Am J Physiol Cell Physiol Journal of Applied Physiology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Cell Physiol 293: C1209-C1215, 2007. First published July 25, 2007; doi:10.1152/ajpcell.00213.2007
0363-6143/07 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Figures
Right arrow All Versions of this Article:
293/4/C1209    most recent
00213.2007v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Imai, K.
Right arrow Articles by D'Armiento, J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Imai, K.
Right arrow Articles by D'Armiento, J.

EXTRACELLULAR MATRIX, CELL INTERACTIONS

Bone growth retardation in mouse embryos expressing human collagenase 1

Kazushi Imai,1 Seema S. Dalal,1 John Hambor,2 Peter Mitchell,2 Yasunori Okada,3 William C. Horton,4 and Jeanine D'Armiento1

1Division of Molecular Medicine, Department of Medicine, College of Physicians and Surgeons of Columbia University, New York, New York; 2Central Research Division, Pfizer Inc., Groton, Connecticut; 3Department of Pathology, School of Medicine, Keio University, Tokyo, Japan; and 4Emory Orthopaedics and Spine Center, Emory University, Atlanta, Georgia

Submitted 23 May 2007 ; accepted in final form 20 July 2007

ABSTRACT

Cellular growth and differentiation are readouts of multiple signaling pathways from the intercellular and/or extracellular milieu. The extracellular matrix through the activation of cellular receptors transmits these signals. Therefore, extracellular matrix proteolysis could affect cell fate in a variety of biological events. However, the biological consequence of inadequate extracellular matrix degradation in vivo is not clear. We developed a mouse model expressing human collagenase (matrix metalloproteinase-1, MMP-1) under the control of Col2a1 promoter. The mice showed significant growth retardation during embryogenesis and a loss of the demarcation of zonal structure and columnar array of the cartilage. Immunological examination revealed increased degradation of type II collagen and upregulation of fibronectin and {alpha}5-integrin subunit in the transgenic cartilage. The resting zone and proliferating zone of the growth plate cartilage exhibited a simultaneous increase in bromodeoxyuridine (BrdU)-incorporated proliferating cells and terminal deoxynucleotidyl transferase-mediated X-dUTP nick-end labeling-positive apoptotic cells, respectively. Chondrocyte differentiation was not disturbed in the transgenic mice as evidenced by normal expression of the Ihh and type X collagen expression. These data demonstrate that type II collagen proteolysis is an important determinant for the skeletal outgrowth through modulation of chondrocyte survival and cartilagenous growth.

chondrocyte; fibronectin; integrin; matrix metalloproteinase; type II collagen

THE EXTRACELLULAR MATRIX (ECM) is an intricate three-dimensional complex comprising structural proteins interspersed with cellular components. During development the ECM provides critical information to the cells for differentiation and survival. To establish a functional organism, biological signals from the ECM are required to coordinate the appropriate cell phenotypes into the correct spatiotemporal pattern. ECM remodeling through proteolytic degradation and new protein deposition accompanies this dynamic feature of the ECM.

The growth plate cartilage in the epiphysis is an important structure in endochondral ossification and longitudinal bone growth. The growth plate consists of three demarcated zones of chondrocytes with characteristic morphology in parallel with their differentiation state, i.e., the resting, proliferating, and hypertrophic zones. Chondrocytes in the resting zone undergo a period of proliferation during which they secrete a matrix rich in type II collagen and aggrecan (14, 33). After proliferating, the cells begin a phase of maturation marked by an increase in cellular volume and the expression of phenotypic markers of hypertrophy including type X collagen. Finally, the mature cartilage matrix surrounding the hypertrophic chondrocytes undergoes calcification, resorption, and replacement with woven bone (2, 46). These structural proteins also play an important role in providing biological information to the chondrocytes. Genetic mutations or deletions of the proteins disturb chondrocyte differentiation, resulting in skeletal malformations (20, 33). These genetic studies provide conclusive evidence that each cartilage component has a critical and intrinsic role in the development of the growth plate. The ECM can transmit signals to the differentiating chondrocytes (6, 11, 34), and therefore alteration of this matrix may regulate chondrocyte and cartilage maturation.

Recent gene-targeting studies of matrix metalloproteinases (MMPs), including gelatinase B (MMP-9), membrane-type 1 MMP (MMP-14), and collagenase 3 (MMP-13), demonstrate involvement of ECM proteolytic degradation in skeletogenesis (16, 18, 23, 41, 45). In these genetic models, the cartilage ECM is not replaced by trabecular bone due to the lack of angiogenesis into the ossification center, resulting in elongation of the cartilaginous zone and skeletal growth retardation. These studies emphasize a critical role for proteolytic activity in organogenesis. However, it is difficult to determine the biological role of ECM degradation using gene-targeting animal models because of redundancy of enzyme expression and sharing of substrate specificity (19, 27, 32, 37, 39, 48). Aggrecan, a major ECM protein besides type II collagen, is a large chondroitin sulfate proteoglycan and forms huge structural aggregates with hyaluronate and cartilage link protein. Although homozygous and heterozygous null mutation of aggrecan gene exhibits dwarfism due to the aberration of cartilaginous development (22, 47), recent evidence demonstrated that aggrecan is primarily degraded by a desintegrin and metalloproteinase with thrombospondin motifs (ADAMTS), but not by MMPs, including human and mouse cartilage (21, 28, 40). To substantiate a biological consequence of type II collagen degradation, we have developed a transgenic mouse model expressing MMP-1, of which its homologue is absent in mice, in chondrocytes, quoting reliable detection of net transgene expression. The mice upregulated fibronectin and {alpha}5-integrin subunit in cartilage and exhibited skeletal growth retardation during embryonic development, suggesting that proteolysis of type II collagen directly orchestrates cartilagenous development.

