Advan. Enzyme Regul., Vol. 32, pp. 255--263, 1992 Britain. All rights reserved

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A sexual difference in the frequency of primary liver cancer in humans as well as a different susceptibility of male and female rats to chemically induced liver cancer emphasizes the importance of investigating the hormonal control of liver functions. Using the rat as an experimental model we are studying the mechanisms of hormonal regulation of the sexual dimorphism in liver metabolism. This line of research has developed from classical endocrine manipulations with subsequent measurements of hepatic enzyme activities, identification of the major common hormonal denominator of sex differentiated metabolism and cloning of genes for steroid metabolizing enzymes to the challenge of understanding the molecular mechanisms of growth hormone (GH) regulation of the expression of cytochrome P450 enzymes (P450). HORMONAL





Cytochrome P450 enzymes are ubiquitous and active in the oxidative metabolism of an extensive range of endogenous and xenobiotic compounds. Today, over 140 P450s have been isolated and, among them, at least 20 are expressed in rat liver (1). Some P450s are constitutively expressed while others are inducible following exposure to certain xenobiotic compounds. Hydroxylation of xenobiotics can lead to biological activation of the parent compound with the formation of mutagenic or carcinogenic derivatives as the result. However, hydroxylated steroid hormones formed by P450 catalyzed reactions in the liver are generally thought to be products of degradative pathways but in view of the precise age- and sex-dependent regulation of many of these enzymes it is likely that also some of these metabolites may exhibit physiological functions. The predominant P450s responsible for the sexual dimorphism in steroid metabolism in the rat are members of the P4502C-gene subfamily (2). The 255


A. MODE, et al.

gene product of CYP2C12, catalyzing 1513-hydroxylation of steroid sulfates, and that of CYP2C11, catalyzing 16ct-hydroxylation of testosterone, are female- and male-specific, respectively. The expression of these enzymes is developmentally regulated and becomes manifest in adult animals (3, 4). The influence of androgens during both the neonatal period and postpubertally is a prerequisite for maintenance of the male phenotype (2Cll > 2C12). Although administration of estrogens to adult male rats feminizes hepatic steroid metabolism completely, estrogens are not necessary for the maintenance of the female phenotype (2C12 > 2 C l l ) in the absence of androgens. These effects of gonadal hormones are, however, indirect in that they arc mediated via the hypothalamo-pituitary axis where growth hormone (GH) is the final denominator (5). The reason why GH, present in both sexes, is the major determinant of sex differences in P450 expression resides in the sex dependent mode of G H secretion (6). The male pattern of G H secretion is characterized by regular peaks every 3-4 hr with low, often undetectable, levels in between. In female rats GH secretion is more irregular with lower peak amplitudes and higher basal levels. In hypophysectomized animals, devoid of GH, the sex specific expression of P4502Cll and P4502C12 is lost. When GH is administered to hypophysectomized rats in a sex characteristic fashion, intermittently to mimic the male pattern, or continuously to mimic the female pattern, the GH treatment reverses the effects of hypophysectomy on hepatic steroid metabolism (7). Thus, the action of gonadal hormones is via the mode of GH secretion. The mechanism(s) mediating these effects are unknown. However, from accumulated data it seems conceivable that the secretion and interaction of the two hypothalamic peptides regulating G H release, somatostatin (SS) and GH releasing factor (GRF), are affected (8, 9). Furthermore, a direct action of sex hormones on the pituitary may also occur. Alternative experimental animal models to the hypophysectomized rat have been used to investigate the role of GH in the control of hepatic steroid metabolism (10, 11). In some cases, the use of such models has led to the controversial suggestion that GH does not regulate the sexual differentiation of rat liver P450s. Thus, a new strain of dwarf rats (NIMR/AS) with an isolated GH deficiency (less than 5% of normal pituitary G H content) has been shown to express sexually differentiated P450s at levels indistinguishable from those in normal rats, apparently indicative of a non GH-dependent expression. However, when the secretory profile of G H was analyzed in dwarf rats, using a highly sensitive radioimmunoassay where the detection limit is brought down to 0.2 ng/ml, the observed pattern in each respective sex is superimposable on the profiles of normal rats (C. Legraverend, A. Mode, T. Wells, I. Robinson and J.-/~. Gustafsson, Fed. Proceed., in press (1992)). That


GH R E G U L A T I O N OF P4502C12


C 100

GH 1200



GH + E

GH + A





Adult male rat hepatocytes were maintained in culture on Matrigel substrata in a serum-free, chemically defined medium containing insulin (1 #g/mi) as the only hormone. Two hr after plating of the cells additions of vehicle (C), bovine GH (50 ng/ml) (GH), diethylstilbestrol 10-8 M (E), methyltrienolone 10--8 M (A) or the combinations (GH + E) or (GH + A) were made. Medium with the respective additions was renewed daily and cells were harvested after 3 days. Total nucleic acid preparations were analyzed for the content of P4502C12 mRNA .using a specific solution hybridization assay.

