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Endocrinology Vol. 144, No. 6 2597-2605
Copyright © 2003 by The Endocrine Society

Expression of a Novel C-Type Lectin in the Mouse Vagina

Yoshinao Katsu, Dennis B. Lubahn and Taisen Iguchi

Center for Integrative Bioscience, Okazaki National Research Institutes (Y.K., T.I.), Okazaki 444-8585, Japan; Core Research for Evolutional Science and Technology of Japan, Science and Technology Corporation (Y.K., T.I.); Department of Molecular Biology, University of Missouri (D.B.L.), Columbia, Missouri 65211; and Department of Molecular Biomechanics, School of Life Science, Graduate University of Advanced Studies (T.I.), Okazaki 444-8585, Japan

Address all correspondence and requests for reprints to: Dr. Taisen Iguchi, Center for Integrative Bioscience, Okazaki National Research Institutes, 5-1 Higashiyama, Myodaiji, Okazaki 444-8585, Japan. E-mail: taisen{at}nibb.ac.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Estrogens regulate the proliferation and differentiation of mouse vaginal epithelial cells. However, the molecular mechanisms underlying estrogen-induced changes have not been elucidated. The goal of this study was to identify estrogen-responsive genes related to the proliferation and differentiation of mouse vaginal epithelial cells. We used differential display to reveal specific genes regulated by estrogens and identified a transcript that was designated DDV10. DDV10 encodes a membrane protein with a C-type lectin domain in the carboxyl-terminal region; thus, we inferred that it belongs to the C-type lectin family. We analyzed the temporal and spatial expression of DDV10 using RT-PCR, quantitative real-time RT-PCR, and in situ hybridization. Ovariectomy decreased DDV10 mRNA levels, whereas 17ß-estradiol treatment increased expression of DDV10 mRNA in vaginas of ovariectomized mice. DDV10 mRNA was first detected between 20 and 30 d after birth and was found in eye, tongue, stomach, and stratified and cornified vaginal epithelial cells, but not in stromal cells or uterus. DDV10 transcripts were not detected in vaginas of estrogen receptor {alpha} knockout mice. Taken together, these data suggest that DDV10 encodes a novel, 17ß-estradiol-regulated, C-type lectin in the mouse vagina. DDV10 may play a role in the stratification and/or cornification of epithelial cells during differentiation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PROLIFERATION and differentiation of the vaginal epithelium are regulated by the natural hormone, 17ß-estradiol (E2), which modifies cellular physiology by modulating the transcriptional activity of specific nuclear estrogen receptors (ERs). Estrogens are believed to stimulate primary response genes, initiating a cascade of transcriptional events, the products of which participate in physiological responses known to be estrogen-dependent events in the target organs in vivo. The multifaceted role of estrogens within the female reproductive tract has been well studied and characterized. However, the regulation of vaginal gene expression by estrogens and the molecular mechanisms underlying estrogen-mediated proliferation remain unclear.

In addition to stimulating vaginal epithelial proliferation, estrogens elicit a complex pattern of differentiative events in vaginal epithelium. The rodent vaginal epithelium exhibits cyclical changes in response to cyclical ovarian secretions, displaying an alternating pattern of keratinization and mucification (1). The vaginal epithelium of the ovariectomized mouse is atrophied to two or three cell layers. In response to estrogens, basal epithelial cells proliferate rapidly, leading to the formation of a highly stratified epithelium. The postmitotic suprabasal cells differentiate as they move outward through the epithelium, becoming enlarged and undergoing structural and morphological changes indicative of cornification. The apical layer becomes heavily keratinized. These morphological changes, in response to estrogens, are accompanied by the production of cytokeratins 1 and 10, markers common to epidermal and vaginal epithelial differentiation (2).

Singh and Gupta (3) showed that treatment with estrogens induces a change in intracellular calcium levels, and calcium is essential for normal epidermal differentiation (4). It is known that normal epidermal cells (keratinocytes) form cornified envelopes in response to calcium as a result of cross-linking of substrates such as involucrin by a specific membrane-bound enzyme, transglutaminase-1 (5, 6). However, the molecular mechanisms underlying calcium-inducible differentiation of keratinocytes is unclear beyond the potential involvement of protein kinase C and/or calcium receptor (7, 8).

