The American Journal of Pathology, Vol. 186, No. 11, November 2016

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MUSCULOSKELETAL PATHOLOGY

Discoidin Domain Receptor 2 as a PotentialTherapeutic Target for Development of

Disease-Modifying Osteoarthritis DrugsLauren B. Manning,* Yefu Li,yz Nithya S. Chickmagalur,y Xiaolong Li,yx and Lin Xuyz

From the Departments of Prosthodontics* and Developmental Biology,y Harvard School of Dental Medicine, Boston, Massachusetts; the Faculty of Medicine,z

Harvard Medical School, Boston, Massachusetts; and the State Key Laboratory of Oral Diseases,x Department of Orthodontics, West China Hospital ofStomatology, Sichuan University, Chengdu, China

Accepted for publication

C

h

June 30, 2016.

Address correspondence toYefu Li, M.D., Ph.D., REBRoom 504, or Lin Xu, M.D.,Ph.D., REB Room 514A, 188Longwood Ave, Boston,MA 02115. E-mail: yefu_li@hms.harvard.edu or lin_xu@hms.harvard.edu.

opyright ª 2016 American Society for Inve

ttp://dx.doi.org/10.1016/j.ajpath.2016.06.023

Osteoarthritis (OA) is the most common form of arthritis disorders, but the identification of therapeutictargets to effectively prevent OA has been increasingly difficult. The goal of this investigation is toprovide experimental evidence that discoidin domain receptor 2 (DDR2) may be an ideal target for thedevelopment of disease-modifying OA drugs. Ddr2 was conditionally deleted from articular cartilage ofadult mouse knee joints. Aggrecan-CreERT2;floxed Ddr2 mice, which were generated by crossingAggrecan-CreERT2 mice with floxed Ddr2 mice, then received tamoxifen injections at the age of 8 weeks.The mice were then subjected to destabilization of the medial meniscus (DMM) surgery. At 8 and 16weeks after DMM, mice were euthanized for the collection of knee joints. In a separate experiment,Aggrecan-CreERT2;floxed Ddr2 mice were subjected to DMM at the age of 10 weeks. The mice thenreceived tamoxifen injections at 8 weeks after DMM. The mice were euthanized for the collection of kneejoints at 16 weeks after DMM. The progressive process of articular cartilage degeneration was signifi-cantly delayed in the knee joints of Ddr2-deficient mice in comparison to their control littermates.Articular cartilage damage in the knee joints of the mice was associated with increased expressionprofiles of both Ddr2 and matrix metalloproteinase 13. These findings suggest that DDR2 may be anideal target for the development of disease-modifying OA drugs. (Am J Pathol 2016, 186: 3000e3010;http://dx.doi.org/10.1016/j.ajpath.2016.06.023)

Supported by a Laboratoires Servier (Surensnes, France) research grant.Disclosures: None declared.

Osteoarthritis (OA) is the most common form of arthritisdisorders.1 Because the molecular mechanism underlyingthe development of OA is largely unknown, it is difficult toidentify therapeutic targets that effectively prevent and treatOA. Currently, existing drugs for the treatment of OAprovide, at best, symptomatic relief from pain and inflam-mation.2 Current pharmacological interventions that addresschronic pain and inflammation do not prevent articularcartilage degeneration, which eventually leads to OA.Therefore, the identification of targets for the developmentof disease-modifying OA drugs (DMOADs) has becomemore pressing than ever. The objective of this presentinvestigation is to determine whether a cell surface receptortyrosine kinase, discoidin domain receptor 2 (DDR2), fornative collagen type II is an ideal target for the developmentof DMOADs.

stigative Pathology. Published by Elsevier Inc

What led us to investigate the role of DDR2 in thedevelopment of OA? The answer is the classic biochem-istry event in living systems, known as enzyme induction.An example of such a system is the inducible lactose-metabolizing enzyme, by which the enzyme is induced byits own substrates. Bacterial Escherichia coli is unable todirectly use disaccharide lactose for consumption. How-ever, under the condition in which lactose is solely present,the enzyme b-galactosidase is induced in E. coli. Theenzyme then breaks down lactose into monosaccharides,galactose and glucose, which the bacteria is able tometabolize. We believe that the induction of matrixmetalloproteinase 13 (MMP-13) in chondrocytes may

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DDR2 as a Target for OA Drugs

represent a similar situation. For cartilage tissue turnover,extracellular matrix molecules, such as type II collagen,need to be degraded. MMP-13 can degrade type IIcollagen. However, type II collagen cannot enter chon-drocytes to physically induce MMP-13. Thus, the collagenhas to interact with a cell surface molecule(s) and sendsignals into chondrocytes to induce the expression ofMMP-13 and the subsequent release of MMP-13 into theextracellular space. If this is the case, the question becomeswhich cell surface molecule(s) transduce the signal intochondrocytes? Initially, we examined whether integrina2b1 was the cell surface receptor responsible for thistransduction.3 However, results from our previous studyindicated that the blockage of the integrin a2b1 did notprevent the induction of MMP-13 in chondrocytes bynative type II collagen.4 Moreover, the activation of theintegrin a2b1 and the stimulation of type II collagen tochondrocytes showed a synergistic effect on the inductionof MMP-13 in the cell. This suggests that there is anothercell surface receptor responsible for transducing the signalto induce MMP-13 expression. We speculated that DDR2might be such a receptor,5,6 and results from our previousinvestigations supported our speculation.2,7,8

