ARTICLE |
Correspondence to: Isabelle RosinskiChupin, Unité de Génétique et Biochimie du Développement, Département dImmunologie, Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris Cedex 15, France.
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Summary |
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The variable coding sequence (VCS) multigene family encodes diverse salivary proteins, such as the SMR1 prohormone and the PR-VB1 proline-rich protein in the rat. In situ hybridization was used to study the cell-specific expression of two new mouse VCS genes, Vcs1 and Vcs2. We show that the Vcs1 transcripts, which code for a proline-rich protein, MSG1, are highly abundant in male and female parotid glands, in which they are specifically detected in acinar cells. No expression was seen in the submandibular or sublingual glands. In contrast, Vcs2 transcripts were found only in the acinar cells of the submandibular glands (SMGs) of male mice, in which they are expressed in response to androgens. Expression was found to be heterogeneous within acinar structures. No Vcs2 transcripts were detected in the SMGs of females or castrated males by Northern blot, RNase protection, or in situ hybridization. Androgen administration to females or castrated males induced expression at a level comparable to that of intact males. The Vcs2 gene is the first example of a mouse androgen-regulated gene that is expressed in SMG acinar cells. This result, in addition to our previous observation on SMR1 expression in rats, demonstrates that both acinar cells and granular convoluted tubule (GCT) cells are target cells for androgen action in rodent SMG. (J Histochem Cytochem 46:669678, 1998)
Key Words: in situ hybridization, submandibular glands, parotid, acini, androgens, proline-rich proteins, multigene family
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Introduction |
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Salivary proteins are involved in buccal and dental tissue protection and maintenance, control of oral microflora and digestion (
The VCS (variable coding sequence) family was recently described in rats (
Recently, we began the characterization of the VCS family in the mouse. We cloned two cDNAs from SMGs, corresponding to the MSG1 and MSG2 proteins (
The Vcs2 gene differs in its structure from other VCS genes; this gene results from the juxtaposition in the same transcription unit, by a complex DNA rearrangement, of sequences from three different VCS genes ( protein, is predicted to be secreted and matured at dibasic sites to generate, like SMR1, a five or six amino acid-long peptide.
Here we study the expression of Vcs1 and Vcs2 mouse genes in the salivary glands, in particular the cellular distribution of the transcripts, by in situ hybridization.We show that the Vcs1 gene, encoding the proline-rich protein MSG1, is mainly expressed in the acinar cells of the parotid glands. In contrast, the Vcs2 gene, encoding MSG2, appears to be expressed only in the submandibular gland acinar cells of male mice.
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Materials and Methods |
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Animals and Tissue Preparation
Male and female Balb/c mice were obtained from Iffa Credo (Lyon, France). To evaluate the effects of androgens on VCS gene regulation, male, female and castrated mice were purchased at 5 weeks and kept in house for 4 weeks. The 9-week-old mice were then injected SC either with dihydrotestosterone (Sigma Chemicals; St Louis, MO) at 6 mg/ml (20 mg/ kg) in sesame oil or with vehicle alone. They received five injections spread over 1 week. At 24 hr after the last injection, the mice were sacrificed by cervical dislocation or CO2 suffocation.
Submandibular, sublingual, and parotid glands were rapidly removed and processed as previously described (
Northern Blot Analysis
RNA from submandibular glands of 8-week-old male (n = 5) and female (n = 5) Balb/c mice were prepared according to cDNA, GenBank accession number U 82380), exon III' (227-BP fragment between nt 310536 of Vcs2ß, GenBank accession number U82376), phospholipase A2 (GenBank accession number X82631), ß-actin (297-BP Pvu II-Kpn I fragment between nt 688985 of ß-actin cDNA, GenBank accession number X03765) were radiolabeled by random priming (
-32P]-dCTP. Hybridization was performed as previously described (
Plasmids for In Vitro Transcription
Two sequences of the Vcs2 gene were used to generate plasmids for in vitro transcription. The first corresponds to a 364-BP Ava II-Eco RI fragment of Vcs2 cDNA and was subcloned between the Eco RI and Eco RV sites of the pcDNAII plasmid (Invitrogen Corporation; Leek, The Netherlands) to generate the pcV2a plasmid. The second sequence corresponds to a 309-BP Hae III-Pvu II fragment of Vcs2ß cDNA and was subcloned into the Eco RV site of pcDNAII to generate the pcV2b plasmid. Probes generated by in vitro transcription of pcV2b recognize exon III'-containing mRNAs (ß,
,
, and
Vcs2 mRNAs). pcV1 was obtained by subcloning a 258-BP Sau 3A-Pvu II fragment of Vcs1 cDNA (GenBank accession number X71629) in the Bam HI-Eco RV sites of pcDNAII. pAct was obtained by subcloning the Pvu II-Kpn I fragment (nt 688985) of ß-actin cDNA into the pcDNAII plasmid.
