Expression of Interstitial Collagenase during Skeletal Development of the Mouse Is Restricted to Osteoblast-like Cells and Hypertrophic Chondrocytes

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1 Vol. 6, , June 1995 Cell Growth & Differentiation 759 Expression of Interstitial Collagenase during Skeletal Development of the Mouse Is Restricted to Osteoblast-like Cells and Hypertrophic Chondrocytes Sabine Gack, Rudiger Vallon, Jorg Schmidt, Agamemnon Grigoriadis,2 Jan Tuckermann, Johannes Schenkel, Hans Weiher, Erwin F. Wagner, and Peter Angel3 Forschungszentrum Karlsruhe, Institut f#{252}r Genetik, P.O. Box 3640, D Karlsruhe, Germany IS. G., R. V., J. T., J. Sche., H. W., P. A.J; Forschungszentrum f#{252}r Umwelt und Gesundheit, Institut f#{252}r Molekulare Virologie, Neuherberg, D Oberschleigheim, Germany [J. Schm.I; and Research Institute of Molecular Pathology, Dr. Bohr-Gasse 7, A-i 030 Vienna, Austria IA. G., E. F. W.I Abstrad We determined the expression pattern of the matrix metalloproteinase interstitial collagenase (MMP-1) during mouse embryo development using in situ hybridization and immunohistochemistry. Localized MMP-1 mrna was first deteded at days postconceptus. The spatial and temporal expression was restrided to areas of endochondral and intramembranous bone formation, such as in the mandibula, maxilla, clavicle, scapula, in the vertebrae, and in the dorsal, but not the ventral part of the ribs. The highest levels of MMP-1 transcripts and MMP-1 protein were found in the metaphyses and diaphyses of the long bones. MMP-1 was expressed by hypertrophic chondrocytes and by osteoblastic cells localized along the newly formed bone trabeculae. No expression was detected in osteoclasts. Two other related members of the MMP family, stromelysin-1 (MMP-3) and stromelysin-2 (MMP-1 0), were not expressed during days 7.5 and of mouse embryogenesis. The tissuespecific expression of MMP-1 and the exclusive ability of interstitial collagenase to digest native collagen of types I, II, Ill, and X, the major components of bone, cartilage, and tendon, strongly suggests an important and specific fundion of this enzyme in bone development and remodeling. Introdudion Continuous changes of the ECM4 are thought to play an essential role during physiological processes that require tissue remodeling, such as morphogenesis, angiogenesis, Received 3/1 /95; accepted 3/31/95. 1 Work was supported by a grant from the Deutsche Forschungsgemeinschaft (An 182/6-2). 2 Present address: United Medical and Dental School, Guy s Hospital, Department of Craniofacial Development, Guy s Tower Floor 28, London Bridge, London SE1 9RT, United Kingdom. 3 To whom requests for reprints should be addressed. 4 The abbreviations used are: ECM, extracellular matrix; MMP, matrix metalloproteinase; p.c., postconceptus; GST, glutathione S-transferase; TRAP, tartrate-resistant acid phosphatase; PTH, parathyroid hormone; PA, plasminogen activator. bone resorption, inflammation, and wound healing (reviewed in Refs. i-s). The tight balance of synthesis and degradation of the ECM is essential for the normal development of an organism (6). Enzymes that initiate the degradation of components of the ECM belong to the family of Zn2-dependent MMPs. The MMPs identified thus far share a common scheme of modular structure of protein domains. They differ, however, in substrate specificity and can, therefore, be divided into different subclasses, including type I collagenases (interstitial and neutrophil, collagenase-3); gelatinases A and B (Mr 72,000 type IV collagenase and Mr 92,000 type IV collagenase, respectively), and stromelysin-i and stromelysin-2 (4, 7, 8). The expression and activity of MMPs is tightly controlled on several levels including regulation of transcription (9- i 3), activation of the latent proenzyme (1 4-i 6), and interaction with specific inhibitors of MMPs (TIMP-i, TIMP-2, and TIMP-3; Refs. i 7-20). Inappropriate and excessive activity has been proposed to be involved in a variety of pathological processes, such as rheumatoid arthritis, corneal ulceration, metastasis of tumor cells, and genetic diseases (reviewed in Refs. 1, 3, 4, 6, 7, and 21). Interstitial collagenase (MMP-i) and neutrophil collagenase (MMP-8) are the only members of the MMP family that are capable of degrading