Expression and Localization of the Transcription Factor JunD in the Duct System of Mouse Submandibular Gland

Abstract

We studied the expression and localization of JunD, a component of the transcription factor activator protein-1 (AP-1), in the mouse submandibular gland with immunoblotting and immunohistochemistry. In adult mice, all seven Jun and Fos family members constituting the AP-1 complex were expressed more abundantly in the female gland than in the male gland, and JunD was the most abundant of the members. Immunoreactivity for JunD was localized exclusively in the duct system of the gland, in which it was localized to the nuclei of intercalated duct (ID) cells and a subpopulation of striated duct (SD) cells located adjacent to ID. In contrast, granular convoluted tubule (GCT) cells, which are much more abundant in the male gland, were devoid of JunD. During postnatal development of the male gland, JunD was lost from the duct cells as they differentiated to GCT cells at 3-5 weeks postpartum. When GCT differentiation was induced in adult female gland by testosterone administration, many JunD-negative SD cells were temporarily induced to express JunD after 6-24 hr, but those cells lost JunD as they completely converted to GCT cells by 48 hr. These results suggested that JunD is involved in the differentiation of the duct system of mouse submandibular gland, in which there is crosstalk between the androgen/androgen receptor system and the AP-1 complex.

Keywords

AP-1 JunD immunohistochemistry submandibular gland duct mouse

The submandibular gland of rodents provides a useful model for investigating the proliferation and differentiation of epithelial cells in vivo because extensive development of both the acinar and the ductal components of this gland takes place postnatally under the control of neuronal and hormonal factors (Jacoby and Leeson 1959; Gresik 1980). The proliferation of acinar cells and their precursors, as well as the differentiation of the former from the latter, occurs during the first few postnatal weeks and is considered to be controlled primarily by the autonomic nervous system through β-adrenergic mechanisms (Chang and Barka 1974; Cutler et al. 1985). Consistent with this, administration of the β-adrenergic agonist isoproterenol induces proliferation and hypertrophy of submandibular gland acinar cells in developing and mature mice and rats. Isoproterenol exerts its effect through binding to the cell surface β-adrenergic receptor and activating the cAMP/protein kinase A (PKA) signaling pathway, which is known to involve activated cAMP response element-binding protein (CREB) as its downstream transcription factor. There is evidence in the rat submandibular gland that CREB is expressed abundantly in the nuclei of acinar cells during the early postnatal weeks and is responsible for the isoproterenol-induced acinar cell proliferation (Amano and Iseki 1998,2001).

In contrast, development of the mature duct system, which is represented by the differentiation of granular convoluted tubule (GCT) cells from striated duct (SD) cells, occurs during puberty under the control of hormonal factors such as androgens, thyroid hormones, and adrenocortical hormones (Chretien 1977; Pinkstaff 1980; Gresik 1994). In particular, androgens are considered the primary factor of GCT differentiation because in the mouse there is a marked sexual dimorphism in the development of GCT that proceeds around 3-5 weeks postpartum. Castration of adult male mice causes marked regression of GCT owing to conversion of GCT cells to SD cells, whereas administration of androgens to castrated male or normal female mice readily causes the opposite phenomenon (Caramia 1966b; Chretien 1977). GCT cells are characterized by large secretory granules that contain a variety of biologically active polypeptides, including nerve growth factor (NGF), epidermal growth factor (EGF), renin, and kallikreins (Barka 1980; Gresik 1994).

The androgens exert their biological functions by binding to the androgen receptor (AR), a member of the nuclear receptors that is believed to act as a transcription factor in itself by binding to androgen response element (ARE) upstream of the androgen-regulated genes (Zhou et al. 1994; Chang et al. 1995; Brinkmann et al. 1999). AR is expressed in both acinar and duct cells of the rodent submandibular gland (Morrell et al. 1987; Zhuang et al. 1996). However, little is known about the mechanisms by which the androgen/AR system causes differentiation of GCT, including the gene product that is induced by androgen and plays a crucial role in GCT cell differentiation.

