Introduction

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Sulfation is a major route of biotransformation of chemicals that contain hydroxyl groups (De Meio, 1975). Steroids such as hydrocortisone (cortisol), DHEA and androstanols are excreted as sulfates in the urine of both experimental animals and humans (Mulder, 1984). These reactions are primarily catalyzed by cytosolic STs² in the livers. Hepatic hydroxysteroid sulfation shows clear sex dimorphism in rat livers, in which higher activity is observed in the females than in the males (Singer et al., 1976).

Multiple forms of the enzyme catalyzing the sulfation of hydroxysteroids have been separated from rat livers using ion-exchange chromatography (Lyon and Jakoby, 1980; Singer et al., 1976). Although these purified preparations were very similar in their chemicophysical properties and substrate specificities (Jakoby et al., 1984), cloning and sequencing of ST cDNA demonstrated that there are multiple forms of hydroxysteroid STs in rat livers. Ogura et al. (1989, 1990) isolated several hydroxysteroid ST cDNAs from rat liver. Furthermore, the senescence marker protein-2 originally reported by Chatterjee et al. (1987) is now known to be a member of hydroxysteroid STs from its deduced amino acid sequence (Ogura et al., 1990).

Yamazoe et al. (1989) studied the mechanisms of sex-related difference in cortisol sulfation in rat livers and demonstrated the role of both gonadal and pituitary glands on the regulation of the hepatic hydroxysteroid ST activity. This study indicates the principal role of pituitary GH on the regulation of cortisol sulfating activity. In preliminary experiments, we examined the effect of GH on DHEA sulfation in rat livers. We found that GH altered hepatic DHEA sulfation, but the response was different from that observed with cortisol sulfation.³ These studies implied that rat livers contain at least two related, but distinct, functionally active genes encoding each of the hydroxysteroid STs. However, there are no reports on the specific form responsible for GH-sensitive sulfation of hydroxysteroids.

In the present study, we examined the effect of GH on the mRNAs of two major hydroxysteroid STs, ST2A1 and ST2A2. The results indicate that GH independently regulates boh ST2A1 and ST2A2. ST2A1 is regulated by GH through a mechanism similar to that is observed in the regulation of CYP2C12, a female-specific P450. On the contrary, a unique suppression by the pulsatile secretion of GH is observed in the regulation of ST2A2.

	Methods

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Chemicals. Human recombinant methionylated GH (Somatonorm, Kabi Vitrum, Stockholm, Sweden) was a generous gift from Sumitomo Pharmaceutical (Osaka, Japan).

Animal treatments. Male and female SD rats were obtained from Clea Japan (Tokyo, Japan). Some rats were hypophysectomized at the age of 7 weeks. After recovery for 1 week, human recombinant methionylated GH was administered by a continuous infusion (0.01 IU/hr) to mimic female secretory pattern or subcutaneous injection (0.2 IU/100 g twice a day) to mimic male secretory pattern for 7 days as previously described (Gong et al., 1992; Yamazoe et al., 1986a). Spontaneous dwarf rats derived from a closed colony of SD rats were maintained in Department of Anatomy, School of Medicine, Jikei University, Tokyo, Japan (Okuma and Kawashima, 1980), and those born after >23 generations were used in this study. Male and female dwarf rats at the age of 44 or 47 days were treated with human recombinant methionylated GH by a continuous infusion (0.01 IU/hr for 7 days). The 40- or 42-day-old male dwarf rats were treated with ovine GH [NIADDK-oGH-15 (AFP-7649C)] by subcutaneous injection (100 µg/head once a day for 9 days).

RNA preparation. Total RNA was prepared from livers by an acid guanidinium thiocyanate-phenol-chloroform method (Chomczynski and Sacchi, 1987). The total RNA content was determined by measuring the absorbance at 260 nm using a spectrophotometer (Beckman DU 7500).

Oligonucleotide probes. The 26-mer oligonucleotide probe for ST2A1 has a sequence of 5-ACAGTGCCTTTCCTCATGAGGCCAGT-3, which is complementary to base pairs 761 to 786 of ST2A1 (ST-20) mRNA (Ogura et al., 1989). The corresponding sequence for ST2A2 (ST-40) shares 38.5% homology. Hybridization was performed at 60°C. The 27-mer oligonucleotide probe for ST2A2 has a sequence of 5-CCCAGTAGTGCCGTTTCTCATGAAAGT-3, which is complementary to base pairs 764 to 790 of ST2A2 (ST-40) mRNA (Ogura et al., 1990). The corresponding sequence for ST2A1 (ST-20) shares 29.6% homology. Hybridization was performed at 65°C. The 26-mer oligonucleotide probe for CYP2C12 has a sequence of 5-TAGCAGCAAAATGTTTTGAATGTGTC-3, which is complementary to base pairs 693 to 718 of CYP2C12 (P450₁₅) mRNA (Zaphiropoulos et al., 1988). Hybridization was performed at 52°C as previously described (Sasamura et al., 1990). The 19-mer oligonucleotide probe for the 18S rRNA has a sequence of 5-TCTTGCGCCGGTCCAAGAA-3, which is complementary to base pairs 969 to 987 of the rat 18S rRNA (Chan et al., 1984). Hybridization was performed at 60°C.