E2 stimulates epithelial cell proliferation in the female genital tract indirectly through stromal ER{alpha} via a paracrine mechanisms. Our objective in this study was to analyze the mechanism of E2-induced proliferation and differentiation of the vaginal epithelium. Using differential display/RT-PCR, we isolated a new member of the C-type lectin family that is expressed in vagina with proliferating and cornified epithelium. Characterization of mRNA expression indicates that estrogen regulates the gene encoding this novel C-type lectin in mouse vagina. Furthermore, this C-type lectin is found in epithelial cells, but not in stromal cells, suggesting that it may be an important factor in the stratification and/or cornification of the vaginal epithelium of mice.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
RNA isolation, Northern blotting, RT-PCR, and quantitative PCR (Q-PCR)
Total RNA was isolated using RNeasy kit (QIAGEN, Chatsworth, CA). For Northern blot analysis, 20 µg total RNA/lane were denatured and separated on a formaldehyde-3-[N-morpholino]propanesulfonic acid agarose gel and transferred to Hybond nylon membranes (Amersham Pharmacia Biotech, Piscataway, NJ). Hybridization was carried out in 5x standard saline citrate (SSC), 1% (wt/vol) sodium dodecyl sulfate, 5x Denhardt’s solution, 100 µg/ml herring sperm DNA, and [{alpha}-32P]deoxy-CTP-labeled DNA probe at 45 C overnight. The membrane was washed in 2x SSC/0.1% (wt/vol) sodium dodecyl sulfate for 30 min, followed by intensive washing in 1x SSC/0.1% (wt/vol) sodium dodecyl sulfate at 45 C for 30 min with two changes of the washing solution. The membrane was exposed to an imaging plate and analyzed using an image analyzer (BAS-2000, Fuji Photo Film Co., Ltd., Tokyo, Japan). For RT-PCR, 4 µg total RNA were reverse transcribed using SuperScript II reverse transcriptase (Invitrogen, Carlsbad, CA) and oligo(deoxythymidine) [oligo(dT)] primer. The following primer sets were used for PCR: DDV10-1, 5'-TCGGTTTGACAACGAGAAGG-3'; and DDV10-2, 5'-CTAGACAGGAACAGGAG-TTG-3' for DDV10; DDV10-3, 5'-TAGGTTTGACAACCAGGATG-3'; and DDV10-4, 5'-CTAGGAAGGAAAAAAAGGAG-3' for OCIL; and GAP-1, 5'-ATGACCACAGTCCATGCCATCAC-3'; and GAP-2, 5'-TCATTGTCATACCAGGAAATGAG-3' for glyceraldehyde phosphate dehydrogenase (GAPDH). Twenty-eight cycles of amplification were carried out under the following conditions: denaturation at 94 C for 30 sec, annealing at 55 C for 30 sec, and extension at 72 C for 1 min. At completion of the PCR, fragments were resolved on 1.5% agarose gels. First strand cDNA was synthesized from 2 µg total RNA for real-time Q-PCR. The following specific primers were used: DDV10-5, 5'-TCTAAGCACCCTTGGATGTGGA-3'; and DDV10-6, 5'-AGCTTTCGCCTTCTTCTGGCT-3' for DDV10; and GAP-3, 5'-AACGACCCCTTCATTGACCTC-3'; and GAP-4, 5'-CCTTGACTGTGCCGTTGAATT-3' for GAPDH. PCR was carried out using ABI PRISM 5700 (PE Applied Biosystems, Foster City, CA) according to the manufacturer’s protocol, except that 40% of the suggested reaction volume (20 µl) was used. PCR conditions were 2 min at 50 C, 95 C for 10 min, and 40 cycles at 95 C for 15 sec and 60 C for 1 min. Gene expression levels were normalized to GAPDH mRNA expression. To establish standard curves for each gene, serial dilutions of a known amount of cDNA sample were used. The CT (threshold cycle) values for each gene were plotted on these standard curves to obtain the amount of copies present in the initial cDNA sample. All samples were run in triplicate and replicated in three independent experiments, and the mean CT for GAPDH was subtracted from the DDV10 mean CT for normalization.

