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Endocrinology Vol. 145, No. 10 4763-4774
Copyright © 2004 by The Endocrine Society

Structural Characterization of Three Novel Rat OKL38 Transcripts, Their Tissue Distributions, and Their Regulation by Human Chorionic Gonadotropin

Choon Kiat Ong, Chuan Young Ng, Caine Leong, Chee Pang Ng, Chye Sun Ong, Thi Thanh Tuyen Nguyen and Hung Huynh

Laboratory of Molecular Endocrinology, Division of Cellular and Molecular Research, National Cancer Centre of Singapore, Singapore 169610

Address all correspondence and requests for reprints to: Hung Huynh, Laboratory of Molecular Endocrinology, Division of Cellular and Molecular Research, National Cancer Centre of Singapore, 11 Hospital Drive, Singapore 169610. E-mail: cmrhth{at}nccs.com.sg.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We previously identified a novel pregnancy-induced growth inhibitory gene, OKL38. To develop a rat model for further characterization of OKL38’s role in the initiation and progression of breast and ovarian cancer, we now report the cloning and characterization of three novel rat OKL38 cDNAs that are derived through alternative splicing and differential promoter usage. These three transcripts differ in their 5' untranslated regions but share a common open reading frame that encoded for a 52-kDa protein. OKL38 is mapped to chromosome 19, spanning a region of approximately 15 kb, and contains eight exons. Differential expression of these three rat OKL38 transcripts was observed in liver, kidney, ovary, mammary gland, and uterus. In situ hybridization localized the rat OKL38 transcripts to the luminal epithelial cells of the rat mammary gland and to the granulosa cells in the rat ovary. In vivo studies showed that the RtOKL38-2.0 transcript and protein were regulated by human chorionic gonadotropin in the rat mammary gland and ovary. Importantly, overexpression of RtOKL38-enhanced green florescence protein fusion protein in Buffalo rat liver cells resulted in growth inhibition and cell death. Our present findings suggest that OKL38 may function as an effector for human chorionic gonadotropin protection against mammary carcinogenesis, and the availability of the three rat OKL38 cDNAs may help to elucidate the possible role of OKL38 in cellular growth, differentiation, and carcinogenesis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
WOMEN WHO HAVE their first pregnancy after 30 yr of age have a 2- to 5-fold higher lifetime risk of developing breast cancer compared with those whose first pregnancy occurs before 20 yr of age (1, 2). The protective effect of pregnancy against the development of cancer is believed to be attributed by an increase in cellular differentiation and the reduced rate of cell proliferation in the mammary epithelium (3). Russo et al. (4, 5, 6) demonstrated that 7, 12-dimethylbenz (a) anthracene (DMBA)-induced mammary carcinogenesis in the female rat is effectively inhibited by either pregnancy or human (h) chorionic gonadotropin (CG) treatment. The protective effect of hCG on the development of mammary tumors has been variously attributed to induction of differentiation, inhibition of cell proliferation, increase in DNA repair capabilities, induction of inhibin by the mammary epithelium, activation of programmed cell death, and down-regulation of IGF-I and IGF-I receptors in mammary epithelial cells (4, 5, 6, 7, 8, 9).

The CG hormone is a heterodimeric placental glycoprotein essential for normal reproductive function (10) and acts to maintain high progesterone levels during pregnancy (11). CG stimulates the production of gonadal steroid hormones via its interaction with the lutropin-choriogonadotropin-receptor (LH-CG-R) present in the granulosa cells of the ovary and in the testicular Leydig cells (12). Upon binding of CG to its receptor, adenylyl cyclase activity is elevated through an intracellular membrane-associated G protein-coupled mechanism that results in an increase in cAMP levels, leading to the synthesis of steroid and polypeptide hormones (12). Recently, Jiang et al. (13) have shown that hCG and inhibin induce histone acetylation in human breast epithelial cells, which could be an alternative route of gene regulation.

We have previously isolated a pregnancy-induced growth inhibitory gene, OKL38 (14), that is ubiquitously expressed in all rat tissues with the highest levels detected in the ovary, kidney, and liver. OKL38 expression was increased during pregnancy and lactation in the rat mammary gland; however, low levels of OKL38 transcripts were observed in various human breast cancer cell lines and barely detectable in DMBA-induced rat mammary tumors. Transfection of MCF-7 cells with OKL38 cDNA resulted in growth inhibition in vitro and reduction in tumor formation in vivo, suggesting that OKL38 may play a vital role in the growth regulation and differentiation of breast epithelial cells during pregnancy and tumorigenesis (14). The role of OKL38 in cancer is further supported by our recent study showing that the protein is lost or down-regulated in kidney tumor (15). The molecular mechanisms by which OKL38 exerts its role in growth inhibition and differentiation are still unknown.

As a first step in understanding function and regulation, herein we report the cloning of three rat OKL38 cDNAs, their tissue distributions, and their function. Because OKL38 has been implicated in tumorigenesis and its expression is induced during pregnancy and lactation (14), the in vivo effect of hCG on its regulation was also investigated.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Animal were maintained and treated according to the protocol approved by the institutional animal care committee. Commercially available hCG (Profasi, Laboratoires Serono S.A., Aubonne, Switzerland) was used for the investigation. Twenty-four female Sprague Dawley rats (50 d old and weighing approximately 250 g) were divided into four groups. Each group (n = 6) received daily ip injection of either PBS or three doses of hCG (10, 20, and 40 IU in 200 µl of PBS) for 21 d. After the last injection, the rats were allowed to rest for an additional 2 d before they were killed. The ovary, kidney, liver, and mammary gland were harvested, frozen in liquid nitrogen, and stored at –80 C.

Probe labeling
All probes used for library screening as well as for Southern and Northern blot analyses were radioactively labeled as described previously (15).