Recently, we found in the rat submandibular gland that CREB is expressed abundantly in the nuclei of intercalated duct (ID) cells and distal SD cells for 3-5 weeks postpartum but is no longer expressed in differentiated GCT cells (Amano and Iseki 1998; Kim et al. 2001). Administration of testosterone to immature or hypophysectomized rats caused a temporary increase in the number of CREB-positive SD cells before their differentiation to GCT cells. Furthermore, inhibition of CREB synthesis by the antisense oligonucleotide led to suppression of GCT cell differentiation. These results suggested the existence of cross-talk between the cAMP/PKA and the androgen/AR signaling pathways in the mechanisms of androgen-dependent GCT differentiation.

Here we examined the expression of another transcription factor, activator protein-1 (AP-1), in the mouse submandibular gland. The AP-1 family is a complex composed of the Jun family (c-Jun, JunB, and JunD) and the Fos family (c-Fos, FosB, Fra1, and Fra2), the proto-oncogene-encoded nuclear proteins. The Jun family members form homo- or heterodimers among themselves or heterodimers with the Fos family members and bind to the AP-1 consensus DNA sequence located in the promoter region of a variety of target genes (Vogt and Bos 1990; Angel and Karin 1991). The activity of AP-1 in transactivating the expression of target genes is regulated at both the transcriptional and the post-translational level. The transcription of c-Jun is positively regulated by binding of c-Jun homodimers to an AP-1 site located in the promoter region of c-Jun gene itself, whereas the transcription of c-Fos is induced by a variety of factors such as serum, hormones, growth factors, and cytokines, through their cognate signaling pathways and cis-acting elements in the c-Fos promoter. On the other hand, the post-translational activation of AP-1 is represented by phosphorylation and/or dephosphorylation of c-Jun protein, which is also regulated by a variety of signaling pathways (Angel and Karin 1991; Karin 1995).

AP-1 has been implicated in the regulation of cell proliferation, differentiation, apoptosis, and transformation in many in vitro and in vivo systems. In the mouse submandibular gland, stimulation of the β-adrenergic receptor by isoproterenol causes a rapid, transient induction of c-Fos expression in both acinar and duct cells, although the biological significance of this phenomenon has not been clarified (Barka et al. 1986). In this study, by use of immunochemical and immunohistochemical methods, we demonstrate that many AP-1 family members, primarily JunD, are expressed exclusively in the duct system of mouse submandibular gland, and that JunD expression and localization are regulated in association with the androgen-dependent duct cell differentiation.

Materials and Methods

Animals and Tissue Preparation

Male and female ddY mice were purchased from Nippon SLC (Hamamatsu, Japan) and grown under standard laboratory conditions with free access to food and water. All experiments were conducted in accordance with the Guidelines for the Care and Use of Laboratory Animals at the Takaramachi Campus of Kanazawa University. At the ages of 1 W, 2 W, 3 W, 4 W, 5 W, 6 W, and 8 W (adult) the animals were sacrificed under pentobarbital anesthesia by transcardial perfusion with physiological saline. For immunoblot analyses, the submandibular glands were removed and frozen immediately in liquid nitrogen. For immunohistochemical (IHC) analyses, the animals were fixed by perfusion with 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.2. The submandibular glands were removed and further fixed by immersion in the same fixative for 4 hr at 4C. In one experiment, groups of three adult female mice were administered a single SC injection with testosterone (Wako Pure Chemical; Osaka, Japan) dissolved at 100 mg/kg body weight in 0.2 ml olive oil and were sacrificed at 6 hr, 24 hr, or 48 hr after the injection. For a negative control, vehicle alone was administered.