In one of our previous investigations, we removed onecopy of Ddr2 in two mouse models of OA.9 We found thatthe reduction in the expression of Ddr2 attenuated articularcartilage degeneration in knee joints of the mouse models.However, several critical questions remain to be addressedbefore we deem DDR2 as an ideal target to developDMOADs. First, in that study, we were unable to evaluatethe chondroprotective effect of the complete removal ofDdr2 from mature articular cartilage of mouse knee joints,as a means of investigating how critical Ddr2 is to theprogression of OA. This is because of the fact that homo-zygous conventional Ddr2 knockout mice that we used inthe study exhibit short stature (dwarfism) and such cannotbe used to study OA. Second, we were unable to determinewhether inhibition of Ddr2, after the onset of articularcartilage degeneration, could still prevent the joint frombeing destroyed. This was because of the lack of a floxedDdr2 mouse strain; therefore, a complete deletion of Ddr2was not possible at a desirable time in articular cartilage.Third, numerous investigations report there is a biologicaleffect secondary to the absence of DDR2 on articularcartilage development in human.10e12 Thus, the fullpotential chondroprotective effect of the Ddr2 deletion by aconventional knockout technique could be compromised ormisleading. To address these questions, in this presentinvestigation, we used a conditional knockout technique todelete Ddr2 from mature articular cartilage of mouse kneejoints to eliminate any potential developmental growthabnormalities that might arise. We then investigated thechondroprotective effect of the deletion of Ddr2 on kneearticular cartilage before and after the onset of cartilagedegeneration induced by destabilization of the medialmeniscus (DMM).

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In this present study, we have investigated what thepotential chondroprotective effect on mouse knee joints is.To address this question, we generated a floxed Ddr2 mousestrain. We then specifically removed Ddr2 from articularchondrocytes before, or after the onset of, articular cartilagedegeneration induced by DMM. We then examined themorphology of articular cartilage in knee joints ofDdr2-deficient mice and their control littermates, forevidence of changes in histology and in protein expressionsof Ddr2 and Mmp-13. We also examined the expressionprofile of Ddr2 in normal adult mice.

Materials and Methods

Generation of the Floxed Ddr2 Mice

All animal experimental procedures were performedafter approval from the Harvard Medical School Institu-tional Animal Care Committee. Embryonic stem cellscontaining the floxed Ddr2 allele were purchasedfrom EUCOMM (Wellcome Trust Genome Campus,Hinxton, Cambridge, UK). The embryonic stem cells wereinjected into blastocysts with C57BL/6 genomic back-ground to generate chimeric mice. The chimeric mice werethen bred with wild-type, C57BL/6, mice to generateheterozygous floxed Ddr2 mice. The floxed Ddr2 allelecontains several elements, including flippase recognitiontarget (FRT)-flanked En2SA-IRES-LacZ-bact-Neo cas-sette, which may have potential effects on the developmentof mice. Thus, by crossing floxed Ddr2 mice with FLPeRmice, the DNA fragment containing these elements wasremoved, eliminating the potential for undesired develop-mental abnormalities.

Mouse Genotyping

Mouse genotyping for Aggrecan-CreERT2 and the floxedDdr2 gene was performed. Genomic DNA was isolatedfrom the mice tails. To determine whether mice wereAggrecan-CreERT2 positive (AgcCreERT2þ/�), the forwardprimer 50-TAACTACCTGTTTTGCCGGG-30 and thereverse primer 50-GTCTGCCAGGTTGGTCAGTAA-30

were used. The PCR for AgcCreERT2 was set for primarydenaturation at 94�C for 3 minutes, followed by 30 cycles ofdenaturation at 94�C for 45 seconds, annealing at 60�C for45 seconds, and elongation at 72�C for 1 minute, withfinal elongation at 72�C for 10 minutes. To determinewhether mice were homozygous for the floxed Ddr2 gene(Ddr2flox/flox), the forward primer 50-AGTAGGTGCTA-GCTACCTCCCACC-30 and the reverse primer 50-CTGCT-TCCTCCCAGGTACCTTCCC-30 were used. The PCR forfloxed Ddr2 was set for primary denaturation at 94�C for 3minutes, followed by 30 cycles of denaturation at 94�C for30 seconds, annealing at 60�C for 30 seconds, and elon-gation at 72�C for 30 seconds, with final elongation at 72�Cfor 10 minutes.

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Characterization of the Floxed Ddr2 Mice

Pregnant AgcCreERT2þ/�;Ddr2flox/flox mice were adminis-tered 3 mg tamoxifen per mouse at 24 hours before givingbirth. The young mice were then examined at the age of 1week old. One of the hind legs from each mouse was usedfor Alcian blue/alizarin red whole-mount skeleton staining.The other hind leg was embedded in paraffin section andstained with Safranin O/Fast Green for growth plateanalysis.13

