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Nucleic Acids Research, 2002, Vol. 30, No. 14 3286-3293
© 2002 Oxford University Press

Regulation of ribonuclease expression by estradiol in Rana catesbeiana (Bullfrog)

Pin-Chi Tang, Huey-Chung Huang1, Sui-Chi Wang, Jen-Chong Jeng and You-Di Liao*

Institute of Biomedical Sciences, Academia Sinica, 128, Yen-Chiu-Yuan Road, Sec. 2, Taipei 115, Taiwan and 1 Institute of Biochemistry, College of Medicine, National Taiwan University, Taipei 100, Taiwan

*To whom correspondence should be addressed. Tel: +886 2 2789 9167; Fax: +886 2 2782 9142; Email: ydliao{at}ibms.sinica.edu.tw
The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors
+AF039104, AF288642.2

Received November 5, 2001; Revised January 2, 2002; Accepted May 23, 2002

DDBJ/EMBL/GenBank accession nos+ .


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Multiple ribonucleases are widely found in living organisms, but the function and regulation of individual ribonucleases are still not clear. In the present study, we found that one oocytic ribonuclease, RC-RNase, is developmentally expressed in the liver and stored in the oocyte of the bullfrog, while another ribonuclease, RC-RNase L1, is constitutively expressed and retained in the liver at all stages. In females, the expression of RC-RNase increased with the degree of maturity and the concentration of plasma estradiol during oogenesis. In males, the RC-RNase gene was activated in the liver and the newly synthesized protein was secreted into plasma if estradiol was administered. To investigate the mechanism of estrogen-mediated activation of ribonuclease expression, we cloned the RC-RNase promoter and analyzed the putative transcription factor binding sites, e.g. TATA box, ERE, AP1 and CAAT box. Using luciferase as a reporter gene, we found that an estrogen response element in the promoter of RC-RNase was essential for both basic transcription and estradiol-mediated gene activation in estrogen receptor-positive MCF7 cells. These results support the hypothesis that RC-RNase is synthesized in the liver upon stimulation by estradiol during oogenesis, then secreted into the bloodstream and stored in oocytes for embryonic development.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Multiple ribonucleases in a living organism are widely found and they are involved in RNA metabolism and other biological functions (1). In humans, RNase 1 from pancreas is involved in RNA degradation, RNase 2 and RNase 3 from eosinophils exert neurotoxicity and RNase 5 from plasma induces blood vessel formation (2). However, the regulation of most ribonuclease gene expressions is still not clear except for human RNase 2 (eosinophil-derived neurotoxin; EDN) because it is activated and stored in differentiated eosinophils and causes allergic diseases (3,4). Recently, a group of cytotoxic ribonucleases was found in the oocytes and embryos of Rana spp. frogs (5–7). These frog ribonucleases differ from those of mammals in several novel properties, e.g. antitumor activities, substrate preference for pyrimidine– guanine, resistance to human ribonuclease inhibitor and specific location in the oocytes and embryos. To investigate the possible function of ribonuclease in bullfrog (Rana catesbeiana) embryonic development, we examined the expression of two distinct ribonucleases in the ribonuclease family (8). We found that one oocytic ribonuclease, RC-RNase, is transcribed in the liver rather than in the oocytes where RC-RNase is stored (9,10). The expression of RC-RNase increased with the maturity and plasma estradiol in females during oogenesis and it could be activated in males by estradiol. The activation of RC-RNase expression was mediated through an estrogen response element (ERE) in the promoter of the RC-RNase gene. In contrast, a liver-specific ribonuclease, RC-RNaseL1, in the family was constitutively expressed irrespective of sex, maturity and estradiol.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals and treatments
Native bullfrogs (R.catesbeiana) at different maturation stages were purchased from a local frog farm. All animals were kept separately with respect to sex and maintained in running water with a depth of 2 inches at constant temperature (25°C). According to physical parameters, female bullfrogs were divided into seven stages, from stage I, immature stage, to stage VII, ovulation stage. Ovulation was induced by intraperitoneal injection of 5–10 homogenized pituitary glands, which were dissected from female bullfrogs, in 0.5 ml phosphate-buffered saline (PBS). Bullfrogs subjected to estradiol treatment were injected (intramuscular) with 1,3,5[10]-estratriene-3,17-diol (estradiol) in propylene glycol (5–8 mg/100 g body weight). In the experiments presented here, at least three frogs were used in each group and all samples were taken during the breeding season (February to April).

