ARTHRITIS & RHEUMATISMVol. 63, No. 12, December 2011, pp 3876–3886DOI 10.1002/art.30607© 2011, American College of Rheumatology

Identification of Cell Surface–Specific Markers toTarget Human Nucleus Pulposus Cells

Expression of Carbonic Anhydrase XII Varies With Age and Degeneration

Karen A. Power,1 Sibylle Grad,2 Joost P. H. J. Rutges,3 Laura B. Creemers,3

Mattie H. P. van Rijen,3 Peadar O’Gaora,1 J. Gerard Wall,4 Mauro Alini,2

Abhay Pandit,5 and William M. Gallagher1

Objective. Back pain is a major cause of disabil-ity, affecting millions of people worldwide. One cause ofaxial back pain is degeneration of the nucleus pulposus(NP) of the intervertebral disc. This study was under-taken to investigate associations of NP cells with cellsurface–specific proteins that differ from proteins inclosely related cell types, i.e., intervertebral disc anulusfibrosus (AF) cells and articular cartilage (AC) chon-drocytes, in order to identify potential surface moleculesfor directed delivery of therapeutic agents.

Methods. We conducted a complementary DNAmicroarray analysis of 16 human samples from 6 do-nors, followed by gene list reduction using a systematicapproach. Genes that were more highly expressed in NPthan AC cells, contained transmembrane domains, andappeared attractive for targeting were assessed by quan-

titative reverse transcription–polymerase chain reaction(RT-PCR). As a viable candidate, carbonic anhydraseXII (CAXII) was analyzed at the protein level by immu-nohistochemistry and functional study.

Results. Microarray results demonstrated a cleardivide between the AC and AF and between the AC andNP samples. However, the transcriptomic profile of AFand NP samples displayed a greater intersubject simi-larity. Of the 552 genes with up-regulated expression inNP cells, 90 contained transmembrane domains, and 28were quantified by RT-PCR. Most intense CAXII label-ing was observed in the NP of discs from young subjectsand in degenerative tissue.

Conclusion. CAXII may be considered for detec-tion or targeting of degenerating disc cells. Further-more, CAXII may be involved in pH regulation of NPcells. Its potential for directed delivery of regenerativefactors and its functional role in NP cell homeostasiswarrant further investigation.

Back pain is a leading cause of disability, anddegeneration of the intervertebral disc (IVD) is believedto be a major cause of neck and lower back pain. TheIVD is composed of 2 distinct but interdependenttissues: a gelatinous center, known as the nucleus pul-posus (NP), and several surrounding coaxial lamellaethat form the inner and outer anulus fibrosus (AF). Thisunique structural feature allows the IVD to constrainmotion at high loads and provide flexibility at low loads.Factors such as abnormal mechanical stresses, biochem-ical imbalances, nutritional factors, and genetic varia-tions are all reported to play a role in degenerative discdisease (1). As the natural aging process continues, thegelatinous NP region of the disc is replaced by a more

Supported by the Science Foundation Ireland grant 07/SRC/B1163, and the Swiss National Science Foundation (research grant320030-116818). Dr. Creemers’ work was supported by the DutchRheumatoid Arthritis Foundation. The University College DublinConway Institute of Biomolecular and Biomedical Research is fundedby the Programme for Research in Third Level Institutions, asadministered by the Higher Education Authority of Ireland.

1Karen A. Power, PhD, Peadar O’Gaora, PhD, William M.Gallagher, PhD: University College Dublin, Dublin, Ireland, andNational University of Ireland, Galway, Ireland; 2Sibylle Grad, PhD,Mauro Alini, PhD: AO Research Institute, Davos, Switzerland, andNational University of Ireland, Galway, Ireland; 3Joost P. H. J. Rutges,MD, Laura B. Creemers, PhD, Mattie H. P. van Rijen, MH: UniversityMedical Center, Utrecht, The Netherlands; 4J. Gerard Wall, PhD:National University of Ireland, Galway, Ireland; 5Abhay Pandit, PhD:Network of Excellence for Functional Biomaterials, National Univer-sity of Ireland, Galway, Ireland.

Drs. Power and Grad contributed equally to this work.Address correspondence to Sibylle Grad, PhD, AO Research

Institute Davos, Clavadelerstrasse 8, 7270 Davos, Switzerland. E-mail:sibylle.grad@aofoundation.org.

Submitted for publication October 6, 2010; accepted in re-vised form August 3, 2011.

3876

solid, less flexible, and ultimately fibrous disc. Thisdegeneration has been suggested to cause vasculariza-tion and the ingrowth of nerves and Schwann cells (2,3),which contribute to back pain that in some cases ischronic.

Currently, there is no definitive cure for thiscellular transition. Depending on the severity of a pa-tient’s symptoms, therapies can include one or a combi-nation of measures, i.e., physiotherapy, pain and antiin-flammatory medications, and finally, surgeries such asspinal fusion, dynamic stabilization, or disc arthroplasty.Although the established surgical treatments generallyyield good results, especially in the short term, they allhave disadvantages. In particular, these treatments canonly relieve symptoms; their curative effects are minimaland they do not restore the functional, original disctissue. Moreover, they are risky and may lead to seriouscomplications (4). Thus, availability of a treatment thatcould prevent mechanical failure of the segment andrestore a functional matrix of the disc using minimallyinvasive techniques would be desirable.

There are relatively recent and novel approachesto IVD regenerative therapy that avoid the above-described disadvantages, including use of injectable bio-materials, growth factors, and gene therapy (5,6). Anattractive strategy is the use of therapeutic cargo–carrying nanoparticles. The use of this strategy for drugand/or gene delivery would limit the need for surgicalprocedures because the treatment would be injectableand could be specifically directed toward cells residing inthe NP, in order to induce endogenous regenerativeactivity. Therefore, a primary goal is to identify asuitable and specific target in NP cells with which to tagactive compounds in the therapeutic cargo–carryingnanoparticles, for local injection. Yet, current knowl-edge about the specific expression of surface proteins inhuman NP cells is limited. The expression of cell surfacereceptors such as integrin subunits was identified ininvestigations of porcine and human IVD tissue (7,8)and in human herniated discs (9); however, these recep-tors were not located on NP tissue exclusively. CD24, acell adhesion molecule, was identified as a specific cellsurface marker for NP cells in the rat (10), and CD56(neural cell adhesion molecule 1) was found to beexpressed in canine NP cells (11), but gene expressionanalysis of human disc cells revealed that CD24 expres-sion was not specific for NP cells and that CD56 wasexpressed only at low levels (12).