Efficiency of Ddr2 Removal from Articular Chondrocytesof Mouse Knee Joints

Three AgcCreERT2þ/�;Ddr2flox/flox mice, at the age of 8weeks old, were injected with tamoxifen at 2 mg/10 g bodyweight daily via i.p. injection for 10 consecutive days, whereasanother three AgcCreERT2þ/�;Ddr2flox/flox mice were injectedwith sunflower seed oil. The mice were then euthanized, andarticular cartilage was collected from the knee joints forisolation of genomic DNA. For detection of the exon 9 ofDdr2, PCR was performed with the forward primer 50-AGTAGGTGCTAGCTACCTCCCACC-30 and the reverseprimer 50-GGTTTCCATGAACCCTACTG-30. For the quan-titative measurement of the exon 9 of Ddr2, a duplex real-timePCR was performed using TaqMan Copy Number Assay.TaqMan probe (catalog number 4400291) for the target gene,Ddr2 exon 9 (transcript: ENSMUST00000027985), waslabeled with FAM, and TaqMan probe (catalog number4458366) for a reference gene, Tfrc, was labeled with VIC.PCR was performed in 20 mL reaction containing 20 ng ofgenomic DNA (Life Technologies Corporation, Carlsbad,CA), according to the company’s protocol. Real-time PCRwas performed at 95�C for 10 minutes, followed by 40 cyclesof 95�C for 15 seconds and 60�C for 60 seconds, usingStepOnePlus real-time PCR System (Thermo Fisher Scientific,Waltham, MA). Each sample was tested in triplicate.

Conditional Removal of Ddr2 in Mature ArticularCartilage of Mouse Knee Joints

The mouse strain, AgcCreERT2, expresses a recombinantprotein consisting of Cre-recombinase and the modifiedestrogen receptor (CreERT2), driven by the endogenousaggrecan promoter. In this mouse strain, the CreERT2 isexpressed in chondrocytes of uncalcified articular cartilage,but not in the chondrocytes of calcified cartilage inadult mice.14 AgcCreERT2þ/� mice were crossed withDdr2flox/flox mice to generate double-heterozygous mutant,AgcCreERT2þ/�;Ddr2þ/flox, mice. The compound mutantmice, AgcCreERT2þ/�;Ddr2flox/flox, were generated bycrossing the double-heterozygous mutant mice and identi-fied by PCR (see previous paragraph). AgcCreERT2þ/�;Ddr2flox/flox mice, and corresponding control mice, wereseparated and maintained in the mouse facility under a cycleof 12-hour light and dark environments.

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One group of AgcCreERT2þ/�;Ddr2flox/flox mice wasinjected, via i.p. injection, with tamoxifen at 2 mg/10 g bodyweight daily for 10 consecutive days at the age of 8 weeksold. The mice were then subjected to DMM surgery. Themice were then euthanized at 8 and 16 weeks after DMM forcollection of the knee joints. Another group ofAgcCreERT2þ/�;Ddr2flox/flox mice was subjected to DMMsurgery at 10 weeks of age. These mice were then injected,via i.p. injection, with tamoxifen at 2 mg/10 g body weightdaily for 10 consecutive days at 8 weeks after DMM. Themice were euthanized at 16 weeks after DMM for collectionof the knee joints. The corresponding controls, DMM onAgcCreERT2þ/�;Ddr2flox/flox mice without removal of Ddr2and sham surgery on AgcCreERT2þ/�;Ddr2flox/flox micewith removal of Ddr2, were also performed.DMM surgery on mice was performed as follows. Briefly,

after the mice were anesthetized via i.p. administration ofketamine (90 mg) and xylazine (10 mg)/kg mouse bodyweight, the right knees were prepared for aseptic surgery.The joint capsule immediately medial to the patellar tendonwas opened and, for DMM, the medial meniscotibial liga-ment was sectioned. The joint capsule was then closed with8-0 Vicryl suture, and the skin was closed with 7-0 suture.Sham surgery, in which the meniscotibial ligament wasvisualized but not transected, was performed as a negativecontrol.

Histology and Immunohistostaining

The knee joints were fixed in 4% paraformaldehyde for 6hours at room temperature, then decalcified in Morse’s so-lution and processed for paraffin embedding. The sampleswere sectioned, by serial sectioning, at a 6 mm thickness inan anterior to posterior direction. Every tenth section wascollected for Safranin O/Fast Green staining. The patho-logical condition of the articular cartilage was evaluated bya scoring system designed to assess the histology of OA inmouse joints; the system is recommended by the Osteoar-thritis Research Society International (OARSI) histopathol-ogy initiative.15 The minimal score of 0 is for normal mousearticular cartilage, and the maximal score of 6 is for verticalclefts/erosion to the calcified cartilage extending >75% ofthe articular surface.For analysis of Ddr2 and Mmp-13 expression, 8 to 10

paraffin sections, distributed throughout each knee joint, ofarticular cartilage from Ddr2-deficicent and control mice at8 weeks after the surgery, were selected for immunohis-tostaining. Thus, there were 32 to 40 (4 � 8 or 4 � 10)paraffin sections in each experiment. Paraffin sections wereincubated with a polyclonal antibody against Ddr2 (1:200dilution, catalog number sc-8989; Santa Cruz Biotech-nology, Inc., Dallas, TX) or a rabbit polyclonal antibody(1 mg/mL at the final concentration of 1:400 dilution) againstMmp-13 (Abcam, Cambridge, MA). After overnight incu-bation at 4�C, the sections were washed and treated with abiotinylated secondary antibody. Color development was

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DDR2 as a Target for OA Drugs

performed using a peroxidase substrate (Vector Labora-tories, Burlingame, CA) after treatment of the sections witha mixture of avidin and biotinylated horseradish peroxidase(Vector Laboratories). Staining without primary antibodywas performed as a negative control.

