Global analysis of gene expression profiles in the submandibular salivary gland of klotho knockout mice

Salivary dysfunction commonly occurs in many older adults and is considered a physiological phenomenon. However, the genetic changes in salivary glands during aging have not been characterized. The present study analyzed the gene expression profile in salivary glands from accelerated aging klotho deficient mice (klotho−/−, 4 weeks old). Microarray analysis showed that 195 genes were differentially expressed (z‐score > 2 in two independent arrays) in klotho null mice compared to wild‐type mice. Importantly, alpha2‐Na+/K+‐ATPase (Atp1a2), Ca2+‐ATPase (Atp2a1), epidermal growth factor (EGF), and nerve growth factor (NGF), which have been suggested to be regulators of submandibular salivary gland function, were significantly decreased. When a network was constructed from the differentially expressed genes, proliferator‐activated receptor‐γ (PPAR γ), which regulates energy homeostasis and insulin sensitivity, was located at the core of the network. In addition, the expression of genes proposed to regulate various PPAR γ‐related cellular pathways, such as Klk1b26, Egfbp2, Cox8b, Gpx3, Fabp3, EGF, and NGFβ, was altered in the submandibular salivary glands of klotho−/− mice. Our results may provide clues for the identification of novel genes involved in salivary gland dysfunction. Further characterization of these differentially expressed genes will be useful in elucidating the genetic basis of aging‐related changes in the submandibular salivary gland.

in manifestations of dry mouth in elderly people (Barka, 1980). Many previous studies have described age-related differences in the rate of flow and volume of saliva, as well as the contents of saliva (Melvin, Yule, Shuttleworth, & Begenisich, 2005;Nagler, 2004). In addition, age-related changes in the salivary glands are consistently associated with a reduction of total and/or secretory protein synthesis (Nakamoto et al., 2007). However, the extent to which genetic alterations affect the function of these glands is unclear.
The klotho gene plays a critical role in regulating aging and the development of age-related diseases in mammals. Life span is extended by up to 30% in transgenic mice over-expressing the klotho gene compared with wild-type mice (Kuro-o, 2009;Kuro-o et al., 1997). Klotho-deficient phenotypes include osteoporosis, skin atrophy, ectopic calcification, pulmonary emphysema, hypogonadism, impaired bone mineralization, and neurodegeneration, which are also observed in the human aging phenotype (Kuro-o et al., 1997). Many researchers have recently demonstrated an association between single nucleotide polymorphisms of the klotho gene and age-related disorders, including coronary artery disease, senile osteoporosis, and stroke (Kawano et al., 2002;Ogata et al., 2002).
A few reports have shown a lack of eosinophilic granules and diminished granular ducts and lobes in the submandibular salivary glands of klotho-deficient mice (Suzuki, Amizuka, Noda, Amano, & Maeda, 2006). In addition, histological observations have shown that the numbers of NGF-and EGF-immunopositive ducts in the submandibular salivary gland are decreased in klotho-deficient mice compared to wild-type mice (Suzuki et al., 2006). However, these gene depletion studies have not provided insights on the regulation of salivary gland function during aging.
The purpose of the present study was to evaluate and compare differences in gene expression associated with aging in the submandibular salivary gland of klotho-deficient mice. We used DNA microarray platforms in combination with functional signaling pathway analysis in this study. In addition, the data provided a network for investigating PARP-γ transcriptional programs in the submandibular salivary gland in klotho-related aging. These differentially expressed genes will contribute to an understanding of the genetic basis of klotho and the elucidation of the mechanism of biological behavior in the submandibular salivary gland. and 0.5 mg/ml proteinase K (Roche) solution at 55°C for at least 1 hr with vigorous shaking. The DNA was purified by phenol/chloroform extraction followed by ethanol precipitation and then dissolved in 0.1 ml of TE solution. We used the following specific primers: wild-type klotho, forward 5′-TTGTGGAGATTGGAAGTGGACGAAAGAG-3′ and reverse 5′-CTGGACCCCCTG-AAGCTGGA-GTTAC-3′; klotho mutant, forward 5′-TTGTGGAGATTGGAAGTGGACGAAAGAG-3′ and reverse 5′-CGCCCCGACCGGAGCTGAGA-GTA-3′. The GAPDH PCR primers were forward 5′-CCAAGGTCATCCATGACAACT-3′ and reverse 5′-GCATTGCTGATGATCTTGAGGCTG-3′. These primers were expected to produce 815 bp (WT) and 419 bp (klotho-deficient) amplification products. The PCR conditions were as follows: denaturation at 94°C for 5 min, 30 cycles of 94°C for 30 s, annealing at 60°C for 1 min, and extension at 72°C for 45 s, and a final extension at 72°C for 10 min.

