SLC26A4 Mutation Promotes Cell Apoptosis by Inducing Pendrin Transfer, Reducing Cl− Transport, and Inhibiting PI3K/Akt/mTOR Pathway

Objective Pendrin is encoded by SLC26A4, which is expressed in the apical membrane of inner ear epithelial cells and drives chloride reabsorption in the apical septum. In the inner ear, pendrin dysfunction and hypofunctional mutations lead to vestibular aqueduct (EVA) enlargement and sensory neural hearing loss. Mutations in SLC26A4 are a common reason of deafness. However, the underlying mechanisms of SLC26A4 mutants in hearing loss remain unknown. Methods In the present study, pEGFP-N1 carrying wild-type and mutant SLC26A4 (c.85G>A, c.2006A>T, and c.853G>A) were transfected into HEK-293T cells. GFP fluorescence and GFP levels were determined. SLC26A4 mRNA levels were examined by quantitative real-time polymerase chain reaction (qRT-PCR). Then, the expression of chloride intracellular channel 1 (CLIC1) and CLIC2 was measured by Immunofluorescence assay. Intracellular chloride concentration and apoptotic rate were analyzed by flow cytometry. The levels of membrane/cytoplasmic pendrin, apoptosis-associated proteins, and the PI3K/Akt/mTOR pathway members were determined by Western blot. Results Constructed SLC26A4 mutant 1 (c.85G>A), SLC26A4 mutant 2 (c.2006A>T), and SLC26A4 mutant 3 (c.853G>A). The wild-type and 3 mutations were stably expressed in HEK-293T. SLC26A4 mRNA expression was significantly increased after transfection with wild-type SLC26A4 and mutant SLC26A4 compared with the untransfected vector group (P < 0.01). Compared with the vector group, the expression levels of membrane pendrin, cytoplasmic pendrin, CLIC1, CLIC2, Bcl-2, p-PI3K, p-Akt, and p-mTOR were upregulated. Compared with the vector group, the chloride concentration, cell apoptotic rate, and the expression levels of caspase-3, caspase-9, and Bax were downregulated. Compared with the vector group, the above effects of SLC26A4 were reversed after the SLC26A4 mutant. Conclusion After SLC26A4 mutation, pendrin was transferred from the membrane, the chloride intracellular channel function was reduced, and the excessive accumulation of chloride in the cytoplasm induced cell apoptosis by inhibited PI3K/Akt/mTOR pathway phosphorylation.


Introduction
Hearing loss is a serious public health issue, affecting nearly 360 million people worldwide according to the report of World Health Organization (WHO) in 2012 [1]. Hearing loss can affect people of all ages. Many risk factors are associated with hearing loss, including genetic mutations, age, noise exposure, ototoxic medication exposure, and infections [2]. More than 120 genes participate in hearing loss, including SLC26A4 gene [3]. SLC26A4 gene, also known as PDS gene, encodes a multifunctional anion exchanger pendrin, a protein with 780 amino acids [4]. Pendrin is expressed in several tissues, including the thyroid and kidney, as well as epithelial cells of the inner ear [5,6]. Mutations in SLC26A4 are the second most frequent cause of hereditary hearing loss in human, after GJB2 gene mutations [7]. It has been reported that mutations in SLC26A4 are responsible for both Pendred syndrome and non-syndromic hearing loss with enlarged vestibular aqueduct [8]. To date, more than 539 mutations in SLC26A4 gene have been identified [9]. However, the possible molecular mechanism of SLC26A4 mutation in hearing loss has not yet been fully elucidated.
The phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of the rapamycin (mTOR) signaling pathway is activated in human cancers, which participates in many cellular processes, such as cell survival, metastasis, autophagy, metabolism, and angiogenesis [10]. Despite the correlation with human cancers, the PI3K/Akt/mTOR pathway is also responsible for the pathogenesis of many other human diseases, such as osteoarthritis, stroke, asthma, and traumatic brain injury [11][12][13][14]. Recently, Li et al. have reported that activation of the Akt/mTOR signaling pathway is associated with cochlear hair cell regeneration from supporting cells [15]. However, it is still not clear whether mutations in SLC26A4 lead to hearing loss via regulation of the PI3K/Akt/mTOR pathway.