Morphological Evaluation of Articular Cartilages ofDdr2þ/� Mice after DMM

Information of the conventional Ddr2 knockout mice hasbeen reported in our previous publication.9 Briefly, Ddr2 isdeleted by the conventional knockout technique in micewith the hybrid genetic background of C57/Black andMouse129. For the histology study, knee joints were cutfrom the lateral to the medial side of the joints at 6 mmthickness by serial sectioning. The sections were stained bySafranin O/Fast Green. The pathological condition of theknee joints was evaluated by the OARSI score system.

Whole-Mount X-Gal Staining of Mouse Tissues

Multiple tissues and organs were collected from the floxedDdr2 mice containing FRT-flanked En2SA-IRES-LacZ-bact-Neo cassette (LacZ driven by endogenous Ddr2 promoter) andtheir control littermates at 2 and 4 months of age. Sampleswere subjected to whole-mount X-Gal staining. Reagents werepurchased from EMD Millipore (Darmastadt, Germany), andthe experimental protocol was from the company (catalognumber mcproto058). Briefly, samples were fixed in tissuefixative for 45 minutes at 4�C and X-gal staining was per-formed at 37�C in the dark until blue color developed. Thesamples were then analyzed under a Leica MZ16 stereomi-croscope (Leica Microsystems Inc., Buffalo Grove, IL).

Wild-type allele

Targeted allele

8 FRT FRT

loxP loxPloxP

9 act::neoEn2 SA IRES lacZ

8 9 10 11

10 11

8 FRT

loxPloxP

9 10 11

Figure 1 Generation of the floxed Ddr2 mice. A flippase recognitiontarget (FRT)-flanked En2SA-IRES-LacZ-bact-Neo cassette and a loxP-flankedexon 9 were recombined into Ddr2 allele. The elements flanked by FRT wereremoved by crossing the floxed Ddr2 mice with FLPeR mice.

Statistical Analysis

A total of 10 to 12 paraffin sections, which were evenlydistributed throughout an entire joint, were examined andscored. The score from the section depicting the worstcondition was selected to represent that joint. There were sixto eight animals in each experimental group; therefore, sixto eight scores were obtained for each group. Then, anaverage score was calculated from six to eight scores foreach group. A Student’s t-test, with a significance level of0.05, was used to determine whether a significant differencebetween any two scores was present.

To determine sample size in this study, a pilot study wasperformed on the effect of Ddr2-deficient mice with DMM.From those results, it was concluded that a sample size witha minimum of five animals is required to achieve thespecified CI (95%) with at least 50% reduction of the scorein the Ddr2-deficient mice.

The t-test was also used to determine the efficiency of theDdr2 removal and in immunohistostaining experiments.

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Notation of Mouse Strains in This Study

We call the floxed Ddr2-containing FRT-flanked En2SA-IRES-LacZ-bact-Neo cassette as Ddr2-LacZ mice, thefloxed Ddr2 without FRT-flanked En2SA-IRES-LacZ-bact-Neo cassette as Ddr2flox/flox mice, tamoxifen-treatedAgcCreERT2þ/�;Ddr2flox/flox as Ddr2D/D mice, oil-treatedAgcCreERt2þ/�;Ddr2flox/flox as Ddr2f/f mice, heterozygousconventional Ddr2 knockout as Ddr2þ/�, and their wild-type littermates as Ddr2þ/þ mice.

Results

Generation of the Floxed Ddr2 Mice

We received three Ddr2 chimeric mice. One of them con-tained the germline integration of the floxed Ddr2 allele.After several rounds of breeding of the chimeric mouse withwild-type C57/BL6 mice, heterozygous floxed Ddr2 micewere generated. We then removed several elements,including FRT-flanked En2SA-IRES-LacZ-bact-Neocassette, in the floxed Ddr2 allele by crossing the floxedDdr2 mice with FLPeR mice. Mice containing homozygousfloxed Ddr2 without the previously mentioned elements(Ddr2flox/flox) were obtained (Figure 1).

Genotyping of AgcCreERT2 and Floxed Ddr2 Mice

To identify AgcCreERT2 mice, the PCR primers from theCre-recombinase gene generated a 544-bp PCR product.Because wild-type mice do not contain the Cre-recombinasegene, there was no PCR product from genomic DNA ofwild-type mice. To identify the floxed Ddr2 allele, PCRprimers crossing one FRT site and one loxP site in the intron8 of Ddr2 generated a PCR product with the size of 260 bpfrom genomic DNA of wild-type mice. However, theseprimers produced a PCR product with the size of 446 bpfrom genomic DNA of mice with the floxed Ddr2 allele.

Characterization of the Floxed Ddr2 Mice

A short stature of the body was observed in Ddr2D/D micecompared with their Ddr2f/f littermates at 1 week after birth

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1650 2000

bp

A B ***

40 60 80

100

ell w

ith D

dr2

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(Figure 2, A and B). Histological examination revealed thedisorganized proliferating chondrocyte columns and thinnerproliferative zone in the growth plate of Ddr2D/D mice(Figure 2C). The phenotype of the Ddr2D/D mice isconsistent with the result from the investigation by anotherindependent research group.10

600 720 0

20

Ddr2f/f

Ddr2/

% c

Figure 3 The removal of Ddr2 in mouse articular cartilage. A: PCRanalysis showed a 720-bp product in articular cartilage of Ddr2D/D mice anda 1650-bp product in Ddr2f/f mice. B: The removal efficiency of the Ddr2exon 9 was measured by real-time quantitative PCR. The exon 9 of Ddr2 hasbeen removed from 75% of chondrocytes in articular cartilage of Ddr2D/D

mice. ***P < 0.001.

