Introduction
Low back pain (LBP) is a condition that affects a significant proportion of the population, with a lifetime incidence rate in excess of 70% in industrialised nations [
1]. It not only impacts on quality of life, but also places a substantial financial burden on the National Health Service and the economy in general due to loss of working days [
1,
2]. Many cases of LBP are attributed to degeneration of the intervertebral disc (IVD) and imaging studies have indicated a link between IVD degeneration and LBP [
3,
4].
To date, no clear mechanism for IVD degeneration has been identified, although the involvement of both environmental and genetic factors has been proposed [
5‐
8]. The occurrence of IVD degeneration increases with age [
9,
10]; however, a subset of individuals appear to exhibit accelerated degeneration that is independent of age [
5,
6]. This has led to speculation that additional factors could play a key role in the development of degeneration in some individuals.
There is increasing evidence that many features of IVD degeneration, including altered matrix synthesis and enhanced matrix degradation, originate at a cellular level [
6,
11,
12]. Cellular senescence is a strong candidate for the prolonged alteration in cellular activity observed during degeneration. Senescence and accompanying alterations in cell function have been implicated in ageing-related, degenerative, and pathological changes in a variety of tissues, including atherosclerotic plaque development within blood vessels and osteoarthritic alterations to cartilage [
13‐
15]. Two groups have shown increased staining for senescence-associated β-galactosidase (SA-β-gal) in cells from prolapsed and degenerate IVD cells, respectively, when compared with non-degenerate discs [
16,
17]. More recently, our group has presented more comprehensive evidence of senescence biomarkers in human IVD samples, demonstrating increased cellular senescence during IVD degeneration [
18]. In particular, cells from degenerate discs exhibited increased SA-β-gal activity, elevated expression of the cell cycle inhibitor p16
INK4a, telomere erosion, and a decrease in replicative potential. Furthermore, a correlation was observed between p16
INK4a expression and the expression of matrix-degrading enzymes matrix metalloproteinase (MMP)-13 and a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-5, suggesting a role for cell senescence in the molecular processes observed during IVD degeneration [
18].
Senescence occurs naturally with ageing but can also occur prematurely in response to stresses (such as exposure to cytokines or oxidative stress) in a number of cell types [
19‐
24]. Since telomeric erosion and p16
INK4a protein expression are increased in degenerate discs compared to non-degenerate age-matched samples [
18], we hypothesised that stress-induced premature senescence (SIPS) occurs within the IVD and may be responsible for the accelerated degeneration observed in some individuals.
Caveolae are plasma membrane compartments found abundantly in terminally differentiated cells such as fibroblasts and endothelial and muscle cells [
25]. The mammalian caveolin gene family codes for three 21 to 25 kDa caveolin proteins, which are integral membrane proteins essential for the structural integrity and function of caveolae [
26]. Expression of caveolin-3 is muscle-specific, whereas caveolin-1 and caveolin-2 are coexpressed in many cell types [
26]. Proposed functions include lipid transport, membrane trafficking, and a role in intracellular signalling pathways which stems from the colocalisation of caveolins with a variety of signal transduction molecules [
25‐
28]. Interestingly, caveolin-1 has been implicated in the senescent phenotype of several cell types, including human fibroblasts, lung adenocarcinoma cells, endothelial cells, and articular chondrocytes [
19,
29‐
33]. Moreover, caveolin-1 has been proposed to mediate SIPS in murine fibroblasts and human articular chondrocytes in response to oxidative stress and the inflammatory cytokine interleukin-1β (IL-1β) (both of which are known to be increased during IVD degeneration) [
19,
31,
34‐
38]. Here, we have investigated the expression of caveolin-1 in human IVDs and correlated its expression with the cell cycle inhibitor and the biomarker of senescence p16
INK4a, focusing on the nucleus pulposus (NP) as this area shows the most evidence of cell senescence in human IVDs [
18].
Discussion
This study has demonstrated for the first time that cells from the NP of human IVDs express caveolin-1 and furthermore that caveolin-1 gene expression and protein expression are elevated in degenerate IVDs, but that this rise in caveolin-1 expression does not correlate with increasing age. This is consistent with a role for caveolin-1 in degenerative rather than age-induced changes in the NP.
Changes associated with tissue ageing and degeneration have been postulated to involve cellular senescence [
41‐
43]. Two major categories of senescence are generally described in the literature as replicative senescence (RS) and SIPS. RS was first described by Hayflick in 1965 [
44] and is widely regarded as one of the main mechanisms underlying the normal ageing process via reduction of telomere length to critical levels following cumulative population doublings. In addition, there are a number of reports describing premature induction of senescence as a result of cellular exposure to stress. Factors linked to the induction of SIPS vary widely, from DNA damage – for example, radiation (bovine aortic endothelial cells [
45]), UV light (human fibroblasts [
46] and human melanocytes [
47]), chemical treatment (nasopharyngeal carcinoma cells [
48] and human fibroblasts [
49,
50]), and oxidative stress (human fibroblasts [
20,
22,
24] and human articular chondrocytes [
19]) – to oncogenic protein overexpression (for example, ras in human fibroblasts [
51]) and exposure to inflammatory cytokines such as IL-1 and tumour necrosis factor-α (human chondrocytes and fibroblasts [
19,
21,
23]). Previous data from our laboratory described accelerated senescence (characterised by a variety of biomarkers, including reduced cell replication potential, elevated levels of the cell cycle inhibitor p16
INK4a, increased SA-β-gal activity, and telomere erosion) in degenerate human IVDs compared with age-matched non-degenerate discs [
18], suggesting that SIPS may be involved in IVD degeneration.
