclear proteins were extracted in buffer C as above. Protein concentration was measured with the BCA protein assay kit from Pierce (Perbio Science, Cheshire, UK).

RPA (akr1b7 ribonuclease protection assay)
A PCR product amplified from the F18 clone (23), corresponding to akr1b7, was joined to a TOPO Tools T7 3'-element and T3 5'-element according to the manufacturer’s instruction (Invitrogen Life Technologies), except that a 1:1:1 molar ratio was used and the joining reaction was carried out for 15 min. Using this linear template, a biotin-labeled antisense probe corresponding to nucleotides 1107–1386 of the akr1b7 cDNA (5) was transcribed using T7 RNA polymerase and the Biotin RNA labeling mix from Roche. As an internal control, a glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) probe was transcribed from a StyI digested pTRI-GAPDH-Rat Antisense Control Template (Ambion, Austin, TX) using T3 RNA polymerase, resulting in a 134-bp protected fragment. The protection assays were performed with 0.5–5 µg of total RNA using the RPA III kit from Ambion, and Ambion’s Bright Star Bio Detection kit was used for detection of protected fragments.

Solution hybridization
The mRNA levels of CYP2C12, IGF-I, suppressor of cytokine signaling (SOCS)-2, cytokine-inducible Src homology (SH)-2-containing protein (CIS) and akr1b7 in tNA or RNA samples were analyzed in solution hybridization assays using specific [35S]uridine triphosphate-labeled cRNA probes essentially according to the method of Durnam and Palmiter (28). The probes and assay conditions for CYP2C12, IGF-I, SOCS-2, and CIS measurements have been described previously (29, 30, 31). The probe used for akr1b7 was transcribed from the pGEM3Z vector (Promega, South Hampton, UK) into which nucleotides 1254–1386 of the akr1b7 cDNA (5) was cloned at the EcoRI/HindIII polylinker sites. mRNA transcribed from the same vector construct served as standard for quantification. Assay conditions used for akr1b7 were 75 C and 25% formamide. Samples were analyzed in triplicate and the results are expressed as attomoles of the specific mRNA per microgram of DNA or RNA.

Western blotting
Samples (5 µg protein) were separated on a 10% SDS-PAGE gel and transferred to PVDF-Plus membrane (Osmonics Inc., Westborough, MA) by semidry blotting. The membrane was blocked overnight in PBS containing 0.1% Tween 20 (PBST) and 5% fat-free milk, incubated for 1 h with rabbit anti CYP3A1 (Human Biologics, Phoenix, AZ) diluted 1:1000 in PBST containing 5% milk. The membrane was washed and exposed for 1 h to horseradish peroxidase-conjugated goat antirabbit IgG diluted 1:2000 in PBST/5% milk. Specific antibody signals were visualized on x-ray film using the ECL Western blotting analysis system (Amersham Biosciences, Uppsala, Sweden). The membrane was reprobed with anti-ß-actin (A5316 Sigma) diluted 1:10 000 in PBST/5% milk.

EMSA
Double-stranded oligonucleotides were end-labeled with [{gamma}-32P]ATP (Amersham) using T4 polynucleotide kinase and purified on a nondenaturing 12% polyacrylamide gel. Oligonucleotides used were: activator protein (AP)-1, 5'-TTTTCCTTGTCTCAAACTGCT-3' and nuclear factor (NF)-{kappa}B, 5'-AGAAACAGGGAGTTTCCCCTTG-3' (sense strands with consensus sites in bold) corresponding to –136 to –116 and –3760 to –3739, respectively, in the akr1b7 gene with –1 according to Val et al. (5). Nuclear extracts (7 µg) were incubated with 1 µg poly(deoxyinosine-deoxycytosine) in binding buffer [100 mM NaCl, 10 mM HEPES (pH 7.9), 2 mM EDTA and 6% glycerol]. For competition and supershift assays, unlabeled probe and antibody were added 10 and 40 min, respectively, before addition of the labeled probe. After incubation with the labeled probe for 30 min at room temperature, DNA-protein complexes were resolved on nondenaturing 4% polyacrylamide gels in buffer containing 25 mM Tris-borate and 0.5 mM EDTA. Gels were dried on Whatman 3MM paper (Whatman Ltd., Maidstone, UK) and exposed to x-ray film. The c-fos antibody (sc-52x) was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).

