Transfection of adult primary rat hepatocytes in culture.

The use of adult primary hepatocytes in culture is of importance for the understanding of hepatic processes at the cellular and molecular levels, and the possibility to employ transient transfection of reporter constructs is invaluable for mechanistic studies on hepatic gene regulation. Although frequently used, there is a lack of reports addressing optimization and characterization of transfection of primary rodent hepatocytes. Here, we have shown that the efficiency of biochemical transfection reagents varies significantly and that Lipofectamine2000 was a superior transfection reagent for adult primary rat hepatocytes when using luciferase reporter vectors. The efficiency increased when the cells were allowed ample time to adapt to the in vitro milieu. Cotransfection of a second reporter gene indicated a risk for promoter competition, and we found that relating reporter activity to total cellular protein content gave consistent and reliable results. Differentiation of the cells, achieved by including biomatrix from the Engelbreth-Holm-Swarm mouse sarcoma in the culture system, was to a larger extent required for hormonal/drug responses of transfected constructs than for responses of endogenous genes and assured responses of transfected constructs. Dexamethasone (Dex) is most often included in hepatocyte culture media, but we could not demonstrate a general beneficial effect of Dex on expression of luciferease reporter contructs. Using the established protocol, we have demonstrated responses of transfected constructs to growth hormone, glucocorticoid and LXR stimuli.

Cloning of a novel growth hormone-regulated rat complementary deoxyribonucleic acid with homology to the human alpha1B-glycoprotein, characterizing a new protein family.

A sex-specific secretion of GH prevails in the rat. This has bearings on the expression of target genes, particularly in the liver. We have used suppressive subtractive hybridization to search for genes expressed in response to the female-characteristic, near-continuous secretion of GH. One sequence was particularly abundant among the obtained clones. After isolation of the corresponding full-length complementary DNA using rapid amplification of complementary DNA ends, it was found to be homologous to the human alpha1B-glycoprotein. Sequence comparisons suggest that the human alpha1B-glycoprotein and the rat homolog are members of a new family of proteins, of which at least four additional forms were found in the databases of human and mouse expressed sequence tags. In situ hybridization confirmed the female-specific expression, and by RNase protection analysis a liver-specific expression was indicated. Up-regulation of the messenger RNA by continuous exposure to GH, but not to the male-characteristic intermittent exposure, was demonstrated in hypophysectomized rats and in cultured primary hepatocytes.

Pulsatile growth hormone secretion decreases S-adenosylmethionine synthetase in rat liver.

S-adenosylmethionine synthetase (AdoMet synthetase) is responsible for the synthesis of the major methyl donor S-adenosylmethionine. The AdoMet synthetase gene was identified by subtractive suppressive hybridization as being expressed at higher levels in the liver of rats continuously exposed to growth hormone (GH) than in rats intermittently exposed to the hormone. Further studies on the regulation of AdoMet synthetase showed that the activity and mRNA levels were higher in female than in male rats. Hypophysectomy increased AdoMet synthetase mRNA in both male and female rats. Combined thyroxine and cortisol treatment of hypophysectomized rats had no effect on AdoMet synthetase mRNA levels. Two daily injections of GH for 7 days, mimicking the male secretory pattern of GH, decreased AdoMet synthetase activity and mRNA levels. A continuous infusion of GH, mimicking the female secretory pattern of GH, had small or no effects on AdoMet synthetase activity and decreased the mRNA levels to a lesser degree than two daily injections. It is concluded that the lower AdoMet synthetase activity in male rats is due to an inhibitory effect of the male characteristic pulsatile secretory pattern of GH on AdoMet synthetase mRNA expression.

Cis desensitizes GH induced Stat5 signaling in rat liver cells.

