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RESEARCH REPORT |
1 Department of Oral Medicine, Infection, and Immunity, Harvard School of Dental Medicine, Boston, MA, USA;
2 Schepens Eye Research Institute, Boston, MA, USA;
3 Department of Ophthalmology, Harvard Medical School, Boston, MA, USA; and
4 Department of Physics, University of Massachusetts-Boston, Boston, MA, USA;
* corresponding author, Division of Oral Medicine and Dentistry, Brigham and Women’s Hospital, 75 Francis Street, Boston, MA 02115, USA; ntreister{at}partners.org
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: salivary glands • microarrays • gene expression • sex differences • androgens
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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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.
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MATERIALS & METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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Molecular Biological Procedures
RNA Isolation
Frozen glands were pulverized by mean of a mortar and pestle, and total RNA was isolated and purified as previously described (Treister et al., 2005).
Microarray Hybridization and Image Processing
RNA samples (2 µg) were prepared and processed for microarray hybridization by the CodeLink Expression Assay Reagent Kit (Amersham, Piscataway, NJ, USA), as previously described (Treister et al., 2005). In brief, cDNA was synthesized from total RNA and purified by a QIAquick purification kit (Qiagen, Valencia, CA, USA). Following cRNA synthesis, the target product was recovered by an RNeasy kit (Qiagen) and quantified by means of a UV photospectrometer.
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
To verify, in part, the differential expression of selected genes, we synthesized cDNA from total RNA, and RT-PCR (50 µL) reactions were carried out as previously described (Treister et al., 2005). Selected genes were highly expressed and exhibited previously demonstrated sex-related differences (Treister et al., 2005).
Microarray Data Analysis and Statistics
Normalized data from the CodeLink software package were uploaded to GeneSifterTM (www.genesifter.net; VizX Labs LLC, Seattle, WA, USA) for analysis. We assessed the differential expression of genes by averaging the normalized (and log-transformed) triplicate samples and running a pair-wise analysis. Statistical significance was determined by Student’s t test (two-tailed, unpaired). Since the low-intensity signals are most susceptible to noise, we filtered the data for spot quality by excluding all genes with signal intensity smaller than 0.75 (compared with the median signal of 1.0) for every sample. Only genes that passed the intensity filter and had p values < 0.05 were included in the analysis. Gene ontology reports (Biological Process, Cellular Component, and Molecular Function, based on the Gene Ontology Consortium, http://www.geneontology.org/GO.doc.html) (Ashburner et al., 2000), including z-score analyses, were generated by GeneSifter.Net. We utilized GeneSifter’s Intersector program (http://public.gensesifter.net/intersector), which allows users to identify genes in common (and unique) from multiple datasets of differentially expressed genes, to find genes that were up- or down-regulated both in testosterone-treated male submandibular glands (present study) and in male submandibular glands compared with females (both untreated, data not shown) (Treister et al., 2005).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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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 magnitude of these differences and significance levels were typically much higher in the up-regulated genes (Table 2, Appendix Tables 1, 4). The genes up-regulated to the greatest extent by androgen treatment were for nerve growth factor ß, followed by those encoding various enzymes. The data from the individual arrays (n = 6) are accessible for download through the National Center for Biotechnology Information’s Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo) via series accession number GSE1582.
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). 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.
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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) (Treister et al., 2005). Of the 412 genes more highly expressed in glands of males, compared with those of females, 260 were up-regulated by testosterone (this study). In addition, of the 304 genes that were less expressed in glands of males, relative to females (Treister et al., 2005), 106 were down-regulated by testosterone (this study). Furthermore, the ontological differences discovered between the glands of male and female mice (e.g., in nucleic acid metabolism, transcription, receptor activity, growth factor activity, nucleus, basement membrane) (Treister et al., 2005) were strikingly similar, in terms of both identity and z scores, to those differences found after hormone treatment. Overall, androgen activity appears to contribute to approximately 50% of the sex-related differences in gene expression of the submandibular gland.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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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 ß (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 ß 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 ß, TGF ß 2, and VEGFA. EGF and NGF ß 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 ß 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, TGFß 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 ß-HSD3 and sulfotransferase family 1E, member 1, both of which were highly up-regulated in the testosterone-treated mice. 17 ß-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 ß 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 ß 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.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Received November 15, 2004; Last revision August 2, 2005; Accepted August 2, 2005
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REFERENCES |
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