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. 2019 Sep 16;15(1):169–177. doi: 10.4103/1673-5374.264463

Claudin-15 overexpression inhibits proliferation and promotes apoptosis of Schwann cells in vitro

Jian-Nan Li 1, Zhan Zhang 1, Guang-Zhi Wu 1, Deng-Bing Yao 2, Shu-Sen Cui 1,*
PMCID: PMC6862392  PMID: 31535666

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Keywords: apoptosis, Bax, cell proliferation, c-Jun, Claudin-15, nerve regeneration, peripheral nerve injury, protein kinase C alpha, Schwann cells, Wallerian degeneration

Abstract

Our previous experiments have discovered that Claudin-15 was up-regulated in Schwann cells of the distal nerve stumps of rat models of sciatic nerve injury. However, how Claudin-15 affects Schwann cell function is still unknown. This study aimed to identify the effects of Claudin-15 on proliferation and apoptosis of Schwann cells cultured in vitro and explore the underlying mechanisms. Primary Schwann cells were obtained from rats. Claudin-15 in Schwann cells was knocked down using siRNA (siRNA-1 group) compared with the negative control siRNA transfection group (negative control group). Claudin-15 in Schwann cells was overexpressed using pGV230-Claudin-15 plasmid (pGV230-Claudin-15 group). The pGV230 transfection group (pGV230 group) acted as the control of the pGV230-Claudin-15 group. Cell proliferation was analyzed with EdU assay. Cell apoptosis was analyzed with flow cytometric analysis. Cell migration was analyzed with Transwell inserts. The mRNA and protein expressions were analyzed with quantitative polymerase chain reaction assay and western blot assay. The results showed that compared with the negative control group, cell proliferation rate was up-regulated; p-AKT/AKT ratio, apoptotic rate, p-c-Jun/c-Jun ratio, mRNA expression of protein kinase C alpha, Bcl-2 and Bax were down-regulated; and mRNA expression of neurotrophins basic fibroblast growth factor and neurotrophin-3 were increased in the siRNA-1 group. No significant difference was found in cell migration between the negative control and siRNA-1 groups. Compared with the pGV230 group, the cell proliferation rate was down-regulated; apoptotic rate, p-c-Jun/c-Jun ratio and c-Fos protein expression increased; mRNA expression of protein kinase C alpha and Bax decreased; and mRNA expressions of neurotrophins basic fibroblast growth factor and neurotrophin-3 were up-regulated in the pGV230-Claudin-15 group. The above results demonstrated that overexpression of Claudin-15 inhibited Schwann cell proliferation and promoted Schwann cell apoptosis in vitro. Silencing of Claudin-15 had the reverse effect and provided neuroprotective effect. This study was approved by the Experimental Animal Ethics Committee of Jilin University of China (approval No. 2016-nsfc001) on March 5, 2016.

Chinese Library Classification No. R453; R363; R364

Introduction

Many patients present with peripheral nerve injury every year (Siemionow and Brzezicki, 2009). This commonly results in Wallerian degeneration (Conforti et al., 2014; Cao et al., 2019), first discovered by Waller (1851). The discovery of WLDS protein in strains of mice resistant to axonal degeneration (Mack et al., 2001; Coleman and Freeman, 2010; Williams et al., 2017) convinced researchers that Wallerian degeneration is a programmed cell event (Gerdts et al., 2016; Wang et al., 2018). In normal mice, immediately after peripheral nerves suffer trauma, Wallerian degeneration begins (Girouard et al., 2018); the disintegration of axonal structures occurs (Geden and Deshmukh, 2016; Simon et al., 2016), atrophy of fiber tracts is observed and macrophages and Schwann cells infiltrate (Liou et al., 2011; Simon et al., 2016; Zigmond and Echevarria, 2019).

In the peripheral nervous system, Schwann cells wrap around the nerve fibers and secrete neurotrophins to protect and nourish the peripheral nerves (Kennedy and Zochodne, 2005; Jortner, 2019). During Wallerian degeneration, Schwann cells play a vital role in the degeneration and regeneration of neurons (Jessen and Arthur-Farraj, 2019; Jessen and Mirsky, 2019; Shefa and Jung, 2019; Yi et al., 2019). The Schwann cells release a variety of factors associated with apoptosis (Li et al., 2018; Zhang et al., 2018) and participate in the clearance of myelin debris (Namikawa et al., 2006; Lindborg et al., 2017). Schwann cells proliferate (Liu et al., 2019), dedifferentiate and migrate, offering regenerating axons both the structural direction and the growth promotion factors (Tetzlaff, 1982; Stoll et al., 2002; Jessen and Mirsky, 2016). Nevertheless, how Schwann cells participate in Wallerian degeneration remains unclear.

