Introduction
Lower back pain affects up to 85 % of people at some point during their lives and causes severe incapacity that increases medical expenses and affects the workforce [
1]. In the United States, lower back pain results in annual health care and related costs of approximately $100 billion [
2]. Although the cause of lower back pain is considered to be an interdependent multifactorial process, intervertebral disc (IVD) degeneration is one of the major pathological processes implicated [
3‐
5].
The IVD is structurally an avascular tissue that includes an inner nucleus pulposus (NP) surrounded by an outer annulus fibrosus (AF), which is sandwiched between the cartilaginous endplates of contiguous vertebral bodies. The nutritional supply to NP cells depends on diffusion, primarily through the endplates, which is reduced by aging- and smoking-induced sclerosis of the subchondral bone [
6]. A loss of nutritional supply ultimately leads to apoptotic cell death in the IVD [
7,
8]. In particular, apoptosis of NP cells is considered to be the first sign of IVD degeneration [
9]. Therefore, treatments targeting cell apoptosis need to be developed to inhibit or prevent degenerative IVD disease.
Silent mating type information regulator 2 homolog 1 (SIRT1), a mammalian homolog of silent information regulator 2, is a nicotinamide-dependent class 3 histone deacetylase that is essential for cell survival and increases the longevity of mammalian cells under calorie restriction [
10]. In response to cellular energy loss, SIRT1 regulates multiple cellular processes, including the cell cycle, metabolism, and apoptosis [
11,
12]. Previously, we hypothesized that SIRT1 might play an important role in homeostasis of IVD tissues, especially during the early phase of IVD degeneration. We subsequently revealed for the first time the expression of SIRT1 in human NP cells, which markedly increases in IVDs during early degeneration stages, thereby promoting extracellular matrix (ECM) metabolism and cell proliferation [
13]. More recently, it has been shown that SIRT1 prevents human chondrocyte apoptosis, suppresses catabolic changes in gene expression, and accelerates osteoarthritis in conditional SIRT1-deficient mice [
14‐
16]. SIRT1 has been considered to inhibit apoptosis by deacetylation of nuclear proteins such as p53 and nuclear factor κB (NF-κB) [
17,
18]. Thus, it is conceivable that SIRT1 is a key regulator of mammalian musculoskeletal cell survival through modulation of apoptosis.
SIRT1 is also known to regulate the autophagy–lysosome pathway [
19]. Autophagy-related genes
Atg5,
Atg7, and
Atg8, which regulate autophagy, are deacetylated by SIRT1 in a nicotinamide-dependent manner, and this deacetylation is accelerated by limited nutrition [
19]. Autophagy is an important cell survival mechanism activated by calorie restriction in which cells digest and recycle their own damaged proteins and organelles [
12]. It has been revealed that autophagy is associated with certain degenerative diseases such as osteoarthritis [
20], Parkinson’s disease [
21], and Alzheimer’s disease [
22]. Autophagy and apoptosis are closely associated in the pathological processes of diverse human diseases [
19].
On the basis of the antiapoptotic and proautophagic roles of SIRT1, we hypothesized that SIRT1 might play a key role in the homeostasis of IVD cells under nutrient deprivation at the early phase of IVD degeneration. Furthermore, exogenous administration of recombinant human silent mating type information regulator 2 homolog 1 (rhSIRT1) protein into an IVD might contribute to cell survival by inhibition of apoptosis though autophagy induction. The purpose of this study was to elucidate the effects of exogenous SIRT1 on autophagy and apoptosis under limited nutrition in human IVD NP cells.
Methods
Ethics statement
The collection and use of human IVD specimens were approved by the ethics committee of Kobe University Hospital. All samples were obtained in accordance with the World Medical Association Declaration of Helsinki Ethical Principles for Medical Research Involving Human Subjects. This study was approved by the institutional review board of Kobe University Graduate School of Medicine. All patients provided written informed consent.