with high efficiency the interstitial collagens of types I, II, III, and X. Although data from cell culture systems indicate a coordinate expression of MMP-i, stromelysin-i and stromelysin-2, and gelatmnase B by a common set of extracellular stimuli, recent analysis of gelatinases A (MMP-2) and B (MMP-9) transcription in the mouse identified clearly separate sites of expression (22-24), suggesting distinct functions of these enzymes in the physiological and pathological processes described above (reviewed in Refs. 1-7). Since the collagens of types I, II, III, and X comprise more than 50% of total collagen, and since they are found primarily in bone, cartilage, and skin (25), interstitial collagenase is thought to play a crucial role in the development and remodeling of these tissues (4, 26). Expression of MMP-i in vitro has been found in cultured cells of the osteogenic lineages in response to hormones regulating bone homeostasis, such as retinoic acid, vitamin D3, and parathyroid hormone (27-33). In many other cell types, e.g., fibroblasts or epithelial cells, MMP-i transcription is strongly induced in response to a broad spectrum of signals including growth factors, hormones, mediators of inflammation, oncogenes, phorbol esters, or radiation (10, 34-36). In most cell systems, induction of MMP-i expression is mediated by the transcription factor AP-i, which is a heterodimeric complex whose subunits are encoded by members of the fun, los and ATF gene families (reviewed in Ref. 37). In line with the requirement of AP-i for MMP-i transcription in tissue culture cells, expression of this gene was recently found to be enhanced in a tissue-specific manner

2 760 MMP Expression during Mouse Development a.s,._ :,. : mx. 5. fe-. i...,: _c--- - :.:.. : , : bq... - :;...,._.#{149}%.,.;.,.....: 0. : ;;:... q #4eO.llt,I; M:r I3ct Fig. 1. Localization of MMP-1 transcripts in a 14.5-day p.c. embryo. a, sagittal section of a day p.c. embryo. b, higher magnification of the basioccipital bone marked with a circle in a. c, darkfield illumination of b. d, higher magnification of collagenase type I expressing cells (arrows). bo, basioccipital bone; fe, femur; mx, maxilla; vc, vertebral column. Bars:a, 1 mm;bandc,250im;d, 100 pm. A control sense-strand probe gave low, nonspecific signals equally distributed on the coverslip and in the sections (data not shown).. # p. - I, 4 t)#{188},.il #{149}I#{149}. #{149}.,.; t-.. I -,s!. il_, in mice overexpressing Fos (38) that identified interstitial collagenase as the first example of an AP-1 -regulated gene in animals. The DNA sequence of murine interstitial collagenase is highly homologous to human collagenase-3 (8, 38) but less similar to human collagenase type I (MMP-1). Both enzymes efficiently degrade collagen type I (8). However, while human collagenase-3 is exclusively expressed in breast carcinomas but is not detectable in any normal tissues (8), the murine homobogue is expressed in many tissues including muscle, kidney, and bone (38). Reminiscent of human MMP-1, the expression of the murine interstitial collagenase gene is inducible in fibroblasts of different origins by phorbol esters, UV, tumor necrosis factor a, interleukin 1, and basic fibroblast growth factor in an AP- 1 -dependent manner (38). Since the human MMP-1 probe does not cross-hybridize with mouse DNA, these data suggest that the murine interstitial collagenase gene very likely represents the functional equivalent of human MMP-1. To define its role in vivo, we analyzed the expression of interstitial collagenase during mouse embryonal development. Despite the many proposed functions of this enzyme in the remodeling of tissues containing collagen types I, II, and Ill, expression of MMP-1 during embryogenesis is restricted to the developing skeleton in the process of ossification. At later stages of embryonic development and in.; B. Baumann and P. Angel, unpublished observations., P. Angel, unpublished observations. postnatal life, expression of interstital collagenase is detected in hypertrophic chondrocytes at the zone of bone growth and mineralization and in bone-lining cells, presumably osteoblasts, but not in osteoclasts. Expression of MMP-1 differs from that of gelatinase B (MMP-9), which preferentially degrades gelatin and which is secreted by osteoclasts (24). Due to its unique substrate specificities, the site-specific expression of MMP-1 imply a critical role of this enzyme in bone homeostasis. Results Expression of Interstitial Collagenase Starts at Day of Mouse Development in Developing Skeletal Tissues. To define the spatial and temporal expression of interstitial collagenase during mouse development, we determined the level of MMP-1 -specific transcripts by in situ hybridization in embryos ranging from days 5.5 to 16.5 p.c. Specific transcripts were first detected with two independent MMP-1 probes at day of gestation (Fig. 1 and data not shown). The onset of MMP-1 expression at this stage of development was confirmed by Northern Blot analysis of poly(a) RNA isolated from embryos from 9.5 to days p.c. (data not shown). The hybridization signals appeared in a few cells in the basioccipital bone (Fig. 1 ), the largest primary center of ossification at this stage. Hybridization signals were also observed in the maxilla, in the mandibular bone, in the palatal shelf of the maxilla, and in the ossification centers (shaft regions) of the humeri and femora (data not shown). Thus, it appears that the spatial expression of interstitial collagenase at this stage of development parallels the pro-

3 Cell Growth & Differentiation 761 a ( :. - T. - I: #{149}4 I,e.. ;.,. I - - I._i I..--. Fig. 2. Localization of interstitial collagenase and collagen X transcripts in a 16.5-day p.c. embryo. a, sagittal section of a 16.5-clay p.c. embryo showing the pattern ot interstitial collagenase expression. b, darkfield illumination of a. c, darkfield illumination of a parallel section of a showing collagen type X hybridization signals. Arrows, the sites of MMP-l and collagen X expression, respectively. d, magnification of the mandible in a. Arrows, bone matrix. e, magnification of d showing high expression ot interstitial collagenase in cells lining the bone matrix (arrows). cl, clavicle; hb, hipbone; ron, mandible; rb, ribs; mc, mandibular condyle; (I), tooth bud. Bars: a-c, 1 mm; ci, 250 pm; e, 1 00 pm. cess of bone formation both in endochondral and intramembranous bone. In 16.5-day p.c. embryos, MMP-1 expression was detected in the mandibular bone, maxilla, clavicle, scapula, in vertebrae, hipbone, in the base of the skull (exoccipitab), and in the dorsal, but not in the ventral parts, of the ribs (Figs. 2 and 3 and data not shown). High bevels of coblagenase I transcripts were found in the diaphyses of the bong bones (Figs. 2 and 3). In nonskebetab tissues, such as skin, kidney, muscle, or heart, MMP-1 transcripts were not detected during embryonic development (Fig. 2). MMP-1 Is Expressed Specifically by Osteoblastic Cells and Hypertrophic Chondrocytes. Detailed analyses of skeletal tissues from day p.c. embryos showed MMP-1 expression in basophilic cells along newly formed bone in the mandible (Fig. 2, d and e), which presumably represent osteobbasts. In bong bones of the ulna and radius, expression of interstitial collagenase was confined to cells localized along bone trabeculae and to cells in the zone of hypertrophic chondrocytes (Fig. 3). Collagen type X, a hallmark of hypertrophic chondrocytes, was also expressed in cells of the upper zone of hypertrophic chondrocytes (Fig. 3). These cells did not express MMP-i (Fig. 3). Neither collagen X nor MMP-1 expression was found in resting or proliferating chondrocytes (Figs. 2 and 3). In agreement with the appearance of an intermediary cartilage model, type X collagen expression was restricted to the sites of endochondrab ossification, such as the bong bones and hip, but was not detectable at sites of intramembranous ossification of mandibular bone and maxibla, both of which expressed high bevels of interstitial cobbagenase (Fig. 2). The presence of MMP-1 protein was shown by immunohistochemicab analyses using a cobbagenase I-specific polycbonal antiserum. This antiserum was raised against a bacterially expressed protein composed of GST fused to a small region of murine MMP-1. Cobbagenase I protein was found in sections from and 17.5-day-old embryos at all sites identified by in situ hybridization, such as the dorsal areas of the ribs, in mandibular bone, in the metatarsals, and in the clavicle (Fig. 4 and data not 7 R. Vallon and P. Angel, unpublished observations.