MATERIALS AND METHODS

Generation of transgenic mice. The 9.3-kb genomic human matrix metalloproteinase (MMP-1) gene fragment including the signal peptide sequence (7) was ligated to the rat Col2a1 gene promoter (1, 17, 24) at the KpnI site in pBluescript II KS +/– (Stratagene, La Jolla, CA). The 12.8-kb Col2a1-MMP-1 transgene was isolated and purified after NotI-SalI enzymatic digestion as previously described (7). The transgene was microinjected into fertilized FVB mouse eggs. Four founder transgenic mice were identified by Southern blot analysis and analyzed in the experiments described below. All animals were housed in accordance with institutional guidelines and utilized in experiments that were reviewed and approved by the Institutional Review Boards of Columbia University.

Analysis of mRNA. Total RNA was prepared from mouse tissues using the guanidium thiocyanate-cesium chloride method (5), and RNase protection analysis was performed as previously described (7, 8). Ten micrograms of each RNA sample was hybridized and a protected fragment of 585 nucleotides was generated after RNase T2 digestion (GIBCO-BRL, Grand Island, NY) for ~16 h at 65°C. A standard amount of RNA from the lungs of mice, which carry an MMP-1 transgene, was used as a positive control (7).

For Northern blot analysis, 15 µg of total RNA was electrophoresed on a 1.2% formaldehyde-agarose gel and transferred to nylon membranes (Hybond-N+, Amersham) followed by cross-linking with ultraviolet light. Filters were hybridized with 32P-labeled probes for the mouse Col1a1, Col2a1, and Col3a1 cDNA as previously described (15).

Histological analysis. Mouse tissues were fixed in 10% buffered formalin for ~16 h at 4°C and embedded in paraffin wax. Tissues containing calcified bones were treated with 0.5 M EDTA, pH 7.4, before paraffin embedding. Tissue sections (4 µm) were stained with hematoxylin and eosin or toluidine blue. For skeletal analysis, animals were euthanized by CO2 asphyxiation, and the skin was removed for isolation of DNA to determine the genotype. The carcasses were fixed in 95% ethanol and stained with 0.015% alcian blue and 0.005% alizarin red (15). For terminal deoxynucleotidyl transferase (TdT)-mediated X-dUTP nick-end labeling (TUNEL) on tissue sections, the In Situ Cell Death Detection Kit (Boeheringer Mannheim, Indianapolis, IN) was used according to the manufacturer's instructions. For bromodeoxyuridine (BrdU) labeling, pregnant mice at 15-day of postcoitus (dpc) and newborn mice were injected intraperitoneally with BrdU at a dose of 50 µg/g body wt 2 h before and 100 µg/g body wt 1 h before being euthanized, respectively. Limbs fixed in 10% formalin and embedded in paraffin wax were examined for BrdU incorporation by using a BrdU staining kit (Zymed, San Francisco, CA). For immunochemical staining, primary antibodies against type II collagen (Rockland, Gilbertsville, PA), type X collagen (Calbiochem, La Jolla, CA), Bcl2 (Santa Cruz Biotechnology, Santa Cruz, CA), or MMP-1 (Fujichemical Industries, Takaoka, Japan) were used. Goat antibodies specific to Ihh, {alpha}1-, {alpha}2-, {alpha}3-, or {alpha}5-integrin subunit, or fibronectin (Santa Cruz Biotechnology) were also used. Tissue specimens were pretreated with protease (type XXIV, Sigma, St. Louis, MO) and testicular hyarulonidase (Sigma) for immunohistochemistry performed with type II and type X collagen antibodies and hyarulonidase for immunohistochemistry with other antibodies (42). Biotinylated goat anti-rabbit IgG or FITC-conjugated rabbit anti-goat IgG were used as secondary antibodies. To immunolocalize human MMP-1, mouse monoclonal antibody and a Mouse-on-Mouse staining kit were used (InnoGenex, San Ramon, CA).

Western blot analysis. The limb cartilage of newborn transgenic mice was obtained and cultured in 1 ml of serum-depleted Neuman and Tytell culture medium containing L-glutamine (GIBCO-BRL) for 3 days at 37°C in a 5% CO2 incubator. Control samples that did not harbor the transgene were obtained from littermates. The culture media (40 µl/mg tissue dry wt) were subjected to SDS-PAGE (10% total acrylamide) under reducing conditions before and after treatment with 1 mM p-aminophenylmercuric acetate (an activator of proMMP-1) for 12 h at 37°C. To prepare the peptic fractions of the cartilage homogenates, limbs from newborn mice were first homogenized in 2 mM sodium phosphate buffer, pH 6.8, containing (in mM) 150 NaCl, 100 6-amino-hexanoic acid, 20 EDTA, 5 benzamidine, 5 N-ethylmaleimide, and 0.1 phenylmethylsulfonyl fluoride, digested with pepsin (Sigma) as previously described (42) and then subjected to SDS-PAGE (8.5% total acrylamide) with 2% beta-mercaptoethanol. Proteins separated on the gels were electrophoretically transferred onto nitrocellulose filters. The filters were then incubated for ~12 h at 23°C with polyclonal antibody specific to human MMP-1 (31) or to COL2A1 (Rockland) following incubation with biotinylated goat (anti-rabbit IgG) IgG, and developed with 3,3'-diaminobenzidine tetrahydrochloride.