Experimental animals
C57BL/6 mice and ER{alpha} knockout ({alpha}ERKO) mice (9) were maintained on a commercial diet (CE-1, CLEA, Tokyo, Japan) and tap water and were kept at 24 C under 12-h light, 12-h dark cycle. Female mice were given five daily sc injections of 3 µg diethylstilbestrol (DES; Sigma-Aldrich Corp., St. Louis, MO) dissolved in sesame oil or of the oil vehicle alone starting on the day of birth. Mice were ovariectomized at 7–8 wk of age, and tissues were dissected 10 d later. Tissues were flash-frozen in liquid nitrogen and stored at -80 C until RNA extraction or fixed for histology. For the study of estrogenic effects, E2 (1.0 or 0.25 µg/animal; Sigma-Aldrich Corp.), progesterone (1.0 µg/animal; Sigma-Aldrich Corp.), or the ER antagonist ICI 182,780 (50.0 µg/animal; TOCRIS, Ellisville, MO) were injected sc into ovariectomized mice. All experiments and animal husbandry protocols were approved by the animal care committee of Okazaki National Research Institutes.

Tissue separation
Tissue separation procedures for vaginal epithelium and stroma have been previously described (10). Briefly, vaginas from mice were trimmed, opened lengthwise, and incubated with 0.5% trypsin (Invitrogen, Carlsbad, CA) in calcium- and magnesium-free Hanks’ balanced salt solution (Invitrogen, Carlsbad, CA) for 90 min at 4 C. Vaginal stroma and epithelium were then separated by removing the epithelium from the underlying stroma using a fine forceps.

Differential display/RT-PCR (DD/RT-PCR) and sequencing of DD/RT-PCR fragments
Total RNA (2 µg) isolated from vagina was reverse transcribed using rhodamine-labeled oligo(dT) primer (5'-rhodamine labeled-TnGC). The resulting cDNAs were amplified by PCR using rhodamine-labeled oligo(dT) downstream primer and upstream arbitrary primers (AP-1, 5'-GATCATGGTC-3'). Samples from each amplification reaction were loaded onto a 5% nondenaturing polyacrylamide gel and electrophoresed at 30 mA for 3 h. The gel image was analyzed to detect differentially expressed, rhodamine-labeled DNA fragments using a Fluoro-Image Analyzer (FLA-3000G, Fuji Photo Film Co., Ltd.). Bands of interest were excised from the gel, and the cDNA was eluted and reamplified by PCR using the same primers as those used in the original PCR amplification. The amplified DNA fragment was recovered from the gel and subcloned into pGEM-T Easy vector using the T/A cloning system (Promega Corp., Madison, WI). The clones were sequenced using the BigDye Terminator Cycle Sequencing Kit (PE Applied Biosystems) with T7 primer and analyzed on the ABI PRISM 377 automatic sequencer (PE Applied Biosystems).

Plasmids
The full-length DDV10 and OCIL cDNAs were isolated from mouse vaginal RNA using a PCR-based technique and subcloned with T/A subcloned as described above. Amplification was performed for 30 cycles of 94 C for 1 min, 55 C for 1 min, and 72 C for 1 min using the 5' primer DDV10-7 (5'-TCTGAGATGAACATTACAAGG-3') and the 3' primer DDV10-2 for DDV10, and the 5' primer DDV10-8 (5'-ATGTGTGTCACAAAGGCTTC-3') and the 3' primer DDV10-4 (for OCIL). All clones were sequenced using the BigDye Terminator Cycle Sequencing Kit (PE Applied Biosystems) with T7 and SP6 primers and analyzed on the ABI PRISM 377 automatic sequencer (PE Applied Biosystems).