In order to gain more comprehensive knowledgeabout the expression of surface proteins in human NPcells, we investigated gene expression profiles of human

NP cells by large-scale microarray analysis, with partic-ular emphasis on cell surface markers. Expression pro-files were compared to those of AF cells and articularcartilage (AC) chondrocytes, which are known to exhibitphenotypes similar to NP cells. The information gainedfrom this study may be used not only to develop newcurative approaches involving targeted delivery of ther-apeutic agents to the NP cell via specific surface mole-cules, but also to acquire a better understanding of thedisc cell phenotype in general.

MATERIALS AND METHODS

Isolation of NP, AF, and AC cells. The study wasapproved by the medical ethics committee of the UniversityMedical Center Utrecht and the scientific committee of theDepartment of Pathology of the University Medical CenterUtrecht. Specimens obtained at autopsy from 6 subjects withno known history of IVD disease were studied. Samples wereobtained within an average of 16 hours after death (range6.25–23.0 hours). Between death and tissue collection thesubjects’ bodies were kept in the mortuary at 4°C. The age ofthe subjects ranged from 25 to 81 years (mean 54.5 years)(Table 1).

The degree of disc degeneration was assessed accord-ing to the Thompson scoring system (13). IVD tissue washarvested from segments between L1 and L5 and was sepa-rated into NP and AF tissue. To exclude any contamination byAF tissue, only the central gel-like or amorphous NP tissue,which could easily be separated from the lamellar structure ofthe AF tissue, was harvested to be assigned to the group of NPtissue samples; the transition zone, including part of the innerAF, was entirely excluded from analysis. This is of particularimportance with regard to aged discs, in which it can bedifficult to clearly distinguish NP and AF tissue. In these casesNP tissue was sampled by conservatively excising tissue fromthe center of the disc. AC was harvested from the patellae ofthe same subjects. Chondrocytes were extracted from full-thickness cartilage, which implies the presence of mixed pop-ulations of superficial, middle, and deep zone cells.

Tissue was cut into small pieces and cells were enzy-matically isolated by sequential Pronase (Roche) and type IIcollagenase (Worthington Biochemical) digestion, with DNaseII (Sigma) added to prevent cell clumping. AC and AF weretreated with 0.2% Pronase/0.004% DNase for 1 hour, and thenwith 200 units/ml collagenase/0.004% DNase overnight. NPwas treated with 0.2% Pronase/0.004% DNase for 1 hour, andthen with 100 units/ml collagenase/0.004% DNase for 8 hours,with stirring at 37°C in a humidified atmosphere. After enzy-matic isolation, cell suspensions were filtered through a 70-�mcell strainer, washed twice with Dulbecco’s modified Eagle’smedium (DMEM), and lysed in TRI Reagent (MolecularResearch Center). Samples were stored at �80°C until RNAisolation.

RNA extraction. RNA was isolated using a modifiedTriSpin method (14,15). Briefly, bromo-chloro-propane(Sigma) was added to the lysate, phases were separated, andethanol (Merck) added to the aqueous phase. Total RNA was

DETECTION OF CAXII AS A NOVEL NP CELL SURFACE MARKER 3877

extracted using the SV Total RNA Isolation System (Pro-mega), which includes on-column DNase digestion, and elutedin 100 �l of RNase-free water.

Quality control. NanoDrop (Thermo Scientific) andBioanalyzer (Agilent) instruments were used to assess thequantity and the quality of the RNA samples, respectively.RNA quality was assessed for all 17 samples (AC was notavailable from 1 subject), using the RNA integrity number(Table 1). All samples were considered of high quality, with anRNA integrity number of �7. GeneChip Test3 Arrays (Af-fymetrix) were used to determine the sample quality in relationto the hybridization of housekeeping genes and the relativequantity of 3� to 5� probes. AC from donor 6 had a 3�:5� ratioon one of the housekeeping genes (AFFX-has) of 15.77, andwas therefore removed from further analysis (as recommendedby Affymetrix if the ratio is �3). Sixteen samples in total wereof suitable quality for microarray analysis.

Microarray data analysis. Human GeneChip U133Plus 2.0 Affymetrix arrays were used for all 16 samples. Thestatistical environment R (http://www.r-project.org) and Bio-conductor (http://www.bioconductor.org) (16,17) were used fordata analysis. Samples were background-corrected, log2-transformed, and quantile-normalized using Robust Multi-array Average within the Bioconductor package Affy (18).Correspondence analysis and hierarchical clustering using the

Euclidean distance measure were also performed, with theMade4 and Stats packages, respectively (19). Differentialexpression analysis was carried out with the Linear Models forMicroarray Data package (20,21), using the false discoveryrate to control for multiple testing (22).

Short-listing of candidate genes. Genes that exhibitedhigher expression in NP compared to AC cells were ranked toidentify potential cell-specific surface marker proteins. Ini-tially, BioMart was used to identify gene products with at least1 transmembrane domain, after which ExPASy (www.expasy.ch), InterPro (www.ebi.ac.uk/interpro/), and primary literaturedatabases were used to eliminate products associated withnuclear or intracellular organelle membranes. Candidate geneswere further eliminated if their products contained only extra-cellular domains of �30 amino acid residues, if they wereknown to be glycosylated, if they contained a high number ofdisulfide linkages, or if they were active as multimers. Thelatter 3 features represent sources of potential bottlenecks inEscherichia coli expression of recombinant proteins, whichwould be necessary to validate a protein as a molecular target.

Real-time reverse transcription–polymerase chain re-action (RT-PCR). TaqMan reverse transcription reagents (Ap-plied Biosystems) were used for complementary DNA synthe-sis. PCR was performed with an SDS 7500 real-time PCRinstrument using TaqMan Gene Expression Master Mix (allfrom Applied Biosystems) and standard thermal conditions (10minutes at 95°C for polymerase activation, followed by 40cycles of 95°C for 15 seconds and 60°C for 60 seconds).Primer-probe systems, purchased as Gene Expression ArrayPlates, were from Applied Biosystems. Expression of targetgenes was normalized to 4 endogenous controls: 18S ribosomalRNA, GAPDH, hypoxanthine phosphoribosyltransferase 1,and glucuronidase �. Gene expression levels were calculatedaccording to the �Ct method and presented as log2-transformed relative messenger RNA (mRNA) amounts (23).Differences between NP and AF and between NP and ACwere assessed in paired samples by 2-tailed t-test, with P valuesless than 0.05 considered significant.

Immunohistochemistry. Human IVD tissue was ob-tained as part of a standard postmortem procedure in which asection of the lumbar and thoracic spine is removed fordiagnostic purposes. IVD between the fourth and fifth lumbarvertebra (spinal motion segment L4–L5), including the adja-cent end plates, was obtained from all subjects. The grade ofdegeneration was scored by 3 individual observers, using theclassification system described by Thompson et al (13).