Claudins are the main constituents of the tight junction membrane proteins. At least 27 Claudins have been discovered (Morita et al., 1999; Van Itallie and Anderson, 2006; Muto, 2017; Tsukita et al., 2019). Claudins are the key molecules that maintain cell polarity and molecular transport (Hagen, 2017). Commonly, Claudins form chains that mediate cell adhesion and function as barriers to cells (Krause et al., 2015; Tsukita et al., 2019). They have also been shown to function as ion channels and regulate homeostasis and inflammation (Garcia-Hernandez et al., 2017; Alberini et al., 2018).

Claudins are expressed in Schwann cells (Manole et al., 2015). Claudin family members Claudin-14 and Claudin-15 are classic tight junction molecules (Krause et al., 2008) that we discovered were expressing in Schwann cells of the sciatic nerve after peripheral nerve injury (Li et al., 2013; Gong et al., 2014). The molecular mechanism underlying how Claudin-15 affects Schwann cell functions was poorly understood. This study explores the functions of Claudin-15 on cell proliferation, apoptosis and migration in cultured Schwann cells.

Materials and Methods

Schwann cells culture and transfection

Schwann cells were cultured according to the protocols described previously (Li et al., 2013; Zhang et al., 2018). Primary Schwann cells were extracted and isolated from the sciatic nerves of one-day old Sprague-Dawley rats (Experimental Animal Center of Jilin University, China; license No. SCXK (Ji) 2002-0002). Primary Schwann cells were cultured in Dulbecco’s modified Eagle’s medium (Sigma, Saint Louis, MO, USA) containing 10% fetal bovine serum (Gibco, New York, USA) and penicillin-streptomycin solution (Beyotime, Jiangsu, China) at 37°C with 5% CO2. Fibroblasts were destroyed by complement cleavage using polyclonal anti-Thy1.1 antiserum (Sigma). Recombinant human NRG1-beta 1/HRG1-beta 1 epidermal growth factor Domain Protein (R&D system, Minneapolis, MN, USA) and forskolin (Sigma) were added to the media for cell proliferation.

Schwann cells were transfected separately with siRNA-1, siRNA-2 and siRNA-3 (RiboBio, Guangzhou, China; Table 1) using Lipofectamine 3000 reagent (Invitrogen, New York, NJ, USA) for Claudin-15 knockdown (n = 3). A negative control siRNA transfection group (Table 1) was used as the control group for Claudin-15 knockdown. Schwann cells were transfected with pGV230-CLDN15 plasmid using Lipofectamine 3000 reagent for overexpression of Claudin-15 (n = 3). Transfection with pGV230 acted as the control group. RNA was collected 48 hours after transfection. Proteins were collected and assessed 72 hours after transfection. Schwann cells were planted on the Transwell insert 48 hours after transfection. Cell proliferation assay and cell apoptosis assay were done 72 hours after transfection. Every experimental procedure and protocol was approved by the Experimental Animal Ethics Committee of Jilin University of China (approval No. 2016-nsfc001) on March 5, 2016.

Table 1.

Claudin-15 siRNA primers

Gene Sequence Product size (bp)
NC Forward: 5′ UUC UCC GAA CGU GUC ACG UTT 3′ 21
Reverse: 5′ ACG UGA CAC GUU CGG AGA ATT 3′ 21
siRNA-1 Forward: 5′ GGA ACG UCA UCA CCC ACU AAC A 3′ 22
Reverse: 5′ UUA GAG GUG AGG ACG UUC CCA 3′ 22
siRNA-2 Forward: 5′ GGA GAG UGU CUA CCG UCC AUG 3′ 21
Reverse: 5′ UGG ACG GUA GAC ACU CUC CAG 3′ 21
siRNA-3 Forward: 5′ GGG UGG GAC UUC CCU ACA AGC 3′ 21
Reverse: 5′ UUG UAG GGA AGU CCC ACC CUG 3′ 21
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siRNA: Small interfering RNA; NC: negative control.

