| Sign In to gain access to subscriptions and/or personal tools. |
Influence of Androgens on Gene Expression in the BALB/c Mouse Submandibular Gland
1 Department of Oral Medicine, Infection, and Immunity, Harvard School of Dental Medicine, Boston, MA, USA; Correspondence: * corresponding author, Division of Oral Medicine and Dentistry, Brigham and Women’s Hospital, 75 Francis Street, Boston, MA 02115, USA; ntreister{at}partners.org
Androgens have profound effects on the murine submandibular gland. Our objective was to determine the nature and extent of androgen control of gene expression in the submandibular gland, and to explore the degree to which this might account for known sex differences. Orchiectomized male BALB/c mice were treated with placebo- or testosterone-containing hormone pellets for 14 days. Glands were collected, and total RNA was isolated. Samples were analyzed for differentially expressed mRNAs by CodeLink microarrays, and the data were evaluated with GeneSifter. Androgens significantly (p < 0.05) influenced the expression of over 1300 genes, and many (n = 366) of the genes differentially regulated by androgen treatment were also differentially expressed in males compared with the females in our previous study. These findings support our hypotheses that testosterone extensively influences gene expression in the male submandibular gland, and that many of the sex differences are due to androgens.
Key Words: salivary glands • microarrays • gene expression • sex differences • androgens
We have recently found that significant sex-related differences exist in gene expression in the submandibular, parotid, and sublingual glands (Treister et al., 2005). Many of these differences are gland-specific and involve genes that regulate numerous biological processes, such as cell proliferation, cell cycle regulation, growth factor activity, immune response, protein glycosylation, lipid metabolism, steroid biosynthesis, pain perception, and angiogenesis. We hypothesized that many of these sex-related differences in gene expression are due to the effects of androgens. In support of this hypothesis, androgen action has been linked to several sexually dimorphic features of salivary tissues (Jayasinghe et al., 1990; Sawada and Noumura, 1991; Cangussu et al., 2002). In fact, castration of male mice leads to a female-like phenotype in the salivary gland, a reaction that may be reversed by androgen treatment (Hosoi et al., 1978). Likewise, the treatment of female mice with testosterone induces a male-like salivary gland pattern (Kim and Ogita, 1981). The impact of androgens on salivary tissues is not limited to an influence on morphology (Jayasinghe et al., 1990; Sawada and Noumura, 1991; Cerbon et al., 1996), but also includes a broad spectrum of effects on glandular physiology (Sims-Sampson et al., 1984; Mizuki and Kasahara, 1992). The purpose of this study was to begin to test our hypothesis. Toward this end, we determined the nature and magnitude of the androgen control of gene expression in the submandibular gland. We also examined the extent to which these results correlated with our previous findings of sex-related differences in salivary gland gene expression.
Animals and Hormone Treatment Young adult male BALB/c mice (n = 30), orchiectomized at age 8 wks, were purchased from Taconic Laboratories (Germantown, NY, USA) and maintained in constant-temperature rooms with fixed light/dark periods of 12 hours’ length. Animals were allowed to recover from surgery for at least 9 days, then were anesthetized intraperitoneally with ketamine (200 mg/kg) and xylazine (10 mg/kg), and subcutaneous implants of placebo (n = 15; cholesterol, methylcellulose, lactose)- or testosterone (n = 15; 10 mg)-containing pellets were placed in the subscapular region. Hormone pellets were obtained from Innovative Research of America (Sarasota, FL, USA) and were designed for slow, continuous release of vehicle or physiologic amounts of testosterone over a two-week period (Ariga et al., 1989). After 2 wks of hormone exposure, the mice were killed in a CO2 chamber, and the submandibular glands were removed and pooled (5 mice or 10 glands per group) according to group treatment. Tissues were immediately frozen and stored in liquid nitrogen until RNA isolation. All animal study protocols ensured that humane practices were followed and were approved by the Institutional Animal Care and Use committee of the Schepens Eye Research Institute.
Molecular Biological Procedures
Microarray Hybridization and Image Processing Fragmented target cRNA (10 µg) was added to the hybridization solution, and a 250-µL quantity of denatured target solution was injected into each hybridization chamber of a CodeLink Mouse Uniset I microarray (Amersham Biosciences/GE Healthcare, Piscataway, NJ, USA), which contained perfect-match, 30-mer, oligonucleotide probes for approximately 10,000 different genes. Hormone (n = 3) and placebo (n = 3) samples were hybridized and scanned as previously described (Treister et al., 2005). A single array was used per pooled group. The 10,000 spot intensities on the microarray image were normalized to a median value of 1, and the data were exported for further analysis.
