Soluble klotho regulates the function of salivary glands by activating KLF4 pathways

KLF4 expression in klotho (-/-) mouse organs

To determine the tissue level expression of KLF in klotho (-/-) mice, we analyzed KLF4 expression in 8 organs (lung, salivary gland, heart, skin, liver, kidney, brain, spleen) harvested from four-week-old klotho (-/-) mice. Quantitative RT-PCR revealed that the mRNA levels of KLF4 were decreased to different extents—from 2- to 7-fold—in several organs. The salivary glands, skin, brain, and spleen exhibited significantly decreased KLF4 expression in klotho (-/-) mice compared to that in these organs of wild-type mice (Figure 1A). Similarly, KLF4 protein expression was also decreased in all tissues of klotho (-/-) mice (Figure 1B).

Figure 1. Kruppel-like factor (KLF) 4 is downregulated in various tissues harvested from klotho (-/-) mice. (A) Expression of mRNA encoding KLF4 in wild type and klotho (-/-) mouse tissues (as measured by qRT-PCR). (B) Protein expression of KLF4 in tissues harvested from wild-type and klotho (-/-) mice. Total protein samples were collected from the skin, liver, kidney, brain, salivary gland, lung, and spleen. The expression of KLF4 protein was determined by Western blot. Actin was used as an internal control.

Klotho induces KLF4 expression

To elucidate the relationship between klotho and KLF4 gene expression, we evaluated KLF4 mRNA expression using real-time quantitative RT-PCR in wild-type and klotho (-/-) mouse embryonic fibroblasts (MEFs). Depletion of klotho led to significantly diminished mRNA and protein levels of KLF4 (Figure 2A–2C), and the promoter activity of KLF4 was significantly increased after soluble klotho transfection. These results indicated that klotho leads to the induction of KLF4 transcription (Figure 2D).

Figure 2. The expression of KLF4 in wild-type and klotho (-/-) MEFs. qRT-PCR analysis of KLF4 and FOXO1 in wild-type and klotho (-/-) MEFs. Total RNA samples were prepared from wild-type and klotho (-/-) MEFs, followed by quantitative RT-PCR analysis to examine the expression levels of KLF4 (A) and FOXO1 (B). Transcript abundances were normalized to the GAPDH RNA level and are expressed as relative values. The mean ± S.D. of three independent experiments is shown. (C) Western blot analysis was performed to assess the KLF4 and FOXO1 protein levels. (D) Soluble klotho induced KLF4 transcriptional activation. Transient cotransfection with the pGL3-KLF4 reporter construct and pcDNA3-soluble klotho was performed in HEK239 cells, followed by the dual-luciferase assay as described in the Materials and Methods. The mean ± S.D. of three independent experiments is shown (*p < 0.05, **p < 0.01).

Soluble klotho induces the KLF4-related pathway

To directly assess the functional contribution of soluble klotho to KLF4 signaling, we investigated the expression changes in KLF4-related genes upon the overexpression of soluble klotho in MEFs. We first evaluated the overexpression of soluble klotho in soluble klotho-transfected MEFs by real-time quantitative RT-PCR and observed abundant KLF4 mRNA expression (Figure 3A–3D). As shown in Figure 3E and 3F, soluble klotho the increased KLF4 protein expression in wild-type and klotho (-/-) MEFs. Immunoblotting analysis also revealed increased expression of KLF4-related genes, such as mTOR/p70s6k, p21, cyclinD1/cyclinB1, and SOD1/SOD2, in soluble klotho-transfected MEFs. The phosphorylation and/or expression of mTOR/p70s6k, p21, AMPK, cyclinD1, cyclinB1, SOD1, and SOD2 were strikingly upregulated in soluble klotho-transfected MEFs. In addition, the effects of soluble klotho protein on the expression of KLF4-related proteins are shown in Supplementary Figure 1 The upregulation of the KLF4 pathway by soluble klotho was further confirmed in HEK293 cells (Supplementary Figure 2).

