Osteoarthritis and Cartilage 20 (2012) 430e439

Osteoarthritis-like changes in the heterozygous sedc mouse associated with theHtrA1eDdr2eMmp-13 degradative pathway: a new model of osteoarthritis

D.W. Holt y, M.L. Henderson y, C.E. Stockdale y, J.T. Farrell y, D.L. Kooyman y, L.C. Bridgewater z,R.E. Seegmiller yx*yDepartment of Physiology and Developmental Biology, Brigham Young University, Provo, UT 84602, USAzDepartment of Microbiology and Molecular Biology, Brigham Young University, Provo, UT 84602, USAxCollege of Dental Medicine, Roseman University of Health Sciences, South Jordan, UT 84095, USA

a r t i c l e i n f o

Article history:Received 20 April 2011Accepted 21 November 2011

Keywords:Spondyloepiphyseal dysplasia congenitaCartilage degradationHtrA1Ddr2Mmp-13OsteoarthritisType II collagen mutation

* Address correspondence and reprint requestsDepartment of Physiology and Developmental BiologProvo, UT 84602, USA. Tel: 1-801-422-2303; Fax: 1-8

E-mail address: robert_seegmiller@byu.edu (R.E. S

1063-4584/$ e see front matter � 2011 Osteoarthritidoi:10.1016/j.joca.2011.11.008

s u m m a r y

Objective: To test the hypothesis that the spondyloepiphyseal dysplasia congenita (sedc) heterozygous(sedc/þ) mouse, a COL2A1 mutant, is a model for the study of osteoarthritis (OA) in the absence ofdwarfism and to investigate the presence of HtrA1, Ddr2, and Mmp-13 and their possible involvement ina universal mechanism leading to OA.Design: Whole mount skeletons of adult animals were analyzed to determine whether sedc/þ miceexhibit dwarfism. To characterize progression of osteoarthritic degeneration over time, knee andtemporomandibular joints from sedc/þ and wild-type mice were analyzed histologically, and severity ofarticular cartilage degradation was graded using the Osteoarthritis Research Society International(OARSI) scoring system. Immunohistochemistry was used to detect changes in expression of HtrA1, Ddr2,and Mmp-13 in articular cartilage of knees.Results: As previously reported, the sedc/þ skeleton morphology was indistinguishable fromwild type, andskeletal measurements revealed no significant differences. The sedc/þ mouse did, however, show signifi-cantly higherOARSI scores in knee (9,12 and 18months) and temporomandibular joints at all ages examined.Histological staining showed regions of proteoglycan degradation as early as 2 months in both temporo-mandibular and knee joints of themutant. Cartilage fissuring and erosionwere observed to begin between 2and 6 months in temporomandibular joints and 9 months in knee joints from sedc/þ mice. Immunohisto-chemistry of mutant knee articular cartilage showed increased expression of HtrA1, Ddr2, and Mmp-13compared to wild type, which upregulation preceded fibrillation and fissuring of the articular surfaces.Conclusions: With regard to skeletal morphology, the sedc/þ mouse appears phenotypically normal butdevelops premature OA as hypothesized. We conclude that the sedc/þ mouse is a useful model for thestudy of OA in individuals with overtly normal skeletal structure and a predisposition for articularcartilage degeneration.

� 2011 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.

Introduction

Osteoarthritis (OA) is a growing problem in the United States asthe population ages, affecting a reported 27 million people1.

Articular cartilage acquires its tensile strength from a densemeshwork of collagen fibrils in the extracellular matrix (ECM),including types II, IX, and XI collagen2. In addition, type VI collagenis present in the region immediately surrounding chondrocytes, thepericellular matrix (PCM), and may help the chondrocytes attach to

to: Robert E. Seegmiller,y, Brigham Young University,01-422-0700.eegmiller).

s Research Society International. P

the framework of the ECM3. An assortment of other ECM and PCMproteins and proteoglycans such as aggrecan, decorin, biglycan,fibromodulin, cartilage oligomeric matrix protein (COMP),fibronectin, and matrilin-3 interact with the collagen network inthe ECM to provide cartilage with its resistance to compression3.