Statistical analysis
Values are expressed as the mean ± SD. Comparisons between groups were made using one-way ANOVA followed by the Neuman-Keul’s test. Samples were considered significantly different at P < 0.05. In hepatocyte experiments, a minimum of three individual dishes per experimental point were used.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression of akr1b7 mRNA in rat liver and adrenals
Regulation of akr1b7 gene expression by trophic pituitary hormones has been demonstrated in adrenals, testis, and ovaries (6, 7). Here we show that GH regulates the expression in rat liver. Using RPA we could show that akr1b7 mRNA expression is sexually dimorphic, present in female rat livers and virtually absent in male livers (Fig. 1AGo). Feminization of the GH-plasma pattern in normal males by continuous GH infusion led to accumulation of akr1b7 mRNA. The lack of akr1b7 expression in livers from Hx males is consistent with the female-characteristic continuous GH pattern being the inducer rather than the male-characteristic intermittent GH pattern causing down-regulation. In accordance with other studies (3), no sex difference in adrenal akr1b7 mRNA levels was observed (Fig. 1BGo). The adrenal expression was not affected by feminization of the GH-plasma pattern in male rats but as expected, Hx, because of the loss of ACTH, substantially decreased the adrenal expression.



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  		FIG. 1. GH regulates akr1b7 mRNA expression in rat liver but not in adrenals. Hepatic (A) and adrenal (B) akr1b7 mRNA expression in rats with different GH status was assessed by RPA. Total RNA, 5 µg, from individual livers (n = 3) or 0.5 µg of pooled adrenal samples were analyzed. F, Normal female; M, normal male; fM, feminized male, i.e. normal males administered GH continuously (0.5 mg/kg·d) for 6 d; HxM, Hx male; Bg, background; Inp, input. GAPDH served as an internal standard.

 
Quantification of akr1b7 mRNA expression using solution hybridization confirmed the GH pattern-dependent regulation in the liver (Fig. 2AGo). The hepatic akr1b7 mRNA level in GH-feminized male rats reached 35% of that in normal females. Continuous GH treatment of Hx female rats induced akr1b7 mRNA to 10% of the level in normal females and upon substitution of Hx females with T4 and glucocorticoids the attained GH-induced level was 20% of the normal female level. The potentiation of the GH effect by the substitution therapy was significant, P = 0.01. The prototypical example of a rat hepatic gene specifically regulated by the female-specific pattern of GH is the cytochrome P450 gene CYP2C12 (18, 19). In this set of animals, the expression of CYP2C12 mRNA in feminized males and Hx females continuously treated with GH was 35% and 16% (data not shown), respectively, of that in the normal females. This was lower than in previous studies. We have no explanation for this low degree of refeminization but the same magnitude of response to the GH treatment of akr1b7 as of CYP2C12 corroborates that continuous GH exposure is a major regulator of rat liver akr1b7 expression.



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  		FIG. 2. Quantification of akr1b7 mRNA expression in liver and adrenal samples from rats with different hormonal status. Liver tNA (A) and adrenal RNA (B) samples were analyzed for akr1b7 mRNA content by solution hybridization. Values are expressed as attomoles of akr1b7 mRNA per microgram of DNA for liver samples or as attomoles akr1b7 mRNA per microgram of RNA for adrenal samples and are the mean ± SD of three to seven rats per group. F, Normal female; M, normal male; fM, feminized male (see Fig. 1Go); HxF, Hx female; HxM, Hx male. One set of Hx animals received substitution treatment with cortisol phosphate, 400 µg/kg·d (C), and 10 µg/kg·d T4. GH (0.5 mg/kg·d) was administered continuously for 6 d. nd, Not detectable.

 
The mRNA levels of IGF-I, another GH target gene that is induced by both the male and female pattern of GH secretion (32), were similar in normal female and male rats and in continuously GH-treated males (185 ± 31, 221 ± 22, and 193 ± 30 amol/µg DNA, respectively). This confirmed that the effect of the low dose of continuous GH treatment (0.5 mg/kg·d for 6 d) was in fact a feminization of the GH pattern and not an effect of total GH exposure.

As shown in Figs. 1BGo and 2BGo, similar levels of akr1b7 mRNA were expressed in the adrenals in normal male and female rats and in feminized male rats. In Hx rats, untreated or treated with GH and/or T4 and glucocorticoid, the adrenal akr1b7 mRNA level was about 24% of that in animals with an intact pituitary (Fig. 2BGo). Comparison of the level of akr1b7 mRNA expression in adrenal and liver in female rats revealed about 7-fold higher levels of expression in the adrenal than in the liver, 548 ± 115 and 76 ± 13 amol/µg RNA, respectively.