Isolation of rat OKL38 cDNA
To clone the rat OKL38 cDNA, the human OKL38 1.6 kb cDNA (GenBank accession no. AF191740) was used as a probe to screen about 1 million plaques generated from the Rat Liver 5'-STRETCH PLUS cDNA library (Clontech, Palo Alto, CA). Positive clones were subjected to a secondary screen, and Southern blot analysis was used to confirm the identity of the clones. Positive clones were fully sequenced using 5' (5'-TCCGAGATCTGGACGAGC-3') and 3' (5'-TAATACGACTCACTATAGGG-3') sequencing primers. The authenticity of the clones was verified using automated sequencing via dideoxy chain termination using the BigDye version 3.0 (PerkinElmer, Boston, MA).

RNA isolation and Northern blot analysis
Total RNA was extracted using Trizol Reagent (Invitrogen, Carlsbad, CA). The poly (A)+ mRNA was isolated from the indicated organs and tissues of female rats using the Oligotex mRNA Kit as described by the manufacturer (Qiagen, GmbH, Hilden, Germany). Northern blot analysis was performed on poly (A)+ mRNA or total RNA as described (16). Blots were hybridized with rat OKL38 probe (nucleotide position 926-2004) that detects all three variants, variant-specific probe (nucleotide position 6–395 of RtOKL38-2.3 transcript), which detects only the RtOKL38-2.3 and -2.3A transcripts, or glyceraldehyde-3-phosphate dehydrogenase (American Type Culture Collection, Manassas, VA). mRNA levels were determined by densitometric scanning of autoradiographs and normalized to the level of glyceraldehyde-3-phosphate dehydrogenase.

Rapid amplification of cDNA end (RACE) analysis
To establish the full-length cDNA of rat OKL38, 5'RACE was performed using the SMART RACE cDNA amplification kit (Clontech) as previously described (15). Gene-specific primer, 1R (Table 1Go), was designed based on the 5' end sequence of the isolated rat OKL38 cDNA. Cloning and sequence analysis of RACE products were performed as previously described (15).


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  		TABLE 1. Sequences of oligonucleotides used for RT-PCR primer, cloning primer, sequencing primer, and 5'RACE primer

 
Cloning cDNA of RtOKL38-2.0, -2.3, and -2.3A cDNAs via PCR
Cloning of the full-length cDNA of the RtOKL38-2.0 transcript was achieved using two PCR primers, forward primer FL-2.0F (nucleotide position 12–34) designed at the 5' end and reverse primer FL-2.0R (nucleotide position 1937–1959; Table 1Go) designed at the 3' end of the 2.0 cDNA. To clone the RtOKL38-2.3 and -2.3A cDNAs, forward primer FL-2.3F (nucleotide position 6–30 of 2.3 transcript; Table 1Go) was used instead of the FL-2.0F primer (see Fig. 2BGo). In all cases, the forward primer carried a HindIII restriction site and the reverse primer carried a NotI restriction site for directional cloning. To generate the cDNA template for PCR cloning, 1 µg of rat kidney poly (A)+ mRNA was reversed transcribed using SuperScript II reverse transcriptase (Life Technologies, Carlsbad, CA) as recommended by the manufacturer. The template was amplified by PCR under the following conditions: denaturation step at 94 C for 2 min, followed by 35 cycles of 94 C for 1 min, 65 C for 1 min, 72 C for 2 min 30 sec, and a final extension at 72 C for 5 min. PCR products of the correct size were cloned into pBluescript SK (Stratagene, La Jolla, CA) for sequencing. Two more internal primers, 2R and 3R (Table 1Go), were also used for sequencing.



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  		FIG. 2. Genomic structure of rat OKL38 gene and its mRNA isoforms. A, Schematic illustration of the genomic structure of rat OKL38 showing differential splicing at the 5' region of the gene. The two forward arrows at the 5' end of exons 1 and 3 indicate the two putative promoters, and the dotted line indicates alternative splicing of exon 3. The exon numbers are indicated on top of the diagram. B, Schematic diagram showing the various rat OKL38 mRNA isoforms. Different shaded boxes below the diagram represent the exons present in each isoform. Arrows indicate the translational start and termination sites. The first shaded box of RtOKL38-2.0 indicates the first 43 nucleotides that lie in intron 2, which is unique to this transcript only. Above are position of primers used in this study.

 
Due to the low expression level of the RtOKL38-2.3A transcript, additional secondary PCR was performed for its cDNA isolation. After PCR amplification, the region below the 2.3-kb band was purified using the gel extraction kit (Qiagen), and the extracted DNA was subjected to a second round of PCR amplification under the same conditions. A 2.2-kb fragment was obtained, cloned, and sequenced as described earlier.

Semiquantitative One Step RT-PCR of rat OKL38 variants
Poly (A)+ mRNA (100 ng) was used as template for One Step RT-PCR (Qiagen), adopting the procedure recommended by the manufacturer. To study the expression of the RtOKL38-2.0 transcript, variant-specific forward primer RTPCR-2.0F (nucleotide position 24–44) and a common reverse primer 2R (Table 1Go) were designed for RT-PCR. To determine the expression of RtOKL38-2.3 and -2.3A transcripts, RT-PCR was performed using the forward primer FL2.3F and reverse primer 2R (Table 1Go). The specificity of the designed primers and PCR conditions were optimized using the three cloned cDNAs, RtOKL38-2.0, -2.3, and -2.3A, as templates. To optimize the number of cycles for RT-PCR to detect the three different variants and to prevent an amplification plateau for any one transcript, kidney mRNA (expressing high levels of all three transcripts) was amplified through 23, 25, 27, 29, 31, and 33 cycles. The One Step RT-PCR was performed as previously described (15). The optimal cycles for detection of the RtOKL38-2.0 and -2.3 transcripts were 25 and 31 cycles, respectively. A pair of tubulin primers, TubF and TubR (Table 1Go), which amplify a 400-bp fragment of rat tubulin cDNA, was used for normalization. The amplified products were separated on a 2.0% agarose gel.