RNA isolation and real-time quantitative polymerase chain reaction (PCR) assay

Total RNA was isolated from Schwann cells following the manufacturer’s protocols. Real-time quantitative PCR was accomplished with TB Green™ Fast qPCR Mix (Takara, Kusatsu, Japan). Primer sequences of Bax: Bcl-2-Associated X protein (Bax, pro-apoptosis factor), B-cell lymphoma 2 (Bcl-2, anti-apoptosis factor), basic fibroblast growth factor (bFGF, neurotrophin), neurofibromin 2 (Nf2, cytoskeleton-associated protein), neurotrophin-3 (NT3), protein kinase C alpha (PKCα, serine threonine kinase) are listed in Table 2. Relative mRNA expression levels were calculated using the relative quantitation comparative CT method (2–ΔΔCt method) (Li et al., 2018; Zhang et al., 2018). Each sample was run in triplicate.

Table 2.

Primers used in real-time quantitative polymerase chain reaction

Name Sequence Product size (bp)
CLDN15 Forward: 5′-CGG GCA GAA GCA ATC AGA C-3′ 93
Reverse: 5′-AAG ACT GAG GAG GGA GAA GGT T-3′
Bax Forward: 5′-TGC AGA GGA TGA TTG CTG AC-3′ 173
Reverse: 5′-GAT CAG CTC GGG CAC TTT AG-3′
Bcl-2 Forward: 5′-GCA GAG ATG TCC AGT CAG C-3′ 129
Reverse: 5′-CCC ACC GAA CTC AAA GAA GG-3′
bFGF Forward: 5′-CCC GCA CCC TAT CCC TTC ACA GC-3′ 130
Reverse: 5′-CAC AAC GAC CAG CCT TCC ACC CAA A-3′
Nf2 Forward: 5′-CTG GGA TTG GGT TCA TGG GTG GAT-3′ 127
Reverse: 5′-AGG AAG CCC GAG AAG CAG AGC G-3′
NT3 Forward: 5′-GAC AAG TCC TCA GCC ATT GAC ATT C-3′ 131
Reverse: 5′-CTG GCT TCT TTA CAC CTC GTT TCA T-3′
PKCα Forward: 5′-GAA CAC ATG ATG GAC GGG GTC ACG AC-3′ 170
Reverse: 5′-CGC TTG GCA GGG TGT TTG GTC ATA-3′
GAPDH Forward: 5′-GCA AGT TCA ACG GCA CAG-3′ 141
Reverse: 5′-CGC CAG TAG ACT CCA CGA C-3′
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Bax: Bcl-2-associated X protein; Bcl-2: B-cell lymphoma 2; bFGF: basic fibroblast growth factor; CLDN15: claudin 15; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; Nf2: neurofibromin 2; NT3: neurotrophin-3; PKCα: protein kinase C alpha.

Cell proliferation assay

The assay of cell proliferation was performed with Cell-Light Apollo567 In Vitro Kit (RiboBio, Guangzhou, China). Complete medium was used to re-suspend the Schwann cells that were then tallied and plated on 96-well poly-L-lysine-coated plates. EdU was applied and the cells were cultured after cell transfection. The cells were fixed with phosphate buffered saline containing 4% formaldehyde and stained with Apollo 567 (RiboBio, Guangzhou, China) and Hoechst 33342 (RiboBio). Schwann cell proliferation analysis was performed using randomly selected images through a fluorescence microscope (Leica, Mannheim, Germany). The proliferating cell numbers were calculated. The average number of proliferating cells in the control group was set as 100%. The cell proliferation rate of p-GV230-Claudin-15 group was obtained by dividing by the average number of proliferating cells in the negative control or pGV230 group. The results were presented as fold change.

Flow cytometric analysis

Cell apoptosis was probed using Annexin V-FITC Apoptosis Detection Kit (Beyotime, Jiangsu, China). The Schwann cells were trypsinized, ultra-centrifuged, and resuspended. Annexin V-FITC solution was dropped onto each sample and left to stand for 15 minutes. Cells were resuspended. Propidium iodide reagent was dropped onto the samples, which were then kept in the dark for 15 minutes at room temperature. The cells were analyzed by Beckman Flow Cytometer (Beckman, Fullerton, CA, USA). The average rate of apoptosis in the control group was set as 100%. The cell apoptotic rate of p-GV230-Claudin-15 group was obtained by dividing it with the average rate in the negative control or pGV230 group. The results were exhibited as fold change.