GH-dependent expression of akr1b7 in primary cultures of rat hepatocytes
Primary hepatocytes treated with GH for 18 h showed a dose-dependent accumulation of akr1b7 mRNA up to 2.5 nM of GH, thereafter the response leveled out (Fig. 3Go). This shows that the GH effect observed in vivo is exerted directly on the hepatocyte. Noteworthy, there was a marked difference in the absolute amount of accumulated akr1b7 mRNA in cells derived from normal female, normal male or GH-treated males despite the fact that no basal expression was detected in any cells cultured without GH. In subsequent experiments, cells from female rats were used. Time-course experiments showed a linear increase of akr1b7 mRNA from 6–32 h of GH treatment, the time period tested (data not shown).



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  		FIG. 3. Dose-dependent GH induction of akr1b7 mRNA expression in primary rat hepatocytes. Cells were isolated from normal female ({blacksquare}), normal male ({bullet}), or feminized male rats ({blacktriangleup}). At 48 h of culture age, the hepatocytes were treated for 18 h with varying concentrations of bGH in the presence of 0.5 nM insulin. tNA samples were analyzed for akr1b7 mRNA content by solution hybridization. Results are expressed as attomoles akr1b7 mRNA per microgram of DNA and values are the mean ± SD of triplicate dishes.

 
To examine whether the GH-induced expression of akr1b7 was sensitive to inhibition of ongoing protein synthesis, cells were treated with GH in the presence of CHX. As shown in Fig. 4Go, CHX at the concentration of 0.1 µg/ml reduced akr1b7 mRNA accumulation by 60%, at 0.5 µg/ml CHX the GH induction was completely abolished. CHX alone had no effect. The corresponding effect on GH induction of IGF-I mRNA was smaller; the accumulation was reduced by 40 and 75% at 0.1 and 0.5 µg/ml of CHX, respectively.



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  		FIG. 4. GH induction of akr1b7 mRNA in primary rat hepatocytes is sensitive to CHX. Female-derived hepatocytes cultured in the presence of 0.5 nM insulin were treated with 4.5 nM bGH for 18 h in the absence or presence of varying concentrations of CHX. tNA samples were analyzed for akr1b7 (open bars) and IGF-I mRNA (gray bars) content by solution hybridization assays. Results are expressed as percent induction with the level in the presence of only GH set to 100%. Values are the mean ± SD of at least triplicate dishes. *, Significantly different from cells treated with GH (P < 0.05), one-way ANOVA followed by Newman Keul’s test. nd, Not detectable.

 
Dex and thyroid hormone exert opposite effects on GH-induced akr1b7 expression
It is well documented that a differentiated hepatic phenotype of cultured hepatocytes is maintained by the use of an extracellular matrix such as the matrigel used in this study (25, 33, 34). Dex and T3 are commonly included in hepatocyte culture medium and recently a critical role for Dex in cooperation with extracellular matrix was demonstrated for phenotypical stability of cultured hepatocytes, including expression of liver-specific transcription factors (35). Moreover, substitution treatment of Hx rats with thyroid and glucocorticoid hormones potentiated the GH induction of akr1b7. We therefore examined the effect of Dex and T3 on the expression of akr1b7 in cultured hepatocytes. Treatment with T3 (0.1 nM–1 µM) or Dex (1 nM–1 µM) alone had no effect (data not shown). Both hormones affected the GH induction dose dependently but in opposite directions (Fig. 5Go). Although T3 augmented the GH response, Dex attenuated it; in fact, at 10 nM Dex the GH response was completely abolished. Consistent with previous observations (26), the GH induction of CYP2C12 mRNA was also reduced by Dex treatment, whereas IGF-I mRNA expression was either not affected or augmented by Dex (Fig. 5BGo). This indicates that Dex treatment interferes with mechanisms specific for gene expression dependent on continuous GH exposure.



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  		FIG. 5. Dose-dependent effects of Dex and T3 on GH-induced gene expression in primary rat hepatocytes. Female-derived hepatocytes cultured in the presence of 0.5 nM insulin were treated with 4.5 nM bGH for 18 h in the absence or presence of T3 (A) or Dex (B). The mRNA levels of akr1b7 (open bars; A and B), IGF-I (B, dark gray bars) and CYP2C12 (B, light gray bars) were analyzed by solution hybridization assays. Results are expressed as percent induction with the level in the presence of only GH set to 100%. Values are the mean ± SD of at least three independent dishes. *, Significantly different from cells treated with GH (P < 0.05), one-way ANOVA followed by Newman Keul’s test. nd, Not detectable.