Efficiency of the Removal of Ddr2 in ArticularChondrocytes of the Knee Joint

We used AgcCreERT2 to remove Ddr2 specifically inarticular chondrocytes of knee joints in adult mice in thisexperiment. Results from our previous experiment indi-cated that CreERT2 was highly inducible in articularchondrocytes of the knee joints in AgcCreERT2 mice at 2months of age.16 We noticed that the induced CreERT2appeared in chondrocytes above the tidemark in articularcartilage, which is consistent with the result from a studyby an independent research group.14 We examined theefficiency of the ablation of Ddr2 by AgcCreERT2 inarticular chondrocytes of adult mouse knee joints.After several rounds of crossing AgcCreERT2 micewith floxed Ddr2 mice, we obtained compound mutantmice, heterozygous CreERT2 driven by the endogenousaggrecan promoter and homozygous floxed Ddr2(AgcCreERT2þ/�;Ddr2flox/flox). PCR analysis usinggenomic DNA from articular cartilages of the kneejoints showed a 720-bp product in tamoxifen-treatedAgcCreERT2þ/�;Ddr2flox/flox (Ddr2D/D) mice, indicatingthat the exon 9 of Ddr2 has been deleted, and a 1650-bpproduct in oil-treated AgcCreERT2þ/�;Ddr2flox/flox

(Ddr2f/f) mice (Figure 3A). We found that the exon 9genomic DNA of Ddr2 floxed by loxP sites was deletedin 75% of articular chondrocytes in adult mouse kneejoints (Figure 3B). The loss of the exon 9 resulted in apremature stop codon in the exon 10 of Ddr2. This resultindicated that Ddr2 was deleted in most articular chon-drocytes of the adult knee joints in Ddr2D/D mice.

Figure 2 Characterization of the hind leg and growth plate of Ddr2D/D micelittermates. B: The hind leg of the Ddr2D/D mice was also shorter. C: The histologicand disorganized columns of the proliferative chondrocytes in the Ddr2D/D mice. WDdr2D/D mice and their control littermates. Dashed lines mark borders of PZ (thlines). Arrow shows chondrocyte columns. Scale bar Z 100 mm (C).

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Chondroprotective Effect on Adult Mouse Knee Jointsagainst the Development of OA by the Genetic Deletionof Ddr2 in Articular Cartilage Chondrocytes

First, we determined whether the deletion of Ddr2 from artic-ular cartilage chondrocytes of adult mouse knee joints couldattenuate the progression of articular cartilage degenerationbefore initiation of cartilage degeneration. We removed Ddr2from the knee joints of adult AgcCreERT2þ/�;Ddr2flox/flox

mice, and then performed DMM surgery on the mice and theircorresponding controls. We found significant disparities in theprogressive process of articular cartilage degeneration in kneejoints between Ddr2D/D and Ddr2f/f mice at 8 and 16 weeksafter DMM (Figure 4, A and B). The progression toward OAwas dramatically delayed in the Ddr2D/D mice after DMMsurgery. Therewere no abnormalmorphological changes in thesham surgery groups.The condition of the articular cartilage was also evaluated

with the scoring system designed to assess the histology ofarticular cartilage in a surgically induced mouse model ofOA. Mice at 8 weeks after sham surgery were used as a

. A: The body size of Ddr2D/D mice was smaller than that of their controlal examination of growth plates revealed the thinner proliferative zone (PZ)e did not see significant differences in the hypertrophic zone (HZ) betweene top and middle dashed lines) and HZ (the middle and bottom dashed

ajp.amjpathol.org - The American Journal of Pathology

Figure 4 Morphology of articular cartilages of mouse knee joints. There were no overt morphological changes seen in the articular cartilage of knee jointsin the sham mice at 8 weeks (A) and 16 weeks (B) after the surgery. However, there were significant disparities in the progressive process of articular cartilagedegeneration in knee joints between Ddr2D/D and Ddr2f/f mice. At 8 weeks after destabilization of the medial meniscus (DMM), fibrillation was seen in thearticular cartilage of Ddr2f/f mice. The damaged area of the articular cartilage was limited to <25% the surface area. A: In Ddr2D/D mice, there was only thelocalized absence of the proteoglycans observed. At 16 weeks after the surgery, the loss of the articular cartilage was evident in both femoral and tibiacondyles of Ddr2f/f mice. B: The missing cartilage was extended to >75% of the surface area. In Ddr2D/D mice (either Ddr2 was deleted before or after DMM),the fibrillation was evident, and the damaged cartilage was only extended to <25% of the surface area. C: We also noticed the osteophyte formation in Ddr2f/f

mice at 16 weeks after DMM. Arrows indicate tidemark. Scale bars Z 100 mm (AeC). Original magnification, �100. f, femur; m, meniscus; os, osteophyte; s,synovial membrane; sb, subchondral bone.

DDR2 as a Target for OA Drugs

normal control (scoreZ 0). At 8 weeks after DMM surgery,the average scores for Ddr2D/D mice and Ddr2f/f littermateswere 0.64 and 1.71, respectively. At 16 weeks after DMMsurgery, the average scores were 1.31 for Ddr2D/D mice and4.57 for Ddr2f/f littermates. The scores were significantlydifferent between the two groups at both time points(Table 1).