4 762 MMP Expression (luring Mouse Development (I...., S.. hc. #{149} I, d.. Fig. 3. Interstitial collagenase and collagen type X mrna distribution in the forelimb of a 16.5-day p.c. embryo. a, sagittal section showing interstitial collagenase expression in the diaphysis of ulna and radius. b, darkfield illumination of a. c, darkfield illumination showing collagen type x expression in hypertrophic chondrocytes. d, magnification of a showing MMP-1 expression in cells of the calcified cartilage and in the area of the primary spongiosa. e, darkfield illumination of d. 1, higher magnification of c. The signal is restricted to the zone of hypertrophic chondrocytes. Arrows, corresponding regions of the sections. hu, humerus; ra, radius; ul, ulna; hc, hypertrophic cartilage; B, bone trabecula. Bars: a-c, 250 pm; d-t 100 pm. shown). Control antiserum recognizing epitopes of the GST protein did not give specific signals (Fig. 4d). The Sites of Expression of Interstitial Collagenase and Other Members of the MMP Family Are Different. To determine whether MMP-1 and other members of the MMP gene family are coexpressed, we determined the sites of expression of gebatinase A (MMP-2), stromelysin-1 (MMP- 3), and stromebysin-2 (MMP-10) in day 16.5 p.c. embryos by in situ hybridization. In agreement with previous findings (23), expression of MMP-2 was detected in various mesenchymal tissues distributed throughout the embryo but not in areas where MMP-1 expression was determined (Fig. 5). The mutually exclusive pattern of expression of the two members of the MMP gene family was particularly evident in the dorsal part of the ribs. MMP-1 transcripts were found in the central part ofthe tissues, whereas MMP-2 transcripts were detected in the periosteab regions (Fig. 5). Similar results were observed in the scapula and clavicle (Fig. 5). In situ hybridization using probes that encode two other members ofthe MMP family, stromebysin 1 (MMP-3) and stromelysin 2 (MMP-1 0), did not show any signals in sections from days 9.5 to embryos (data not shown). These data indicate that related members of the MMP gene family, interstitial collagenase, gelatinase A, stromelysin 1, and stromebysin 2, were not expressed coordinately but differed in cell type-specific and temporal expression. Of the four MMP genes analyzed at this stage of mouse development, only MMP-1 appeared to be invariably associated with skeletal development. Hypertrophic Chondrocytes and Osteoblastic Cells but not Osteoclasts Express Interstitial Collagenase in Young Adult Bone Tissue. In the developing skeleton during embryogenesis, MMP-1 expression was only found in areas undergoing bone formation (Figs. 1 and 2). A very similar staining pattern was detected in young adult bone tissue from 2-week-old mice (Fig. 6). A high level of MMP-1 transcripts was detected in cells of the bower zone of hypertrophic chondrocytes in the calcified cartilage (Fig. 6, a and b). This region partially overlapped with the region containing collagen type X-expressing cells (Fig. 6c). To determine the cells that express MMP-1, bone sections of 1 -, 2- and 4-week-old mice were examined by in situ hybridization and enzyme histochemical analysis of TRAP, a marker of osteoclast precursor cells and mature osteoclasts (39). MMP-1 transcription was particularly high in basophibic cells localized along the newly formed bone trabeculae (Fig. 7, a and b). Distinct hybridization signals were also detected in osteobbasts in the diaphyseal perios-

5 Cell Growth A Difterentiation 763 a Fig. 4. Localization ot MMP-1 protein in tissues ot a clay p.c. enli)ryo. t, sagittal section showing i nterstitial collagenase Protein in the diaphysis (arrow) of nietatarsal bone ot the hindlimb. I), higher magniticalion 01 a. c and d, parallel sagittal sections of the clavi(le. For ininiunohistochemical analysis, antisera raised against GST-l lagc nase ) a-c) or GST d) were used. nit, nietatarsal i)one; (1, clavicle. Bars: a. 1 mm; 1)-il, 1 00 pn. 0.; teuni and in a few osteocytes of the peripheral area of the cortex (Fig. 7c). Cobbagenase I was preferentially expressed in basophibic cells on osteoid surfaces located adjacent to multinuclear TRAP-positive cells (Fig. 7b). In TRAP-positive mononuclear osteoclast precursor cells and mubtinucleated osteoclasts, MMP-1 expression was not detected (Fig. 7, a, b, and d). Discussion In this study, we demonstrate that interstitial cobbagenase is first expressed