Culture of human nucleus pulposus cells
Thirty human NP tissue samples were obtained from consenting patients during surgical procedures (lumbar disc herniation, 13 cases; lumbar spinal stenosis, 10 cases; idiopathic scoliosis, 9 cases). There were 18 males and 14 females with a mean age of 32.4 years (range 11–80 years). Degeneration grades of the IVDs were determined by magnetic resonance imaging and classified according to the Pfirrmann grading system [
23]. The degeneration grades were represented by 9 cases of grade II IVD and 23 cases of grade IV IVD. All Pfirrmann grade II IVDs were obtained from young patients with idiopathic scoliosis (Table
1). To clearly compare early degenerative IVDs (Pfirrmann grade II) with advanced degenerative IVDs (Pfirrmann grade IV), moderate degenerative IVDs of Pfirrmann grade III were not analyzed in the present study.
Table 1
Human nucleus pulposus tissue sampling
Mean age (yr) | 13.6 ± 1.5 | 39.8 ± 11.5 | <0.01 |
Sex (male:female) | 2:7 | 16:7 | <0.01 |
Diseases | Idiopathic scoliosis: 9 | Lumbar disc herniation: 13 | |
Lumbar spinal stenosis: 10 |
The NP was carefully separated from the AF immediately after surgery. The tissue specimens were centrifuged and then subjected to sequential enzyme digestions at 37 °C with 0.025 % (w/v) collagenase P (Roche Applied Science, Indianapolis, IN, USA) and 0.001 % (w/v) deoxy-ribonuclease 2 (DNase 2) (Sigma-Aldrich, St Louis, MO, USA) for 12 h. The digested NP tissues were placed in complete tissue culture medium consisting of Dulbecco’s modified Eagle’s medium (DMEM; Sigma-Aldrich) supplemented with 10 % (v/v) heat-inactivated fetal bovine serum (FBS; Sigma-Aldrich), 100 U/ml penicillin, and 100 mg/ml streptomycin. When confluent, the NP cells were released and transferred to 35-mm culture dishes for 3 days of preculture at 37 °C in 20 % (v/v) O2, 5 % (v/v) CO2, and 1000 mg/L glucose. These isolated cells were defined as NP cells.
Treatment
To induce nutritional stress, NP cells were cultured in DMEM supplemented with 1 % (v/v) FBS, defined as the low nutritional (LN) condition, compared with 10 % (v/v) FBS defined as the normal nutritional (N) condition. In this study, to extrapolate possible clinical applications of SIRT1 to IVDs, exogenous supplementation of rhSIRT1 (Sigma-Aldrich) and not transfection of the SIRT1-encoding gene was employed. To inhibit autophagy, we used 3-methyladenine (3-MA; Santa Cruz Biotechnology, Santa Cruz, CA, USA), a specific inhibitor of the early stages of the autophagic process.
After 3 days of preculture, the NP cells were assigned to one of four culture groups (Table
2) as follows: group N, normal nutritional (N) condition cultured with 10 % (v/v) FBS; group LN, low nutritional (LN) condition cultured with 1 % (v/v) FBS; group LN + SIRT1, low nutritional (LN) condition cultured with 1 % (v/v) FBS and rhSIRT1 (10 μM); and group LN + SIRT1 + 3-MA, low nutritional (LN) condition cultured with 1 % (v/v) FBS, rhSIRT1 (10 μM), and 3-MA (5 mM). Introduction of rhSIRT1 using a protein delivery reagent with culture medium was repeated every 3 days at the same time as media changes.
Table 2
Experimental groups
Group N | DMEM with 10 % (v/v) FBS |
Group LN | DMEM with 1 % (v/v) FBS |
Group LN + SIRT1 | DMEM with 1 % (v/v) FBS + 10 μM rhSIRT1 |
Group LN + SIRT1 + 3-MA | DMEM with 1 % (v/v) FBS + 10 μM rhSIRT1 + 5 mM 3-MA |
Delivery efficacy of rhSIRT1
rhSIRT1 was introduced into NP cells using Pro-DeliverIN™ (OZ Biosciences, Marseille, France), a protein delivery reagent, according to the manufacturer’s instructions. The delivery efficiency of Pro-DeliverIN™ in NP cells was measured using rhSIRT1 labeled with a HiLyte Fluor™ 555 Labeling Kit-NH
2 (Dojindo Molecular Technology, Rockville, MD, USA). A microscopic visual count was performed as described previously to calculate the delivery efficiency of rhSIRT1 [
13].
