Responses and adaptations of intervertebral disc cells to microenvironmental stress: a possible central role of autophagy in the adaptive mechanism

Abstract

Intervertebral discs comprise the largest avascular cartilaginous organ in the body, and its nutrient condition can be impaired by degeneration, aging and even metabolic disease. The unique microenvironment brings special stresses to various disc cell types, including nucleus pulposus cells, notochordal cells, annulus fibrosus cells and endplate chondrocytes. These cells experience nutrient starvation, acidic stress, hypoxic stress, hyperglycemic stress, osmotic stress and mechanical stress. Understanding the detailed responses and complex adaptive mechanisms of disc cells to various stresses might provide some clues to guide therapy for disc degeneration. By reviewing the published literatures describing disc cells under different hostile microenvironments, we conclude that these cells exhibit different responses to microenvironmental stresses with different mechanisms. Moreover, the interaction and combination of these stresses create a complex environment that synergistically increase or decrease influences on disc cells, compared with the effects of a single stress. Interestingly, most of these stresses activate autophagy, a self-protective mechanism by which dysfunctional protein and organelles are degraded. It is becoming clear that autophagy facilitates the cellular adaptation to stresses and might play a central role in regulating the adaptation of disc cells under stress. Therefore, autophagy modulation might be a potential therapeutic method to treat disc degeneration.

• Adaptations
• autophagy
• intervertebral disc
• microenvironmental stress
• responses

Introduction

Intervertebral disc degeneration (IDD), the primary cause of low back pain, is considered to arise from abnormal mechanical loading, unfavorable inheritance, smoking, obesity and aging (1–4). However, cell death has recently been considered an important contributor to IDD (5). Disc cells, including nucleus pulposus (NP) cells, annulus fibrosus (AF) cells, notochordal cells (NCs) and endplate chondrocytes, produce extracellular matrix (ECM) and maintain biomechanical function. To maintain the balance of ECM synthesis and degradation in discs, these cells secrete matrix metalloproteinases (MMPs) and disintegrin and metalloproteinase with thrombospondin motifs (ADAMTSs), as well as their inhibitors, tissue inhibitors of metalloproteinases (TIMPs) (6). Excessive levels of MMPs and ADAMTSs disturb the balance by degrading collagen II and aggrecan. Given the importance of disc cells, understanding the responses, adaptations and related mechanisms of these cells to microenvironmental stresses is crucial for developing new therapeutic approaches to treat IDD.

Lying between the vertebrae, intervertebral discs (IVDs) comprise the largest avascular cartilaginous organ in the body, and its nutrient supply depends on the diffusion and convection through the cartilaginous endplate (CEP) and outer AF, neither of which are efficient transport mechanisms (7,8). Material transport, including the import of essential nutrients and the export of metabolic wastes, can be interrupted by endplate calcification and mechanical loading (9). In addition, the extracellular environment can be further damaged by degeneration, aging and even metabolic diseases, such as diabetes mellitus. Given the unique microenvironment, these cells, especially the NP cells, experience various stresses including starvation, acidic stress, hyperglycemia, hypoxia and even mechanical stress. Under these stresses, cellular physiological processes such as proliferation, growth and protein synthesis could be impaired to some extent, resulting in a series of changes, such as cell cluster formation, viability loss, cell senescence, apoptosis and autophagy (10).

In order to relieve stress and reinstate homeostasis, most mammalian cells rely on intrinsic adaptive mechanisms, such as cell cycle arrest, programed cell death and macroautophagy referred to simply as “autophagy” (11). Autophagy can be induced in cultured mammalian cells by a variety of stress stimuli, including hypoxia, starvation, endoplasmic reticulum stress and acidic stress (12).

Although there are many reviews regarding IVD nutrition (7,13,14), there are few reviews detailing the responses and adaptations of disc cells. Hence, we examined the existing evidence for the response and adaptation of disc cells under different microenvironmental stresses, as well as the role of autophagy in disc cell adaptation, to enhance the understanding of the mechanisms involved in IDD, which will hopefully guide future research and identify novel treatments.

