Microtubule destabilization caused by silicate via HDAC6 activation contributes to autophagic dysfunction in bone mesenchymal stem cells
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Silicon-modified biomaterials have been extensively studied in bone tissue engineering. In recent years, the toxicity of silicon-doped biomaterials has gradually attracted attention but requires further elucidation. This study was designed to explore whether high-dose silicate can induce a cytotoxicity effect in bone mesenchymal stem cells (BMSCs) and the role of autophagy in its cytotoxicity and mechanism.
Morphologic changes and cell viability of BMSCs were detected after different doses of silicate exposure. Autophagic proteins (LC3, p62), LC3 turnover assay, and RFP-GFP-LC3 assay were applied to detect the changes of autophagic flux following silicate treatment. Furthermore, to identify the potential mechanism of autophagic dysfunction, we tested the acetyl-α-tubulin protein level and histone deacetylase 6 (HDAC6) activity after high-dose silicate exposure as well as the changes in microtubule and autophagic activity after HDAC6 siRNA was applied.
It was found that a high dose of silicate could induce a decrease in cell viability; LC3-II and p62 simultaneously increased after high-dose silicate exposure. A high concentration of silicate could induce autophagic dysfunction and cause autophagosomes to accumulate via microtubule destabilization. Results showed that acetyl-α-tubulin decreased significantly with high-dose silicate treatment, and inhibition of HDAC6 activity can restore microtubule structure and autophagic flux.
Microtubule destabilization caused by a high concentration of silicate via HDAC6 activation contributed to autophagic dysfunction in BMSCs, and inhibition of HDAC6 exerted a cytoprotection effect through restoration of the microtubule structure and autophagic flux.
KeywordsBMSCs Silicate Autophagic flux Microtubule HDAC6
- Baf A1
Bone mesenchymal stem cells
Histone deacetylase 6
Synaptosome associated protein 29
Vesicle-associated membrane protein 8
Bone mesenchymal stem cells (BMSCs), which are derived from the bone marrow, have the capacity for multidirectional differentiation within special culture conditions [1, 2]. BMSCs play an important role in the process of bone growth, development, and repair and are indispensable to bone formation. BMSCs act both as an important source of osteoblasts and in the synthesis and secretion of various growth factors . Silicate-doped biomaterials can induce the differentiation of BMSCs and enhance bone formation in a certain range [4, 5, 6]. In recent years, the cytotoxicity of silicate-doped biomaterials has gradually attracted attention, and studies have found that silicate-doped bioceramics could promote the caspase-dependent apoptosis of macrophages via altering the ionic microenvironment between the implants and hosts . In clinical practice, it was found that primary total hip arthroplasty (THA) using bioactive bone cement (SiO2 34.0%) showed an early radiological loosening after long-term follow-up, and the mechanism still remained unclear ; several researches identified that intracortical silicon microelectrode implants could cause blood-cerebral barrier dysfunction and neuronal cell loss [9, 10]. Moreover, studies have confirmed that a high concentration of silicate could inhibit the viability of human BMSCs . Our previous study also identified that a high concentration of silicate could induce autophagic flux blockage and cellular apoptosis in human umbilical vein endothelial cells . However, whether silicate has a cytotoxic effect on BMSCs and its mechanism remains to be further studied. Furthermore, silicon ion concentrations in different biomaterials range from 0.03 mM at the lowest up to 50 mM at the highest . Silicon is a trace element in the human body, and the silicon content of most implants is significantly higher than the normal range of the human body; the potential toxicity of silicate cannot be ignored, and its logical range in BMSCs still needs further identification [13, 14].
Autophagy is a functionally and evolutionarily preserved process that degrades and recycles harmful proteins or injured organelles in eukaryotic cells . Autophagy broadly includes macroautophagy, microautophagy, and chaperone-mediated autophagy. This study mainly focuses on macroautophagy, which is also the most studied. Autophagy is an adaptive response to maintain cell homeostasis and survival in the face of adverse environmental threats or stress. Disruption of autophagy can induce cells to self-repair disorders and further fall into apoptosis or necrosis [16, 17]. Moreover, Yang et al. have found that activation of autophagy could partially reverse the aging of BMSCs and increase osteogenic differentiation capacity; furthermore, autophagy could maintain the osteogenic differentiation ability of mesenchymal stem cells under adverse conditions [18, 19].