at day 14.5 during mouse development and that expression is restricted to areas of bone formation. No other sites of expression were detectable, suggesting that MMP-1 plays an important robe during the process of bone remodeling and that it may not be essential for remodeling processes of other tissues. High expression of MMP-1 was found in the zone of hypertrophic chondrocytes along the growth plate-metaphyseab junction and in basophibic cells, most likely osteobbasts, lining the newly formed bone trabecubae in the primary and secondary spongiosa and the metaphyseab and diaphyseab periosteum. In addition, osteocytes were also found to express MMP-1, however, to a lesser extent. In contrast, we did not find expression of MMP-1 in TRAP-positive mono- or mubtinuclear osteoclasts. Recently, Debaisse et a!. (40) described the immunobocabization ofcoblagenase I protein in 5-day-old mice in the subosteoclastic bone-resorbing compartment. Using in vitro cultured bone cells isolated from the bong bones of newborn rats, MMP-1 protein was detected in osteoblasts, chondrobbasts, and other, uncharacterized mononuclear cells but only aftertreatment ofthe sections with chondroitinase. High bevels of MMP-1 protein were also found in resorbing osteoclasts. The reason for this apparent contradiction on MMP-1 expression in osteoclasts is unknown. It is possible that in vitro culturing of bone cells for several hours on devitalized cortical bone slices (40) induces MMP-1 expression in osteoclasts. It is also possible that the antiserum raised against affinity-enriched complete cobbagenase I protein from mouse calvaria (40) recognizes additionab members of the MMP family that are immunobogicabby rebated to MMP-1. Since the cell-specific expression of MMP-1 described here was determined by in sitti hybridization at high stringency using two different antisense probes that cover separate regions of the MMP-1 coding region, we consider hypertrophic chondrocytes, osteobbastbike cells, and osteocytes, but not osteoclasts, to be the source of MMP-i production. The same pattern of spatial and temporal expression was observed by immunohistochemical analysis using an MMP-1 antiserum raised against a small region of murine MMP-1 (Fig. 4),7 Our data are in

6 764 MMP Expression during Mouse Development Fig. 5. Comparison of interstitial collagenase and M, 72,000 type IV collagenase expression in the 16.5-day p.c. embryo. a, darkfield illumination showing interstitial collagenase expression in the ossifying tissues of an embryo. b, darkfield illumination of an adjacent section showing M, 72,000 type IV collagenase expression in mesenchymal-derived tissues. Arrows, nonoverlapping sites of MMP-1 and MMP-2 expression in the ribs, in the hindlimb, in the scapula, maxilla, and in the mandibular bone. Bars: a and b, 1 mm. a Fig. 6. Expression of MMP-1 and collagen type X transcripts in the bone tissue of 2-week-old mice. a, sagittal section of the distal femur of a 2-week-old mouse showing interstitial collagenase expression in the epiphysis and the metaphysis. b,darkfield illuminationofa. c, darkfield illumination of a parallel section of a showing collagen type x expression in the zone of hypertrophic cartilage in the growth plate. Arrows, corresponding regions of the sections. Bm, bone marrow; gp, growth plate. Bar: a-c, 1 mm. agreement with previous fi nd i ngs demonstrati ng immunoreactive cobbagenase in osteoblasts but not in osteocbasts (27). The exact function of MMP-1 in bone formation and remodeling is presently unknown. During endochondrab ossification, collagens type lb and X, the major components of cartilage, have to be degraded and replaced by collagen type I, which is synthesized by osteobbasts (41 ). Since interstitiab colbagenase is able to degrade efficiently native cobbagens type lb and X (reviewed in Ref. 4), it is tempting to speculate that expression of this enzyme in the calcified zone of hypertrophic chondrocytes may be essential for the cartilage/bone transition. The possible robe of MMP-1 expressed by osteobbastic cells and osteocytes is far less obvious. In general, bone remodeling is a dynamic process whereby osteobbastic bone formation is tightly coupled to osteoclastic bone resorption (39, 42). Despite the fact that MMP-1 is not produced by osteoclasts, the proposed function of MMP-1 in bone remodeling by degrading native collagen type I is compatible with earlier findings suggesting a functional