Autophagy

As a self-protective mechanism, autophagy degrades dysfunctional cellular organelles and proteins by lysosomal enzymes (15). When confronted with dramatic extracellular environment changes or pathogen invasion, mammalian cells can create double-membraned vesicles named autophagosomes in the cytoplasm, sequestering the organelles and protein. Autophagosomes fuse with lysosomes to produce autophagolysosomes and then degrade the sequestered contents with proteolytic enzymes in the lysosomes (Figure 1) (16). Generally, the basal level of autophagy controls cellular quality; however, the exact role of autophagy in stressed cells is extremely complicated and has aroused great interest in skeletal biology (15). Hostile environments induce cells to upregulate autophagy to remove toxic protein aggregates and provide amino acids and fatty acids. Autophagy activation has been shown to ameliorate the progression of experimental rat osteoarthritis (17). Conversely, loss of autophagy can lead to Parkinson's disease and cardiac dysfunction (18,19). However, autophagy plays dichotomous roles in several diseases. Excessive autophagy might exacerbate the death of chondrocytes and synovial fibroblasts in rheumatoid arthritis (20,21). “Autophagic cell death”, which refers to cell death-associated autophagy, is extensively used in studies about autophagy, though direct and precise evidence demonstrating that autophagy leads to the execution of cell death remains limited (22).

Figure 1. Autophagy induction and execution in disc cells. Under different stresses and following rapamycin treatment, disc cells produce a double-membrane structure termed as a phagophore, which engulfs dysfunctional organelles and proteins. As the vesicle elongates, the phagophore becomes the autophagosome, which fuses with the lysosome, producing the autolysosome and degrading the sequestered contents with proteolytic enzymes.

Depending on whether the mammalian target of rapamycin (mTOR) is involved or not, autophagy activation pathways are divided into mTOR-dependent and mTOR-independent (23). mTOR inhibition could promote autophagy; mTOR in the mTOR complex 1 (mTORC1), a highly conserved serine/threonine protein kinase, is associated with cell growth, proliferation and survival (24). Microenvironmental stress suppresses the phosphorylation of mTOR and its substrates, including the 4E-binding protein 1 (4E-BP1) and ribosomal S6 protein kinase (p70S6K), impairing the signal translation and protein production (25). mTOR combines with autophagy-related genes (ATGs) to regulate autophagy initiation, formation and degradation (23). Atg5, 6, 8 (Atg5, Beclin-1 and LC-3 in mammals) are the main regulators of autophagic formation; therefore, they are usually utilized as markers to detect autophagy.

Apoptosis and autophagy, both forms of programed cell death, can be sequentially induced by the same stress, but there are many differences and interactions between the two processes (26). Compared with the intracellular organelle changes that occur during autophagy activation, the process of apoptosis involves a series of changes that affect cellular morphology, ultimately resulting in apoptotic body formation (27). The activation of apoptosis accompanied by increased levels of caspases and catabolic hydrolases leads to cell death; however, proper autophagy activation protects cells against stresses (27). Only when the stress reaches a lethal level does the over-activation of autophagy promote cell death. Within the same environment, autophagy interacts with apoptosis, forming a complex network to help cells confront and adapt to hostile stress (26).

Starvation stress

Cell starvation is the first and main stress experienced by disc cells due to decreased nutritional diffusion (for small molecules) and convection (for large molecules) through the CEP and AF in the disc. Because it is difficult to perform nutrition research in vivo, glucose- or serum-free medium is usually used to assess the response and adaptation of AF and NP cells to starvation conditions (28,29) (Table 1).

Table 1. Responses and adaptations of disc cells to starvation.