| Tissue preparation and histological examination
At 4 weeks of age, all animals were killed under ether anesthesia, and the submandibular salivary glands were dissected. The submandibular salivary gland tissue and tongue were fixed in 10% formalin, embedded in paraffin and cut into 4 μm-thick sections for staining. All sections were stained with hematoxylin and eosin. Sections of tongue were also stained with von Kossa, Elastin, and Congo Red to detect histological alterations such as calcification, fibrosis, and amyloid accumulation.

| RNA purification and RT-PCR
Total RNA was isolated from the salivary glands of wild or klotho−/− mice (4 weeks old) using TRIzol reagent (Invitrogen, Calsbad, CA). To avoid genomic DNA contamination, the extracted RNA was purified using an RNeasy kit (Invitrogen). The quantity and quality of the RNA were determined by measuring the optical density (OD) at 260 and 280 nm. A 2 µg of RNA were used for cDNA synthesis using an oligo-(dt) 15 primer and M-MLV reverse transcriptase. The reverse transcription (RT) reaction included an initial 10 min incubation at room temperature, followed by 60 min at 42°C and 10 min at 70°C to terminate the reaction. Subsequently, a 2 µl aliquot of cDNA was PCR amplified in a total volume of 25 µl containing 2.5 µl of 10 × PCR buffer followed by 30 cycles of 95°C for 30 s, 55-60°C for 30 s, and 72°C for 30 s, with a final extension step at 72°C for 10 min. The specific primers for RT-PCR are described in Table 1. The PCR products were then electrophoresed on a 2% acrylamide gel and visualized using a gel documentation system (Bio-Rad, Hercules, CA).

| Microarray raw data preparation and statistical analysis
Total RNA was amplified and purified using an Ambion Illumina RNA amplification kit (Ambion, Austin, TX) to obtain biotinylated cRNA according to the manufacturer's instructions. The array signal was detected using an Illumina MouseRef-8 v2 Expression BeadChip The quality of hybridization and overall chip performance were monitored by visual inspection of both internal quality control checks and raw scanned data. The raw data were extracted using the software provided by the manufacturer (Gene Expression Module v1.5.4, Illumina, Inc., San Diego, CA). The array data were filtered by a detection p-value <0.05 (similar to signal to noise) in at least 50% samples (we applied a filtering criterion for data analysis; a higher signal value was required to obtain a detection p-value <0.05). The selected gene signal value was transformed by logarithm and normalized by the quantile method. Comparative analysis between the control group and test group was performed using fold-change.

| Functional and network analysis
We used Ingenuity Pathway Analysis (Ingenuity Systems, Inc., Redwood city, CA) to determine the statistically significant pathways, functions, and networks in which the identified genes regulated by klotho may be involved. Fisher's exact test was used to identify the significant functions and pathways represented within the respective gene sets. First, to confirm the depletion of klotho gene expression in the salivary glands of Klotho mutant mice, we performed RT-PCR analysis. As shown in Figure 1a, the mRNA expression levels of Klotho were suppressed in the salivary glands of Klotho mutant mice compared with wild-type mice.
Histological analysis of the salivary glands of wild-type and klothodeficient mice was performed at 4 weeks of age. In the klotho wild-type and hetero mice, normally duct cells were found in the submandibular salivary gland, and in particular, there were many granular convoluted tubules with abundant granules. Compared to wild-type mice, klotho-deficient mice had less tubules and connective tissue in the submandibular salivary gland. In addition, the submandibular salivary gland was composed of serous acini and mucous acini in the wild-type mice. However, in the klotho-deficient mice, the submandibular salivary gland was composed of only mucous acini, and no serous acini were present ( Figure 1b).