Cell migration assay

Cell migration was assayed using Transwell inserts (Corning Inc, Corning, NY, USA) (Mantuano et al., 2008). The membrane of each insert was coated with fibronectin (Sigma). Schwann cells were planted in the top chamber with Dulbecco’s modified Eagle’s medium. The lower chambers contained complete medium. After 24 hours, the migrated Schwann cells were fixed with methanol and stained with crystal violet solution. The non-migrated cells in the upper chamber were wiped with cotton swabs. Migrated cells were imaged and tallied using a DMR inverted microscope (Leica, Mannheim, Germany). The migrated cell numbers were calculated, taking the average number of migrated cells in control group as 100%. The cell migration rate of the p-GV230-Claudin-15 group was obtained by dividing it with the average number of negative control or pGV230 group. The results were exhibited as fold changes.

Western blot assay

Schwann cells were prepared with RIPA lysis buffer (Sangon Biotech, Shanghai, China) and their protein concentrations were evaluated by BCA Protein Assay Kit (Beyotime, Jiangsu/Shanghai, China). The protein was electrophoresed through a 12% sodium dodecyl sulfate polyacrylamide gel and then transferred to polyvinylidene fluoride membranes. The membranes were blocked by 5% bovine serum albumin in Tris-buffered saline Tween-20 for 1 hour at room temperature. The membranes were incubated with primary antibodies at 4°C overnight: rabbit polyclonal anti-Claudin 15 antibody (1:200; Santa Cruz Biotechnology, Santa Cruz, CA, USA); rabbit monoclonal anti-AKT antibody (1:1000; CST, Boston, MA, USA) (AKT pathway pro-survival factor); rabbit monoclonal anti-phospho-AKT antibody (1:1000; CST) (AKT pathway pro-survival factor); rabbit monoclonal anti-ERK1/2 antibody (1:1000; CST) (ERK pathway pro-survival factor); rabbit monoclonal anti-phospho-ERK1/2 (1:1000; CST) (ERK pathway pro-survival factor); mouse monoclonal anti-c-Jun antibody (1:200, Santa Cruz Biotechnology) (JNK pathway pro-apoptosis factor); mouse monoclonal anti-p-c-Jun antibody (1:200; Santa Cruz Biotechnology) (JNK pathway pro-apoptosis factor); rabbit monoclonal anti-β-Catenin antibody (1:5000; Abcam, Cambridge, MA, USA) (WNT pathway cell adhesion factor); rabbit polyclonal anti-c-Fos antibody (1:250; Abcam) (JNK pathway transcription factor); rabbit monoclonal anti-GAPDH antibody (1:10,000; Abcam). The next day, the membranes were incubated with secondary antibody, either horseradish peroxidase-labeled goat anti-mouse IgG (1:1000; Beyotime) or horseradish peroxidase-labeled goat anti-rabbit IgG, for 2 hours at room temperature. Protein expression levels were normalized with reference to GAPDH. Images were scanned by a GS800 Densitometer Scanner. The blot intensity was evaluated by Multi Gauge software (Fuji, Tokyo, Japan). The blot intensity was first normalized with respect to that of GAPDH in each group. The average blot density of control group was set as 100%. The relative optical density value of p-GV230-Claudin-15 group was obtained by dividing them with the optical density values of the negative control or pGV230 group.

Statistical analysis

All data are expressed as the mean ± SD, and analyzed using SPSS 15.0 software (SPSS, Chicago, USA). The data were analyzed with two tailed, unpaired Student’s t-test and one-way analysis of variance followed by Tukey’s post hoc test. P < 0.05 was considered statistically significant.

Results

siRNA and plasmid selection

Our previous research showed that the expression of Claudin-15 was up-regulated in Schwann cells of rat sciatic nerve after peripheral nerve injury (Li et al., 2013). The three specific Claudin-15 siRNAs listed in Table 1 were synthesized by RiboBio. Claudin-15 expression in cultured Schwann cells was significantly decreased at the mRNA level by siRNA-1 transfection (Figure 1). The expression of Claudin-15 declined more than 80% with siRNA-1 (Figure 1). Hence, siRNA-1 was chosen for following experiments. We used the pGV230-Claudin-15 plasmid for Claudin-15 overexpression analysis (Figure 1).