Recently a novel family of proteins, the Cis/Socs family, has been shown to constitute negative regulators of cytokine-induced Jak/Stat signaling. Here we demonstrate that Socs-2 and Cis mRNA expression in rat liver is dependent on the presence of growth hormone (GH), and that GH induce Cis mRNA expression in cultures of primary rat hepatocytes. Furthermore, cotransfection studies in the rat liver cell line, BRL-4, revealed that constitutive expression of Cis, but not Socs-2, inhibited the GH-induced transactivation of a Stat5-responsive reporter gene construct. This indicates a functional role for Cis in the desensitization of GH activated Jak/Stat5 signaling in rat liver cells. In response to the intermittent pattern of GH secretion in male rats, GH activates Stat5b signaling whereas this activation is blunted in female rats having a continuous pattern of GH secretion. We hypothesize that GH induction of Cis could be one mechanism by which sexually dimorphic GH signaling via Stat5b is achieved in the rat liver.

IRE-ABP (insulin response element-A binding protein), an SRY-like protein, inhibits C/EBPalpha (CCAAT/enhancer-binding protein alpha)-stimulated expression of the sex-specific cytochrome P450 2C12 gene.

In primary hepatocytes, overexpression of an insulin response element-A binding protein (IRE-ABP), a member of the SRY family of high-mobility group (HMG) proteins, inhibits CCAAT/enhancer-binding protein alpha (C/EBPalpha)-mediated activation of the female-specific cytochrome P450 2C12 (CYP2C12) gene, but not the male-specific cytochrome P450 2C11 (CYP2C11) gene. IRE-ABP and C/EBPalpha have overlapping specificity for the C/EBPalpha target site in the CYP2C12 promoter and compete for binding to CYP2C12 DNA in vitro. In contrast, IRE-ABP and C/EBPalpha bind distinct sequences in the CYP2C11 promoter. A single amino acid substitution in the HMG domain of IRE-ABP impairs its ability to bind DNA and to inhibit the effect of C/EBPalpha on CYP2C12 gene expression. Therefore, the ability of IRE-ABP to inhibit C/EBPalpha-stimulated CYP2C12 gene expression requires a functional DNA-binding domain. Taken together, our findings suggest that SRY-like proteins can bind to a subset of sequences recognized by the C/EBP family of DNA-binding proteins and modulate gene transcription in a context-specific manner.

Down-regulation of cytochrome P450 2C family members and positive acute-phase response gene expression by peroxisome proliferator chemicals.

In this study, we show that peroxisome proliferator chemical (PPC) exposure leads to alterations in the expression of genes in rat liver regulated by the sex-specific growth hormone secretory pattern and induced during inflammation. Expression of the male-specific cytochrome P450 (P450) 2C11 and alpha2 urinary globulin (alpha2u) genes and the female-specific P450 2C12 gene was down-regulated by some PPC. Expression of P450 2C13, also under control by the sex-specific growth hormone secretory pattern, was not altered by PPC treatment, indicating that regulation of CYP2C family members does not involve perturbation of the growth hormone secretory pattern. In contrast to the increases in expression observed when inflammation was induced in male rats, two positive acute-phase response genes, alpha1-acid glycoprotein and beta-fibrinogen, were decreased by PPC exposure. The down-regulation of the P450 2C11 by WY-14,643 could be reproduced in cultured rat hepatocytes, indicating the down-regulation is a direct effect. Experiments in wild-type mice and mice that lacked a functional peroxisome proliferator-activated receptor-alpha gene showed that down-regulation by WY of alpha1-acid glycoprotein, beta-fibrinogen, and a mouse homologue of alpha2u was dependent on peroxisome proliferator-activated receptor-alpha expression. Our results demonstrate that PPC exposure leads to down-regulation of diverse liver-specific genes, including CYP2C family members important in hormonal homeostasis and acute-phase response genes important in inflammatory responses.

Desensitization of the growth hormone-induced Janus kinase 2 (Jak 2)/signal transducer and activator of transcription 5 (Stat5)-signaling pathway requires protein synthesis and phospholipase C.