Figure 3. Effects of soluble klotho on the expression of proteins belonging to the KLF4 pathway in wild-type and klotho (-/-) MEFs. (A–D) qRT-PCR analysis of soluble klotho and KLF4. Total RNA samples were prepared from soluble klotho-transfected MEFs, and quantitative RT-PCR analysis was performed using the primers described. (E, F) The expression of proteins related to the KLF4 pathway. Wild-type and klotho (-/-) MEFs were transfected with soluble klotho expression plasmids (pcDNA3-soluble klotho). At 48 h after transfection, Western blot analysis was performed to assess the KLF4, mTOR, p70S6K, p21, AMPK, cyclin D1, cyclin B1, SOD1, and SOD2 levels. The mean ± S.D. of three independent experiments is shown (*p < 0.05, **p < 0.01).

Soluble klotho and KLF4 regulate the p53/p21 and SOD1/2 pathways

We next assessed the effect of soluble klotho depletion on KLF4-related protein expression. The inhibition of soluble klotho by siRNA was detected by real-time PCR and Western blot analysis in HEK293 cells, and reduced expression of KLF4 and FOXO1 was observed. KLF4 protein expression was also inhibited in siRNA soluble klotho-transfected cells (Figure 4A–4D).

Figure 4. Effects of si-klotho and siKLF4 on KLF4-related protein expression. (A–D) Expression of klotho, FOXO-1, and KLF4 in si-klotho-overexpressing HEK293 cells. Cells were transfected with siRNA (0.5 or 1.0 nM) for 48 h. Comparisons of the si-klotho silencing efficiency by qRT-PCR and Western blot. (E) The KLF4 mRNA levels in HEK293 cells treated with KLF4 siRNA as measured by RT-PCR. (F) Western blot analysis of protein extracted from KLF4 siRNA (0.1, 0.5 or 1.0 nM)-transfected cells in a concentration-dependent manner. The expression levels of KLF4-related proteins, such as mTOR, p70S6K, p53, p21, AMPK, cyclin D1, cyclin B1, SOD1, SOD2, and actin (as a control), were determined. (G) Schematic diagram of the cell signaling pathway regulated by soluble klotho/KLF4. The mean ± S.D. of three independent experiments is shown (*p < 0.05, **p < 0.01).

To further clarify whether KLF4 depletion modulates soluble klotho-induced KLF4 signaling, we knocked down KLF4 by siRNA in HEK293 cells. As shown in Figure 4E–4F, the expression of KLF4 was dramatically downregulated in siKLF4-transfected cells. Interestingly, the expression of SOD1, SOD2, and P53 was strikingly downregulated in siKLF4-transfected cells in a concentration-dependent manner. However, the soluble klotho-induced expression/activation of mammalian target of rapamycin (mTOR), cyclin D1, and cyclin B1 was not changed in siKLF4-transfected cells compared to that in control siRNA-transfected cells. Together, these results showed that soluble klotho directly regulates KLF4 expression and may modulate the cell cycle and antioxidant signaling by regulating p53/p21 and SOD1/2 through KLF4 signaling pathways (Figure 4G).

Soluble klotho induces the function of primary salivary gland cells (PSGCs)

Single-cell suspensions obtained by mechanical and enzymatic dissociation of klotho (-/-) mouse salivary glands were grown in our culture system as described in the Materials and Methods. In klotho (-/-) PSGCs, we examined the expression of KLF4 by real-time quantitative RT-PCR and Western blot. Similar to the results shown in Figure 1, KLF4 expression was decreased in klotho (-/-) PSGCs (Figure 5A and 5B).

Figure 5. The expression of salivary gland functional proteins in primary salivary gland cells (PSGCs). (A, B) The protein and mRNA expression of KLF4 in KL(+/+) PSGCs and KL(-/-) PSGCs. (C–E) Comparison of functional protein or mRNA (aquaporin 5, EGFBP2, ZO-1, and amylase) expression between KL(+/+) PSGCs and KL(-/-) PSGCs by RT-PCR, qRT-PCR, and immunoblot analyses. (F–H) Effects of soluble klotho on functional protein expression. Cells were transfected with pcDNA3-soluble klotho for 48 h. Comparisons of salivary gland functional proteins by RT-PCR, qRT-PCR, and Western blot. The actin protein was used as an internal control. The results are reported as the mean ± SD (*p < 0.05, **p < 0.01).