| Strategy to identify genes related to salivary gland dysfunction in klotho−/− aging mice
The aim of this study was to identify salivary gland dysfunction-related  The mRNA expression levels of the Egf, Ngf, Atp1a2, Atp2a1, and Gpx3 genes significantly decreased in klotho-deficient mice compared with wild-type mice. However, the Cxcl9 and Ctgf genes significantly increased in klotho-deficient mice (Figures 3a and 3b). Immunoblotting constantly showed that klotho and ATP1α2 were upregulated in submandibular tissues of wild-type mice. The level of CTGF protein was downregulated in wild-type mice compared with klotho-deficient mice ( Figure 3c).

| Ingenuity pathway analysis in klotho−/− mice submandibular salivary dysfunction
Ingenuity pathway analysis (IPA) was performed on all genes identified as regulated in the submandibular salivary gland in klotho−/− mice.
Fisher's exact test was applied, and we identified the top 20 significant canonical pathways based on p-value <0.05 and a threshold value of log (p-value) of 0.05. The significant pathways, which included fatty acid metabolism, calcium signaling, AMPK signaling, endoplasmic reticulum stress pathway, glycerolipid metabolism, and type II diabetes mellitus signaling, are shown in Figure 4. The most significant canonical pathway was energy metabolic signaling, followed by lipid metabolism, glycerolipid metabolism, hepatic fibrosis, and differential regulation of cytokine production in intestinal epithelial cells. Among the genes belonging to energy metabolic signaling, peroxisome proliferatoractivated receptor gamma (PPAR γ), which is a ligand-activated transcription factor that mainly regulates genes responsible for cellular differentiation, development, fatty acid (FA) storage, and energy metabolism, was significantly differentially expressed between klotho +/+ and klotho−/− mice. The expression of this gene was 2.4-fold lower in klotho−/− mice compared with klotho+/+ mice (Table 4). We specifically focused on several genes linked to the PPAR γ networks.
Gene network analysis also revealed overlapping network connectivity for 42 of the differentially expressed genes in the klotho−/− submandibular gland ( Figure 5).  klotho-deficient submandibular tissues (Figure 6a). PPAR γ-related genes involved in "signaling transduction," especially oxidative phosphorylation (Cox8b), proteolysis (Klk1b26 and Egfbp2), ion transport (Atp1α2 and Atp2α1), and stress response (Gxp3), were down-regulated. By contrast, the expression of genes (Lcn2, IL-10, and IL-1β) involved in "small molecular transport and cytokine" was increased in klotho−/− mice (Figure 6b). In addition, several key players in the water channel (Aqp3, Aqp4, and Aqp5) and endocrine signaling (FGF15 and FGF23) were differently regulated in klotho-deficient submandibular tissues (data not shown). The changes in the expression of TLR5, TLR7, TLR9, and NODR2, toll-like receptor (TLR) genes that play a key role in the innate immune system, were also validated

