Epidermal Growth Factor Receptor-deficient Mice Have DelayedPrimary Endochondral Ossification Because of DefectiveOsteoclast Recruitment*

Received for publication, March 19, 2004, and in revised form, September 8, 2004Published, JBC Papers in Press, September 28, 2004, DOI 10.1074/jbc.M403114200

Ke Wang‡§, Hiroaki Yamamoto‡§, Jennie R. Chin‡§, Zena Werb¶, and Thiennu H. Vu‡§�

From the Departments of ‡Medicine and ¶Anatomy, and §Lung Biology Center, University of California,San Francisco, California 94143

The epidermal growth factor receptor (EGFR) and itsligands function in diverse cellular functions includingcell proliferation, differentiation, motility, and survival.EGFR signaling is important for the development ofmany tissues, including skin, lungs, intestines, and thecraniofacial skeleton. We have now determined the roleof EGFR signaling in endochondral ossification. We an-alyzed long bone development in EGFR-deficient mice.EGFR deficiency caused delayed primary ossification ofthe cartilage anlage and delayed osteoclast and osteo-blast recruitment. Ossification of the growth plates wasalso abnormal resulting in an expanded area of growthplate hypertrophic cartilage and few bony trabeculae.The delayed osteoclast recruitment was not because ofinadequate expression of matrix metalloproteinases, in-cluding matrix metalloproteinase-9, which have previ-ously been shown to be important for osteoclast recruit-ment. EGFR was expressed by osteoclasts, suggestingthat EGFR ligands may act directly to affect the forma-tion and/or function of these cells. EGFR signaling regu-lated osteoclast formation. Inhibition of EGFR tyrosinekinase activity decreased the generation of osteoclastsfrom cultured bone marrow cells.

Skeletal elements develop by two distinct mechanisms: in-tramembranous and endochondral ossification (1). Endochon-dral ossification is a process by which a cartilaginous templateis first formed and then replaced by bone. During embryogen-esis, condensations of mesenchymal cells form, within whichchondrocytes develop, proliferate, and differentiate to form acartilage template that contains distinct zones of resting, pro-liferative, and hypertrophic chondrocytes. The proliferationand differentiation of chondrocytes within the cartilage tem-plate are spatially ordered, with proliferating cells at the twoends of the template and progressively more mature cells form-ing hypertrophic cartilage in the middle. Hypertrophic chon-drocytes secrete a specialized extracellular matrix (ECM)1 con-taining collagen X, which becomes calcified. Endochondral

ossification begins with the invasion of the calcified hyper-trophic cartilage by blood vessels, accompanied by osteoclastsand osteoblasts (primary ossification center). The function ofosteoclasts is to remove the hypertrophic cartilage ECM andthat of osteoblasts is to replace it with bone ECM. Longitudinalbone growth is accomplished by the continuing proliferationand maturation of chondrocytes at the ends of the cartilagetemplate (the growth plates) to form more hypertrophic carti-lage and its continual removal and replacement by bone(growth plate ossification or formation of primary spongiosa).Normal endochondral bone development requires the exquisitecoordination of hypertrophic cartilage formation, vascular in-vasion, and the development and function of osteoclasts andosteoblasts (2, 3).

The epidermal growth factor receptor (EGFR) family of re-ceptor tyrosine kinases includes EGFR/ErbB1, HER2/ErbB2,HER3/ErbB3, and HER4/ErbB4 (4, 5). EGFR binds severalligands including epidermal growth factor (EGF), transforminggrowth factor-�, betacellulin, epiregulin, and amphiregulin.During mouse development, EGFR and ligands are expressedin many tissues, including skeletal tissues such as embryonicmandible, Meckel’s cartilage, and limbs (6–10). EGFR-defi-cient mice have abnormal craniofacial cartilage and intramem-branous bone formation resulting in abnormal developmentwith narrow, elongated snouts, underdeveloped jaw, and a highincidence of cleft palate, as well as abnormal development inmany epithelial organs including skin, intestines, and lungs(11–16).

EFGR expression has been detected in the axial and appen-dicular skeleton at the bone-cartilage junction (17), suggestinga role for this signaling pathway in endochrondral bone forma-tion. Overexpression of EGF in transgenic mice using the �-ac-tin promoter results in growth retardation and overprolifera-tion of osteoblasts, consistent with a role for EGFR inosteoblastic cell growth (18). A recent study showed thatEGFR-deficient mice have impaired endochondral ossification,probably secondary to a defect in hypertrophic chondrocytematuration and osteoblastic cell proliferation (19). However, incultured fetal rat long bones, EGF stimulates bone resorption,suggesting that EGFR signaling also plays a role in osteoclastfunction (20). In this study, we showed that impaired recruit-ment of osteoclasts contributed to the impaired endochondralbone formation in EGFR-deficient mice and that EGFR signal-ing is necessary for osteoclast formation from bone marrowprogenitors.

* This work was supported by National Institutes of Health GrantAR46238 (to T. H. V. and Z. W.) and a grant from the Sandler FamilySupporting Foundation (to T. H. V.). The costs of publication of thisarticle were defrayed in part by the payment of page charges. Thisarticle must therefore be hereby marked “advertisement” in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.

� To whom correspondence should be addressed: Box 2911, Universityof California, San Francisco, CA 94143-2911. Tel.: 415-514-4266; Fax:415-514-4365; E-mail: thiennu@itsa.ucsf.edu.

1 The abbreviations used are: ECM, extracellular matrix; EGFR,epidermal growth factor receptor; M-CSF, monocyte-colony stimulatingfactor; MEM, minimal essential medium; MMP, matrix metalloprotein-ase; TRAP, tartrate-resistant acid phosphatase; RT, reverse tran-

scriptase; HC, hypertrophic cartilage; VEGF, vascular endothelialgrowth factor; E, embryonic day.