Real-time PCR
Microarray Data Analysis and Statistics
To study the effects of androgens on gene expression in the submandibular gland, we collected glands from testosterone-and placebo-treated castrated male mice and processed them for analysis of differentially expressed genes using CodeLink Mouse Uniset I microarrays and GeneSifter analytical software.
Our results showed that androgens significantly (p < 0.05) influenced the expression of over 1300 genes (out of 5954 genes that passed the intensity filter; or 22% of all possible genes). This hormone effect involved the up- and down-regulation of 813 and 491 genes, respectively, and frequently resulted in two- (> 300 genes) to 10- (> 10 genes) fold changes in gene expression (Table 1
The nature of androgen influence on submandibular gland gene expression encompassed a diverse array of biological, molecular, and cellular ontologies (Table 3  ). Testosterone treatment significantly altered the expression of genes associated with receptor activity, signal transduction, transferase activity, cell growth, cell communication, protein metabolism, protein modification, and transcription regulation.
For further evaluation of the microarray results, 9 genes that previously demonstrated sex-related differences were selected for RT-PCR confirmation (Treister et al., 2005) (Appendix Tables 2, 3). These analyses verified the androgen up-regulation of genes for epidermal growth factor (EGF), hydroxysteroid (17β) dehydrogenase 3 (17 β-HSD3), nerve growth factor β (NGF β), prostaglandin D2 synthase, sulfotransferase family 1E, member 1, transforming growth factor β2 (TGF β 2), and vascular endothelial growth factor A (VEGFA), as well as the hormonal down-regulation of genes encoding sialyltransferase 4a and stearoyl-coenzyme A desaturase 1 (SCD1).
Of particular interest, androgen action may account for many of the sex-related differences previously found in the gene expression of the submandibular gland (Table 4
Our study demonstrates that testosterone exerts a significant influence on gene expression in murine submandibular glands. Androgen treatment altered the expression of over 1300 genes, with the majority being up-regulated by hormone exposure. Many of these genes, in turn, are involved in multiple biological, cellular, and molecular processes, suggesting that androgens have a considerable impact on the structure and function of the submandibular gland. Additionally, our findings indicate that androgen action may account for many of the sex-related differences in gene expression in submandibular tissue. Other investigators have also reported androgen effects on specific gene expression in the mouse submandibular gland. For example, androgens have been shown to up-regulate the expression of genes encoding EGF and NGF β (Tuomela et al., 1990; Siminoski et al., 1993), which our results confirmed, and to down-regulate the activity of other genes (e.g., mouse kallikreinin 1, proteinase F) (Hosoi et al., 1984; Kurihara et al., 1999). Moreover, several of these androgen actions may be tissue-specific, given that androgens have no effect on NGF β mRNA in the bladder (Bjorling et al., 2002) and even suppress EGF mRNA in the kidney (Pascall et al., 1989). It is important to note, though, that the magnitude of androgen influence on gene expression in submandibular tissue, as shown in the present study, has not been previously realized. The mechanism(s) by which androgens regulate gene expression in the submandibular gland, and contribute to sex-related differences in this tissue, may be related to the presence of androgen receptors, levels of tissue androgens, and/or effects on mRNA stabilization. Androgen receptors have been identified in the salivary glands of males and females (Laine et al., 1993), with a higher concentration of nuclear androgen-binding sites in males (Kyakumoto et al., 1986). Androgen interaction with its receptor is known to modulate gene transcription (Lee and Chang, 2003). In addition, tissue androgen levels in rodents have been shown to be high in males and very low in females, with levels highest in the submandibular gland and low or non-existent in the sublingual and parotid glands (Morrell et al., 1987). Androgens may also affect mRNA stabilization, by regulating the 3' untranslated regions of certain genes and specific proteins that bind these genetic elements (Sheflin et al., 2001). It is unlikely that testosterone effects are due to aromatization to estrogens, because many of the up-regulated genes are expressed to a greater degree in glands of males, compared with those of females. The expression of various genes that were up- or down-regulated by androgens was confirmed by RT-PCR. These genes were associated with three major categories: growth factor, sex steroidogenic enzyme, and other enzyme mRNAs. Growth factor mRNAs included EGF, NGF β, TGF β 2, and VEGFA. EGF and NGF β mRNAs were both highly up-regulated in the testosterone-treated mice, which is consistent with previous studies of these two genes (Siminoski et al., 1993). TGF β 2 and VEGFA mRNAs were also both up-regulated in the testosterone-treated mice. Both genes have been demonstrated to be under the influence of androgens in tissues other than the submandibular gland. For instance, TGFβ 