Signal transducers and activators of transcription (Stat) proteins are latent cytoplasmic transcription factors that are tyrosine phosphorylated by Janus kinases (Jak) in response to GH and other cytokines. GH activates Stat5 by a mechanism that involves tyrosine phosphorylation and nuclear translocation. However, the mechanisms that turn off the GH-activated Jak2/Stat5 pathway are unknown. Continuous exposure to GH of BRL-4 cells, a rat hepatoma cell line stably transfected with rat GH receptor, induces a rapid but transient activation of Jak2 and Stat5. GH-induced Stat5 DNA-binding activity was detected after 2 min and reached a maximum at 10 min. Continued exposure to GH resulted in a desensitization characterized by 1) a rapid decrease in Stat5 DNA-binding activity. The rate of decrease of activity was rapid up to 1 h of GH treatment, and the remaining activity declined slowly thereafter. The activity of Stat5 present after 5 h is still higher than the control levels and almost 10-20% with respect to maximal activity at 10 min; and 2) the inability of further GH treatment to reinduce activation of Stat5. In contrast, with transient exposures of BRL-4 cells to GH, Stat5 DNA-binding activity could repeatedly be induced. GH-induced Jak2 and Stat5 activities were independent of ongoing protein synthesis. However, Jak2 tyrosine phosphorylation and Stat5 DNA-binding activity were prolonged for at least 4 h in the presence of cycloheximide, which suggests that the maintenance of desensitization requires ongoing protein synthesis. Furthermore, inhibition of protein synthesis potentiated GH-induced transcriptional activity in BRL-4 cells transiently transfected with SPIGLE1CAT, a reporter plasmid activated by Stat5. GH-induced Jak2 and Stat5 activation were not affected by D609 or mepacrine, both inhibitors of phospholipase C. However, in the presence of D609 and mepacrine, GH maintained prolonged Jak2 and Stat5 activation. Transactivation of SPIGLE1 by GH was potentiated by mepacrine and D609 but not by the phospholipase A2 inhibitor AACOCF3. Thus, a regulatory circuit of GH-induced transcription through the Jak2/Stat5-signaling pathway includes a prompt GH-induced activation of Jak2/Stat5 followed by a negative regulatory response; ongoing protein synthesis and intracellular signaling pathways, where phospholipase C activity is involved, play a critical role to desensitize the GH-activated Jak2/Stat5-signaling pathway.

Bile acid synthesis in primary cultures of rat and human hepatocytes.

The regulation of hepatic bile acid formation is incompletely understood. Primary cultures of mammalian hepatocytes offer an opportunity to examine putative regulatory factors in relative isolation. Using rat and human hepatocytes in primary culture, we examined bile acid composition and the expression of the rate-limiting enzyme of formation, cholesterol 7alpha-hydroxylase. Control rat hepatocytes showed a declining bile acid production over 4 days, from 156 +/- 24 ng/mL (67% cholic acid) on day 1 to 55 +/- 11 ng/mL (55% cholic acid) on day 4. In addition to cholic acid, chenodeoxycholic acid, alpha-muricholic acid, and beta-muricholic acid were formed. Treatment with triidothyronine (T3) or dexamethasone alone had no significant effect on bile acid production. A combination of T3 and dexamethasone significantly increased the total bile acid production on day 4 (224 +/- 54 ng/mL) and resulted in a marked change in composition to 23% cholic acid and 77% non-12alpha-hydroxylated bile acids. Control rat hepatocytes had a cholesterol 7alpha-hydroxylase activity of 3.3 +/- 0.6 pmol/mg protein/min after 4 days in culture. Cells treated with the combination of dexamethasone and T3 had an activity of 16.4 +/- 3.6 pmol/mg protein/min. The cholesterol 7alpha-hydroxylase messenger RNA (mRNA) levels, determined by solution hybridization after 4 days of culture, showed results similar to those for the activity data; control cells had 5.3 +/- 0.9 cpm/microg total nucleic acids (tNAs). T3 or dexamethasone-treated cells did not differ from control cells, whereas the combination of T3 and dexamethasone increased the mRNA levels to 20.6 +/- 2.8 cpm/microg tNAs. In human hepatocytes, isolated from donor liver, bile acid formation increased from 206 +/- 79 ng/mL on day 2 to 1490 +/- 594 ng/mL on day 6 and then declined slightly. Cholic acid and chenodeoxycholic acid were formed, constituting about 80% and 20%, respectively. The combined addition of T3 and dexamethasone had a tendency to decrease rather than increase bile acid formation. Also, mRNA levels of the cholesterol 7alpha-hydroxylase increased severalfold in the human hepatocytes from day 2 to day 4 and then declined. The addition of T3 or dexamethasone did not effect the mRNA levels in any consistent way. It is noteworthy that the capacity of the cultured human hepatocytes to produce bile acids was higher than that of cultured rat hepatocytes, in spite of the fact that the production of bile acids in rat liver is 3- to 5-fold higher than that in human liver in vivo. It is also evident that while hormonal factors appear to regulate bile acid synthesis in the rat, no evidence for this was found in human hepatocytes. As the composition of bile acids secreted by human hepatocytes in primary culture closely resembles that found in vivo, this represents a useful model for further studies of the synthesis and regulation of bile acids.