In vitro transcription and translation (TNT) study and Western blot analysis
To verify the predicted molecular weight of the putative open reading frame (ORF) of rat OKL38, the TNT Coupled Reticulocyte Lysate System (Promega, Madison, WI) was used to transcribe and translate the RtOKL38-2.0, -2.3, and -2.3A cDNAs according to the manufacturer’s instructions as previously described (15). The synthesized protein was labeled with [35S] L-methionine (ICN, Costa Mesa, CA), and 5 µl of the reaction was loaded and electrophoresed on 8% SDS-PAGE. The synthesized protein was transferred onto a nitrocellulose membrane and exposed to film. To confirm the identity of the in vitro synthesized protein, Western blot analysis was performed on the TNT products using rabbit anti-OKL38 antibodies. This antiserum was obtained from rabbits immunized with a polypeptide generated from amino acid position 343–366 (QMMRDQSILSPSPYEGYRSLPEHQ) of rat OKL38. Western blot analysis was performed as described previously (15). The blots were incubated with 1:2000 anti-OKL38 and 1:7500 horseradish peroxidase-conjugated donkey antirabbit secondary antibodies. After washing, the blots were then visualized with a chemiluminescent detection system (Amersham Pharmacia Biotech, Arlington Heights, IL) as described by the manufacturer.

In situ hybridization
To determine the cell-specific expression of OKL38, in situ hybridization was performed using sections derived from mammary tissues and ovaries of controls and 20 IU hCG-treated rats. To generate sense and antisense rat OKL38 probes, a 542-bp fragment (nucleotides position 925-1466) of rat OKL38 cDNA (GenBank accession no. AY081218) was cloned into the pBluescript SK plasmid. The plasmid construct was linearized, and digoxigenin-labeled sense and antisense RNA probes were synthesized using the digoxigenin RNA labeling kit (Roche Molecular Biochemicals, Indianapolis, IN) as described by the manufacturer. Fresh tissues were treated with PBS containing 0.5% active diethyl pyrocarbonate for 10 min, embedded in Jung-tissue freezing medium (Leica Instruments, Nussloch, Germany), and stored at –80 C. Serial 7- to 8-µm sections were heated at 50 C for 2 min to immobilize the tissue onto the slide. To minimize nonspecific background caused by lipid vesicles, the sections were delipidized for 5 min in chloroform, dried at room temperature, and fixed in PBS containing 4% paraformaldehyde. Prehybridization, hybridization, washing, and immunological detection were performed as described by Braissant and Wahli (17).

Generating sense RtOKL38-enhanced green florescence protein (eGFP)- and eGFP-Antisense-RtOKL38-pcDNA3.0 constructs
The rat OKL38 cDNA contained an ORF of 478 amino acids. To fuse this OKL38 protein to the eGFP via PCR, four primers (478-F, 478-eGFP-R, eGFP-F, and eGFP-R; Table 1Go) were designed. Two separate PCR reactions were performed using the primers 478-F and 478-eGFP-R to amplify the ORF of OKL38 and primers eGFP-F and eGFP-R to amplify the ORF of eGFP. The amplified products from the two PCR reactions were then mixed and reamplified using primers 478-F and eGFP-R. The PCR reaction was performed as follows: 95 C for 5 min, followed by 25 cycles of 94 C for 1 min, 55 C for 1 min, 72 C for 2 min, and final extension at 72 C for 5 min. The recombinant products were cloned into pCR-Blunt-II-TOPO vector (Invitrogen), screened for orientation, and subsequently cloned into pcDNA3.0 (Invitrogen) mammalian expression vector. The positive control eGFP-pcDNA3.0 was constructed with the same strategy using only the eGFP-F and eGFP-R primers for PCR cloning. As for the eGFP-Antisense-RtOKL38-pcDNA3.0 construct, the rat OKL38 ORF was inserted in the reverse order into the C terminus of eGFP protein from the eGFP-pcDNA3.0 construct. All three constructs were fully sequenced, and endotoxin-free plasmid for transfection was prepared using Maxi-prep kit (Qiagen).

Cell culture and transfection
Buffalo rat liver (BRL) cells were maintained as monolayer cultures at 37 C (5% CO2) in DMEM plus phenol red, supplemented with 10% fetal bovine serum, 1% penicillin, and 1% streptomycin (Life Technologies, Grand Island, NY). BRL cells were seeded at 2 x 105 in 100-mm culture dishes containing cover slip swab with ethanol and grew to 70% confluence before transfection. Cells were transfected with 10 µg of eGFP-, RtOKL38-eGFP-, or eGFP-Antisense-RtOKL38-pcDNA3.0 plasmid DNA and 12 µl of Lipofectamine reagent (Life Technologies) following the manufacturer’s recommendations. Each cover slip was removed at 24, 48, 72, 96, and 120 h post transfection using sterile forceps. The cover slip with cells was fixed with 10% formalin, washed with PBS, and mounted onto slide for observation using a microscope (Olympus, Tokyo, Japan) equipped with epifluorescence optics and appropriate filters for fluorescein isothiocyanate.

Statistics analysis
Differences in OKL38 gene expression were analyzed by the Mann-Whitney U test.

