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Cloning of a Novel Growth Hormone-Regulated Rat Complementary Deoxyribonucleic Acid with Homology to the Human 1B-Glycoprotein, Characterizing a New Protein Family¹

Department of Medical Nutrition (C.G., A.M.), Karolinska Institutet, Novum, S-14186 Huddinge, Sweden; and Stockholm Bioinformatics Centre and Department of Biochemistry and Biophysics (B.P.), Karolinska Institutet, S-171 77 Stockholm, Sweden

Address all correspondence and requests for reprints to: Agneta Mode, Ph.D., Department of Medical Nutrition, Karolinska Institutet, Novum, S-14186 Huddinge, Sweden. E-mail: agneta.mode{at}mednut.ki.se

	Abstract

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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 1B-glycoprotein. Sequence comparisons suggest that the human 1B-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.

	Introduction

Top Abstract Introduction Materials and Methods Results and Discussion References

 
GH HAS A BROAD RANGE of physiological actions, including major effects on postnatal growth and intermediary metabolism (1, 2). A major GH target organ is the liver, in which GH regulates the transcription of genes encoding a variety of proteins ranging from hormone and growth factor receptors to enzymes, secretory proteins, and transcription factors (3). It is not only the absolute presence or absence of GH, but also the temporal pattern of hormonal exposure that has bearings upon gene transcription and determines the cellular response. In mammals, the GH secretion pattern is sexually dimorphic, and in the rat this is particularly marked (4). This has been shown to lead to sex-specific expression of several hepatic GH-target genes (5, 6, 7). Male rats secrete GH intermittently in high amplitude peaks every third to fourth hour, with undetectable GH levels in between peaks. In contrast, the female GH secretion pattern is characterized by frequent low amplitude GH peaks and a high basal level, resulting in a continuous presence of GH in serum (4). The effects of the male pattern of GH secretion can be mimicked in hypophysectomized rats by giving one or two daily sc injections of GH, and the effects of the female pattern of GH secretion can be mimicked by a continuous administration of GH (5, 8, 9, 10, 11). Important knowledge about GH pattern-dependent mechanisms of action has been gained by studies using the rat as a model. However, it is evident that much remains to be found out regarding the effects of GH both from a physiological and a molecular point of view. Finding of new GH target genes dependent on the sex-specific secretory pattern will aid in understanding the diverse actions of GH and provide additional tools for mechanistic studies.

There exist several techniques for the study of differential gene expression. One of these techniques, suppression subtractive hybridization (SSH) (12), has been reported to give rise to a relatively low frequency of false positives, especially when the samples contain many differentially expressed sequences, as can be expected for the liver. To search for rat liver genes up-regulated by the continuous, female-characteristic pattern of GH secretion, we have used the SSH technique. A sequence highly represented among the obtained clones showed homology to the human 1B-glycoprotein (1B) (13). Following isolation of the full-length complementary DNA (cDNA) the structural homology to human 1B was further strengthened. Furthermore, our data indicate that these two proteins constitute members of a new family of proteins.

	Materials and Methods

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Animals and hormone therapy
Normal and hypophysectomized Sprague Dawley rats were obtained from Møllegaard (Møllegaard Breeding Center Ltd., Ejby, Denmark). Hypophysectomy was performed at 6 weeks of age by the breeder. All rats were maintained under standardized conditions of temperature (24-26 C) and humidity (50–60%) with lights on between 0500 h and 1900 h, and had free access to standard laboratory chow and water. Rats treated with bovine GH (bGH) received the hormone either continuously by means of an Alzet minipump (Alza Corp., Palo Alto, CA) for 7 days or as two daily sc injections for 7 days. Normal GH-treated male rats received 0.5 mg bGH/kg·day continuously. Hypophysectomized rats treated with GH, either continuously or intermittently were given 0.7 mg bGH/kg·day. In hypophysectomized rats, the GH treatment was started at the same time as substitution therapy with glucocorticoid and thyroid hormone. Recombinant bGH was a generous gift from American Cyanamid (Princeton, NJ). The osmotic mini-pumps were implanted sc on the back of the rats under light anesthesia. All hypophysectomized rats were substituted with cortisol phosphate (400 µg/kg·day; Solu-Cortef, Upjohn, Puurs, Belgium) and L-thyroxine (10 µg/kg·day; Nycomed, Oslo, Norway) as a daily sc injection commencing 7 days after hypophysectomy (9). The South Ethical Committee of the Swedish National Board for Laboratory Animals approved this study.