At present, there is no systematic report on the effect of soluble silicate on autophagy. Although some studies have pointed out that silicon nanoparticles can block autophagy flux by affecting the lysosome function, it is almost always caused by the structure of the nanomaterials themselves [20, 21, 22]. Whether autophagy is related to silicon ions remains to be further studied. Here, based on the treatment of BMSCs with different concentrations of sodium metasilicate representing silicon ions, the effect of free silicate on autophagy was determined through the detection of autophagy flux. Simultaneously, a reference was provided to clarify the reasonable and safe range of silicon ions in BMSC-related biomaterials.
Reagents and antibodies
Chloroquine (CQ) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Bafilomycin A1 (Baf A1) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Cell Counting Kit-8 (CCK-8) was purchased from Dojindo Molecular Technologies (Shanghai, China). LC3 (#3868), HDAC6 (#7612), acetyl-α-tubulin (#5335), and α-tubulin (#2125) primary antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). SQSTM1/p62 (#ab56416), LAMP1 (#ab24170), and LAMP2 (#ab13524) primary antibodies were purchased from Abcam (Cambridge, UK). GAPDH (#60004-1-lg), VAMP8 (#15546-1-AP), SNAP29 (#12704-1-AP), and STX17 (#17815-1-AP) primary antibodies were purchased from Proteintech Group (Boston, MA, USA). The second antibodies (peroxidase-conjugated goat anti-rabbit IgG and peroxidase-conjugated goat anti-mouse IgG) were purchased from Yeasen Biotechnology (Shanghai, China). The sodium metasilicate solution was prepared as previously reported at a stock solution of high concentration (100 mM) with some modification [23, 24]. In brief, sodium metasilicate solution (Sigma-Aldrich, #338443) was diluted into alpha-MEM to prepare a stock solution. The pH was adjusted using HCl to a physiological range (pH about 7.4) before 10% fetal bovine serum (FBS; ScienCell, Carlsbad, CA, USA) was added.
Cell culture and treatment
BMSCs were harvested and identified according to our previous studies . In brief, the cells were extracted from the bone marrow of rats less than a month old and were identified with cell surface marker such as CD73, CD90, and CD105 as well as multilineage differentiation ability (osteogenesis, adipogenesis, and chondrogenesis). The cells were cultured and purified in alpha-MEM (Keygentec, Nanjing, China), which contained 10% FBS and 1% penicillin/streptomycin solution (P/S), in an incubator at 37 °C with 5% CO2. Cell dissociation and passages were performed for 3 days each time. Primary BMSCs are prone to aging, and we generally only use the cells in four generations. The medium was replaced with different concentrations of chemicals when the cells achieved 80% density and continued to culture for 24 h. Cells were pretreated with CQ or Baf A1 for 2 h and continued in the following 24 h in the corresponding group.
Inductively coupled plasma atomic emission spectrometry (ICP-AES, NexION 300X ICP-MS, PerkinElmer, Waltham, MA, USA) was applied to evaluate the cellular silicon level. BMSCs were treated with various concentrations (0, 0.1, 0.5, 1.5 mM) of silicate for 24 h. Cell samples were collected and washed with phosphate-buffered saline (PBS) twice and subsequently resuspended in PBS for cell counting. After that, the cells were centrifuged and lysed with 5 ml RIPA overnight at room temperature. The silicon standard series was prepared, and the silicon level in the samples was measured by ICP-AES. The results are presented as the silicon level in each 106 cells (umol/106 cells).