Analysis of total NP cell numbers
NP cells that were approximately 60–70 % confluent were treated with 10 μM rhSIRT1 or 5 mM 3-MA under N or LN conditions in six-well plates at a density of 1.5 × 105 cells/well. Briefly, after 3, 7, and 14 days of initial treatment, images of five random fields were obtained with Motic Images Plus 2.2 software (Shimadzu, Kyoto, Japan). The number of NP cells in each well was then calculated and represented as the percentage of the baseline cell number.
Immunohistochemical staining
After 72 h of treatment, the cells were fixed in 4 % (w/v) paraformaldehyde/phosphate-buffered saline (PBS) for 15 minutes at room temperature, permeabilized, and blocked in 0.2 % (v/v) Triton X-100 in PBS for 30 minutes. To analyze autophagy, the cells were incubated with a primary antibody against human light chain 3 (LC3; Santa Cruz Biotechnology) overnight at 4 °C, followed by an Alexa Fluor 594–conjugated secondary antibody (Invitrogen, Carlsbad, CA, USA) for 30 minutes at room temperature. Images were obtained using a BZ-9000 microscope (Keyence, Osaka, Japan).
Analysis of autophagy activity
Autophagy activity was assessed by using an autophagy staining kit (Cayman Chemical, Ann Arbor, MI, USA). The absorbance of monodansylcadaverine (MDC), which enters multilamellar bodies by both an ion-trapping mechanism and interactions with membrane lipids as a probe to detect autophagic vacuoles, was measured using a microplate reader after 6, 12, 24, 48, and 72 h of treatment. Autophagy activity was calculated as the MDC absorbance normalized to the cell number.
Analysis of apoptosis
Apoptosis was assessed using a MEBCYTO apoptosis kit (MBL International, Nagoya, Japan) and flow cytometry. Early apoptotic cells stained only with annexin V conjugated with fluorescein isothiocyanate (annexin V-FITC), and late apoptotic/necrotic cells stained with propidium iodide (PI) as well as annexin V-FITC. After 3, 7, and 14 days of treatment, treated NP cells were isolated, centrifuged, and then incubated in 100 μl binding buffer containing annexin V-FITC and PI. Apoptotic cells, indicated by positive staining for annexin V-FITC only or annexin V-FITC and PI, were counted by flow cytometry and represented as the percentage of the total cell number.
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting
Treated NP cells were lysed on ice for 30 minutes in lysis buffer containing protease and phosphatase inhibitors (Roche Diagnostics, Basel, Switzerland). Total cellular proteins were centrifuged and collected. The separated proteins were transblotted electrically onto a blotting membrane (Amersham Biosciences, Arlington Heights, IL, USA). The membranes were probed with primary antibodies followed by a horseradish peroxidase–conjugated secondary antibody. Proteins were visualized with ECL Plus reagent (Amersham Biosciences) and a Chemilumino analyzer LAS-3000 mini (Fujifilm, Tokyo, Japan). The expression of acetylated and total p53, as well as of acetylated and total NF-κB proteins, was detected to elucidate the deacetylation potential of SIRT1. LC3 and p62 protein/sequestome 1 (p62/SQSTM1) were detected to monitor autophagy. In addition, Bcl-2-associated X protein (Bax), B-cell lymphoma (Bcl-2), caspase 3, and caspase 9 proteins were analyzed to determine the involved pathways of SIRT1 during apoptosis. The intensities of the bands were quantified using ImageJ analysis software, version 1.45 (NIH Image; National Institutes of Health, Bethesda, MD, USA). The antibodies used in this study were as follows: rabbit monoclonal or polyclonal antibodies against acetylated and total p53, acetylated and total NF-κB, LC3, p62/SQSTM1, Bax, Bcl-2, caspases 3 and 9, and cleaved caspases 3 and 9 (Cell Signaling Technology, Danvers, MA, USA); a mouse-monoclonal antibody against α-tubulin (Sigma-Aldrich); and horseradish peroxidase–conjugated anti-rabbit and anti-mouse antibodies (Amersham Biosciences).