RESULTS

Generation of transgenic mice and expression of the transgene. The tissue-specific expression pattern of MMP-1 in the transgenic mice under the control of the Col2a1 promoter was identical in all four founder mice in agreement with previous studies using this promoter in transgenic mice (1, 17, 24), and all four founders were analyzed in each experiment described below. Tissue specificity was confirmed when transgene mRNA expression in four lines harboring the Col2a1-MMP-1 transgene was examined (Fig. 1A). The predominant site of expression of the transgene was observed in the joints and tail with a low level of expression seen in the heart and eye in an agreement with endogenous type II collagen expression (4). Immunohistochemical analysis indicated that the MMP-1 protein localized to the chondrocytes in the resting zone and upper part of the proliferating zone of the transgenic mouse cartilage (Fig. 1B). Culture media of the cartilage explants of the humerus and tibiae from newborn mice were subjected to Western blot analysis by using a human MMP-1-specific antibody. As shown in Fig. 1C, a specific reaction was observed in the transgenic samples for both the 52-kDa and 42-kDa form of MMP-1 (Fig. 1C). Thus the human MMP-1 transgene was secreted and activated in the transgenic cartilage but not in wild-type mouse cartilage.


Figure 1
View larger version (76K):
[in this window]
[in a new window]

  		Fig. 1. Expression of the Col2a1-MMP-1 transgene. A: top, expression of matrix metalloproteinase-1 gene (MMP-1) in the joint and tail in four transgenic lines examined by RNase protection analysis. The protected fragment of 585 nucleotides (nt) from RNase T2 digestion was represented as MMP-1. Col 50 control indicates positive control RNA samples isolated from the lung of MMP-1-expressing mice as previously reported (Ref.7). Bottom, expression of MMP-1 mRNA in multiple tissues in the line 104 examined by RNase protection analysis. B: immunohistochemical localization of human MMP-1 in tibial growth plates of newborn transgenic embryos (line 104). Wt, wild-type mouse; Tg, transgenic mouse. Bar indicates 150 µm. C: Western blot analysis demonstrates the latent form (52 kDa) and/or active form (42 kDa) of human MMP-1 in culture media of cartilage explants of newborn mice from three different transgenic founders (lines 104, 106, and 121). Partially activated human recombinant MMP1 reacts with the MMP-1 antibodies (MMP-1). A wild-type mouse sample did not react with the antibody (negative control).

 
Skeletal growth retardation in transgenic mouse embryos. During embryogenesis the transgenic mice were discernibly smaller than wild-type embryonic littermates. To analyze the skeletal growth and ossification pattern of these mice, embryos and neonates were subjected to alizarin red and alcian blue staining (Fig. 2A). Alizarin red deposited with Ca2+ within calcified bone and alcian blue localized to the cartilage matrix. Although the shape and ossification patterns of each skeletal component, including the limbs, rib cage, axial skeleton, and hyoid bone, were not different between transgenic and wild-type mice, bone growth in the transgenic animals was significantly delayed as indicated by the femoral length (Fig. 2B). The cranial and facial structure was indistinguishable between mice with or without transgene expression (data not shown). No obvious gross and histological abnormalities were observed in the joints of the transgenic mice compared with wild-type littermates throughout their lifetime (analyzed at ages 3, 6, 12, and 18 mo) (data not shown).


Figure 2
View larger version (16K):
[in this window]
[in a new window]

  		Fig. 2. Embryonic growth retardation of the MMP-1 Tg mice (line 104). A: skeletal staining with alizarin red and alcian blue showed growth retardation of the Tg mice at 15 day of postcoitus (dpc). B: growth retardation as indicated by the femoral length was significant in the embryos but compensated postnatally (closed circle, Tg mice; open circle, Wt littermates; pp, day of postpartum; bars indicate means ± SD; *P < 0.05) Number of observation in each circle is at least five embryos.

 
Microscopic structure of the growth plates. Histological examination was undertaken on the tibial and femoral growth plates. The typical growth plate in wild-type embryos exhibits a clear demarcation of the cartilaginous zones with distinct cellular morphology. In the resting zone, the chondrocytes are round or oval in shape. The proliferating chondrocytes are flattened and organized into linear clusters, and the hypertrophic chondrocytes are vacuolated and larger in size (Fig. 3, A, B, D, and F). However, in the transgenic mice, the chondrocytes in the resting zone appear to extend into the proliferating zone, and the proliferating chondrocytes are more homogeneously dispersed and less flattened (Fig. 3, A and E). These cellular changes result in a loss of the demarcation between these two layers in the transgenic cartilage. There was no difference in the hypertrophic chondrocytes between the transgenic and wild-type mice (Fig. 3, F and G). Although these changes were still present in the newborn mice, they were less pronounced than in the 15 dpc embryos (data not shown). Endochondral and subperiosteal ossification in the longitudinal bones was not disturbed by MMP-1 expression, and the manifestation of angiogenesis in forming the ossification center in epiphysis and diaphysis was also indistinguishable between transgenic and wild-type mice (data not shown). The morphological difference in the growth plate ceased in the postnatal growth period. All other tissues, including the eyes, heart, and tail in which MMP-1 was expressed, did not exhibit any histological anomalies in the transgenic mice compared with wild-type mice (data not shown). All of the subsequent studies were performed with 15 dpc embryos to gain insight into the mechanism of abnormal growth.