Research style	Species	Animal or cell model	Starvation time	Cell specify	Key findings	Authors
In vitro 3-D culture	Bovine	Special chamber Treated with no-glucose medium	0 d–12 d	NP cells	Viability and GAG production decreased by distance, density and glucose-free medium	Horner et al. (32)
In vitro 3-D culture	Bovine	No-glucose medium	5 h–48 h	NP cells	No glucose inhibited cell viability	Bibby et al. (33)
In vitro	Wistar rats	Serum-free medium	12 h–24 h	NP cells	No serum induced cell apoptosis	Risbud et al. (34)
In vitro	SD rats	Serum -free medium	6 h, 48 h	NP cells	Starvation induced apoptosis, cycle arrest and a group of related genes	Sudo et al. (37)
In vitro	Rats	Serum -free medium	6 h, 48 h	NP cells	Starvation inhibited expression of Col-II and aggrecan and induced apoptosis Overexpression of bcl-2 reversed the effect	Sudo et al. (38)
In vitro	Human	Serum-free medium	0 h–48 h	NP cells	Transfection of hTERT attenuated the negative influence of starvation	Liang et al. (35)
In vitro	SD rats	Serum-free medium EBSS	12 h, 24 h 2 h	AF cells NP cells	Autophagy protected disc cells from apoptosis induced by starvation	Jiang and Shen (28,45)

NP, nucleus pulposus; AF, annulus fibrosus; GAG, glycosaminoglycans; hTERT, human telomerase reverse transcriptase; EBSS, Earle’s Balanced Salt Solution.

Cell numbers decrease from the periphery of the AF to the NP as the nutrition level declines toward the NP (30). Glycolysis is the main process by which NP cells obtain energy (31); therefore, glucose levels in discs determine NP cell viability. Using agarose gels and chambers to mimic nutrient diffusion through the endplate, Horner et al. (32) found that the bovine NP cell viability decreased with the increased cell density and greater distance from the medium, and viability was also significantly suppressed by glucose-free medium (32,33). When subjected to serum starvation, apoptosis and G1 arrest were induced in AF and NP cells isolated from rat and human discs (28,34–36). However, transfection of recombinant human telomerase reverse transcriptase (hTERT) gene attenuated the apoptosis and cell arrest previous negative influences, suggesting the importance of senescence in the cellular response to starvation (35). Additionally, microarray analysis identified a group of genes that was upregulated by starvation, mainly including genes involved in DNA damage checkpoints (37). Therefore, it is reasonable to conclude that starvation could induce disc cells to become senescent. In terms of cell function, serum starvation inhibited the expression of collagen II and aggrecan (38).

The balance of nutrient supply and cellular demand determines cell viability under starvation conditions. When nutrients, especially amino acids, are scarce in culture medium, some cancer cells respond by impeding the biosynthetic machinery via inhibiting mTOR and activating autophagy to maintain the energy balance (39). In chondrocytes, AMP-activated protein kinase (AMPK), an enzyme that could regulate cellular energy status, was also involved in autophagy upregulation (40). Although multiple lines of evidence have suggested a role for sirtuins in the induction of autophagy, no similar study has yet been reported in the cartilage or disc (41). The sirtuins, a conserved family of class III histone deacetylases, regulate a variety of cellular processes, including autophagy, senescence and apoptosis by consuming the nicotinamide adenine dinucleotide (NAD⁺) (42). During the process of disc aging and degeneration, the nutrition supply declines due to calcification of endplate cartilage; therefore, autophagy increased in the aged and degenerated NP to address the tension between nutrient supply and demand (43,44). In vitro, rat AF cells subjected to starvation with Earle's balanced salts solution and 1% fetal bovine serum (FBS) in Dulbecco’s minimum essential medium (DMEM) expressed higher levels of autophagy compared to cells grown in control conditions, and autophagy inhibited starvation-induced apoptosis (28,43), indicating that autophagy might be a major mechanism to improve the adaptive ability of disc cells in low-nutrient microenvironments. We also found a similar adaptive mechanism in rat NP cells (45). Furthermore, autophagy could even supply amino acids to yeast cells under starvation, which might be an important energy source (46). However, the role of mTOR in disc cells experiencing nutritional changes is not fully understood. Meanwhile, some other adaptive mechanisms have also been reported in discs. Transfection of the bcl-2 gene could directly inhibit apoptosis, inhibit caspase3 activation, and increase the expression of collagen II and aggrecan mRNA (38). In addition, the above-mentioned genes identified with microarray analysis might be promising targets to protect NP cells under starvation conditions.