Histological observation
For immunocytochemistry, vaginas were fixed in 10% formaldehyde neutral buffer solution (Nacalai, Kyoto, Japan) embedded in paraffin, serially sectioned at 6 µm, deparaffinized, rehydrated, and washed twice in PBS for 10 min each time. Sections were then stained with Histofine (Nichirei, Tokyo, Japan) according to the manufacturer’s instructions. Antikeratin polyclonal antibody (Nichirei) and antimouse keratin 1 antibody (BabCo, Richmond, CA) were used as primary antibodies. The expression of DDV10 mRNA was examined by in situ hybridization using digoxygenin-UTP-labeled probes translated in vitro the from DDV10 clone. We selected the cytoplasmic domain of DDV10 as a probe to distinguish between DDV10 and OCIL. Vaginas were fixed with 4% paraformaldehyde in PBS for 16 h at 4 C. Preparation of the digoxygenin-labeled probe, conditions of hybridization and washing, and detection of the signal were performed according to the manufacturer’s instructions (Roche Molecular Biochemicals, Indianapolis, IN).

Data analysis
Statistical analysis was carried out using t test or Welch’s test. All data are reported as the mean ± SEM. In all cases, means were considered significantly different at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Identification of a novel C-type lectin by differential display/RT-PCR
The proliferation and cornification of vaginal epithelium are regulated by estrogen; however, the molecular mechanisms underlying estrogen-dependent cell proliferation and differentiation of vaginal epithelium are unclear. Neonatal DES treatment causes estrogen-independent cell proliferation and differentiation (11, 12, 13, 14, 15). Therefore, the vaginas of DES-treated mice provide a good system for analyzing the proliferation and cornification of vaginal epithelium. We compared the transcripts from ovariectomized (OVX) mouse vaginas treated neonatally with either oil vehicle (oil-OVX-vagina) or DES (DES-OVX-vagina) using DD/RT-PCR. A differentially expressed DNA fragment was detected in samples from DES-OVX mice (Fig. 1AGo, designated DDV10) and was isolated from the gel, reamplified, subcloned, and sequenced. This cDNA fragment is 598 bp long (Fig. 1BGo; DDBJ/EMBL/GenBank accession no. AB091386). BLAST analysis of this partial cDNA against the nonredundant GenBank/EMBL database revealed that the DDV10 clone is identical with a cDNA (4632413B12 gene) of unknown function that was isolated by the RIKEN cDNA project (16). DDV10 encodes a 269-amino-acid putative membrane protein with structural homology to the C-type lectin family. The predicted DDV10 protein has a 142-amino-acid extracellular domain, a 21-amino-acid transmembrane domain, and a 106-amino-acid cytoplasmic domain. The extracellular domain contains five cysteine residues and three predicted N-linked glycosylation sites at residues 137, 163, and 221. A myristoylation motif is also predicted in the intracellular domain. As shown in Fig. 2Go, the amino acid sequence of DDV10 is similar to that of mouse OCIL (osteoclast inhibitory lectin) that was isolated as an inhibitor of osteoclast formation (17) (81% identical in extracellular domain, 60% overall). As DDV10 was identified as a differentially expressed band by DD/RT-PCR, we confirmed its expression by RT-PCR using gene-specific primer sets. As expected, the expression level of DDV10 was high in DES-OVX-vagina. In contrast to DDV10 expression, OCIL was high in oil-OVX-vagina in which the epithelium is composed of two or three layers of cuboidal cells (Fig. 3Go).



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  		Figure 1. Identification of DES-induced mRNA by DD/RT-PCR. A, Mice were neonatally treated with oil or DES for the first 5 d after birth. Mice were OVX 7 d after birth, and vaginas were dissected from animals 10 d later. Total RNA was isolated, and DD/RT-PCR/gel analysis was performed. The arrow indicates the band that is differentially expressed in the vaginas of neonatally DES-treated mice. B, Nucleotide sequence of DDV10. Two underlined sequences were used as primers for DD/RT-PCR as upstream and oligo(dT) primer, respectively.

 


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  		Figure 2. Sequence alignment of DDV10 and OCIL. Deduced amino acid sequences of DDV10 (RIKEN cDNA 4632413B12, GenBank accession no. AK014570) and mouse OCIL (accession no. AF 321553) are shown. The cytoplasmic, transmembrane, and extracellular domains are indicated. Identical residues are marked with an asterisk.

 


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  		Figure 3. Verification of the gene expression of the transcript identified by DD/RT-PCR. Total RNA was isolated from vaginas of neonatally DES-treated and oil-treated mice. RT-PCR was used to determine the expression levels of DDV10, mouse OCIL, and GAPDH mRNAs. As a control, RT (-) reaction mixture was used.