Statistical analysis
Data are expressed as the mean ± standard deviation. Three-way analysis of variance with the Tukey–Kramer post hoc test was used to assess differences in experimental groups. Student’s t test was used to evaluate differences between the Pfirrmann grades II and IV groups. P values <0.05 were regarded as statistically significant. Statistical analyses were performed using PASW Statistics 18 (IBM SPSS, Armonk, NY, USA).
Discussion
In the present study, we demonstrate that reduced nutrition induced apoptosis through the mitochondrial pathway in human IVD NP cells and that rhSIRT1 prevented this apoptosis as well as induced autophagy. Pharmacological inhibition of autophagy resulted in a marked increase in apoptosis, suggesting an antiapoptotic effect of rhSIRT1 through induction of autophagy. Furthermore, these effects of rhSIRT1 on autophagy and apoptosis in human NP cells were more prominent in Pfirrmann grade IV IVDs than in Pfirrmann grade II IVDs.
Published evidence suggests that excessive apoptosis of IVD cells is a potential cause of IVD degeneration [
3,
5,
24,
25]. Sudo et al. demonstrated that serum starvation and annular needle puncture models induce apoptosis leading to IVD degeneration, and they also revealed that both Bcl-2 overexpression and caspase 3 knockdown prevent apoptosis and degenerative alterations [
26,
27]. We also found that apoptosis of both AF and NP cells is mediated through the mitochondrial pathway in a rat tail static compression model of disc degeneration [
28]. In surgically obtained herniated IVDs, Park et al. revealed that NP cell apoptosis is also induced by the mitochondrial pathway [
9]. Consequently, prevention of apoptosis in NP cells is considered to be a therapeutic strategy for IVD regeneration. In the present study, reduced serum concentrations induced apoptosis in both early and advanced degenerative IVDs. This proapoptotic effect induced by nutritional stress was more remarkable in advanced degenerative IVDs than in early degenerative IVDs. On the basis of these results, advanced degenerative NP cells are supposed to be more sensitive to nutritional stress than early degenerative NP cells, leading to apoptosis more readily.
Researchers in several studies have reported that prevention of apoptosis by platelet-derived growth factor [
29], Bcl-2 [
27], caspase 3 small interfering RNA (siRNA) [
26], or a caspase inhibitor [
30] has positive effects on ECM metabolism in IVD cells. In the current study, introduced rhSITR1 reduced apoptosis under LN conditions in human NP cells from Pfirrmann grades II and IV IVDs. Furthermore, this antiapoptotic effect of rhSIRT1 was attenuated by 3-MA, an inhibitor of autophagy. Therefore, introduced rhSIRT1 prevented apoptosis through autophagy induction. In addition, apoptosis inhibition was more obvious in Pfirrmann grade IV IVDs than in Pfirrmann grade II IVDs. We also found that introduced rhSIRT1 reduced acetylation of p53, decreased proapoptotic Bax expression, apoptotic cleavage of caspases 3 and 9, and increased antiapoptotic Bcl-2 expression in human NP cells. It has been shown that deacetylation of p53 by SIRT1 contributes to inhibition of p53-dependent mitochondrial apoptosis [
18,
31]. Thus, introduced rhSIRT1 potentially contributes to NP cell survival by modulation of the mitochondrial apoptotic pathway.
Autophagy is one type of cytoprotective process and ameliorates various types of cell stress. Activation of autophagy improves cell survival, whereas inhibition of autophagy promotes cell death [
32]. Autophagy is known to be activated by a variety of stress stimuli, including calorie restriction [
33], hypoxia [
34], and DNA damage [
35]. In addition, SIRT1 serves to activate autophagy under limited nutrition [
19]. In the present study, introduced rhSIRT1 activated autophagy in NP cells under nutrient starvation and increased the number of NP cells through suppression of apoptosis that was inhibited by autophagy, which is in line with previous evidence.