Expression of hepatocyte nuclear factor 6 in rat liver is sex-dependent and regulated by growth hormone.

Growth hormone (GH) binding to its receptor modulates gene transcription by influencing the amount or activity of transcription factors. In the rat, GH exerts sexually dimorphic effects on liver gene transcription through its pattern of secretion which is intermittent in males and continuous in females. The expression of the CYP2C12 gene coding for the female-specific cytochrome P450 2C12 protein is dependent on the continuous exposure to GH. To identify the transcription factor(s) that mediate(s) this sex-dependent GH effect, we studied the interactions of the CYP2C12 promoter with liver nuclear proteins obtained from male and female rats and from hypophysectomized animals treated or not by continuous GH infusion. GH treatment induced the binding of a protein that we identified as hepatocyte nuclear factor (HNF) 6, the prototype of a novel class of homeodomain transcription factors. HNF-6 competed with HNF-3 for binding to the same site in the CYP2C12 promoter. This HNF-6/HNF-3 binding site conveyed both HNF-6- and HNF-3-stimulated transcription of a reporter gene construct in transient cotransfection experiments. Electrophoretic mobility shift assays showed more HNF-6 DNA-binding activity in female than in male liver nuclear extracts. Liver HNF-6 mRNA was barely detectable in the hypophysectomized rats and was restored to normal levels by GH treatment. This work provides an example of a homeodomain-containing transcription factor that is GH-regulated and also reports on the hormonal regulation of HNF-6.

CYP2C7 expression in rat liver and hepatocytes: regulation by retinoids.

Rats deficient in vitamin A express low levels of P4502C7 mRNA in the liver. Administration of all-trans retinoic acid (at-RA) or growth hormone (GH) to deficient animals only partially restored the expression whereas the combined treatment returned the P4502C7 mRNA levels to that observed in normal rats. That a retinoid is the predominant inducer of P4502C7 at the cellular level is evident from studies performed with primary hepatocytes, but it became clear that GH is a prerequisite for the vitamin A effect in vivo. The at-RA induction of P4502C7 mRNA in primary rat hepatocytes was inhibited by ketoconazole, an inhibitor of P450 activity, and by cycloheximide, blocking ongoing protein synthesis. In contrast, the at-RA induction of RAR-beta2 mRNA was not affected by any of these compounds. This could indicate previously not recognized mechanisms of at-RA action. Interestingly, at-4-oxo-RA, an at-RA metabolite formed by a P450 catalyzed reaction, also induced P4502C7 mRNA. Induction of P4502C7 mRNA by the retinoic acid receptor (RAR) selective agonist TTNPB indicated that this pathway is preferred over the retinoid X receptor (RXR) pathway. In addition, analysis of RA metabolites in liver cell extracts revealed the formation of several as yet unidentified metabolites. The formation of some of these metabolites was inhibited by ketoconazole and they could therefore constitute potential inducers of CYP2C7. We suggest that metabolism of at-RA, possibly by a P450 enzyme, is an important step in the at-RA induction of P4502C7.

Zonation of cytochrome P450 enzyme expression in rat liver. Isozyme-specific regulation by pituitary dependent hormones.