 
Insulin modulates GH induction of akr1b7
In addition to Dex and T3, insulin is also suggested to contribute to hepatocyte morphological integrity in culture and can affect GH responsiveness in hepatocytes and hepatic cell lines (26, 34, 36). The experiments described above were carried out with 0.5 nM insulin in the medium, and next we investigated the effect of varying concentrations of insulin on the GH induction of akr1b7 mRNA. Induction was obtained both in the absence and presence of insulin (0–100 nM), however, higher levels of akr1b7 mRNA were attained in the presence of insulin (Fig. 6Go). At physiologically relevant concentrations of insulin, 0.5–5 nM, enhanced induction was significant at 2.5 and 5 nM. No effect was observed by insulin alone. In subsequent experiments, cells were cultured in the presence of 2.5 nM insulin.



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  		FIG. 6. Insulin modulates GH-induced akr1b7 mRNA expression in primary rat hepatocytes. Female-derived hepatocytes were treated with 4.5 nM bGH for 18 h in the presence of varying concentrations of insulin. tNA samples were analyzed for akr1b7 mRNA content by solution hybridization. Results are expressed as attomoles akr1b7 mRNA per microgram of DNA and values are the mean ± SD of at least triplicate dishes. *, Significantly different from cells treated with GH in the absence of insulin (P < 0.05), one-way ANOVA followed by Newman Keul’s test.

 
GH-induced expression of akr1b7 mRNA in hepatocytes is inhibited by the LXR agonist T1317
The LXR regulation of the akr1b7 gene demonstrated in mouse intestine (8), also indicated to occur in mouse liver (9), prompted us to treat hepatocytes with increasing concentrations of the LXR agonist T1317 in the absence or presence of GH. T1317 had no effect on its own (data not shown) but dose-dependently reduced the GH induction of akr1b7 mRNA (Fig. 7Go). GH-stimulated CYP2C12 mRNA expression, but not IGF-I mRNA expression, was reduced by T1317 but to a lesser extent than the akr1b7 expression.



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  		FIG. 7. Effects of LXR-ligand treatment on GH-induced gene expression in primary rat hepatocytes. Female-derived hepatocytes cultured in the presence of 2.5 nM insulin were treated for 18 h with 4.5 nM bGH in the absence or presence of varying concentrations of T1317. The mRNA levels of akr1b7 (open bars), IGF-I (dark gray bars) and CYP2C12 (light gray bars) were analyzed by solution hybridization. Results are expressed as percent induction with the level in the presence of only GH set to 100%. Values are the mean ± SD of at least three independent dishes. *, Significantly different from cells treated with GH (P < 0.05), one-way ANOVA followed by Newman Keul’s test.

 
LXRs and most nuclear receptors for nonsteroidal ligands form obligate heterodimeric complexes with retinoid X receptors (RXRs) (37), and we next examined the effect of the RXR agonist 9-cis RA. Concentrations of 9-cis RA from 10 nM to 1 µM did not mitigate the GH induction of akr1b7, only at 10 µM was a reduction of the GH response observed (Fig. 8Go) and then only by 25%. In comparison, this level of reduced GH response was obtained with concentrations of T1317 of 50 nM. These results are in line with the major effect of T1317 being elicited by LXRs but do not exclude involvement of other RXR partners.



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  		FIG. 8. Effect of 9-cis RA on GH-induced akr1b7 mRNA levels in primary rat hepatocytes. Female-derived hepatocytes cultured in the presence of 2.5 nM insulin were treated with 4.5 nM bGH for 18 h in the absence or presence of varying concentrations of the RXR-agonist 9-cis RA. tNA samples were analyzed for akr1b7 mRNA content by solution hybridization. Results are expressed as percent induction with the level in the absence of 9-cis RA set to 100%. Values are the mean ± SD of at least three independent dishes. *, Significantly different from cells treated with GH (P < 0.05), one-way ANOVA followed by Newman Keul’s test.

 
During the course of this study, it became evident that T1317 is capable of activating pregnane X receptors (PXR) in rodent hepatocytes (38). Furthermore, Dex, which even more efficiently than T1317 attenuated the GH induction of akr1b7, can also activate PXR. This prompted us to investigate the effect of the PXR ligand PCN on akr1b7 mRNA expression in the absence or presence of GH. PCN at concentrations of 10 nM to 10 µM did not abrogate the GH induction of akr1b7 (Fig. 9Go) and had no effect on its own (data not shown). PCN and Dex, via activation of PXR, are known inducers of CYP3A1/2 (39, 40) and at 10 µM both ligands produced a pronounced increase of immunoreactive CYP3A1/2 amount (Fig. 10Go). However, at 10 nM Dex, which completely blunted the GH induction of akr1b7, no induction of CYP3A1/2 was seen. Treatment of the cells with 500 nM T1317 that reduced the GH induction of akr1b7 by 70% led to a modest induction of CYP3A1/2 amounts. Thus, the lack of effect of PCN on GH induction of akr1b7 but efficient induction of CYP3A1/2 together with the pronounced effect of T1317 on GH induction of akr1b7 and modest effect on CYP3A1/2 suggest that the T1317 perturbation of the GH response is not mediated via PXR.