RNase Protection
Three RNA probes were generated by in vitro transcription in the presence of [-32P]UTP, according to manufacturer's instructions (Stratagene; La Jolla, CA) of (a) pcV2a from the SP6 promoter after linearization by Xba I, (b) pcV2b from T7 promoter after linearization by Bam HI, and (c) pAct from SP6 promoter after Xba I linearization.
RNase protection assay was performed using RNase I (Promega; Madison, WI) according to the manufacturer's instructions. Briefly, 20 µg of total RNA was hybridized with a mixture of the three probes (200,000 cpm of each; specific activity of each probe 160 Ci/mmol) for 22 hr at 45C in 30 µl of hybridization buffer (80% deionized formamide, 40 mM PIPES, pH 6.4, 0.4 M sodium acetate, pH 7, 1 mM EDTA). After hybridization the samples were digested by 5 U of RNase I for 1 hr at 37C in 300 µl of 10 mM Tris-HCl, pH 7.5, 5 mM EDTA, 200 mM sodium acetate, pH 7). Digestions were stopped by addition of SDS (final concentration 0.07%) and tRNA (40 µg/ml final). After ethanol precipitation, the samples were resolved in a 6% polyacrylamide sequencing gel. For each group of animals, five individual preparations of RNA were separately analyzed.
In Situ Hybridization
35S-Labeled RNA probes were synthesized in a 25-µl volume, using 500 ng of linearized plasmid and 50 µCi of [-35S]-UTP (1000 Ci/mmol) in the presence of 40 mM Tris-HCl, pH 7.5, 2 mM spermidine, 6 mM MgCl2, 10 mM NaCl, 10 mM dithiotreitol, 0.6 mM each of ATP, GTP, CTP, and 40 U of T7 or SP6 RNA polymerase. No hydrolysis of the probes was performed. Slides were postfixed by immersion in a 4% paraformaldehyde solution in PBS, pretreated with 1 µg/ml proteinase K at 37C in 20 mM Tris-HCl, pH 7.5, 20 mM CaCl2 for 25 min, and acetylated before hybridization. Hybridization was performed overnight at 52C as previously described, using 50,000 cpm/µl of probe in 50% formamide, 0.3 M NaCl, 20 mM Tris-HCl, pH 7.4, 5 mM EDTA, 10 mM NaH2PO4, pH 8.0, 10% dextran sulfate, 1 x Denhardt's solution, and 0.5 mg/ml yeast RNA. Washings were as previously described (
In situ hybridization experiments with Vcs1 digoxigenin-labeled RNA probes were performed as previously described (
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Results |
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Expression of the Mouse Vcs1 Gene in Mouse Parotids
In situ hybridization experiments were performed on mouse salivary glands sections using a Vcs1 cRNA probe into which either a radiolabeled (Figure 1a) or a digoxigenin-labeled (Figure 1b) ribonucleotide was incorporated. This probe, designed to specifically detect Vcs1 transcripts, corresponds to the coding region of Vcs1 exon III. Parotid sections were positive in both male and female mice after a 1-day exposure time. No sex-linked difference in expression was observed (data not shown). The signal was completely abolished by RNase pretreatment and was not detected after hybridization with sense Vcs1 RNA probe (data not shown), demonstrating that this signal is specific for Vcs1 transcripts. Acinar cells were determined as the site of Vcs1 expression in mice parotids. No expression was detected on the parotid ductal cells (Figure 1b). No effect of castration or androgen injection was observed on the intensity of the signal in parotid acinar cells (data not shown).