IVD tissue from 21 human subjects ages 3–72 years(mean � SD 36 � 21 years, median 35 years) was evaluated.Sagittal slices of the motion segments were fixed in formalin,decalcified for 6 hours with Kristensen’s solution (50% formicacid and 68 gm/liter sodium formiate) in a microwave at 150Wand 50°C, dehydrated in graded ethanol series, and embeddedin paraffin. Sections were deparaffinized, treated with 3%hydrogen peroxide in methanol for 30 minutes, and thentreated with heated (95°C) citrate buffer (10 mM sodiumcitrate, 0.05% Tween 20 [pH 6.0]) for 15 minutes, for antigenretrieval. They were then blocked for 1 hour with 5% normalhorse serum, and were incubated with 0.04 mg/liter rabbitanti–carbonic anhydrase XII (anti-CAXII) precursor antibody(catalog no. HPA008773; Prestige Antibodies, Sigma) at a 1:60dilution overnight at 4°C. Negative control sections were

Table 1. Data on autopsy subjects from whom samples were ob-tained, and sample characteristics*

Subject/age/sex,tissue type

Thompsongrade

RNA integritynumber

1/61/FNP 3 9.2AF 3 9.2AC 3 8.9

2/72/MNP 4 8.6AF 4 8.7AC 4 9.1

3/81/FNP 3 9.3AF 3 9.1AC 3 9.4

4/25/MNP 1 8.7AF 1 8.1

5/56/MNP 3 8.5AF 3 7.8AC 3 8.5

6/32/MNP 1–2 7.9AF 1–2 8.5AC† 1–2 8.9

7/43/M‡NP 2 NA

* Articular cartilage (AC) was not available from subject 4. NP �nucleus pulposus; AF � anulus fibrosus; NA � not applicable.† Sample had a 3�:5� ratio of �3 on one of the housekeeping genes,and was therefore excluded from further analysis.‡ Sample was included for reverse transcription–polymerase chainreaction studies, but not for microarray analysis.

3878 POWER ET AL

incubated without primary antibody. Biotinylated secondaryanti-rabbit antibody diluted 1:200 (Vectastain ABC kit Elite,catalog no. PK-6102; Vector) was applied, followed by additionof ABC complex and chromogen development using diamino-benzidine (DAB Kit, catalog no. SK-4100; Vector). Sectionswere counterstained with Mayer’s hematoxylin. To evaluatethe composition of the extracellular matrix (ECM) in theimmunostained IVD sections, separate sections were alsostained with Alcian blue/picrosirius red and observed both inbrightfield and polarized light. The Alcian blue/picrosiriusmethod is known to produce distinctive staining of collagen(red) and proteoglycans, including hyaluronan (blue) (24).

Effect of CAXII inhibition. NP cells were isolated, asdescribed above, from the tails of 3–4-month-old cows. Cellswere seeded in 24-well plates at a density of 50,000 per welland cultured in low-glucose DMEM containing 10% fetal calfserum at an oxygen concentration of either 5% or 21%. After1 day of culture the medium was changed and 4-aminobenzenesulfonamide (Sigma), a CA inhibitor with specificity forCAXII, was added at concentrations of 1, 10, 100, 1,000, and10,000 nM (25). The lactate concentration in the culturemedium was measured 24 hours after addition of4-aminobenzene sulfonamide (26) and was normalized to theconcentration in the control medium without CA inhibitortreatment. Cytotoxicity of 4-aminobenzene sulfonamide (atconcentrations of up to 10,000 nM) toward NP cells wasdetermined using WST-1 Cell Proliferation Reagent accordingto the protocol of the manufacturer (Roche). A general linearmodel with multivariate tests and Bonferroni adjustment wasused for statistical analysis of the effect of CA inhibitortreatment.

RESULTS

Unsupervised clustering. Hierarchical clusteringand correspondence were used to identify gene expres-sion profiles that differed between the tissue types. TheAC samples were clearly separated from the AF and NP

samples using both of these unsupervised clusteringtechniques. However, there was no clear separationbetween AF and NP samples. Results of the hierarchicalclustering analysis are shown in Figure 1. When the datawere sorted and viewed by subject, a trend toward anintrasubject gene profile similarity between the AF andNP samples was observed. Correspondence analysisresults are available from the corresponding authorupon request.

Differential expression. With the use of a strict Pvalue cutoff of P � 0.01, there were no genes that weredifferentially expressed between NP and AF. Increasingthe cutoff to P � 0.05 resulted in the identification of6,611 genes that were differentially expressed betweenNP and AF, NP and AC, or AF and AC; however only3 of these were differentially expressed between NP andAF. These 3 probe sets were annotated to SPARCL1,NFATC4, and one that only had a GenBank accessionnumber (AW003173). In summary, the lack of globalgene expression differences between NP and AF, asdisplayed by the unsupervised clustering techniques, wasconfirmed by the lack of highly significant P valuesbetween these groups.

Cell surface markers. In order to focus theexperiment toward cell surface markers within the NPsamples, transmembrane domain–containing genes wereidentified from genes that were found to be up-regulatedin the NP samples using BioMart (27). Starting with acutoff of P � 0.01, 552 genes were up-regulated in NPsamples in comparison to AC samples, 90 of whichcontained transmembrane domains (Table 2).

Figure 1. Hierarchical clustering results, by sample type (articular cartilage [AC], anulus fibrosus[AF], and nucleus pulposus [NP]) (A) and by subject number (B). Each sample type and subject isrepresented by a different color in the bars below the diagrams. Color figure can be viewed in theonline issue, which is available at http://onlinelibrary.wiley.com/journal/10.1002/(ISSN)1529-0131.