Computational analysis
The nucleotide and protein sequences were analyzed as previously described (15).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Isolation of rat OKL38 cDNA
We have previously reported the cloning of human OKL38 cDNA (GenBank accession no. AF191740) from a human ovarian cDNA library. OKL38 transcripts were detected at high levels in the ovary, kidney, and liver (14, 15). To develop a rat model for further characterization of the role of OKL38 in growth, differentiation, and during tumorigenesis, we proceeded to isolate the rat OKL38 cDNA. The human OKL38 cDNA was used as a probe to screen rat liver cDNA library. Four positive plaques were isolated, with the longest insert of 1093 bp. Blast search with the nonredundant nucleotide database (GenBank) showed that the nucleotide sequence of the isolated rat OKL38 cDNA was 85% and 90% identical to our previously published human OKL38 cDNA (14, 15) and the mouse counterpart IMAGE:37844 (GenBank accession no. BC022135), respectively.

Expression of OKL38 in various rat tissues
To determine the tissue distribution of rat OKL38 transcripts, Northern blot analysis was performed using 5 µg of poly (A)+ mRNA isolated from various rat tissues and hybridized with rat OKL38 probe. Transcripts of approximately 2.0 kb and 2.3 kb were detected in the kidney, whereas the 2.0-kb transcript was ubiquitously expressed in liver, ovary, mammary gland, and uterus. Larger putative transcripts ranging from 4.0–9.0 kb were also observed (Fig. 1AGo). The overall expression pattern of rat OKL38 mRNA coincided with our previous reports (14, 15).



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  		FIG. 1. Expression of OKL38 transcripts in the rat tissues. Five micrograms of poly (A)+ mRNA derived from indicated tissues were subjected to Northern blot analysis. Blots were hybridized with rat OKL38 cDNA probe, which detects all possible variants (A), variant-specific cDNA probe, which detects the RtOKL38-2.3 and -2.2 transcripts (B) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA (C). Larger transcripts ranging from 4.0–9.0 kb were also detected. D, Schematic diagram of One Step RT-PCR strategy. The sequence from nucleotide position 1–43 of RtOKL38-2.0 was unique to this transcript and thus variant-specific forward primer, RTPCR2.0F, was designed in this region for expression study. FL2.3F forward primer and the common reverse primer 2R were used to determine the expression of RtOKL38-2.3 and -2.3A. Tissue distributions of rat OKL38 variants using One Step RT-PCR are shown (F–H). E, Result of PCR verifying the specificity of primers used for One Step RT-PCR. Lanes 1 and 4, RtOKL38-2.3 cDNA; lanes 2 and 5, RtOKL38-2.2 cDNA; and lanes 3 and 6, RtOKL38-2.0 cDNA. Reactions in lanes 1–3 contained both FL2.3F and 2R primers, whereas lanes 4–6 contained the RTPCR2.0F and 2R primers. Note that primers were specific for detection of all three variants. One Step RT-PCR was performed using 100 ng of poly (A)+ mRNA obtained from rat tissues. The 432-, 791-, and 690-bp fragments correspond to the RtOKL38-2.0, -2.3, and -2.3A variants, respectively. RT-PCR was performed using primers RTPCR2.0F and 2R (F), primers FL2.3F and 2R (G), and a pair of tubulin primers (I) as internal control. The amplified products were separated on a 2.0% agarose gel and transferred onto nylon membrane. Southern blot analysis was performed using variant-specific probe (H). Li, Liver; Ki, kidney; Ov, ovary; Mg, mammary gland; Ut, uterus.

 
5'RACE
The Northern blot results showed that the rat OKL38 transcript is approximately 2 kb in size, and the longest cDNA we isolated was 1093 bp. To establish the full-length transcripts, 5'RACE was performed using poly (A)+ mRNA derived from both rat liver and kidney tissues. Two major fragments of approximately 1.1 and 1.4 kb were obtained. The 1.1-kb fragment was detected in both liver and kidney, whereas the 1.4-kb fragment was only found in the kidney (data not shown). Sequence alignment analysis using the ClustalW program from EMBL-EBI (European Molecular Biology Laboratory-European Bioinformatics Institute, Cambridge, UK; program available from http://www.ebi.ac.uk/clustalw/) showed that the 1.1-kb fragment from the liver was identical to that cloned from the kidney. The sequence of the 1.1-kb fragment aligned perfectly from the 3' end with the 1.4-kb fragment except for the first 43 nucleotides, which were found uniquely in the 5' end of the 1.1-kb fragment.

Cloning of RtOKL38-2.0 and -2.3 cDNAs
Using RT-PCR amplification, the full-length cDNA belonging to the 1.1-kb RACE fragment was cloned using forward primer FL-2.0F (Table 1Go) that resided within the unique 43-bp region of the 1.1-kb fragment and a reverse primer FL-2.0R (Table 1Go). The cloning of the cDNA belonging to the 1.4-kb RACE fragment was amplified using forward primer FL-2.3F primer residing in the 5' end of the 1.4-kb fragment together with FL-2.0R primer (Fig. 2BGo). Because the RtOKL38-2.0 and -2.3 kb transcripts were found abundantly in the kidney, we proceeded to clone these transcripts from the kidney. Two PCR products of 2.0 and 2.3 kb were cloned, and five clones from each were sequenced, which enabled us to establish two rat OKL38 cDNAs of 2003 and 2355 bp. The sizes of these two cDNAs were in agreement with the transcript sizes observed in Northern blot analysis (Fig. 1Go, A and B). We named the 2.0-kb cDNA as RtOKL38-2.0 (GenBank accession no. AY081218) and the 2.3-kb cDNA as RtOKL38-2.3 (GenBank accession no. AF549441). The results confirmed the existence of OKL38 variants.