Estimation of mRNA levels. For Northern blotting, 20 µg of total RNA was subjected to electrophoresis on 1% agarose gel and transferred to nylon membrane (Hybond-N Plus, Amersham Japan, Tokyo, Japan). Oligonucleotide probes were 5-end-labeled with [-³²P]ATP (Amersham Japan). After prehybridization with 15 mM sodium chloride containing 1.5 mM sodium citrate and 0.5% SDS, the membrane was hybridized with ³²P-labeled probe (1 × 10⁶ cpm/ml) in 0.5 M sodium phosphate, pH 7.2, containing 7% SDS, 1% bovine serum albumin and 1 mM EDTA. After washing with 20 mM sodium phosphate, pH 7.2, containing 1% SDS and 1 mM EDTA, the membrane was exposed to x-ray film (Kodak X-Omat AR film) at 80°C. For slot blotting, 10 µg of total RNA was blotted onto nylon membrane using MINIFOLD S-SRC60D (Schleicher & Schuell, Keene, NH). The membrane was hybridized as described for Northern blotting. After scanning of the x-ray film using a Nikon AX-1200 flatbed scanner, the intensity of the autoradiographic spots was quantified using Adobe Photoshop 2.5J and NIH Image 1.55. The levels of ST2A1, ST2A2 and CYP2C12 mRNAs were determined after the correction from the abundance of 18S rRNA. Statistical significance was determined using analysis of variance followed by a post hoc test with Fisher's PLSD.

	Results

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Effect of GH treatment on hepatic ST2A1, ST2A2 and CYP2C12 mRNA levels in hypophysectomized rats. The effect of GH treatment on hepatic ST2A1 and ST2A2 mRNA levels in hypophysectomized rats was investigated using specific oligonucleotide probes to distinguish the hydroxysteroid STs. To compare GH susceptibility, mRNA level of a female-specific P450, CYP2C12, was also estimated in this experiment.

View larger version (61K): [in this window] [in a new window]   Fig. 1.   Northern blot analyses of ST2A1 (A), ST2A2 (B) and CYP2C12 (C) mRNAs in three of male and female Sprague-Dawley rat livers.

View larger version (40K): [in this window] [in a new window]   Fig. 2.   Effect of GH treatment on hepatic levels of ST2A1 (A), ST2A2 (B) and CYP2C12 (C) mRNAs in hypophysectomized rats. GH was given to hypophysectomized rats (Hx) by continuous infusion [GH(i)] or subcutaneous injection [GH(s)] as described in Methods. Columns and bars indicate the mean (specific mRNA levels/18S rRNA levels) and S.D. values, respectively, from three different rats except GH-infused male hypophysectomized rats (two rats). The values are expressed as relative levels compared with normal female rats. Data for male rats treated with GH(i) are median values of two animals. * and , significantly different from the corresponding control (Cont) and hypophysectomized rats, respectively (P < .05). N.D., not detected.

Effect of GH treatment on hepatic ST2A1, ST2A2 and CYP2C12 mRNA levels in spontaneous dwarf rats. A SD strain-derived spontaneous dwarf rat has a specific loss of GH caused by a point mutation of its GH gene (Takeuchi et al., 1990), although it contained other pituitary hormones. To more precisely verify the influence of GH, the spontaneous dwarf rats were used to examine the effect of GH treatment on hepatic mRNA levels of ST2A1, ST2A2 and CYP2C12.

View larger version (43K): [in this window] [in a new window]   Fig. 3.   Effect of GH treatment on hepatic levels of ST2A1 (A), ST2A2 (B) and CYP2C12 (C) mRNAs in spontaneous dwarf rats. GH was given to dwarf rats (DW) by continuous infusion [GH(i)] or intermittent injection [GH(s)] as described in Methods. Columns and bars indicate the mean (specific mRNA levels/18 S rRNA levels) and S.D. values, respectively, from three or four rats. The values are expressed as relative levels compared with normal female Sprague-Dawley rats (SD Female). * and , significantly different from the corresponding normal and dwarf rats, respectively (P < .05). N.D., not detected.

	Discussion

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Watabe et al. (1994) isolated five different cDNAs encoding hydroxysteroid STs, including ST2A1 (ST-20) and ST2A2 (ST-40). Two other cDNAs, ST-21 and ST-41, are considered to be allelic variants of ST2A1 (ST-20) and ST2A2 (ST-40), respectively (Ogura et al., 1994). The exact relationship between hydroxysteroid ST cDNAs and isolated proteins, however, has not been completely determined because of the similarity of substrate specificity and electrophoretic mobility on SDS-PAGE. In addition, the entire amino-terminal sequences of individual forms of hydroxysteroid ST have not yet been determined. Therefore, we investigated the effect of GH on each form of hydroxysteroid STs only on the mRNA levels by using a oligonucleotide probe specific for each form. In Northern blot analysis with a specific oligonucleotide probe for ST2A5 (ST-60), we could hardly detect a hybridizable band in livers of either sex of rats at 1 day to 24 months of age. These data suggested that ST2A1 and ST2A2 are the major forms of hydroxysteroid STs in rat livers.