 
Expression of DDV10 and OCIL mRNAs in vagina
To analyze the effect of estrogen on DDV10 expression, oil vehicle, E2, or E2 plus the ER antagonist ICI 182,780 were injected into mice 10 d after ovariectomy (OVX). Thirty hours after injection, vaginas were dissected, total RNA was isolated, and potentially contaminating genomic DNA was removed by deoxyribonuclease treatment, cDNAs were synthesized and used for Q-PCR. The steady-state levels of DDV10 transcripts were increased by E2 treatment, and this increase was blocked when E2 was accompanied by ICI 182,780 (Fig. 4AGo). We found that, like E2, DES can induce the expression of DDV10 (data not shown), whereas progesterone treatment does not (Fig. 4BGo). To more precisely analyze estrogenic effects on DDV10 expression, mice were OVX, and vaginas were dissected at 24-h intervals after OVX. Transcript levels were examined by Northern blot or Q-PCR. DDV10 transcripts were decreased by OVX; however, OCIL mRNA increased slightly (Fig. 5Go, A and B). E2 administration 10 d after OVX led to an increase in the levels of DDV10 transcripts approximately 12 h after treatment (Fig. 5CGo), whereas E2 treatment slightly decreased OCIL mRNA levels (Fig. 5DGo).



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  		Figure 4. Induction of DDV10 expression by E2. A, Expression level of DDV10 from vaginas of OVX mice after vehicle treatment (lane 1, oil), ICI plus E2 (lane 2, 50 µg ICI and 0.25 µg E2/animal), and E2 (lane 3, 0.25 µg E2/animal). *, P < 0.01 vs. oil and ICI plus E2. B, Expression level of DDV10 from vaginas of OVX mice after vehicle (lane 1, oil), progesterone (P4; lane 2, 1.0 µg P4/animal), and E2 (lane 3, 1.0 µg E2/animal). Results are the mean ± SEM of three experiments, each performed in triplicate. *, P < 0.01 vs. oil and P4.

 


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  		Figure 5. Regulation of DDV10 and OCIL mRNA expressions by E2. Mice were OVX at 50 d of age (designated d 0), and vaginas were dissected from animals 0, 1, 3, 6, and 10 d later (A and B). Total RNA was isolated from vaginas, and Q-PCR analysis (A for DDV10 mRNA) and Northern blot analysis (B for mOCIL mRNA) were carried out. Fifty-day-old mice were OVX, and a single injection of E2 (1 µg/animal) was given 10 d later. Vaginas were dissected from animals 0, 6, 12, 15, 18, 21, and 24 h after the injection (C) and 0, 1, 3, 6, 12, and 24 h after the injection (D). Total RNA was isolated from vaginas, and mRNA levels were analyzed by Q-PCR (C for DDV10 mRNA) or Northern blot (D for mOCIL mRNA). Results are the mean ± SEM of three experiments, each performed in triplicate. *, P < 0.05 vs. before treatment.

 
Tissue distribution of DDV10 and OCIL mRNAs
To determine the distribution of DDV10 and OCIL mRNAs, total RNA was isolated from various tissues from male and female mice, and RT-PCR was performed using gene-specific primers. Intriguingly, OCIL was expressed in both vagina and uterus, whereas DDV10 was expressed only in vagina (Fig. 6AGo). We could not detect DDV10 in the uterus of E2-treated mice (data not shown). OCIL mRNA was detected in all tissues examined as described by Zhou et al. (17) (Fig. 6BGo). DDV10 mRNA was detected in eye, tongue, stomach, and spleen (Fig. 6BGo). These results suggested that DDV10 may be involved in the stratification and/or cornification of the epithelium. However, the spleen does not have a stratified and cornified epithelium, and the function of DDV10 in spleen will probably be different from that in epithelia.



