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p70S6K signaling pathway
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Silencing of KPNA2 inhibits high glucose-induced podocyte injury via inactivation of mTORC1/p70S6K signaling pathway Xiaobao Fan a, b, Zhenjiang Li b, Xiaoming Wang b, Jing Wang b, Zhiming Hao a, * a
Department of Rheumatology and Immunology, The First Affiliated Hospital of Medical College of Xi ‘an Jiaotong University, Xi’an City, Shaanxi Province, 710061, China b Nephrotic Hemodialysis Center, Shaanxi Provincial People’s Hospital, Xi’an City, Shaanxi Province, 710068, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 17 October 2019 Accepted 31 October 2019 Available online xxx
Dysregulation of apoptotic and autophagic function are characterized as the main pathogeneses of diabetic nephropathy (DN). It has been reported that Karyopherin Alpha 2 (KPNA2) contributes to apoptosis and autophagy in various cells, but its role in DN development remains unknown. The purpose of present study was to explore the function and underling mechanisms of KPNA2 in development of DN. In this study, 30 mM high glucose (HG)-evoked podocytes were used as DN model. The expression of KPNA2 was detected by qRT-PCR and Western blot assays. The cell viability was tested by CCK-8 kit, the apoptosis was measured using flow cytometry assay, the apoptotic and the autophagy related genes was detected by Western blot. Our results indicated that KPNA2 was significantly increased after HG stimulation. Knockdown of KPNA2 inhibited apoptosis, and promoted cell viability and autophagy in HGtreated podocytes. In addition, silencing of KPNA2 deactivated mTORC1/p70S6K pathway activation via regulating SLC1A5. Further results demonstrated that activating mTORC1/p70S6K pathway strongly ameliorated the effect of KPNA2 on cell viability, apoptosis and autophagy. Therefore, our study suggested that knockdown of KPNA2 rescued HG-induced injury via blocking activation of mTORC1/p70S6K pathway by mediating SLC1A5. © 2019 Published by Elsevier Inc.
Keywords: Diabetic nephropathy Podocyte KPNA2 SLC1A5 mTORC1/p70S6K pathway
1. Introduction Diabetic nephropathy (DN) is a major microangiopathy of diabetes mellitus and one of the important inducements leading to end stage of renal disease (ESRD) [1,2]. The prevalence of DN in Chinese is increasing rapidly, which is second only to diabetic cardiovascular disease [3]. With the occurrence and development of DN, the renal function of diabetic patients will gradually deteriorate, which will bring great economic burden to the society and individual patients [4]. The pathogenesis of DN is extremely complex, and it is well accepted that DN is mainly associated with metabolic disorders, hemodynamic disorders, oxidative stress injury, inflammation and genetic factors [5,6]. Recently, large number of studies demonstrate that DN pathogenesis is closely related to inhibition of autophagy in renal cells [7,8].
* Corresponding author. Department of Rheumatology and Immunology, The First Affiliated Hospital of Medical College of Xi ‘an Jiaotong University, No 277 yanta west road, Xi’an City, Shaanxi Province, 710061, China. E-mail address: [email protected] (Z. Hao).
Autophagy, which is self-phagocytosis, is a unique life phenomenon of eukaryotic cells and has important physiological significance for maintaining intracellular homeostasis [7]. In the state of diabetes mellitus, the autophagy function in renal cells is suppressed by a negative regulatory molecule, mammalian target of rapamycin (mTOR) [8,9]. Subsequently, the damaged organelles and cytotoxic substances are failing to be removed timely and effectively, but accumulated in the cells, which affects the normal function of cells to accelerate cell damage and apoptosis [10]. What is more, the activation of mTOR pathway by HG environment also promotes the expression of Atg5 (autophagy-related gene 5), which can cause mitochondria-targeted translocation, release cytochrome C and activate caspase family proteins, thereby promoting cell apoptosis [11,12]. Therefore, deactivation of mTOR pathway is an effective way to alleviate the pathogenesis of DN [13,14]. AATs are transmembrane proteins that participate in a series of cellular processes by regulating the transport of amino acids in cells or organelles [15]. Solute-linked carrier family A1 member 5 (SLC1A5/ASCT2) is a cell surface Naþ-dependent neutral AAT [16]. Loss of SLC1A5 results in inhibition of glutamine uptake, thereby reducing mTORC1 activity in Myc ± mice [17]. Nicklin et al. also
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Please cite this article as: X. Fan et al., Silencing of KPNA2 inhibits high glucose-induced podocyte injury via inactivation of mTORC1/p70S6K signaling pathway, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.10.200
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revealed that knockdown of SLC1A5 attenuates inhibition of mTORC1 on cellular autophagy by down-regulating L-glutamine (LG) uptake [18]. A previous study indicates that SLC1A5 is hypomethylated in the blood of diabetic patients, which suspected that SLC1A5 might play a role in the development of diabetes [19]. In addition, SLC1A5-mediated mTORC1 signaling pathway has been proved as a downstream target of various transcription factors in multiple diseases, such as TEAD1 (TEA Domain Transcription Factor 1) in vascular injury, PDZ domain of sorting nexin 27 (SNX27) in cancer [20e22]. It has been reported that SLC1A5 is hypomethylated in diabetic patients via RNA-seq analysis [23]. Karyopherin family is a class of protein that plays an important role in protein transport between cytoplasm and nucleus. Karyopherin a2 (KPNA2) is a member of the karyopherin family, which is composed of 529 amino acids [24]. Given its function in nucleocytoplasmic transport, KPNA2 mediates the translocation of various proteins and is involved in numerous