In the present study, by using two GH-deficient animals (hypophysectomized rats and spontaneous dwarf rats), we provide evidence that pituitary GH independently regulates hepatic ST2A1 and ST2A2 at the pretranslational level in rats.

As shown in figures 1A and 2A, hepatic ST2A1 mRNA levels were 5 times higher in female rats than males. ST2A1 mRNA was not detected in both GH-deficient models, but the level increased by continuous infusion of GH (figs. 2A and 3A). These results indicate that GH regulates ST2A1 in a manner similar to the female-specific P450, CYP2C12, in which a continuous GH secretory profile (female secretory pattern) has a stimulative effect on the gene expression of ST2A1 as well as CYP2C12.

As shown in figures 1B and 2B, ST2A2 mRNA was detected in livers of female rats but not in males. ST2A2 mRNA was also detectable in both sexes of hypophysectomized rats and spontaneous dwarf rats (figs. 2B and 3B). This is in clear contrast to the responses of ST2A1 and CYP2C12 to GH. Intermittent injection of GH clearly decreased the level of ST2A2 mRNA in both GH-deficient models. These results indicate that pulsatile secretory profile (male secretory pattern) suppresses the levels of female-specific ST2A2 mRNA.

Levels of ST2A1 mRNA varied depending on GH states, whereas the levels of ST2A2 mRNA were not consistent between hypophysectomized rats and dwarf rats. Hepatic ST2A2 mRNA was detected in both GH-deficient rats but differed in their levels. Continuous infusion of GH to hypophysectomized rats did not restore significantly ST2A2 mRNA levels, whereas similar treatment to dwarf rats significantly increased ST2A2 mRNA levels. Although the reason for the difference is unclear, differences in other hormone states may cause the inconsistent results between these GH-deficient rats. Hypophysectomized rats lacking all pituitary hormones show atrophy of organs that produce peripheral hormones, whereas dwarf rats have a specific loss of GH. However, the involvement of factors other than hormones should not be excluded.

As shown in figure 1, clear sex-related differences are observed on the levels of hepatic ST2A1 and ST2A2 mRNAs. Both ST2A1 and ST2A2 mRNA levels were higher in females than in males, but we observed a clear difference in GH-mediated regulation toward ST2A1 and ST2A2. Pituitary GH is essential for the appearance of ST2A1 mRNA, and the continuous presence of GH is necessary for the maximal expression. On the contrary, the level of ST2A2 mRNA decreases but is still detectable in GH-deficient states, and the level is rather suppressed by male type of GH secretion. Previous reports showed that pituitary GH suppresses hepatic levels of several cytochrome P450s, including CYP3A2 (Waxman et al., 1988; Yamazoe et al., 1986b) and CYP2B1 (Yamazoe et al., 1987). Their suppression is more evident in GH-deficient animals treated with GH via continuous infusion than in those treated via intermittent injection. Suppression of ST2A2 mRNA is observed in GH-deficient animals after intermittent injection of GH but is rather refractory to GH infusion. The susceptibility of ST2A2 is unique among GH-sensitive drug-metabolizing enzymes in rat livers.

GH pulse suppression has been reported in the expression of female-specific cyp2a-4 (steroid 15-hydroxylase) and cyp2b-13 (steroid 16-hydroxylase) in mouse livers (Noshiro and Negishi, 1986). These mRNAs are also detected in GH-deficient mouse (Little mouse), but the levels are higher than those in the normal female mouse. This is in clear contrast to ST2A2 mRNA in GH-deficient model rats. These results suggest that the mechanism of GH regulation differs between the expression of ST2A2 and that of cyp2a-4 and cyp2b-13.

Although no sex-related difference was observed in the effect of GH continuous infusion on both ST2 mRNAs in GH-deficient rats in the present study, Labrie et al. (1994) reported a clear difference in the effect of continuous infusion of GH on hepatic DHEA ST mRNA levels. The reason for the difference is unclear, but they used a PCR product corresponding to nucleotides 179 to 531 for ST2A1 (ST-20) cDNA sequence as a probe, which shared 97.2% homology with ST2A2 (ST-40) cDNA.

In conclusion, the present study indicates that pituitary GH regulates two hydroxysteroid STs, ST2A1 and ST2A2, in rats through distinct modes of GH secretion. A clear sex-related difference in hydroxysteroid ST that we observed in rats is useful in understanding the underlying mechanism of regulation of mammalian STs. Distinct susceptibility toward GH between ST2A1 and ST2A2 may imply that these two enzymes have different functional and physiological roles in rat livers. We are currently investigating the functional properties and regulation of ST2A1 and ST2A2.