Table 1 Evaluation of Articular Cartilage Condition in KneeJoints of Ddr2D/D Mice

Time pointsafter DMM(weeks)

Ddr2f/f Ddr2D/D

P valuen Means � SD n Means � SD

8 7 1.71 � 0.36 7 0.64 � 0.23 <0.00116 7 4.57 � 0.49 8* 1.31 � 0.79 <0.001

5y 1.10 � 0.58 <0.001

*Ddr2 deleted before DMM.yDdr2 deleted after DMM.DMM, destabilization of the medial meniscus.

The American Journal of Pathology - ajp.amjpathol.org

In addition, we noticed the appearance of osteophytes inDdr2f/f littermates at 16 weeks (Figure 4C). However,there was no evidence of osteophyte formation in Ddr2D/D

littermates. We did not observe other abnormalmorphology in the subchondral bone and synovial tissue ineither Ddr2D/D or Ddr2f/f mice.

Second,we reevaluated thepathological conditionof articularcartilages of knee joints in Ddr2þ/� mice after DMM by usingtheOARSI system. At 8weeks after DMMsurgery, the averagescores forDdr2þ/�mice andDdr2þ/þ littermateswere 0.81 and1.88, respectively. At 16weeks after DMMsurgery, the averagescores were 2.14 for Ddr2þ/� mice and 4.63 for Ddr2þ/þ lit-termates. The scores were significantly different between thetwo groups at both time points (Table 2). Previously, we applieda different scoring system to the Ddr2þ/� mice,9 because thescoring system that we used in this present study was notavailable at that time. We compared the result from Ddr2þ/�

mice with Ddr2D/D mice. Results showed that there was a sig-nificant difference (P < 0.05) in the scores between Ddr2þ/�

andDdr2D/Dmice at 16weeks afterDMMsurgery, scoring 2.14

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Table 2 Evaluation of Articular Cartilage Condition in KneeJoints of Ddr2þ/� Mice

Time pointsafter DMM(weeks)

Ddr2þ/þ Ddr2þ/�

P valuen Means � SD n Means � SD

8 8 1.88 � 0.57 8 0.81 � 0.47 <0.0116 8 4.63 � 0.81 7 2.14 � 0.60 <0.001

DMM, destabilization of the medial meniscus.

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versus 1.31, but there was no significant difference between thescores at 8 weeks, scoring 0.81 versus 0.64.

Third, we determinedwhether the deletion ofDdr2 after theinitiation of cartilage degeneration could delay the progres-sive process of degeneration. We removed Ddr2 from theknee joints of adult mice at 8 weeks after the DMM surgery.Mice were then allowed to survive 8 additional weeks. Wefound significant disparities in the progressive process ofcartilage degeneration in knee joints between Ddr2D/D andDdr2f/f mice (Figure 4B); the progression was significantlydelayed in the Ddr2D/D mice, with an average score of 1.10(Table 1). This is similar to what was seen in mice with DMMsurgery after the deletion of Ddr2 was completed.

Immunohistostaining of Ddr2 and Mmp-13 in ArticularCartilage of Knee Joints

We examined the protein expression of Ddr2 and Mmp-13in the articular cartilage of knee joints from mice at 8 weeksafter DMM. The positive staining cells were counted in eachparaffin section and summed to determine the total numberof positive cells from all of the sections in each group. Ddr2and Mmp-13 positive staining cells were detected at 4%(means � SD Z 0.04 � 0.02) and 5% (means �SDZ 0.05 � 0.02), respectively, in the articular cartilage ofknee joints from Ddr2-deficient mice (Figure 5A). More-over, Ddr2 and Mmp-13 positive staining cells were presentat 20% (means � SD Z 0.20 � 0.03) and 24%(means � SD Z 0.24 � 0.02), respectively, in the articularcartilage of knee joints from their control littermates(Figure 5A). The number of positive staining cells wassignificantly different between the two groups (P < 0.001).The location of positive cells was randomly scattered in thesuperficial layer of the articular cartilage. We did notobserve the increase in the expression of Ddr2 in thesynovial membranes in the sham, Ddr2-deficient mice andtheir control littermates (Figure 5B).

Expression Profile of Ddr2 in Adult Mice

We investigated the expression profile of Ddr2 in adult miceusing Ddr2-LacZ mice containing LacZ element driven byendogenous Ddr2 promoter. Tissues and organs expressingDdr2 will develop blue color by whole-mount X-Galstaining. Results demonstrate that Ddr2 is not expressedbroadly in adult mouse tissues. Expression was detected in

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the renal pelvis and testis in adult mice at 2 and 4 months ofage (Figure 6).