Western Blotting Analysis

Rabbit polyclonal IgG antibodies against the synthetic peptides of the Jun family members (c-Jun, JunB, and JunD), Fos family members (c-Fos, FosB, Fra1, and Fra2), mouse NGF, and actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The frozen mouse submandibular glands were homogenized in a lysis buffer composed of 1% Nonidet P40, 0.5% sodium deoxycholate, 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, and the proteinase inhibitor cocktail (Roche; Mannheim, Germany). After centrifugation at 3000 rpm, the supernatants were examined for the protein concentration using a BCA protein assay kit (Pierce; Rockford, IL) and used as cell lysates. The cell lysates at 20 μg protein/lane were separated by electrophoresis in a 15% polyacrylamide gel in the presence of 0.1% SDS and then transferred to PVDF membranes (BioRad Laboratories; Hercules, CA). After treatment with 4% non-fat skimmed milk in PBS plus 0.01% Tween-20, the membranes were incubated with the rabbit antibodies against the primary antibodies at 0.05 μg/ml for 1 hr at room temperature (RT). This concentration of the antibodies was determined in preliminary experiments to give the maximal immunoreactions for all antigens. After washing with PBS plus 0.01% Tween-20, the membranes were incubated with horseradish peroxidase-conjugated anti-rabbit IgG antibody (DAKO; Glostrup, Denmark) at 1:3000 dilution for 1 hr. The immunoreactions were detected with X-ray films (Kodak X-OMAT AR) after treatment of the membranes with the chemiluminescence kit ECL-plus (Amersham Pharmacia Biotech; Uppsala, Sweden). For a negative control, the antibodies were absorbed with a tenfold amount of the peptide antigen used for immunization (Santa Cruz Biotechnology) for 1 hr at 4C before use.

Immunodot-blot Assay

Five μl of the cell lysates of submandibular glands from three mice in each experimental condition were serially diluted, spotted in duplicate onto methanol-activated PVDF membranes, and dried. The blots were then immunostained with anti-JunD antibody and visualized using chemiluminescence in the same way as for Western blotting. The developed X-ray films were scanned with an Epson GT-9800F scanner and the densitometric analysis of blots was performed with Image Gage V3.41 software (Fuji Photo Film; Tokyo, Japan). Within the dilutions of lysate showing proportional increase in optical density, the relative immunore-activity of each sample was determined in terms of the mean optical density of duplicate blots. The values were expressed as the mean ± SD of three samples, and the difference between two values with p<0.05 was considered significant on Student's t-test.

Immunohistochemistry

The fixed submandibular glands were dehydrated with a graded ethanol series, cleared with xylene, and embedded in paraffin. Five-mm sections were cut from the paraffin blocks and mounted on gelatin-coated glass slides. The sections were treated successively with 0.3% Tween-20 in PBS for 1 hr for cell permeabilization, 0.3% H₂O₂ in methanol for 10 min to inhibit intrinsic peroxidase activity, and 3% normal swine serum for 30 min to prevent nonspecific antibody binding. They were then incubated overnight at RT with rabbit anti-JunD antibody or rabbit anti-NGF antibody at a concentration of 0.5 μg/ml in PBS. To confirm the specificity of immunoreactions, the antibodies were absorbed with a tenfold amount of the corresponding peptide antigen for 1 hr at 4C before use. After washing, the sites of immunoreactions were visualized by incubating the sections successively with biotinylated anti-rabbit IgG antibody (Vector Laboratories; Burlingame, CA) at 1:200 dilution for 1 hr, horseradish peroxidase-conjugated streptavidin (DAKO) at 1:300 dilution for 1 hr, and 0.01% diaminobenzidine tetrahydrochloride in the presence of 0.02% H₂O₂ in 50 mM Tris-HCl, pH 7.5, for about 10 min. The sections were observed under an Olympus BX50 microscope connected to a CCD video camera system. At 480-fold magnification, three fields were selected randomly from each of the sections obtained from three animals at each experimental condition. The percentage of JunD-immunopositive cell nuclei to the total cell nuclei in the duct system (ID, SD, GCT, and excretory ducts), defined as the ratio of immunopositive nuclei, was calculated for each field and expressed as the mean ± SD of nine fields. The difference between two values with p<0.05 was considered significant on Student's t-test. In one experiment, fluorescent double immunostaining for JunD and NGF was performed. The sections were incubated with a mixture of rabbit anti-JunD antibody (4 μg/ml) and sheep anti-NGF antibody (1 μg/ml; Chemicon, Temecula, CA) overnight at RT. After washing, the sections were incubated with a mixture of Cy3-labeled anti-rabbit IgG (6.5 μg/ml; Jackson ImmunoResearch Laboratories, West Grove, PA) and Cy2-labeled anti-goat IgG (32.5 μg/ml; Jackson ImmunoResearch Laboratories) for 1 hr. They were then mounted in glycerol and observed with an Olympus BX50/BX-FLA fluorescent microscope using green emission for Cy3 and blue emission for Cy2.