Several different human chondrodysplasias are caused by muta-tions in the cartilage collagen genes. These include spondyloepiphy-seal dysplasia (SED), spondyloepimetaphyseal dysplasia(SEMD), oto-spondylo-megaepiphyseal dysplasia (OSMED), multipleepiphyseal dysplasia (MED), Kniestdysplasia,Marshall syndrome, andStickler syndrome4. These chondrodysplasia syndromes all featurevarying degrees of disproportionately short stature, craniofacialabnormalities, hearing loss, eye problems, and cleft palate and insome, degenerative changes in articular cartilage. For example, in an

ublished by Elsevier Ltd. All rights reserved.

D.W. Holt et al. / Osteoarthritis and Cartilage 20 (2012) 430e439 431

inherited form of OA associated with mild chondrodysplasia inhumans, the expression in cartilage of a type II collagen mutation atthe protein level has been documented5. Our biochemical studiesusingmousemodels of chondrodysplasia have also shown that whendefective type II collagen6 ordefective typeXI collagen7 is deposited inthe matrix of diseased cartilage, collagen fibrils are abnormal andpremature degeneration of articular cartilage results8e10.

While chondrodysplasia models of OA have been important inadvancing our understanding of OA, the majority of OA cases inhumans are not associated with any recognizable features of chon-drodysplasia. This raises the question of whether the cartilagecollagen genes play a significant role in the majority of human OA, orare only relevant in the minority of cases associated with chon-drodysplasias. A recent study by Kannu et al.11 demonstrated that, infact, at least two different heterozygous point mutations in the triplehelical domain of the COL2A1 gene can cause degenerative jointdisease in humans in the absence of other phenotypic abnormalities.

In the present study, we report OA-like changes in a mousemodel that, like the human families presented by Kannu et al., bearsa mutation in the Col2a1 gene that behaves as a recessive mutationresulting in no obvious phenotype in the heterozygote. The mousemutation was named spondyloepiphyseal dysplasia congenita(sedc) by Donahue et al.12 because when homozygous it producesfeatures that resemble themost common clinical phenotypes of SEDcongenita in humans. The sedc mutation results from an R992Csubstitution within the triple helix of the collagen a1 (II) chains.Biochemical studies of pepsin extracted collagen clearly show thepresence of disulfide bonded collagen a1 (II) chains in adult sedcmouse rib cartilage and absence in þ/þ (wild type) cartilage (Drs.D.R. Eyre and R.J. Fernandes, personal communication). In humans,an analogous mutation R519C in the triple helix of collagen a1 (II)chain has been shown to cause an inherited form of OA with mildchondrodysplasia5 and another similar mutation, R789C, also in thetriple helical region ofa1 (II) chains has been shown to cause SEDC13.Disulfide bondedmutant a1 (II) collagen chains, not normally foundin type II collagen, were detected in cartilage from both patients12.

To look for signs of OA in the overtly normal heterozygous sedc(sedc/þ) mice, we examined knee and temporomandibular jointsover the course of several months and found premature andprogressive OA as demonstrated by loss of proteoglycans in thearticular cartilage ECM,fibrillation of the articular surfaces,fissuringof the cartilage, and ultimately separation of decalcified fromcalcified articular cartilage. Furthermore, we saw increased levels ofHtrA1, a serine protease that is believed to contribute to OA bydegrading components of the PCM;Ddr2, a cell surface receptor thatbinds type II collagen and up regulates Mmp-13 expression; andMmp-13, a matrix metalloproteinase proposed to play a key role inOA progression by digesting type II collagen. Previous studies haveshown that Mmp-13 and Ddr2 knockout mice are resistant tosurgically inducedOA14,15. In the present studywe establish the sedcheterozygousmouse as amodel of OA and confirm the presence andpossible involvement of HtrA1, Ddr2, and Mmp-13 in its progres-sion. We conclude that the sedc/þ mouse is a useful model for thestudy of OA in individuals with normal skeletal structure who arepredisposed to articular cartilage degeneration.