synergism between osteobbasts and osteoclasts (39, 42). Possibly, osteoblasts initiate the resorptive process by dissolving osteoid by MMP-1 and thereby expose the underlaying mineralized bone surface to osteoclasts. Subsequently, the 3/4 and 1/4 fragments ofcolbagen type I may be further degraded to low molecular weight fragments by the Mr 92,000 gebatinase B (MMP-9), which is specifically expressed by osteoclasts (24, 43). The concept of synergistic action of MMP-1 and MMP-9 does, however, not exclude the requirement of other proteinases, such as cysteine proteinases and thiob proteinases (e.g., cathepsin B) in bone resorption (44-46). Osteobbasts secrete MMP-1 in response to stimulators of bone resorption, including PTH, vitamin D5, interbeukin 1, and tumor necrosis factor a (28-33). Whether one or more ofthese factors directly regulates MMP-1 in vivo is presently unknown. In addition to specific extraceblubar signals, such as hormones and cytokines, cell/cell and cell/matrix interactions may play an important robe in MMP-1 expression. Most interestingly, we noted very strong expression of

7 Cell Growth & Differentiation 765 C.- : - -: - Fig. 7. Expression of interstitial collagenase in osteoblasts and osleocytes but not in osteoclasts. a, sagittal section of a long bone from a 4-week-old mouse showing expression of interstitial collagenase and TRAP-positive cells in the region of the growth plate and the metaphysis. Red cells represent mononuclear osteoclast precursors and multinucleated osteoclasts located along bone trabeculae. b, higher magnification of a showing MMP-1 expression in basophilic osteoblastic cells but not in osteoclasts, which are characterized by several nuclei and high TRAP activity. c, MMP-1 expression in osteocyles (arrowhead) in a diaphyseal area of the femur. d, TRAP-positive mononuclear cells in the bone marrow do not express collagenase I. Bars: a, 100 pm; b-d, 50 pm. On the other hand, mice that are deficient in c-fos expression suffer from an imbalance in bone remodeling and develop osteopetrosis (55, 56) due to the absence of osteoclasts (57). It will be interesting to determine whether the back of osteoclasts, the bow bevel of bone turnover, and/or the boss of Fos expression in osteoblasts and chondrocytes abrogates MMP-1 expression in Fos-deficient mice and whether these alterations are associated with the development and maintenance of the osteopetrotic phenotype. It should be noted that, in addition to transcriptional regulation and protein synthesis, MMP-1 activity also depends on the activation of the latent enzyme by proteobytic enzymes, such as the PA/plasmin system and on the bevel of the inhibitors of MMPs, TIMP-1, TIMP-2, and TIMP-3. In line with the proposed function of PA/plasmin, expression of PA is stimulated in cultured osteobbasts in response to PTH (58). While the patterns of TIMP-2 and TIMP-3 expression are presently unknown, TIMP-1 expression seems not to overlap completely with MMP-1 expression but resembles expression of the Mr 72,000 gebatinase A (MMP-2), suggesting that TIMP-1 might be the physiological inhibitor of MMP-2 (59, 60). The distinct expression of interstitial coblagenase in hypertrophic chondrocytes, osteoblasts, and osteocytes during skeletal development strongly suggests an important function of this enzyme during this process. Mutant mice in which the MMP-1 gene has been inactivated by homobogous recombination would provide powerful means to study the robe of MMP-1 during bone development and turnover and to unravel the participation of MMP-1 in genetic bone diseases. MMP-1 in osteoblastic cells located adjacent to mature osteoclasts. In contrast, cells that are associated with TRAPpositive mononuclear cells do not express MMP-1. These data suggest that terminably differentiated osteoclasts induce or enhance MMP-1 expression in osteobbastic cells via either cell to cell contact by a juxtacrine or by a paracrine mechanism. Modulation of MMP-1 expression in response to regubators of bone remodeling is likely to include alterations in the activity of the transcription factor AP-1 (Fos/Jun). Based on in vitro studies of the promoter of the human cobbagenase I gene in tissue culture cells, the Fos and Jun proteins are the main regulators that mediate enhanced transcription of MMP-1 by growth factors, cytokines, various oncoproteins, and genotoxic agents (reviewed in Ref. 37). Most importantly, murine c-jun and c-fos are