Morphologic changes in BMSCs under the microscope (× 40) were recorded and compared after being treated with different concentrations of silicate (0, 0.1, 0.5, 1.5, 3, 5 mM). CCK-8 was used to evaluate cell viability at different time points (6 h, 24 h, 72 h). In brief, BMSCs were cultured in a 96-well plate with a density of 1 × 105, and they were treated with different concentrations of silicate (0.1, 0.5, 1.5, 3, 5 mM). When the cells achieved 80% density, each group was set with 6 repeat wells. To each well, 10 μl of CCK-8 was added and cells were incubated at 37 °C for 2 h. The absorbance values were measured at a 460-nm wavelength with a spectrophotometer. The viability rate was calculated using the following formula:
Viability rate (%) = (OD treatment group − OD blank group) / (OD control group − OD blank group) × 100%
Western blot analysis
Western blot was conducted as reported previously [12, 25]. In brief, RIPA with protease inhibitor (PMSF) was applied to lyse BMSCs on ice. A total of 30 μg protein was loaded and separated with 12.5% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (12.5%; Epizyme Biotech, Shanghai, China) and then transferred to a polyvinylidene fluoride membrane (0.2 μm; Millipore, Darmstadt, Germany). The membrane was blocked with 5% nonfat milk for 1 h at room temperature and incubated with primary antibodies at 4 °C for the night. Primary antibodies, including LC3, p62, HDAC6, acetyl-α-tubulin, α-tubulin, VAMP8, SNAP29, and STX17, were diluted at 1:1000; LAMP1 and LAMP2 primary antibodies were diluted at 1:500; and GAPDH antibody was diluted at 1:5000. The secondary antibodies (HRP-1inked rabbit or mouse at 1:3000 dilution) were applied at room temperature for 2 h after TBST washing. Enhanced chemiluminescence system reagent was applied for imaging exposure, and results of the images were analyzed by Quantity One software. The experiments were repeated at least four times for each protein.
Immunofluorescence assay was performed as previously reported with some modifications . For LC3 staining, the slide of the BMSCs was fixed in 4% paraformaldehyde for 10 min and subsequently permeabilized in 0.25% Triton X-100 for 15 min. After that, the slide was blocked with 3% bovine serum albumin for 30 min at room temperature and then incubated with rabbit anti-LC3 primary antibody (1:200, Cell Signaling Technology) overnight at 4 °C. Then, the cell slice was incubated in FITC-AffiniPure Goat Anti-Rabbit IgG (1:50, Yeasen Biotechnology, Shanghai, China) for 1 h at room temperature and subsequently with DAPI for 5 min. The slide was reviewed and recorded under a fluorescence microscope (Nikon, Tokyo, Japan). LC3 puncta indicated autophagosomes; the number of LC3 puncta was counted and compared in 30 random fields (× 400), and the data were presented as LC3 dots count/cell. Moreover, specific BMSCs with stable expression of the mRFP-GFP-LC3 were constructed; BMSCs were inoculated with Ad-mRFP-GFP-LC3 (#HB-AP210, Hanbio, Shanghai, China) for 48 h and then treated with corresponding reagents. The samples were reviewed and recorded under a fluorescence microscope. For the lyso-tracker, BMSCs were cultured in a 12-well plate and divided into the following groups: control group, silicate (3 mM)-treated group, Baf A1 (100 nM) or CQ (20 uM) group. Lyso-tracker solution (Keygentec, Nanjing, China) was diluted in alpha-MEM at 1:500 and co-cultured with BMSCs at 37 °C for 30 min. The slide was reviewed and recorded under a fluorescence microscope (Nikon, Tokyo, Japan). The fluorescence intensity was evaluated and analyzed using ImageJ software in 30 random cells, and the average fluorescence intensity per cell was compared. For the tubulin-tracker assay, BMSCs were cultured in slides and treated with different concentrations of silicate or interference for 24 h in the corresponding group. The slide of BMSCs was fixed in 4% paraformaldehyde for 10 min and subsequently washed twice in 0.1% Triton X-100 for 5 min. The tubulin-tracker solution (Keygentec, Nanjing, China) was diluted in alpha-MEM in 1:200 and co-cultured with BMSCs at 37 °C for 1 h. The slide was reviewed and compared via laser scanning confocal microscopy (Leica, Wetzlar, Germany).
Real-time quantitative polymerase chain reaction
Total RNA from treated cells was extracted via RNA Extraction Reagent (Yeasen Biotech, Shanghai, China) according to the manufacturer’s instructions. The concentration and quality of RNA were evaluated by spectrophotometric determination at 260/280 nm, and equal amounts of RNA from each sample were used for cDNA reverse-transcription following the PrimeScript RT Reagent Kit with gDNA Eraser (Takara Biomedical Technology, Beijing, China). cDNA was further applied as a template for real-time quantitative polymerase chain reaction (RT-qPCR) following the manufacturer’s instructions (Mastercycler Gradient, Eppendorf, Germany). The primers were listed as follows:
Syntaxin 17 (STX17): 5′-GAGGGTCCGTCAGTCAAGTT-3′ (forward), 5′-CTGACCCTCAGGCATCCAAT-3′ (reverse)
Synaptosome associated protein 29 (Snap29): 5′-CCCTTCCTGCTTCCAAGGTT-3′ (forward), 5′-CCCTGCGTAACACCTCTTGT-3′ (reverse)
Vesicle-associated membrane protein 8 (Vamp8): 5′-GGAAGCCACGTCTGAACACTT-3′ (forward), 5′-GATGGTGCCCGTAGCAAAGA-3′ (reverse).