Figure 1.

Figure 1

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Claudin-15 mRNA expression changes after siRNA knockdown and overexpression in cultured Schwann cells.

(A) Relative levels of Claudin-15 mRNA expression after siRNA transfection compared to the negative control. (B) Western blot assay of Claudin-15 after siRNA transfection. The upper panel shows target bands of Claudin-15; the lower panel shows the loading control GAPDH. (C) Relative levels of Claudin-15 protein expression after siRNA transfection. (D) Relative levels of Claudin-15 mRNA expression after pGV230-Claudin-15 transfection. (E) Western blot assay of Claudin-15 after pGV230-Claudin-15 transfection. The upper panel shows target bands of Claudin-15; the lower panel shows the loading control GAPDH. (F) Relative levels of Claudin-15 protein expression after pGV230-Claudin-15 transfection. *P < 0.05, vs. NC group; #P < 0.05, vs. pGV230 group. Average blot density of control group blot was set as 100%. The relative optical density value of p-GV230-Claudin-15 group was obtained by dividing its optical density with the optical density values of the NC or pGV230 group. Assays were performed in triplicate. Data are shown as the mean ± SEM (A: Unpaired Student’s t-test; C, D, F: one-way analysis of variance followed by Tukey’s post hoc test). GAPDH: Gyceraldehyde-3-phosphate dehydrogenase; NC: negative control.

Claudin-15 affects several gene expression changes of cultured Schwann cells

To further investigate the functions of Claudin-15 on expressions and relative factors release in Schwann cells, we analyzed Bcl-2, Bax, bFGF, PKCα, NT3 and Nf2 expressions. These factors are related to nerve degeneration and regeneration after Claudin-15 overexpression and knockdown in transfected Schwann cells. In Claudin-15 knockdown Schwann cells, the expressions of Bax, Bcl-2 and PKCα decreased, whereas bFGF and NT3 were up-regulated (Figure 2A). The expressions of Bax and PKCα increased, whilst NT3 and bFGF were down-regulated in Schwann cells with over expressed Claudin-15 (Figure 2B). These results suggest that differential expression of Claudin-15 resulted in expression changes of other factors in Schwann cells.

Figure 2.

Figure 2

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Gene expression changes in Claudin-15 siRNA-1 knockdown and pGV230-Claudin-15 overexpresed Schwann cells in vitro.

(A) Relative levels of Bax, Bcl-2, bFGF, NT3, Nf2 and PKCα mRNA expression of siRNA-1 transfected Schwann cells. GAPDH was used as normalizer. (B) Relative levels of Bax, Bcl-2, bFGF, NT3, Nf2 and PKCα mRNA expression of pGV230-Claudin-15 transfected Schwann cells. GAPDH was used as normalizer. *P < 0.05, vs. NC group; #P < 0.05, vs. pGV230 group. All experiments were performed in triplicate. Data are shown as the mean ± SEM (unpaired Student’s t-test). Bax: Bcl-2-associated X protein; Bcl-2: B-cell lymphoma 2; bFGF: basic fibroblast growth factor; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; NC: negative control; Nf2: neurofibromin-2; NT3: neurotrophin-3; PKCα: protein kinase C alpha.

Claudin-15 inhibits Schwann cells proliferation in vitro

We analyzed the action of Claudin-15 on the proliferation of Schwann cells. After transfection with Claudin-15 siRNA-1, pGV230-Claudin-15 plasmid or negative control, EdU proliferation assay demonstrated that the proliferation proportion of cultured Schwann cells after siRNA transfection significantly increased compared with that of the negative control (P < 0.05; Figure 3A, B, and E). On the other hand, when Schwann cells were transfected with pGV230-Claudin-15 plasmid, their proliferation rate significantly decreased compared with the pGV230 group (P < 0.05; Figure 3C, D, and F). Schwann cells were stained with Hoechst 33342 (blue), and the proliferating cells were stained with Apollo 567 (red) (Figure 3). These results suggested that Claudin-15 expression inhibited Schwann cells proliferation in vitro.

Figure 3.