| DISCUSSION
We have performed gene profiling of the salivary gland to provide a database for the interpretation of age-dependent alterations. Because  The submandibular salivary glands have been considered an agestable organ (Ramirez & Soley, 2011). However, we and others have reported that submandibular salivary glands in accelerated aging klothodeficient mice show a loss of granular ducts and mucous acini compared to wild type mice (Suzuki et al., 2006). Except for a loss of ducts in the submandibular salivary gland, we observed a comparatively stable structure of the submandibular gland, parotid, and sublingual gland in aging klotho-deficient mice. The mouse submandibular gland contains various biological molecules such as epidermal growth factor (EGF), nerve growth factor (NGF), renin, kallikreins, and proteases (Atkinson & Wu, 1994;Sabbadini & Berczi, 1995). EGF and NGF have been reported to be biosynthesized in granular convoluted tubule (GCT) cells of the Comparison of gene expression in wild-type and klotho-deficient mice. (a,b) Total RNAs were extracted from submandibular gland tissues isolated from individual mice. cDNA was synthesized by reverse transcription-polymerase chain reaction (RT-PCR). mRNA levels were normalized to GAPDH. The bar graph represents expression relative to GAPDH. The data are reported as the mean ± SD of three independent experiments. *p < 0.05, **p < 0.001. (c) Expression of klotho, ATP1α2, and CTGF protein in submandibular gland tissues of wild-type and klotho-deficient mice. The total protein was extracted, and klotho, ATP1α2, and CTGF protein levels were measured by Western blot, respectively. Actin was used as a loading control FIGURE 4 Ingenuity Pathway Analysis of the genes that were regulated in the klotho-deficient salivary gland. (a) The significance of each function or canonical pathway was determined based on the p-values determined using Fisher's exact test and a threshold less than 0.05. The top 20 possible functions and canonical pathways are shown submandibular ducts and secreted in the saliva of mice (Gresik, 1994). A previous study showed that the granular ducts of the salivary glands exhibited remarkably decreased immunoreactivities for NGF and EGF in klotho-deficient mice (Suzuki et al., 2006). Interestingly, we also confirmed that the expression of EGF and NGF was inhibited in the submandibular glands of klotho-deficient mice.
Our data analysis suggested that ATP1α2 and ATP2α1/SERCA1, subtypes of Na + /K + -ATPase and Ca 2+ ATPase, were downregulated in the submandibular salivary glands of aging Klotho-deficient mice. In the salivary glands, ATP1α2, a subtype of Na + /K + -ATPase, is localized mainly at basal infolding of the striated duct and excretory ducts, though it is also weakly expressed on the membranes of granular convoluted tubules (GCT) and on acinar basolateral membranes (Sims-Sampson, Gresik, & Barka, 1984). The activity of ATP1α2 establishes transmembrane ion gradients and is essential to cell function and survival (De Lores Arnaiz & Ordieres, 2014).
However, the link between disruption of Na + /K + -ATPase activity and salivary gland dysfunction in aging remains to be clarified. The FIGURE 5 Network connectivity of differentially regulated genes in the salivary glands of klotho-deficient mice. Ingenuity Pathway Analysis was applied to genes showing significant dysregulation, with filtering on their relative distance from the mean ratio of the population. The first main pathway that appeared to be differentially expressed was PPAR γ signaling. Genes belonging to this pathway were significantly differentially down-regulated salivary fluid secretion is dependent on Cltransport across the apical membrane of acinar cells. Intracellular accumulation of Clrequires a Na + gradient (Chaib, Kabre, Metioui, Franco, & Dehaye, 1999). Thus, saliva secretion from the salivary gland may be dependent on Na + /K + -ATPase activity.
Additionally, in the salivary gland, saliva secretion is initiated by activation of phospholipase C, generation of inositol 1,4,5 trisphosphate (IP3), and release of Ca 2+ from the ER to the cytosol. The cytosolic Ca 2+ is taken up by sarco/endoplasmic reticulum Ca 2 + -ATPases (SERCA1-3) (Homann, Kinne-Saffran, Arnold, Gaengler, & Kinne, 2006). The sarco/endoplasmic reticulum Ca 2+ -ATPase (SERCA) is the active Ca 2+ transporter in the sarcoplasmic reticulum (SR), and regulation of its function is a key mechanism of Ca 2+ homeostasis and depends on the cell type and state of differentiation (Homann et al., 2006). A significant age-dependent loss in Ca 2+ -ATPase activity and Ca 2+ -uptake rate has been observed specifically in the rat skeletalmuscle SR (Schöneich, Viner, Ferrington, & Bigelow, 1999). Despite these changes in aging, whether Ca 2+ -ATPase (SERCA) affects salivary gland function in aged mice is unclear. However, in the salivary gland, cytosolic calcium or sodium reduction may be important for salivary gland dysfunction during aging as well as in dystrophic pathological conditions. Further studies are needed to precisely elucidate the functional significance of changes in the ion efflux pump as well as salivary gland dysfunction both in aging and in diseases.