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  		FIG. 9. Effect of PCN on GH-induced akr1b7 mRNA expression in primary rat hepatocytes. Female-derived hepatocytes cultured in the presence of 2.5 nM insulin were treated with 4.5 nM bGH for 18 h in the absence or presence of the PXR-agonist PCN. tNA samples were analyzed for akr1b7 mRNA content by solution hybridization. Results are expressed as percent induction with the level in the absence of PCN set to 100%. Values are the mean ± SD of at least three independent dishes.

 


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  		FIG. 10. Analysis of CYP3A protein levels in primary rat hepatocytes treated with LXR, GR, or PXR ligands. Female-derived primary rat hepatocytes cultured in the presence of 2.5 nM insulin were treated with 0.5 µM T1317, 0.01 µM or 10 µM Dex, or 10 µM PCN for 18 h. Control cells received vehicle, 0.1% DMSO. Cellular extracts were prepared and 5 µg of protein was subjected to SDS-PAGE and Western blot analysis using an antibody recognizing CYP3A1/2. The filter was reprobed with an antibody against ß-actin as a loading control.

 
Mechanisms of GR- and LXR-mediated inhibition of GH-stimulated akr1b7 expression
In attempts to elucidate the mechanisms of the repressive effects of LXR and GR activation, we first measured SOCS-2 and CIS mRNA levels. SOCS and CIS proteins constitute components of a negative feedback loop of GH signaling (41), and it has been demonstrated that 100 nM Dex increases the GH induction of CIS and SOCS-2 mRNA in primary rat hepatocytes cultured essentially as in this study (31). We found that GH-induced CIS and SOCS-2 mRNA levels were differently affected by 10 nM Dex, whereas CIS levels were increased SOCS-2 levels were decreased (Fig. 11Go). T1317 also decreased the GH effect on SOCS-2 mRNA but did not significantly affect CIS mRNA levels. The SOCS-2 and CIS mRNA levels in cells treated with T1317 or Dex alone were not different from those in control cells (data not shown).



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  		FIG. 11. SOCS-2 and CIS mRNA levels in primary rat hepatocytes in response to GH, Dex, and T1317. Female-derived primary hepatocytes cultured in the presence of 2.5 nM insulin were treated for 18 h with 4.5 nM bGH and/or 500 nM T1317 or 10 nM Dex. tNA samples were analyzed for SOCS-2 (light gray bars) and CIS (dark gray bars) mRNA contents by solution hybridization assays. Results are expressed as percent with the levels in the presence of GH alone set to 100%. Values are the mean ± SD of three independent dishes. *, Significantly different from cells treated with GH (P < 0.05), one-way ANOVA followed by Newman Keul’s test.

 
Mechanisms for negative GR effects on transcription include inhibition of AP-1 and NF-{kappa}B and GH has the ability to activate both pathways (42, 43). Furthermore, LXR-dependent repression of cytokine induction of the matrix metalloproteinase-9 gene involves perturbation of NF-{kappa}B signaling (44). Using EMSA, we analyzed binding to DNA sequences in the akr1b7 promoter harboring consensus sites for AP-1 or NF-{kappa}B. Nuclear extracts from hepatocytes untreated or treated for 30 min or 3 h with GH in the absence or presence of Dex or T1317 were used. Binding to both sites was slightly stimulated using extracts from cells treated with GH for 30 min compared with extracts from control cells. (Fig. 12Go). Complexes specifically competed for by excess unlabeled probe are marked with arrowheads. The complex formed on the AP-1 probe was attenuated by including an c-fos antibody in the EMSA reaction, verifying AP-1 binding. Neither Dex nor T1317 affected GH-stimulated binding to the AP-1 site (Fig. 12AGo, compare lanes 2, 4, and 6). We observed a slight reduction in GH-stimulated binding activity to the NF-{kappa}B probe upon cotreatment of the cells with Dex but not with T1317 (Fig. 12BGo, lanes 3, 7, and 5). After 3 h of cell treatment, binding activities were similar in all extracts. For comparative purposes, we also analyzed binding of nuclear extracts from normal female and Hx female rats (lanes 13 and 14 in Fig. 12AGo and lanes 14 and 15 in Fig. 12BGo). Complexes with the same migration were formed with nuclear extracts from hepatocytes and liver on both probes. With liver nuclear extracts, the specific complexes were more prominent using extracts from intact female rats than from Hx female rats, demonstrating a pituitary-dependent binding.