Figure 3
View larger version (112K):
[in this window]
[in a new window]

  		Fig. 3. Histological examination of the tibial growth plates of 15 dpc embryos (line 104). A: growth plate is less demarcated and chondrocytes in the resting zone extend into the proliferating zone of Tg mice compared with Wt mice. R, resting zone; P, proliferating zone; H, hypertrophic zone. In the high-power view, chondrocytes in the resting zone of the Tg mice exhibited hypercellularity and bigger nuclei (C) when compared with the chondrocytes of the Wt mice (B). Cells are less flattened and the columnar array disorganized in the proliferating zone (E) of the Tg mice compared with the Wt mice (D). In the hypertrophic zone, there is no obvious histological difference between the Wt and Tg plates (G and H). Data were reproducible between lines and representative mice are shown from line 104. Bar indicates 150 µm (A) and 20 µm (B).

 
Collagen expression and degradation. Anti-type II collagen antibody stained the cartilage matrix throughout the growth plate of both the transgenic and wild-type mice (online supplementary Fig. 1). The MMP-1 protein was shown to be active since quantitative Western blot analysis demonstrated an increased amount of TCA fragment (3/4 fragment of collagen fibrils) in the transgenic cartilage extract, which is concomitant with the decrease in the intact type II collagen molecule (Fig. 4A). Measurement of band intensity demonstrated that the amount of TCA fragment was increased (from 4.18- to 33.41-fold) in transgenic cartilage compared with wild-type counterparts (Fig. 4B). Interestingly, along with the degradation of type II collagen protein, the expression of Col2a1 was upregulated in the transgenic cartilage (Fig. 4C). The type I and III collagen genes were not expressed in the mouse cartilage (data not shown).


Figure 4
View larger version (50K):
[in this window]
[in a new window]

  		Fig. 4. Type II collagen degradation and expression in the cartilage (line 104). A: peptic fraction of cartilage homogenate were electrophoresed by SDS-PAGE under reducing conditions and transferred onto nitrocellulose membranes. The membranes were then stained with Coomassie brilliant blue (left) or reacted with an anti-COL2A1 antibody for Western blot analysis (right). The extracts from Wt cartilage and Tg cartilage were loaded in lanes 1 and 2, and lanes 3 and 4, respectively. In right, collagenase-specific digestion was demonstrated by the increase of TCA fragments and by the reduction of intact Col2a1 chain in the Tg cartilage samples (lanes 3 and 4) compared with the Wt extracts (lanes 1 and 2). B: degradation of type II collagen was quantitatively analyzed by measurement of band intensity presented in A. Protein amount applied for each lane was normalized by the intensity of 68-kDa band in the gel stained with Coomassie brilliant blue. Degradation products of type II collagen (closed bars, TCA fragment in A) were divided by intact type II collagen band (grayed bars, Col2a1 in A). The ratio of degradation calculated by TCA fragment per Col2a1 was represented at the top of each lane. Lanes 1 and 2: Wt extracts; lanes 3 and 4: Tg cartilage samples. C: Northern blot analysis exhibited upregulation of Col2a1 in the Tg compared with the Wt mouse. 28S indicates the internal control of RNA samples used.

 
Fibronectin and integrin expression. Since the ECM in a proper balance of constitutive proteins is required for the development of cells and tissues, aberrant degradation of the ECM protein may have a magnitude of impacts on the balance. Expression of type X collagen and Ihh are specific molecular markers of hypertrophic chondrocytes and are expressed in the cells between the proliferating and hypertrophic zones (prehypertrophic chondrocytes), respectively (14). Immunolocalization of type X collagen in the hypertrophic chondrocytes (online supplementary Fig. 1) and Ihh in the prehypertrophic chondrocytes (online supplementary Fig. 1) was unchanged between transgenic and wild-type cartilage. However, fibronectin immunoreactivity was remarkably increased in the extracellular space of the resting zone and lower part of the proliferating zone in the transgenic growth plate (Fig. 5, A and B) . Also, immunostaining for the {alpha}5-integrin subunit that colocalized with fibronectin was more prominent in the transgenic mouse cartilage (Fig. 5D) than that in the wild-type littermates (Fig. 5C). Although chondrocytes express {alpha}-subunits that can associate with beta1, including {alpha}1, {alpha}2, {alpha}3, {alpha}6, and {alpha}10, the only known partner for {alpha}5 is the beta1-subunit (35). The reactivity and localization pattern of the collagen integrin receptors ({alpha}1- and {alpha}2-subunits) was indistinguishable between transgenic and wild-type mice (online supplementary Fig. 2). Another integrin-type collagen receptor ({alpha}3-subunit) was not expressed in any of the animals (online supplementary Fig. 2).


Figure 5
View larger version (105K):
[in this window]
[in a new window]

  		Fig. 5. Immunolocalization of fibronectin and {alpha}5-integrin subunit. Fibronectin (A and B) and {alpha}5-integrin subunit (C and D) were localized in the resting zone and lower part of the proliferating zone in the tibiae of Wt (A and C) and Tg mice (B and D) with an increase in both fibronectin (B) and {alpha}5-integrin (D) immunoreactivity in the cartilage of the Tg mice (line 104). Bar indicates 150 µm.