Tissue distribution of rat OKL38 variants via semiquantitative One Step RT-PCR and cloning of a novel RtOKL38-2.3A cDNA
The distribution of rat OKL38 variants in various rat tissues was determined via One Step RT-PCR using variant-specific primers (Fig. 1DGo). Three RT-PCR products of 432, 791, and 690 bp were amplified, and these corresponded to the RtOKL38-2.0, -2.3, and -2.3A transcripts, respectively (Fig. 1Go, E–H). The RtOKL38-2.0 transcript was detected at high levels in the liver, kidney, and ovary. The observation was in agreement with the results from Northern blot analysis (Fig. 1AGo). Low levels of RtOKL38-2.0 transcript were detected in mammary gland and uterus (Fig. 1FGo). The RtOKL38-2.3 transcript was specifically detected in the kidney (Fig. 1Go, A and G), and its identity was verified via direct sequencing and Southern blot analysis (Fig. 1HGo). Southern blot analysis revealed a very low level of the RtOKL38-2.3 cDNA in the liver, and no expression was detected in the ovary, mammary gland, and uterus (Fig. 1HGo). A smaller fragment of 690 bp was repeatedly amplified in RNA sample derived from kidney (Fig. 1Go, G and H), and we believed that this fragment could belong to the existing OKL38 cDNAs or another novel variant. To investigate this possibility, we proceeded to clone this 690-bp fragment, and sequence analysis found that it belonged to an alternate spliced form of the RtOKL38-2.3 variant. The full-length cDNA of this spliced variant was annotated as RtOKL38-2.3A (GenBank accession no. AF549442).

Genomic organization of rat OKL38 gene
Using the National Center for Biotechnology Information (NCBI; Bethesda, MD) BLAST algorithm, the sequence of the three cloned rat OKL38 cDNAs was used to search the GenBank sequence database. The rat OKL38 gene was mapped onto the rat chromosome 19 (WGS supercontig; NW_043217.1). Using the sequence information, the genomic structure of the rat OKL38 gene was deduced (Fig. 2AGo), and the exon/intron boundaries were established. Each of the 5'-donor and 3'-acceptor splice sites conformed to the consensus sequences with the highly conserved, invariable GT/AG dinucleotides present at the immediate exon/intron boundaries (Table 2Go). The rat OKL38 gene spanned a genomic region of approximately 15 kb and contained eight exons with sizes ranging from 92–1314 bp (Table 2Go).


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  		TABLE 2. Exon-intron junctions of rat OKL38 gene

 
The 5'RACE results and cloning of the rat OKL38 mRNA isoforms suggested that differential promoter usage and alternative splicing at the 5' region of OKL38 gene might be responsible for the variants. Two putative promoters were also identified via 5'RACE analysis and sequence comparison in this study (Fig. 2AGo). The RtOKL38-2.3 cDNA contained all the eight exons, whereas the RtOKL38-2.3A cDNA was derived by alternative splicing of exon 3 (Fig. 2Go). Both the RtOKL38-2.3 and -2.3A transcripts were likely arrived at as a result of upstream promoter usage, whereas the cryptic promoter in intron 2 might have given rise to the RtOKL38-2.0 transcript, which only consists of exons 3–8 (Fig. 2BGo).

Sequence analysis of RtOKL38-2.0, -2.3, and -2.3A cDNAs
Sequence alignment study of RtOKL38-2.0, -2.3, and -2.3A cDNAs showed that the three variants shared the same 3' end, and the differences were only observed at the 5' end. Inspecting the 5' untranslated region (UTR) of the three RtOKL38 cDNAs revealed the presence of an inframe stop codon (TGA) 18 bp upstream of the predicted translational start site (ATG) (Fig. 3AGo). Despite the differences in the 5' end, the RtOKL38-2.0, -2.3, and -2.3A variants have the same ORF. Inframe small ORF that encoded for 66 and 33 amino acids were also detected in the 5'UTR of the RtOKL38-2.3 and -2.3A variants, respectively. Multiple sequence alignment study showed that the rat and mouse OKL38 (GenBank accession no. BC006032) shared a high identity of 91 and 93% at the nucleotide and amino acid levels, respectively. The identity between the rat and human OKL38 was only 80% at the nucleotide level and 85% at the amino acid level.



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  		FIG. 3. Nucleotide and deduced amino acid sequences of RtOKL38-2.0 cDNA and comparison of pyr-redox domain of other protein. A, The deduced amino acid sequence in single-letter code are shown below the nucleotide sequence. Nucleotides are numbered on the left, and the amino acids are numbered on the right. The translational start site at the 5' end and the polyadenylation signal in the 3'UTR are in bold. The inframe translation terminating codon (TGA) upstream of ATG and the two predicted glycosylation sites, Ser-217 and Thr-285, are in black box. The region in box was predicted to be the pyr-redox domain. The amino acid residues in italic (residues 343–366) were used to generate OKL38 antibodies. B, A graphical representation of pyr-redox domain from rat OKL38, thioredoxin reductase, and glutathione reductase. Shaded boxes represent the pyr-redox domain, and the white boxes represent the dimerization domain, which was not found in rat OKL38 protein.

 
Computational analysis of the predicted amino acid sequence using Pfam CDS-Conserved Domain Search (NCBI) detected a putative domain belonging to that of the pyridine nucleotide-disulfide oxidoreductase (pyr-redox), which was also present in glutathione reductase and thioredoxin reductase (Fig. 3Go). The pyr-redox domain resided in exon 8 of rat OKL38 gene. The hydrophobicity average of rat OKL38 was 0.200209, and it was predicted to be a cytosolic protein using the SOSUI (18) and Tmpred (19) programs from ExPASy Proteomics tools (http://ca.expasy.org/tools). Two putative N-linked glycosylation sites at Ser-217 and Thr-285 were identified using NetOGlyc 2.0 software (Center for Biological Sequence Analysis, Lyngby, Denmark) (20) as indicated in Fig. 3AGo.