cellular processes, such as cellular differentiation, proliferation and apoptosis, transcriptional regulation, immune response, and viral infection [25,26]. For example, knockdown of KPNA2 inhibits cell proliferation and promoted apoptosis in bladder cancer via interacting with OCT4 nuclear transportation, KPNA2 contributes to apoptosis in human tongue squamous cell carcinoma via activating Bax and caspase-3 and down-regulating Bcl-2 [27,28]. Notably, recent studies have reported that overexpression of KPNA2 promotes cellular autophagy in human oral squamous cell carcinoma, glioblastoma, adipose-derived stem cells, and so on [29e31]. However, the role of KPNA2 in autophagic function of DN pathogenesis is still unclear. This study aims to verified the effect of KPNA2 and the relationship between KPNA2, SLC1A5 and mTORC1/p70S6K signaling in the course of DN. The results showed that expression of KPNA2 was significantly increased in high glucose-induced podocytes. Further, we found that downregulation of KPNA2 facilitated apoptosis and autophagy through inactivating mTORC1/p70S6K signaling pathway by mediating SLC1A5. This finding provides an understanding of KPNA2 in DN, and might serve a therapeutic target for DN patients. 2. Materials & methods 2.1. Podocytes culture and treatment The human podocytes were obtained from Celprogen Inc. (Torrance, USA), and were cultured in RMPI 1640 medium (Gibco; Thermo Fisher Scientific, Inc., USA) supplemented with 10% fetal bovine serum (FBS; Gibco), in a 5% CO2, 33 C humidified incubator. After maintained in normal glucose medium (NG, 5 mM) for 7 days, podocytes were cultured with NG, or medium added mannitol (MA, 30 mM) or high glucose (HG, 30 mM). 2.2. Transfection The small interfering RNAs of KPNA2 (KPNA2 siRNA1, KPNA2 siRNA2) and overexpression plasmid of SLC1A5 (pcDNA- SLC1A5) was constructed by the manufacturer’s instructions (Generay Biotech, Shanghai, China). Treated or non-treated podocytes were transfected by Lipofectamine 3000 Kit (Invitrogen, Carlsbad, CA) for 48 h. 2.3. Cell viability assessment Podocytes were seeded in 96-well plate with density of 2 104 cells per well, then subsequently maintained in NG, MA or HG medium for 24 h. Thereafter, 10 ml Cell Counting Kit-8 (CCK-8,
Dojindo Molecular Technologies, Inc.) solution was added to each well and incubated at 37 C for 2 h, and OD values were measured at 450 nm. 2.4. Apoptosis assessment The HG treated podocytes were harvested and resuspended in binding buffer, and then incubated with 0.25 mg/ml Annexin VFITC (Dojindo, Japan) and 10 mg/ml propidium iodide (PI) (Dojindo, Japan) at 25 C in the dark for 30 min. Finally, apoptosis evaluated by a flow cytometry (BD Biosciences, USA). 2.5. Quantitative real-time PCR (qRT-PCR) Total RNA of podocytes was extracted using Trizol reagent (Invitrogen, USA) referring to the manufacturer’s protocol, and then reverse transcribed complementary DNA by the PrimeScriptVR RT reagent kit (Takara Bio, Inc., Tokyo, Japan). The cDNAs were quantified through DyNamo SYBR1 Green qPCR Kit (Takara), and b-actin was used as the internal control. The relative expression of KPNA2 was evaluated by the 2DD CT. The primer of KPNA2 is Forward: 50 ATTGCAGGTGATGGCTCAGT-3’; Reverse: 50 -CTGCTCAACAGCATCTAT CG-3’. 2.6. Western blot assay Podocytes were harvested and lysed Radio Immunoprecipitation Assay (RIPA) buffer (Thermo Scientific) with a protease inhibitor cocktail (Millipore, MA, USA). The protein extraction resolved by sodium dodecyl sulphate polyacrylamide gel electrophoresis, and subsequently transferred onto a PVDF membrane (Millipore). After blocked by 5% nonfat milk for 1 h at room temperature, the membrane was incubated with primary antibody at 4 C overnight. The primary antibodies including KPNA2 (1:1000, Abcam, ab84440); SLC1A5 (1:1000, Abcam, ab237704), Bcl-2 (1:1000, Abcam, ab32370), pro-caspase3 (1:1000, Abcam, ab13847), cleaved caspase3 (1:1000, Abcam, ab49822), Beclin 1 (1:1000, Abcam, ab207612), LC3 II (1:1000, Abcam, ab51520), p62 (1:1000, Abcam, ab56416), p-mTORC1 (1:1000, Cell Signaling Technology, Inc, #2983), p-p70S6K (1:1000, Abcam, ab59208) and b-actin (1:1000, Abcam, ab179467). Then, the membrane was treated with secondary antibody conjugated to horseradish peroxidase for 2 h at room temperature. 2.7. Statistical analysis All experiments were repeated in triplicates. Statistical analysis was performed by SPSS 22.0 software (SPSS Inc., USA). The data are all indicated as the mean ± SD. The result value of *p < 0.05, # p < 0.05, or &p < 0.05 was considered statistically significant. 3. Results 3.1. The expression of KPNA2 was up-regulated in HG-induced podocytes Firstly, the result of CCK-8 assay indicated that high glucose (HG, 30 mM) significantly reduced the cell viability of human podocytes, compared with normal glucose (NG, 5 mM) and mannitol (MA, 30 mM) treatment (Fig. 1A, p < 0.05). Then, the results of qRT-PCR and Western blot assays indicated that HG remarkably increased KPNA2 expression in podocytes (Fig. 1BeC, p < 0.05). Therefore, KPNA2 might play roles in the HG-evoked induced podocytes.
Please cite this article as: X. Fan et al., Silencing of KPNA2 inhibits high glucose-induced podocyte injury via inactivation of mTORC1/p70S6K signaling pathway, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.10.200
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Fig. 1. The expression of KPNA2 was up-regulated in HG-induced podocytes (A) Human podocytes were treated with normal glucose (NG, 5 mM), mannitol (MA, 30 mM) or high glucose (HG, 30 mM) for 3 days. The cell viability was detected using CCK-8 assay assessment. Normal glucose treatment was performed as control, mannitol treatment was performed as negative control. (B, C) After NG, MA, and HG treatment for 24 h, the mRNA and protein expression of KPNA2 in podocytes were measured using qRT-PCR and Western blot assays, respectively. *p < 0.05 vs NG or MA.