Results

Sexual Dimorphism in the Expression and Localization of AP-1 factors

The cell lysates from adult male and female submandibular glands were analyzed with Western blotting using antibodies specific for the AP-1 factors, diluted to provide the maximal intensity of immunoreactions for individual factors (Figure 1). In the female gland, immunopositive bands for all three Jun family members (c-Jun, JunB, and JunD) and four Fos family members (c-Fos, FosB, Fra1, and Fra2) were detected, with predicted molecular weights. In particular, JunD was seen with much higher intensity than any other factors. In contrast, in the male gland only very small amounts of JunD and Fra2 were seen, but the other factors were hardly detectable. This sexual dimorphism in the expression of AP-1 factors was in sharp contrast to that of NGF, which was much more abundant in the male gland. The equality of protein loading was confirmed for actin, a housekeeping gene product. The specificity of the immunoreactions was confirmed by disappearance of the bands when the antibodies were absorbed with the corresponding antigen peptides before use (not shown).

Figure 1

Western blotting analysis showing the expression of the members of Jun (c-Jun, JunB, and JunD) and Fos (c-Fos, FosB, Fra1, and Fra2) families constituting the AP-1 complex in the submandibular glands of male and female mice. Cell lysates at 20 μg protein per lane were electrophoresed, blotted onto PVDF membrane, and incubated with the corresponding antibodies. For comparison, incubation with anti-NGF antibody and anti-actin antibody was also performed. Molecular weights of the immunoreactive bands are indicated at right.

The paraffin sections of adult male and female submandibular glands were then analyzed for localization of AP-1 factors with IHC (Figure 2). We show only the results for JunD, which provided the most intense and clear immunostaining, but the results for other factors were essentially the same as those for JunD. The male and female glands had apparent sexual dimorphism, as demonstrated by NGF immunostaining. The duct system of the male gland was composed mostly of GCT cells that contained NGF-positive secretory granules, whereas SD cells occupied the largest part of the duct system of the female gland, with only a small number of NGF-positive GCT cells scattered in the middle to distal portions of SD (Figures 2a and 2b). In the male gland, the immunoreactivity for JunD was localized exclusively in the nuclei of ID cells and was not detected in GCT cells nor in acinar cells (Figure 2c). In the female gland, JunD immunoreactivity was localized to the nuclei of both ID cells and the cells occupying the distal end portions of SD adjacent to ID. A small number of JunD-positive cells were also found in other portions of SD, but they appeared to be distinct from GCT cells with clear secretory granules. The majority of SD cells and acinar cells were immunonegative for JunD (Figure 2d). When the antibody was preabsorbed with the antigen polypeptide, no immunostaining was obtained in any cell or structure (Figures 2e and 2f).

Figure 2

IHC localization of JunD and NGF in adult male and female submandibular glands. The sections of male (a,c,e) and female (b,d,f) glands were immunostained with anti-NGF antibody (a,b), anti-JunD antibody (c,d), and anti-JunD antibody preabsorbed with the JunD antigen (e,f). (a,b) NGF immunoreactivity is localized to the cytoplasm of GCT cells, which occupy most of the duct portions in the male but are scattered in small numbers among immunonegative SD cells in the female. (c,d) JunD immunoreactivity is localized to the nuclei of ID cells in the male, whereas in the female it is localized to the nuclei of both ID cells and the cells located in the distal portions of SD adjacent to ID. GCT cells and acinar cells (A) are immunonegative. (e,f) No JunD immunoreactivity is present in any cells. Bars = 50 μm.

