Caveolin-1 expression and stress-induced premature senescence in human intervertebral disc degeneration

Introduction Chronic and debilitating low back pain is a common condition and a huge economic burden. Many cases are attributed to age-related degeneration of the intervertebral disc (IVD); however, age-related degeneration appears to occur at an accelerated rate in some individuals. We have previously demonstrated biomarkers of cellular senescence within the human IVD and suggested a role for senescence in IVD degeneration. Senescence occurs with ageing but can also occur prematurely in response to stress. We hypothesised that stress-induced premature senescence (SIPS) occurs within the IVD and here we have investigated the expression and production of caveolin-1, a protein that has been shown previously to be upregulated in SIPS. Methods Caveolin-1 gene expression in human nucleus pulposus (NP) cells was assessed by conventional and quantitative real-time polymerase chain reaction (PCR), and caveolin-1 protein expression was examined within human IVDs using immunohistochemistry. The correlation between caveolin-1 and p16INK4a (biomarker of cellular senescence) gene expression was investigated using quantitative real-time PCR. Results Caveolin-1 gene expression and protein expression were demonstrated within the human IVD for the first time. NP cells from degenerate discs exhibited elevated levels of caveolin-1 which did not relate to increasing chronological age. A negative correlation was observed between gene expression for caveolin-1 and donor age, and no correlation was found between caveolin-1 protein expression and age. A positive correlation was identified between gene expression of caveolin-1 and p16INK4a. Conclusion Our findings are consistent with a role for caveolin-1 in degenerative rather than age-induced changes in the NP. Its expression in IVD tissue and its association with the senescent phenotype suggest that caveolin-1 and SIPS may play a prominent role in the pathogenesis of IVD degeneration.


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].
(page number not for citation purposes) 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][14][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][20][21][22][23][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 stressinduced 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][26][27][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][30][31][32][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][35][36][37][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].

Tissue samples
Human IVD tissue was obtained either at post mortem (PM) examination or from patients undergoing surgery, where patients were selected on the basis of magnetic resonance imaging-diagnosed degeneration and progression to anterior resection either for spinal fusion or disc replacement surgery for chronic LBP. Local research ethics committee approval was obtained together with informed consent from the patient or relatives. Disc tissue was removed as detailed previously [37].
General procedure for tissue specimens A block of tissue (incorporating annulus fibrosus [AF] and NP in continuity) was fixed in 10% vol/vol neutral buffered formalin and embedded in paraffin wax. Four micron sections were stained with haematoxylin and eosin to grade the degree of morphological degeneration according to previously published criteria that assess the demarcation between NP and AF, proteoglycan content of the NP, presence and extent of structural fissures, and cell cluster formation [39]. Potential grades range between 0 and 12. A grade of 0 to 3 indicates a histologically non-degenerate IVD, 4 to 7 indicates evidence of intermediate (or moderate) degeneration, and 8 to 12 indicates severe degeneration. Further tissue sections were taken for immunohistochemical analysis of caveolin-1.

Isolation of nucleus pulposus cells
To obtain NP cells from human IVD tissue, NP tissue was identified and dissected from AF. NP tissue was finely chopped and digested in a solution of 2 U/mL protease (Sigma-Aldrich, Gillingham, UK) in Dulbecco's modified Eagle's medium plus Ham's F-12 nutrient medium (DMEM + F-12) (Gibco BRL, now part of Invitrogen, Paisley, UK) for 30 minutes at 37°C. NP cells were washed twice with DMEM + F-12 prior to cell isolation with collagenase type I treatment (0.4 mg/mL; Invitrogen).

Conventional reverse transcription-polymerase chain reaction
To investigate gene expression of caveolin-1 in human NP cells, RNA was extracted from isolated cells following the standard procedure for TRIzol ® reagent (Invitrogen). cDNA was then synthesised using Superscript II in accordance with the instructions of the manufacturer (Invitrogen). A standard Platinum Taq (Invitrogen) method was used for conventional polymerase chain reaction (PCR), using a concentration of 1.5 mM MgCl 2 . Primers specific for caveolin-1 [19] and the housekeeping gene 18S (Invitrogen) are detailed in Table 1. All primers were confirmed for gene specificity using BLAST (Basic Local Alignment Search Tool) (Genbank database sequences). Reactions, including non-template controls, were conducted for 35 cycles, including the annealing temperature of 58°C on a thermal cycler (MJ Research, now part of Bio-Rad Laboratories, Hercules, CA, USA), and products were analysed alongside a 100-base pair DNA ladder (Hyperladder IV; Bioline, London, UK) by electrophoresis on a 1.5% wt/vol agarose gel containing 0.2 μg/mL ethidium bromide (Sigma-Aldrich). Product bands were visualised by UV transillumination and images were captured using Gene Snap software (Syngene, Cambridge, UK).

Quantitative real-time polymerase chain reaction
Quantitative real-time reverse transcription-PCR (qRT-PCR) was performed to further examine caveolin-1 gene expression in human NP cells and to investigate any correlation between caveolin-1 and p16 INK4a gene expression in isolated NP cells using the standard curve method of analysis as described previously [18].