BioMed Research International
Wild-type   2.9. Statistical Analysis. Data were expressed as mean ± standard deviation (SD). Statistical analysis was conducted using ANOVA followed by Tukey's test. P < 0:05 was defined as statistically significant. All experiments were repeated independently three times.

Construction of Recombinant Plasmids Carrying Wild-Type and Mutant SLC26A4.
To investigate the effects of these mutants on the function of SLC26A4 gene and possible mechanism of these mutants in hearing loss, SLC26A4 mutants were inserted into pEGFP-N1 to generate mutant 1 (c.85G>A), mutant 2 (c.2006A>T), and mutant 3 (c.853G>A). Meanwhile, wild-type SLC26A4 was inserted into pEGFP-N1 to serve as the corresponding control. DNA sequencing analysis revealed that the G>A substitution at nucleotide 85 of SLC26A4 resulted in amino acid change at codon 29 (p.E29K) (Figure 1(a)  The results revealed that HEK-293T cells transfected with empty vector pEGFP-N1, wild-type SLC26A4, mutant 1, mutant 2, or mutant 3 exhibited more obvious GFP fluorescence (Figure 1(d)). In addition, after transfected plasmids 24 h and 48 h, compared with the control group, the protein levels of GFP have significant increase in vector group, wildtype group, mutant 1 group, mutant 2 group, and mutant 3 group (P < 0:01) (Figure 1(e)). Interestingly, compared with the vector group, the SLC26A4 mRNA expression has significant increase in wild-type group, mutant 1 group, mutant 2 group, and mutant 3 group (P < 0:01), but in mutant 1 group and mutant 3 group, the SLC26A4 mRNA expression was lower than wild-type group and mutant 2 group (Figure 1(f)). These results indicated that the plasmids of SLC26A4 (wild-type), SLC26A4 (E29K mutant), SLC26A4 (D669V mutant), and SLC26A4 (V285I mutant) were successfully transfected in HEK-293T cells.

The Effects of SLC26A4 Mutations in Cell's Pendrin and
Chloride. After transfected with wild-type SLC26A4, com-pared with the vector group, the protein levels of membrane pendrin and cytoplasm pendrin have significant increase in wild-type group (P < 0:01). SLC26A4 mutations led to significant reduction in membrane pendrin, while cytoplasmic levels of pendrin significantly increased as compared to those of the wild-type group (P < 0:01) (Figure 2(a)). Compared with the control group, chloride concentration has significant decrease in wild-type group (P < 0:01) (Figure 2(b)). Compared with the wild-type group, chloride concentration has significant increase in mutant 1 group, mutant 2 group, and mutant 3 group (P < 0:01) (Figure 2(b)). Furthermore, we found that transfection with wild-type SLC26A4 greatly upregulated CLIC1 and CLIC2 levels compared to the vector. However, as compared to the wild-type, SLC26A4 mutants decreased both CLIC1 and CLIC2 expression ( Figure 3). These results indicated that SLC26A4 mutations induce the transfer of pendrin from cell membrane to cytoplasmic, decrease chloride channel proteins, and increase chloride concentration.

The Effects of SLC26A4 Mutations in Cell
Apoptosis and PI3K/Akt/mTOR Pathway. In wild-type group, cell apoptosis rate lower than vector group, compared with the wild-type group, cell apoptosis rate has significant increase in mutant 1 group, mutant 2 group, and mutant 3 group (P < 0:01) (Figure 4(a)). Meanwhile, compared with the vector group, the protein levels of caspase-3, caspase-9, and Bax were greatly downregulated, while Bcl-2 levels were upregulated in wildtype group (P < 0:01); compared with the wild-type group, the protein levels of caspase-3, caspase-9, and Bax were greatly upregulated, while Bcl-2 levels were downregulated in mutant   (Figure 4(b)). The results indicated that SLC26A4 decreases cell apoptosis, and SLC26A4 mutations induce cell apoptosis. And finally, we found that p-PI3K/PI3K, p-Akt/Akt, and p-mTOR/mTOR ratios were significantly higher in wild-type group, compared with the vector group (P < 0:01). However, compared with the vector group, the ratios of p-PI3K/PI3K, p-Akt/Akt, and p-mTOR/mTOR were decreased in mutant 1 group, mutant 2 group, and mutant 3 group (P < 0:01) ( Figure 5). This means that SLC26A4 mutations can be suppressed the PI3K/Akt/mTOR pathway.