Methods and materials

Experimental animals and genotyping

Mice (<5 per cage) were maintained in 5� 8 inches (40 squareinches) cages containing paper bedding, fed a standard mouse diet,and provided with a 12 h light/12 h dark cycle. All experimentswere approved by the Brigham Young University IACUC.

Heterozygous sedc mice (JAX� Mice # 003916 The Jackson Labo-ratory, Bar Harbor, ME) originally derived from a mixed backgroundcomprising C57BL/6J, C3H/HeJ, and AKR/J strains were crossed toproduce the þ/þ and sedc/þ mice used in this study. Animalswere identified using an ear punch system and DNA for genotypingwas isolated from tail biopsies. Genotyping was performed usingpolymerase chain reaction (PCR) amplification with the followingprimers: forward: TTGGTCCCTCTGGCAAAG; reverse: AACAGGGCCTGTTTCTCCTG. The amplified DNA was digested with BtsCI, whichcleaves the mutated nucleotide sequence, and then visualized usingagarose gel electrophoresis12.

Morphometric analysis

Male þ/þ mice (average age 10.2 weeks� 2.7) and sedc/þ mice(average 10.5 weeks� 2.5) were euthanized by CO2 inhalation andeviscerated, soft tissues were removed in 1% KOH, and skeletonswere stained in a 1% KOH, 0.004% Alizarin red solution. Specimenswere cleared in a graded glycerol series (50%, 80% and 100%). Thelength, condylar and midshaft widths of the femor, and the length,condylar, midshaft and malleolar widths of the tibia were measuredfrom both hind limbs with a dial caliper (Tresna, Essen, Germany) to0.01 mm. Length from the nose to the base of the tail was alsomeasured. The sample size was n¼ 6 for þ/þ controls and n¼ 4 forsedc/þmice. Statistical analysis was done using SPSS 18 independentsample t-test where P-values of <0.01 were considered significant.

Tissue processing

Wild-type and sedc/þ mice at 2, 6, 9, and 12 months of age wereeuthanized by CO2 inhalation, and knees (R & L) and temporoman-dibular joints were excised and fixed in 4% paraformaldehydeovernight. The tissues were decalcified in a formic acid solution thatwas changed every 2e3 days for a period of 2e3weeks. Tissues wereembedded in paraffin wax using an automated tissue processor(ThermoFisher Scientific, MA). Paraffin blocks were prepared withthe knee joint bent at a 120� angle, with the anterior tibial surfaceflush with the cutting surface. TM joints were embedded in paraffinwax with the joint flush with the cutting surface8.

Knee and TM joint blocks were sectioned at 6 mm through theentire joint from the anterior surface to the posterior. Four to fivesections were placed per glass microscope slide, yielding approxi-mately 35e50 slides or approximately 160e225 sections per joint.

Histological analysis

Histopathology was documented by Safranin O and fast greenstaining of every sixth slide of 2-, 6-, 9-, and 12-month kneesamples (2 monthsþ/þ, 2 months sedc/þ, 6 monthsþ/þ all n¼ 7, 6months sedc/þ n¼ 9, 9 months þ/þ n¼ 8, 9 months sedc/þ, 12months þ/þ, 12 months sedc/þ all n¼ 7) The same was done for 2-,6-, 9-, and 12-month temporomandibular joints (n¼ 4 for each agegroup). In the above analyses, “n” refers to the number of mice fromwhich joints were examined. Using a light microscope equippedwith a digital camera, photographs of each knee joint were taken at200� and 400� magnifications.