Caveolin-1 forms homodimers, or heterodimers with its family member caveolin-2, that insert into the plasma membrane of terminally differentiated cells [
25]. The caveolin-1-rich areas termed caveolae and the caveolin proteins themselves are proposed to regulate cellular processes, including membrane traffic, signal transduction, and cellular senescence [
25‐
28,
52]. Caveolin-1 was investigated here due to its possible role in cellular senescence, in particular SIPS [
19,
31,
52]. Here, we show that caveolin-1 gene expression and protein expression are increased during IVD degeneration, but not in a manner that is associated with increasing chronological age.
Moreover, we demonstrate a correlation between caveolin-1 and p16
INK4a gene expression. p16
INK4a is a cyclin-dependent kinase inhibitor that prevents retinoblastoma phosphorylation and arrests the cell cycle in the G
0/G
1 phase prior to entry into the synthesis phase [
53,
54]. Many studies have shown increased levels of p16
INK4a alongside the occurrence and maintenance of permanent growth arrest and senescence, including a rodent model of ageing [
55‐
57]. Previous studies by our group and others strongly suggest a role for p16
INK4a in cellular senescence within degenerate tissue when compared with age-matched controls [
18,
58]. Furthermore, elevated p16
INK4a expression has been described in the premature senescence of human fibroblasts and leukaemic cells exposed to oncogenic ras and DNA double-strand breaks [
51,
59,
60], strengthening the reports that p16
INK4a is a biological marker for senescence. The present study demonstrated that the increased expression of caveolin-1 seen in the degenerate NP positively correlated with gene expression for p16
INK4a, suggesting that caveolin-1 expression is linked to the senescent phenotype observed in these cells.
The literature describes evidence linking cell exposure to stressful stimuli to both caveolin-1 expression and cellular senescence. In mouse NIH 3T3 fibroblasts, administration of subcytotoxic levels of H
2O
2 to experimentally mimic oxidative stress induced cellular senescence and increased caveolin-1 expression. Treatment with H
2O
2 in the presence of caveolin-1 antisense oligonucleotides reduced expression of senescence biomarkers, whereas transgenic overexpression of caveolin-1 induced SIPS [
31]. In human endothelial cells, isolated from atherosclerotic patients and induced to senesce, caveolin-1 expression was correlated with senescence biomarkers and with expression of 4-hydroxynonenal expression (a marker of lipid peroxidation and thus oxidative stress) independently of an effect on telomere length [
31]. These studies strongly support a role for caveolin-1 in SIPS induced by oxidative stress and this is further strengthened by work conducted on osteoarthritic articular chondrocytes. Administration of H
2O
2 to these chondrocytes induced cellular senescence via expression of the caveolin-1 protein, a mechanism reversed by antisense oligonucleotide-mediated downregulation of the caveolin-1 gene [
19]. The same study demonstrated an identical role for the inflammatory cytokine IL-1β.
Articular chondrocytes and the degenerative process observed during osteoarthritis share many characteristics with IVD cells and IVD degeneration [
12,
43]. Interestingly, IVD cells are subjected to both oxidative stress and catabolic cytokines, which have been implicated in the induction of SIPS [
19‐
22,
24]. Work published by our group suggests that IL-1β not only is increased in degenerate discs but is an important factor involved in catabolic events during IVD degeneration, including decreased matrix production and increased MMP and ADAMTS expression [
37,
38,
61,
62]. Moreover, advanced glycation endproducts (AGEs) such as carboxymethyl-lysine (CML) and the receptor for AGEs (RAGE) have been localised to the NP of degenerate IVD [
34‐
36]. CML is a tissue marker for accumulated oxidative stress [
35]; therefore, its presence and that of its receptor RAGE are highly significant for both mechanisms underlying IVD degeneration and the likelihood that they could cause SIPS in human NP cells. Furthermore, RAGE has been localised to caveolin-1-rich membranes in endothelial cells [
63]. This gives evidence, together with studies involving IL-1, that there are factors in the degenerate disc that may induce caveolin-1 expression and thus lead to the senescent phenotype described in IVD cells [
16‐
18].
Caveolin-1-rich regions of the plasma membrane have been associated with several receptors and signalling molecules, predominantly through isolation of caveolae and colocalisation studies. These studies have highlighted a subset of proteins that are relevant to IVD degeneration and to SIPS. First, RAGE, described above, is known to regulate several intracellular signalling pathways, including the nuclear factor-kappa-B pathway, which is essential for the expression of MMPs present in the degenerate IVD [
34,
64]. Second, there is evidence suggesting that caveolin-1, β1 integrin, and urokinase plasminogen activator receptor (uPAR) colocalise in human articular chodrocytes [
65]. uPAR has an integral role in plasmin activation and thereby promotes catabolic events through initiation of a proteolytic cascade through which matrix-degrading enzymes described in IVD degeneration such as MMPs are activated [
66]. Both could conceivably be pathways via which elevated caveolin-1 levels exert aspects of the senescent cellular phenotype observed in IVD degeneration.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
SKH participated in the design of the study, performed the majority of the laboratory work and analysis, and drafted the manuscript. CLM helped to secure funding, participated in the design of the study and the interpretation of data, and assisted in the preparation of the final manuscript. JAH conceived the study, secured funding, contributed to the design and coordination of the study, and participated in the interpretation of data and extensive preparation of the final manuscript. All authors read and approved the final manuscript.