Acidic stress

Due to ischemia and hypoxia, disc cells, especially NP cells, mainly rely on glycolysis for ATP production, even under aerobic conditions (13,14). Therefore, extracellular acidity is an inevitable consequence of increased lactic acid produced by anaerobic glycolysis and the slow diffusion of lactic acid across the matrix. In addition, free cations, including H⁺ ions, could be attracted by the numerous negative-charged groups in glycosaminoglycan (GAG) in the disc, leading to a high concentration of free cations and low PH around NP cells (47). Compared with that in cartilage, the matrix acidity in a degenerated disc is even more pronounced (48), with pH value dropping dramatically from 7.0 to 7.2 in healthy discs to 5.7–6.5 in degenerated ones (49–51). In addition, the intracellular pH of NP cells is typically maintained at 6.0 as a result of high lactic acid production.

The viability and function of different disc cells can be impaired by the acidic microenvironment (Table 2). Our previous study demonstrated that acid promoted apoptosis of rat endplate chondrocytes via the ovarian cancer G protein-coupled receptor 1 (OGR1) (52). Similarly, articular chondrocytes also underwent apoptosis due to the acidic microenvironment (53). On the other hand, Razaq et al. (54) found that when the pH of the extracellular environment decreased from 7.4 to 6.3, the production of sulfated GAG and TIMP-1 in bovine NP cells was significantly inhibited. Consistent with this, Ohshima et al. (55) found that proteoglycan synthesis in human NP peaked at pH 7.2–6.9 and declined steeply below pH 6.8, resulting in only 20% of normal production at pH 6.3. Importantly, the research excluded the effect of lactic acid itself on the acid-induced negative influence (55). When analyzing the influence of interactions of different microenvironments on NP cells, Neidlinger-Wilke et al. (56) found that ECM mRNA levels were suppressed by acid. However, while TIMP was inhibited in an acidic microenvironment, MMPs were found to be insensitive to the pH change, leading to an imbalance of pro- and anti-ECM degradation (54).

Table 2. Responses and adaptations of disc cells to acidic stress.

Research style	Species	Animal or cell model	Cell specify	Key finding	Authors
In vitro	SD rats	Cells treated with HCl pH: 6.4, 7.4	Endplate chondrocyte	Acid promoted apoptosis via OGR1	Yuan et al. (52)
In vitro 3-D culture	Bovine	Treated with HCl pH: 6.3–7.5	NP cells	Acid inhibited GAG, TIMP-1 production	Razaq et al. (54)
In vitro 3-D culture	Bovine	Treated with HCl pH: 6.5, 6.8, 7.2	NP cells	Acid inhibited mRNA of aggrecan, collagen-I, collagen-II	Neidlinger-Wilke et al. (56)
In vitro 3-D culture	Bovine	Adjusted to pH: 6.2, 6.7, 7.4	NP cells	Low pH inhibited cell viability with or without oxygen	Bibby et al. (33)
Ex-vivo	Human and bovine	Treated with lactic acid or HCl pH: 6.3–7.4	NP cells	Acid suppressed proteoglycan synthesis	Ohshima et al. (55)
In vitro 3-D culture	Bovine	Adjusted to pH: 6.0, 7.4	NP cells	Low PH inhibited cell viability	Horner et al. (32)
In vitro	SD rats	Treated with lactic acid 2 mM, 6 mM	NP cells	Lactic acid promoted autophagy	Wu et al. (69)

NP, nucleus pulposus; OGR1, ovarian cancer G protein-coupled receptor 1; GAG, glycosaminoglycans; TIMPs, tissue inhibitors of metalloproteinases.

Recent studies focusing on the effect of acid on chondrocytes have revealed an important role of acid-sensing ion channels (ASICs) (57,58). ASICs are a superfamily of amiloride-sensitive cationic channels and have six subunits: ASIC1a, ASIC1b, ASIC2a, ASIC2b, ASIC3, ASIC4. In the rat chondrocyte, blocking ASICs with amiloride prevented apoptosis in vitro (59). ASIC3 was detected in NP cells and was suggested to act as a protective mediator in acidic microenvironments (60,61). However, there is limited research into whether ASICs mediate the influence of acidic environments on disc cells. Interestingly, ASICs are involved in the nociceptive signaling of nerves stimulated by acid and inflammatory factors, both of which can be found in fissured and degenerated AF (62,63), indicating a potential protective role of ASICs in discogenic pain.