 
Enhanced chondrocyte proliferation and death. BrdU incorporation in chondrocytes at the S phase during cell cycle demonstrates proliferation is occurring in both the resting and proliferating zones of the cartilage of both wild-type and transgenic mice. Transgenic mice demonstrated an increased number of BrdU labeled cells particularly in the resting zone of the cartilage (Fig. 6, B and D) compared with those in the wild-type littermates (Fig. 6, A and C). BrdU was less incorporated into cells in the lower part of the proliferating zone than the resting chondrocytes in both the transgenic or wild-type mice. TUNEL labeling associated with cellular death was found to be increased in the proliferating zone of the transgenic growth plate (Fig. 6F) but not changed at the bottom line of hypertrophic zone as is typically seen in the wild-type cartilage growth plate (Fig. 6E). This increase in apoptosis correlated with expression of Bcl2, an anti-apoptotic protein, which normally is localized to chondrocytes in the proliferating zone of wild-type mice (Fig. 6G). In the transgenic cartilage the expression was dramatically reduced in the transgenic cartilage (Fig. 6H).


Figure 6
View larger version (56K):
[in this window]
[in a new window]

  		Fig. 6. Chondrocyte proliferation and death in the tibial growth plate. Bromodeoxyuridine (BrdU) was incorporated into the resting and proliferating chondrocytes in the 15-dpc embryo tibial growth plates (line 104). The incorporation was enhanced in the Tg growth plate (B and D) as compared in the Wt growth plate (A and C). Terminal deoxynucleotidyl transferase-mediated X-dUTP nick-end labeling (TUNEL) labeling was observed in the bottom rows of the hypertrophic zone in the Wt mice (E). In the Tg mice, the labeling was increased in the proliferating zone (F). The Bcl2 immunoreactivity in the Wt proliferating zone (G) was remarkably diminished in the Tg mice (H). Bar indicates 250 (A and B), 60 (C and D), and 150 µm (E–H).

 
DISCUSSION

In the present study, we demonstrated that inappropriate degradation of type II collagen during embryogenesis by MMP-1 retards embryonic bone growth. The chondrocytes in the transgenic embryonic epiphyseal growth plate of these mice exhibited 1) increased cellularity in the resting zone; 2) disorganized column structures in the proliferating zone; 3) less demarcation between the resting and proliferating zones; and 4) no obvious impediment of cells progressing from prehypertrophic to hypertrophic cells. Our analyses suggest that MMP-1 degradation of type II collagen (a major cartilage ECM protein) enhanced chondrocyte proliferation and apoptosis during embryogenisis and led to an upregulation of fibronectin and {alpha}5beta1-integrin expression. These cellular and matrix changes then resulted in disruption of the organized development of the growth plate. These data are the first to demonstrate that proteolytic degradation of the ECM directs chondrocyte proliferation and death.

A number of studies on gene targeting and positional cloning of human heritable diseases revealed a critical role of cartilage ECM proteins in chondrocyte proliferation and differentiation (20, 33). These genetic mutations often result in severe dwarfism or embryonic death. The transgenic mice generated in the present study exhibited embryonic growth retardation that was then postnatally compensated. This compensation is compatible with the results of mouse models carrying missense mutation in Col2a1 (12, 30). Although the Col2a1 null mice die at birth, the heterozygote in which type II collagen production is reduced exhibits embryonic growth retardation and as seen in our study the size difference in the heterozygous mice becomes less apparent as they grow older (26). The histological features of the Col2a1 mice are also similar to those observed in this study. Their proliferating chondrocytes are disorganized in a columnar array and less extended in contrast to the well-organized and flattened columnar stacks in the normal mice (12, 26, 30). Such a change in morphology results in less demarcation between the resting and proliferating zones. Restoration of type II collagen in the Col2a1 null mice rescues the animals from the lethal phenotype and from the chondrocyte disorganization (36). Overexpression of normal type II collagen in the cartilage is also responsible for embryonic dwarfism (13). Thus the integrity of type II collagen metabolism in the cartilage ECM is critical for the normal development of the cartilage.

The present study showed the enhanced degradation of type II collagen and upregulation of fibronectin and {alpha}5beta1-integrin in the transgenic mouse. Because the loss of tissue integrity by proteolytic enzymes impacts on cell phenotypes, aberrant degradation of type II collagen may result in the phenotypic alteration of chondrocytes. Previous studies (3, 38) had shown that degradation of type II collagen and its proteolytic fragments inhibit mitogen-activated protein kinase pathways, which are required for survival and change the integrin-mediated adhesion of chondrocytes. Binding of chondrocytes to intact type II collagen is mediated by {alpha}2beta1-integrin, whereas binding to denatured collagen is mediated by {alpha}5beta1-integrin-fibronectin complexes (43). Fibronectin is highly expressed in the early phase of chondrogenesis but declined in parallel with chondrocyte differentiation (25). Binding of fibronectin via {alpha}5beta1-integrin prolongs survival and stimulates proliferation of chondrocytes (10, 35). In fact, this transgenic mouse model increased BrdU incorporation and chondrocyte cellularity in the resting zone of growth plate, which increased expression of fibronectin and {alpha}5beta1-integrin. In contrast, in chondrocytes of the proliferating zone there was increased labeling by the TUNEL reaction but decreased staining of Bcl2, confirming a previous study that downregulation of Bcl2 in the Col2a1 null mice is associated with chondrocyte apoptosis (49). Disruption of integrin-mediated cell-ECM adhesion is reported to decrease Bcl2 expression and induce apoptosis (50). The differentiation markers of the prehypertrophic and hypertrophic chondrocytes Ihh and type X collagen (9, 44) did not change in their distribution, indicating the pathway of terminal differentiation was only minimally affected. Collectively, the enhanced type II collagen degradation in the present transgenic mice may provoke a significant alteration in the constituent of ECM and has an impact on survival, proliferation, and apoptosis of chondrocytes, resulting in the disalignment of zonal and columnar structures in the cartilage.