Discussion

We generated floxed Ddr2 mice. Before we used these micefor our study, we first removed several elements within thefloxed Ddr2 construct from the mice to avoid any potentialbiological influence of those particular elements on micedevelopment. We then characterized the mice to knowwhether the inserted loxP sites were accessible to the fusedrecombinant protein, Cre-recombinase with the modifiedhuman estrogen receptor (CreERT2). Results from our ex-periments demonstrated that Ddr2D/D mice revealed a shortstature (dwarfism). The growth plate of these mice showedthe disorganized proliferating chondrocyte columns and athinner growth plate. This phenotype is consistent with thegross appearance of the skeleton and the morphologicalalteration of the growth plate in homozygous Ddr2knockout mice generated by the conventional knockouttechnique,10 suggesting that the loxP sites in our floxedDdr2 mice were accessible to CreERT2. By use of thesemice, we were able to conditionally delete Ddr2 fromarticular chondrocytes of knee joints in adult mice. Wefound that Ddr2 was deleted/removed in 75% of articularchondrocytes and that the progressive progression of carti-lage degeneration was significantly delayed by the deletionof Ddr2. This suggests that a decrease in the expression andactivity of Ddr2 can decelerate the progression of cartilagedegeneration, which is consistent with our previous obser-vation.9 Why was Ddr2 not removed completely fromarticular chondrocytes? There are two possible explanations.First, in our previous experiments, we found that chon-drocytes below the tidemark of articular cartilage of mouseknee joints did not express aggrecan.16 To examine the ef-ficiency of Ddr2 removal from adult articular chondrocytes,articular cartilage was trimmed from the surface of mouseknee joints. This included chondrocytes both above andbelow the tidemark. However, because Cre-recombinasewas under the control of the endogenous aggrecanpromoter, Cre-recombinase is only expressed in aggrecan-producing cells. That may, in part, account for the 25% ofcells (noneaggrecan-producing cells) in the articular carti-lage that contained Ddr2. Second, it may be difficult toremove Ddr2 from every aggrecan-producing chondrocytevia tamoxifen injection.We are aware of the report of a predisposition to the

development of articular cartilage degeneration in differentinbred mouse lines.17 However, in the present study, wecompared the pathological condition of articular cartilage,induced by DMM, in Ddr2þ/� mice (the hybrid genomebackground of C57/Black and Mouse129) with the patho-logical condition in Ddr2D/D mice (the genome backgroundof C57/Black), and we did not observe a significant differ-ence between these two groups. In addition, we did not see a

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Figure 5 Immunohistostaining of Ddr2 and matrix metalloproteinase 13 (Mmp-13) in articular cartilages of knee joints in mice at 8 weeks after destabilization ofthe medial meniscus (DMM). A: Each image is one representative section. Therewere approximately 20% of Ddr2-positive cells (brown) and 24% of Mmp13-positive cells (brown) in control mice after DMM surgery. But there were 4% ofDdr2-positive cells and 5% of Mmp13-positive staining cells in Ddr2D/D mice afterDMM surgery. There were hardly Ddr2- and Mmp13-positive cells detected in shamsurgery mice. The positive background staining (brown staining cells) in the bonemarrow of all mice indicated that the paraffin sections were treated under aconsistent immunostaining condition. Boxed areas are shown at higher magnifi-cation in the insets. B: We did not see the increase in the expression of Ddr2 insynovial membrane. Scale bars Z 100 mm (A and B). Original magnification: �100(A, main images); �200 (insets). m, meniscus; s, synovial membrane; t, tibia.

DDR2 as a Target for OA Drugs

significant difference in the predisposition to articularcartilage degeneration in mice knee joints among mice withC57/Black and Mouse129; C57/Black genome backgroundsin our other studies.9,16 In our previous study with Ddr2þ/�

mice,9 we found that reduction in the expression of Ddr2delayed the progression of articular cartilage degeneration.

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In the present investigation, we used a scoring system,recommended by OARSI, to reevaluate the cartilage of kneejoints from the previously mentioned study, as well as toevaluate the cartilage of knee joints in our present study. Asa result, we found that a larger number of chondrocyteswithout Ddr2 exhibit greater chondroprotective efficiency in

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Kidney

Testis

Control Ddr2-LacZ

2 months

Renal pelvis

4 months

Renal pelvis

Control Ddr2-LacZ

Figure 6 Whole-mount X-Gal staining of or-gans from Ddr2-LacZ mice. Expression of Ddr2 wasdetected in renal pelvis and testis in adult mice atthe ages of 2 and 4 months old.

Manning et al

adult mouse knee joints. This information is critical in theconsideration of Ddr2 as a target for the development ofDMOADs.

Why is DDR2 a potential target for the development ofDMOADs? First, on the basis of results from our studiesand others’ investigations,18e20 it is clearly shown that in-hibition of the activity of DDR2 can protect articularcartilage from being degraded. In the present study, wewere able to delete Ddr2 before, and after the onset of,articular cartilage degeneration. Our results indicate thatarticular cartilage was protected under both conditions;thus, inhibition of DDR2 activity has both prophylactic andtreatment effects on articular cartilage degeneration. Thisalso implies that even individuals at high risk for thedevelopment of OA, such as athletes participating in contactsports with high injury rates, would not need to receivedrugs that inhibit DDR2 activity before injury, becausearticular cartilage degeneration could still be delayed afterthe injury occurs. Hence, a small-molecule inhibitor ofDDR2 can inhibit the activation of DDR2 to prevent theinduction of MMP-13 against the development of OA.Second, although there is interest from pharmacologicalcompanies in inhibiting the activity of MMP-13 as a meansof delaying the progression of OA, the broad biologicaleffects of MMP-13 limit its application as a target enzymeof inhibitory drugs in the treatment of OA.21 However, thisdoes not apply to DDR2 because the activation of DDR2 bythe interaction of the receptor with type II collagen occurslocally in the pericellular matrix of chondrocytes. Wespeculate that excessive mechanical force can incite chon-drocytes to degrade the cartilaginous matrix through aprocess known as chondrocytic chondrolysis. As thedegenerative process progresses over time, the activatedchondrocytes synthesize and release matrix-degrading en-zymes. As a consequence of enzyme release, there isdisruption of the pericellular matrix of chondrocytes, whichnormally separates the surface of the chondrocytes fromtype II collagen fibrils present within the territorial andinterterritorial locations of normal articular cartilage.22e25