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  		FIG. 12. Binding of hepatocyte nuclear proteins to akr1b7 DNA-sequences harboring consensus motif for AP-1 (A) or NF-{kappa}B (B). EMSA reactions were carried out with nuclear extracts prepared from female-derived primary hepatocytes cultured with 2.5 nM insulin and treated for 30 min or 3 h with 4.5 nM bGH and/or 500 nM T1317 or 10 nM Dex as indicated in the figure, control cells (Ctrl) received vehicle. Nuclear extracts (7 µg) from normal female (F) and Hx female rat livers were also included in the analyses. An antibody directed against c-fos (panel A, lane 17), was incubated with the nuclear extract before addition of the probe. Addition of 100-fold molar excess of unlabeled probes competed for the formation of the complexes marked with arrowheads [panel A, data not shown; panel B, (100x comp)].

 
Next, we examined the GH induction of akr1b7 mRNA in the presence of PDTC, an inhibitor of NF-{kappa}B signaling (45). The doses used have been shown to inhibit NF-{kappa}B nuclear translocation in cultured mouse hepatocytes (46). As shown in Fig. 13Go, PDTC did not inhibit the GH-stimulated akr1b7 mRNA expression, on the contrary, the expression was increased. We did not detect any akr1b7 mRNA in the presence of PDTC alone (data not shown). To this end, we have no evidence that GR- and LXR-mediated inhibition of GH-stimulated akr1b7 expression is because of perturbation of AP-1 or NF-{kappa}B signaling.



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  		FIG. 13. Effects of the NF-{kappa}B inhibitor PDTC on GH-stimulated induction of akr1b7 mRNA levels in primary rat hepatocytes. Female derived primary hepatocytes were cultured in the presence of 2.5 nM insulin and treated with 4.5 nM bGH for 18 h in the absence or presence of varying concentrations of PDTC. tNA samples were analyzed for akr1b7 mRNA content by solution hybridization. Results are expressed as percent induction with the level in the absence of PDTC set to 100%. Values are the mean ± SD of three independent dishes. *, Significantly different from cells treated with GH alone (P < 0.05), one-way ANOVA followed by Newman Keul’s test.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The number of rodent hepatic genes shown to be regulated by the sex-specific temporal pattern of GH secretion increases continuously (23, 47, 48), and in this study we have shown that akr1b7 belongs to this group of genes and that its expression is dependent on the female-specific continuous presence of GH. The approximately 7-fold lower expression of akr1b7 mRNA in the liver compared with the adrenal is consistent with the liver being a nonsteroidogenic tissue and thereby not active in producing the substrate isocaproaldehyde. The female-specific liver expression may relate to the stimulatory effect of continuous GH exposure on hepatic lipogenic enzymes (47, 48). Peroxidation of {omega}-6 lipids such as arachidonic acid yields 4-HNE, also a substrate of AKR1B7, and interestingly, we have previously found that continuous GH exposure of hepatocytes stimulates cytosolic phospholipase A2 and the release of arachidonic acid (49). Thus, a concomitant GH-induced expression of akr1b7 with the GH-stimulated release of arachidonic acid may constitute a protective mechanism against cytotoxic 4-HNE that eventually can be formed. In mouse liver in which GH- and sex-dependent gene expression also is pronounced, the expression of akr1b7 has been reported to be nonsexually dimorphic (3). Hepatic akr1b7 expression was, however, not the major subject of that study and further investigations are required to ascertain whether the sex-specific GH regulation of hepatic akr1b7 is unique to the rat. We found no evidence for a sex-differentiated adrenal expression of akr1b7 mRNA, which is consistent with observations in the mouse (3).

Our data using primary hepatocytes demonstrate that GH exerts a direct effect on the hepatocyte in inducing akr1b7 expression and that the GH induction occurs without any other hormonal addition to the medium. Furthermore, the dose-dependent sensitivity to CHX may indicate that the induction is dependent on de novo protein synthesis. However, nonspecific effects of CHX and other inhibitors of protein synthesis have been demonstrated in studies on phenobarbital-induced CYP gene expression in primary rat hepatocytes (50) and cannot be excluded. When GH-responsive cis-acting elements in the akr1b7 gene have been identified, the CHX effect is worth reassessing.