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 279, No. 51, Issue of December 17, pp. 53848–53856, 2004© 2004 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

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EXPERIMENTAL PROCEDURES

Reagents—AG1478 was purchased from Calbiochem (San Diego,CA). Mouse macrophage colony stimulating factor (mouse M-CSF) waspurchased from R&D (Minneapolis, MN). Recombinant murine RANKligand (rm-sRANKL) was purchased from PeproTech, Inc. (Rocky Hill,NJ). Minimum essential (MEM) �-medium with ribonucleosides andfetal bovine serum were purchased from Invitrogen and Ficoll-Hypaquewas purchased from Amersham Biosciences.

Histological Analyses—The generation of the EGFR-null allele byhomologous recombination in ES cells was previously described (11).EGFR�/� and EGFR�/� mice were genotyped by PCR for the targetedallele. Mice heterozygous for the null allele (EGFR�/�) were mated andthe day of the vaginal plug is designated as E0.5. Pregnant mice weresacrificed at E16.5 and E18.5, the embryos were removed, and the longbones dissected for analyses. In some experiments, long bones fromnewborn pups from heterozygous mating are collected. EGFR�/� em-bryos or newborn pups were recognized by their obvious phenotype ofopen-eyed (15). Bones were fixed in 4% paraformaldehyde in phosphate-buffered saline overnight and decalcified in 0.5 M EDTA (pH 7.4) for 1–3days at 4 °C prior to processing for paraffin embedding. E16.5 boneswere not decalcified. For general morphology, sections were stainedwith Masson Trichrome stains using a kit from Sigma according to theinstructions provided by the manufacturer.

In Situ Hybridization—Complementary DNAs corresponding toCbfa-1 (Runx2), osteocalcin, collagen-type I, matrix metalloproteinase-9(MMP-9), MMP-13 (collagenase-3), and MMP-14 (MT1-MMP) wereused to generate [35S]UTP-labeled antisense riboprobes using a tran-scription kit from Promega (Madison, WI). In situ hybridization wasperformed as described previously (21). Briefly, slides were deparaf-finized, treated with proteinase K (20 �g/ml) for 5 min at ambienttemperature, and hybridized with 35S-labeled antisense riboprobes inhybridization buffer (50% deionized formamide, 300 mM NaCl, 20 mM

Tris-HCl (pH 8.0), 5 mM EDTA, 0.5 mg/ml yeast tRNA, 10% dextransulfate, and 1� Denhardt’s) in a humidified chamber at 55 °C over-night. Following hybridization, the slides were treated with RNase A,washed to a final stringency of 50% formamide, 2� SSC at 60 °C,dipped in emulsion, exposed for 1–4 weeks, developed, and counter-stained with hematoxylin and eosin.

Gelatin Substrate Gel Analyses—The long bones were dissected fromE16.5 and E18.5 EGFR�/� and EGFR�/? embryos and homogenized inlysis buffer (50 mM Tris-HCl, pH 7.4, 1% Nonidet P-40, 0.25% sodiumdeoxycholate, 150 mM NaCl, 1 mM EGTA, 1 mM phenylmethylsulfonylfluoride, 1 �g/ml each aprotinin, leupeptin, pepstatin). Insoluble aggre-gates and nuclei were removed by centrifugation and protein concen-tration of the supernatants was quantified using the BCA protein assayreagent kit (Pierce). Samples (10 �g of protein) were added to non-denaturing loading buffer and separated on 10% SDS-polyacrylamidegels containing 1 mg/ml gelatin (Sigma). Gels were washed two times in2.5% Triton X-100 at ambient temperature for 30 min each, then incu-bated in substrate buffer (50 mM Tris-HCl, pH 8.0, 5 mM CaCl2, 0.02%sodium azide) at 37 °C overnight. Gels were then fixed and stained in30% isopropyl alcohol, 10% acetic acid, and 0.1% Coomassie Blue.

Isolation of Bone Marrow Mononuclear Cells and Osteoclast Forma-tion Assay—Bone marrow mononuclear cells were isolated from 6–8-week-old CD1 mice by a modified procedure from a previously describedmethod (22). Briefly, mice were killed by cervical dislocation. Femoraand tibiae were dissected free of soft tissue. The ends of the bone werecut, and bone marrow cells were flushed out into a cell culture dish byslowly injecting MEM at one end of the bone using a sterile 27-gaugeneedle. The cell suspension was filtered through a cell strainer (Falcon,70 �m nylon) and pelleted by centrifugation at 1000 rpm at 4 °C. Cellswere then resuspended in MEM containing 10% fetal calf serum, platedin a 100-mm cell culture dish at a density of 1 � 107 cells/ml, andincubated at 37 °C in 5% CO2 overnight. The next morning the non-adherent cells were collected, centrifuged, and purified on a Ficoll-Hypaque gradient. The monocyte cell layer was aspirated carefullyfrom the medium and washed with phosphate-buffered saline. The cellswere counted, resuspended in MEM containing 2.5% fetal bovine se-rum, and placed in a 24-well plate at 4 � 104 cells/ml. To each of thesewells, growth factor (25 ng/ml M-CSF and 25 ng/ml RANKL) and eithervehicle (Me2SO) or AG1478 at different concentrations (1.25, 2.5, and 5�M) were added. The culture medium was changed every 3 days. After6 days of culturing, osteoclast formation was evaluated by quantifica-tion of tartrate-resistant acid phosphatase (TRAP)-positive multinucle-ated osteoclast cells as described below.

TRAP Staining—The osteoclast preparations were stained for TRAPactivity using a leukocyte acid phosphatase kit from Sigma. Briefly,

after culturing for 6 or 8 days, cells were rinsed with phosphate-buffered saline, fixed with 37% formaldehyde (formalin) in acetone-citrate buffer for 1 min, and stained according to the instructionsprovided by the manufacturer. All the osteoclasts in one well werecounted under the microscope after counterstaining with hematoxylinas TRAP� cells containing at least three nuclei. The results wereexpressed as mean � S.D. of triplicate samples. TRAP staining was alsoperformed on the paraffin sections according to the instructions pro-vided by the manufacturer. The determination of the numbers anddistribution of TRAP� cells in longitudinal sections of bones wereperformed as described previously (23).