3.2. KPNA2 siRNA inhibited apoptosis and promoted autophagy function Next, we investigated the effect of KPNA2 on HG-induced apoptosis. Two small interfering RNAs against KPNA2 (KPNA2
siRNA1 and KPNA2 siRNA2) were transfected into podocytes. After HG treatment, the expression of KPNA2 were significantly enhanced by KPNA2 siRNAs, and transfection efficiency of siRNA1 was more significant than siRNA2 (Fig. 2A, p < 0.05). The results of CCK-8 and Annexin V-FITC/PI assays indicated that KPNA2 siRNAs
Fig. 2. KPNA2 siRNA inhibited apoptosis and promoted autophagy function (A, B) Podocytes were transfected with constructed KPNA2 small interfering RNAs (KPNA2 siRNA1 and siRNA2) or its negative control (si-NC) for 48 h before HG treatment. Then, qRT-PCR was used to assess the mRNA expression of KPNA2. (B, C) The cell viability and apoptosis of transfected podocytes were tested by CCK-8 assay and Annexin V-FITC/PI assay, respectively. (DeF) The Western blot analysis of protein expression of apoptosis related genes Bcl-2, Pro/Cleaved-Caspase-3. Bcl-2: B-cell lymphoma-2. (D, G-I) The Western blot and quantification analysis of protein expression of autophagy related genes, including Beclin 1, LC3 II/LC3 I, and p62*p < 0.05 vs control or NC, #p < 0.05 vs KPNA2 siRNA1.
Please cite this article as: X. Fan et al., Silencing of KPNA2 inhibits high glucose-induced podocyte injury via inactivation of mTORC1/p70S6K signaling pathway, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.10.200
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conspicuously promoted the cell viability and reduced apoptosis rate in HG-induced podocytes. What is more, the effect of siRNA1 on cell viability and apoptotic function more significant than siRNA2 (Fig. 2B and C, p < 0.05). Therefore, KPNA2 siRNA1 was chose in the following experiments. As shown in Fig. 2DeF, KPNA2 siRNA1 dramatically increased the protein expression of Bcl-2, and decreased the protein expression of Pro/Cleaved-Caspase-3 (p < 0.05). In addition, KPNA2 siRNA1 significantly promoted Beclin 1 and LC3 II/LC3 I expression, and inhibited p62 expression (Fig 2D and G-I, p < 0.05). Thus, our data indicated that silencing of KPNA2 could inhibit apoptosis and rescue autophagic function in HG-injured podocytes.
3.3. KPNA2 siRNA deactivated mTORC1/p70S6K pathway in podocytes via regulating SLC1A5 A recent study indicated that SLC1A5 was a potential target of KPNA2 [32], and it is well known that the activation of mTORC1/ p70S6K signaling pathway is dependent on SLC1A5, therefore, we speculated that KPNA2 might regulate mTORC1/p70S6K signaling pathway through SLC1A5. To verified our speculation, HG-induced podocytes were transfected with KPNA2 siRNA1 and SLC1A5 overexpression vector (pcDNA-SLC1A5). The result of Western blot showed that KPNA2 siRNA strongly decreased the expression of SLC1A5, p-mTORC1 and p-p70S6K, but pcDNA-SLC1A5 attenuated this decrease (Fig. 3AeD, p < 0.05). These data suggested that KPNA2 siRNA blocked the activation of mTORC1/p70S6K pathway in podocytes via suppressing SLC1A5 expression.
3.4. Activation of mTORC1/p70S6K pathway ameliorated the inhibition of KPNA2 siRNA on HG-induced apoptosis and autophagy To explore the role of mTORC1/p70S6K pathway in the regulation of KPNA2 on HG-induced injury, pcDNA-SLC1A5 and mTORC1/ p70S6K pathway activator (MHY1485) were used to treat HGinduced podocytes after transfected with KPNA2 siRNA. Our data showed that pcDNA-SLC1A5 and MHY1485 significantly counteracted the effect of KPNA2 siRNA on cell viability and apoptosis in HG-induced podocytes (Fig. 4A and B, p < 0.05). Besides, the results of autophagy related genes expression indicated that pcDNASLC1A5 and MHY1485 remarkably suppressed the increase of KPNA2 siRNA on Beclin 1 and LC3 II/LC3 I, and the decrease of KPNA2 siRNA on p62 in HG-induced podocytes (Fig. 4CeF, p < 0.05). Thus, our data revealed that knockdown of KPNA2 inhibited HGinduced injury though the deactivation of mTORC1 pathway by mediating SLC1A5. 4. Discussion Podocyte, the epithelial cell of the glomerulus, is one of the important components of the glomerular filtration barrier. Numerous studies have demonstrated that high glucose (HG)induced podocyte injury is a major cause in the progression of chronic renal nephropathy, which is an important target for clinical treatment. Consistent with the discoveries in previous studies, our data showed that HG stimulation reduced cell viability in podocytes. In HG-induced podocytes, we found that KPNA2 expression was significantly elevated, and knock downing KPNA2 promoted
Fig. 3. KPNA2 siRNA deactivated mTORC1/p70S6K pathway in podocytes via regulating SLC1A5 (AeD) Podocytes were transfected with KPNA2 siRNA1 and/or SLC1A5 overexpression vector (pcDNA-SLC1A5). The Western blot analysis of SLC1A5, p-mTORC1 and p-p70S6K protein expression. Non-treated podocytes as control*p < 0.05 vs control, # p < 0.05 vs KPNA2 siRNA1, &p < 0.05 vs pcDNA-SLC1A5.