SSH and cloning
The messenger RNA (mRNA) was isolated from 8-week-old rats by the use of PolyATtract System 1000 (Promega Corp., Madison, WI). SSH was performed using the PCR-Select cDNA Subtraction Kit, (CLONTECH Laboratories, Inc., Palo Alto, CA) essentially according to the manufacturer’s instructions. cDNA made from feminized male rat liver mRNA samples, i.e. normal male rats treated continuously with bGH (0.5 mg/kg·day for 7 days), was used as tester. cDNA made from normal male hepatic mRNA was used as driver. The number of cycles in the PCR were optimized to 27 cycles in the first PCR amplification and to 15 cycles in the second amplification using nested primers. The subtracted cDNA products were cloned into an T/A vector (AdvanTage PCR Cloning Kit; CLONTECH Laboratories, Inc.) after gel purification using the QiaQuick Gel Purification Kit (QIAGEN, Hilden, Germany). Sequence analysis of expressed cDNA products was performed using cycle sequencing with dye-labeled nucleotides (Big-Dye Terminator; Perkin-Elmer Corp., Norwalk, CT) and the gels were run at Cybergene (Huddinge, Sweden).

Northern blot
Total RNA was isolated from rat liver according to Chomczynski and Sacchi (14) with the addition of a wash in 4 M sodium acetate (pH 5.0) to remove glycogen. Twenty micrograms of RNA were run in formaldehyde containing 1% agarose gel. The RNA was blotted onto Hybond N nylon membranes (Amersham Pharmacia Biotech, Aylesbury, Buckinghamshire, UK) and covalently linked to the membrane by UV irradiation (UV Stratalinker 2400; Stratagene, La Jolla, CA). The membranes were prehybridized at 50 C for at least 3 h in a solution containing 5x SSPE, 50% formamide, 5x Denhardt’s solution, 1% SDS, 10% dextransulphate, and 150 µg denatured salmon sperm DNA per milliliter. The same solution without salmon sperm DNA was used for hybridization. The C44 probe template, corresponding to nucleotides 543–997, was made by amplification of the T/A vector insert using the nested primers in a PCR and gel purified using the QIAquick Gel Extraction Kit (QIAGEN). Membranes were reprobed with glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) as an internal standard. The GAPDH sequence was the almost full-length cDNA (15). The probes were labeled with [³²P]dATP (Amersham Pharmacia Biotech) using a random prime kit, Strip-EZ DNA (Ambion, Inc., Austin, TX) and this kit was also used for the stripping procedure. The final wash of the membranes was carried out in a solution containing 0.05x SSPE and 0.1% SDS at 65 C for 60 min. Autoradiography was used for detection.

Rapid amplification of cDNA ends (RACE)
The SMART RACE cDNA Amplification Kit (CLONTECH Laboratories, Inc.) was used according to the manual. Hepatic mRNA from 8-week-old female rats, isolated by the PolyATtract System 1000 kit (Promega Corp.), was used in the cDNA reaction. For the 5' RACE the following primers were used: GSP1, 5'-CAGCTCTACGGGCTTGCTCTCCTCCG-3'; and NGSP1, 5'-GCATACGGTAGCGGCAGGTAAAAGGAC-3'. For the 3' RACE the following primers were used: GSP2, 5'-CCTGATGTCCAGCACAAGGGAACGGC-3'; and NGSP2, 5'-GCTACCTAACCCATGCAGGAGGCGAACC-3'. The RACE products were gel purified using the QiaQuick Gel Purification Kit (QIAGEN) and cloned into an T/A vector (AdvanTage PCR Cloning Kit; CLONTECH Laboratories, Inc.) before being sequenced.