The effect of hypophysectomy and subsequent infusion of growth hormone (GH) or injections of triiodothyronine (T3) on the acinar expression pattern of four homonally regulated P450 isozymes was studied to elucidate the involvement of pituitary dependent hormones in regulating the characteristic centrilobular expression pattern of most members of the cytochrome P450 (CYP) gene family in rat liver. Hypophysectomy was previously observed to allow high expression of CYP2B1/2 and 3A1/2 in the normally silent periportal region. In the present study, it had much less effect on the zonation of the ethanol-inducible P450 2E1 form: only a moderate shift of 2E1 staining towards the periportal region was observed by immunohistochemistry. Subsequent injections with T3 moderately decreased CYP2E1 expression in the periportal region and no significant countereffect of GH was discerned. T3 treatment, previously observed to block only the periportal expression of CYP3A1/2, counteracted the increased CYP2B1/2 expression caused by hypophysectomy equally in the periportal and perivenous region. This was true both at the protein and mRNA level, as analysed from cell lysates obtained by in situ perfusion of livers by zone-restricted digitonin treatment. Thus, although hypophysectomy and subsequent GH and T3 treatment affect the total expression of CYP2B1/2, 2E1, and 3A1/2 similarly, the zonal effects were isozyme-specific. In contrast, the perivenous zonation normally seen for the dioxin-inducible P450 1A2 form was steepened rather than diminished by hypophysectomy, both in male and female rats. Administration of GH by the female-type continuous infusion had no effect in male rats, but partially counteracted the effect of hypophysectomy in females, suggesting an involvement of GH. In contrast to other CYP genes investigated, the female-characteristic expression of CYP2C12 was found to be completely non-zonated. Hypophysectomy and continuous GH administration dramatically affected the amount of mRNA of both P450 2C12 and the male-specific 2C11 form, but analysis of periportal and perivenous cell lysates indicated that these effects were not zone-specific. The distribution of the GH receptor was investigated to explain the zonal effects of GH. Immunohistochemically, a moderate perivenous dominance was observed, whereas the mRNA abundance of both GH receptor and GH binding protein was slightly higher in the periportal region. Thus, zonal regulation by GH does not appear to result from a GH receptor zonation; rather, a sinusoidal GH gradient may be involved. These data, combined with our previous results, indicate that pituitary-dependent hormones regulate the zone-specific expression of some P450 forms strongly (i.e. 2B1/2 and 3A1/2), and other forms are moderately regulated (i.e. 1A2 and 2E1), or are affected across the whole acinus (i.e. 2C11, 2C12).

The human growth hormone (hGH) antagonist G120RhGH does not antagonize GH in the rat, but has paradoxical agonist activity, probably via the prolactin receptor.

Human GH (hGH) acts by dimerizing two hGH receptors that bind to different sites in hGH. G120RhGH, an analog mutated in the second binding site to prevent receptor dimerization, acts as an antagonist in vitro. We have now tested the activity of this analog in vivo in rats with low or absent endogenous GH secretion. Surprisingly, treatment with G120RhGH failed to antagonize the effects of infusions or injections of hGH in hypophysectomized (Hx) rats and had little effect on hepatic GH-sensitive CYP2C transcripts in GH-deficient dwarf (dw) rats. Paradoxically, G120RhGH stimulated skeletal growth when infused into Hx rats; a pulsatile iv infusion was more effective than a continuous pattern. Coinfusion of G120RhGH with hGH produced an additive effect on growth. In addition, continuous, but not pulsatile, G120RhGH infusion elevated hepatic 2C12 messenger RNA (mRNA) expression and reduced 2C11 mRNA expression in Hx rats. The direct effects of G120RhGH on hepatic CYP2C transcripts were confirmed in cultured hepatocytes in vitro, which also revealed a significant action of PRL in elevating 2C12 mRNA expression. Binding studies revealed that G120RhGH bound preferentially to hepatic PRL receptors, as [125I]G120hGH was completely displaced by ovine PRL but was unaffected by bGH, a specific GH receptor ligand. The weak growth-promoting effects of G120RhGH were similar to those induced by recombinant hPRL in Hx rats. Our results show that G120RhGH is a poor in vivo GH antagonist in the rat, but shows a paradoxical agonist effect, probably mediated by PRL receptors in this species.