extremely low levels of G H are sufficient to cause an effect on Sex differentiated P450 expression is consistent with our previous data showing that hypophysectomized rats with a pituitary graft under the kidney capsule are feminized with regard to hepatic steroid metabolism (12). A direct effect of G H on the liver has been firmly established since we have demonstrated that primary adult rat hepatocytes in culture, maintained in a completely hormone free medium on a biomatrix, respond to G H treatment with an increased expression of the female specific P4502C12 (13). Furthermore, as shown in Table 1, estrogen (diethylstilbestrol) or androgen (methyltrienolone) treatment of primary hepatocytes alone or in combination with G H suggests that these hormones may have minor modulatory effects on the expression of P4502C12. The significance of these observations is yet to be determined. However, the effects of gonadal steroids as well as of other hormones such as thyroid hormones, glucocorticoids, insulin and insulin-like growth factor-I are essentially modulatory in comparison to the G H effects and may in part be indirect (14-16). In addition, it should be mentioned that induction of the male-specific P4502Cll by an intermittent administration of G H to primary hepatocytes has not as yet been possible to achieve. In conclusion, available data are consistent with the hypothesis that G H is the principal regulator of sexually dimorphic aspects of hepatic metabolism and that it is the circulating GH level recognized by the liver as presence or absence of GH, that is the underlying cause of expression of a particular subgroup of microsomal P450 isozymes. MOLECULAR




It has become evident from several studies that GH exerts its effects on P450 pretranslationally. By comparing steady-state mRNA levels of the P450 forms 2 C l l and 2C12 with the rate of transcriptional initiation of the respective gene in livers from rats with different G H status we


a. MODE, et al.

conclude that the expression of these genes is regulated by GH at the level of transcription (C. Legraverend, A. Mode, S. Westin, A. StrOm, H. Eguchi, P. Zaphiropoulos and J.-/~. Gustafsson, Mol. Endocrinol. in press (1992)). An increase in the transcriptional activation of the female-specific CYP2C12 gene studied in primary hepatocytes in Culture is evident already after 1 hr and reaches an ll-fold induction after 20 hr. The intracellular events evoked by binding of GH to its hepatic receptor are not known, nor have any GH-responsive cis-regulatory sequences in P450 target genes been identified. Interestingly, Yoon et al. have presented evidence for a GH activated hepatic nuclear factor interacting with a regulatory sequence in the serine protease inhibitor 2.1 gene (17). The activity of this as yet unidentified factor is not blocked by protein synthesis inhibition, suggesting that a pre-existing factor mediates this effect of GH. This is in contrast to our findings regarding transcriptional activation of the CYP2C12 gene. GH-induced transcription of this gene in primary hepatocytes is completely blocked by cycloheximide (18). Thus, it would appear that GH can activate more than one signalling pathway in the hepatocyte. The Growth Hormone Receptor The tissue response to GH is of course dependent on the expression of GH receptors (GHR). Whether a sex difference in G H R expression exists in the rat is a controversial issue. Some studies show slightly higher levels (1.5-3-fold) in female than in male rat livers but this observation appears to be related to the strain of rat investigated (19, 20). Furthermore, epitope mapping studies indicate the presence of more than one subpopulation of G H R (21). In addition to the membrane localized G H R on target cells, a soluble GH binding protein (GHBP) has been identified in several species. The GHBP protein is encoded by a shorter but highly homologous mRNA to the receptor mRNA. In the rat, the G H R and the GHBP are thought to be synthesized from different mRNAs generated by alternative splicing of a primary transcript (22). G H R / G H B P levels are to a certain extent dependent on the pituitary and appear to increase in response to a female secretory pattern of GH. The co-expression of GHBP and G H R suggests a functional role for the GHBP in the tissue regulation of GH action. Data have started to emerge which may help to clarify the function of the GHBP. Signalling Pathways of the Growth Hormone Receptor Cloning of the G H R from several species, including the rat, has placed the G H R in the super-family of cytokine receptors (23). These receptors span the plasma membrane once and have previously generally been thought