The growth retardation in the present transgenic mouse lines is most pronounced in a C57BL/6J genetic background. Although the mechanism of phenotypic consequences is not clear at present, the level of tissue inhibitor of metalloproteinases-1 (Timp-1) mRNA expression in the C57BL/6J mouse cartilage is sevenfold less than in cartilage of FVB mice as determined by RNase protection analysis (unpublished data). It is possible that variation in TIMP-1 levels accounts for the phenotypic differences seen in different genetic backgrounds.

Mice that harbor an MMP-1-resistant type I collagen have suggested a physiological role of collagen degradation in adult mice without affecting developmental cell differentiation and survival (29). In the present MMP-1-expressing transgenic mice, type II collagen degradation results in the less-matured chondrocyte-ECM adhesion, which stimulates cell proliferation. In addition, the excess collagen degradation also causes chondrocyte death, which leads to a disorganization in the cellular structure of the growth plate. This disorganization of cartilage development appears to be responsible for the embryonic growth retardation. Our transgenic mouse model indicates an involvement of the ECM protein metabolism in cellular survival. These experiments emphasize that a balance between ECM production and proteolysis during embryogenesis is indispensable for coordinated expression of ECM proteins and appropriate development to occur.

ACKNOWLEDGMENTS

We are grateful to Drs. Y. Yamada (National Institute of Dental Research, Bethesda, MD) for the kind gift of mouse Col2a1 gene cDNA.

FOOTNOTES


Address for reprint requests and other correspondence: K. Imai, Dept. of Biochemistry, Nippon Dental Univ., 1-9-20 Fujimi, Chlyoda-ku, Tokyo, Japan (e-mail: kimai{at}tky.ndu.ac.jp)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

REFERENCES

1. Bruggeman LA, Hou-Xiang X, Brown KS, Yamada Y. Developmental regulation for collagen II gene expression in transgenic mice. Teratology 44: 203–208, 1991.[CrossRef][Web of Science][Medline]

2. Cancedda R, Descalzi-Cancedda F, Castagnola P. Chondrocyte differentiation. Int Rev Cytol 159: 265–358, 1995.[Web of Science][Medline]

3. Carragher NO, Levkau B, Ross R, Raines EW. Degraded collagen fragments promotes rapid disassembly of smooth muscle focal adhesions that correlates with cleavage of pp125 (FAK), paxillin, and talin. J Cell Biol 147: 619–630, 1999.[Abstract/Free Full Text]

4. Cheah KS, Lau ET, Au PK, Tam PP. Expression of the mouse alpha 1(II) collagen gene is not restricted to cartilage during development. Development 111: 945–953, 1991.[Abstract/Free Full Text]

5. Chirgwin J, Prxybyla A, MacDonald R, Rutter WJ. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 18: 5294–5299, 1979.[CrossRef][Medline]

6. Chung UI, Lanske B, Lee K, Li E, Kronenberg H. The parathyroid hormone/parathyroid hormone-related peptide receptor coordinates endochondral bone development by directly controlling chondrocyte differentiation. Proc Natl Acad Sci USA 95: 13030–13035, 1998.[Abstract/Free Full Text]

7. D'Armiento J, Dalal SS, Okada Y, Berg RA, Chada K. Collagenase expression in the lungs of transgenic mice causes pulmonary emphysema. Cell 71: 955–961, 1992.[CrossRef][Web of Science][Medline]

8. D'Armiento J, DiColandrea T, Dalal SS, Okada Y, Huang MT, Conney AH, Chada K. Collagenase expression in transgenic mouse skin causes hyperkeratosis and acanthosis and induces susceptibility to tumorgenesis. Mol Cell Biol 15: 5732–5739, 1995.[Abstract]

9. Elima K, Eerola I, Rosati R, Metsaranta M, Garafalo S, Perela M, deCrombrugghe B, Vuorio E. The mouse collagen X gene: complete nucleotide sequence, exon structure and expression pattern. Biochem J 289: 247–253, 1993.[Web of Science][Medline]

10. Enomoto-Iwamoto M, Iwamoto ML, Nakashima K, Mukaida Y, Boettiger D, Pacifici M, Kuris K, Suzuki F. Involvement of {alpha}5beta1 integrin in matrix interactions and proliferation of chondrocytes. J Bone Miner Res 12: 1124–1132, 1997.[CrossRef][Web of Science][Medline]

11. Enomoto-Iwamoto M, Iwamoto M, Mukaida Y, Kawakami Y, Nohno T, Higuchi Y, Takemoto S, Ohuchi H, Noji S, Kuris K. Bone morphogenetic protein signaling is required for maintenance of differentiated phenotype, control of proliferation, and hypertrophy in chondrocytes. J Cell Biol 140: 409–418, 1998.[Abstract/Free Full Text]

12. Garofalo S, Vuorio E, Metsaranta M, Rosati R, Toman D, Vaughan J, Lozano G, Mayne R, Ellard J, Horton W, deCrombrugghe B. Reduced amount of cartilage collagen fibrils and growth plate anomalies in transgenic mice harboring a glycin-to-cysteine mutation in the mouse type II procollagen {alpha}1-chain gene. Proc Natl Acad Sci USA 88: 9648–9652, 1991.[Abstract/Free Full Text]

13. Garofalo S, Metsäranta M, Ellard J, Smith C, Horton W, Vuorio E, deCrombrugghe B. Assembly of cartilage collagen fibrils is disrupted by over expression of normal type II collagen in transgenic mice. Proc Natl Acad Sci USA 90: 3825–3829, 1993.[Abstract/Free Full Text]

14. Hering TM. Regulation of chondrocyte gene expression. Frontier Biosci 4: 743–761, 1999.[CrossRef]

15. Hogan B, Beddigton R, Costantini F, Lacy E. Manipulating The Mouse Embryo (2nd ed.), New York: Cold Spring Harbor Press, 1994.