Figure 3

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Claudin-15 knockdown and overexpression affect Schwann cells proliferation in vitro.

(A–D) Proliferation of Schwann cells in the NC (A), siRNA-1 (B), pGV230 (C), and pGV230-Claudin-15 (D) groups was determined by EdU staining. Images are taken by a fluorescence microscope. Merged image of EdU-positive Schwann cells (Red) and cell nuclei labeled by Hoechst 33342 (blue). Scale bar: 100 μm. (E) Cell proliferation rate of siRNA-1 group compared with NC group. (F) Cell proliferation rate of pGV230-Claudin-15 group compared with pGV230 group. **P < 0.01, vs. NC group; #P < 0.05, vs. pGV230 group. All experiments were performed in triplicate. Data are shown as the mean ± SEM (unpaired Student’s t-test). EdU: 5-Ethynyl-20-deoxyuridine; NC: negative control.

Claudin-15 promotes Schwann cells apoptosis in vitro

To detect the apoptosis function of Claudin-15 on Schwann cells, siRNA-1, pGV230-Claudin-15 plasmid or negative control was used to transfect primary Schwann cells. The apoptotic rate of Schwann cells was lower in the siRNA-1 group than in the negative control group (P < 0.05; Figure 4A, B, and E). On the contrary, apoptotic rate was higher in the pGV230-Claudin-15 group than in the pGV230 group (P < 0.05; Figure 4C, D, and F). The results suggested that Claudin-15 promotes Schwann cells apoptosis.

Figure 4.

Figure 4

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Claudin-15 knockdown and overexpression affect Schwann cells apoptosis (Annexin V-FITC/PI Assay) in vitro.

(A–D) Apoptosis of Schwann cells in the NC group (A), siRNA-1 group (B), pGV230 group (C) and p-GV230-Claudin-15 group (D) was measured by Annexin V-FITC/PI assay. (E) The rate of apoptosis of Schwann cells in the siRNA-1 group compared with NC group. (F) The rate of apoptosis of Schwann cells in pGV230-Claudin-15 group compared with pGV230 group. *P < 0.05, vs. NC group; ##P < 0.01, vs. pGV230 group. Average number of proliferating cells in NC or pGV230 group was set as 100%. The cell proliferation rate of p-GV230-Claudin-15 group was obtained by dividing its number of proliferating cells with the average number of proliferating cells in the NC or pGV230 group. The results were exhibited as fold change. All experiments were performed in triplicate. Data are shown as the mean ± SEM (unpaired Student's t-test). FITC: Fluorescein isothiocyanate; NC: negative control; PI: propidium iodide.

Claudin-15 expression affects Schwann cells migration in vitro

To explore the effectiveness of Claudin-15 on Schwann cells in vitro further, we used the Transwell method to investigate the effects of Claudin-15 on Schwann cells migration. After transfection with siRNA, pGV230-Claudin-15 plasmid or negative control, the data showed that no significant changes were detected between the siRNA-1 and negative control groups (Figure 5A, B, and E); nor between the pGV230-Claudin-15 and pGV230 groups (Figure 5C, D, and F).

Figure 5.

Figure 5

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Claudin-15 knockdown and overexpression affect Schwann cells migration in vitro.

(A–D) Migrated Schwann cells in NC group (A), siRNA-1 group (B), pGV230 group (C) and pGV230-Claudin-15 group (D). Migrated Schwann cells were stained with crystal violet solution. Images are taken by an inverted light microscope. Scale bar: 50 μm. (E) Cell migration rate of Schwann cells in siRNA-1 group compared with NC group. (F) Cell migration rate of Schwann cells in pGV230-Claudin-15 group compared with pGV230 group. Assays were performed in triplicate. Data are shown as the mean ± SEM (unpaired Student’s t-test). NC: Negative control.

Silencing Claudin-15 downregulates c-Jun and AKT pathways in vitro

To identify the effect of Claudin-15 knockdown in Schwann cells, the signaling pathways and their phosphorylated (activated) ratios were investigated. The results showed that silencing Claudin-15 in cultured Schwann cells down-regulated c-Jun and AKT pathway expression (Figure 6A, B, and D). However, it had no effect on the ERK, β-catenin or c-Fos expression (Figure 6C, E, and F).

Figure 6.

Figure 6

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Silencing of Claudin-15 down-regulates AKT and c-Jun signaling pathways in vitro.