Systems in which a differentiated state of primary hepatocytes in culture can be maintained are particularly valuable for the elucidation of cellular and molecular mechanisms of liver biology. There are almost as many ways to maintain cultured hepatocytes differentiated for a specific purpose as there are laboratories performing the studies, but consensus regarding the importance of extracellular matrix prevails (25, 33, 34). A thorough investigation carried out by the Omiecinski group (35) clearly shows that morphological integrity of cultured rat hepatocytes and the phenotypic expression of the liver-enriched transcription factors CCAAT/enhancer binding protein {alpha}, HNF-1{alpha}, -3{alpha}, and -4{alpha}, and RXR{alpha} require the presence of nanomolar concentrations of Dex in addition to extracellular matrix. Therefore, the totally blunted GH response of akr1b7 in the presence of 10 nM Dex was not expected. However, we have previously observed an inhibitory effect of glucocorticoids on the GH-induced expression of the CYP2C12 gene (26), which was verified in this study. In contrast, the GH induction of IGF-I mRNA was significantly stimulated at 1 nM Dex, also consistent with previous observations (26). Of note is that the IGF-I gene is induced by both intermittent and continuous patterns of GH exposure, whereas akr1b7 and CYP2C12 expression is restricted to continuous exposure. Whether Dex activation of GR specifically interferes with mechanisms solely activated by continuous GH or if these two target genes display a specific setting of GH response elements is an intriguing question. In GR-deficient mice, GH activation of the IGF-I gene mediated by the transcription factor Stat5 is impaired (51). This and other studies support the model of GR acting as a coactivator for Stat5 (52, 53). Characterization of the GH transcriptional regulation of the CYP2C12 gene by in vivo transfection has revealed a cooperative effect of Stat5, HNF-4, HNF-6, and one or more unknown factors, possibly cAMP response element binding protein (CREB) (22). Apparently, GR is not an obligatory coactivator of Stat5 and could possibly act as a repressor if GR/Stat5 interaction prevents Stat5 from cross talk with other transcription factors such as HNF-4 or HNF-6. Such a mechanism could apply for the Dex effect on the GH regulation of CYP2C12 and akr1b7. It is evident that glucocorticoids exert dose- and time-dependent effects on GH responsiveness (54, 55). Because the IGF-I response was not reduced by the used doses of Dex, we do not interpret our results in terms of reduced GH receptor expression or GH binding. In this respect, it should be mentioned that the EC50 values for akr1b7 and IGF-I induction by GH were similar, whereas the EC50 value for the induction of CYP2C12 was 2-fold lower (data not shown and Ref.24).

As mentioned above, cis-acting elements and trans-acting factors conveying GH regulation of the CYP2C12 gene have been identified; however, the dependency on continuous GH has not been possible to explain in terms of these factors because the analyzed constructs were expressed in both male and female rats (22). One possibility could be that epigenetic mechanisms, not revealed by transfection experiments, are involved in the transcriptional regulation of genes by continuous GH. The different levels of akr1b7 induced in hepatocytes isolated from rats with different GH status (Fig. 3Go) would be consistent with involvement of such long-lasting effects.

In contrast with the negative effect of Dex on GH-stimulated akr1b7 expression, including insulin or T3 in the hepatocyte culture medium augmented the GH response. An impact of insulin on both hepatocyte differentiation and on hepatic GH receptors is apparent from several studies (26, 34). Furthermore, GH binding to rat liver membranes has been shown to be directly related to the thyroid status of the animals (56). Thus, it is reasonable to suggest that the effects of insulin and T3 on GH-stimulated akr1b7 expression relate to an increased interaction of GH with its receptor.

GH stimulation of the IGF-I gene is conveyed by activation of the Jak2/Stat5 signaling pathway (57). Because IGF-I induction was not mitigated by GR or LXR activation, it is unlikely that this pathway was repressed. The inhibitory effect of SOCS/CIS proteins on GH signaling via the Jak2/Stat5 pathway has been shown to involve SOCS-1 and -3 but not, or to a lesser extent, SOCS-2 and CIS (41, 58). Nevertheless, as shown in this and previous studies, GH induces both SOCS-2 and CIS in rat hepatocytes (31, 59), and it is reasonable to assume that they could be involved in mitigation of other GH signaling pathways than the Jak2/Stat5 pathway. Because GH stimulation of SOCS-2 mRNA was reduced in the presence of Dex or T1317, this factor is not implicated as a mediator of the inhibition of akr1b7 expression. The discrepancy of the effect of Dex on GH-stimulated SOCS-2 mRNA expression between our study and the study by Tollet-Egnell et al. (31) in which SOCS-2 mRNA expression was increased could relate to that different doses of Dex were used, 10 nM and 100 nM, respectively. The 2-fold increase in GH-stimulated CIS mRNA expression by Dex, but not by T1317, could possibly suggest an involvement of CIS in the GR-mediated inhibition of GH-stimulated akr1b7 expression and would then constitute a distinct mechanism elicited by GR activation.