To confirm the function of PSGC populations, we examined the expression of salivary gland functional markers, such as α-amylase (AC secretion product), aquaporin 5 (water channel protein), and ZO-1 (tight junction protein). Quantitative reverse transcription PCR (qRT-PCR) was used to validate the altered expression of these genes. Klotho (-/-) PSGCs showed reduced expression of α-amylase and aquaporin-5 compared to that in klotho (+/+) PSGCs. The tight junction protein ZO-1 was also inhibited in klotho (-/-) PSGCs (Figure 5C–5E). In contrast, the effect of soluble klotho overexpression on KLF4-related protein expression was further confirmed by RT-PCR. The expression of α-amylase, aquaporin-5, and ZO-1 was dramatically upregulated in soluble klotho-expressing klotho (-/-) PSGCs (Figure 5F–5H). Consistent with those in klotho (-/-) PSGCs, the mRNA levels of aquaporin 5 and EGFBP2 were increased in soluble klotho-transfected klotho (+/+) PSGCs. The mRNA expression of α-amylase, aquaporin-5, and ZO-1 in KLF4-overexpressing klotho (+/+) PSGCs was also confirmed by real-time quantitative RT-PCR. In addition, salivary gland functional marker expression in PSGCs was confirmed by Western blot analysis of soluble klotho- or KLF4-expressing wild-type PSGCs (Supplementary Figure 3).

Soluble klotho-induced KLF4 induces differentiation into acinar-like cells

To determine whether PSGCs induced by soluble klotho or KLF4 transfection could differentiate into functional activating cells, such as salivary gland ACs, we utilized a 3D culture system. First, we observed the induction of typical mesenchymal stem cell (MSC) surface antigen markers, such as CD44 and CD90, which are known to interact with the extracellular matrix, in soluble klotho- or KLF4-expressing wild-type PSGCs (Figure 6A). In addition, salivary gland functional marker expression in soluble klotho- or KLF4-expressing wild-type PSGCs was confirmed by Western blot (Figure 6B). Then, 4D cultured PSGCs were immunostained for four AC markers, α-amylase, Aqua-5, and ZO-1, to confirm AC differentiation. Under soluble klotho or KLF4 transfection conditions, small colonies of epithelial-shaped cells were observed on the culture substrate within 5 days after seeding. Cells with a homogeneous polygonal shape proliferated and expanded to form an epithelial-like monolayer. The cells in the 3D culture substrate were generally covered by monolayered cells. In this crowded region, 3D cell aggregations and spherical shapes were observed (Figure 6C). As shown by immunofluorescence analysis of 3D cultured PSGCs, these spherical cells were positively labeled with AQP-5 and α-amylase. Notably, strong staining of ZO-1 was observed. These findings suggested that soluble klotho-induced PSGCs exhibited increased functional characteristics. The acceleration of the KLF4 pathway upon expression of soluble klotho or KLF4 was further confirmed in salivary gland ACs (Supplementary Figure 4).

Figure 6. Functional restoration of PSGCs by soluble klotho and KLF4 expression. (A) Detection of the CD90 and CD44 protein levels in soluble klotho- or KLF4-expressing PSGCs. Cells were transfected with soluble klotho or KLF4 expression plasmids. At 48 h after transfection, total protein was prepared and subjected to immunoblotting. (B) Expression of KLF4, aquaporin 5, ZO-1, and amylase in PSGCs after transfection with soluble klotho or KLF4 expression plasmids. (C) Immunofluorescence staining of aquaporin 5, ZO-1, and amylase in PSGCs cultured on 3D Matrigel at day 5.

Cell cultures

MEFs were derived and cultured from SAMP1/kl+/+ and SAMP1/kl-/- 13-day-old embryos that were obtained by mating SAMP1/kl+/- mice with SAMP1/kl+/- mice [32] and maintained in Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco, NY) containing gentamycin, glutamine, MEM nonessential amino acids, 2-mercaptoethanol, and 10% fetal bovine serum (FBS, Gibco, NY). The human embryonic kidney 293 cell line and MEFs isolated from wild-type or klotho knockout mice were grown in DMEM supplemented with 10% FBS, 100 units/ml penicillin, and 100 μg/ml streptomycin (Invitrogen, CA) in a humidified 5% CO₂/95% air atmosphere at 37°C. Immortalized human salivary gland ACs were cultured on keratinocyte serum-free medium (K-SFM, Gibco/Life Technologies, Grand Island, NY) containing 100 U/ml penicillin and 100 μg/ml streptomycin.