To demonstrate the sexual dimorphism in JunD expression and localization quantitatively, we used two parameters, i.e., the relative immunoreactivity and the ratio of immunopositive nuclei (Figures 3a and 3b). Because Western blotting of cell lysates with the present anti-JunD antibody formed a single immunoreactive band with low background reaction, the relative immunoreactivity for JunD was quantified for multiple cell lysate samples using the immunodot-blot assay (De León et al. 1995). On the other hand, the ratio of the number of immunopositive nuclei against that of the total nuclei in the duct system was evaluated in tissue sections. For relative immunoreactivity, the female gland was 12-fold higher than the male gland (p<0.05) (Figure 3a). The ratio of immunopositive nuclei in the female gland was 29%, which was higher than 17% in the male gland by ~1.7 fold (p<0.05) (Figure 3b). These results suggested that the lower expression of JunD in the male submandibular gland is explained, at least partly, by the absence of JunD in GCT cells that are developed preferentially in the male gland.

Figure 3

Quantitative analysis of the expression of JunD in male and female submandibular glands. (a) Relative immunoreactivity for JunD in cell lysates of male and female submandibular glands as measured by immunodot-blot assay. The mean of the values for female glands is set at 1.0. ∗Significantly higher than the male; p<0.05, n = 3. (b) Ratio of immunoreactive nuclei in the duct system of male and female submandibular glands as measured in JunD-immunostained sections. ∗Significantly higher than the male; p<0.05, n = 9.

Postnatal Changes in the Expression and Localization of JunD

We then examined the changes in expression and localization of JunD during the postnatal development of male and female submandibular glands. In the early postnatal ages of 1-2 W postpartum, the duct system was composed of the ID and SD. JunD immunoreactivity was present in most of cell nuclei in the duct system in both male and female glands, although the intensity of the immunostaining varied among the nuclei (Figures 4a and 4b). There was no significant difference between sexes in the relative immunoreactivity or the ratio of immunopositive nuclei (Figures 4e and 4f). At 3-5 W, extensive differentiation of SD cells into GCT cells occurred preferentially in the male duct system, whereas the female duct system remained composed of mostly SD cells. The relative immunoreactivity for JunD in the female gland was almost unchanged during this period, whereas that in the male gland showed a sharp decline, reaching a value about 12% of that in the female gland at 6 W (p<0.05) (Figure 4e). The ratio of immunopositive nuclei decreased in both male and female glands, but the male gland showed a significantly larger decrease than the female gland in this parameter, reaching a value about 66% of that in the female gland at 6 W (p<0.05) (Figure 4f). In the sections of 5-W submandibular glands, nuclear JunD immunoreactivity remained present in most SD cells but was no longer present in the differentiated GCT cells, which had large clear secretory granules and were much more abundant in the male gland (Figures 4c and 4d). These results suggested that JunD disappears from the duct cell nuclei in association with the differentiation of SD cells into GCT cells, a phenomenon known to be androgen-dependent. After 6 W postpartum, the nuclear immunoreactivity in the remaining SD cells of the female gland also became weaker except in the distal end portions of SD (not shown).

Figure 4

Expression and IHC localization of JunD in the duct system of male and female submandibular glands during postnatal development. Sections of male (a,c) and female (b,d) glands at 2 W (a,b) and 5 W (c,d) postpartum were immunostained with anti-JunD antibody. The relative immunoreactivity in the cell lysates (e) and the ratio of immunoreactive nuclei in the duct system (f) during postnatal development are also shown. (a,b) The duct system is composed of ID cells and SD cells in both male and female glands. The immunoreactivity is positive in the nuclei of almost all duct cells, whereas it is negative in acinar (A) cells. (c) The duct system is composed of ID cells, some remaining SD cells adjacent to ID, and many GCT cells. The immunoreactivity is positive in the nuclei of ID and SD cells but is negative in GCT cells. (d) The duct system is composed of ID cells, many remaining SD cells, and a small number of GCT cells. The immunoreactivity is positive in the nuclei of ID cells and a majority of SD cells but is negative in GCT cells. Bars = 50 μm. (e) The mean of the values for female glands at 1 W is set at 1.0. ∗Significantly higher than the male; p<0.05, n = 3. (f) ∗Significantly higher than the male; p<0.05, n = 9.