CCAAT/enhancer-binding protein-alpha-dependent transactivation of CYP2C12 in rat hepatocytes.

Expression of the rat CYP2C12 gene is liver specific and is induced by GH at the transcriptional level. In primary cultures of rat hepatocytes, GH inducibility of CYP2C12 and the presence of C/EBP alpha protein were demonstrated to be equally dependent on attachment of the cells to an extracellular matrix gel (Matrigel). Transient transfection of a C/EBP alpha expression vector into hepatocytes, cultured without Matrigel, increased the cellular P4502C12 messenger RNA levels 10-fold. Cotransfection studies using deletion constructs of the CYP2C12 promoter fused to the luciferase reporter gene localized the C/EBP alpha response to the region -250 to -180. Sequence comparisons and deoxyribonuclease I footprinting using rat liver nuclear extracts indicated two potential C/EBP binding sites in this region. Mutagenesis of the most upstream element (-229 to -207) abolished transactivation by C/EBP alpha. Using gel mobility supershift assays, this element was demonstrated to bind C/EBP alpha and C/EBP beta in liver nuclear extracts and in lysates from hepatocytes cultured on Matrigel. GH treatment of the cells did not alter the C/EBP protein levels or the C/EBP-binding activity to this element. Neither did GH increase the expression of CYP2C12 reporter gene constructs regardless of the presence of different amounts of cotransfected C/EBP alpha. We conclude that C/EBP alpha is a potent transactivator of the CYP2C12 gene and most likely contributes to its liver-specific expression. Although the results presented here do not exclude the possibility of a GH-enhanced transactivating ability of C/EBP alpha, the mechanism of GH-induced levels of P4502C12 is not through increased levels of C/EBP alpha or via enhanced DNA-binding activity of this transcription factor.

Hormonal regulation of the female enriched GH receptor/binding protein mRNA in rat liver.

At least two classes of mRNA for the GH receptor (GHR) and GH binding protein (GH BP) with different 5' untranslated first exons exist in the rat. One such class, the GHR1 is predominantly expressed in the liver of female rats. The hepatic expression of the GHR1 mRNA in normal and hypophsectomized rats of both sexes was studied by employing an RNase protection/solution hybridization assay. Normal females expressed 10-fold more GHR1 mRNA than males, hypophysectomy of female rats decreased the GHR1 level to that observed in male rats. Continuous GH treatment of hypophysectomized male and female rats for 6 days increased the expression of GHR1 mRNA to levels found in normal females, whereas intermittent GH treatment without effect. Bovine GH(bGH) induced the GHR1 expression in a time- and dose-dependent manner in primary cultures of adult rat hepatocytes as determined by solution hybridization. Maximal induction was achieved after 72 h of treatment with 50 ng bGH/ml medium. Female enriched expression of receptor and binding protein mRNAs raises the possibility that they participate in determining the ability of the liver to respond differently to the male and female GH secretory patterns. Our in vitro model utilizing cultures of primary adult rat hepatocytes could be used to address this issue as well as explore a hormonal interplay in regulation of GHR1 expression.

Characterization of orphan nuclear receptor binding elements in sex-differentiated members of the CYP2C gene family expressed in rat liver.

The HepG2-specific P450 2C factor motif (HPF1-motif) is conserved in many hepatic cytochrome P450 genes (CYP). Its functional importance for rabbit CYP2C genes has led to the proposal that the HPF1 motif acts as a common regulator for the liver-specific expression of CYP2 genes with hepatic nuclear factor (HNF)-4 being the corresponding trans-activator. The HPF1-like elements in the rat CYP2C genes 2C7, 2C11, 2C12, and 2C13 have been studied with regard to functional importance and binding of the orphan receptors HNF-4, apoAI regulatory protein-1 (ARP-1), and v-erbA-related receptors (EAR) 3 and 2. Binding activity in rat liver nuclear extracts includes these orphan receptors as judged from electromobility supershift experiments and from results obtained with expressed receptors, although the element in CYP2C11 did not bind HNF-4. Mutations of the HPF1-like elements in the CYP2C7, CYP2C11, and CYP2C12 promoters had marginal effects on the expression of luciferase reporter gene constructs transiently transfected into HepG2 cells, whereas for CYP2C13 the activity was reduced to 60% of the wild type construct. Coexpression of HNF-4 in COS-7 cells had limited effect on the luciferase activity generated from the 2C promoters, maximally 3-fold. Our data indicate that the HPF1 elements in the rat CYP2C genes have limited functional importance and that HNF-4 is not a major trans-activator for any of these genes.