Discussion
Our previous study has reported two novel mutations (c.85G>A and c.853G>A) and one reported mutation (c.2006A>T) in SLC26A4 in children with non-syndromic hearing loss compared to normal controls (data not shown).  The levels of caspase-3, caspase-9, Bax, and Bcl-2 were determined by Western blot. GAPDH served as an internal control. # P < 0:05, ## P < 0:01 compared to the vector group. * * P < 0:01 compared to the wild-type group. 6 BioMed Research International However, whether these mutations in SLC26A4 are related to hearing loss remains unknown. In the present study, we intended to investigate the relationship between SLC26A4 mutations and hearing loss and to further clarify the possible underlying mechanism.
To investigate the possible role of these three mutations in hearing loss, SLC26A4 mutants were made by a commercial kit and then inserted into pEGFP-N1 vector. These plasmids were subjected to DNA sequencing. In this study, DNA sequencing analysis suggests that we successfully obtained recombinant plasmids carrying wild-type SLC26A4, mutant 1 (c.85G>A), mutant 2 (c.2006A>T), and mutant 3 (c.853G>A). pEGFP-N1, a eukaryotic expression vector encoding GFP, is commonly used to detect the expression of inserted genes via examination of GFP by fluorescence microscopy and Western blot [16]. After transfection of HEK-293T cells with these plasmids, we found that HEK-293T cells transfected not only with empty vector but also with wild-type SLC26A4 and mutants exhibited obvious GFP fluorescence and upregulated GFP expression compared to control cells, indicating successful transfection of these plasmids. Then, qRT-PCR was performed to verify SLC26A4 expression in HEK-293T cells. Transfection with wild-type and mutant SLC26A4 significantly upregulated SLC26A4 levels compared to the vector. These results demonstrated that wild-type SLC26A4 and all mutants were successfully transfected into HEK-293T cells and overexpressed SLC26A4 in the cells.
Pendrin, an anion exchanger that mediates the transportation of chloride, iodide, bicarbonate, and formate, is localized at the plasma membrane [17]. Generally, transmembrane proteins synthesized by ribosomes translocate from the ER to the Golgi apparatus via transport vesicles and then anchor to the plasma membrane [18,19]. Then, membrane and cytoplasmic pendrin levels were determined. We found that transfection with wild-type SLC26A4 significantly increased pendrin levels in both plasma membrane and cytoplasm compared to vector-transfected cells. Mutations in SLC26A4 abolished the recruitment of pendrin mutants to the plasma membrane in HEK-293T cells. Similar findings are reported by previous studies [20]. In non-syndromic hearing loss patients, p.L445W and p.M147T muta-tions in SLC26A4,followed by cell experiments, proved that the mutation prevented pendrin from targeting to the plasma membrane [21]. This may be a crucial mechanism of Pendred syndrome [20,22]. All the three SLC26A4 mutants may inhibit localization of Pendrin to the plasma membrane and they may be degraded via the ERAD pathway. Further studies are required to verify our hypothesis.
Pendrin, a chloride/bicarbonate exchanger with higher affinity for chloride, bicarbonate, and iodide, participates in regulation of chloride reabsorption and bicarbonate secretion [23]. To investigate whether SLC26A4 mutants affect chloride reabsorption, we examined the concentration of intracellular chloride with MQAE. The results showed that intracellular chloride concentrations were significantly elevated by wild-type SLC26A4, while reduced by SLC26A4 mutants in HEK-293T cells, which is consistent with previous study [24]. CLIC1 and CLIC2, two members of chloride intracellular channel family, are evolutionary conserved proteins and possess chloride channel activity [25,26]. However, no study has reported the relationship between SLC26A4 mutation and CLIC proteins. Maybe CLIC1 and CLIC2 are downstream regulators of SLC26A4. In our study, CLIC1 and CLIC2 expression were examined by Immunofluorescence assay. We found that wild-type SLC26A4 increased both CLIC1 and CLIC2 expression, while mutations in SLC26A4 decreased in their levels in HEK-293T cells. These results suggest that SLC26A4 mutants may directly reduce intracellular chloride concentration or indirectly regulate this via CLIC1 and CLIC2.