Primers and probe design
Primers and FAM-MGB probe specific for human caveolin-1 were designed by Applied Biosystems (ABI) (Warrington, UK) upon provision of caveolin-1-specific exon sequence (Gene expression assays) ( Table 1). p16 INK4a primers and probe were as described previously [18], and 18S primer/VIC-TAMRA probe set was a pre-developed assay reagent (PDAR) purchased from ABI.

Genomic curve standards
Genomic DNA (gDNA) was used to create standard curves for absolute quantification of copy number per reaction. gDNA (Promega Corporation, Southampton, UK) was homogenised, diluted to 100 ng/μL, and sonicated on ice. Serial dilutions of gDNA were prepared to generate standards with gene copy numbers of 75,000, 7,500, 750, 75, and 0 copies per 25 μL reaction.
Quantitative real-time reverse transcription-polymerase chain reaction amplification qRT-PCRs were carried out in triplicate in a 96-well plate. Reactions contained 12.5 μL of mastermix (Taqman ® Universal PCR mastermix; ABI) and 2.5 μL of template cDNA or gDNA. Primers were added to a final concentration of 900 nM and probe to a concentration of 250 nM, and molecular-grade water was added to a total reaction volume of 25 μL. A gDNA standard curve for each gene was included on each plate. Real-time PCR was performed using an ABI Prism 7000 sequence detection system (ABI). Reactions consisted of an initial Taq activation step of 95°C for 10 minutes to denature DNA and activate Taq polymerase followed by 40 cycles of 95°C for 15 seconds and 60°C for 1 minute.

Quantitative real-time reverse transcription-polymerase chain reaction analysis
Following amplification, an auto-baseline was set using the ABI 7000 sequence detection software and a threshold was set for each gene, above background levels and within the exponential phase. From these, a cycle threshold (Ct) was obtained for each well and data exported into Microsoft Excel Table 1 Details of polymerase chain reaction (PCR) primers, probes, and amplicon sizes (Microsoft Corporation, Redmond, WA, USA), where the three Ct values for each sample were averaged. Data were analysed as described previously [18] and results were expressed as copy number of target gene per 100 ng cDNA normalised to 18S.
Immunohistochemistry Immunohistochemistry (IHC) was used to determine the expression and localisation of caveolin-1 protein in the NP of 28 paraffin-embedded disc samples ( Sections were visualised using a Leica RMDB microscope (Leica Camera Limited, Knowlhill, Milton Keynes, UK), and images were captured using a digital camera and Bioquant Nova image analysis system (Bioquant Image Analysis Corporation, Nashville, TN, USA). For analysis, the NP was identified morphologically within each disc section. Within each section, a minimum of 200 NP cells were analysed from at least five different fields of view and immunopositivity was calculated as a percentage of the total cell population.

Statistical analysis
Data were non-parametric and thus Mann-Whitney U tests were conducted to compare gene copy number and numbers of caveolin-1-immunopositive cells in non-degenerate NP (grades 0 to 3) and degenerate NP (grades 4 to 7 and 8 to 12). Non-parametric linear regression analysis was performed to analyse the correlation between copy numbers of different genes and between gene copy numbers and subject age or number of caveolin-1-immunopositive cells and subject age.   Figure  1). This was supported by qRT-PCR analysis (Figure 2a

Immunohistochemical detection of caveolin-1 protein in human nucleus pulposus
Caveolin-1 protein expression was investigated in 28 IVD samples (for sample details, see Table 2). Immunohistochemical analysis for caveolin-1 demonstrated cytoplasmic/membrane staining within the chondrocyte-like cells of the NP (Figure 3). The percentage of immunopositive cells for caveolin-1 increased from 2.59% ± 1.01% (mean ± standard error of the mean [SEM]) in non-degenerate discs to 13.62% ± 6.51% in severely degenerate samples (Figure 4a). All IgG1 controls were negative. It must be noted that the majority of patients with severely degenerate discs were above 50 years of age; however, in the 24 samples of all grades for which the chronological age of individuals was known, no correlation was observed between caveolin-1 immunopositivity and age of the donors (P = 0.6609) (Figure 4b).

Correlation between caveolin-1 gene expression and gene expression of the senescence biomarker p16 INK4a
Seventeen NP samples were analysed for both caveolin-1 and p16 INK4a gene expression using qRT-PCR. Analysis of p16 INK4a expression agreed with our previous study [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.  [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.

Figure 4
Analysis of caveolin-1 immunohistochemistry Analysis of caveolin-1 immunohistochemistry. (a) Percentage of cells immunopositive for caveolin-1 protein in non-degenerate, moderately degenerate, and severely degenerate intervertebral discs. Data are shown as mean ± SEM. (b) Correlation of caveolin-1 protein expression with age of subject. Non-parametric linear regression analysis (P = 0.6609; R 2 = 0.0089).

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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][26][27][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][56][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. . 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 2 O 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][20][21][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 ; 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][17][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 factorkappa-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.

Conclusion
This study has shown that caveolin-1 expression in human NP cells is linked to IVD degeneration and is associated with the senescent phenotype as depicted by increased expression of p16 INK4a . Caveolin-1 expression was not linked to increasing chronological age, suggesting a role in accelerated degeneration which could be due to SIPS, rather than RS. Further work will elucidate the role of caveolin-1 in these related areas.