Growth hormone signaling leading to CYP2C12 gene expression in rat hepatocytes involves phospholipase A2.

The expression of CYP2C12 is liver-specific and regulated at the transcriptional level by growth hormone (GH). In attempts to elucidate the nature of signaling molecules mediating the GH regulation of this gene in rat hepatocytes, a role for phospholipase A2 (PLA2) as a transducer of GH-induced levels of P4502C12 mRNA was investigated. GH was shown to induce tyrosyl-phosphorylation of p42 and p44 microtubule-associated protein (MAP) kinases and to reduce the electrophoretic mobility of a 100-kDa protein, immunologically related to cPLA2. These events were observed in parallel with GH-stimulated release of [3H]arachidonic acid ([3H]AA) from cellular phospholipids of rat hepatocytes labeled with [3H]AA. These rapid effects of GH action, as well as the GH-induced expression of CYP2C12, were inhibited in cells treated with the tyrosine kinase inhibitor herbimycin A. Similarly, when the GH-induced liberation of [3H]AA was blocked by the PLA2 inhibitor mepacrine or the Ca2+ channel blocker verapamil, GH-induced accumulation of P4502C12 mRNA was absent. These results suggest a correlation between PLA2 activity and GH regulation of the CYP2C12 gene. The inhibitory effect of mepacrine on GH induction of P4502C12 mRNA was reversed by AA addition, further supporting a role for eicosanoids in the regulation of CYP2C12. Finally, inhibitors of P450-mediated AA metabolism, SKF-525A and ketoconazole as well as eicosatetraynoic acid, blocked the GH-mediated induction of P4502C12 mRNA, whereas more specific inhibitors of cyclooxygenase or lipoxygenase metabolism did not. Based on these results, we suggest that GH signaling in rat hepatocytes, leading to increased expression of CYP2C12, involves PLA2 activation and subsequent P450-catalyzed formation of an active AA metabolite.

Transactivation of the rat CYP2C13 gene promoter involves HNF-1, HNF-3, and members of the orphan receptor subfamily.

The rat CYP2C13 gene (2C13) encodes one of the constitutive male forms of cytochrome P-450 that are involved in steroid metabolism. In addition to being developmentally regulated, the expression of 2C13 is restricted to the liver and suppressed by the female pattern of growth hormone (GH) secretion at the transcriptional initiation level. In this study, we show that the liver-specific expression, but not the regulation by GH, can be reconstituted with 117 bp to 2 kb of 2C13 5' flank. Transactivation of the 2C13 promoter requires both HNF-1 and HNF-3 and is influenced by members of the orphan receptor subfamily of transcription factors. Although HNF-4, ARP-1, EAR-2, and COUP-TF bind to the 2C13 promoter in vitro, overexpression of EAR-2 and COUP-TF, but not of HNF-4 or ARP-1, results in the potentiation of the HNF-3- and HNF-1-supported activity in non-liver cells.

Characterization of the proximal promoter and two silencer elements in the CYP2C11 gene expressed in rat liver.