Analysis of EGFR mRNA in Osteoclasts by RT-PCR—Total RNAsfrom cultures of osteoclast cells, and from EGFR�/� and EGFR�/�embryonic heads were isolated using TRIzol Reagent (Invitrogen) ac-cording to the instructions provided by the manufacturer and dissolvedin 50 �l of nuclease-free water. Concentration of the RNA preparationswas quantified by absorbance at 260 nm. EGFR mRNA expression wasdetermined using one tube access reverse transcription (RT)-PCR sys-tem (Promega). Total RNA (1 �g) was added to a RT mixture containing1� AMV/Tfl reaction buffer, 0.2 mmol/liter dNTP mixture, 1 mmol/literMgSO4, 0.1 units/�l AMV reverse transcriptase, 0.1 units/�l Tfl DNApolymerase, and 1 �mol/liter of each EGFR primer in a total volume of50 �l. The EGFR forward primer (5�-CTGCCAAGGCACAAGTAACA-3�) spans nucleotides 304–323 of the mouse EGFR gene and the reverseprimer (5�-ATTGGGACAGCTTGGATCAC-3�) spans nucleotides 783–802. The RT reaction was carried out at 48 °C for 40 min. PCR wascarried out for 40 cycles of 94 °C for 30 s, 56 °C for 30 s, 72 °C for 30 s.PCR products were analyzed on a 1.5% agarose gel.

Northern Blotting—Total RNAs from cultured osteoclasts, EGFR�/�and EGFR�/� embryonic heads were isolated as described above. TotalRNA (20 �g) was electrophoresed through a 1% denaturing formalde-hyde-agarose gel, transferred to Hybond-N� membrane (AmershamBiosciences), and then cross-linked by UV irradiation. Blots were pre-hybridized for 1 h at 68 °C in QuikHybTM reagent (Stratagene, La Jolla,CA) and then hybridized with a random primed 32P-labeled EGFRprobe overnight at 68 °C. Blots were washed at a final stringency of60 °C in 0.2� SCC � 0.1% SDS and then exposed to Hyperfilm MP(Amersham Biosciences) at �70 °C. The EGFR probe used was providedby Dr. Janice Liu at the University of Washington, Seattle, WA (24).The probe contains residues 969–1242 of the rat EGFR cDNA.

Bone Marrow Cell Culture Proliferation Assay—Bone marrow mono-nuclear cells were isolated as described above and seeded in 96-wellculture plates at a density of 20,000 cells/well with MEM supplementedwith 2.5% fetal bovine serum, 25 ng/ml M-CSF, 25 ng/ml RANKL.Either vehicle (Me2SO) or different concentrations of AG1478 (1.25, 2.5,and 5 �M) were added to the wells. Cells were then incubated at 37 °Cin 5% CO2. After 48 h, the number of viable cells was measured by the3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide uptakemethod. Briefly, 10 �l of a 5 mg/ml 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide solution was added to 100 �l of culturemedium in each well and the cells were incubated at 37 °C in 5% CO2

for 4 h. 100 �l of 10% SDS in 0.01 N HCl was then added to each wellovernight and the color reaction was determined by absorbance at 570nm. Six wells were used for each treatment, and experiments wererepeated 3 times.

Bone Resorption Assay—Osteoclast resorption was performed on cal-cium phosphate-coated discs (BD Biosciences Biocoat Osteologic discs).Bone marrow cells were isolated and cultured on osteologic discs in thepresence of M-CSF and RANKL in MEM with 10% fetal calf serum.After osteoclasts form, the medium was changed to resorption medium(MEM adjusted to pH 7.0 with HCl with 10% fetal calf serum), andeither Me2SO or AG1478 (5 �M) was added. After 2 days, the discs werebleached to remove cells, washed in water, and air-dried. Resorptionpits were visualized under light microscopy. Images were taken with adigital camera and analyzed using Adobe Photoshop. Images werevisualized in gray scale and inverted. Areas of resorption pits wereoutlined, and under the histogram function, the percentage of pixels inthe top 25% of the gray scale range contained in the outlined area wascalculated as the percent resorption area. Statistical analysis was doneusing the Student’s t test.

RESULTS

EGFR�/� Mice Show Delayed Primary Endochondral Ossi-fication and Lengthened Growth Plate Hypertrophic CartilageZones—EGFR-null mice generally die within the first postnatalday because of severe respiratory distress (14). Therefore, westudied skeletal development during embryonic development.

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In the data that follows, we show the humerus; however, sim-ilar phenotypes were seen in the radius, ulna, and in the hindlimbs. Primary ossification in the wild type and EGFR�/�bones occurred normally with humeri at E16.5 showing com-pleted invasion of capillaries into the calcified hypertrophiccartilage (HC), with the resultant removal of the middle sectionof the HC and replacement of this area with vascularizedtissues (Fig. 1A). In contrast, in the EGFR�/� humeri, themiddle section of the EGFR�/� HC remained intact, indicatingdelayed primary ossification (Fig. 1B).