Effect of Testosterone on Expression and Localization of JunD

To confirm the above issue, we examined the expression and localization of JunD in adult female submandibular gland induced for GCT differentiation by testosterone administration. Six to 24 hr after SC injection of a single dose of 100 mg/kg testosterone, there was a temporary and significant rise in the relative immunoreactivity and the ratio of immunopositive nuclei in the gland (p<0.05) (Figures 5e and 5f). In the tissue sections, many immunopositive cell nuclei newly appeared in the portions of SD other than its distal end, which, together with ID, continued to have immunopositive cell nuclei (Figures 5a-5c). By 48 hr after the injection, the majority of SD cells differentiated to GCT cells with clear secretory granules similar to those of the male gland. JunD immunoreactivity was no longer present in the nuclei of differentiated GCT cells (Figure 5d). The relative immunoreactivity and the ratio of immunopositive nuclei also showed a significant decline from those at 24 hr to values comparable to or even lower than those before the injection of testosterone (p<0.05) (Figures 5e and 5f).

Figure 5

Expression and IHC localization of JunD in female submandibular glands stimulated with testosterone. Sections of adult (8-W) female glands at 0 hr (a), 6 hr (b), 24 hr (c), and 48 hr (d) after testosterone injection were immunostained with anti-JunD antibody. The relative immunoreactivity in the cell lysates (e) and the ratio of immunoreactive nuclei in the duct system (f) after testosterone injection are also shown. (a) JunD immunoreactivity is localized to the nuclei of ID cells and cells located in the distal portions of SD adjacent to ID. Acinar (A) cells are immunonegative. (b,c) Many SD cells in the middle and distal portions of SD have become positive for nuclear immunoreactivity. (d) The immunoreactivity has disappeared from most of the differentiated GCT cells. Bars = 50 μm. (e) The mean of the values at 0 hr is set at 1.0. ∗Significantly higher than the 0-hr value and ∗∗significantly lower than the 24-hr value; p<0.05, n = 3. (f) ∗Significantly higher than the 0-hr value and ∗∗significantly lower than the 24-hr value; p<0.05, n = 9.

To further clarify the relationship between GCT differentiation and nuclear JunD expression, the double immunostaining for NGF and JunD was performed on the gland 24 hr after testosterone injection. Most of the cells with positive cytoplasmic NGF were negative for nuclear JunD, and most of the cells with positive nuclear JunD were negative for cytoplasmic NGF (Figures 6a-6c). This result suggested that JunD is temporarily induced in the nuclei of SD cells by testosterone but is lost from the nuclei of differentiated GCT cells.

Figure 6

Double immunostaining for NGF and JunD in the female gland 24 hr after testosterone injection. (a) Cytoplasmic NGF shown with green fluorescence. (b) Nuclear JunD shown with red fluorescence. (c) Merged a and b. Note that cytoplasmic NGF and nuclear JunD do not occur in the same cells. Bars = 50 μm.

Discussion

The present study demonstrated that JunD, a member of the Jun family composing the transcription factor AP-1 complex with the Fos family (Hirai et al. 1989; Ryder et al. 1989), is expressed in the duct system of adult mouse submandibular glands, with higher immunoreactivity in the female than in the male gland. The duct system of adult mouse submandibular gland is composed of the ID, SD, GCT, and interlobular excretory duct (Pinkstaff 1980). The sexual dimorphism of the duct system is best represented by the difference in the proportion of GCT to SD, which is much higher in the male than in the female gland (Caramia 1966b; Gresik 1994). Unlike GCT cells, the majority of SD cells lack secretory granules but instead have extensive basal striations. However, a subpopulation of SD cells situated between ID and SD in the female or between ID and GCT in the male gland is characterized by the presence, in addition to basal striations, of apical secretory granules similar in appearance to those of GCT but smaller in size and number (Caramia 1966a). These cells are designated as striated granular duct (SGD) cells. SGD cells are much higher in number in the female gland than in the male gland, representing another sexual dimorphism of the duct system (Denny et al. 1990). In the present study, JunD immunoreactivity was localized to the nuclei of most ID cells in both male and female glands and of a subpopulation of SD cells situated at the distal end of SD in the female gland, whereas it was almost absent in the nuclei of GCT cells in the male gland. The immunopositive SD cell population in the female gland most likely represents SGD cells, which is justified by the absence or very small numbers of immunopositive cells between ID and GCT in the male gland. The absence of immunoreaction in GCT cells seems to account, at least in part, for the lower expression of JunD in the male gland compared with the female gland. The other six members of the Jun and Fos families, although much lower in immunoreactivity than JunD, showed similar expression and localization patterns to those of JunD, suggesting that the AP-1 complex is operating as a transcription factor in the duct system of mouse submandibular gland.