An increasing number of studies have demonstrated an association between acidic stress and autophagy (64–66). Acidic microenvironments promote autophagy in melanoma cells, while inhibition of autophagy with small interfering RNA for ATG5 significantly increased cell death (64). ATG5 is an essential protein for the formation of pre-autophagosomes (67). It has been reported that extracellular acidification could inhibit the phosphorylation of mTOR, which controls protein synthesis and energy expenditure (68). AMPK and Ca²⁺ are the main messengers mediating the effect of acid on mTOR (68). Under acidic stress, inhibition of mTOR might induce disc cells to restrain energy-consuming anabolic processes to adapt to inhospitable microenvironments. We recently found that lactic acid could enhance the level of autophagy in rat NP cells, providing further support for this hypothesis (69). The further revelation of a possible protective role of autophagy in disc cells against acidic microenvironments might be an important step to prevent IDD.

Hyperglycemia stress

Disc cell viability is inhibited by both low and high glucose (Table 3). Clinically, the incidence of diabetes mellitus in patients with lumbar or cervical IVD herniation who undergo surgery is significantly higher than that in the age-matched patients (70,71). The outcome of spinal surgery for patients with co-morbid diabetes and degenerated spinal disease is poorer than those without diabetes (72). We previously reported that IVDs from rats with type I diabetes mellitus had reduced mRNA and protein levels of aggrecan and collagen II compared with normal discs (73). The spontaneously diabetic Otsuka-Long-Evans-Tokushima fatty (OLETF) rats exhibited a higher degree of transition to fibrocartilaginous NP, indicating that NP in the diabetic rat was more inclined to be premature (74). Consequently, disc height and NP and AF cell morphologies and viabilities were altered by diabetes (75).

Table 3. Responses and adaptations of disc cells to hyperglycemia stress.

Research style	Species	Animal or cell model	Cell specify	Key findings	Authors
In vitro	SD rats	Cells treated with high glucose	Notochordal cells NP cells	High glucose promoted apoptosis and MMPs, inhibited cell proliferation	Park, Yang et al. (76,80)
In vitro	SD rats	Cells treated with high glucose	Notochordal cells	High glucose impaired mitochondria and enhanced ROS and autophagy	Park, Yanget et al. (81)
In vitro	SD rats	Streptozotocin-induced Type I diabetes	NP cells	Diabetes induced apoptosis, senescence, and autophagy	Jiang et al. (73)
In vitro	OLETF rats	Spontaneously diabetes	Notochordal cells	Diabetes induced apoptosis and MMPs expression	Won et al. (74)
In vitro	SD rats human	Cells from diabetic discs	NP cells	AGEs increased RAGE, MMP2 and p-ERK	Tsai et al. (79)
In vivo	ROP Os/+ mice	Streptozotocin-induced Type I diabetes	NP cells and AF cells	PYR and PPS attenuated disc degeneration	Illien-Junger et al. (75)

MMPs, matrix metalloproteinase; ROS, reactive oxygen species; AGEs, advanced glycation end products; PYR, Pyridoxamine; PPS, Pentosan Polysulphate; NP, nucleus pulposus; AF, annulus fibrosus.

NC proliferation was inhibited by high glucose from 0.1 to 0.4 M in a dose- and time-dependent manner (76). It has been demonstrated that diabetes or high glucose could enhance apoptosis of rat NPs and NCs both in vitro and in vivo (73,74,76). The specific mechanism underlying the induction of apoptosis by high glucose might be the mitochondrial (intrinsic) pathway, evidenced by increased levels of Bid, cytochrome-c and caspase-9 proteins rather than caspase-8 protein (76). However, we found that caspase-8 was also enhanced in diabetic rats, indicating the differences of apoptotic signal pathways between in vitro and in vivo conditions (73). The role of the endoplasmic reticulum pathway in hyperglycemia-induced apoptosis remains to be elucidated (77,78).