expressed in osteogenic tissues during mouse embryonic development (47-SO), and expression can be induced in tissue culture cells in response to osteotropic hormones and cytokines, including PTH, insulin-bike growth factors, retinoic acid, and vitamin D3 (reviewed in Ref. 51), and in osteobbasts of the long bones of rats after s.c. administration of PTH (52). Furthermore, we have recently demonstrated that expression of interstitial cobbagenase is enhanced more than 200-fold in bones of c-fos-transgenic mice (38). Interestingly, enhanced MMP-1 expression in bone correlates with the development of osteosarcomas.7 These Fos-induced bone tumors were found to be derived from osteoblasts (53). Based on the identification of the cell type expressing MMP-1, it is possibbe that c-fos directly enhances MMP-1 expression in osteosarcomas of c-fos transgenic mice by binding to the AP-1 site in the MMP-1 promoter (54). Materials and Methods Embryo Collection. Embryos were recovered from naturally mated CFW x BALB/c female hybrid mice. Noon of the day of vaginal plug was designated 0.5 days p.c. Embryos were collected in sterile PBS and dissected from the extraembryonic tissues. Bone tissue of postnatal CS7BL/6 mice were dissected and prepared as described previously (56). Isolation of RNAs from Embryos and Northern Blot Analysis. Embryos were homogenized in guanidinium thiocyanate (61 ), and total RNA was purified by centrifugation through a 5.7 M CsCI-2S mi sodium acetate (ph 5.0) cushion in a Beckman SW4O rotor spun at 31,000 rpm for 22 h. Poby(A) RNA was obtained by retention on an obigo(dt)- cellulose column, and 1 0 pg of each sample were analyzed by Northern blot hybridization as described previously (38). In Situ Hybridization and Immunostaining. To generate [35S]UTP-babebed sense and antisense probes for in situ hybridization, the following MMP-specific cdnas (38) were subcboned into the pgem4 in vitro transcription vector (Promega): a 362-bp EcoRl/Drab 3 -fragment of collagenase I (positions ); a 223-bp BamHl/EcoRl fragment of stromelysin 2 (positions ); a 302-bp HindIlI/Sacl fragment of stromebysin 1 (positions ); and a 340-bp Pvull/Sacl fragment of cobbagenase IV (Mr 72,000; positions ). The positions are according to the cdna sequences described by Gack et a!. (38). The 1-kb ctl type X collagen antisense probe was generated from plasmid pxl (kindly provided by Dr. Klaus van der Mark), which contains a 3.2-kb fragment of the human ai(x) gene (62). The plasmids were linearized with the appropriate restriction enzymes, and single-stranded

8 766 MMP Expression during Mouse Development RNA probes were synthesized using either T7 or 5P6 RNA polymerase. To prepare the sections for in situ hybridizations, embryos were fixed overnight in 4% paraformaldehyde in PBS and embedded in paraffin. Sections were cut at 6 pm and placed onto 3-aminopropyl-triethoxysilane-coated slides. The sections were either subjected to in situ hybridizations as described (63) or assayed for TRAP activity prior to hybridization. In brief, deparaffinized sections were pretreated with proteinase K, hybridized with the labeled probe (1-2 X i0 cpm/pl) in a humid chamber overnight at 50#{176}C, followed by extensive washing at high stringency. The slides were dried, dipped in autoradiographic emulsion (LM-i ; Amersham), and exposed for 1 0 days at 4#{176}C. After developing the film, the sections were stained with hematoxylin (0.1%; Sigma Chemical Co.) and eosin (1% final concentration) and mounted. To detect MMP-1 protein, deparaffinized slides were analyzed by immunohistochemical staining as described (64) using a MMP-1 -specific rabbit antiserum raised against bacterially expressed murine MMP-1. Control sections were incubated with GST-specific rabbit antiserum. Staining for TRAP. To identify mononuclear osteoclast precursor cells and multinuclear (three or more nuclei/cell) osteoclasts in skeletal tissues, paraffin sections were dewaxed in xylene, rehydrated, and incubated at 37#{176}C in 50 ms sodium acetate (ph 5.2) containing 0.1 5% Naphtol-AS-TRphosphate (dissolved in N,N-dimethylformamide), 50 mtvi sodium tartrate, and 0.1% Fast Red T.R. (Sigma). Acknowledgments We are particularly grateful to Anette NeubOser for providing RNA from early-stage mouse embryos and to Haruhiko Koseki and Rudi Balling for their initial help in performing in situ hybridization analysis. 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