Please cite this article as: X. Fan et al., Silencing of KPNA2 inhibits high glucose-induced podocyte injury via inactivation of mTORC1/p70S6K signaling pathway, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.10.200
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Fig. 4. Activation of mTORC1/p70S6K pathway ameliorated the inhibition of KPNA2 siRNA on HG-induced apoptosis and autophagyPodocytes were divided into 4 groups, HG stimulation group (as control), HG and KPNA2 siRNA1 treatment group, HG and KPNA2 siRNA1 and pcDNA-SLC1A5 treatment group, HG and KPNA2 siRNA1 and mTORC1/p70S6K pathway activator (MHY1485) treatment group. (A, B) The cell viability and apoptosis of each group of podocytes. (CeF) The Western blot analysis of Beclin 1, LC3 I, LC3 II, and p62 protein expression in each group of podocytes.*p < 0.05 vs control, #p < 0.05 vs HG þ KPNA2 siRNA group.
cell viability and autophagic function while inhibited cell apoptosis. Further results indicated that suppression of KPNA2 plays a protective role against HG-induced podocyte apoptosis and autophagic function arrest through mTORC1/p70S6K pathway by regulating SLC1A5 expression. Emerging evidences showed that aberrant expression of KPNA2 is frequently observed in a variety of cancerous patients. Recent studies have showed that up-regulation of KPNA2 aggravates the development of esophageal squamous cell carcinoma and epithelial ovarian carcinoma by promoting cell proliferation, glioblastoma and oral squamous cell carcinoma by enhancing cellular autophagy [30,31,33,34]. In addition, Gao et al. revealed that KPNA2 induces
apoptosis of tongue squamous cell carcinoma through downregulating Bcl-2 expression and activating the caspase-dependent pathway [28]. Growing studies suggest that KPNA2 functions its specific role in distinct cells by involving in cellular growth and autophagy. In the present study, downregulating KPNA2 enhanced cell viability and autophagy, and repressing cell apoptosis in podocyte after HG stimulation. What is more, KPNA2 expression was found elicits various effects in different diseases by regulating distinct genes or signaling pathways. For instance, the involvement of KPNA2 in cancer cell proliferation by regulating FGF1 signaling pathway, KPNA2 contributes to epithelial ovarian cancer cell proliferation via up-regulating c-Myc [33,35]. This study demonstrated
Please cite this article as: X. Fan et al., Silencing of KPNA2 inhibits high glucose-induced podocyte injury via inactivation of mTORC1/p70S6K signaling pathway, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.10.200
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that silencing KPNA2 down-regulated SLC1A5 expression, which seems to contradict with data form a previous study that overexpression of KPNA2 inhibits SLC1A5 expression in glioblastomas. It has been proved that KPNA2 enhances c-Myc translation by directly binding with it, and c-Myc is a direct activator of SLC1A5 translation [32,33,36,37]. Therefore, we speculated that knockdown of KPNA2 repressing SLC1A5 expression through c-Myc axis. mTORC1 is one of the functional protein complexes of serine/ threonine kinase mTOR, which is activated via glutamine transported by SLC1A5 [38]. mTORC1 functions important regulatory role in cell growth, proliferation and protein synthesis via phosphorylating its two downstream effectors, p70S6K and 4EBP1 [39]. Numerous evidences have proved that the activation of mTORC1/ p70S6K pathway plays an important regulatory role in the growth and apoptosis of podocytes [39,40]. In addition, mTORC1/p70S6K axis achieves the effect of inhibiting autophagy of podocytes by suppressing the activity of the promoter of the autophagosome [41]. The previous studies proved that inhibition of mTORC1/ p70S6K signaling pathway can ameliorate the down-regulated autophagy in DN renal cells, and mTORC1/p70S6K signaling pathway activator MHY1485 can aggravate DN development by repressing autophagy, which suggests mTORC1/p70S6K pathway can serve as a target to inhibit the DN development [14,42]. Here, we also report that overexpressed KPNA2 ameliorated HG-induced autophagy via inactivation of mTORC1/p70S6K signaling pathway in podocytes. Moreover, it has been proved that the inhibition of mTORC1/p70S6K signaling ameliorates apoptosis in DN [43]. Moreover, our data also showed that KPNA2 retarded HG-induced apoptosis through SLC1A5-mediated mTOR signaling pathway. In conclusion, the present study indicated that KPNA2 was down-regulated in HG-induced podocytes. Silencing of KPNA2 ameliorated HG-induced injury through suppressing apoptosis and enhancing autophagy. Further data showed that KPNA2 functions its effect on DN via blocking mTOR signaling pathway activation by regulating SLC1A5. Thus, KPNA2 might act a therapeutic strategy for DN treatment. Author contribution statement All authors have contributed significantly, and that all authors agree with the content of the manuscript. Funding information No funding. Declaration of competing interest We have read and understood the conflicts of interest and declare that we have none. Transparency document Transparency document related to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.10.200. References [1] D.T. Talsma, K. Katta, M.A.B. Ettema, B. Kel, M. Kuschegullberg, M.R. Daha, C.A. Stegeman, J.V.D. Born, L. Wang, Endothelial heparan sulfate deficiency reduces inflammation and fibrosis in murine diabetic nephropathy, Laboratory investigation, J. Tech. Methods Pathol. 98 (2018). [2] M. Hu, R. Wang, X. Li, M. Fan, J. Lin, J. Zhen, L. Chen, Z. Lv, LncRNA MALAT1 is dysregulated in diabetic nephropathy and involved in high glucose-induced podocyte injury via its interplay with b-catenin, J. Cell Mol. Med. 21 (2017) 2732e2747. [3] L. Zhu, J. Han, R. Yuan, L. Xue, W. Pang, Berberine ameliorates diabetic