DETECTION OF CAXII AS A NOVEL NP CELL SURFACE MARKER 3879

Table 2. Top 90 transmembrane domain–containing genes that were found to be up-regulated in nucleus pulposus*

Symbol IDLog foldchange Adjusted P†

Average log2

expression‡ Description

ODZ3§ 219523_s_at –2.17 0.0000015 6.77 Odz, odd Oz/10-m homolog 3 (Drosophila)DNER§ 226281_at –3.5 0.0000015 10.23 Delta/notch-like epidermal growth factor repeat–

containingFLRT2§ 204358_s_at –2.43 0.0000087 6.69 Fibronectin leucine-rich transmembrane protein 2PERP 217744_s_at –1.59 0.0000164 9.1 p53 apoptosis effector related to peripheral

myelin protein 22, TP53 apoptosis effectorCHPT1 221675_s_at –1.2 0.0000361 10.83 Choline phosphotransferase 1CLEC2B§ 209732_at –4.07 0.0000996 8.02 C-type lectin domain family 2, member BJPH3 229294_at –2.53 0.0001339 5.5 Junctophilin 3SLC14A1 229151_at –3.63 0.0001348 8.16 Solute carrier family 14 (urea transporter),

member 1 (Kidd blood group)CANX 200068_s_at –1.07 0.0001402 11.37 CalnexinCLDN11 228335_at –3.4 0.0001573 7.04 Claudin 11PTPLA 219654_at –1.94 0.0001622 6.91 Protein tyrosine phosphatase–like (proline instead

of catalytic arginine), member ARNF128 219263_at –2.05 0.0001637 5.71 Ring finger protein 128BACE2 217867_x_at –1.91 0.0001637 7.55 �-site APP-cleaving enzyme 2DSG2§ 1553105_s_at –2.26 0.0001956 7.52 Desmoglein 2ZDHHC9 222451_s_at –0.8 0.0002071 8.73 Zinc finger, DHHC-type–containing 9ATRNL1§ 1569796_s_at –1.97 0.000345 6.49 Attractin-like 1WFS1 1555270_a_at –1.26 0.0004581 7.33 Wolfram syndrome 1 (wolframin)SLC44A1§ 228486_at –1.72 0.0004744 7.11 Solute carrier family 44, member 1CRTAP 1555889_a_at –0.86 0.0004947 10.57 Cartilage-associated proteinPTGFR 207177_at –2.03 0.0005338 6.24 Prostaglandin F receptor FPCDS1 205709_s_at –1.63 0.0006395 6.66 CDP-diacylglycerol synthase (phosphatidate

cytidylyltransferase) 1CA12§ 203963_at –3.1 0.0006808 7.09 Carbonic anhydrase XIIRNF130§ 217865_at –0.82 0.0006808 9.88 Ring finger protein 130ORAI3 221864_at –1.24 0.0006934 9.01 Calcium release–activated calcium modulator 3TMEM106A§ 1552302_at –2.16 0.0007158 5.28 Transmembrane protein 106AINSIG1 201625_s_at –2.02 0.0007539 7.29 Insulin-induced gene 1LRRN4CL§ 1556427_s_at –1.88 0.0007752 8.27 Leucine-rich repeat neuronal 4, C-terminal–likeLAMP2 200821_at –0.66 0.0008272 9.76 Lysosomal-associated membrane protein 2AGPAT9 224480_s_at –1.96 0.0008453 8.56 1-acylglycerol-3-phosphate O-acyltransferase 9KCNS3 205968_at –2.11 0.0009038 5.67 Potassium voltage–gated channel, delayed-

rectifier, subfamily S, member 3CDH19§ 206898_at –2.15 0.0009091 8.97 Cadherin 19, type 2TMEM27§ 223784_at –2.16 0.0010235 4.8 Transmembrane protein 27MXRA7§ 212509_s_at –1.04 0.0011493 11.91 Matrix remodeling–associated 7F2RL1 213506_at –2.37 0.0012699 4.25 Coagulation factor II (thrombin) receptor–like 1BCL2L13 217955_at –0.83 0.0013199 8.9 Bcl-2–like 13 (apoptosis facilitator)IGF1R§ 203627_at –1.3 0.0013493 7.97 Insulin-like growth factor 1 receptorNETO2§ 218888_s_at –2.28 0.0013561 5.23 Neuropilin and tolloid–like 2KCNMB4 222857_s_at –1.47 0.0013864 6.13 Potassium large conductance calcium-activated

channel, subfamily M, beta member 4SLC38A7 218727_at –0.68 0.0014735 5.85 Solute carrier family 38, member 7TYRO3§ 211432_s_at –1.06 0.0014962 6.21 TYRO3 protein tyrosine kinasePDIA4 208658_at –1.4 0.0015233 8.45 Protein disulfide isomerase family A, member 4REEP1 204364_s_at –1.29 0.001669 4.86 Receptor accessory protein 1SLC27A2 205769_at –1.61 0.0016925 5.17 Solute carrier family 27 (fatty acid transporter),

member 2SLC7A7 204588_s_at –1.32 0.0018381 7.04 Solute carrier family 7 (cationic amino acid

transporter, y� system), member 7GCNT1 205505_at –1.64 0.0018832 7.98 Glucosaminyl (N-acetyl) transferase 1, core 2

(�-1,6-N-acetylglucosaminyltransferase)CRHR1§ 214619_at –0.55 0.0020058 5.08 Corticotropin-releasing hormone receptor 1SEL1L 202064_s_at –0.56 0.0020525 6.72 Sel-1 suppressor of lin 12–like (Caenorhabditis

elegans)ABCG1 204567_s_at –2.5 0.0020546 5.05 ATP-binding cassette, subfamily G, member 1SLC7A1 206566_at –0.83 0.0020546 4.86 Solute carrier family 7 (cationic amino acid

transporter, y� system), member 1MTDH 212250_at –0.47 0.002086 10.12 MetadherinARMCX6§ 214749_s_at –0.92 0.00217 10.13 Armadillo repeat–containing, X-linked 6

3880 POWER ET AL

Gene list reduction. Further reduction of thegene list was required in order to obtain the most worthycandidates for laboratory validation and for potentialuse as an NP target using phage display and cloning.

Gene list reduction according to the methods describedabove for short-listing of candidate genes generated afinal short list of 28 candidates from the original 90, ofwhich 23 were “independent” candidates and the other 5

Table 2. (Cont’d)

Symbol IDLog foldchange Adjusted P†

Average log2

expression‡ Description

GPR175 218855_at –0.73 0.0022858 7.62 G protein–coupled receptor 175NPAL3 214579_at –0.55 0.0024259 6.48 Nuclear interacting partner of anaplastic

lymphoma kinase–like domain–containing 3SSR2 200652_at –0.73 0.0025448 11.26 Signal sequence receptor, beta (translocon-

associated protein �)LRRC15 213909_at –2.87 0.0025708 6.76 Leucine-rich repeat–containing 15HS6ST3 232276_at –1.01 0.0028393 5.13 Heparan sulfate 6-O-sulfotransferase 3FZD5 221245_s_at –1.73 0.0031143 8.5 Frizzled homolog 5 (Drosophila)SLC22A17 218675_at –1.07 0.0035063 7.3 Solute carrier family 22, member 17MYADM 224920_x_at –1.52 0.003514 7.96 Myeloid-associated differentiation markerDUOX1 219597_s_at –0.55 0.0035606 4.34 Dual oxidase 1NRXN3§ 229649_at –1.21 0.0041662 4.52 Neurexin 3TSPAN31§ 203227_s_at –0.83 0.0041663 9.57 Tetraspanin 31KCNE3 227647_at –1.42 0.0041803 6.24 Potassium voltage–gated channel, Isk-related

family, member 3YME1L1 201352_at –0.7 0.0044695 10.73 Yeast mitochondrial escape–like 1