(A) Western blot assay of Claudin-15, p-AKT, AKT, p-ERK, ERK, c-Jun, p-c-Jun, β-catenin and c-Fos expression after siRNA-1 transfection. The top panels show target bands of Claudin-15, p-AKT, AKT, p-ERK, ERK, c-Jun, p-c-Jun, β-catenin and c-Fos. The bottom panel shows the loading control GAPDH. (B–D) Relative levels of p-AKT/AKT (B), p-ERK/ERK (C) and p-c-Jun/c-Jun (D) protein activation ratio after siRNA-1 transfected Schwann cells. (E, F) Relative levels of β-catenin (E) and c-Fos (F) protein expression of siRNA-1 transfected Schwann cells. *P < 0.05, vs. NC group. Average blot density of control group was set as 100%. The relative optical density value of p-GV230-Claudin-15 group was obtained by dividing its optical density with the optical density values of NC or pGV230 group. Assays were performed in triplicate. Data are shown as the mean ± SEM (unpaired Student’s t-test). ERK: Extracellular signal-regulated kinase; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; NC: negative control.

Claudin-15 overexpression upregulates c-Jun and c-Fos pathways in vitro

To identify the effect of Claudin-15 overexpression in Schwann cells, the signaling pathways and their phosphorylated (activated) ratios were investigated. The results showed that Claudin-15 overexpression up-regulated c-Jun and c-Fos expression in Schwann cells in vitro (Figure 7A, D, and F). However, there was no effect on AKT, ERK or β-catenin (Figure 7B, C, and E).

Figure 7.

Figure 7

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Overexpression of Claudin-15 activates c-Jun and c-Fos signaling pathways in vitro.

(A) Western blot assay of Claudin-15, p-AKT, AKT, p-ERK, ERK, c-Jun, p-c-Jun, β-catenin and c-Fos expression after pGV230-Claudin-15 transfection. The top panels show target bands of Claudin-15, p-AKT, AKT, p-ERK, ERK, c-Jun, p-c-Jun, β-catenin and c-Fos. The bottom panel shows the loading control GAPDH. (B–D) Relative levels of p-AKT/AKT (B), p-ERK/ERK (C) and p-c-Jun/c-Jun (D) protein activation ratio after pGV230-Claudin-15 transfected Schwann cells. (E, F) Relative levels of β-catenin (E) and c-Fos (F) protein expression of pGV230-Claudin-15 transfected Schwann cells. #P < 0.05, vs. pGV230 group. Average blot density of control group blot was set as 100%. The relative optical density value of p-GV230-Claudin-15 group was obtained by dividing its optical density with the optical density values of NC or pGV230 group. Assays were performed in triplicate. Data are shown as the mean ± SEM (unpaired Student’s t-test). ERK: Extracellular signal-regulated kinase; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; NC: negative control.

Discussion

In this study, we explored the effects of Claudin-15 on cell proliferation, apoptosis, migration ability and the signaling pathways of Schwann cells. The results suggested that Claudin-15 overexpression inhibited Schwann cell proliferation and promoted Schwann cell apoptosis, thereby activating PKCα, c-Jun, and Bax. Whereas silencing Claudin-15 reversed the above effects and provided neuroprotection for Schwann cells.

Schwann cells are the primary mediators of Wallerian degeneration after peripheral nerve injury. The proliferation, apoptosis and migration abilities of Schwann cells are crucial to Wallerian degeneration. Previously, we reported that Claudin-15 is up-regulated in Schwann cells during Wallerian degeneration (Li et al., 2013). Claudin-15 is mainly associated with the permeability and selectivity of cellular tight junctions (Tamura et al., 2011; Wada et al., 2013; Samanta et al., 2018). Other scientists found that Claudin-19 was not involved in the polarized morphogenesis of Schwann cells in peripheral myelinated axons (Miyamoto et al., 2005). We predicted Claudin-15 and Claudin-19 have similarities in their functions in Schwann cells.

Claudin-15 has been shown to play a role in cell migration (Takehara et al., 2009). β-Catenin, as part of the Wnt pathway, is also involved in cell migration (Yin et al., 2015). We used this to explore the effect of Claudin-15 on the migration of Schwann cells. However, our results did not show any association of Claudin-15 with the migration of Schwann cells. Moreover, there was no noticeable change in expression of β-catenin. Further experiments need to be carried out.