The RtOKL38-2.0 cDNA harbored an ORF of 1434 bp. The conceptual translation of this cDNA predicted a protein of 478 amino acids, with a calculated molecular mass of 52 kDa (Fig. 3Go) and a predicted isoelectric point of 6.83. In vitro TNT study using the RtOKL38-2.0, -2.3, and -2.3A cDNAs resulted in the synthesis of an approximately 52-kDa protein (Fig. 4AGo), and its identity was confirmed by Western blot analysis using anti-OKL38 antibodies (Fig. 4BGo).



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  		FIG. 4. In vitro TNT study of rat OKL38 cDNAs. In vitro TNT was performed using [35S] L-methionine and cDNA templates from the three rat OKL38 variants. Lanes consist of RtOKL38-2.0 (lane 1), -2.3 (lane 2), and -2.3A (lane 3) cDNA as template, water as the negative control (lane 4), and luciferase construct as a positive control (lane 5). A, The TNT reaction containing the synthesized protein was separated on 14% SDS-PAGE gel, transferred onto nitrocellulose membrane, and exposed to Kodak film (Kodak, Rochester, NY) overnight. B, The same membrane was then subjected to Western blot analysis using anti-OKL38 antibody. Note that a radiolabeled protein of approximately 52 kDa was detected and also recognized by OKL38 antibody.

 
Regulation of rat OKL38 protein during pregnancy
We have previously shown that the OKL38 transcripts were up-regulated in the rat mammary gland during pregnancy (14). Using the OKL38 antibody, we proceeded to determine whether the rat OKL38 protein was also up-regulated during pregnancy. Western blot analysis showed that OKL38 protein was highly expressed in the mammary glands of the pregnant and lactating rats, whereas the protein was barely detectable in the nonpregnant mammary glands (Fig. 5EGo). The level of OKL38 protein returned to basal level during involution.



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  		FIG. 5. Effect of hCG and progression of pregnancy on OKL38 expression in the rat mammary gland and ovary. Female rats were treated with indicated concentrations of hCG over a period of 3 wk to emulate pregnancy in rat. Five micrograms of poly (A)+ mRNA derived from mammary tissues (A) and 50 µg of total RNA derived from ovary (C) were subjected to Northern blot analysis. Blots were hybridized with OKL38 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNAs. Densitometric scanning of the OKL38 mRNA band in mammary gland (B) and ovary (D) after being normalized to the levels of GAPDH mRNA is shown. Expression of rat OKL38 protein in mammary gland during the progression of pregnancy (E) and in ovary treated with different concentration of hCG (G) is also shown. One hundred micrograms of total protein from the indicated tissues were used for Western blot analysis. Anti-RtOKL38 antibody recognized the 52-kDa rat OKL38 protein in both the mammary gland and ovary. The ß-actin indicates equal loading in all lanes (F and H).

 
Induction of rat OKL38 expression by hCG
It is well established that CG production by placenta increases during pregnancy. Russo et al. (4, 5, 6) have shown that pregnancy and human CG could confer protection of breast against carcinogenesis. Because the onset and advancement of pregnancy up-regulated OKL38 expression in the mammary gland, it was thus important to determine whether a hormone of pregnancy, such as CG, was able to up-regulate OKL38. Total RNA derived from ovary and mammary gland of hCG-treated rats was used for Northern blot analysis. OKL38 transcript was significantly increased in a dose-dependent manner by hCG treatment, whereas a low level of transcript was observed in the mammary gland and ovary of vehicle-treated rats (Fig. 5Go, A–D). In the ovary, hCG also induced the expression of OKL38 protein (Fig. 5GGo). Unlike the ovary and the mammary gland, OKL38 expression in the liver and kidney was not regulated by hCG (data not shown).

In situ hybridization study was performed to determine the cell-specific expression of OKL38 in both mammary gland and ovary after hCG treatment. Our previous studies showed that OKL38 was expressed at a low level in undifferentiated mammary epithelial cells of control rats (14). In this study, sections derived from mammary tissues and ovaries of controls and hCG-treated rats (20 IU/d) were hybridized with a sense and antisense OKL38 RNA digoxigenin-labeled probes. Intense signal was observed in the differentiated secretory epithelial cells of the mammary gland. In contrast, little or no signal was detected in the undifferentiated controlled mammary gland, blood vessels, and connective tissues (Fig. 6Go, A and B, indicated by arrow). Low OKL38 signal was detected in the granulosa cells of the ovary in the control vehicle-treated rats (Fig. 6DGo). Expression of OKL38 was greatly increased in large corpora lutea and granulosa cells in the follicles of hCG-treated rats (Fig. 6EGo). No signal was detected in the connective tissues of the ovary (Fig. 6EGo, indicated by arrow). The control sense OKL38 RNA probe gave only background signals in the mammary tissue (Fig. 6CGo) and ovary (Fig. 6FGo).



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  		FIG. 6. In situ hybridization with sense and antisense RNA probes for OKL38 expression in rat mammary gland and ovary. Sections derived from mammary and ovary of untreated rats (A and D) and rats treated with 20 IU of hCG (B and E) were subjected to in situ hybridization using a sense control (C and F) and antisense OKL38 probe (A, B, D, and E). Low OKL38 mRNA was detected in alveolar epithelial cells of the mammary gland from untreated rat but not in stromal cells or blood vessels (A; arrow). B, High levels of OKL38 mRNA were detected in the secretory epithelial cells of mammary tissue derived from rats treated with 20 IU of hCG. No staining signal was detected in the connective tissue or blood vessels (B; arrow). In the ovary, higher levels of OKL38 mRNA were detected in the granulosa cells of the ovaries derived from hCG-treated animals (E) compared with those of vehicle-treated animals (D). No staining signal was detected in the blood vessels and the connective tissues between the ovaries (E; arrow). Sense OKL38 probe showed no staining signal in the mammary gland and ovary of hCG-treated rat (C and F). Note that hCG-treated animals have larger ovaries due to enlargement of corpora lutea. Magnification, x400 (A), x100 (B and C), and x40 (D–F).