We also observed that CXCL9 was increased in the klotho−/− salivary gland. CXCL9 proteins are predominantly expressed in the ductal epithelium adjacent to lymphoid infiltrates in the Sjögren's syndrome salivary gland but are not expressed in the normal salivary  Table 1. (d) Expression of endogenous ATP1α2 and ATP2α1 (SERCA1) in klotho-overexpressing AC and HSG salivary gland cells. Cells were transfected with klotho expression plasmids. A total of 48 hr after transfection, total RNA was prepared and subjected to RT-PCR. (e) HSG cells were transfected with pcDNA3.1-klotho for 24 hr, treated to PPARG antagonist BADGE (30 µM) and incubated for another 20 hr. A Western blot analysis was performed to assess the PPARG, SCD, ATP1α2, and CIDEA levels gland (Ogawa, Ping, Zhenjun, Takada, & Sugai, 2002). Therefore, in our study, the up-regulation of CXCL9 might reflect a higher proportion of T cells in aged inflammatory tissue compared with healthy controls.
CXCL9 is a validated biomarker of the development of tissue dysfunction, suggesting an underlying pathophysiological relation between the levels of these chemokines and the development of aged salivary dysfunction.
In our downregulated gene lists (  (Antonelli et al., 2014;Beauregard & Brandt, 2003;Shen et al., 2014). In addition, PPAR γ ameliorates Sjögren's syndrome through regulation of the expression of cytokines in peripheral blood and/or salivary gland in non-obese diabetic mice (Li, Xu, Wang, & Wei, 2014). PPAR α and PPAR γ can inhibit IL-1β-induced NO production in cultured lacrimal gland acinar cells, suggesting that PPAR may be useful therapeutic target for preventing NO-mediated gland damage.
However, the effects of PPAR α and PPAR γ on the progression of aged salivary gland dysfunction are not clear.
Many specific genes targeting various metabolic pathways are modulated by both PPAR γ and PPAR α/γ in the aged klotho−/− salivary gland ( Figure 5). Thus, many genes involved in PPAR-targeted functions were regulated, including lipid metabolism (CIDEA, SCD, and Fabp3), chronic kidney disease (FGF23), ion transport (Atp2a1 and Atp1a2), mitochondrial oxidative phosphorylation (Cox8b), stress response (Gpx3), inflammation (IL-10 and IL-1β), immunity (TLR2-9 and NODR1-2), and water channel (AQP3-5). Therefore, IPA and gene network analysis indicated that the nodal point in this cross-talk in aged salivary gland dysfunction may be PPARα and/or PPARγ. Further studies are also needed to precisely elucidate the functional significance of changes in PARPs in aged-salivary gland dysfunction. The function of the salivary glands is to produce saliva, which is crucial for digestion, taste, the maintenance of tooth integrity, and anti-microbial. Previous studies have indicated that anatomical changes in salivary gland with age is accompanied by atrophy of the acinar cells and replacement of the normal gland parenchyma with fibrous and/or adipose tissue (Azevedo, Damante, Lara, & Lauris, 2005;Choi, Park, Kim, Lim, & Kim, 2013;Syrjanen, 1984). A reduction in saliva leads to xerostomia or dry mouth. Xerostomia, or chronic dry mouth, is a common syndrome caused by a lack of saliva that can lead to severe eating difficulties, dental caries, and oral candida infections (Gupta, Epstein, & Sroussi, 2006). In our study was designed to investigate the effects of klotho depletion salivary gland dysfunction on certain aspects of the morphology and cell proliferation rate of mouse tongue tissues. We found that the excessive calcification was observed in the tongue muscle of klotho−/− mice. Therefore, increased elastin fiber in the blood vessel wall and amyloidosis were observed in the tongues of klotho−/− mice compared to klotho+/+ mice. We also demonstrated that cell death induces the tongues in klotho−/− mice and that cell death in tongue may be associated with calcification and fibrosis in muscle and blood vessel wall. These observed klotho may be important to salivary gland function, and may contribute to maintenance of oral health.
In this study, we detected changes in global gene expression patterns in the submandibular glands of wild-type and klothodeficient mouse. This is the first investigation to use genome-wide screening by cDNA microarray technology to identify changes in gene expression in aged submandibular gland tissue, which consists of mixed cell types such as acinar, ductal, stroma, and fatty, in klotho-deficient mice.