HDAC6 activity assay
HDAC6 activity assay was conducted according to the manufacturer’s instructions (# 50076, BPS Bioscience, San Diego, CA USA). In brief, cell lysates of BMSCs were diluted in an HDAC assay buffer and subsequently mixed with the substrate. After that, a HDAC developer was added and the cell lysates continued to incubate. The values were measured with a spectrofluorometer with excitation at a wavelength of 380 nm and detection of emitted light of 460 nm.
The cells of each group were inoculated in 6-well plates 1 day before transfection. Alpha-MEM free of antibiotics was used for transfection dilution. The primer sequence of siRNA-HDAC6 (siHDAC6, RiboBio Co. Guangzhou, China) was designed as follows:
SiHDAC6 and its negative control sequences were mixed with Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Following a 20-min incubation at 37 °C, the mixture was added to plates and continued to culture for 4–6 h and then used in subsequent experiments (nsRNA: vehicle group and silicate group (3 mM); SiHDAC6: vehicle group and silicate group (3 mM)). The transfection efficiency was calculated according to the mRNA expression of HDAC6 after the cells were treated with 50 nM siRNA for 48 h.
Data are presented as mean ± standard deviation (SD) and analyzed with GraphPad Prism 5.0 software (San Diego, CA, USA) or SPSS 20.0 (IBM Corp., Armonk, NY, USA). Multigroup comparisons of the means were carried out by one-way analysis of variance (ANOVA) test with post-test and by Student–Newman–Keuls (S-N-K) multiple comparison test, and p < 0.05 indicated statistical significance.
High concentration of silicate affected morphologic changes and cell viability of BMSCs
High concentration of silicate impaired autophagic flux in BMSCs
Silicate impaired autophagic flux via disrupting microtubule stability
Inhibition of HDAC6 activity restored microtubule stability and autophagic activity
Bone formation mainly involves two major processes: osteogenesis and angiogenesis. BMSCs are indispensable in the process of bone formation and play an accelerative role in promoting angiogenesis . Biocompatibility of biomaterials with the host microenvironment plays a crucial role in maintaining the adhesion, proliferation, and differentiation of BMSCs. Silicon is a kind of trace element in the human body and is especially important for bone growth and development . Extensive studies have confirmed that silicon-based biomaterials can not only promote osteogenesis formation but also stimulate angiogenesis via BMSC interaction [4, 32]. In recent years, an increasing number of researchers have paid attention to the cytotoxic mechanisms of silicon-doped materials; whether silicon nanomaterials or high concentration of free silicate, both can affect the cell viability to some extent . In this study, it was found that treating BMSCs with a high concentration of soluble silicate could affect the viability of BMSCs and cause the interruption of autophagic flux of BMSCs, and the interruption of autophagic flux was related to the destabilization of microtubule structure. Furthermore, microtubule destabilization caused by silicate was induced by HDAC6 activation via α-tubulin histone deacetylation.
Autophagy is a self-protective mechanism of cells under stress. The interruption of autophagic flux can further cause a large accumulation of autophagosomes, leading to an imbalance of cell homeostasis. On the one hand, damaged organelles inside cells cannot be removed, especially mitochondria and endoplasmic reticulum, which can directly trigger apoptosis; on the other hand, the interruption of the cell energy cycle leads to an energy crisis . In this study, it was found that a high concentration of silicate can lead to a simultaneous increase of LC3-II and p62; LC3-II is the marker protein of autophagosomes, while p62 protein can bridge the binding of ubiquitination protein and LC3 to promote clearance of the targeted ubiquitination protein through autophagic flux; the simultaneous increase of both often indicates the interruption of autophagy flux . Furthermore, we detected the LC3-II and p62 expression with LC3 turnover test via CQ and Baf A1 application. The data confirmed that a high concentration of silicate could lead to disruption of the autophagic flux (>1.5 mM), causing a large accumulation of autophagosomes via LC3 fluorescence detection. Accordingly, the cell viability test found that with a further increase in the high concentration of silicate, the intracellular silicon increased, and cell viability gradually decreased, suggesting that the interruption of autophagic flux may affect cell viability .