The articular cartilage in two representative sections from eachstained slide was analyzed using the Osteoarthritis ResearchSociety International (OARSI) scoring system to quantify the path-ological state of each joint, with a score of zero representingunaltered articular cartilage and six representing severe OA. OverallOARSI scoring was based on an osteoarthritic damage 0e6subjective scoring system applied to all four quadrants of theknee16. The OARSI scoring system was applied to the TM joint asa whole rather than quadrants. Immunohistochemistry and

Fig. 1. Heterozygous sedc skeletons appear morphologically normal compared to þ/þ. Alizarin red-stained skeletons from 3-month-old þ/þ and sedc/þ mice demonstrate that sedc/þmice show none of the skeletal changes typically associated with chondrodysplasia and are not morphometrically different from þ/þ.

D.W. Holt et al. / Osteoarthritis and Cartilage 20 (2012) 430e439432

histological analysis focused on different parts of the joint todemonstrate the universal nature of the OA in the sedc model.

Statistical analysisAmixedmodels analysis of variance (ANOVA)was performed on

the OARSI score data. The dependent variables were the OARSIscores for both the knee and TMJ. The independent variables wereage and genotype along with their interaction. Blocking was oneach animal to account for their multiple measures. Post-hoc t-testswere performed to determine differences in genotype at each age,P-values of <0.05 were considered significant.

Table Isedc/þ skeletons are not morphometrically different from þ/þ

Measurement (mm) þ/þ (n¼ 6) sedc/þ (n¼ 4)

Femur length 14.8� 0.5 14.6� 0.7Femoral midshaft width 1.5� 0.5 1.5� 0.3Tibia length 18.9� 0.6 18.8� 0.7Tibial condylar width 3.0� 0.2 3.0� 0.1Tibial midshaft width 1.1� 0.1 1.2� 0.1Tibial malleolar width 1.9� 0.2 1.9� 0.1Nose to base of tail 82.6� 1.2 80.8� 1.4

Mean� standard deviation; n¼ number of mice.

Immunohistochemical analysis

Immunohistochemistry was performed on sections of mouseknee joints from every sixth slide from 4 þ/þ and 4 sedc/þ animalsat 2-, 6-, 9-, and 12-month time points. Separate slides were stainedwith antibodies against HtrA1, Ddr2, Mmp-13 and type VI collagen.Each slide was deparaffinized and then blocked with bovine serumalbumin for 1 h. Primary antibodies against HtrA1 (ab38611)(Abcam, Cambridge, MA), against Ddr2 (SC-8989) (Santa CruzBiotechnology, Santa Cruz, CA), against Mmp-13 (AB8120)(Chemicon, Temecula, CA) and against type VI collagen (SC-9855)(Santa Cruz Biotechnology, Santa Cruz, CA). All antibodies werediluted 1:200, applied to specimens, and incubated overnight at4 �C. On the second day, samples were rinsed with PBS and thenincubated with an avidin/biotin ABC mix (Vectastain elite ABC Kit).Slides were rinsed again with PBS and incubated with biotinylatedsecondary antibody. After a third rinse, a color reaction was

initiated using a peroxidase substrate (Vector Labs, NovaRED).Negative controls were prepared by staining without the additionof primary antibody. Differences in staining intensity werecompared qualitatively with þ/þ controls. Counting of stained cellswas performed using ImageJ (NIH, Bethesda, MD).

Results

The sedc/þ mouse shows normal skeletal morphology

Consistent with that previously reported by Donahue et al.,when compared with þ/þ controls, the adult sedc/þ mouseskeleton showed normal morphology and size (Fig. 1). Morpho-metric analysis of the femor and tibia and other skeletal structuresrevealed no statistically significant differences between sedc/þ andcontrols, and the overall length of the mice from the tip of the noseto the base of the tail was also similar to control (Table I).