Preparation of serum, plasma and tissue extracts
Animals were bled to obtain plasma and sera before being killed. Tissues were excised from frogs and homogenized in 5 vol extraction buffer (20 mM HEPES pH 8.0, 0.5 M NaCl, 0.5 mM EDTA). After centrifugation twice at 12 000 g for 30 min, the supernatant was collected and stored at –20°C before use. The concentration of protein was determined by the Bradford method (11). The levels of bullfrog plasma estradiol were determined by an ACS:180® Automated Chemiluminescence System (Bayer, NY) performed by United Clinical Laboratory (Taipei, Taiwan).

Preparation of total RNA
Tissues were dissected and cut into pieces <0.5 cm3 in size and kept in 5 vol RNAlaterTM (Ambion, Inc., TX) at 4°C before further processing. Total RNA from tissues was extracted by RNAzolTM B (TEL-TEST, Inc., TX) according to the manufacturer’s instructions.

Western blotting
Tissue extracts and sera were separated by 13.3% SDS–PAGE and contact-transferred to nitrocellulose membranes (Hybond-C extra, Amersham Pharmacia) in transfer buffer (10 mM Tris–HCl pH 7.5, 50 mM NaCl, 2 mM EDTA, 0.5 mM 2-mercaptoethanol) overnight (10). The membranes with immobilized proteins were washed briefly with PBS, blocked with 5% skim milk in PBS for 1 h, followed by incubation with rabbit antiserum against the N-terminus of RC-RNase (1:100) or the whole recombinant RC-RNaseL1 (rRC-RNase L1) (1:100) in blocking buffer (5% skim milk in PBS) for 1 h. After three washes in PBS, the membranes were incubated with goat anti-rabbit IgG antibodies conjugated with alkaline phosphatase (1:3000; Promega Corp., Madison, WI) in blocking buffer for 1 h, followed by three washes in 50 mM Tris–HCl pH 7.5, 150 mM NaCl. Bands were visualized by the addition of a bromochloroindolyl phophate/nitro blue tetrazolium substrate (9). The N-terminal 18mer oligopeptitide of RC-RNase ending with pyroglutamate was synthesized directly onto the branching lysine arms to form an immunogenic macromolecule without conjugating to a carrier protein.

Ribonuclease activity assay
Ribonuclease activity was analyzed by zymogram assay on RNA-casting 13.3% SDS–PAGE. After electrophoresis, the gels were washed twice with 25% isopropyl alcohol in 10 mM Tris–HCl pH 7.4 for 15 min each to remove SDS for protein renaturation. Following three washes in 10 mM Tris–HCl pH 7.5 at room temperature, the activity was visualized by 0.2% Toluidine blue O staining (9).

Reverse transcription (RT) and PCR
cDNA was synthesized by M-MuLV reverse transcriptase (RNase H–; New England Biolabs, Inc.) at 37°C for 1.5 h. The analysis of ribonuclease gene expression was performed by RT–PCR and the following specific primers were used to amplify the individual ribonuclease genes from cDNA: (i) RC 5' primer 5'-CAGAACTGGGCAACATTTCAG-3', nucleotides 310–330 of AF039104; (ii) RC 3' primer 5'-CTAAGGACATCGTCCTATTCC-3', nucleotides 645–625 of AF039104; (iii) RCL1 5' primer 5'-CAAGCACATCGAGCATCGATT-3', nucleotides 317–337 of AF288642.2; (iv) RCL1 3' primer 5'-CTTATTATTATCCGGGCTGGGGTC-3', nucleotides 558–535 of AF288642.2. The protocols for RT–PCR applied to the amplification of the ribonuclease gene were 95°C for 5 min, 30 cycles of 95°C for 40 s, 55°C for 40 s, 72°C for 2 min, and completed with 72°C for 2 min.