At E18.5, ossification in the wild type/heterozygous humericontinued in the longitudinal direction resulting in the forma-tion of an area of trabecular bone (primary spongiosa) and agrowth plate that contained an area of hypertrophic cartilage of

relatively small size (Fig. 1C). However, at E18.5, theEGFR�/� humeri showed a lengthened HC zone at the growthplate and ossification that had not proceeded very far longitu-dinally (Fig. 1D). This delay in ossification continued untilbirth, with continuing accumulation of HC at the growth platesin newborn EGFR�/� mice compared with their wild type orheterozygous littermates (Fig. 1, E and F). Trabecular boneformation was also impaired in EGFR�/� mice. In E18.5 wildtype or heterozygous humeri, the primary spongiosa area waslarge and contained many long trabeculae (Fig. 1G), but in theEGFR�/� humeri, there were only a few short trabeculae (Fig.1H). This impairment in bone formation persisted until birth,and the differences in trabecular bone were also seen in new-born mice (Fig. 1, I and J). We concluded that primary ossifi-cation of the cartilage templates and the subsequent ossifica-tion of the growth plates of the long bones are impaired in theEGFR�/� mice. This occurs in the absence of overall growthretardation in utero. Because the EGFR�/� mice die soon afterbirth, and those that survive for a few days to weeks areseverely growth retarded, we did not analyze postnataldevelopment.

Because heterogeneous mice did not show a bone phenotype,the EGFR wild type and heterozygote embryos were used in-terchangeably in the results that follow. For simplicity they arereferred to as wild type.

EGFR�/� Mice Have Delayed Osteoclast Recruitment intoHypertrophic Cartilage—We next determined the EGFR-de-pendent mechanisms in endochondral ossification. A key eventin primary ossification of the hypertrophic cartilage anlage isthe recruitment of osteoclasts. Mononuclear hematopoietic pre-cursors are disseminated via the bloodstream and deposited inthe mesenchyme surrounding the bone rudiments. There, theyproliferate and differentiate into TRAP� cells that are theprecursors of multinucleated osteoclasts (3). These (pre)oste-oclasts invade the HC together with blood vessels and initiatethe resorption of HC (25). During this migration into HC themononucleated (pre)osteoclasts fuse together to form themultinucleated mature osteoclasts. We saw many TRAP� cellswithin the middle section of the HC in the E16.5 wild typehumeri (Fig. 2A). In contrast, TRAP� cells were found mainlyat the periphery of the HC in the EGFR�/� humeri (Fig. 2B).Quantification of the number of these cells on serial sectionsshowed a significant difference in the number of TRAP� cellsinside versus outside the calcified HC between wild type andEGFR�/� bones (Fig. 2G). There were no apparent differencesin the size of the TRAP� cells. There were also no differencesin the number of nuclei per TRAP� cell between wild type andEGFR�/� mice (data not shown). The difference in the numberof TRAP� cells inside the calcified HC between EGFR�/� andwild type bones diminished by E18.5 (Fig. 2, C–G). Theseresults indicate that the delayed primary ossification of EGFR-deficient HC is coupled with a delay in osteoclast recruitment.

Impaired Bone Formation in EGFR�/� Mice Is Due in Partto Delayed Osteoblast Recruitment—During primary ossifica-tion, concurrent with vascular invasion and osteoclast recruit-ment, osteoblasts also migrate from the bony collar into thehypertrophic cartilage. Because bone formation was also im-paired in the EGFR�/� humeri, we asked whether the recruit-ment of osteoblasts into the EGFR�/� HC was also delayed.We assayed for the expression of the transcription factorCbfa-1, a marker of osteoblast differentiation (26–28) and forthe expression of osteocalcin, a marker of mature osteoblasts(29). In E16.5 wild type and heterozygous humeri, many Cbfa-1-positive cells were found in the middle of the HC zone,whereas in the EGFR�/� bones, these cells were found more atthe periphery (Fig. 3, A and B). Similarly, osteocalcin-positive

FIG. 1. Long bone development in wild type and EGFR�/�mice. Trichrome Masson-stained tissue sections of the humerus ofE16.5 (A and B), E18.5 (C and D, G and H), and newborn (E and F, I andJ) wild type or heterozygous (A, C, E, G, and I) and EGFR�/� (B, D, F,H, and J) mice. At E16.5, vascularization of the calcified hypertrophiccartilage zone has already occurred in the wild type humerus withvascularized tissues replacing hypertrophic cartilage in the diaphysis(A, arrows), whereas invading capillaries remain at the outer edge ofthe calcified hypertrophic cartilage zone in EGFR�/� humerus (B,arrows). At E18.5, endochondral ossification has continued in the wildtype humerus resulting in an area of trabecular bone and a normalsized growth plate (C), whereas there is still a large area of un-ossifiedhypertrophic cartilage in the EGFR�/� humerus (D). In newborn mice,there continues to be a large area of hypertrophic cartilage at thegrowth plate of EGFR�/� humerus (F) compared with wild type (E).Many long bony trabeculae are present in the metaphysis in wild typehumerus at E18.5 (G, arrows) and at birth (I, arrows), but the bonytrabeculae in EGFR�/� littermates are very few and short (H and J,arrows). Bar, A–F, 200 �m; G–J, 100 �m. Because heterogeneous micedid not show a bone phenotype, the EGFR wild type and heterozygoteembryos were used interchangeably and are indicated as EGFR�/?.

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cells were found in the middle of the HC zone of E16.5 wild typehumeri, but they were largely limited to the periphery in theEGFR�/� bones (Fig. 3, C and D). Thus the delay in primaryossification of EGFR�/� HC is also coupled to a delay inrecruitment of osteoblasts. However, by E18.5, there weremany osteocalcin-positive cells in EGFR�/� bones at the car-tilage bone junction (Fig. 3, E and F). Yet, there were still fewtrabecular spicules in these bones, and few osteocalcin-positivecells on these spicules (Fig. 3, E and F). The deficiency intrabecular spicules persisted until birth (see Fig. 1, G–J). Thesedata suggested that development of osteoblast in EGFR defi-ciency might be normal, but their subsequent proliferation,

survival, and/or function might be impaired. The functionalimpairment is not because of a deficiency in osteoblast differ-entiation as measured by collagen I expression, however, be-cause in situ hybridization showed abundant collagen type ImRNA expression at the EGFR�/� growth plates (Fig. 3, Gand H).