During postnatal development of the rodent submandibular gland, it is generally accepted that SD cells differentiate to GCT cells in an androgen-dependent manner (Cutler and Chaudhry 1975; Gresik and MacRae 1975; Srinivasan and Chang 1975). GCT cells first occur in the distal portions of SD by 2 W postpartum and show a much more rapid increase in numbers in the male than in the female at around 3-5 W in mice and 4-8 W in rats. ID cells in the developing gland are considered to serve as the stem cells that proliferate and give rise to both SD and acinar cells. On the other hand, in adult male mouse gland, the number of SD cells is very small, and 70% of the renewal of the GCT cell population is estimated to occur by self-proliferation. The other 30% is believed to occur by the differentiation of ID and SGD cells, which have high proliferating activity, into GCT cells (Denny et al. 1993). ID cells in adult glands are considered to give rise exclusively to GCT cells and not to acinar cells. These facts, together with the morphological features of the duct cell populations, suggest that SGD cells are the intermediate between ID and GCT cells and there is a cell lineage that originates in ID and sequentially differentiates to GCT cells (Denny et al. 1999). In this regard, the higher proportion of SGD cells in the female gland relative to the male gland may be the result of a prolonged or abortive differentiation of ID cells into GCT cells, due to low plasma androgen levels. The present study demonstrated that nuclear JunD immunoreactivity is present in most ID and SD cells in the early postnatal weeks, whereas in adults it is present in ID and SGD cells but is absent in GCT cells and most SD cells. These results suggest that JunD is required in the cells on the course of their differentiation to GCT cells but is no longer required in differentiated GCT cells.

In the present study, administration of testosterone to female mice caused almost complete conversion of SD cells to GCT cells in 48 hr, in agreement with the past literature. The temporary rise in JunD immunore-activity at 6-24 hr was associated with the temporary appearance of nuclear immunoreactivity in SD cells. This newly induced immunoreactivity disappeared as those SD cells converted to GCT cells, a phenomenon confirmed by the double immunostaining showing the lack of co-existence of nuclear JunD and cytoplasmic NGF in the same cell. We hypothesize that adult female SD cells, unlike ID and SGD cells, normally do not undergo differentiation to GCT cells but are induced by testosterone to enter this process. The expression of nuclear JunD is critical for this process but is no longer required in differentiated GCT cells. In this context, the quantitative gap between the two parameters of JunD expression obtained in the male and female glands is noteworthy. In adults, the ratio of female to male glands was 12-fold in terms of the relative immunoreactivity quantified by immunoblotting, whereas it was only 1.7-fold in terms of the ratio of immunopositive nuclei. This may be interpreted by assuming that the female SD cells produce a considerable amount of immunoreactive JunD but that this JunD is not localized in the nuclei for some reason, e.g., it may be inactive and not bound to the AP-1 element on DNA. Testosterone might cause activation of JunD in SD cells so that it translocates to the nuclei and binds to DNA. However, the possibility cannot be ruled out that the quantitative difference in the two parameters is due to a difference in the sensitivity of the two techniques of JunD detection. Further study employing molecular biological techniques is required to clarify this issue.