A few studies have shown that the Claudin family of proteins is associated with cell death and proliferation (Tsukita et al., 2008; Shimobaba et al., 2016). Our findings demonstrated that Claudin-15 induced Schwann cell apoptosis and inhibited their proliferating process. The quantitative polymerase chain reaction evidence showed that overexpression of Claudin-15 up-regulated PKCα and Bax and the activation ratio of p-c-Jun/c-Jun was up-regulated. PKCα is a serine threonine kinase upstream of the JNK pathway (Lampasso et al., 2002; Abou-Kandil et al, 2012; Jiang et al., 2017; Takami et al., 2018). Activating PKCα can induce apoptosis in many cell lines (Powell et al., 1996). The c-Jun and c-Fos participate in forming the transcription factor AP-1 downstream of the JNK pathway (Eferl and Wagner, 2003; Bi et al., 2018; Jin et al., 2018). Activation of c-Jun induced rapid cell death in cultured cells (Lei et al., 2002; Wang et al., 2017; Li et al., 2018). Bax is a key regulator of cell apoptosis (Campbell and Tait, 2018) and is essential for JNK-induced cell apoptosis (Lei et al., 2002; Weston and Davis, 2007). We predicted that Claudin-15 promotes Schwann cell apoptosis through the PKCα/JNK/Bax pathway.

AKT and ERK were key pro-survival factors inducing cell proliferation (Song et al., 2005; Huang et al., 2017). However, our study showed that the expression of activated AKT was down-regulated, whereas cell proliferation was upregulated by Claudin-15 knockdown. Furthermore, there was no statistical difference in ERK activation. Down-regulating PKCα may be responsible for the inactivation of AKT, since AKT can be activated by PKCα in conjunctival goblet cells (Li et al., 2013). We will continue to explore the pathways modifying cell proliferation of Schwann cells.

The main nerve regeneration regulators are bFGF, NT3 and Nf2 (Godinho et al., 2013; Truong et al., 2018; Wu et al., 2018). The up-regulation of bFGF and NT3 gene expression after Claudin15 knockdown showed that silencing Claudin-15 can be neuroprotective during Wallerian degeneration and/or nerve regeneration.

However, our results did not clarify how Claudin-15 affects Wallerian degeneration or nerve regeneration in vivo. Since Schwann cells have a decisive role in Wallerian degeneration and nerve regeneration, we predict that Claudin-15 may be a vital factor in Wallerian degeneration and nerve regeneration. Further studies of Claudin-15 functions in vivo may open new possibilities for providing insight into the mechanism of Claudins on nerve degeneration and/or regeneration.

Acknowledgments:

We greatly appreciate the editorial assistance of Te Zhang from China-Japan Union Hospital of Jilin University, China.

Footnotes

Conflicts of interest: The authors declare that they have no competing interests.

Financial support: This study was supported by the National Natural Science Foundation of China, No. 81671220 (to SSC); the Scientific and Technological Development Program of Jilin Province of China, No. 20160101077JC (to SSC); the School Joint Construction Special Project of Jilin Province of China, No. SXGJQY2017-13 (to SSC). The funding sources had no role in study conception and design, data analysis or interpretation, paper writing or deciding to submit this paper for publication.

Institutional review board statement: All experimental procedures and protocols were approved by the Experimental Animal Ethics Committee of Jilin University of China (approval No. 2016-nsfc001) on March 5, 2016. The experimental procedure followed the United States National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publication No. 85-23, revised 1996).

Copyright license agreement: The Copyright License Agreement has been signed by all authors before publication.

Data sharing statement: Datasets analyzed during the current study are available from the corresponding author on reasonable request.

Plagiarism check: Checked twice by iThenticate.

Peer review: Externally peer reviewed.

Funding: This study was supported by the National Natural Science Foundation of China, No. 81671220 (to SSC); the Scientific and Technological Development Program of Jilin Province of China, No. 20160101077JC (to SSC); the School Joint Construction Special Project of Jilin Province of China, No. SXGJQY2017-13 (to SSC).

C-Editor: Zhao M; S-Editors: Wang J, Li CH; L-Editors: Dawes EA, Haase R, Qiu Y, Song LP; T-Editor: Jia Y

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