In terms of in vivo cell function, levels of MMPs, including MMP-1, -2, -3 and -13, were all enhanced in diabetic rats (74,75). Compared with the significant increase of MMP expression, TIMP-2 was only slightly elevated in rats, which resulted in a lower net inhibitory effect on MMP activity (74). The results of in vitro and in vivo studies were similar (76). The increased levels and activities of MMPs in diabetic discs directly inhibited GAG expression (75).

In order to further determine the mechanisms and materials involved in the above-described effects of high glucose levels, rat and human NP cells were treated with advanced glycation end products (AGEs) (79). AGEs are the products of glycation reactions in the setting of high glucose concentrations. As expected, AGEs increased the expressions of receptor of AGE (RAGE), MMP2 and phospho-extracellular-signal-regulated kinase (P-ERK), suggesting that the interaction between AGEs and RAGE activated the ERK pathway and led to an increased MMP expression (79). Similarly, in vivo research revealed that AGE accumulation in diabetes-induced degenerated discs was associated with the high expression of tumor necrosis factor (TNF)-α in the NP and AF of streptozotocin-treated mice (75). Therefore, inflammation might also mediate disc degeneration in diabetic subjects. Furthermore, the mitochondrial transmembrane potential of both NC and NP cells could be impaired by high glucose levels due to increased production of reactive oxygen species (ROS) (80–82). Generally, AGEs bind with RAGEs to increase ROS levels, causing oxidative stress and activating mitochondria-mediated apoptosis. In addition, the expressions of manganese superoxide dismutase (MnSOD) and catalase were also adaptively increased (81). Illien-Junger et al. (75) found that the combination of pyridoxamine (PYR, an AGE inhibitor) and pentosanpolysulfate (PPS, a broad acting anti-inflammatory) could maintain disc height, attenuate NP cell morphology, and decelerate the increased expression of MMP-13 and ADAMTS-5 in diabetic mice. The results indirectly demonstrated the role of inflammation and AGEs in diabetes-induced degeneration. In conclusion, the binding of AGEs and RAGEs might induce both inflammation and oxidative stress, leading to a sequence of consequences, including apoptosis, autophagy and catabolic metabolism.

When confronted with hyperglycemia stress, different disc cells could rely on their intrinsic adaptive mechanisms. In response to mitochondrial damage and apoptosis, the levels of antioxidants and autophagy in NC cells were adaptively enhanced by high glucose (83). Similarly, our research also showed that NP cell autophagy was increased in the type I diabetic rat disc (73). Recently, autophagy has been hypothesized to prevent oxidative stress and inflammation in the setting of diabetes (84). By degrading and recycling damaged proteins and organelles, autophagy could maintain mitochondrial integrity and even engulf impaired mitochondria to regulate oxidative stress and inflammation (85,86). In addition, the inhibitory effect of autophagy on apoptosis has been demonstrated with convincing research in osteoarthritis and IDD (28,87). However, the protective role of autophagy in diabetes-induced disc degeneration remains unclear and requires further elucidation. Given the above evidence, drugs that could both promote autophagy and exert anti-inflammation, anti-oxidative and anti-AGEs effects might be useful for regulating homeostasis within disc cells under hyperglycemic stress.

Hypoxic stress

Given its limited vascular distribution, the disc has a slower oxidative metabolism than other tissues (7,88). However, the rate of oxygen consumption is still significant in the disc. The partial pressure of oxygen (pO₂) value in the normal outer AF is 7.5 kPa, leaving only 0.5 kPa oxygen in the NP (88,89). Therefore, different disc cells, especially NP cells, are exposed to hypoxia, even in normal conditions. NP cells have adapted to the hypoxia microenvironment, which is to some extent a protective factor. Compared with a normoxic environment, hypoxia ensures a higher survival rate, less apoptosis and greater production of GAG and collagen II in NP cells through a hypoxia inducible factor (HIF)-dependent mechanism (33,90). Because the response of NP cells to hypoxia has been described in detail in previous reviews (10,91), this review mainly focuses on the association between autophagy and hypoxia.