Osteoclasts Express EGFR—The delay in osteoclast recruit-ment into EGFR�/� HC may be because of either a directeffect of EGFR signaling in osteoclasts and/or osteoclast pre-cursors or an indirect effect because of EGFR function on othercells that in turn regulate osteoclasts. To determine whetherEGFR ligands can act directly on osteoclasts, we determinedexpression of EGFR by osteoclasts. We isolated wild type bonemarrow cells from CD1 mice and cultured them in vitro withM-CSF and RANK ligand to induce the formation of oste-oclasts. We then determined the expression of EGFR in cul-tured osteoclasts by RT-PCR and Northern blotting. TotalRNAs were isolated from primary cultures of osteoclasts andfrom the heads of wild type and EGFR�/� E18.5 embryos forpositive and negative controls, respectively. RT-PCR was per-formed using a forward primer in exon 1 and a reverse primerin exon 4 to distinguish wild type EGFR mRNA and any resid-ual mRNA from the targeted allele, which has a disrupted exon2. We observed the expression of EGFR mRNA in osteoclasts

FIG. 2. Effect of EGFR deficiency on the number and distribu-tion of TRAP� cells in the developing humerus. Tissue sections ofthe humerus of E16.5 (A and B), E18.5 (C and D), and newborn (E andF) wild type or heterozygous (A, C, and E) and EGFR�/� (B, D, and F)mice stained for TRAP activity. In E16.5 wild type humerus, manyTRAP� cells were detected in the vascularized hypertrophic cartilage(A, arrows). However, in E16.5 EGFR�/� humerus, most of the TRAP�cells were found at the periphery of the hypertrophic cartilage (B,arrows). In E18.5 (C and D) and newborn mice (E and F), there werejust as many TRAP� cells inside the bone rudiment in EGFR�/�humerus (D and F, arrows) as in the wild-type humerus (C and E,arrows). Bar, 100 �m. G, quantification of the number of TRAP� cellsin wild type and EGFR�/� humeri at different stages. Horizontal barsshow mean counts of TRAP� cells found either outside the calcifiedhypertrophic cartilage at the perichondrium/periosteum or inside thecalcified hypertrophic cartilage. At E16.5, there is a significant differ-ence in the total number of TRAP� cells found outside versus inside thecalcified hypertrophic cartilage between wild type and EGFR�/� mice(p � 0.05).

FIG. 3. Expression of Cbfa-1, osteocalcin, and collagen type I inthe humerus of wild type and EGFR�/� mice. A–H, bright fieldimages of tissue sections of E16.5 (A–D) or E18.5 (E–H) humeri fromwild type (A, C, E, and G) or EGFR�/� (B, D, F, and H) mice hybridizedwith 35S-labeled Cbfa-1 (A and B), osteocalcin (C–F), and collagen typeI (G and H) antisense probes. In E16.5 wild type humerus, manyCbfa-1-positive cells were found in the middle section of the hyper-trophic cartilage (A, arrows), whereas these cells are found largely atthe periphery of the hypertrophic cartilage in EGFR�/� humerus (B,arrows). Similarly, osteocalcin (oc) expressing cells are found at theperiphery of hypertrophic cartilage in E16.5 EGFR�/� (D, arrows) andin the middle of wild type hypertrophic cartilage (C, arrows). By E18.5,there were abundant osteocalcin-positive cells in the metaphysis ofEGFR�/� humerus. However, these cells are found mainly at thecartilage-bone junction (F, arrows), and very few are found in theprimary spongiosa, which also contain very few trabecular spicules (F,arrowhead). In contrast, in the E18.5 wild type humerus there areabundant osteocalcin-positive cells both at the cartilage-bone junction(E, arrows) and in the primary spongiosa on trabecular spicules (E,arrowheads). Collagen type I expression was found in the metaphysis inboth wild type and EGFR�/� E18.5 humerus (G and H, arrows). Bar,200 �m.

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and wild type embryonic heads (Fig. 4A). As expected, no PCRproduct corresponding to EGFR was seen in the EGFR�/�embryonic heads. On Northern blot, two transcripts of �9.6and 5 kb were found in osteoclast cultures and wild type em-bryonic heads (Fig. 4B). No transcripts were found inEGFR�/� embryonic heads. These data suggest that EGFRligands may act directly on osteoclasts to affect their function.

Delayed Primary Osteoclast Recruitment in EGFR-null MiceIs Not Because of Deficiency in MMPs—Previous studiesshowed that MMPs were necessary for the migration of (pre)os-teoclasts into calcified hypertrophic cartilage during primaryendochondral ossification (23). In particular, MMP-9 (gelatin-ase B) is required for the timely recruitment of these cells.MMP-9-deficient mice show a delay in osteoclast recruitmentinto the calcified hypertrophic cartilage during primary endo-chondral ossification of the metatarsals (25). Because EGFRsignaling can modulate the cellular expression of MMPs, thedelay in osteoclast recruitment into the EGFR�/� HC may bebecause of deficiency in the expression of MMPs. To test thishypothesis, we analyzed the expression of several MMPs by insitu hybridization. The expression of MMP-9 and MMP-14

(MT1-MMP), which are highly expressed in osteoclasts (30–32), was consistent with the TRAP staining results. In E16.5wild type humeri, MMP-9 expression was found in cells corre-sponding to osteoclasts in the vascularized bone marrow cavityand at the cartilage-bone junction (Fig. 5, A and C). In theE16.5 EGFR�/� humeri, MMP-9 expression was found in cellsat the periphery of the HC, consistent with the location ofosteoclasts in these skeletal elements at this time (Fig. 5, B andD). There were no significant differences in the amount ofMMP-9 mRNA per cell, judged by the intensity of the signal. ByE18.5, there were just as many MMP-9 expressing cells at thecartilage-bone junction of the growth plate and in the bonemarrow cavity in the EGFR�/� bones compared with wild typebones. This is consistent with the observation that the differ-ences in the number of osteoclasts between wild type andEGFR�/� bones have diminished by this time. Substrate gel

FIG. 4. RT-PCR and Northern blot analysis of EGFR mRNA incultured osteoclasts. A, RT-PCR with EGFR-specific primers of totalRNA isolated from cultured osteoclasts, wild type embryonic heads, andEGFR�/� heads. The expected 499-bp PCR product was seen in RNAfrom osteoclast and wild type embryonic head, but not in EGFR�/�head. B, Northern blot of total RNA isolated from cultured osteoclasts,wild type embryonic heads, and EGFR�/� heads hybridized with a32P-labeled EGFR probe. Two transcripts, 9.6 and 5.0 kb in size, weredetected in RNA from osteoclasts and wild type embryonic head, but notfrom EGFR�/� head.