Bioinformatics
The GCG program package (Wisconsin Package, version 10.1; Genetics Computer Group, Madison, WI) was used for DNA sequence analysis. FASTA3 (16) and TBLASTN (17) were used for protein sequence comparisons toward the Swissprot (18) and the database at expressed sequence tags (dbEST) (http://ftp.ncbi.nlm.nih.gov/blast/db) databases, respectively. Multiple sequence alignments were constructed using ClustalW (19), into which the sequences were positioned according to results from the TBLASTN runs.

Solution hybridization
Total nucleic acids (tNA) were prepared from tissue samples and cultured hepatocytes as previously described (20). The concentration of nucleic acids in tNA samples was measured spectrophotometrically and the DNA concentration was quantified using a fluorometric assay (21). The level of C44 mRNA was analyzed using [³⁵S]uridine triphosphate-labeled complementary RNA (cRNA) transcribed from the pGEM3Z vector (Promega Corp.) into which the C44 sequence 786–991 was cloned at the EcoRI/HindIII polylinker sites. mRNA transcribed from the same vector construct was used as standard. The solution hybridization assay was carried out as previously described (22). Results are expressed as attomoles per microgram DNA.

In situ hybridization
The in situ hybridization was carried out on liver tissues from 8-week-old normal rats of both sexes as previously described (23) with minor modifications; the cryosections (8 µm) were air dried for 5 min, the hybridizations were performed directly after the wash in SSC, and the slides were exposed for 1 week. The same cRNA probe was used as for the solution hybridization assay.

Hepatocyte cultures
Hepatocytes were isolated from 8-week-old normal female rats and cultured on a substratum extracted from the Engelbreth-Holm-Swarm sarcoma as previously described (20), except that the medium used was William’s E. The medium was supplemented with insulin (0.1 µg/ml) and antibiotics. Cells were treated with varying doses of bGH at 72 h of culture age. After 20 h of the treatment, cells were harvested and tNA were prepared.

	Results and Discussion

Top Abstract Introduction Materials and Methods Results and Discussion References

 
To isolate rat liver transcripts dependent on a continuous female-characteristic exposure to GH, we used normal males exposed to sustained exogenous GH as tester, feminized males, and normal male rats as driver in a SSH experiment. The rational for using feminized males as tester instead of normal females was to obtain transcripts solely due to the presence of continuous GH, and not sex-dimorphic transcripts independent of GH status. We isolated 250 clones in the SSH assay as being potentially more expressed in the feminized male rat liver. Sequence determination of the cDNAs showed that 12 of the clones corresponded to the prototypical example of a highly expressed gene specifically induced by continuous GH, namely the CYP2C12 gene (24).This implied a positive outcome of the SSH experiment. One particular sequence named C44, distinct from CYP2C12, was found in 22 of the clones indicating that the corresponding gene would be expressed at high amounts in response to continuous GH. This was confirmed by Northern blot analysis of liver mRNA from normal rats of both sexes and from feminized males using the C44 sequence as probe (Fig. 1). An mRNA of about 1.8 kb was detected in female and feminized male samples (arrow), but not in normal male samples. After exposing the film four times longer, i.e. for 8 days, a very weak signal appeared in the male sample (data not shown). Thus, the C44 clone represents a sex-specific liver transcript regulated by GH.

View larger version (41K): [in this window] [in a new window]   Figure 1. Sex-differentiated and GH-dependent expression of C44 mRNA in rat liver. Total liver RNA (20 µg) from three normal females (F, lanes 1–3), three feminized males, i.e. normal males given GH continuously for 1 week (fM, lanes 4–6), and three normal males (M, lanes 7–9) was analyzed by Northern blot using a cDNA probe for C44. The arrow indicates C44, corresponding to approximately 1.8 kb. Reprobing the membrane with GAPDH served as an internal standard.