During evolution, disc cells have developed adaptive responses to sustained hypoxia, and these adaptive mechanisms protect discs against further damage. HIF-1, -2 and -3 are transcription factors with important roles in this process (91,92). The expressions of HIF-1α, β and HIF-2α have been detected in NP cells under hypoxia stress. However, only HIF-1β was found in rat AF cells in vivo (93). In a hypoxic microenvironment, the α-subunits of HIF are stable and dimerize with the other subunits to bind hypoxia response elements (HREs) and promote the expression of protective mRNAs and proteins, such as vascular endothelial growth factor (VEGF), cited2 and galectin-3 to inhibit apoptosis (94–96). With regard to the ECM, HIF-1 was reported to promote the synthesis of aggrecan, which is the major proteoglycan expressed in discs (97).

Despite intensive research into the role of HIF in disc cell survival, there are few studies regarding the association between HIF and autophagy in disc cells. HIF-1 activates autophagy in the mouse chondrocyte by promoting mitogen-activated protein kinase (MAPK) and inhibiting mTOR (40,98). By contrast, suppression or knockout of HIF-2 enhanced autophagy, suggesting the inverse regulation of HIF-1 and HIF-2 in autophagy (99). The biochemical characteristics of NP cells are similar to those of chondrocytes; therefore, the relationship between autophagy and HIF in chondrocytes might also exist in NP cells. As mentioned by Risbud et al. (92), the regulation of oxemic conditions within the environment could be a promising approach to restore NP cell function, but the importance of autophagy in the hypoxic disc remains to be elucidated.

Mechanical stress

Embedded in the space between the adjacent vertebrae that bear weight, IVDs experience hydrostatic pressure and tensile stress under normal physiological conditions (100). The mechanics are changed dramatically in degenerated discs; hence, biomechanical alterations have always been regarded as an important cause of IDD.

Unphysiological loading reduces cell viability and stimulates NP and AF cell apoptosis (101–104) (Table 4). Under axial 20.8 -N compression for 3 days, the apoptotic incidence of rat tail NP cells was significantly increased (101). Similar to NP cells, the 20% elongation with a frequency of 0.5 Hz could induce apoptosis in ∼28% of rat AF cells after 48 h (105). The pathways involved in disc cell apoptosis induced by mechanical stress mainly include the mitochondrial and endoplasmic reticulum stress pathways (102,105,106). Static compression of 1.3 MPa on rat tail discs for 7 days could even reproduce the process of disc degeneration with the graduate loss of NC cells (107). In addition, dynamic and static compression both promoted the production of total ATP and lactate in porcine AF and NP cells, leading to decreased disc pH (108). Following cell viability changes, the ECM and matrix-degrading enzyme could also be altered by loading changes (107,109). For example, human NP cells in tissue under dynamic compression for 2 h exhibited increased ADAMTS expression and decreased aggrecan expression (109).

Table 4. Responses and adaptations of disc cells to mechanical stress.

Research style	Species	Animal or cell model	Cell specify	Key findings	Authors
In vivo and in vitro	SD rats	Compressive loading on the tail disc and scaffold	NP cells	Excessive magnitude, duration and frequency of compressive loading accelerated the apoptosis via mitochondrial pathway	Kuo et al. (100)
In vitro	SD rats	Cultured cells treated with air pressure	NP cells	1.0 Mpa static compression reduced cell viability and induced apoptosis in a time-dependent manner via mitochondrial pathway	Ding et al. (101)
In vitro	SD rats	Compressive loading on the tail disc	Notochordal cells	1.3 MPa static compression reduced notochordal cells, increased apoptosis and induced catabolic metabolism	Hirata et al. (106)
In vitro	Pigs	Compressive loading on the cultured disc	NP and AF cells	10% compressive static and dynamic strain increased the total ATP and lactate content in the IVD	Wang et al. (107)
In vitro	Bovine	Cyclic compression and torsion on the cultured disc	NP and AF cells	Combined dynamic compression and axial torsion promoted NP cell death compared with the pure loading	Chan et al. (102)
In vitro	SD rats	Cultured cells treated with cyclic tension loading	AF cells	0.5 Hz 20% stretch induced AF cells apoptosis via endoplasmic reticulum stress through nitric oxide production	Zhang et al. (104)
In vitro	Human	Cyclic compression on the cultured NP tissues	NP ells	The dynamic compression induced the increase of ADAMTS and decrease of aggrecan	Huang et al. (108)

NP, nucleus pulposus; AF, annulus fibrosus; IVD, intervertebral disc; ATP, adenosine 5′-triphosphate; ADAMTS, a disintegrin and metalloproteinase with thrombospondin motifs; ATP, adenosine 5′-triphosphate.