Preparation of primary submandibular salivary gland cells from mice and in vitro culture

Submandibular gland tissues were harvested from 4- or 8-week-old wild-type and klotho (-/-) mice. The tissues were dissociated from cervical fascia and connective tissues under a dissecting microscope and gently collected in phosphate-buffered saline (PBS). Freshly dissociated tissues were washed twice with DMEM and Ham’s F-12 medium (DMEM/F12 medium, 1:1, Gibco, USA) supplemented with 100 U/mL penicillin G and 100 μg/ml streptomycin (Gibco), minced with scissors, and incubated with DMEM/F12 medium containing collagenase type II (0.025%), hyaluronidase (0.04%) and CaCl₂ (6.25 mM) at 37°C for 40 min. Dissociated cells were centrifuged at 1000 rpm for 5 min, and the cell pellet was resuspended in the culture medium. The cells were plated in a 12-well plate at a density of 5 × 10⁴ cells/well in DMEM/F-12 (Gibco) medium supplemented with 100 U/mL penicillin G, 100 μg/ml streptomycin, 20 ng/mL EGF (Sigma-Aldrich), 20 ng/mL basic fibroblast growth factor (Gibco), 1/100 N2 supplement (Gibco), 10 μg of insulin-transferrin-selenium (Gibco), and 1 μM dexamethasone (Sigma-Aldrich) and incubated in 5% CO₂ at 37°C. Fresh medium was added every three days. After 4-5 passages, the expression of salivary gland functional markers, such as α-amylase (AC secretion product), aquaporin 5 (water channel protein), and ZO-1 (tight junction protein), was examined.

Real-time quantitative PCR

Total RNA was extracted with TRIzol reagent (Invitrogen). Reverse transcription was performed on 1 μg of total RNA using oligo dT primers and M-MLV Reverse Transcriptase (Invitrogen) in a final volume of 20 μl for 5 min at 65°C followed by 1 h at 37°C. Real-time quantitative PCR was performed using the SYBR Green kit (Thermo Fisher, USA) according to the manufacturer’s instructions. The expression levels of soluble klotho, KLF4, and FOXO1 mRNA were normalized to those of GAPDH for each sample. The following primers were used for real-time quantitative PCR detection: soluble klotho, 5′-GCTCTGCTTCCGCC ACTT-3′ (forward) and 5′-GCATGAGCCAGGAGGAG G-3′ (reverse); KLF4, 5′-TTCCAACTCGCTAACCCAC C-3′ (forward) and 5′-GCTCGGGACTCAGTGTAG-3′ (reverse); FOXO1, 5′-AGACCCGGGTTCTTTGACAC-3′ (forward) and 5′-TTGTCATAGGCTTCCCACCA-3′ (reverse); amylase, 5′-GGTGCAACAATGTTGGTGTC-3′ (forward) and 5′-ACTGCTTTGTCCAGCTTGAG-3′ (reverse); Aqua5, 5′-CGACCGTGTGGCTGTGGTCA-3′ (forward) and 5′-GTGCCGGTCAGTGTGCCGTC-3′ (reverse); ZO-1, 5′-CGAGGCATCATCCCAAATAAGA AC-3′ (forward) and 5′-TCCAGAAGTCTGCCCGATC AC-3′ (reverse). The samples were quantified based on the threshold cycle by interpolation from the standard curve. The reliability of the PCR results was evaluated by a solubility curve as follows: ΔCt = CT _{(target gene)} − CT _{(internal reference)} and ΔΔCt = ΔCt _{(experimental group)} − ΔCt _{(control group)}. The relative expression levels of related genes were determined by the 2^−ΔΔCT method, and the experiments were repeated three times.

siRNA interference

KLF4 siRNA was obtained in the form of Silencer Select Validated siRNA (Applied Biosystems). The sense sequence of the KLF4 siRNA was 5′-CUCGUUCUCG GCUUGUCUA (dTdT)-3′, and the antisense sequence was 5′-UAGACAAGCCGAGAACGAG (dTdT)-3′. The cells were transfected with siRNA (20 nM) using Lipofectamine 2000 siRNA Transfection Reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. The cells were harvested 48 h after transfection. Total cell lysates were separated by SDS-PAGE and analyzed by Western blot as described above.