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  		Figure 6. Tissue distribution of DDV10 and OCIL mRNA. A, Total RNA was isolated from uteri (UT) and vaginas (VA). cDNA was synthesized with (RT+) or without (RT-) reverse transcriptase, and expressions of DDV10, mouse OCIL, and GAPDH mRNAs were analyzed by RT-PCR. B, Total RNA was prepared from brain (B), eye (E), tongue (TO), heart (H), lung (LU), liver (LI), stomach (ST), spleen (SP), kidney (K), and testis (TE). GAPDH was used as a positive control. mRNAs of DDV10 and mOCIL were detected by RT-PCR.

 
DDV10 expression during postnatal ontogeny
The vaginal epithelium of newborn mice is a pseudostratified epithelium composed of two or three layers, and this state continues to change about 20 d after birth (Fig. 7AGo). Keratins constitute a family of approximately 20 related {alpha}-helix-rich structural proteins of 40–70 kDa that are subunits of the intermediate filaments present in all epithelial cells (18). Keratin 1 is the predominant cytoskeletal intermediate filament protein expressed in epithelial cells during the transition from the proliferative to the terminal differentiation stage. Antikeratin 1 antibody-positive cells in vaginal epithelium were not found 5 and 20 d after birth (Fig. 7AGo). The production of E2 begins approximately 20 d after birth, and the vaginal epithelium proliferates and differentiates in response to increased E2 levels. Accordingly, antikeratin 1 antibody-positive cells are detected in the epithelium by 30 d after birth (Fig. 7AGo). Almost all vaginal epithelial cells were recognized by antikeratin 1 antibody (Fig. 7AGo). Q-PCR analysis did not detect DDV10 mRNA 20 d after birth, but by 30 d of age, the vaginal epithelium was multilayered, and a high level of DDV10 expression was observed (Fig. 7BGo).



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  		Figure 7. Expression of DDV10 during postnatal ontogeny. A, Histological changes in vaginal epithelium. Vaginas were dissected from animals 5, 20, and 30 d after birth. Vaginas were fixed and stained with hematoxylin-eosin or immunostained with antikeratin antibody ({alpha}-kera) and antimouse keratin 1 antibody ({alpha}-MK1). Magnification, x200. B, Total RNA was isolated from vagina 5, 10, 20, and 30 d after birth, and the DDV10 mRNA level was analyzed by Q-PCR. Results are the mean ± SEM of three experiments, each performed in triplicate. *, P < 0.05 vs. 5 d.

 
DDV10 is not expressed in vaginas of {alpha}ERKO mice
Estrogens promote vaginal epithelial proliferation and differentiation (19). These events require ER{alpha}, as shown by the lack of vaginal epithelial proliferation, stratification, and cornification in E2-treated {alpha}ERKO mice (20). Antikeratin antibody recognized the vaginal epithelium of {alpha}ERKO mice. However, antikeratin 1-positive staining was not detected in epithelial cells (Fig. 8AGo). RNAs from vaginas and tongues of wild-type and {alpha}ERKO mice were analyzed for the expression of DDV10 mRNA by RT-PCR. As shown in Fig. 6BGo, DDV10 mRNA was expressed in both vagina and tongue of wild-type mice. In {alpha}ERKO mice, DDV10 mRNA was detected in tongue, but not in vagina (Fig. 8BGo). E2 injection into {alpha}ERKO mice was unable to elicit detectable expression of DDV10 in vagina (data not shown).



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  		Figure 8. DDV10 is not expressed in vaginas of {alpha}ERKO mice. A, Immunohistochemistry was carried out on a section of vagina from {alpha}ERKO mice. Antikeratin antibody ({alpha}-keratin; upper panel) and antimouse keratin 1 antibody ({alpha}-MK1; lower panel) were used. Magnification, x200. B, Total RNA was prepared from vagina (V) and tongue (T) of wild-type and {alpha}ERKO mice, and the cDNA was synthesized. The expression of DDV10 and GAPDH mRNAs was analyzed by RT-PCR.