FIG. 5. Expression of MMP-9 in the humeri of wild type/ho-mozygous and EGFR�/� mice. A–D, bright field (A and B) and darkfield (C and D) images of tissue sections of E16.5 humerus from wildtype (A and C) or EGFR�/� (B and D) mice hybridized with 35S-labeledMMP-9 antisense probe. MMP-9 expression was found in cells insidethe vascularized hypertrophic cartilage, including at the cartilage-bonejunction in wild type humerus (A and C, arrows), and in cells at theouter edge of the calcified hypertrophic cartilage in EGFR�/� humerus(B and D, arrows). E–H, bright field (E and F) and dark field (G and H)images of tissue sections of E18.5 humerus from wild type (E and G) orEGFR�/� (F and H) mice hybridized with 35S-labeled MMP-9 anti-sense probe. Similar number and distribution of MMP-9 expressingcells were found in both wild type and EGFR�/� humerus. I, gelatinzymogram of tissue lysates from the long bones of wild type andEGFR�/� mice showing a slightly decreased level of both latent andactivated gelatinase B (Gel Ba) in EGFR�/� bones and a normal levelof latent and activated gelatinase A (Gel Aa). Bar (A–H), 200 �m.

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analyses showed that there was slightly less MMP-9 protein inextracts of EGFR-null bones compared with wild type (Fig. 5I).There was no apparent change in the ratio of active versuslatent forms of MMP-9 proteins. These results indicated thatMMP-9 expression and activity in osteoclasts were not signifi-cantly altered by EGFR deficiency, even though there might bean overall decrease in the level of MMP-9 because of decreasedosteoclast number. Thus deficiency in MMP-9 activity was un-likely to be the primary cause for the delayed recruitment ofosteoclasts into EGFR-null HC. Similar results were observedfor the expression of MMP-14 (MT1-MMP). In E16.5 wild typehumeri, MMP-14 was expressed by cells with similar distribu-tion to osteoclasts, at the cartilage-bone junction and in thebone marrow cavity (Fig. 6, A and C). In contrast, cells express-ing MMP-14 were located at the periphery of the unvascular-ized HC in E16.5 EGFR�/� bones (Fig. 6, B and D). At E18.5the number and location of MMP-14 expressing cells werecomparable in wild type, heterozygous, and EGFR�/� bones(data not shown).

MMP-13 (collagenase-3) may also be important for the vas-cularization of hypertrophic cartilage during primary endo-chondral ossification. It can degrade native collagen, which is amajor component of the hypertrophic cartilage ECM (33, 34).MMP-13 is highly expressed in hypertrophic chondrocytes andosteoblasts (35) and therefore may act to degrade collagens inthe hypertrophic cartilage ECM during endochondral ossifica-tion. In E16.5 wild type humeri, MMP-13 was expressed by latehypertrophic chondrocytes and by cells at the vascularizationfront, some of which may be osteoblasts (Fig. 6, E and G). In the

E16.5 EGFR�/� bones, there was high expression of MMP-13in hypertrophic chondrocytes in the calcified HC (Fig. 6, F andH). These results indicated that deficiency in MMP-13 expres-sion was not the cause for the delayed primary ossification andosteoclast recruitment into EGFR�/� hypertrophic cartilage.

Osteoclast Formation from Bone Marrow Cells Is Attenuatedby EGFR Inhibitor—The delay in osteoclast recruitment intoEGFR�/� hypertrophic cartilage may be because of deficiencyin either their formation or their migration. To address the roleof EGFR signaling in osteoclast formation, we inhibited EGFRsignaling during the formation of osteoclasts from bone marrowcells in vitro using the EGFR tyrosine kinase inhibitor AG1478(36). Wild type bone marrow cells from CD1 mice were isolatedand cultured in the presence of M-CSF and RANK ligand toinduce the formation of osteoclasts. The addition of AG1478 inthese cultures attenuated osteoclast formation (Fig. 7). Cul-tures treated with vehicle showed the formation of multinucle-ated TRAP� cells characteristic of osteoclasts (Fig. 7A). Addi-tion of increasing concentrations of AG1478 decreased thenumber of multinucleated TRAP� cells that were formed in adose-dependent manner (Fig. 7, B–D), reaching over 90% inhi-bition at 5 �M AG1478 (Fig. 7E). Addition of exogenous EGF ortransforming growth factor-� had no additional effects on theformation of osteoclasts in these cultures (data not shown).