Cloning and mutation of the RC-RNase promoter
The promoter-like fragment of the RC-RNase gene was obtained using the Universal GenomeWalkerTM Kit and Advantage® Genomic PCR Kit (Clontech, Palo Alto, CA). After hanging with KpnI and HindIII recognition sites on the 5' and 3' ends, respectively, the promoter-like sequence was subcloned into a pGL3-basic luciferase reporter vector (Promega Corp.). Substitution mutants were obtained by the site-directed mutagenesis method using the following mutagenic primers: 5'-CAACTGTCAATTCCCCAATTTGGC-3' (169–192) for the pAP1 clone; 5'-CAATGACCTGATTTGGCTACAGAAG-3' (176–201) for the pCAAT clone; 5'-GGGAAATTCCAGCAGTTTGTGACACTTATTTAG-3' (232–264) for the pERE1 clone; 5'-TCCAGGTCATTGGTCAACTTATTTAGATAATG-3' (239–270) for the pERE2 clone; 5'-CACAACTTGCATATCCATACACATAATG-3' (460–433) for the pTATA clone. The recombinant vectors were isolated from Escherichia coli MC10–1693 by a Qiagen Plasmid Mega Kit (Hilden, Germany) and their sequences were confirmed by DNA sequencing.

Transient expression and luciferase activity assay
Human breast carcinoma cells MCF-7 were cultured in phenol red-free Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS at 37°C under 5% CO2. For transient expression, MCF-7 cells were seeded in DMEM with charcoal-stripped FBS in six-well plates. The individual recombinant vector containing the RC-RNase promoter and luciferase gene (0.4 µg) was cotransfected with a 0.03 µg pSV-ß-galactosidase vector (Promega Corp.) using EffecteneTM transfection reagent (Qiagen) according to the manufacturer’s instructions. Estradiol (1 nM, dissolved in DMSO) or DMSO only (0.1% v/v) was added into medium 4 h after transfection and cells were cultured for an additional 8 h before harvesting. Cells were washed twice with PBS and subjected to activity assays for luciferase and ß-galactosidase. The activity of luciferase was assayed using a Luciferase Assay System (Promega Corp.) and quantitated on a Lumat LB9507 luminometer (EG &G Berthold, Germany). The activity of ß-galactosidase was determined by ß-Gal reagents (o-nitrophenyl-ß-D-galactopyranoside in 0.1 M phosphate buffer, pH 7.5) and the absorbance at 420 nm was read. The promoter activity is expressed as a fold value by comparing the luciferase activity of the experimental promoter with the basic vector only and normalizing by the activity of ß-galactosidase.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Expression of ribonucleases in bullfrog tissues/organs
The ribonucleolytic activity of female bullfrog tissues/organs was detected by zymogram on RNA-casting SDS–PAGE. The ribonucleolytic activity of the ovary was the highest, followed by kidney, skin and pancreas in descending order of activity. At least two distinguishable ribonuclease species were revealed on the zymogram (Fig. 1A). Because the catalytic activities of some ribonucleases are very low compared to that of RC-RNase, we analyzed these ribonucleases by western blotting using specific antibodies raised against the N-terminus of RC-RNase or whole recombinant RC-RNase L1. The results shown in Figure 1B indicated that antibodies against the N-terminal peptide of RC-RNase recognized most purified bullfrog ribonucleases, RC-RNase, RC-RNase L1, RC-RNase 2, RC-RNase 3, RC-RNase 4 and RC-RNase 6 in descending order of recognition (8), whereas antibodies against rRC-RNase L1 recognized RC-RNase L1 and RC-RNase only. Consistent with the result of the zymogram assay, western blot using antibodies against RC-RNase also showed that the ovary and kidney contained RC-RNase and several other abundant ribonucleases with larger molecular size than that of RC-RNase, whereas the liver and serum contained only very small amounts of ribonuclease (Fig. 1C, top panel). Using antibodies raised against rRC-RNase L1, RC-RNase was found predominantly in the ovary and possibly in the spleen, but not in liver and serum, whereas RC-RNase L1 was widely distributed in most organs except in the skin and plasma based on the relative mobility on the blot (Fig. 1C, bottom panel).