16. Holmbeck K, Bianco P, Caterina J, Yamada S, Kromer M, Kuznetsov SA, Mankani M, Robev PG, Poole AR, Pidoux I, Ward JM, Birkedal-Hansen H. MT1-MMP-deficient mice develop dwarfism, osteopenia, arthritis, and connective tissue disease due to inadequate collagen turnover. Cell 99: 81–92, 1998.[CrossRef][Web of Science]

17. Horton W, Miyashita T, Kohno K, Hassel JR, Yamada Y. Identification of a phenotype-specific enhancer in the first intron of the rat collagen II gene. Proc Natl Acad Sci USA 84: 8864–8868, 1987.[Abstract/Free Full Text]

18. Inada M, Wang Y, Byrne MH, Rahman MU, Miyaura C, López-Otín C, Krane SM. Critical role for collagenase-3 (Mmp13) in development of growth plate cartilage and in endochondral ossification. Proc Natl Acad Sci USA 101: 17192–17197, 2004.[Abstract/Free Full Text]

19. Itoh T, Ikada T, Gomi H, Nakano S, Suzuki T, Itohara S. Unrelated secretion of beta-amyloid precursor protein in gelatinase A (matrix metalloproteinase-2)-deficient mice. J Biol Chem 272: 22389–22392, 1997.[Abstract/Free Full Text]

20. Jacenko O, Olsen BR. Transgenic mouse models in studies of skeletal disorders. J Rheumatol 43: 39–41, 1995.

21. Jones GD, Riley GC. ADAMTS proteinases: a multi-domain, multi-functional family with roles in extracellular matrix turnover and arthritis. Arthritis Res Ther 7: 160–169, 2005.[CrossRef][Web of Science][Medline]

22. Kimata K, Barrach HJ, Brown KS, Pennypacker JP. Absence of proteoglycan core protein in cartilage from the cmd/cmd (cartilage matrix deficiency) mouse. J Biol Chem 256: 6961–6968, 1981.[Abstract/Free Full Text]

23. Kosaki N, Takaishi H, Kamekura S, Kimura T, Okada Y, Mingi L, Amizuka N, Chung UI, Nakamura K, Kawaguchi H, Toyama Y, D'Armiento J. Impaired bone fracture healing in matrix metalloproteinase-13 deficient mice. Biochem Biophys Res Commun 354: 846–851, 2007.[CrossRef][Web of Science][Medline]

24. Krebsbach PH, Nakata K, Bernier SM, Hatano O, Miyashita T, Rhoder CS, Yamada Y. Identification of a minimum enhancer sequence for the type II collagen gene reveals several core sequence motifs in common with link protein gene. J Biol Chem 271: 4298–4303, 1996.[Abstract/Free Full Text]

25. Kulyk WM, Upholt WB, Kosher RA. Fibronectin gene expression during limb cartilage differentiation. Development 106: 449–455, 1989.[Abstract]

26. Li SW, Prockop D, Helminen H, Fässler R, Lapveteläiven T, Kirally K, Peltarri A, Arokoski J, Lui H, Arita M, Khillan JS. Transgenic mice with targeted inactivation of the Col2a1 gene for collagen II develop a skeleton with membranous and peritoneal bone but no endochondral bone. Genes Dev 9: 2821–2830, 1995.[Abstract/Free Full Text]

27. Lijnen HR, van Hoef B, Vanlinthout I, Verstreken M, Rio MC, Collen D. Accelerated neointima formation after vascular injury in mice with stromelysin-3 (MMP-11) gene inactivation. Arterioscler Thromb Vasc Biol 19: 2863–2870, 1999.[Abstract/Free Full Text]

28. Little CB, Meeker CT, Hembry RM, Sims NA, Lawlor KE, Golub SB, Last K, Fosang AJ. Matrix metalloproteinases are not essential for aggrecan turnover during normal skeletal growth and development. Mol Cell Biol 25: 3388–3399, 2005.[Abstract/Free Full Text]

29. Liu SX, Wu H, Byrne M, Jeffrey J, Krane S, Jaenish R. A targeted mutation at the known collagenase cleavage site in mouse type I collagen impairs tissue remodeling. J Cell Biol 130: 227–237, 1995.[Abstract/Free Full Text]

30. Maddox BK, Garofalo S, Smith C, Keene DR, and Horton WA. Skeletal development in transgenic mice expressing a Gly574Ser of type II collagen. Dev Dyn 7: 170–177, 1997.

31. Mitchel PG, Magna HA, Reeves LM, Lopresti-Morow LL, Yocum SA, Rosner PJ, Geoghegan KF, Hambor JE. Cloning, expression, and type II collagenolytic activity of matrix metalloproteinase-13 from human osteoarthritic cartilage. J Clin Invest 97: 761–768, 1996.[Web of Science][Medline]

32. Mudgett JS, Hutchinson NI, Chartain NA, Forsyth AJ, McDonnell J, Singer II, Bayne EK, Flanagan J, Kawka D, Shen CF, Stevens K, Chen H, Trumbauer M, Visco DM. Susceptibility of stromelysin 1-deficient mice to collagen-induced arthritis and cartilage destruction. Arthritis Rheum 41: 110–121, 1998.[CrossRef][Web of Science][Medline]

33. Mundlos S, Olsen BR. Heritable disease of the skeleton. Part I: molecular insights into skeletal development-transcription factors and signaling pathway. FASEB J 1: 125–132, 1997.