 
DDV10 mRNA is expressed in vaginal epithelial cells, but not stromal cells
Our data suggested that DDV10 may be involved with the proliferation, stratification, and cornification of vaginal epithelium; therefore, we next examined whether DDV10 mRNA is expressed in epithelium or stroma. The vaginal epithelium was separated from the stroma, and RNA was prepared from each tissue and analyzed by Q-PCR. We found that DDV10 mRNA was present in epithelium, but not in stroma (data not shown). We confirmed this localization using in situ hybridization. To distinguish DDV10 from OCIL, an RNA probe complementary to amino acids 1–98 of DDV10, was used. As expected, DDV10 mRNA was detected in epithelium, but not in stroma (Fig. 9Go). Furthermore, DDV10 was localized in suprabasal cells, but not in basal cells that proliferate in response to estrogen. These results suggest that DDV10 may be involved in stratification and/or cornification, but not proliferation, of vaginal epithelial cells.



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  		Figure 9. Expression of DDV10 in vaginal epithelial cells. In situ hybridization was carried out on a section of vagina at the stage of estrus. DDV10 mRNA expression was observed in suprabasal cells in vaginal epithelium (upper panel, antisense probe; lower panel, sense probe). Magnification, x100.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Estrogens regulate the proliferation and differentiation of cells in the mouse vagina. However, the molecular mechanism underlying estrogen-induced cell proliferation and differentiation have not been defined. In this study we reported the molecular cloning and characterization of a putative C-type lectin, termed DDV10, which may be involved in the stratification and/or cornification of vaginal epithelium. The predicted structure suggests that DDV10 encodes a type II transmembrane protein containing a carboxyl-terminal extracellular domain and an amino-terminal cytoplasmic domain. We showed that DDV10 is expressed in stratified, cornified epithelia of eye, tongue, stomach, and epithelial cells, but not stromal cells, in vagina. DDV10 is not expressed in vagina until 20 d after birth; moreover, vaginal expression is drastically decreased after OVX, but returns approximately 12 h after E2 treatment. DDV10 mRNA is undetectable in vaginas of ER{alpha}KO mice. We infer from these results that DDV10 is a novel, E2-regulated, C-type lectin.

Lectins are nonenzymatic sugar-binding proteins that bind with considerable specificity to complex carbohydrates found on secreted glycoproteins, cell surface glycoproteins, and extracellular matrix proteins. Lectins are involved in numerous cellular processes, such as host-pathogen interactions, targeting of proteins within cells, and cell-cell interactions (21, 22). The calcium-dependent (C-type) lectin family consists of two subtypes: soluble C-type lectin and transmembrane C-type lectin. C-Type lectins, include cell adhesion molecules such as selectins, which target leukocytes to lymphoid tissues and sites of inflammation (23, 24); mannose-binding proteins, which function in antibody-independent host defense against pathogens (25, 26); and leticans, a family of chondroitin sulfate proteoglycans including aggrecan, versican, neurocan, and brevican (27, 28). Each of these proteins contains a C-type carbohydrate recognition domain attached to other domains responsible for the physiological functions of the molecule (29). A new C-type lectin related to DDV10 called OCIL (osteoclast inhibitory lectin) was recently cloned and characterized (17). DDV10 has a high homology to mouse OCIL. The C-type lectin domains of DDV10 and OCIL amino acid sequences are 83% identical in the C-type lectin domain, but only 32% identical in the cytoplasmic domain. No further similarity to other proteins or putative conserved domains has been detected. We note that the cytoplasmic domain of DDV10 consists of 106 amino acids (Fig. 2Go); therefore, DDV10 may interact with another protein(s) through its cytoplasmic domain and could be involved in the regulation of calcium-dependent signal transduction. We compared the expression of DDV10 and OCIL in vagina and other tissues and found that DDV10 is expressed in proliferated vaginal epithelium. DDV10 mRNA levels rapidly decrease, whereas the expression of OCIL gradually increased after OVX. E2 treatment induced DDV10 expression and repressed OCIL expression. DDV10 mRNA levels change during the normal reproductive cycle and are high at estrus (data not shown). Taken together, these results suggest that estrogens may positively regulate DDV10 while negatively regulating expression of OCIL. It is currently unknown whether the effects of estrogens on the expression of DDV10 are direct or indirect. Unlike c-Jun and c-Fos, which rapidly increase after E2 treatment (30, 31, 32), DDV10 expression was not detectable until 12 h after E2 treatment in OVX mice. The putative DDV10 promoter does not contain a consensus E2 response element (data not shown), and the expression of DDV10 in stomach appears to be E2 independent (data not shown). Therefore, it is likely that DDV10 is indirectly regulated by estrogens, but further study is required to establish this point.