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  		Figure 1. Expression of ribonucleases in adult female bullfrog. (A) Zymogram. Tissue extracts (1 µg each) and purified ribonucleases (0.025 µg each) were separated by RNA-casting 13.3% SDS–PAGE and stained by Toluidine blue O. (B) Western blotting of purified ribonucleases. Purified ribonucleases (0.15 µg each) were separated by 13.3% SDS–PAGE, transferred to a nitrocellulose membrane and probed with antibodies raised against the N-terminus of RC-RNase (left) and whole rRC-RNase L1 (right). (C) Western blotting of bullfrog ribonucleases. Tissue extracts (20 µg each) and purified ribonucleases (0.25 µg each) were separated by 13.3% SDS–PAGE, transferred to a nitrocellulose membrane and probed with anti-N-terminus of RC-RNase (top) and anti-rRC-RNase L1 (bottom) antibodies. (D) Fidelity of PCR. The coding regions of six bullfrog ribonuclease genes were cloned into pGEM-T vectors and used as DNA templates (1 ng each) with pairs of RC-RNase and RC-RNase L1 specific primers for PCR amplified with 30 cycles. (E) RT–PCR. cDNA from various tissues were used as templates and amplified with pairs of specific primers. RC, RC-RNase; RC2, RC-RNase 2; RC3, RC-RNase 3; RC4, RC-RNase 4; RC6, RC-RNase 6; RCL1, RC-RNase L1; Liv, liver; Ova, ovary; Kid, kidney; Pan, pancreas; Spl, spleen; Lun, lung; Hrt, heart; Int, intestine; Ski, skin; Ser, serum; pRC, cloned RC-RNase gene; pRC2, cloned RC-RNase 2 gene; pRC3, cloned RC-RNase 3 gene; pRC4, cloned RC-RNase 4 gene; pRC6, cloned RC-RNase 6 gene; pRCL1, cloned RC-RNase L1 gene; M, 100 bp marker; +, respective plasmid DNA.

 
Although most of ribonucleases were mainly localized in the ovary and kidney, the expression of individual ribonucleases in tissue was still not clearly identified because of their similar antigenicities and differential catalytic activities. We therefore designed pairs of specific primers to detect the specific mRNA of RC-RNase and RC-RNase L1 by RT–PCR. Figure 1D shows that only the specific RC-RNase gene product was obtained by PCR from individual or six mixed gene templates (to mimic total c-DNAs from tissues) using RC-RNase specific primers in the ribonuclease family (8), and similarly for RC-RNase L1. The PCR products were further confirmed by direct DNA sequencing. These results demonstrate that only authentic gene products were obtained by the respective pair of primers. Using these specific pairs of primers in RT–PCR, the mRNAs of the RC-RNase gene were found exclusively in the liver but not in other organs of adult female bullfrogs (Fig. 1E, top panel) and not in the liver of adult males (see Fig. 3B, top panel, lane C). The mRNA of RC-RNase L1 was detected in most organs of the female bullfrog, e.g. liver, kidney, spleen, lung, heart and intestine (Fig. 1E, bottom panel).



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  		Figure 3. Activation of ribonucleases by estradiol in bullfrog. (A) Zymogram. Tissue extracts (testis/ovary, 1 µg; serum, 6 µg; liver, 1.5 µg) of bullfrog after estradiol administration and purified ribonucleases (0.025 µg each) were analyzed for ribonuclease activity by RNA-casting SDS–PAGE and Toluidine blue O staining. (B) RT–PCR. Liver cDNAs from male and female bullfrogs were amplified by RT–PCR using pairs of specific primers. (C and D) Western blotting analysis of ribonucleases. Tissue extracts (testis/ovary, 40 µg; serum, 60 µg; liver, 40 µg) of bullfrog after estradiol treatment and purified ribonucleases (0.25 µg each) were separated by 13.3% SDS–PAGE, transferred to a nitrocellulose membrane and probed with antibodies raised against the N-terminus of RC-RNase and whole rRC-RNase L1. C represents bullfrog without estradiol treatment. E1, E2, E4, E7 represent 1, 2, 4 and 7 days after estradiol administration. Abbreviations as Figure 1.

 
Expression of ribonucleases at different maturation stages
To detect when RC-RNase gene expression is activated in female bullfrog development, three organs/tissues at different maturation stages were taken for analysis: ovary where RC-RNase is stored, liver where ribonucleases may be produced and plasma into which ribonucleases may be secreted. The development of female bullfrogs was divided into seven maturation stages based on morphology and weight of the ovary, and body weight (Table 1). Ovulating frogs were defined as being at the seventh stage. The result showed that ribonuclease activity began to appear in the ovary at stage II and in serum at stage III. These ribonucleases were probably RC-RNase, since RC-RNase is the most catalytically active in the family (8) and RC-RNase L1 is shown to be absent in oocytes and serum (Fig. 1C). One additional ribonuclease with low catalytic activity and slow mobility was found in the plasma after stage V (Fig. 2A, middle panel). With regard to the ribonuclease activities in the liver, a predominant ribonuclease was expressed from stage I to stage VI and did not fluctuate with maturity until stage VII (Fig. 2A, bottom panel).