Apoptosis, a form of programmed cell death that is critical for tissue homeostasis, is regulated by extrinsic and intrinsic (mitochondrial/ER) pathways [27]. It has been reported that apoptosis of cochlear hair cells is associated with hearing loss [28]. Tang et al. have revealed that highly expressed SLC26A4 is associated with inhibition of cardiomyocyte apoptosis [29]. However, whether mutations in SLC26A4 result in hearing loss through regulation of cell apoptosis remains unknown. Interestingly, apoptosis was inhibited by wild-type SLC26A4. We also found that SLC26A4 mutants significantly promoted cell apoptosis compared to the wild-type, indicating that mutations in  Figure 5: Mutations in SLC26A4 suppress the PI3K/Akt/mTOR signaling pathway. The phosphorylated and total levels of PI3K, Akt, and mTOR were examined by Western blot. The ratios of p-PI3K/PI3K, p-Akt/Akt, and p-mTOR/mTOR were calculated. GAPDH served as an internal control. ## P < 0:01 compared to the vector group. * * P < 0:01 compared to the wild-type group.
SLC26A4 may be associated with cell apoptosis. Upon apoptotic stress, Bax and Bak are oligomerized in the mitochondrial outer membrane, resulting in cytochrome c release and subsequent activation of caspase-9/-3. Bax/Bak oligomerization during apoptosis can be suppressed by anti-apoptotic protein Bcl-2 [30]. As expected, wild-type SLC26A4 decreased caspase-3, caspase-9, and Bax levels, whereas increased Bcl-2 levels in HEK-293T cells. SLC26A4 mutants markedly increased pro-apoptotic protein levels whereas decreased anti-apoptotic protein levels. These results suggest that regulation of cell apoptosis may be an important mechanism of SLC26A4 mutants in hearing loss. The PI3K/Akt/mTOR signaling pathway plays an important role in cell proliferation, metabolism, and metastasis [31]. Moreover, activation of the Akt/mTOR pathway contributes to cochlear hair cell regeneration [15]. However, whether SLC26A4 mutants result in hearing loss via the PI3K/Akt/mTOR pathway remains unknown. In our study, we found that wild-type SLC26A4 significantly activated, while SLC26A4 mutants inhibited the PI3K/Akt/mTOR pathway in HEK-293T cells, suggesting that this pathway may be another important mechanism of SLC26A4 mutants in hearing loss. Mounting evidences have revealed that GSK-3β inhibition attenuates cochlear destruction and hearing loss in vivo [32,33]. Moreover, SLC26A4 can regulate GSK-3β expression in H9c2 cells [29]. Maybe the GSK-3β signaling pathway is involved in hearing loss induced by SLC26A4 mutants and this hypothesis needs to be verified.
In conclusion, wild-type SLC26A4 increased membrane and cytoplasmic pendrin, intracellular chloride concentration, and CLIC1 and CLIC2 expression. Cell apoptosis was inhibited and the PI3K/Akt/mTOR signaling pathway was activated by wild-type SLC26A4. However, SLC26A4 mutants abolished membrane targeting, reduced intracellular chloride concentration, decreased CLIC1 and CLIC2 expression, enhanced cell apoptosis, and suppressed the PI3K/Akt/ mTOR signaling pathway. The present study elucidated the possible mechanism and provides novel therapeutic strategy for hearing loss due to mutations in SLC26A4.

Data Availability
The data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
There are no conflicts of interest.