Fig. 2. Osteoarthritic degradation of cartilage is accelerated in sedc/þ compared toþ/þ knee joints. Large panel images were captured at 200�magnification and show the temporalprogression of OA in þ/þ and sedc/þ knee joints at the time points indicated. Insets are 400� magnifications. In 2-month-old sedc/þ joints, a localized region of proteoglycandegradation is seen in the superficial zone, as demonstrated by decreased Safranin O staining (arrow). Six-month-old sedc/þ joints show increased Safranin O staining of the PCM,accompanied by decreased Safranin O staining in the interterritorial matrix region suggestive of proteoglycan degradation (400� insets). Proteoglycan degradation is also seen inthe superficial zone (arrow). In joints from 9-, 12- and 18-month-old sedc/þ animals, large regions of the superficial zone are entirely lacking proteoglycan staining (arrows).

D.W. Holt et al. / Osteoarthritis and Cartilage 20 (2012) 430e439 433

D.W. Holt et al. / Osteoarthritis and Cartilage 20 (2012) 430e439434

Evidence of OA in sedc/þ knee joints

We examined both right and left knees. No histological differ-ence was observed between left and right knees. For purposes ofuniformity, all pictures are of right knees only.

Wild-type controls showed normal histological staining of theknee articular cartilage (Fig. 2). Only slight staining with Safranin Owas observed in the PCM whereas in the interterritorial ECMintense staining was seen in the superficial zone and moderatestaining was observed in the deep zone as an overall trend in theþ/þ age-matched controls. Decreased Safranin O staining of þ/þsamples occurred as animals aged starting sometime between 2and 6 months primarily in the deep zone adjacent to the middlezone. A decrease of proteoglycan staining was seen in the superfi-cial zone of þ/þ mice at 9 months of age.

Histological analysis of sedc/þ knee joints revealed a significantincrease of Safranin O stain in the PCM compared to controls(Fig. 2). This abnormally intense Safranin O staining in the PCMindicates an increased quantity of proteoglycan, first observed at 6months, which gradually decreased as animals aged. In spite of thisincreased quantity of proteoglycan, localized decreases in SafraninO staining were observed in the ECM of the superficial zone rep-resenting possible lesions in the articular cartilage of sedc/þ micestarting as early as 2 months (Fig. 2). The regions of decreasedSafranin O staining in the superficial zone expanded and deepenedin older sedc/þ animals, with the most significant signs of proteo-glycan degradation appearing at 12 months of age.

The OARSI scoring of sections from þ/þ and sedc/þ knee jointsrevealed that þ/þmice showed a slight progression of OAwith ageuntil 9months inwhich case progressionwas significantly elevated.However, OA in sedc/þ temporomandibular joints was more severethanþ/þmice at all ages examined [Fig. 3(A) and (B), respectively].

Fig. 3. OARSI scoring demonstrated a significant increase in the severity of OA in sedc/þcompared to þ/þ mice at 9, 12 and 18 months in knee joints [(A) n¼ 7e9 per group] and2, 6, 9 and 12 months in temporomandibular joints [(B) n¼ 4 per group]. The “n” refers tothe number of mice. P< 0.05.

Evidence of OA in the sedc/þ temporomandibular joint

Analysis of Safranin O and fast green stained temporomandib-ular joints showed that þ/þ joints had intense Safranin O stainingin both superficial and deep zones at 2 months, which wasdecreased in the superficial zone at 6 months of age and continuedto decrease at later time points (Fig. 4). In sedc/þ temporoman-dibular joints at 2 months of age, proteoglycan staining in thesuperficial zone was less intense compared with controls, and wasseen to decrease until 12 months. In sedc/þ joints at 6 and 9months, increased Safranin O staining was apparent in the PCM butwas decreased in the interterritorial ECM. By 12 months, Safranin Ostaining was nearly absent in both superficial and deep zones ofsedc/þ temporomandibular joints, except in the PCM around somechondrocytes.

The þ/þ temporomandibular joint surface was intact at 2 and 6months, and showed some mild fissuring at 9 and 12 months. Thesedc/þ temporomandibular joint showed mild fissuring at 6months of age and deep fissures with separation of uncalcified fromcalcified articular cartilage at 9 and 12 months of age (Fig. 4). TheOARSI scoring showed that OA was significantly more advanced insedc/þ than in control temporomandibular joints at all time pointsexamined [Fig. 3(B)].