(Saccharomyces cerevisiae)BMPR1B 229975_at –1.32 0.0044806 4.91 Bone morphogenetic protein receptor, type 1BFAM62B§ 1558511_s_at –0.81 0.0044892 9 Family with sequence similarity 62 (C2 domain–

containing) member BRELL1 226430_at –1.17 0.0047258 10.36 Receptor expressed in lymphoid tissues–like 1FKBP11 219117_s_at –1.45 0.0050497 8.88 FK-506 binding protein 11, 19 kdSGCG§ 207302_at –1.83 0.0050613 4.55 Sarcoglycan, gamma (35-kd dystrophin-associated

glycoprotein)UBE2V1 201001_s_at –0.5 0.0051113 8.95 Ubiquitin-conjugating enzyme E2 variant 1NHLRC3 227040_at –1.39 0.005253 6.81 NHL repeat–containing 3ITGB8§ 205816_at –1.89 0.0053165 5.46 �8 integrinTOMM20 200662_s_at –0.79 0.0057177 11.29 Translocase of outer mitochondrial membrane 20

homolog (yeast)CLGN 205830_at –0.66 0.0059216 3.65 CalmeginTMEM71 238429_at –2.06 0.0061826 5.76 Transmembrane protein 71GPR133 232267_at –2.66 0.0062381 6.18 G protein–coupled receptor 133FNDC3B§ 229865_at –1.08 0.0063478 7.49 Fibronectin type III domain–containing 3BSLC9A1 209453_at –0.72 0.0065287 8.38 Solute carrier family 9 (sodium/hydrogen

exchanger), member 1 (antiporter, Na�/H�,amiloride sensitive)

PPAPDC1B 226150_at –1.02 0.0069389 9.67 Phosphatidic acid phosphatase type 2 domain–containing 1B

C1orf9§ 203429_s_at –0.8 0.0072998 9.45 Chromosome 1 open-reading frame 9SLC24A1 206081_at –0.86 0.0073022 5.83 Solute carrier family 24 (sodium/potassium/

calcium exchanger), member 1MFAP3L§ 210493_s_at –1.33 0.0073299 6.51 Microfibrillar-associated protein 3–likeNRM 225592_at –0.62 0.007528 7.08 Nurim (nuclear envelope membrane protein)TFR2 210215_at –0.71 0.0079535 5.36 Transferrin receptor 2SLC22A23 223194_s_at –1.42 0.0086094 7.35 Solute carrier family 22, member 23IL31RA 243541_at –0.76 0.0091668 3.51 Interleukin 31 receptor AFZD3§ 219683_at –1.11 0.0094652 5.39 Frizzled homolog 3 (Drosophila)MAGT1 221553_at –0.96 0.0096616 8.77 Magnesium transporter 1WNT11 206737_at –0.93 0.0097059 5.49 Wingless-type murine mammary tumor virus

integration site family, member 11POR 208928_at –0.71 0.0099655 7.39 Cytochrome P450 oxidoreductase

* Genes are listed in order of P value.† Adjusted for multiple testing.‡ Normalized fluorescence intensity units.§ Analyzed by reverse transcription–polymerase chain reaction.

DETECTION OF CAXII AS A NOVEL NP CELL SURFACE MARKER 3881

were made up of 3 genes containing fibronectin type IIIdomains and 2 containing Ig-like C2 type I domains.While the latter groups may not provide the level of cellspecificity required, they were retained for RT-PCRanalysis to quantify their expression level.

RT-PCR results. Due to small sample volume,real-time PCR was carried out on NP, AF, and ACsamples from only 3 donors (subjects 4, 6, and 7). Sincethere was no AC available from subject 4, the ACsample from subject 5 was used, in order to obtain 3representative values. Of the 28 genes analyzed, 27 weredetectable in all samples (MFAP3L was undetectable),although some of them were expressed at very low levels,especially in the AC. Genes were normalized to theaverage of 4 housekeeping genes. For comparison of NPand AC an average from the 3 AC samples was taken forsubject 4, while the corresponding AC was consideredfor subjects 6 and 7. For most of the genes, the expres-sion differences between NP and AC cells found in themicroarray analysis was mirrored. Significantly higherexpression in NP versus AC cells was found forCLEC2B, ATRNL1, SLC44A1, CA12, TMEM27,

TSPAN31, and SGCG (all P � 0.01) and for DNER,DSG2, CDH19, MXRA7, NETO2, CRHR1, and TYRO3(all P � 0.05), while the differences between NP and ACcells were close to significant for ODZ3, FLRT2, andITGB8 (P � 0.07). Of these genes, CLEC2B and CA12(P � 0.01), and SGCG and TYRO3 (P � 0.05) wereadditionally found to be expressed more highly in NP ascompared to AF cells (Figure 2).

To determine potential correlations between therelative mRNA expression and the age or disc degener-ation grade of the donor, the most significantly differ-entially expressed gene, CA12, was chosen for assess-ment by real-time PCR in samples from all 7 donors.CA12 mRNA expression in NP cells was found to bestrongly negatively correlated with both age (r � �0.923)and Thompson grade (r � �0.981), as shown by Pearsoncorrelation analysis (P � 0.01). In accordance with previ-ous reports (13), there was a strong positive correlationbetween age and Thompson grade (r � 0.74, P � 0.01).These results indicated an obvious reduction in CA12mRNA expression with degrading NP cells. This signif-icant result suggested that CAXII could possibly be used

Figure 2. Transmembrane domain–containing proteins exhibiting significantly higher relativegene expression in nucleus pulposus (NP) cells compared to articular cartilage (AC) cells. Geneexpression was normalized to the average of 4 housekeeping genes and is presented as log2-transformed values. Data are shown as box plots, where the boxes represent the 25th to 75thpercentiles, the lines within the boxes represent the median, and the lines outside the boxesrepresent the 10th and 90th percentiles. � � genes more highly expressed in NP cells than in anulusfibrosus (AF) cells as well as AC cells.

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as a sensitive biomarker of degradation and as a targetfor NP cells, as per the primary aim of this study.

Immunohistochemistry results. CAXII was se-lected for immunohistochemical evaluation because ofits relatively high mRNA expression level in the NP andits markedly stronger expression in NP compared toboth AF and AC cells. The results of CAXII immuno-labeling of NP tissue sections are summarized in Table 3.Although CAXII is known to be specifically expressed atthe cell surface (28,29), its transmembrane localizationcannot be recognized using conventional immunohisto-chemical methods, as the appearance of the staining ishighly dependent on the exact sectioning plane of thesample.