not to be tyrosine kinases. However, a primary event after G H R occupancy seems to be increased phosphorylation of the receptor on tyrosyl residues and, furthermore, the tyrosine kinase activity is evidently tightly associated with the receptor (24). Whether the G H R possesses intrinsic kinase activity and whether phosphorylation of the receptor is necessary for signalling remains to be elucidated. Other receptors of the cytokine family, such as the interleukin-6 receptor (IL-6R), have been shown to associate with a signal transducer in the membrane upon ligand binding (25). Of interest is that only the extra cellular portion of the IL-6R is necessary for this association and triggering of signals. If a similar system is operating for transduction of the GH signal and if the GHBP, constituting the extra cellular portion of the GHR, is enough to evoke cellular signalling are most intriguing questions. Several investigations suggest that phospholipid hydrolysis is of importance in the GH signalling process. In tissues and cells studied so far, G H treatment leads to diacylglycerol (DAG) formation (26-28). In cultured primary hepatocytes we observed a 5-fold increase in D A G production 30 sec after GH addition (18). The phospholipid hydrolyzed by activation of the G H R appears to vary, depending on what tissue GH acts on. No formation of inositol phosphates occurs concomitantly with D A G formation in freshly isolated hepatocytes treated with GH (27). In line with that, we have preliminary data showing that phosphorylcholine formation is stimulated by G H in primary hepatocytes in culture. The fact that D A G is a potent stimulator of protein kinase C (PKC) activity together with observations that inhibitors of PKC block GH effects in adipose cells (29, 30) prompted us to investigate a possible involvement of PKC in the GH mediated induction of P4502C12 mRNA in primary hepatocytes (18). Stimulation of PKC by phorbol ester treatment, in dose and time-course experiments in the presence or absence of a Ca2÷-ionophore, failed to induce the P4502C12 message. However, down regulation of PKC by phorbol ester treatment, i.e., 24-hr pretreatment with 100 nM 4[3-phorbol 12-myristate 13-acetate, did reduce the G H induction of P4502C12 mRNA by 50%. Furthermore, treatment with the potent kinase inhibitor staurosporin in combination with G H completely blocked the induction of P4502C12 mRNA. These results indicate that PKC has a permissive role in G H signalling and that some other kinase(s) is a determining transducer in induction of P4502C12. In this context, a role for protein kinase A was investigated, cAMP-dependent signalling was found to reduce the expression of P4502C12 mRNA but apparently independently of GH. In the overall regulation of P4502C12 expression, hormones affecting the cellular cAMP level must also be taken into consideration.



14 + 5 18 + 2

170 _+ 7 345 + 21

Cells were maintained, treated with GH for 3 days and analyzed as described in Table 1.



As mentioned above, no GH-responsive c/s-regulatory sequences in P450 target genes have been identified so far. Recent studies in our laboratory indicate that female hypophysectomized rats respond more efficiently to continuous GH treatment than hypophysectomized males. When primary hepatocytes from male or female rats are put in culture and subsequently treated with GH, a higher expression of P4502C12 mRNA is observed in cells from female rats (Table 2). Organization of chromatin structure has been considered as an important determinant in the differential sensitivity of specific genes to various regulatory signals (31). For example, sex differences in DNase I hypersensitivity or hypomethylation of cytosine residues of a gene may be



REGION P~latlve ~T-sct4vity




15 FIG. 1. Relative CAT-activity in primary hepatocytes transfected with 5'-flank constructs of the P4502C12 gene in front of a CAT-reporter. 5'-flank constructs extending -2kb, - l k b or -0.Skb upstream of the initiation site in the P4502C12 gene were put in front of a CAT-reporter gene and transfected into primary hepatocytes via electroporation. After 48 hr in culture, as described in Table 1, cells were harvested and analyzed for CAT-activity. The activity in cells transfected with the - l k b construct was set to 100%.

GH R E G U L A T I O N OF P4502C12


important to consider in this respect. With regard to the P4502C12 gene, we have not so far been able to demonstrate such differences (32). Genomic cloning of the CYP2C12 gene has allowed us to sequence 2 kb of the 5'-flanking region. Computer searches for homologies with consensus sequences of identified transcription factor binding sequences in this region have revealed the presence of liver specific as well as other consensus elements. Whether any of these are of importance for transcriptional activation of the P4502C12 gene remains to be elucidated. Much effort is now concentrated o n making deletion constructs of the 2 kb 5'-flank in front of the reporter gene chloramphenicol acetyl transferase (CAT) and to transfect these constructs into our system of primary hepatocytes. Results obtained are so far preliminary and confined to elements important for basal transcription. As shown in Figure 1, the sequence between-0.5 kb to -1 kb appears to harbor an "enhancer" sequence. This sequence is now being mapped further and its possible response to G H investigated. SUMMARY

GH by means of its sexually differentiated secretory pattern is the predominant regulator of the expression of cytochrome P450 enzymes responsible for a sexual dimorphism of hepatic steroid metabolism. Other hormones, such as gonadal, thyroid and glucocorticoid hormones, as well as insulin appear to modulate the sexually differentiated expression of these enzymes. The major constitutively expressed sex specific forms of P450, belonging to the P4502C-subfamily, have been shown to be regulated by G H at the level of transcription. However, the G H postreceptor events leading to increased or decreased transcriptional activity are essentially unknown. Neither is the functional role of the soluble GH binding protein yet resolved. On-going protein synthesis is a prerequisite for G H transcriptional activation of the female specific P4502C12 but not for all GH effects in the hepatocyte. With regard to signalling mechanisms PKC activity appears to be permissive for the GH induction of P4502C12 but some as yet unidentified factor/kinase(s) may also be activated. The transcriptional control exerted on the rat P4502C-gene subfamily by the pattern of G H secretion offers a versatile tool to elucidate the molecular mechanisms of G H regulation of cytochrome P450 expression. ACKNOWLEDGEMENT

We are indebted to Ms Eva Floby and Ms AnnGerd Nilsson for skilful technical assistance. This work was supported by grants from the Swedish Medical Research Council (no. 03X-06807), the Magnus Bergvall Foundation and funds from the Karolinska Institute.



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Growth hormone regulation of hepatic cytochrome P450 expression in the rat.

GH by means of its sexually differentiated secretory pattern is the predominant regulator of the expression of cytochrome P450 enzymes responsible for...
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