JunD is expressed much more abundantly than c-Jun and JunB in quiescent 3T3 cells and is constitutively expressed in high levels in a variety of mouse tissues (Hirai et al. 1989; Ryder et al. 1989). In comparison with c-Jun and JunB expression, which is rapidly and transiently induced by such agents as serum, growth factors, and phorbol ester, JunD expression is believed to be relatively constant in amount and unresponsive to extracellular signals (De León et al. 1995). Nevertheless, there is a growing body of evidence for the involvement of JunD as the main component of AP-1 in many biological phenomena, including growth factor-induced proliferation of lung fibroblasts (Eickelberg et al. 1999), gonadotropin-induced luteinization of ovarian granulosa cells (Sharma and Richards 2000), decidual formation by endometrial stromal cells (Watanabe et al. 2001), and transformation of hepatic stellate cells to a myofibroblast-like phenotype (Smart et al. 2001). The present study has pointed to differentiation of the duct system in the mouse submandibular gland as a candidate for a JunD-dependent phenomenon.

The activity of the Jun family members is post-translationally regulated by their phosphorylation states. Two types of phosphorylation sites exist in the Jun proteins, one adjacent to the DNA-binding domain and the other in the N-terminal transactivating domain (Angel and Karin 1991; Karin 1995). Phosphorylation of the former site negatively regulates Jun activity, and protein kinase C causes dephosphorylation of this site through either activation of a phosphatase or inhibition of a protein kinase, and thereby increases the DNA-binding activity of AP-1 (Boyle et al. 1991; Nikolakaki et al. 1993). Phosphorylation of the latter site, in contrast, positively regulates Jun activity, and activation of Jun N-terminal kinase (JNK) through Ras-MAP kinase pathways causes phosphorylation of this site and thereby increases the transactivating potential of AP-1 (Pulverer et al. 1991; Karin 1995). On the other hand, the promoter region of Jun genes contains not only an AP-1 site but also multiple cis-acting elements that differ among the three members of Jun family, through which expression of the Jun proteins is potentially regulated at the transcriptional level (de Groot et al. 1991a-c). For the JunD promoter, these elements include a CRE, SP-1 binding site, an octamer motif, a CAAT box, a Zif268-binding site, a TATA box, and a variant AP-1 site, of which the octamer motif is unique to the JunD promoter and is postulated to be responsible for the constitutive transcription of JunD protein (de Groot et al. 1991c). The present pattern of JunD expression in the sub-mandibular gland duct system consists of constitutive expression in ID and SGD cells, permanent downregulation in GCT cells, and temporary upregulation in SD cells stimulated with testosterone. Which of the regulatory mechanisms in JunD expression accounts for this pattern remains to be revealed.

The present study has also suggested a cross-talk between the androgen/AR system and the AP-1 system. Androgens belong to the family of hydrophobic ligands whose biological effects are mediated by their cognate nuclear receptors (Laudet et al. 1992), which serve as transcription factors themselves by binding to their cognate hormone-response elements in the promoter region of target genes (Nelson et al. 1999). Thus far, estrogen has been shown to increase c-jun expression in the uterus (Weisz et al. 1990; Chiappetta et al. 1992), which can be explained by transactivation by the estrogen receptor through an estrogen response element-like consensus sequence in the c-Jun gene (Hyder et al. 1995). In the present phenomena, however, AR-mediated transactivation of JunD expression through ARE is unlikely because no ARE-like consensus sequence has been found in the JunD gene. The present effects of androgen on JunD expression in the submandibular duct system, i.e., upregulation in SD cells and downregulation in GCT cells, can be exerted either by the ligand androgen, AR, or the early responsive products of androgen-regulated genes, at the level of either transcription of JunD or post-translational activation of JunD, and should be clarified in the future study. In addition, the target gene(s) activated by JunD that is responsible for the androgen-dependent differentiation of GCT cells, as well as the role of the Jun and Fos family members other than JunD in the androgen effects, remain to be investigated.

Footnotes

Acknowledgements

Supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan (SI).

We wish to thank Mr S. Yamazaki and Ms Y. Akabori for their technical and secretarial assistance.

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