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Please cite this article as: X. Fan et al., Silencing of KPNA2 inhibits high glucose-induced podocyte injury via inactivation of mTORC1/p70S6K signaling pathway, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.10.200
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Silencing of KPNA2 inhibits high glucose-induced podocyte injury via inactivation of mTORC1/p70S6K signaling pathway Xiaobao Fan a, b, Zhenjiang Li b, Xiaoming Wang b, Jing Wang b, Zhiming Hao a, * a
Department of Rheumatology and Immunology, The First Affiliated Hospital of Medical College of Xi ‘an Jiaotong University, Xi’an City, Shaanxi Province, 710061, China b Nephrotic Hemodialysis Center, Shaanxi Provincial People’s Hospital, Xi’an City, Shaanxi Province, 710068, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 17 October 2019 Accepted 31 October 2019 Available online xxx
Dysregulation of apoptotic and autophagic function are characterized as the main pathogeneses of diabetic nephropathy (DN). It has been reported that Karyopherin Alpha 2 (KPNA2) contributes to apoptosis and autophagy in various cells, but its role in DN development remains unknown. The purpose of present study was to explore the function and underling mechanisms of KPNA2 in development of DN. In this study, 30 mM high glucose (HG)-evoked podocytes were used as DN model. The expression of KPNA2 was detected by qRT-PCR and Western blot assays. The cell viability was tested by CCK-8 kit, the apoptosis was measured using flow cytometry assay, the apoptotic and the autophagy related genes was detected by Western blot. Our results indicated that KPNA2 was significantly increased after HG stimulation. Knockdown of KPNA2 inhibited apoptosis, and promoted cell viability and autophagy in HGtreated podocytes. In addition, silencing of KPNA2 deactivated mTORC1/p70S6K pathway activation via regulating SLC1A5. Further results demonstrated that activating mTORC1/p70S6K pathway strongly ameliorated the effect of KPNA2 on cell viability, apoptosis and autophagy. Therefore, our study suggested that knockdown of KPNA2 rescued HG-induced injury via blocking activation of mTORC1/p70S6K pathway by mediating SLC1A5. © 2019 Published by Elsevier Inc.
Keywords: Diabetic nephropathy Podocyte KPNA2 SLC1A5 mTORC1/p70S6K pathway
1. Introduction Diabetic nephropathy (DN) is a major microangiopathy of diabetes mellitus and one of the important inducements leading to end stage of renal disease (ESRD) [1,2]. The prevalence of DN in Chinese is increasing rapidly, which is second only to diabetic cardiovascular disease [3]. With the occurrence and development of DN, the renal function of diabetic patients will gradually deteriorate, which will bring great economic burden to the society and individual patients [4]. The pathogenesis of DN is extremely complex, and it is well accepted that DN is mainly associated with metabolic disorders, hemodynamic disorders, oxidative stress injury, inflammation and genetic factors [5,6]. Recently, large number of studies demonstrate that DN pathogenesis is closely related to inhibition of autophagy in renal cells [7,8].
* Corresponding author. Department of Rheumatology and Immunology, The First Affiliated Hospital of Medical College of Xi ‘an Jiaotong University, No 277 yanta west road, Xi’an City, Shaanxi Province, 710061, China. E-mail address: [email protected] (Z. Hao).
Autophagy, which is self-phagocytosis, is a unique life phenomenon of eukaryotic cells and has important physiological significance for maintaining intracellular homeostasis [7]. In the state of diabetes mellitus, the autophagy function in renal cells is suppressed by a negative regulatory molecule, mammalian target of rapamycin (mTOR) [8,9]. Subsequently, the damaged organelles and cytotoxic substances are failing to be removed timely and effectively, but accumulated in the cells, which affects the normal function of cells to accelerate cell damage and apoptosis [10]. What is more, the activation of mTOR pathway by HG environment also promotes the expression of Atg5 (autophagy-related gene 5), which can cause mitochondria-targeted translocation, release cytochrome C and activate caspase family proteins, thereby promoting cell apoptosis [11,12]. Therefore, deactivation of mTOR pathway is an effective way to alleviate the pathogenesis of DN [13,14]. AATs are transmembrane proteins that participate in a series of cellular processes by regulating the transport of amino acids in cells or organelles [15]. Solute-linked carrier family A1 member 5 (SLC1A5/ASCT2) is a cell surface Naþ-dependent neutral AAT [16]. Loss of SLC1A5 results in inhibition of glutamine uptake, thereby reducing mTORC1 activity in Myc ± mice [17]. Nicklin et al. also
https://doi.org/10.1016/j.bbrc.2019.10.200 0006-291X/© 2019 Published by Elsevier Inc.