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  		Table 1. Physical parameters of bullfrog at different maturation stages
 


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  		Figure 2. Expression of ribonucleases in female bullfrog at different maturation stages. (A) Zymogram. Ribonuclease activities of tissue extracts (ovary, 0.1 µg; serum, 3 µg; liver, 1.5 µg) at different maturation stages and purified ribonucleases (0.025 µg each) were assayed by RNA-casting 13.3% SDS–PAGE and Toluidine blue O staining. (B) RT–PCR. Liver cDNAs from bullfrog at different maturation stages were analyzed for the expression of ribonuclease genes using pairs of specific primers. (C and D) Western blotting analysis of ribonucleases. Tissue extracts (ovary, 40 µg; serum, 60 µg; liver, 40 µg) at different maturation stages and purified ribonucleases (0.25 µg each) were separated by 13.3% SDS–PAGE, transferred to a nitrocellulose membrane and probed with antibodies raised against the N-terminus of RC-RNase and whole rRC-RNase L1. Classification of maturation stages is described in Table 1. Abbreviations as Figure 1.

 
Western blotting using antibodies against RC-RNase showed that at least two abundant ribonucleases appeared in the ovary after stage II and some minor ribonucleases appeared in plasma after stage III. Several ribonucleases existed in the liver at all maturation stages (Fig. 2C). Using antibodies against rRC-RNase L1, we found that RC-RNase, which was recognized by the antibody, accumulated in the ovary after stage II, while RC-RNase L1 was present in the liver at all stages. However, neither proteins were found in plasma at all stages probably due to their concentrations being too low to be detected by western blotting (Fig. 2D).

We next analyzed the mRNA of RC-RNase and RC-RNase L1 in female bullfrog liver at different maturation stages, by RT–PCR using pairs of specific primers. We found that the transcription of the RC-RNase gene was activated in the liver after stage II, while the RC-RNase L1 gene was constitutively expressed at all stages (Fig. 2B). Transcription of the RC-RNase gene in the liver and storage of RC-RNase protein in the ovary, but not in serum, at stage II suggests that only a small amount of RC-RNase begins to be activated and synthesized in the liver, secreted into bloodstream and immediately stored in the oocytes at stage II. The concentration of RC-RNase in serum at this stage is still too low to be detected.

Activation of RC-RNase expression by estradiol
Activation of RC-RNase gene expression in the liver and appearance of RC-RNase in the ovary are consistent with the threshold increase of ovary weight from 2 to 10 g per frog and change of ovary morphology from transparent to gray following stage II. Plasma estradiol also increased from ~200 pg/ml at stage II to ~300 pg/ml at stage III and reached a plateau ~1800 pg/ml at stage VI (Table 1). Because of the absence of RC-RNase and a low level of plasma estradiol (<200 pg/ml) in males, estradiol was administered into male bullfrogs with body weights ranging from 550 to 600 g. Females with similar body weights classified as approximately stages V–VI were used for comparison.

According to our zymogram assay, the ribonuclease activity in the plasma of male bullfrogs gradually increased in the first 4 days after estradiol treatment and reached a maximum at day 7 (Fig. 3A, middle panel), then decreased slightly at day 14 (data not shown). The activity in liver and testis of males only slightly increased after 7 days of treatment. In females, the ribonuclease activity in the plasma of females also increased 7 days after treatment, but the activity in the ovary did not significantly increase because the endogenous ribonucleases of untreated frogs had already been expressed and stored by this stage (Fig. 3A, top panel). The major induced ribonuclease shown by the zymogram was probably RC-RNase because of its high specific activity in the ribonuclease family and relative mobility on the gel.