Because recent studies have identified LXR as a positive regulator of mouse akr1b7 expression (8, 9), we were surprised to find that activation of LXR by T1317 markedly reduced the GH induction of akr1b7 in rat hepatocytes. However, of note is the observation that lxr-deficient mice express elevated amounts of AKR1B7 in duodenum (8). One of the three LXR response elements (LXRE) identified in the proximal promoter of the mouse akr1b7 gene, LXRE3, is fully conserved in the rat, the LXRE1 and LXRE2 sequences show two and three mismatches, respectively. Protein binding to these elements in relation to GH status of the animal and LXR-activation will be of interest to investigate. However, the similar negative effect of GR or LXR activation on akr1b7 and CYP2C12 induction by GH may indicate that GH signaling pathway(s) and not nuclear receptor DNA binding is the mechanism of the inhibition. LXR-dependent repression of cytokine-induced expression of the matrix metalloproteinase-9 gene has been demonstrated in macrophages, and this antagonism is accomplished through inhibition of the NF-{kappa}B signaling rather than by direct binding of LXR/RXR to LXREs (44). Furthermore, repressive actions on immune target genes by GR activation are well documented, and the mechanisms include inhibition of the transcription factors NF-{kappa}B, AP-1, or CREB (Ref.60 and references therein). Our results, EMSA and pharmacological intervention, do not support that GR- and LXR-mediated inhibition of GH-stimulated akr1b7 expression is because of perturbation of NF-{kappa}B signaling. Further experiments on AP-1 as a possible target for the repression will be required, particularly because altered transcriptional activity because of posttranslational modifications of transcription factors or tethering mechanisms is not necessarily revealed by EMSA.

The mechanisms of nuclear receptor action involve ligand-dependent recruitment of coactivators that act in concert with yet other coregulators to modulate gene transcription (Ref.61 and references therein). Efficient recruitment of the coactivator CREB binding protein (CBP/p300) upon LXR activation by T1317 occurs in HepG2 cells (62). CBP has intrinsic acetyl transferase activity and has been shown to enhance the activity of the transcription factors Stat5 and HNF-4, which are implicated in the GH induction of CYP2C12 (22, 63, 64). Moreover, liganded GR can act both as a direct inhibitor of CBP-associated histone acetylase activity and by recruitment of a histone deacetylase complex (65). Thus, both competition for limited amounts of coregulators, such as CBP, and/or an altered balance of acetylation/deacetylation of histones are repressive mechanisms worth pursuing in future experiments on GR- and LXR-mediated inhibition of GH-stimulated akr1b7 expression.

In summary, our results show that rat liver akr1b7 expression is up-regulated by the female-characteristic pattern of GH secretion, and we have found that activation of GR or LXR represses GH-induced akr1b7 and CYP2C12 expression in primary rat hepatocytes. GH induction of AKR1B7, a lipid peroxide detoxifying enzyme, could be one explanation for the lower incidence of hepatocellular carcinomas in female than in male rodents (66, 67). Furthermore, a cross talk between GH receptor and LXR signaling is of interest from a metabolic point of view in that activation of either receptor has major impact on lipid metabolism.


   Acknowledgments
 
We thank P. Tollet-Egnell for the gift of SOCS-2 and CIS plasmids.


   Footnotes
 
This work was supported by grants from the Swedish Research Council (13146), Center for Gender Related Research, Karolinska Institutet and Karolinska Institutet funds.

Abbreviations: AKR, Aldo-keto reductase; AP, activator protein; bGH, bovine GH; CBP, CREB binding protein; 9-cis RA, 9-cis retinoic acid; CHX, cycloheximide; CIS, cytokine-inducible Src homology 2-containing protein; CREB, cAMP response element binding protein; CYP, cytochrome P450; Dex, dexamethasone; DMSO, dimethyl sulfoxide; GAPDH, glyceraldehyde-3-phosphate-dehydrogenase; GR, glucocorticoid receptor; 4-HNE, 4-hydroxynonenal; HNF, hepatic nuclear factor; Hx, hypophysectomy/hypophysectomized; Jak, Janus kinase; LXR, liver X receptors; LXRE, LXR response element; NF, nuclear factor; PBST, PBS containing 0.1% Tween 20; PCN, pregnenolone 16{alpha}-carbonitrile; PDTC, ammonium pyrolidinedithiocarbamate; PXR, pregnane X receptor; RPA, akr1b7 ribonuclease protection assay; RXR, retinoid X receptor; SOCS, suppressor of cytokine signaling; Stat5, signal transducer and activator of transcription 5; tNA, total nucleic acids.

Received April 30, 2004.

Accepted for publication September 1, 2004.


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