Several genes have been shown to be important for osteoclastformation from hematopoietic stem cells (37). We askedwhether any of these genetic pathways was downstream ofEGFR signaling. Quantitative real time RT-PCR of RNA iso-lated from bone marrow cells cultured in the presence of M-CSF and RANK ligand with or without AG1478 showed nochange in the expression of PU.1, c-fos, TRAF-6, MITF, andc-src with AG1478 treatment (data not shown). Thus the reg-ulation of osteoclast formation by EGFR signaling does notappear to be mediated through these pathways. To determinewhether the inhibition of osteoclast formation by AG1478 wasbecause of an effect on cell proliferation, we assayed for cellproliferation in the AG1478-treated bone marrow cell cultures.Wild type bone marrow cells were isolated and cultured for 2days in the presence of M-CSF and RANK ligand with orwithout AG1748. Treatment with AG1478 resulted in a dose-dependent decrease in 3-(4,5-dimethylthiazol-2-yl)-2,5-diphe-nyltetrazolium bromide uptake, indicative of decreased cellnumber (Fig. 7F). This suggested that inhibition of EGFR sig-naling by AG1478 led to decreased cell proliferation. This mayaccount in part for the decrease in osteoclast formation causedby AG1478 treatment.

Osteoclast Resorptive Activity Is Not Modulated by EGFRInhibitor—To determine whether EGFR signaling modulatesosteoclast function, we tested whether osteoclast resorptiveactivity is inhibited in the presence of the EGFR tyrosinekinase inhibitor AG1478. Osteoclasts derived from bone mar-row progenitor cells were allowed to form on calcium phos-phate-coated discs and subsequently switched to resorptivemedia in the presence of AG1478 or vehicle control for 2 days.Quantitative analyses of the areas of resorption showed thatthere were no significant differences in the percent resorptionarea between control and treated osteoclasts (Fig. 8). ThereforeEGFR signaling does not appear to modulate osteoclastic boneresorption.

DISCUSSION

In this study we have investigated the function of EGFRsignaling in endochondral bone formation by analyzing longbone development in EGFR-null mice. Our data showed that inEGFR-null mice primary ossification of the calcified hyper-trophic cartilage anlage was delayed and was associated with adelay in the recruitment of osteoclasts and osteoblasts. Forma-

FIG. 6. Expression of MMP-14 (MT1-MMP) and MMP-13 (colla-genase 3) in the wild type and EGFR�/� humeri. A–D, bright field(A and B) and dark field (C and D) images of tissue sections of E16.5humeri from wild type (A and C) or EGFR�/� (B and D) mice hybrid-ized with 35S-labeled MMP-14 antisense probe. Similar to MMP-9,MMP-14 expression was found in cells inside the vascularized hyper-trophic cartilage, including at the cartilage-bone junction in wild typehumerus (A and C, arrows), and in cells at the outer edge of the calcifiedhypertrophic cartilage in EGFR�/� humerus (B and D, arrows). E–H,bright field (E and F) and dark field (G and H) images of tissue sectionsof E16.5 humeri from wild type (E and G) or EGFR�/� (F and H) micehybridized with 35S-labeled MMP-13 antisense probe. MMP-13 expres-sion was found in the lower hypertrophic chondrocytes adjacent to thevascularized area in wild type humerus (E and G, arrows), and inchondrocytes of the calcified hypertrophic cartilage in EGFR�/� hu-merus (F and H, arrows). Bar: 200 �m.

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tion of the primary spongiosa was also abnormal leading toaccumulation of growth plate HC and decreased trabecularbone mass. The delay in osteoclast recruitment might be be-

cause of inadequate formation of osteoclasts resulting fromEGFR deficiency.

EGFR Signaling Is Necessary for Normal Osteoclast Recruit-ment and Function during Endochondral Ossification—A crit-ical step in endochondral ossification is the invasion of capil-laries into the calcified hypertrophic cartilage zone in thediaphysis of the cartilage anlage. Vascularization of calcifiedHC is accompanied by the recruitment of osteoclasts and os-teoblasts. We found that EGFR deficiency resulted in delayedrecruitment of osteoclasts into calcified HC during primaryossification. However, this delay was only temporary, consist-ent with the model that the lack of EGFR signaling causesdefective formation of osteoclasts from precursor cells sur-rounding the bone rudiments, thus requiring a longer time forsufficient osteoclasts to accumulate. Alternatively, the migra-tion of osteoclasts into calcified HC may also be defective.

Even though osteoclast recruitment into EGFR�/� HCreached the same level as wild type mice at E18.5, ossificationof growth plate HC was still not complete, as evidenced by thelengthened HC zones of the EGFR�/� growth plates up untilbirth. This may be because of the accumulation of HC caused bythe delay in primary ossification, and the inability of the sub-sequently normal number of osteoclasts to overcome the initialdifference and therefore ossification in the longitudinal direc-tion would always be behind in the EGFR-null bones. Alterna-tively, growth plate ossification may also be abnormal becauseof either abnormal formation of growth plate HC or abnormal-ity in its removal. A recent study also reported increasedgrowth plate HC in the EGFR�/� mice (19). The authors foundexpression of EGFR in chondroblasts but no differences ingrowth plate chondrocyte proliferation, and suggested thatEGFR negatively regulated hypertrophic chondrocyte matura-tion. On the other hand, the removal and ossification of EGFR-

FIG. 7. Effect of the inhibition of EGFR signaling on osteoclastformation. A–D, TRAP staining of bone marrow cells cultured for 6 dayswith RANKL (25 ng/ml), M-CSF (25 ng/ml), and either vehicle or increas-ing concentrations of the EGFR tyrosine kinase inhibitor AG1478 (1.25,2.5, and 5 �M). In vehicle-treated cultures, there were a large number ofmultinucleated TRAP� cells characteristic of osteoclasts (A). Treatmentwith AG1478 caused a dose-dependent decrease in the number ofmultinucleated TRAP� cells (B–D). E, quantification of the number ofosteoclasts developed in control cultures and cultures with different con-centrations of AG1478. Each histogram represents the mean number oftotal osteoclasts counted in three wells of a 24-well plate. F, effects ofEGFR signaling inhibition on proliferation of bone marrow mononuclearcells. Wild type bone marrow mononuclear cells were isolated and cul-tured for 2 days in the presence of RANKL (25 ng/ml), M-CSF (25 ng/ml),and either vehicle or increasing concentrations of the EGFR tyrosinekinase inhibitor AG1478 (1.25, 2.5, and 5 �M). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide uptake was assessed by absorbanceand used as a measure of cell number. AG1478 caused a dose-dependentdecrease in cell number as reflected by the decrease in absorbance. Eachhistogram represents the mean value of six replicates. The experimentswere repeated three times with similar results.