Please cite this article as: X. Fan et al., Silencing of KPNA2 inhibits high glucose-induced podocyte injury via inactivation of mTORC1/p70S6K signaling pathway, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.10.200
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revealed that knockdown of SLC1A5 attenuates inhibition of mTORC1 on cellular autophagy by down-regulating L-glutamine (LG) uptake [18]. A previous study indicates that SLC1A5 is hypomethylated in the blood of diabetic patients, which suspected that SLC1A5 might play a role in the development of diabetes [19]. In addition, SLC1A5-mediated mTORC1 signaling pathway has been proved as a downstream target of various transcription factors in multiple diseases, such as TEAD1 (TEA Domain Transcription Factor 1) in vascular injury, PDZ domain of sorting nexin 27 (SNX27) in cancer [20e22]. It has been reported that SLC1A5 is hypomethylated in diabetic patients via RNA-seq analysis [23]. Karyopherin family is a class of protein that plays an important role in protein transport between cytoplasm and nucleus. Karyopherin a2 (KPNA2) is a member of the karyopherin family, which is composed of 529 amino acids [24]. Given its function in nucleocytoplasmic transport, KPNA2 mediates the translocation of various proteins and is involved in numerous cellular processes, such as cellular differentiation, proliferation and apoptosis, transcriptional regulation, immune response, and viral infection [25,26]. For example, knockdown of KPNA2 inhibits cell proliferation and promoted apoptosis in bladder cancer via interacting with OCT4 nuclear transportation, KPNA2 contributes to apoptosis in human tongue squamous cell carcinoma via activating Bax and caspase-3 and down-regulating Bcl-2 [27,28]. Notably, recent studies have reported that overexpression of KPNA2 promotes cellular autophagy in human oral squamous cell carcinoma, glioblastoma, adipose-derived stem cells, and so on [29e31]. However, the role of KPNA2 in autophagic function of DN pathogenesis is still unclear. This study aims to verified the effect of KPNA2 and the relationship between KPNA2, SLC1A5 and mTORC1/p70S6K signaling in the course of DN. The results showed that expression of KPNA2 was significantly increased in high glucose-induced podocytes. Further, we found that downregulation of KPNA2 facilitated apoptosis and autophagy through inactivating mTORC1/p70S6K signaling pathway by mediating SLC1A5. This finding provides an understanding of KPNA2 in DN, and might serve a therapeutic target for DN patients. 2. Materials & methods 2.1. Podocytes culture and treatment The human podocytes were obtained from Celprogen Inc. (Torrance, USA), and were cultured in RMPI 1640 medium (Gibco; Thermo Fisher Scientific, Inc., USA) supplemented with 10% fetal bovine serum (FBS; Gibco), in a 5% CO2, 33 C humidified incubator. After maintained in normal glucose medium (NG, 5 mM) for 7 days, podocytes were cultured with NG, or medium added mannitol (MA, 30 mM) or high glucose (HG, 30 mM). 2.2. Transfection The small interfering RNAs of KPNA2 (KPNA2 siRNA1, KPNA2 siRNA2) and overexpression plasmid of SLC1A5 (pcDNA- SLC1A5) was constructed by the manufacturer’s instructions (Generay Biotech, Shanghai, China). Treated or non-treated podocytes were transfected by Lipofectamine 3000 Kit (Invitrogen, Carlsbad, CA) for 48 h. 2.3. Cell viability assessment Podocytes were seeded in 96-well plate with density of 2 104 cells per well, then subsequently maintained in NG, MA or HG medium for 24 h. Thereafter, 10 ml Cell Counting Kit-8 (CCK-8,
Dojindo Molecular Technologies, Inc.) solution was added to each well and incubated at 37 C for 2 h, and OD values were measured at 450 nm. 2.4. Apoptosis assessment The HG treated podocytes were harvested and resuspended in binding buffer, and then incubated with 0.25 mg/ml Annexin VFITC (Dojindo, Japan) and 10 mg/ml propidium iodide (PI) (Dojindo, Japan) at 25 C in the dark for 30 min. Finally, apoptosis evaluated by a flow cytometry (BD Biosciences, USA). 2.5. Quantitative real-time PCR (qRT-PCR) Total RNA of podocytes was extracted using Trizol reagent (Invitrogen, USA) referring to the manufacturer’s protocol, and then reverse transcribed complementary DNA by the PrimeScriptVR RT reagent kit (Takara Bio, Inc., Tokyo, Japan). The cDNAs were quantified through DyNamo SYBR1 Green qPCR Kit (Takara), and b-actin was used as the internal control. The relative expression of KPNA2 was evaluated by the 2DD CT. The primer of KPNA2 is Forward: 50 ATTGCAGGTGATGGCTCAGT-3’; Reverse: 50 -CTGCTCAACAGCATCTAT CG-3’. 2.6. Western blot assay Podocytes were harvested and lysed Radio Immunoprecipitation Assay (RIPA) buffer (Thermo Scientific) with a protease inhibitor cocktail (Millipore, MA, USA). The protein extraction resolved by sodium dodecyl sulphate polyacrylamide gel electrophoresis, and subsequently transferred onto a PVDF membrane (Millipore). After blocked by 5% nonfat milk for 1 h at room temperature, the membrane was incubated with primary antibody at 4 C overnight. The primary antibodies including KPNA2 (1:1000, Abcam, ab84440); SLC1A5 (1:1000, Abcam, ab237704), Bcl-2 (1:1000, Abcam, ab32370), pro-caspase3 (1:1000, Abcam, ab13847), cleaved caspase3 (1:1000, Abcam, ab49822), Beclin 1 (1:1000, Abcam, ab207612), LC3 II (1:1000, Abcam, ab51520), p62 (1:1000, Abcam, ab56416), p-mTORC1 (1:1000, Cell Signaling Technology, Inc, #2983), p-p70S6K (1:1000, Abcam, ab59208) and b-actin (1:1000, Abcam, ab179467). Then, the membrane was treated with secondary antibody conjugated to horseradish peroxidase for 2 h at room temperature. 2.7. Statistical analysis All experiments were repeated in triplicates. Statistical analysis was performed by SPSS 22.0 software (SPSS Inc., USA). The data are all indicated as the mean ± SD. The result value of *p < 0.05, # p < 0.05, or &p < 0.05 was considered statistically significant. 3. Results 3.1. The expression of KPNA2 was up-regulated in HG-induced podocytes Firstly, the result of CCK-8 assay indicated that high glucose (HG, 30 mM) significantly reduced the cell viability of human podocytes, compared with normal glucose (NG, 5 mM) and mannitol (MA, 30 mM) treatment (Fig. 1A, p < 0.05). Then, the results of qRT-PCR and Western blot assays indicated that HG remarkably increased KPNA2 expression in podocytes (Fig. 1BeC, p < 0.05). Therefore, KPNA2 might play roles in the HG-evoked induced podocytes.
Please cite this article as: X. Fan et al., Silencing of KPNA2 inhibits high glucose-induced podocyte injury via inactivation of mTORC1/p70S6K signaling pathway, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.10.200
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Fig. 1. The expression of KPNA2 was up-regulated in HG-induced podocytes (A) Human podocytes were treated with normal glucose (NG, 5 mM), mannitol (MA, 30 mM) or high glucose (HG, 30 mM) for 3 days. The cell viability was detected using CCK-8 assay assessment. Normal glucose treatment was performed as control, mannitol treatment was performed as negative control. (B, C) After NG, MA, and HG treatment for 24 h, the mRNA and protein expression of KPNA2 in podocytes were measured using qRT-PCR and Western blot assays, respectively. *p < 0.05 vs NG or MA.