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Once the pericellular matrix is destroyed, chondrocytesare able to bind to type II collagen, which results inenhanced molecular signaling, mediated by DDR2. Theactivation of DDR2 then induces expression of MMP-13.Accordingly, interruption of the interaction of DDR2 withtype II collagen by either small molecules or biologicalreagents can inhibit the activation of DDR2 to prevent in-duction of MMP-13. Third, one of the major concerns in thedevelopment of DMOADs is in regard to off-target effectsof a drug. The next question becomes, if a small-molecularinhibitor of DDR2 is identified, which tissues can beaffected by the inhibitor under normal physiological con-ditions? Results from the expression profile of Ddr2 in thisstudy demonstrated that Ddr2 was not expressed broadly inadult mouse tissues. Expression was detected in the renalpelvis and testis of adult mice, which is consistent with theobservation that males of homozygous knockout mice wereinfertile.10 This information is of particular importance inthe consideration of potential off-target effects of DDR2inhibitors in adults, since such effects would be signifi-cantly limited because of the tissue-specific expression ofDDR2. One study reports that the deletion of Ddr2 causes adefect of dermal wound healing associated with defectiveextracellular matrix remodeling.26 Fourth, another inter-esting observation in this study is that articular cartilagecould be protected by the conditional (local) removal ofDdr2 only in the articular cartilage of adult mouse kneejoints. This suggests that intra-articular injections of aDDR2 inhibitor, or biological reagents that interrupt theinteraction of DDR2 with type II collagen, could have anadequate protective effect on joints against the developmentof OA.In the past 10 to 15 years, results from many independent

research groups have demonstrated that the removal orinhibition of genes can protect articular cartilages frombeing degraded; such genes include a disintegrin and met-alloproteinase with thrombospondin motifs 5 (Adamts-5),hypoxia-inducible factor-2a (HIF-2a), Wnt/b-cateninsignaling, salt-inducible kinase 3 (Sik3), and the Zn2þ

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DDR2 as a Target for OA Drugs

importer ZIP8. The chondroprotective effect by the removalof Adamts-5 could in fact be because of the prevention ofsynovial fibrosis and stabilization of subchondral bone.27,28

It has been suggested that the deficiency of Hif-2a couldpossibly inhibit chondrocyte hypertrophy, resulting in thedelay of articular cartilage degeneration in joints.29 Withregard to Wnt/b-catenin signaling, there is a report that theinhibition of b-catenin in chondrocytes results in OA inmice30; however, results from another study indicate that thestabilization of b-catenin causes OA in mice.31 The con-flicting results may be because of the developmental-stagedependent manner of b-catenin function. Sik3 may play arole in regulation of homeostasis of articular cartilage, andthe conditional removal of Sik3 from articular cartilage canprotect knee joints against the development of OA inmice.32 Data from a recent investigation demonstrate thatthe deletion of Zip8, a Zn2þ importer, can delay the pro-gression of OA induced by DMM.33 Still, although thepreviously mentioned genes could be potential targets forthe treatment of OA, further investigations are needed todetermine the precise biological effects of these genes, ortheir down-stream molecules, on articular cartilages andother joint tissues if they are to be targets for the treatmentof OA.

With regard to the development of novel therapeuticprotocols to prevent healthy or degenerating joints frombecoming OA joints, we may have to consider anapproach to protect a whole joint, instead of targeting aparticular joint tissue, such as articular cartilage or sub-chondral bone.34 Results from the present study indicatethat articular cartilage is still damaged continuallywithout Ddr2 present in the cartilage. One possiblereason is that different signaling pathways are involved inthe induction of other extracellular matrix-degrading en-zymes. Another plausible explanation is that the activa-tion of Ddr2 in other joint tissues may also play a role inthe destruction of articular cartilage. Data from severalstudies demonstrate DDR2 expression in bone tissue,35,36

and that DDR2 activation causes osteoblast differentia-tion, which, in turn, may affect the remodeling and sta-bility of the subchondral bone.37,38 Another goodexample of a whole joint treatment approach is the role oftransforming growth factor b1 signaling in the patho-genesis of OA. Results from our previous investigationindicated that the removal of Tgf-b1 signaling frommature articular cartilage could delay the degenerativeprogression of articular cartilage in mice.16 Another in-dependent research group reports that inhibition of Tgf-b1 signaling in subchondral bone can protect articularcartilage from being degenerated.39 Data from otherstudies demonstrate that an increase in the expression ofTgf-b1 signaling results in the hyperplasia of the synovialmembrane and the damage of joint ligaments in mice.40,41

Clearly, it may be more effective to inhibit Tgf-b1signaling in multiple joint tissues to protect the jointagainst the development of OA.

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In summary, the results from the present investigationprovide further information supporting that DDR2 is apotential target for the development of DMOADs.

Acknowledgments

We thank Dr. Stephen Henry and Dr. Benoit de Crom-brugghe (Anderson Cancer Center, Houston, TX) forproviding the AgcCreERT2þ/� mouse strain.

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