HtrA1, Ddr2, and Mmp-13 are upregulated in sedc/þ knee jointcartilage

Immunohistochemical staining for HtrA1, Ddr2, and Mmp-13was virtually absent in knee articular cartilage from 2- and 6-month-oldþ/þ controls. At 9 months, HtrA1 staining was apparentin two of the four samples, and at 12 months, all three proteinswere mildly elevated in three of the four control animals. In sedc/þknee samples, HtrA1 and Ddr2 stainingwas detected at 2months ofage, but Mmp-13 was not detectable. By 6months, HtrA1, Ddr2, andMmp-13 were all markedly increased in sedc/þ samples (Fig. 5). At9 months, the expression of Ddr2 and Mmp-13 was elevated insedc/þ compared to controls, but HtrA1 levels were comparable tocontrols. At 12 months, only Mmp-13 expression remainedelevated compared to controls.

In addition to observing an increase in staining intensity, wealso noted a significant difference in the number of cells staining forHtrA1, Ddr2 and Mmp-13 in sedc/þmice compared to þ/þ controlsat 6 months (Table II). The number of cells per unit area was notsignificantly different between þ/þ and sedc/þ in any of the jointsexamined (Table II).

Cells expressing HtrA1 show less pericellular type VI collagenstaining

Knee articular cartilage from 6-month þ/þ and sedc/þ animalswas stained by immunofluorescence using antibodies against HtrA1(green) and type VI collagen (red). Consistent with the results shownin Fig. 5, sedc/þ samples showed increased HtrA1 staining comparedto þ/þ [compare Fig. 6(E) to Fig. 6(B)]. This increased HtrA1 stainingcorrelated with decreased type VI collagen in the PCM, as demon-strated by red staining that appeared to be primarily intracellular insedc/þ [Fig. 6(D)], rather than surrounding the cells in a ring as wasvisible in þ/þ [Fig. 6(A), arrows]. The same trend was observed at 9months (data not shown).

Discussion

We have demonstrated that sedc/þmice show evidence of OA inhistological analyses of knee and temporomandibular joints. Inboth joints of the mutant, decreased proteoglycan staining of the

Fig. 4. Osteoarthritic degradation of cartilage is accelerated in sedc/þ compared to þ/þ TM joints. The TM joint in sedc/þ animals showed mild signs of proteoglycan degradation asearly as 2 months. Fissuring was seen in sedc/þ joints at 6, 9, and 12 months (arrows). At 12 months of age, fissuring and separation of decalcified from calcified cartilage weresevere, and Safranin O staining was only detectable in the PCM of a limited number of chondrocytes of sedc/þ mice. Pictures were taken at 100� magnification to visualize thewhole TM joint.

D.W. Holt et al. / Osteoarthritis and Cartilage 20 (2012) 430e439 435

Fig. 5. Six-month-old sedc/þ mice show detectable HtrA1, Ddr2, and Mmp-13. Samples were stained with antibodies against HtrA1, Ddr2, and Mmp-13. Pictures were taken at200� magnifications with the insets taken at 400� magnifications. All three OA-biomarkers are present in the sedc/þ animal and virtually absent in þ/þ controls.

D.W. Holt et al. / Osteoarthritis and Cartilage 20 (2012) 430e439436

superficial zone appeared as early as 2 months. At 6- and 9-monthtime points, the frequency of surface fibrillations increased and theintensity of proteoglycan staining decreased, except in the regionimmediately surrounding chondrocytes, the PCM, where stainingintensity increased. By 12 months, fissures were observed andseparation of decalcified from calcified cartilagewas present in fourof the four temporomandibular joints from sedc/þ animals. Whilethe earliest signs of OA were present in both knee and temporo-mandibular joints at 2 months, our observations suggest thatcartilage degradation progresses more rapidly in the temporo-mandibular than knee joints of both the sedc/þ and þ/þ animals(Fig. 3). This may be because mice can choose to curtail theirmovement but not to stop eating. Hence the temporomandibularjoint is used more than the knee. Alternatively, it could be that thecartilage in the temporomandibular joint is inherently differentthan that in the knee. We are currently examining thesehypotheses.