3.2. KPNA2 siRNA inhibited apoptosis and promoted autophagy function Next, we investigated the effect of KPNA2 on HG-induced apoptosis. Two small interfering RNAs against KPNA2 (KPNA2
siRNA1 and KPNA2 siRNA2) were transfected into podocytes. After HG treatment, the expression of KPNA2 were significantly enhanced by KPNA2 siRNAs, and transfection efficiency of siRNA1 was more significant than siRNA2 (Fig. 2A, p < 0.05). The results of CCK-8 and Annexin V-FITC/PI assays indicated that KPNA2 siRNAs
Fig. 2. KPNA2 siRNA inhibited apoptosis and promoted autophagy function (A, B) Podocytes were transfected with constructed KPNA2 small interfering RNAs (KPNA2 siRNA1 and siRNA2) or its negative control (si-NC) for 48 h before HG treatment. Then, qRT-PCR was used to assess the mRNA expression of KPNA2. (B, C) The cell viability and apoptosis of transfected podocytes were tested by CCK-8 assay and Annexin V-FITC/PI assay, respectively. (DeF) The Western blot analysis of protein expression of apoptosis related genes Bcl-2, Pro/Cleaved-Caspase-3. Bcl-2: B-cell lymphoma-2. (D, G-I) The Western blot and quantification analysis of protein expression of autophagy related genes, including Beclin 1, LC3 II/LC3 I, and p62*p < 0.05 vs control or NC, #p < 0.05 vs KPNA2 siRNA1.
Please cite this article as: X. Fan et al., Silencing of KPNA2 inhibits high glucose-induced podocyte injury via inactivation of mTORC1/p70S6K signaling pathway, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.10.200
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conspicuously promoted the cell viability and reduced apoptosis rate in HG-induced podocytes. What is more, the effect of siRNA1 on cell viability and apoptotic function more significant than siRNA2 (Fig. 2B and C, p < 0.05). Therefore, KPNA2 siRNA1 was chose in the following experiments. As shown in Fig. 2DeF, KPNA2 siRNA1 dramatically increased the protein expression of Bcl-2, and decreased the protein expression of Pro/Cleaved-Caspase-3 (p < 0.05). In addition, KPNA2 siRNA1 significantly promoted Beclin 1 and LC3 II/LC3 I expression, and inhibited p62 expression (Fig 2D and G-I, p < 0.05). Thus, our data indicated that silencing of KPNA2 could inhibit apoptosis and rescue autophagic function in HG-injured podocytes.
3.3. KPNA2 siRNA deactivated mTORC1/p70S6K pathway in podocytes via regulating SLC1A5 A recent study indicated that SLC1A5 was a potential target of KPNA2 [32], and it is well known that the activation of mTORC1/ p70S6K signaling pathway is dependent on SLC1A5, therefore, we speculated that KPNA2 might regulate mTORC1/p70S6K signaling pathway through SLC1A5. To verified our speculation, HG-induced podocytes were transfected with KPNA2 siRNA1 and SLC1A5 overexpression vector (pcDNA-SLC1A5). The result of Western blot showed that KPNA2 siRNA strongly decreased the expression of SLC1A5, p-mTORC1 and p-p70S6K, but pcDNA-SLC1A5 attenuated this decrease (Fig. 3AeD, p < 0.05). These data suggested that KPNA2 siRNA blocked the activation of mTORC1/p70S6K pathway in podocytes via suppressing SLC1A5 expression.
3.4. Activation of mTORC1/p70S6K pathway ameliorated the inhibition of KPNA2 siRNA on HG-induced apoptosis and autophagy To explore the role of mTORC1/p70S6K pathway in the regulation of KPNA2 on HG-induced injury, pcDNA-SLC1A5 and mTORC1/ p70S6K pathway activator (MHY1485) were used to treat HGinduced podocytes after transfected with KPNA2 siRNA. Our data showed that pcDNA-SLC1A5 and MHY1485 significantly counteracted the effect of KPNA2 siRNA on cell viability and apoptosis in HG-induced podocytes (Fig. 4A and B, p < 0.05). Besides, the results of autophagy related genes expression indicated that pcDNASLC1A5 and MHY1485 remarkably suppressed the increase of KPNA2 siRNA on Beclin 1 and LC3 II/LC3 I, and the decrease of KPNA2 siRNA on p62 in HG-induced podocytes (Fig. 4CeF, p < 0.05). Thus, our data revealed that knockdown of KPNA2 inhibited HGinduced injury though the deactivation of mTORC1 pathway by mediating SLC1A5. 4. Discussion Podocyte, the epithelial cell of the glomerulus, is one of the important components of the glomerular filtration barrier. Numerous studies have demonstrated that high glucose (HG)induced podocyte injury is a major cause in the progression of chronic renal nephropathy, which is an important target for clinical treatment. Consistent with the discoveries in previous studies, our data showed that HG stimulation reduced cell viability in podocytes. In HG-induced podocytes, we found that KPNA2 expression was significantly elevated, and knock downing KPNA2 promoted
Fig. 3. KPNA2 siRNA deactivated mTORC1/p70S6K pathway in podocytes via regulating SLC1A5 (AeD) Podocytes were transfected with KPNA2 siRNA1 and/or SLC1A5 overexpression vector (pcDNA-SLC1A5). The Western blot analysis of SLC1A5, p-mTORC1 and p-p70S6K protein expression. Non-treated podocytes as control*p < 0.05 vs control, # p < 0.05 vs KPNA2 siRNA1, &p < 0.05 vs pcDNA-SLC1A5.