34. Oh C, Chang S, Yoon Y, Lee S, Lee Y, Kang S, Chun J. Opposing role of mitogen-activated protein kinase subtypes, Erk-1/2 and p38, in the regulation of chondrogenesis of mesenchyme. J Biol Chem 275: 5613–5619, 2000.[Abstract/Free Full Text]

35. Pulai JI, Del Carlo M Jr, Loeser RF. The {alpha}5beta1 integrin provides matrix survival signals for normal and osteoarthritic human articular chondrocytes in vitro. Arthritis Rheum 46: 1528–1535, 2002.[CrossRef][Web of Science][Medline]

36. Rani PU, Stringa E, Dharmavaram R, Chatterjee D, Tuan RS, Khillan JS. Restoration of normal bone development by human homologue of collagen type II (COL2A1) gene in Col2a1. Dev Dyn 214: 26–33, 1999.[CrossRef][Web of Science][Medline]

37. Rudolf-Owen LA, Hulboy DL, Wilson CL, Mudgett J, Matrisian LM. Coordinate expression of matrix metalloproteinase family members in the uterus of normal, matrilysin-deficient, and stromelysin-deficient mice. Endocrinology 138: 4902–4911, 1997.[Abstract/Free Full Text]

38. Shakibaei M, Schulze-Tanzil G, de Souza P, John T, Rahmanzadeh M, Rahmanzadeh R, Merker HJ. Inhibition of mitogen-activated protein kinase kinase induces apoptosis of human chondrocytes. J Biol Chem 276: 13289–13294, 2001.[Abstract/Free Full Text]

39. Shipley JM, Wesselschmid RL, Kobayashi DK, Ley TJ, Shapiro SD. Metalloelastase is required for macrophage-mediated proteolysis and matrix invasion in mice. Proc Natl Acad Sci USA 93: 3942–3946, 1996.[Abstract/Free Full Text]

40. Song RH, Tortorella MD, Maifait AM, Alston JT, Yang Z, Arner EC, Griggs DW. Aggrecan degradation in human articular cartilage explants is mediated by both ADAMTS-4 and ADAMTS-5. Arthritis Rheum 56: 575–585, 2007.[CrossRef][Web of Science][Medline]

41. Stickens D, Behonick DJ, Ortega N, Heyer B, Hartenstein B, Yu Y, Fosang AJ, Schorpp-Kistner M, Angel P, Werb Z. Altered endochondral bone development in matrix metalloproteinase 13-deficient mice. Development 131: 5883–5895, 2004.[Abstract/Free Full Text]

42. Ueda Y, Oda Y, Kawashima A, Tsuchiya H, Tomita K, Nakanishi I. Collagenous and basement membrane proteins of chondroma: immunohistochemical analysis. Histopathology 21: 345–352, 1992.[Web of Science][Medline]

43. Tuckwell DS, Ayad S, Grant ME, Takigawa M, Humphries MJ. Conformation dependence of integrin-type II collagen binding. J Cell Sci 107: 993–1005, 1994.[Abstract]

44. Vortkamp A, Lee K, Lanske B, Segre GV, Kronenberg HM, Tabin CJ. Regulation of rate of cartilage differentiation by Indian hedgehog and PTH-related protein. Science 273: 613–622, 1996.[Abstract]

45. Vu TH, Shipley JM, Bergers G, Berger JE, Helms JA, Hanahan D, Shapiro SD, Senior RM, Werb Z. MMP-9/gelatinase B is a key regulator of growth plate angiogenesis and apoptosis of hypertrophyc chondrocytes. Cell 93: 411–422, 1998.[CrossRef][Web of Science][Medline]

46. Wallis GA. Bone growth: coordinating chondrocyte differentiation. Curr Biol 6: 1577–1580, 1996.[CrossRef][Web of Science][Medline]

47. Watanabe H, Nakata K, Kimata K, Nakanishi I, Yamada Y. Dwarfism and age-associated spinal degeneration of heterozygote cmd mice defective in aggrecan. Proc Natl Acad Sci USA 94: 6943–6947, 1997.[Abstract/Free Full Text]

48. Wilson CL, Heppner KJ, Labosky PA, Hogan BLM, Matrisian LM. Intestinal tumorgenesis is suppressed in mice lacking the metalloproteinase matrilysin. Proc Natl Acad Sci USA 94: 1402–1407, 1997.[Abstract/Free Full Text]

49. Yang C, Li SW, Helminen HJ, Khillan JS, Bao Y, Prockop DJ. Apoptosis of chondrocytes in transgenic mice lacking collagen II. Exp Cell Res 235: 370–373, 1997.[CrossRef][Web of Science][Medline]

50. Zhang Z, Vuori K, Reed J, Ruoslahti E. The {alpha}5beta1 integrin supports survival of cells on fibronectin and up-regulates Bcl-2 expression. Proc Natl Acad Sci USA 92: 6161–6165, 1995.[Abstract/Free Full Text]





This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Figures
Right arrow All Versions of this Article:
293/4/C1209    most recent
00213.2007v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Imai, K.
Right arrow Articles by D'Armiento, J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Imai, K.
Right arrow Articles by D'Armiento, J.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2007 by the American Physiological Society.