 
Overexpression of rat OKL38 protein was lethal to BRL cells
Because the human OKL38 cDNAs were postulated to be involved in cell growth and differentiation (14, 15), we proceeded to investigate the cellular localization and function of rat OKL38 protein. We generated a RtOKL38-eGFP-pcDNA3.0 construct, which fused the reporter gene eGFP to the C terminus of OKL38 protein. Our preliminary transfection study showed that RtOKL38-eGFP fusion protein was localized to the cytosol and formed an aggregation in the BRL cells as early as 24 h post transfection (Fig. 7AGo). Cells expressing the fusion protein gradually rounded up and died, and almost all of the RtOKL38-eGFP-expressing cells were disintegrated after 120 h post transfection. On the other hand, cell growth was observed in the cells transfected with the control eGFP or eGFP-Antisense-RtOKL38 constructs. The presence of the fusion protein (RtOKL38-eGFP) was verified via Western blot analysis performed on the transfected cell using the antirat OKL38 antibody (Fig. 7CGo). The results suggest that overexpression of OKL38 protein was lethal to BRL cells.



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  		FIG. 7. Transfection study of OKL38-eGFP recombinant constructs. BRL cells were transfected with controlled eGFP, eGFP-Antisense-RtOKL38, and Sense-RtOKL38-eGFP recombinant constructs. The transfected cells were harvested at 24, 48, and 72 h post transfection (A). Cells expressing Sense-RtOKL38-eGFP protein were visualized using a microscope equipped with epifluorescence optic. White arrows indicate that the RtOKL38-eGFP recombinant protein forms speckles and eventually leads to cell death. The three constructs cloned in pcDNA3.0 vector used for this transfection are shown in B. C, Western blot analysis verifying the presence of RtOKL38-eGFP recombinant protein (lane 3). Untransfected BRL cells are in lane 1, and BRL cells transfected with empty vector, Sense-RtOKL38-eGFP, and eGFP-Antisense-RtOKL38 are in lanes 2, 3, and 4, respectively. Magnification, x200.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have previously isolated a novel pregnancy-induced growth inhibitory gene, OKL38 (14, 15). Our previously findings demonstrated that OKL38 is involved in the progression of breast and kidney carcinogenesis. To develop a rat model for further characterization of OKL38’s role in breast and ovarian cancer, we isolated and cloned three rat OKL38 cDNA variants, RtOKL38-2.0, -2.3, and -2.3A. These three cloned rat OKL38 cDNAs showed variations in the 5' end similar to that observed in the human (15) but encoded a common ORF of 478 amino acids with a predicted molecular mass of 52 kDa. The molecular mass and identity of the predicted rat OKL38 protein was verified by TNT and Western blot analysis, respectively. The small ORF detected in the 5'UTR of the RtOKL38-2.3 and -2.3A variants may be involved in posttranscriptional regulation of protein synthesis because small upstream ORFs are often used to down-modulate the production of potent proteins such as cytokines, growth factors, protein kinases, and transcription factors (21). The cytotoxicity of OKL38 when overexpressed and the existence of different mRNA isoforms that encoded for the same protein suggest that translation-regulatory cis-elements may exist in the 5'UTR of the mRNA to fine-tune the regulation of OKL38 protein.

The NCBI annotation project predicted the human OKL38 to be about the same length as the RtOKL38-2.0. We have recently cloned and reported three human OKL38 cDNAs of 1.9, 2.2, and 2.4 kb in length (15). Two mouse OKL38 cDNAs of 1982 bp (GenBank accession no. BC022135) and 2383 bp (GenBank accession no. BC006032) have also been reported. Sequence analysis of these cloned rat OKL38 cDNAs with those from the human and mouse showed that the transcripts contained different 5'UTRs. The results from 5'RACE analysis, genomic sequence analysis, and the above observations indicated that alternative splicing of the mRNA and differential usage of multiple promoters were involved in modifying the 5'UTRs.

Several larger transcripts ranging from 4.0–9.0 kb were also detected in liver and kidney by Northern blot analysis. In this present study, these larger transcripts were not characterized. Our 5'RACE analysis did not amplify any larger transcripts other than the 1.1- and 1.4-kb fragment. However, we did not exclude the possibility that they arrive as a result of differential splicing at the 3' end of the gene. Analysis and characterization of the 3' end of the OKL38 gene will be performed in a future study.

Four human OKL38 variants were identified, of which three were cloned, whereas only three variants were cloned from the rat. None of the OKL38 variants identified so far contain all of the eight exons, with the exception of RtOKL38-2.3, which was identified in this report. The expression profiles of these transcripts were quite different in the two species except for RtOKL38-2.0 (or HuOKL38-1a), which showed similar expression profiles. The RtOKL38-2.0 and HuOKL38-1a transcripts encoded for proteins of 478 and 477 amino acids, respectively. The human OKL38 variants were predicted to encode different protein isoforms of 61, 60, and 52 kDa (15). In the rat, all the transcripts encoded for a single protein of 52 kDa. Interestingly, sequence comparison study performed between the human and rat OKL38 variants showed that only the coding region for the 52-kDa protein was conserved, suggesting that the protein has a conserved function. This postulation was supported by our present study, in which overexpression of the rat OKL38-eGFP (52 kDa) in BRL cells showed the same phenomenon as that of the human OKL38-eGFP (52 kDa) in A498 (15). No alignment was obtained in the 5'UTR and 3'UTR of OKL38 in the two species, indicating that the posttranscriptional regulation of OKL38 protein may be different.