FIG. 8. Effect of the inhibition of EGFR signaling on osteoclastfunction. A, images of the resorption pits formed by osteoclasts oncalcium phosphate-coated discs. Bone marrow cells were cultured oncalcium phosphate-coated discs in the presence of M-CSF and RANKLto form osteoclasts before switching to resorptive media containingvehicle control (Me2SO) or AG1478 (5 �M) for 2 days. B, quantitativeanalyses of the area of resorption as a percentage of the total areacorresponding to an osteoclast. Data are presented as mean � S.D.(error bars) of 35 osteoclasts analyzed. There was no significant differ-ence between Me2SO (DMSO) and AG1478 treated groups (p � 0.36)

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null growth plate HC may also be abnormal, proceeding at aslower rate resulting in accumulation of growth plate HC. Wefound expression of EGFR on mature osteoclasts, suggestingthat these cells can respond directly to EGFR ligands. Thefunction of EGFR in osteoclasts is not known, but it may be tostimulate their proteolytic activity. EGFR ligands have beenfound to stimulate osteoclastic bone resorption in vivo and invitro in bone organ cultures (20, 38, 39).

MMPs are necessary for the recruitment of osteoclasts intocalcified hypertrophic cartilage during primary endochondralossification (25). The craniofacial and lung developmental de-fects of EGFR-null mice may be because of alteration in MMPactivity (15, 16). We found that down-regulation MMP-9, MMP-13, and MMP-14 was unlikely to be the primary mechanism forthe delayed recruitment of osteoclasts into EGFR�/� HC.However, this does not exclude EGFR regulation of other ECMdegrading proteinases that are important in osteoclast func-tion. In addition, because MMP-13 acts synergistically withMMP-9 (25), the initial delayed recruitment of osteoclasts ex-pressing MMP-9 could functionally blunt HC ECM degradationand exacerbating the delay in HC vascularization.

Vascular endothelial growth factor (VEGF) is important forvascularization of and osteoclast recruitment into calcified hy-pertrophic cartilage of the developing bones (25, 40). We foundno significant difference in VEGF expression in EGFR-nullbones by in situ hybridization.2 This suggests that the delayedosteoclast recruitment into EGFR-null HC is not because ofdeficiency in VEGF expression. However, because MMP-9 mayregulate the bioavailability of VEGF, the initial delay in oste-oclast recruitment may cause an initial decrease in MMP-9activity leading to decreased bioavailable VEGF, which leads tofurther delay in vascularization and recruitment of osteoclasts.

EGFR Deficiency Leads to Impaired Trabecular Bone Forma-tion—Trabecular bone mass is decreased in the EGFR�/�bones. This may partly be because of a delay in the initialrecruitment of osteoblasts during primary ossification. How-ever, trabecular spicules continued to be deficient until birth.Fewer osteoblasts were found in the EGFR�/� primary spon-giosa even though they are abundant at the cartilage bonejunction. This suggests that formation of osteoblasts in theEGFR�/� bones may be normal, but that their subsequentproliferation/survival and/or function may be impaired. Studiesof the LRP5 (low density lipoprotein receptor-related protein5)-deficient mice showed that osteoblast differentiation andproliferation were differentially regulated (42). Sibilia et al.(19) reported that primary EGFR�/� calvarial osteoblast cul-tures showed decreased proliferation potential and increaseddifferentiation as measured by their ability to form bone nod-ules in vitro (19). Thus the decreased trabecular bone mass inthe EGFR�/� mice was likely because of decreased osteoblastproliferation. A direct effect of EGFR signaling on osteoblasts issupported by previous studies showing that EGFR was ex-pressed in osteoblasts in vivo (17, 43), and that EGF stimulatedosteoblast proliferation in vitro (44, 45).

EGFR Signaling Is Necessary for the Formation of Oste-oclasts—Previous studies have identified two essential factorsfor osteoclastogenesis: M-CSF and RANKL (37). M-CSF is nec-essary for the generation of the monocyte/macrophage cell lin-eage and RANKL for their differentiation into osteoclasts. Wefound that induction of osteoclast formation from bone marrowcells in the presence of RANKL and M-CSF was significantlyinhibited in the presence of the EGFR tyrosine kinase inhibitorAG1478. This is consistent with previous studies suggesting arole for EGFR ligands in osteoclast formation. Addition of ei-

ther transforming growth factor-� or EGF increased the forma-tion of multinucleated osteoclasts from cultured human bonemarrow cells (46). In our bone marrow cell cultures addition ofexogenous EGF or transforming growth factor-� had no addi-tional effect on osteoclast formation. This may be because therewere already saturating amounts of endogenous EGFR ligandsin these cultures, or that the endogenous EGFR was trans-activated by other ligand-receptor signaling pathways (47). Therole of EGFR signaling in osteoclast formation may be a directeffect on osteoclast precursors to stimulate either their prolif-eration or differentiation. Alternatively, EGFR ligands may acton other bone marrow cells to stimulate production of eithersecreted or cell-surface factors that in turn act in a paracrinefashion on osteoclast precursors to effect their growth anddifferentiation. Further studies are needed to distinguish be-tween these two possibilities.

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Ke Wang, Hiroaki Yamamoto, Jennie R. Chin, Zena Werb and Thiennu H. VuEndochondral Ossification Because of Defective Osteoclast Recruitment

Epidermal Growth Factor Receptor-deficient Mice Have Delayed Primary

doi: 10.1074/jbc.M403114200 originally published online September 28, 20042004, 279:53848-53856.J. Biol. Chem. 

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