The increased proteoglycan staining in and around the sedc/þPCM was particularly noticeable at the 6-month time point in bothknee and temporomandibular joints. Fissuring was also apparent at

Table IIsedc/þ cells stained with biomarkers were significantly increased compared withþ/þ

þ/þ (n¼ 4) sedc/þ (n¼ 4)

HtrA1Tibia total cells 124� 13 130� 8% Tibia labeled cells 8 48*Femor total cells 112� 9 106� 4% Femor cells labeled 6 47*

Ddr2Tibia total cells 135� 5 124� 4% Tibia labeled cells 8 49*Femor total cells 116� 3 111� 5% Femor cells labeled 5 49*

Mmp-13Tibia total cells 126� 14 135� 9% Tibia labeled cells 4 54*Femor total cells 135� 9 110� 5% Femoral cells labeled 2 54*

*P< 0.0001; mean� standard deviation; n¼ number of mice.

the same time point in temporomandibular joints, while kneejoints showed less severe damage. This increased proteoglycanstaining in the immediate vicinity of chondrocytes suggests thatthe cells responded to damage in the ECM by increasing proteo-glycan synthesis.

We have demonstrated herein that OA occurs in sedc/þ animalswith overtly normal skeletal structure and no evidence of thedisproportionately short stature that typifies most chon-drodysplasias in humans and mice. This shows that type II collagenmutations that do not cause abnormal skeletal phenotypes can stilllead to degeneration of the articular cartilage. This observation isimportant because past research linking relatively rare chon-drodysplasia/OA syndromes to cartilage collagen gene mutationshas suggested that cartilage collagen mutations are not likely to bea common cause of OA in the general human population17. Ourwork suggests otherwise, and it is consistent with the recent reportby Kannu et al.11 that two different glycine substitution mutationsin the triple helical domain of human type II collagen can predis-pose individuals to early onset OA with no other characteristics ofchondrodysplasia. The sedc heterozygous mouse, therefore, isa useful mouse model for the study of OA caused by non-dominantmutations that lead to overtly normal skeletal phenotypes.

OA researchers in recent years have taken notice of HtrA1,a member of the high temperature requirement family of serineproteases. HtrA1 levels are elevated in synovial fluid from OApatients, and HtrA1 is known to digest severalmajor components ofthe articular cartilage PCM including aggrecan, decorin, fibromo-dulin, fibronectin, and biglycan18e22. The PCM has been shown tobe integral in the biomechanical environment of thechondrocyte23e25. In the cho/þ mouse, which bears a prematuretermination mutation in the Col11a1 type XI collagen gene that isassociated with premature OA, chondrocytes with increased HtrA1immunostaining were shown to lack pericellular type VI collagenstaining26. Those cells also showed increased expression of Ddr2,a cell membrane receptor that binds native type II collagen and, inresponse, induces expression of the matrix metalloproteinase,Mmp-13, which degrades type II collagen26e31.

Based on these observations, Polur et al.26 proposed a model fora potential molecular pathway of articular cartilage degeneration

Fig. 6. Increased HtrA1 staining in 6-month sedc/þ animals coincides with decreased type VI collagen staining in the PCM. Type VI collagen staining (A) and HtrA1 staining (B) ofa 6-month þ/þ tibia. (C) Merge of (A) and (B). Type VI collagen staining (D) and HtrA1 staining (E) of a 6-month sedc/þ tibia. HtrA1 staining of a 6-month sedc/þ tibia surface (E). (F)Merge of (D) and (E).