To identify the expression of individual ribonuclease in the ribonuclease family, western blotting was employed using antibodies against the N-terminus of RC-RNase or rRC-RNase L1. Similar to the results of the zymogram assay, RC-RNase appeared gradually in the plasma in the first 4 days after estradiol treatment and reached a maximum at day 7 (Fig. 3C, middle panel), then decreased slightly at day 14. The ribonucleases in the testis and liver of the male bullfrog appeared only at day 7 (Fig. 3C). In females, one ribonuclease with similar mobility to that of RC-RNase in serum and liver increased at day 7, but two abundant ribonucleases in the ovary did not significantly increase. In contrast, RC-RNase L1 was constitutively expressed in the liver of both male and female bullfrogs, but not in the testis and plasma of male bullfrogs after estradiol treatment (Fig. 3D). The ribonuclease in the ovary was suspected to be RC-RNase rather than RC-RNase L1 because the latter was absent in the ovary.

To further identify the expression of the individual ribonuclease gene activated by estradiol, RT–PCR was used to detect specific mRNA of the RC-RNase or RC-RNase L1 gene in the liver. The expression of RC-RNase was slightly activated 1 day after estradiol treatment, reached a maximum at day 4 and began to decline at day 7. Similar to our findings from western blotting, the RC-RNase L1 gene was constitutively expressed in the liver of both male and female frogs (Fig. 3B).

In conclusion, the transcription of the RC-RNase gene in the liver, subsequent appearance of RC-RNase in the plasma and the accumulation of RC-RNase protein in specific organs after estradiol treatment suggest that the RC-RNase gene is transcribed and translated in the liver, then secreted into the bloodstream and designated to specific organs, e.g. ovary and kidney in females. In contrast, the RC-RNase L1 is constitutively expressed and compartmentalized in the liver irrespective of sex or estradiol treatment.

Cloning and analysis of the RC-RNase promoter
To further elucidate the mechanism of estradiol-responsive activation of RC-RNase gene expression, we cloned a 493 bp fragment upstream from the RC-RNase gene from bullfrog genomic DNA. The putative promoter sequence contained several potential transcriptional factor binding sites, e.g. AP1, CAAT box and TATA box, as shown in Figure 4. Furthermore, we also found in this fragment a 13mer ERE, GGTCATTGTGACA, with an inverted repeat sequence. The promoter activity of the DNA fragments was assayed by the relative luciferase activity driven by the DNA fragment in pGL3 to the control pGL3 basic vector in estrogen receptor (ER)-positive MCF7 cells. The results showed that promoter activity of the 493 bp fragment was 37-fold greater than that of control pGL3 vector, and that it increased further to 97-fold under 1 nM estradiol treatment.



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  		Figure 4. Nucleotide sequence of RC-RNase promoter. The possible sites for AP1, CAAT box, TATA box and ERE in the RC-RNase promoter sequence are shown in boxes and mutations were made for function assay. Bases substituted are shown in bold and italics: mutation of GA->TC at the AP1 site; mutation of CA->TG at the CAAT box; mutation of AA->GG at the TATA box, while four bases were substituted at the first half (GTCA->CAGT, named ERE1) and second half (TGAC->GTCA, named ERE2) of ERE. The RC-RNase promoter fragment and its mutants were subcloned into a pGL3-basic vector containing a luciferase reporter gene through restriction enzyme sites, KpnI and HindIII (underlined).

 
To further analyze the detailed structure of the promoter sequence, we mutated and subcloned the ERE and potential binding sites in the pGL3 vector and referred to these clones as follows: pRC for the wild type RC-promoter; pERE1,2 for the modification of the left and right inverted repeat sequence of the ERE; pAP1, pCAAT and pTATA for the modification of the AP-1 binding site, CAAT box and TATA box, respectively. The promoter activities of both pERE1 and pERE2 decreased from 37- to 3-fold and were not restored upon estradiol treatment (Fig. 5). These results indicate that both the left GTCA and right TGAC inverted repeat sequence of the ERE are essential for basic transcription and responsiveness to estradiol activation. The promoter activity of pTATA decreased from 37- to 3-fold, but it was reactivated to 8-fold by estradiol. These findings suggest that the TATA box is essential for basal transcription but not involved in estradiol responsiveness because the mutated pTATA clone is still responsive to estradiol activation with an ~3-fold greater stimulation than that of wild type RC-RNase promoter. The promoter activities of mutated pCAAT and pAP1 decreased from 37- to 22- and 15-fold, respectively, and they were reactivated to 70- and 41-fold, respectively, by estradiol treatment. These results suggest that the CAAT box and AP1 site are also involved in the transcription of the RC-RNase gene, but not involved in estradiol responsiveness. Together, our findings demonstrate that the 493 bp fragment is the promoter of the RC-RNase gene, and that the 13mer ERE is essential for both transcription and responsiveness for estradiol activation.