The cytochrome P450 gene CYP2C11, expressed in the liver of male rats, is transcriptionally regulated in a dual fashion by the sexually dimorphic secretion pattern of growth hormone. To enable analysis of transcriptional regulatory DNA elements, rat genomic sequences were cloned. DNase I hypersensitivity analysis of rat liver nuclei revealed the existence of two hypersensitive sites whose presence in the vicinity of the transcription start site correlates to high transcriptional activity of the gene. Deletion mutants of the 5' flank were fused to reporter genes and transiently transfected into HepG2 cells or into primary adult rat hypatocytes. Transfection experiments in combination with DNase I footprinting analysis in vitro led to the identification of two negative regulatory regions spanning nucleotides -1,230 to -1,188 and -409 to -368 and designated (SIL1200) and (SIL400), respectively. When placed in front of the heterologous thymidine kinase promoter, SIL1200 and SIL400 reduced the activity of the chloramphenicol acetyl transferase reporter gene to 13% and 23% of the control value, respectively. No sex-dependent binding of liver nuclear extracts to the two silencers could be detected by in vitro footprinting or gel retardation assays. However, a sex-dependent footprint consistently stronger with male liver nuclear extracts than with female extracts was observed in the -320 to -294 region. A significant level of identity was found between the DNA sequence corresponding to this footprint and that of orphan steroid receptor elements as well as with that of a basal transcription element common to several CYP2C genes. However, the identity of a potential trans-acting factor binding between -320 and -294 or response of this element to growth hormone is as yet unknown. A sex- and GH secretory profile-dependent protein-DNA interaction in vitro was observed in the -107 to -95 region. In spite of the sequence similarity that exists between this region and the consensus binding site for HNF-1, this region does not bind HNF-1 alpha. This element acted as a repressor on the heterologous thymidine kinase promoter. To date, the two silencer elements and possibly also the HNF-1-like element are the only functional elements defined in the CYP2C11 gene, and it is conceivable that induction of the gene involves derepression of the silencer elements.

The sensitivity of hepatic CYP2C gene expression to baseline growth hormone (GH) bioactivity in dwarf rats: effects of GH-binding protein in vivo.

Hepatic mRNA transcripts for the steroid-metabolizing enzymes cytochrome P4502C11 (male specific) and P4502C12 (female specific) differ in abundance by 10- to 20-fold in male and female rats and are regulated by their different patterns of GH secretion. This sex difference is also found in dwarf rats with low GH secretion, implying that these transcripts may be very sensitive to low level GH exposure. This has now been characterized in normal and dwarf rats. Continuous i.v. infusion of recombinant human (h) GH (0, 3, 12, and 48 micrograms/day) in both dwarf and normal male rats caused a dose-dependent decrease in P4502C11 and an increase in P4502C12, so that the 2C11/2C12 ratio fell from 17.9 +/- 1.3 to 1.5 +/- 1.0 in normal males and from 6.5 +/- 0.9 to 0.4 +/- 0.3 in dwarf males (0 vs. 48 micrograms hGH/day); over this dose range of hGH, body weight gain, total hepatic insulin-like growth factor-I mRNA levels, and plasma GHBP levels were largely unaffected. These effects of hGH were pattern dependent. The 2C11/2C12 ratio in dwarf males was feminized (from 11.9 +/- 1.3 to 0.08 +/- 0.03) by continuous infusion of hGH (36 micrograms/day), whereas a pulsatile infusion (3-min pulses every 3 h) of the same daily hGH dose was much less effective. Neither continuous nor pulsatile hGH affected P4502C11 or P4502C12 transcripts in dwarf females, although pulsatile hGH infusion caused a significant weight gain. To test whether baseline GH levels could be modified by circulating GH-binding protein (GHBP), hGH infusions were given with and without recombinant hGHBP in different patterns. Pulsatile infusions of recombinant hGHBP (42 micrograms/day, i.v.) did not prevent the feminizing effect of continuously infused hGH (36 micrograms/day, sc) in dwarf males (2C11/2C12 ratios were 0.08 +/- 0.01 and 0.09 +/- 0.01 for hGH vs. hGH plus hGHBP, respectively). This suggested that intermittent complex formation with GHBP did not prevent continuous access of hGH to the hepatic GH receptors. Furthermore, pulses of hGH complexed with GHBP significantly reduced the 2C11/2C12 ratio in dwarf males (from 21.5 +/- 3.9 with pulsatile hGH alone to 9.2 +/- 2.5 with pulses of hGH plus hGHBP), indicating that GHBP prolongs the exposure to hGH. Thus, 2C11/2C12 expression is very sensitive to basal GH levels in dwarf rats, and GHBP can alter hepatic gene expression by modifying the pattern of GH exposure.