The C44 cDNA sequence did not correspond to any known rat gene and bioinformatic analyses were used in attempts to elucidate its nature. Comparison of the deduced amino acid sequence of the C44 cDNA with sequences in protein databases revealed 46% residue identity to the human 1B- glycoprotein (1B) (Accession No. P04217) (13). The deduced amino acid sequence of the C44 cDNA is depicted in Fig. 2 together with sequences of human 1B and opossum proteinase inhibitor (oprin). Oprin has been shown to have 35% residue identity with human 1B (26). Analysis of the human 1B sequence has indicated five repeating structural domains, with homology to the variable heavy and light chains of immunoglobulins, each containing about 95 amino acids and one disulfide bond. The translated full-length C44 sequence showed a similar architecture as the human 1B with five putative immunoglobulin-like domains, each containing two cysteins at identical positions as in the 1B. Furthermore, the consensus carbohydrate attachment sites in human 1B are also conserved in the rat sequence. This indicates that C44 is a rat homolog to the human 1B or a human 1B-like protein. The residue identity of 46% between the translated C44 cDNA and human 1B is relatively low. Evolutionary conservation of functional and structural properties is usually accompanied by protein identities of 60% or more (27). This leads to the question whether a family of 1B-like proteins may exist with homology to the immunoglobulin superfamily. Available sequences for pig, horse, and donkey are restricted to short amino-terminal sequences. For oprin, where a longer sequence is available, the amino-terminal part shows a higher degree of residue identity with human 1B than the complete sequence, 46% and 35%, respectively (26). If this also applies for the pig, horse, and donkey sequences, they will show a lower overall identity than what is stated in Table 1.

View larger version (88K): [in this window] [in a new window]   Figure 2. Multiple sequence alignment of 1B-homologous proteins. Residues given against the black background show identities between rat C44 (Accession No. AJ302031) and human 1B (Accession No. P04217) or oprin (Accession No. Q28359). Any of these residues also conserved with any of the EST or human Celera sequences are similarly shown against the black background. Other residues conserved between rat C44 and any of the EST or human Celera sequences are given against the gray background. Positional numbers are given above for the rat C44 sequence. The black bars indicate consensus carbohydrate attachment sites. Human ESTs 1+2+3 are W25099.1 (zb68b07.r1 Soares_fetal_lung_NbHL19W), AA484435.1 (nf07c12.s1 NCI_CGAP_Li1 IMAGE: 913078), and AW139382.1 (UI-H-BI1-adq-b-06–0-UI.s1 NCI_CGAP_Sub3). Human EST 4 is AA442553.1 (zv75e03.r1 Soares_total_fetus_Nb2HF8_9w). Mouse ESTs 1+2 are AA571166.1 [vl89 g07.r1 Stratagene mouse diaphragm (937303)] and AI390309.1 (mx01c08.y1 Soares mouse NML IMAGE: 678926). Rat EST 3+4 are AA250460.1 (mw99e06.r1 Soares mouse NML IMAGE: 678850) and AI509050.1 (va80 g10.y1 Soares mouse NML IMAGE:737730). Sequences hCP33335 and hCP38577 refer to putative proteins in the human genome from Celera in December 2000.

View this table: [in this window] [in a new window]   Table 1. Comparisons of 1B-homologous proteins of different species to the rat C44, given as percent identity (excluding gaps)

Searching the human and mouse expressed sequence tag (EST) databases and the Celera human genome database for sequences homologous to the C44 cDNA revealed four human and four rat ESTs and two Celera sequences (Fig. 2), which all upon comparison against the Swissprot database scored best with human 1B sequence (expect values typically ranging from 10^-60 to 10^-13). The distant homology between human 1B and immunoglobulin receptors (13) was also noticed in these comparisons, but expected values between any of the proteins in Fig. 2 and immunoglobulin receptors were only 10^-6 or higher, compatible with such a distant relationship. Thus we can conclude that the rat C44, human 1B, oprin, the Celera sequences and the human and mouse ESTs in Fig. 2 form a new family of 1B-related proteins. Even if the EST sequences contain sequence errors, the overall picture would not change, because the relationships between human 1B and the homologous EST sequences cover long segments with good confidence levels as judged by the low expected values from FASTA3 runs. Considering the relatively high rate of sequencing errors in EST end sequences it is conceivable that the human ESTs named 1+2+3 are the human 1B. Furthermore, the Celera sequence hCP33335 seems to be identical with human EST 4. From the alignment in Fig. 2 it can be seen that the mouse ESTs 3+4 show 85% residue identity with the rat C44, which indicates that these ESTs represent the mouse C44 ortholog (Table 1). Taken together these results have revealed C44 species orthologs and indicated the existence of a family of 1B-related proteins.