Strong immunoreactivity for CAXII was ob-served in the large notochord-like cells of the NP of the3-year-old female donor (Figures 3A and B). Also in thissection, some cells of the inner AF stained positive,whereas CAXII immunoreactivity was not noted ineither AF or the cartilaginous end plate of any otherIVD section probed. In healthy discs (Thompson grade1–2) from adolescent and young adult donors, only a fewpositive cells were identified. Immunoreactive cells wereeither located as single cells or occasionally arranged incell clusters (Figures 3C and D). In discs with more

degeneration (Thompson grade 3–4) from older donors,the cells that stained for CAXII were more abundant(Figures 3E and F). Finally, the most intense immuno-labeling was visible in the tissue of severely degenerateddiscs (Thompson grade 5) (Figures 3G and H). To verifythe specificity of the reaction, corresponding negativecontrol sections for all positive areas were examined,and staining was not found in any.

Figure 3. Immunolabeling of nucleus pulposus (NP) cells for carbonicanhydrase XII (CAXII). A and B, NP of a 3-year-old female subject.Fine granular cytoplasmatic CAXII labeling is seen. C and D, NP of a17-year-old male subject. Clusters of CAXII-positive cells are evident.E and F, NP of a 47-year-old female subject with Thompson grade 3disc degeneration. Perinuclear CAXII labeling is seen. G and H, NP ofa 62-year-old male subject with Thompson grade 5 disc degeneration.Intense positive labeling for CAXII can be detected in cells of diseasedtissue. B, D, F, and H are higher-magnification views of the boxedareas in A, C, E, and G, respectively. Bars in A, C, E, and G � 50 �m;bars in B, D, F, and H � 10 �m.

Table 3. Immunohistochemical results on intervertebral discsections*

Section no.(donor age/sex)

Thompsongrade

CAXII stainingof NP

1 (3/F) 1 ���2 (6/F) 1 (�)3 (14/M) 1 ��4 (14/F) 1 (�)6 (18/M) 1 –7 (19/F) 1 �8 (21/M) 1 �9 (22/F) 1 �5 (17/M) 2 ��10 (35/F) 2 �11 (35/M) NA �12 (36/M) 2 �13 (41/M) 2 (�)15 (51/F) 2 (�)14 (47/F) 3 ��19 (63/M) 3 ��16 (54/F) 4 ��17 (57/M) 4 �18 (62/M) 5 ���20 (67/F) 5 ���21 (72/M) 5 ���

* Nucleus pulposus (NP) tissue of discs between L4 and L5 wasassessed. (�) � 1–2 positive cells per field of view; � � 3–4 positivecells; �� � 5–10 positive cells; ��� � �10 positive cells. CAXII �carbonic anhydrase XII; NA � not available.

DETECTION OF CAXII AS A NOVEL NP CELL SURFACE MARKER 3883

In Alcian blue/picrosirius red–stained sections,mainly blue labeling of proteoglycans was observed inThompson grade 1–2 discs from young subjects, redstaining was apparent in grade 3 moderately degener-ated discs, and only pathologic tissue was detected inseverely degenerated discs. Observations with polarizedlight confirmed the increasing amount of collagenousfibers with increased degeneration. In degenerated discs,a detailed image of the collagen fibers could be obtainedunder polarized light only (images available from thecorresponding author upon request).

Effect of CAXII inhibition. In low concentrations(1 nM and 10 nM), 4-aminobenzene sulfonamide did notaffect the lactate level in NP cell–conditioned media.However, at concentrations of 100, 1,000, and 10,000nM, respectively, the CA inhibitor significantly (P �0.01) reduced the relative lactate level to 0.891 � 0.045,0.880 � 0.060, and 0.867 � 0.059 (mean � SD valuesrelative to the concentration in control medium [set at1]) in the medium with NP cells cultured under 5%oxygen conditions; the lactate level in NP cells culturedat 21% oxygen tension was affected by CA inhibitor inhigh concentration only (10,000 nM). No cytotoxic effectof 4-aminobenzene sulfonamide on NP cells was ob-served at the concentrations studied.

DISCUSSION

Conceptually, as NP cells are considered both aunique and a contained cell type as well as the major,constant contributor to IVD degeneration, localizedtargeting is a viable option for IVD therapy. Thisapproach requires cell surface–specific markers, and wehence adopted a systematic approach to carry out theirdetection. DNA microarrays were used to facilitate aglobal transcriptomic approach to identifying transmem-brane domain–containing genes that are differentiallyexpressed between NP cells and AF/AC cells. Theunsupervised clustering transcriptomic results revealedsimilarity between the NP and AF cell types and adistinctive difference between the NP/AF and AC sam-ples. The former result was unexpected for 2 reasons: 1)the different origins of the tissue types, with the ongoing(but nonexclusive) hypothesis that NP is the remnant ofthe notochord (30) and AF is from the mesoderm (31),and 2) a clear morphologic distinction, as visible by earlyThompson grades.

The NP contains mostly type II collagen andproteoglycans in a random orientation, whereas the AFconsists of lamellae composed of highly orientated typesI and II collagen and proteoglycans (32). Looking spe-

cifically at our data set, this profile is consistent withCOL1A1 down- and COL2A1 up-regulation in the NPin comparison to AF; however, for both genes, thedifference in expression between NP and AF did notreach statistical significance (data not shown). Whilethere may be clear phenotypic differences between NPand AF cells that are healthy or only mildly degenerated,this distinction becomes less clear in older, more highlydegenerated discs. However, the observed lack of differ-ences between the 2 IVD tissue types cannot be attrib-utable solely to the similarity between the tissues duringlater-stage disc degeneration, since differences betweenNP and AF were also not observed in samples with earlydegeneration. It rather indicates the similarity of the AFand NP cell phenotypes in spite of different cell mor-phology and ECM composition of the tissues, which maybe related to the different mechanical environment towhich the cells are exposed. More subtle and individualgene differences must be attributable to the morpho-logic differences between the IVD cell types.