Western blot analysis

Cells were lysed, and proteins were extracted in a radio immunoprecipitation assay buffer (RIPA buffer, 25 mM Tris-HCl [pH 7.6], 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS; Pierce) and complete protease inhibitor cocktail (Sigma-Aldrich). The lysates were obtained after centrifugation, and the protein concentration was measured using the BCA protein assay kit (Pierce, Thermo Scientific). The lysates were separated by 12% SDS-PAGE. After transfer to PVDF membranes (Hybond-C; GE Amersham), the membranes were blocked for 1 h at room temperature with 5% nonfat dry milk in TBS-T buffer. The blocked membranes were then incubated with primary antibodies (1:1000 dilution) against klotho (10E2), KLF4 (3F3), FOXO1 (7G6C5), phospho-mTOR (D15C3), phospho-AMPK (D2E5), and p21 (D1F2), which were purchased from Cell Signaling Technology. The cyclin D1, cyclin B1, SOD1, SOD2, p70S6K, and actin antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The membranes were washed 3 times with TBS-T buffer and then incubated with secondary antibodies conjugated to HRP (1:5,000 dilution) for 1 h at room temperature. The immunoreactive proteins were visualized with ECL detection reagents (Thermo).

Luciferase activity assay

The pGL4-KLF4 promoter (-2.046 bp from the start codon) was generously provided by Dr. Yoshida (Jikei University, Tokyo, Japan). HEK293 cells were transfected with 1 μg of the pGL4-KLF4 Luc plasmid and 1 μg of Tk-Renilla by Fugene 6 HD (Promega) reagent and harvested after 48 h. Additionally, the cells were transfected with pcDNA3-sKL and/or pcDNA3-KLF4 using Fugene 6 HD reagent for 48 h. Transfected cells were lysed and measured with the Dual-Luciferase Reporter Assay System (Promega, WI, USA) according to the manufacturer’s instructions. Relative luciferase units were normalized by the Tk-Renilla luciferase value in each sample. Each experiment was performed in triplicate and repeated twice.

Three-dimensional Matrigel spheroid cultures

One hundred microliters of Matrigel (Nitta Gelatin, Inc., Osaka, Japan; 8:1 Cellmatrix type-1: concentrated media) in 48-well chambers was allowed to solidify in a 37°C incubator for 1 h. Upon solidification, 5 x 10⁴ cells/well were seeded on Matrigel, and 3D spheroids were grown at 37°C in 5% CO₂. For the generation of single-cell suspensions from 3D spheroids, spheres were isolated by removing media and adding 0.5 ml of Dispase (1 mg/ml) directly to the Matrigel followed by incubation for 1 h at 37°C in 5% CO₂ to digest the Matrigel. The spheres were washed in PBS, centrifuged at 1200 rpm for 5 min, dissociated with TrypLE Express (Gibco) and passed through a 40 μm filter to remove clumps and obtain single-cell suspensions. The cells were then plated and grown as monolayers in either serum-free medium (SFM) or serum-containing medium (SCM).

Immunofluorescence analysis

The presence of aquaporin-5 and ZO-1 in 3D cultured cells was confirmed by indirect immunofluorescence staining as described previously [33]. Briefly, samples were fixed with 10% formaldehyde/PBS for 10 min and permeabilized by PBS containing 1% BSA/0.2% Triton X-100 for 15 min at room temperature. The samples were incubated with primary antibodies against aquaporin-5 and ZO-1 (1:50 dilution) overnight at 4°C. After three washes with PBS/0.05% Tween 20, the samples were incubated with a secondary antibody (Alexa Fluor 488-conjugated goat anti-mouse IgG [H+L]; Invitrogen, CA), diluted 1:500 for 2 h at room temperature. The cell nuclei were counterstained by propidium iodide for 5 min and subsequently washed three times with PBS/0.05% Tween 20. The stained cells were visualized using a Nikon A1⁺ confocal microscope (Japan). The average intensity value of the nonspecifically bound secondary antibody was subtracted from all images using the ImageJ program.

Statistical analysis

Data are presented as the mean ± SEM. Statistical significance between groups was determined by Student’s t test and one-way analysis of variance (ANOVA). P-values <0.05 were considered statistically significant.