Despite intensive research into the responses of different disc cells under mechanical stress, there are few studies of autophagy under loading. Ma et al. (110) found that autophagy triggered by compression in rat NP cells was associated with apoptosis and intracellular ROS production. However, elucidation of the exact role of autophagy and the precise mechanism mediating autophagy activation require further investigation.

Interactions of starvation, hypoxia, acid and mechanics

Given the complexity of discs in vivo, none of the above factors act alone. For example, in vitro cell viability is further impaired by a combination of glucose starvation and extracellular acidity, compared with the effect of a single variable (33). In contrast, a hypoxic microenvironment could prevent apoptosis and protect NP cells in starvation and acidic microenvironments (33,34). Furthermore, these stresses could also influence each other. Porcine AF cells cultured in higher glucose levels exhibited lower oxygen consumption in sealed metabolism chambers (111). An acidic microenvironment was also shown to reduce the oxygen consumption of bovine NP cells (112). However, porcine NP cell oxygen consumption was insensitive to glucose level changes (111). Conversely, the lactic acid production of bovine NP cells, the main source of a low pH microenvironment, was inhibited by high-glucose medium (33). Experiments with a poromechanical finite element model of a human disc revealed that external loading regulates the regional distributions of oxygen and lactate in discs, which was more obvious in the healthy disc (113). Taken together, these different factors form a unique and complex microenvironment, in which different disc cells and even transplanted stem cells must respond to different stresses (10).

The central role of autophagy in the adaptation

Based on the previous findings, autophagy plays a central role in disc cell adaptation to different extracellular microenvironment stresses (Figure 2). Autophagy can be activated by starvation, acid, hypoxia and hyperglycemia, as well as mechanical loading, suggesting that autophagy activation might be a common adaptive mechanism of stressed disc cells. In a hostile microenvironment, autophagy inhibits apoptosis, regulates energy metabolism and even supplies amino acids to different disc cells to decelerate IDD progression. However, the degree of autophagic activation should be controlled to avoid negative influences on cell viability. It is still unknown whether autophagy plays dual roles in discs, and future studies should investigate this interesting point. Senescence, another important change of NP and AF cells, has also been demonstrated to participate in the degeneration process (73,114,115). However, research regarding the association between autophagy and senescence in discs remains limited. Given that cellular starvation occurs even in normal discs, the role of autophagy in normal and hostile microenvironments must be elucidated. Moreover, further explorations of autophagy in discs, especially in vivo studies, might identify useful therapeutic targets for IDD.

Figure 2. The central role of autophagy in regulating the adaptive mechanism of disc cells under different stresses. Compression, starvation, acid, hypoxia and high glucose have all been reported to induce autophagy in disc cells. The dotted line indicates that the pathway has been demonstrated in chondrocyte but not yet in disc cells.

In summary

As a unique organ in the body, the extracellular microenvironment of IVDs can be influenced by aging, degeneration and even metabolic diseases, and the cells must endure starvation, acidity, hypoxia and high glucose. Under these stresses, different types of disc cells exhibit various responses, including apoptosis, senescence, decreased viability and loss of matrix secretion. However, these different stresses have one thing in common – most induce autophagy, which might serve as a central regulator to protect cells against the hostile microenvironment. One shortcoming of the existing literature is that studies were carried out in rodents, and the cells populated in the NP of these animals are NC cells, which are different from those found in adult human discs. Future research, especially in human discs, is required to provide more information about disc cell survival in hostile environments and identify novel treatments for IDD.

Declaration of interest

The authors declare that they have no competing interests. All authors were involved in drafting the article, and all authors approved the final version to be published.

The research was supported by the Natural Science Foundation of China (81372002), the Natural Science Foundation of China (31170925), and the “Technology Innovation Action Plan” Key Project of Shanghai Science and Technology Commission (12411951300).