We have previously deciphered the genomic structure of the human OKL38 gene (15). The OKL38 gene from both species contains eight exons, and the sizes of exons 5, 6, and 7, which are part of the coding region, are the same. The predicted pyr-redox domain resides in exon 8, the last exon in both species. Differential promoter usage and alternative splicing were used by both the human and rat to regulate OKL38 transcription. Recent evolution may have fine-tuned the regulation of OKL38 in each individual species, but the structure and protein are still conserved. Extensive studies are required to illustrate the functions of this essential gene.

Multiple sequence alignment (EMBL-EBI) delineates the regions of similarity for OKL38 protein from rat, mouse, and human. The OKL38 protein was highly conserved in all the three species, suggesting the importance of this protein in cellular functions such as growth and differentiation. A putative pyr-redox domain was identified in OKL38, which is also present in the well-characterized glutathione reductase and thioredoxin reductase. BLAST search (NCBI) using the deduced amino acid sequence against the nonredundant database identified several proteins of unknown functions, such as human C8ORF1 (GenBank accession no. NP_004328) and bacteria-conserved protein, BH1623 (GenBank accession no. NP_242489) from Bacillus halodurans. The C8ORF1 is a predicted protein from the human chromosome 8, with an identity of 49% and a similarity of 62% to rat OKL38 protein. An unknown conserved protein, BH1623 from B. halodurans, shows a lower homology to that of the rat OKL38, with an identity of 39% and a similarity of 44%. These two proteins and OKL38 may belong to the larger family of pyr-redox and may share some similar functions from this family of proteins.

Our previous in vitro and in vivo studies demonstrated the growth-inhibitory property of OKL38 (14), and loss of transcript was observed in several breast cancer cell lines, suggesting its importance in the process of tumorigenesis. It has been demonstrated that pregnancy and hCG protect the breast from carcinogenesis (4, 5, 6). In this study, we showed that hCG could induce OKL38 expression in mammary epithelial cells from which most of the breast cancer are derived. It is tempting to speculate that OKL38 may be the downstream effector protein involved in the pregnancy and hCG-induced protection of the breast against carcinogenesis. Although high expression of OKL38 was detected in the granulosa cells in the present study, its role in granulosa cell-derived tumor is not known. It has been reported that only 1–2% of the ovarian cancers are derived from the granulosa cell, and the recurrence of this cancer after resection is very high (22). We are in the process of determining the function of OKL38 in granulosa-derived ovarian cancer.

One Step RT-PCR and Northern blot analysis showed that only RtOKL38-2.0 was induced by hCG in the mammary and ovary. The expression of OKL38 protein in mammary gland was observed to be high during pregnancy and throughout lactation, which were stages when the epithelial cells were highly differentiated. During involution, the mammary epithelial cells undergo apoptosis and remodulation. At this stage, OKL38 expression was observed to be reduced, suggesting that the protein may function in maintaining mammary epithelial cell differentiation. This postulation was further supported by the fact that OKL38 is highly expressed in the liver and kidney, which is characterized by relatively low cellular turnover and extensive differentiation.

In the present study, OKL38 expression was also induced by hCG in the ovary. The signal was localized mainly in the granulosa cells and the corpus luteus. CG levels rise during the advancement of pregnancy, and recombinant hCG has been shown to induce enlargement of the rat ovary due to the formation of corpora lutea (Russo, I. H., personal communication). In the ovary, hCG binds to the granulosa cells of the ovary through LH-CG-R, a seven-transmembrane G protein-coupled receptor, and this induces ovulation and maintains the corpus luteum that is essential for maintenance of pregnancy until the placenta becomes fully functional. These observations suggest that the induction of OKL38 expression by hCG could be through the LH-CG-R signaling pathway. Because CG is required for maintenance of progesterone hormone production during pregnancy, it is also possible that hCG-induced OKL38 expression is mediated through progesterone production. Although no cAMP-responsive element or progesterone-responsive element were detected within the 1-kb region of putative promoter P1 (RtOKL38-2.0), other cis-elements, such as activation protein 1, activation protein 4, nuclear factor {kappa}B, and CCAAT enhancer-binding protein ß, were detected using the program MatInspector V2.2 from Genomatix (http://www.genomatix.de/cgi-bin/matinspector/matinspector.pl), and these may play a role in the induction of OKL38 by hCG.

In summary, we have cloned three rat OKL38 cDNAs, established their genomic structure, and shown their distribution in various rat tissues. Several lines of evidences indicated that OKL38 is a conserved essential protein and may belong to the pyr-redox family of proteins. OKL38 expression is up-regulated by hCG in the rat mammary tissue and ovary. The positive correlation between hCG-induced OKL38 expression and hCG protection against mammary carcinogenesis suggests that OKL38 may function as the downstream effector protein of hCG-mediated protection against mammary carcinogenesis by maintaining cellular differentiation. Definite functions of OKL38 are still unknown, although we have shown that overexpression of the protein may be involved in cell death. Further study with the use of transgenic knockout mice may help to illustrate the intrinsic function of OKL38. The present study provides the basic foundation for future study on the role of OKL38 in differentiation, growth, and tumorigenesis.


   Acknowledgments
 
We are grateful to Dr. Irma H. Russo for her valuable advice in the hCG aspect of this work.


   Footnotes
 
This work was supported by Grant LS/00/019 from A*STAR-BMRC (Agency for Science, Technology and Research-Biomedical Research Council) (to H.H.).

Abbreviations: BRL, Buffalo rat liver; CG, chorionic gonadotropin; DMBA, 7, 12-dimethylbenz (a) anthracene; eGFP, enhanced green florescence protein; h, human; LH-CG-R, lutropin-choriogonadotropin-receptor; NCBI, Center for Biotechnology Information; ORF, open reading frame; pyr-redox, pyridine nucleotide-disulfide oxidoreductase; RACE, rapid amplification of cDNA end; TNT, transcription and translation; UTR, untranslated region.

Received April 7, 2004.

Accepted for publication June 18, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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