With regard to the difference between the NPand AC profiles, NP cells are morphologically similar toAC cells, both having a round, chondrocytic morphology(33), from which one might assume a functional similar-ity. However, it has been reported that the ECM inwhich they reside differs in terms of proteoglycan andcollagen content (34–36), and Nettles et al (8) suggestedthat there is a strong physical interaction between cellsand ECM and hypothesized that the composition of theECM may contribute to the differing cellular responsesto mechanical loading between anatomic zones (37).This difference is obvious in our analyses and maypartially explain the number of differentially expressedgenes between these cell types. Only 3 probe sets werefound that fit (albeit marginally) under our significancethreshold of P � 0.05 for the difference between AF andNP, with each having a P value of 0.049, further illus-trating the heterogeneous nature of the samples fromdifferent donors. Pursuing a search for transmembrane-associated genes meant treating the NP and AF popu-lations as one and searching for significant differencesfrom the cartilage samples. CAXII was the most viablecandidate, as it had a high level of gene expressionrelative to the housekeeping genes and was more abun-dantly expressed in the NP than AF and AC cells. Infact, CA12 was recently described by Minogue et al as amarker gene for human NP cells (38).

The mRNA and protein data for CAXII did notdirectly correlate. Moreover, mRNA levels in Thompsongrade 5 samples could not be assessed to corroborate theimmunohistochemistry results because the low cell den-

3884 POWER ET AL

sity did not allow for gene expression profiling; thus, it isnot clear to what extent the disparity is present at thisstage of degeneration. However, the finding of decreas-ing mRNA and protein levels with early aging combinedwith a reexpression of the protein in degenerated tissueparallels observations on other potential NP markers,such as keratin 19 (12). Carbonic anhydrases catalyzethe reversible hydration of carbon dioxide into protonsand bicarbonate. In tumor cells, hypoxia-inducibleCAIX and CAXII have been shown to promote cellsurvival and growth through maintenance of the intra-cellular pH (39). There is little available informationabout the pH regulation in IVD cells. Ion transportpathways have been investigated (40), and distinct mol-ecules that allow for survival in a low-pH environmenthave been identified (41). We found reduced lactaterelease upon CA inhibition in samples cultured underlow oxygen conditions similar to those in IVD cells invivo, and this suggests that intracellular pH regulationmay be disturbed, leading to intracellular acidosis. Athigher concentrations the CAXII inhibitor used in thisstudy may also affect other CAs (25), leading to dysregu-lation of the cell metabolism under normoxic conditionsas well.

CAXII may thus function to counteract cellularacidosis in the hypoxic NP, which results from lactic acidaccumulation following glucose metabolism and lack ofwaste product removal. While CAXII expression in cellsof young growing NP may be necessary for cellular pHregulation during high metabolic activity during devel-opment, the induction of this molecule in degenerativediscs may indicate a response to increased metabolicactivity following inflammation or catabolic changes.Such cells with enhanced CAXII expression could betargeted for delivery of anabolic, anticatabolic, and/orantiinflammatory agents. Considering the intense stain-ing in 6 of 7 Thompson grade 3–4 discs in the presentstudy, CAXII may be suitable for targeting discs withmoderate to severe degenerative changes.

Although at present it is still not clear whichdegenerating discs will actually result in back pain,treatment at adjacent levels after spinal fusion will beamong the first clinical targets, as this surgical procedurecommonly leads to adjacent-level degeneration. In ad-dition, the application of therapeutic cargo–carryingnanoparticles in patients with NP protrusion (anothercondition that invariably leads to disc degeneration) isenvisaged. Studies to verify whether cells in the NP ofthese patients also produce CAXII are ongoing.

In summary, we have applied standard molecularbiology tools to identify cell surface proteins that are

specific for IVD cells. We used a sample set of humantissue rather than tissue from other mammals, as inter-species variations are common and the research ques-tion directly aims to advance toward application in thehuman patient. Ultimately, we created a short list ofcandidate cell surface markers that can be targeted infuture studies, with CAXII showing promise as anintermediate-to-late–stage degeneration target, whichmay be of crucial importance in the preventive treatmentof disc degeneration leading to chronic low back pain.

ACKNOWLEDGMENTS

The authors thank Dr. Stefan Milz and ChristophSprecher for their assistance with immunohistochemicalanalysis and Dr. F. Cumhur Oner for support with samplescoring.

AUTHOR CONTRIBUTIONS

All authors were involved in drafting the article or revising itcritically for important intellectual content, and all authors approvedthe final version to be published. Dr. Grad had full access to all of thedata in the study and takes responsibility for the integrity of the dataand the accuracy of the data analysis.Study conception and design. Power, Grad, Wall, Alini, Pandit,Gallagher.Acquisition of data. Power, Grad, Rutges, Creemers, van Rijen,Pandit.Analysis and interpretation of data. Power, Grad, O’Gaora, Wall,Alini, Pandit, Gallagher.

REFERENCES

1. Adams MA, Roughley PJ. What is intervertebral disc degenera-tion, and what causes it? Spine 2006;31:2151–61.

2. Coppes MH, Marani E, Thomeer RT, Groen GJ. Innervation of“painful” lumbar discs. Spine 1997;22:2342–9.

3. Johnson WE, Evans H, Menage J, Eisenstein SM, El Haj A,Roberts S. Immunohistochemical detection of Schwann cells ininnervated and vascularized human intervertebral discs. Spine2001;26:2550–7.

4. Khan SN, Stirling AJ. Controversial topics in surgery: degenera-tive disc disease: disc replacement. Against. Ann R Coll Surg Engl2007;89:6–11.

5. Fassett DR, Kurd MF, Vaccaro AR. Biologic solutions for degen-erative disk disease. J Spinal Disord Tech 2009;22:297–308.

6. Kalson NS, Richardson S, Hoyland JA. Strategies for regenerationof the intervertebral disc. Regen Med 2008;3:717–29.

7. Gilchrist CL, Chen J, Richardson WJ, Loeser RF, Setton LA.Functional integrin subunits regulating cell-matrix interactions inthe intervertebral disc. J Orthop Res 2007;25:829–40.

8. Nettles DL, Richardson WJ, Setton LA. Integrin expression incells of the intervertebral disc. J Anat 2004;204:515–20.

9. Xia M, Zhu Y. Expression of integrin subunits in the herniatedintervertebral disc. Connect Tissue Res 2008;49:464–9.

10. Fujita N, Miyamoto T, Imai J, Hosogane N, Suzuki T, Yagi M, etal. CD24 is expressed specifically in the nucleus pulposus ofintervertebral discs. Biochem Biophys Res Commun 2005;338:1890–6.

11. Sakai D, Nakai T, Mochida J, Alini M, Grad S. Differential

DETECTION OF CAXII AS A NOVEL NP CELL SURFACE MARKER 3885

phenotype of intervertebral disc cells: microarray and immunohis-tochemical analysis of canine nucleus pulposus and anulus fibro-sus. Spine 2009;34:1448–56.

12. Rutges J, Creemers LB, Dhert W, Milz S, Sakai D, Mochida J, etal. Variations in gene and protein expression in human nucleuspulposus in comparison with annulus fibrosus and cartilage cells:potential associations with aging and degeneration. OsteoarthritisCartilage 2010;18:416–23.