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Surprisingly, although Vcs1 cDNA was cloned from mouse submandibular gland tissue (
Vcs2 mRNAs Are Differentially Expressed in Submandibular Glands of Male and Female Balb/c Mice
Vcs2 transcripts were previously characterized from SMG of male mice (
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To analyze the role of androgens in this sex-linked difference of expression, we performed castration and androgen injection experiments. SMG RNA preparations were analyzed by Northern blot with both probes. As shown in Figure 3B (Lanes 3, and 6), castration results in the disappearance of SMG-specific Vcs2 transcripts. Androgen injection for 1 week to castrated males (Figure 3B, Lanes 4 and 7) or to females (Figure 3B, Lane 2) results in the induction of expression to a level similar to males. RNase protection experiments confirmed that the signals were specific to Vcs2 transcripts and gave the same results as Northern blot experiments (Figure 3C). Bands specific for both probes were only seen in males (Figure 3C, Lanes 9 and 10), androgen-treated females (Figure 3C, Lanes 3 and 4), or androgen-treated castrated males (Figure 3C, Lanes 7 and 8) and not in females (Figure 3C, Lanes 1 and 2) or castrated males (Figure 3C, Lanes 5 and 6) treated with vehicle alone.
Hybridization of male Balb/c mouse SMGs with a 35S-radiolabeled RNA probe corresponding to exon III of the Vcs2 gene resulted in a signal localized in acinar cells (Figure 4). Not all the acinar cells were equally labeled, and inside a single acinus there was cell-to-cell variations. No signal above background could be detected either in the ducts of the SMGs or in the adjacent sublingual and parotid glands. The specificity of the signal for SMG acinar cells was assayed by hybridization with a sense probe and no signal was detected under these conditions (Figure 5e).
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In situ hybridization of mouse SMGs with an anti-sense RNA probe corresponding to exon III' of Vcs2 gave similar results as with the exon III probe (Figure 5b). In addition, a similar pattern of positive cells was revealed by hybridizing serial sections of SMGs with either probe, showing that different types of Vcs2 transcripts are found in the same cells (Figure 5a and Figure 5b).
In contrast, no signal was detected in the glands of female mice using either probe (Figure 5c and Figure 5d). Castration resulted in the almost total disappearance of positive cells on sections of male SMGs (Figure 5f). Treatment with dihydrotestosterone of either females (Figure 5g) or castrated males (Figure 5h) led to the induction of expression of Vcs2 transcripts in a relatively high number of acinar cells, but not all acinar cells were positive.
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Discussion |
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Our aim was to localize, by in situ hybridization, transcripts of two genes of the mouse VCS family. These two genes encode proteins differing in their sequence and probably in their function. Here we show that these two genes are differentially expressed in the salivary glands. Whereas Vcs1 transcripts are expressed at a high level in the acinar cells of parotids, Vcs2 transcripts are expressed at a low level in the acinar cells of the SMG.
The Vcs1 gene was previously shown to encode a proline-rich protein. Proline-rich proteins are abundant in the saliva, well-characterized in humans, rats and mice (
No effect of androgens on the expression of the Vcs1 gene in the parotids has been detected. Whether this result is due to the lack of androgen responsive regulatory sequences in Vcs1 gene or to the absence of androgen responsiveness of the parotids is not known. To date, to our knowledge, no sex-linked difference of gene expression in the parotids has been demonstrated, and attempts to demonstrate the presence of the androgen receptor in mouse parotid glands have failed (
In contrast, the mouseVcs2 gene is differentially expressed in the SMGs of males and females in response to androgens. Expression in males can be abolished by castration and expression in females can be induced by androgen injection. Therefore, the Vcs2 gene is reminiscent of the rat VCSA1 gene, although both genes differ in their level of expression. Whereas VCSA1 mRNAs represent more than 10% of adult male rat SMG mRNAs, Vcs2 transcripts are low-abundance mRNAs. In females, Vcs2 transcripts were not detected by Northern blot, RNase protection, or in situ hybridization. In contrast, low but detectable levels of VCSA1 mRNAs are present in female rat SMG. We also show that, like the rat VCSA1 gene (
Cell specificity of expression among acinar cells is revealed because the level of Vcs2 transcript accumulation varied among cells. The reasons for such differences are not known, and could include different accessibility of cells to androgens or other hormones, and differences in the state of differentiation or in the cell cycle. The persistence of a perinatal cellular phenotype in some acinar cells of adult rat SMG has been reported (
In conclusion, our results show that the VCS genes are differentially regulated, suggesting that regulation of expression has evolved together with the diversification of function, as has also been observed in a number of other multigene families (
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Acknowledgments |
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Supported by grants from the Institut Pasteur, from the GREG (Groupement d'Etudes et de Recherches sur le Genome), and from the DRET (contract 93 113/DRET).
We especially thank Dr B. Laoide for critical reading of this manuscript and Dr C. Rougeot for help and suggestions in experimental care of mice.
Received for publication July 21, 1997; accepted December 11, 1997.
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