Vaginal expression of DDV10 mRNA was rarely detected 20 d after birth, but was routinely detected 30 d after birth. We found that the expression of DDV10 began approximately 20 d after birth, and that DDV10 expression increased as the epithelial cells proliferated in response to increasing endogenous E2 levels (data not shown). In agreement with this model, DDV10 mRNA was not detected in vaginas of {alpha}ERKO mice, which have two or three layers of atrophied, noncornified epithelium. In situ hybridization revealed that DDV10 was expressed in vaginal epithelial cells of the suprabasal cell layers, but not the proliferating basal cell layer, suggesting that it functions during epithelial differentiation, but not proliferation. We previously showed that neuropsin/kallikrein 8 is expressed in the mouse vagina and could not be detected until 24 h after E2 treatment (33) in contrast with DDV10. Thus, like neuropsin/kallikrein 8, DDV10 is a marker of epithelial differentiation and may act as a differentiation-regulating factor, perhaps playing a role in the initial events during the differentiation of vaginal epithelium.

Calcium is an important regulator of keratinocyte differentiation (7). Incubation of cultured keratinocytes with calcium increases differentiation and the expression of differentiation-associated genes (34, 35, 36). Within hours of calcium treatment, keratinocytes shift from production of the basal keratins K5 and K14 to keratins K1 and K10, followed, subsequently, by increased levels of profilaggrin (the precursor of filaggrin, an intermediate filament-associated protein), involucrin and loricrin (precursors for the cornified envelope) (37, 38). An in vivo epithelial gradient of calcium has been identified with increasing calcium levels in the more differentiated layers. This suggests that calcium plays an important role in regulating epidermal differentiation (39). However, the mechanism by which the increase in extracellular free calcium triggers differentiation is not well understood. One possible mechanism involves the calcium-dependent activation of protein kinase C (PKC) (40, 41, 42). PKC{alpha} and PKC{delta} are abundant in keratinocytes and have been implicated as regulators of differentiation (43). Previous studies suggest that calcium regulates involucrin gene expression at the mRNA and protein levels (44, 45), and PKC{delta} was recently reported to enhance the calcium-dependent activation of involucrin promoter activity and endogenous gene expression (46). It is not known whether DDV10 gene expression is regulated by calcium, and there are no data concerning the biological role of DDV10 in epithelial differentiation. DDV10 is a calcium-dependent, carbohydrate-binding protein; hence, it could play an important role in epithelial differentiation via cell-cell interactions and/or signal transduction. This is the first evidence for the existence of an estrogen-regulated C-type lectin in vaginal epithelial cells, and we hypothesize that DDV10 may play an important role in regulating the estrogen-induced differentiation of vaginal epithelium in cooperation with calcium. Therefore, future studies aimed at delineating the biological function of DDV10 and its regulation by E2 will improve our understanding of how E2 regulates epithelial differentiation.


   Acknowledgments
 
We are grateful to Drs. Bruce Blumberg (Department of Developmental and Cell Biology, University of California, Irvine, CA) and Raphael Guzman (Cancer Research Laboratory and Department of Molecular Cell Biology, University of California, Berkeley, CA) for critical reading of the manuscript. We thank the members of the Iguchi laboratory for technical advice and encouragement.


   Footnotes
 
This work was supported in part by a Grant-in-Aid for Scientific Research from Ministry of Education, Science, Sports, and Culture of Japan and a Health Sciences Research Grant from the Ministry of Health, Labor, and Welfare of Japan.

Abbreviations: CT, Threshold cycle; DD/RT-PCR, differential display/RT-PCR; DES, diethylstilbestrol; E2, 17ß-estradiol; ER, estrogen receptor; ER{alpha}KO, ER{alpha} knockout; GAPDH, glyceraldehyde phosphate dehydrogenase; OCIL, osteoclast inhibitory lectin; oligo(dT), oligo(deoxythymidine); OVX, ovariectomized, ovariectomy; PKC, protein kinase C; Q-PCR, quantitative PCR; SSC, standard saline citrate.

Received September 18, 2002.

Accepted for publication February 21, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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