13. Thompson JP, Pearce RH, Schechter MT, Adams ME, Tsang IK,Bishop PB. Preliminary evaluation of a scheme for grading thegross morphology of the human intervertebral disc. Spine 1990;15:411–5.

14. Reno C, Marchuk L, Sciore P, Frank CB, Hart DA. Rapidisolation of total RNA from small samples of hypocellular, denseconnective tissues. Biotechniques 1997;22:1082–6.

15. Lee CR, Sakai D, Nakai T, Toyama K, Mochida J, Alini M, et al.A phenotypic comparison of intervertebral disc and articularcartilage cells in the rat. Eur Spine J 2007;16:2174–85.

16. R Development Core Team. R: a language and environment forstatistical computing. Vienna (Austria): R Foundation for Statis-tical Computing; 2009.

17. Gentleman RC, Carey VJ, Bates DM, Bolstad B, Dettling M,Dudoit S, et al. Bioconductor: open software development forcomputational biology and bioinformatics. Genome Biol 2004;5:R80.

18. Irizarry RA, Hobbs B, Collin F, Beazer-Barclay YD, AntonellisKJ, Scherf U, et al. Exploration, normalization, and summaries ofhigh density oligonucleotide array probe level data. Biostatistics2003;4:249–64.

19. Culhane AC, Thioulouse J, Perriere G, Higgins DG. MADE4: anR package for multivariate analysis of gene expression data.Bioinformatics 2005;21:2789–90.

20. Smyth GK. Linear models and empirical Bayes methods forassessing differential expression in microarray experiments. StatAppl Genet Mol Biol 2004;3:Article3.

21. Smyth GK. Limma: linear models for microarray data. In: Gen-tleman R, Carey V, Huber W, Irizarry R, Dudoit S, editors.Bioinformatics and computational biology solutions using R andBioconductor. New York: Springer; 2005. p. 397–420.

22. Benjamini Y, Hochberg Y. Controlling the false discovery rate: apractical and powerful approach to multiple testing. J R Stat SocSer B 1995;57:289–300.

23. Livak KJ, Schmittgen TD. Analysis of relative gene expressiondata using real-time quantitative PCR and the 2���CT method.Methods 2001;25:402–8.

24. Gruber HE, Ingram J, Hanley EN. An improved staining methodfor intervertebral disc tissue. Biotech Histochem 2002;77:81–3.

25. Vullo D, Innocenti A, Nishimori I, Pastorek J, Scozzafava A,Pastorekova S, et al. Carbonic anhydrase inhibitors. Inhibition ofthe transmembrane isozyme XII with sulfonamides—a new targetfor the design of antitumor and antiglaucoma drugs? Bioorg MedChem Lett 2005;15:963–9.

26. Heywood HK, Bader DL, Lee DA. Rate of oxygen consumption byisolated articular chondrocytes is sensitive to medium glucoseconcentration. J Cell Physiol 2006;206:402–10.

27. Smedley D, Haider S, Ballester B, Holland R, London D, Thoris-son G, et al. BioMart—biological queries made easy. BMC Genom-ics 2009;10:22.

28. Tureci O, Sahin U, Vollmar E, Siemer S, Gottert E, Seitz G, et al.Human carbonic anhydrase XII: cDNA cloning, expression, andchromosomal localization of a carbonic anhydrase gene that isoverexpressed in some renal cell cancers. Proc Natl Acad Sci U SA 1998;95:7608–13.

29. Karhumaa P, Parkkila S, Tureci O, Waheed A, Grubb JH, Shah G,et al. Identification of carbonic anhydrase XII as the membraneisozyme expressed in the normal human endometrial epithelium.Mol Hum Reprod 2000;6:68–74.

30. Rutges JP, Nikkels PG, Oner FC, Ottink KD, Verbout AJ,Castelein RJ, et al. The presence of extracellular matrix degradingmetalloproteinases during fetal development of the intervertebraldisc. Eur Spine J 2010;19:1340–6.

31. Dudek R, Fix J. Embryology. Philadelphia: Lippincott Williams &Wilkins; 2004.

32. Schollmeier G, Lahr-Eigen R, Lewandrowski KU. Observationson fiber-forming collagens in the anulus fibrosus. Spine 2000;25:2736–41.

33. Sive JI, Baird P, Jeziorsk M, Watkins A, Hoyland JA, FreemontAJ. Expression of chondrocyte markers by cells of normal anddegenerate intervertebral discs. Mol Pathol 2002;55:91–7.

34. Mwale F, Roughley P, Antoniou J. Distinction between theextracellular matrix of the nucleus pulposus and hyaline cartilage:a requisite for tissue engineering of intervertebral disc. Eur CellMater 2004;8:58–63.

35. Clouet J, Grimandi G, Pot-Vaucel M, Masson M, Fellah HB,Guigand L, et al. Identification of phenotypic discriminatingmarkers for intervertebral disc cells and articular chondrocytes.Rheumatology (Oxford) 2009;48:1447–50.

36. Vonk LA, Kroeze RJ, Doulabi BZ, Hoogendoorn RJ, Huang C,Helder MN, et al. Caprine articular, meniscus and intervertebraldisc cartilage: an integral analysis of collagen network and chon-drocytes. Matrix Biol 2010;29:209–18.

37. Errington RJ, Puustjarvi K, White IR, Roberts S, Urban JP.Characterisation of cytoplasm-filled processes in cells of theintervertebral disc. J Anat 1998;192:369–78.

38. Minogue BM, Richardson SM, Zeef LA, Freemont AJ, HoylandJA. Characterization of the human nucleus pulposus cell pheno-type and evaluation of novel marker gene expression to defineadult stem cell differentiation. Arthritis Rheum 2010;62:3695–705.

39. Chiche J, Ilc K, Laferriere J, Trottier E, Dayan F, Mazure NM, etal. Hypoxia-inducible carbonic anhydrase IX and XII promotetumor cell growth by counteracting acidosis through the regulationof the intracellular pH. Cancer Res 2009;69:358–68.

40. Razaq S, Urban JP, Wilkins RJ. Regulation of intracellular pH bybovine intervertebral disc cells. Cell Physiol Biochem 2000;10:109–15.

41. Uchiyama Y, Cheng CC, Danielson KG, Mochida J, Albert TJ,Shapiro IM, et al. Expression of acid-sensing ion channel 3(ASIC3) in nucleus pulposus cells of the intervertebral disc isregulated by p75NTR and ERK signaling. J Bone Miner Res2007;22:1996–2006.

3886 POWER ET AL