2 mRNA is down-regulated, and VEGFA mRNA is up-regulated, by androgens in the prostate (Sordello et al., 1998; Desai and Kondaiah, 2000). Sex steroidogenic enzyme mRNAs confirmed by RT-PCR included 17 β-HSD3 and sulfotransferase family 1E, member 1, both of which were highly up-regulated in the testosterone-treated mice. 17 β-HSD3 plays a critical role in the formation of androgens and is known to be important in peripheral sex steroid formation (Martel et al., 1992). Sulfotransferase family 1E, member 1 (formerly known as sulfotransferase, estrogen-preferring), is known to be androgen-dependent, although, interestingly, this enzyme regulates local estrogen activity (Song, 2001). Other enzyme mRNAs that were confirmed by RT-PCR included prostaglandin D2 synthase, sialyl-transferase 4a, and SCD1. Prostaglandin D2 synthase is regulated by sex hormones (Mong et al., 2003) and was the most highly up-regulated gene in submandibular tissues of the testosterone-treated mice. Both sialyltransferase 4a and SCD1 were down-regulated in the testosterone-treated mice, which is interesting, since our previous findings demonstrated that these genes are both down-regulated in the submandibular glands of males compared with those of females (Treister et al., 2005). The gene ontologies and corresponding z-score analyses were of particular interest in explaining how androgens may be influencing activity of the submandibular gland, and how they may account for the sex differences noted. Genes involved in the extracellular matrix, cytoskeleton, basement membrane, collagen, angiogenesis, growth factor activity, and enzyme inhibitor activity were all expressed significantly more than expected. However, given the fact that androgen-treated males (compared with placebo) and males (compared with females) have physically larger glands, and also have many more genes up-regulated (by androgen and/or sex) than down-regulated, it was somewhat surprising to find, in both the current as well as the sex differences studies, that genes involved in nucleic acid metabolism, transcription, nucleic acid binding, and nuclear location were all expressed significantly less than expected. Why these disparities exist and why androgens would affect these groups so differently is unclear, but the answers may provide insight into differences in the salivary glands between the sexes during healthy and disease states. The present findings of sex-related differences in salivary glands (Treister et al., 2005), as well as findings from our previous study, may, in part, help elucidate the underlying pathophysiology of certain autoimmune diseases, such as Sjögren’s syndrome (SS). SS is an autoimmune condition that affects the exocrine tissues, including the salivary glands, causing glandular hypofunction and severe dry mouth (Sullivan, 1997). The most striking feature of this disease is that it affects women at a rate of 9:1 compared with men (Whitacre, 2001). Of particular interest, androgens have been shown to reduce glandular inflammation and restore secretory function in mouse models of SS (Sullivan and Edwards, 1997). Several of the genes that were found to be influenced by androgen exposure and sex (Treister et al., 2005), such as EGF and TGF β 2, have also been reported to be involved in SS (Ogawa et al., 1995; Koski et al., 1997). While levels of EGF have been shown to be decreased in the tears of patients with SS, we found that androgens increase EGF levels (in the SMG), such that lower levels may be an indicator of disease activity, and higher levels may have a protective effect. TGF β 2 has similarly been found to be decreased in salivary glands of patients with SS, and was also up-regulated by androgen treatment in the present study, possibly reflective of the anti-inflammatory properties of androgens. Analysis of these data provides a critical foundation for future studies of mouse models of SS to determine the extent to which the sex- and androgen-associated genes and pathways are involved in suppression of inflammation in the salivary glands. In summary, our study shows that testosterone has an extensive effect on gene expression in the submandibular gland, and that this hormone action may mediate many of the sex-related and molecular biological differences encountered in this tissue.
We thank Patricia Rowley and Michael Lombardi from the Center for Neurologic Diseases, Brigham and Women’s Hospital and Harvard Medical School, Cambridge, MA, for their assistance and expertise in processing the CodeLink microarrays and preparing the data for analysis. We also thank Dr. Christian B. Wade and the personnel at GeneSifter for their technical support, assistance, and expertise. This research was supported by NIH grants EY05612 and K16 and by Allergan, Inc. (USA & Japan).
A supplemental appendix to this article is published electronically only at http://www.dentalresearch.org. Received for publication November 15, 2004. Revision received August 2, 2005. Accepted for publication August 2, 2005.
Journal of Dental Research, Vol. 84, No. 12,
1187-1192 (2005) This article has been cited by other articles:
|
|
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
|
|
 |
|
Sign In to gain access to subscriptions and/or personal tools.
TOP
ABSTRACT
INTRODUCTION