Please cite this article as: X. Fan et al., Silencing of KPNA2 inhibits high glucose-induced podocyte injury via inactivation of mTORC1/p70S6K signaling pathway, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.10.200
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Fig. 4. Activation of mTORC1/p70S6K pathway ameliorated the inhibition of KPNA2 siRNA on HG-induced apoptosis and autophagyPodocytes were divided into 4 groups, HG stimulation group (as control), HG and KPNA2 siRNA1 treatment group, HG and KPNA2 siRNA1 and pcDNA-SLC1A5 treatment group, HG and KPNA2 siRNA1 and mTORC1/p70S6K pathway activator (MHY1485) treatment group. (A, B) The cell viability and apoptosis of each group of podocytes. (CeF) The Western blot analysis of Beclin 1, LC3 I, LC3 II, and p62 protein expression in each group of podocytes.*p < 0.05 vs control, #p < 0.05 vs HG þ KPNA2 siRNA group.
cell viability and autophagic function while inhibited cell apoptosis. Further results indicated that suppression of KPNA2 plays a protective role against HG-induced podocyte apoptosis and autophagic function arrest through mTORC1/p70S6K pathway by regulating SLC1A5 expression. Emerging evidences showed that aberrant expression of KPNA2 is frequently observed in a variety of cancerous patients. Recent studies have showed that up-regulation of KPNA2 aggravates the development of esophageal squamous cell carcinoma and epithelial ovarian carcinoma by promoting cell proliferation, glioblastoma and oral squamous cell carcinoma by enhancing cellular autophagy [30,31,33,34]. In addition, Gao et al. revealed that KPNA2 induces
apoptosis of tongue squamous cell carcinoma through downregulating Bcl-2 expression and activating the caspase-dependent pathway [28]. Growing studies suggest that KPNA2 functions its specific role in distinct cells by involving in cellular growth and autophagy. In the present study, downregulating KPNA2 enhanced cell viability and autophagy, and repressing cell apoptosis in podocyte after HG stimulation. What is more, KPNA2 expression was found elicits various effects in different diseases by regulating distinct genes or signaling pathways. For instance, the involvement of KPNA2 in cancer cell proliferation by regulating FGF1 signaling pathway, KPNA2 contributes to epithelial ovarian cancer cell proliferation via up-regulating c-Myc [33,35]. This study demonstrated
Please cite this article as: X. Fan et al., Silencing of KPNA2 inhibits high glucose-induced podocyte injury via inactivation of mTORC1/p70S6K signaling pathway, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.10.200
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that silencing KPNA2 down-regulated SLC1A5 expression, which seems to contradict with data form a previous study that overexpression of KPNA2 inhibits SLC1A5 expression in glioblastomas. It has been proved that KPNA2 enhances c-Myc translation by directly binding with it, and c-Myc is a direct activator of SLC1A5 translation [32,33,36,37]. Therefore, we speculated that knockdown of KPNA2 repressing SLC1A5 expression through c-Myc axis. mTORC1 is one of the functional protein complexes of serine/ threonine kinase mTOR, which is activated via glutamine transported by SLC1A5 [38]. mTORC1 functions important regulatory role in cell growth, proliferation and protein synthesis via phosphorylating its two downstream effectors, p70S6K and 4EBP1 [39]. Numerous evidences have proved that the activation of mTORC1/ p70S6K pathway plays an important regulatory role in the growth and apoptosis of podocytes [39,40]. In addition, mTORC1/p70S6K axis achieves the effect of inhibiting autophagy of podocytes by suppressing the activity of the promoter of the autophagosome [41]. The previous studies proved that inhibition of mTORC1/ p70S6K signaling pathway can ameliorate the down-regulated autophagy in DN renal cells, and mTORC1/p70S6K signaling pathway activator MHY1485 can aggravate DN development by repressing autophagy, which suggests mTORC1/p70S6K pathway can serve as a target to inhibit the DN development [14,42]. Here, we also report that overexpressed KPNA2 ameliorated HG-induced autophagy via inactivation of mTORC1/p70S6K signaling pathway in podocytes. Moreover, it has been proved that the inhibition of mTORC1/p70S6K signaling ameliorates apoptosis in DN [43]. Moreover, our data also showed that KPNA2 retarded HG-induced apoptosis through SLC1A5-mediated mTOR signaling pathway. In conclusion, the present study indicated that KPNA2 was down-regulated in HG-induced podocytes. Silencing of KPNA2 ameliorated HG-induced injury through suppressing apoptosis and enhancing autophagy. Further data showed that KPNA2 functions its effect on DN via blocking mTOR signaling pathway activation by regulating SLC1A5. Thus, KPNA2 might act a therapeutic strategy for DN treatment. Author contribution statement All authors have contributed significantly, and that all authors agree with the content of the manuscript. Funding information No funding. Declaration of competing interest We have read and understood the conflicts of interest and declare that we have none. Transparency document Transparency document related to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.10.200. References [1] D.T. Talsma, K. Katta, M.A.B. Ettema, B. Kel, M. Kuschegullberg, M.R. Daha, C.A. Stegeman, J.V.D. Born, L. Wang, Endothelial heparan sulfate deficiency reduces inflammation and fibrosis in murine diabetic nephropathy, Laboratory investigation, J. Tech. Methods Pathol. 98 (2018). [2] M. Hu, R. Wang, X. Li, M. Fan, J. Lin, J. Zhen, L. Chen, Z. Lv, LncRNA MALAT1 is dysregulated in diabetic nephropathy and involved in high glucose-induced podocyte injury via its interplay with b-catenin, J. Cell Mol. Med. 21 (2017) 2732e2747. [3] L. Zhu, J. Han, R. Yuan, L. Xue, W. Pang, Berberine ameliorates diabetic
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Please cite this article as: X. Fan et al., Silencing of KPNA2 inhibits high glucose-induced podocyte injury via inactivation of mTORC1/p70S6K signaling pathway, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.10.200