D.W. Holt et al. / Osteoarthritis and Cartilage 20 (2012) 430e439 437

which involves HtrA1, Ddr2, and Mmp-13. They suggested, basedon observations in both genetic and surgical/injury murine modelsof OA, that HtrA1-mediated degradation of proteins in the PCMmaybe one of the early steps in the pathogenesis of OA. Disruption ofthe PCM could position type II collagen fibrils, which are normallyseparated from chondrocytes by a thin layer of PCM, into directcontact with Ddr2 receptors. Because Ddr2 signaling leads toupregulation of both Ddr2 andMmp-13, it may initiate a downwardspiral of cartilage degradation27,32,33. This model is supported bya recent study showing that Mmp-13 as well as Ddr2 deficient miceare resistant to genetically and surgically induced OA14,15.

The immunohistochemical results presented herein coupledwith histopathological changes in the sedc/þ mutant suggest thatthe articular cartilage degeneration we have observed in sedc/þmice is consistent with the model proposed by Polur et al. At 2months of age, when only small regions of proteoglycan degrada-tion in the superficial zone were apparent, HtrA1 and Ddr2 weredetectable in sedc/þ knees. By 6 months, when proteoglycandegradation was more widespread and chondrocytes appeared tohave increased their proteoglycan secretion to compensate,Mmp-13 was easily detectable as well. In þ/þ knee joints, HtrA1was not detectable until 9months, and Ddr2 andMmp-13 elevationfollowed at 12 months of age. Our work validates that which hasbeen previously done showing the presence of HtrA1, Ddr2, andMmp-13 inmutant and surgically induced OAmousemodels and inhuman OA patients, in comparison to the absence thereof in the þ/þ controls14,15,20,26,27,34e36. This mounting evidence suggests thatthese three proteins are involved in the pathogenesis of OA inde-pendent of the event or underlying cause that triggers the onset ofthe disease.

In conclusion, we submit that the sedc/þ mouse is a useful newmodel of OA because, unlike models or conditions that expressdwarfism in the heterozygote (mouse Dmm/þ and human Stickler

syndrome), it exhibits normal skeletal stature and structure.The sedc/þ mouse supports the hypothesis that OA cases in thegeneral population may be triggered or exacerbated by cartilagecollagen mutations, specifically type II collagen mutations, thathave subtle effects on the structure of cartilage. An important focusin the field of OA research at present is determining whether thedifferent forms of OA that develop as a result of genetic predispo-sition, chronic joint stress, or acute injury all follow the samemolecular pathway of articular cartilage degradation once OA isinitiated. HtrA1, Ddr2, and Mmp-13 are potential components ofsuch a common molecular pathway. Previous work has demon-strated their elevation in surgically induced OA and in a geneticchondrodysplasia mouse model35,36. Here we have demonstratedthat HtrA1, Ddr2, and Mmp-13 are also elevated in a non-dwarfismmouse model of OA. The cartilage degradation pathway involvingHtrA1, Ddr2, and Mmp-13 is a mechanism that merits furtherinvestigation, especially because it points to Ddr2 as a potentialtarget for therapeutic intervention. The sedc/þmousewill be usefulto compare with chondrodysplasia, surgical, and other geneticmodels of OA to further establish possible common molecularpathways, and ultimately, to develop novel therapeutic approaches.

Author contributions

LCB and RES wrote the grants that supported this work. Theyalong with DLK oversaw experimental design, execution andinterpretation of data. All authors contributed to writing themanuscript. DWH, MLH, CES and JTF conducted key experimentsand assisted in the interpretation of results.

Conflict of interestThe authors have no financial or personal relationship with otherpeople or organizations.

D.W. Holt et al. / Osteoarthritis and Cartilage 20 (2012) 430e439438

Acknowledgments

This work was supported by NIH grant R15AR056861 (RES, LCB),NIH grant R01AR048839 (LCB), and by Brigham Young UniversityOffice of Research grants (DWH), (CES). The authors thank DavidMcDonald, Darin Johnston, Joshua Lloyd and Trent Johnson for theirexpert assistance. The authors also thank Dr Dennis Eggett of theBYU Department of Statistics for his expert help.

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