The SSH experiment would not only enrich for mRNAs up-regulated by the female-characteristic exposure to GH, but also for mRNAs down-regulated by the male GH secretion pattern. The Northern blot experiment in Fig. 1 also fails to clarify in what way the different GH profiles regulate the C44 expression. Therefore, the expression of the C44 mRNA was examined in hypophysectomized animals, devoid of GH, following sex-characteristic GH replacements. As shown in Fig. 3, only the hypophysectomized males given GH continuously, mimicking the female GH secretory pattern, and the normal females expressed the mRNA. An identical experiment using hypophysectomized females was also carried out and showed the same results (data not shown). Importantly, no difference was seen between normal and hypophysectomized males verifying the expression of rat C44 mRNA to be up-regulated by continuous GH, and not down-regulated by the male pattern of GH secretion.

View larger version (60K): [in this window] [in a new window]   Figure 3. GH-pattern-dependent induction of C44 mRNA. Total liver RNA from two normal males (M, lanes 1 and 2), two hypophysectomized males (HxM, lanes 3 and 4), two hypophysectomized males given GH continuously (HxMc, lanes 5 and 6), two hypophysectomized males given GH intermittently (HxMi, lanes 7 and 8), and two normal female rats (F, lanes 9 and 10) was analyzed by Northern blot using a cDNA probe for C44. The arrow indicates C44. Reprobing the membrane with GAPDH served as an internal standard.

There is no apparent sex difference in the secretory pattern of GH in rats younger than 25 days of age. The sexual difference starts to develop during the prepubertal period (25–30 days of age) and continues to mature during puberty (4). To examine whether there was a concomitant development of C44 mRNA expression, we analyzed the expression in livers from rats of different age. As shown in Table 2, no C44 mRNA was detected in male livers of any age investigated. In females, a low expression was detected at 35 days of age but not in younger animals. At 56 days of age, when the sexually dimorphic secretion of GH is manifest, the females expressed high levels of the C44 mRNA. A similar ontogenesis has been described for the female-specific and GH-regulated CYP2C12 (28).

View this table: [in this window] [in a new window]   Table 2. Liver expression of rat 1B-glycoprotein mRNA in normal animals of different age

View larger version (155K): [in this window] [in a new window]   Figure 4. Expression of C44 mRNA in situ. Light field images of hematoxylin-stained sections of normal female (A) and normal male (C) livers. Dark field image of in situ hybridization with C44 cRNA probe of normal female (B) and normal male (D) liver sections. A appears darker than C; this is due to the silver grains being visible in light field microscopy.

View larger version (12K): [in this window] [in a new window]   Figure 5. Effects of bGH on C44 mRNA expression in cultured hepatocytes from normal female rats. The hepatocytes were exposed to bGH for 20 h. The C44 mRNA levels were assessed by solution hybridization. Data, expressed as attomoles mRNA per microgram DNA, are the mean ± SD from two dishes, which were analyzed in triplicate.

	Acknowledgments

 
We are indebted to Dr. Michael Andäng for valuable help with the in situ hybridizations, and we thank Mrs. Elisabeth Wiersma-Larsson for skillful technical assistance.

	Footnotes

 
¹ This work was supported by grants from the Foundation for Strategical Research and the Swedish Medical Research Council (72XS-13146), the Swedish Research Council, the Novo Nordisk Foundation, and Karolinska Institutet.

Received December 18, 2000.

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