Autophagic flux is the process of the formation of autophagosomes within cells and eventual fusion with lysosomes. The regulation of autophagic flux involves various molecules and microstructures, among which the function of the lysosome is of vital importance and is the key point for the smooth flux of autophagy. Lyso-tracker is a lysosomal fluorescent probe that can selectively remain in acidic lysosomes, thus achieving specific fluorescent labeling of living cell lysosomes . Our study indicated that a high concentration of silicate did not affect the acidic environment in lysosomes; moreover, CQ and Baf A1 were set as the positive control groups, and the study further confirmed that high-concentration silicate may interfere with autophagy flux in a way similar to CQ. Mauthe et al. found that the autophagic flux blocking mechanism of CQ and Baf A1 may not be the same: Baf A1 blocks autophagic flux mainly by altering lysosomal function, whereas CQ blocks the activity of autophagic flux by affecting membrane fusion between autophagosomes and lysosomes , which further suggests that a high concentration of soluble silicate may disrupt autophagic flux by affecting membrane fusion. In addition, we found that the expression of LAMP1 and LAMP2, two lysosomal membrane proteins , was increased after exposure to silicate. The inhibition of autophagy may promote lysosomal synthesis through a feedback mechanism that needs further research. We further investigated the protein and gene expression of membrane fusion assistance proteins including SNAP29, VAMP8, and STX17 . The results showed that the silicate did not affect the expression of these proteins. However, it is still uncertain whether silicate can interfere with the protein interaction of different membrane fusion assistance proteins; this topic needs further study.
Microtubule structure plays an important role as a bridge in the combination of autophagosomes and lysosomes. In recent years, numerous studies have identified that hyperacetylation of α-tubulin is necessary for the stimulation of autophagy. It was found that a decrease in α-tubulin acetylation induced by an acidic environment inhibited autophagic activity and induced rat cardiomyocyte injury [41, 42, 43]. Wang et al. reported that rats with silicosis caused by silica solution (50 mg/rat, 1 ml) via trachea instillation displayed a significant decrease in the expression of acetyl-α-tubulin. This loss of deacetylation was associated with activation of HDAC6 ; thus, we further detected changes in the microtubule structure as well as hyperacetylation of α-tubulin. We demonstrated that the microtubule structure was obviously disordered after the intervention of high-concentration silicate in BMSCs. It was found that acetyl-α-tubulin, which maintained microtubule homeostasis, was significantly reduced, which also preliminarily suggested that a high concentration of silicate might affect the acetylation of microtubule proteins, further leading to the disintegration of microtubules and thereby blocking the trafficking of autophagosomes to lysosomes. α-Tubulin acetylated modification was regulated by HDAC6 [45, 46], and our further research also confirmed that a high concentration of silicate can promote the expression of HDAC6. After using siRNA to interfere with HDAC6 expression, α-tubulin acetylation was enhanced significantly compared with silicate treatment alone, and the microtubule structure was also partially recovered with more beam-like divergent structures. We then verified that autophagic flux was partially restored with reduced autophagosome accumulation via inhibiting HDAC6 expression. Simultaneously, cell viability increased significantly via HDAC6 suppression after high-dose silicate exposure, which indicated that the combination with siHDAC6 may provide a feasible way to protect BMSCs from the toxicity of high silicate exposure.
Our study proves that a high concentration of silicate can affect the viability and cause the blockage of autophagic flux in BMSCs, and the blockage of autophagic flux was related to the destabilization of the microtubule structure. Furthermore, microtubule destabilization caused by silicate was induced by HDAC6 activation via α-tubulin histone deacetylation. This study from the perspective of autophagy redefines the safety range of silicate in biomaterials research for BMSCs; silicate may interfere with autophagic flux activity when the concentration exceeds 1.5 mM.
We appreciate the instructive suggestions of Professor Changsheng Liu and his fellows from East China University of Science and Technology.
LZ, LS, and FT conducted the experiments. PY analyzed the results and designed the figures. LZ wrote the manuscript. ZJ conceived the idea and directed the experiments. All authors read and approved the final manuscript.
This work was supported by the Key Project of Natural Science Foundation of China (Grant No. 31330028).
Ethics approval and consent to participate
All animals procedures were reviewed and approved by the Ethics Committee of Zhongshan Hospital affiliated to Fudan University.
Consent for publication
The authors declare that they have no competing interests.
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