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  		Figure 5. Promoter activity assay by transcient expression of luciferase in MCF-7 cells. The wild type RC-RNase promoter (pRC) and its mutant constructs (pAP1, pCAAT, pERE1, pERE2 and pTATA; as described in Fig. 4) were cotransfected with a pSV-ß-galactosidase vector into MCF-7 cells in phenol red free DMEM medium supplemented with 10% charcoal-stripped FBS. Estradiol in DMSO (1 nM) or DMSO only (0.1% v/v) was added 4 h after transfection and incubated for another 8 h before harvesting. The increase of luciferase activity is expressed as a fold value by comparing the luciferase activity of the experimental promoter to the basic vector only and normalizing by the activity of ß-galactosidase.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Ribonucleases are widely distributed in living organisms and suggested to be involved in RNA metabolism and other biological functions. Some of them are well-characterized biochemically, e.g. RNase A from bovine pancreas and EDN from human eosinophils (1,2). Multiple ribonucleases are found in the same organism and characterized, e.g. six ribonucleases from humans (2), but the regulation of most ribonuclease gene expression is still not clear. In bullfrog, there are at least 10 ribonuclease genes in the ribonuclease family (12). In the present study, we demonstrate for the first time that the expression of one oocytic ribonuclease, RC-RNase, is activated in the liver of either male or female bullfrog by estradiol, whereas one liver ribonuclease, RC-RNase L1, is constitutively expressed in the liver. The present study also showed that the expression of oocytic ribonucleases is regulated by estradiol and synthesized in the liver of the bullfrog at late stages of oogenesis, whereas the liver ribonuclease is constitutively expressed in the liver irrespective of sex, maturity and estradiol. Consequently, our discovery of an ERE in the promoter of RC-RNase could explain the regulatory expression of RC-RNase during oogenesis and the inducible expression of RC-RNase in male frogs.

The biosynthetic pathway of RC-RNase is quite similar to that of vitellogenin, an abundant storage protein, in Xenopus laevis oocytes. In addition to their co-localization in yolk granules, both RC-RNase and vitellogenin are activated by estradiol, produced in liver, secreted into bloodstream and then stored in the yolk granule of oocytes (13). Both genes have a 13mer ERE in their promoters just with one nucleotide difference (14). It is generally accepted that the estrogen responsive activation of vitellogenin gene is mediated through the activation of an ER which contains both DNA-binding and hormone-binding sites. The binding of estradiol to the ER increases the affinity between the ER and the ERE in the promoter of target gene, i.e. vitellogenin, and then activates the expression of the target gene (15). Based on our results on RC-RNase and its resemblance to vitellogenin, it is suggested that RC-RNase expression is developmentally regulated in the liver by estradiol through the ER and the 13mer ERE, then the newly synthesized RC-RNase is secreted into the bloodstream and stored in the yolk granules of oocytes possibly through the presence of RC-RNase-specific receptors for embryonic development.

Since multiple and homologous ribonucleases exist in the same organism, e.g. 10 homologous ribonuclease genes in bullfrogs, it is difficult to define the function and analyze the expression of each individual ribonuclease unless the mRNA, protein and catalytic activity of each gene are specifically identified. In the present study, we demonstrate that RC-RNase gene transcription is activated 1 day after estradiol treatment in the liver detected by RT–PCR, while RC-RNase protein is also synthesized in the liver and then secreted into the bloodstream, detected by western blotting and zymogram. The work presented here regarding the regulation of ribonuclease gene expression, together with our development of a sensitive and specific detection method, will enable us to further investigate the particular function of ribonucleases in bullfrog development.


   ACKNOWLEDGEMENTS
 
We thank Drs Y. S. Lin, T. C. Lee, S. Lin-Chao and Mr D. J. Platt for their critical reading of the manuscript. This work was supported by Academia Sinica, the National Science Council of the Republic of China (NSC88-2316-B-001–007) and National Health Research Institutes.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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