Mounting evidence has indicated possible crosstalk between autophagy and apoptosis pathways. The interplay between autophagy and apoptosis may be important throughout development and homeostasis [
36,
37]. Inhibition of autophagy by 3-MA or siRNA targeting genes related to autophagy triggers apoptosis in human HeLa cells [
33]. Shen et al. reported activation of autophagy in IVDs by interleukin-1β in rat AF cells under serum deprivation. Upon inhibition of autophagy by 3-MA, apoptosis was increased in AF cells. They concluded that autophagy might protect against apoptosis of AF cells as well as against IVD degeneration [
38]. The results of the present study demonstrate that autophagy, which was activated by rhSIRT1, inhibited apoptosis, leading to increases in the number of human NP cells. Notably, this effect of autophagy was more prominent in advanced degenerative IVDs. These findings suggest that autophagy plays an important role in the IVD degeneration process.
Distinct apoptotic and autophagic responses to rhSIRT1 in NP cells from Pfirrmann grade IV IVDs, compared with those from Pfirrmann grade II IVDs, may be due to different disease types and degeneration severity. We have previously reported that SIRT1 mRNA expression is higher in Pfirrmann grade III IVDs than in Pfirrmann grades II and IV IVDs [
13]. We considered that SIRT1 expression might be induced in the early stage of IVD degeneration when the internal environment of the IVD begins to deteriorate because of disturbances in the nutrient supply and that it then decrease in the advanced stage of IVD degeneration. The role of SIRT1 was altered according to IVD degeneration severity. The results, which show that the sensitivity to rhSIRT1 was different between Pfirrmann grades II and IV IVDs, indicate the safety and effectiveness of rhSIRT1 as a therapeutic agent for the treatment of degenerative IVD disease.
The present study may be limited regarding the type of cells employed in experiments. We used only NP cells in this study, but both NP and AF cells are involved in the mechanisms of IVD degeneration. Moreover, the experiments were conducted using monolayer cultures with 1000 mg/L glucose and 10 % (v/v) FBS, 20 % (v/v) oxygen, and a neutral pH. However, in human IVDs, the central NP is hypoxic and the oxygen concentration is <1 % (v/v) [
8]. Therefore, our culture conditions do not reflect the in vivo situation. It is now evident that nutrients, oxygen levels, and pH are powerful regulators of cellular activity and gene expression in IVDs, which can strongly influence the rates of ECM synthesis and degradation, and even cell viability [
7,
39,
40]. This is a limitation of the present study. In addition, we chose a low serum environment of 1 % (v/v) FBS supplementation as a condition of nutrient deprivation because serum interacts with other nutrients and plays a major role in IVD cells [
40]. Serum withdrawal to 1 % (v/v) FBS supplementation has been used as a positive apoptosis control for human AF cells in vitro and resulted in a significantly greater apoptosis incidence compared with the 20 % (v/v) FBS negative control [
29]. However, it remains to be elucidated whether this serum concentration is an acceptably low nutritional condition for human IVD cells. Therefore, the effects of rhSIRT1 should be confirmed in vivo. In addition, SIRT1 is widely involved in the regulation of cellular processes [
11,
12]. SIRT1 may also be directly involved in cell proliferation, as shown by the increases in cell number, or it may affect apoptosis through pathways other than autophagy. Further investigations need to be conducted.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
SM carried out all studies; participated in study conception and design, acquisition of data, analysis and interpretation of data; and wrote the manuscript. KK participated in study conception and design, secured funding, cowrote the manuscript, and helped to revise the manuscript. TY participated in the design of the study, conducted experiments, and contributed to drafting and revising the manuscript. KM and TT participated in the design of the study, conducted experiments, performed statistical analyses, and contributed to drafting and revising the manuscript. ZZ, YT, TK, MI, and TU acquired and interpreted data and helped with drafting the manuscript. TM, RK, and MK participated in study conception and contributed to drafting the final manuscript. KN participated in study conception and design and contributed to drafting and revising the manuscript. All authors read and approved the final manuscript.