Mouse Mandibular Condyle Research Articles

Age-Related Gene and Protein Expression in Mouse Mandibular Condyle Analyzed by Cap Analysis of Gene Expression and Immunohistochemistry

Introduction: Aging, an inevitable physiological process, leads to morphological and histological degenerative changes in the mandibular condylar cartilage (MCC); however, the molecular mechanism has not yet been elucidated, and little information is available on age-related factors. Therefore, this study was designed to identify age-related factors by investigating the age-related differentially expressed genes (DEGs) and localization of their translated protein expression in the mandibular condyle. Methods: Mandibular condyles were collected from 10- and 50-week-old mice. Total RNA was extracted from the samples and then analyzed using cap analysis of gene expression (CAGE) to identify age-related DEGs. Gene ontology (GO) enrichment analysis was performed to determine which biological processes were most affected by aging in terms of gene expression using Metascape. The mandibular condyle samples were processed for histology to investigate morphological changes caused by aging and for immunohistochemistry to localize the protein expression encoded by age-related genes identified with CAGE. Semi-quantitative immunohistochemistry was performed to assess age-related extracellular matrix (ECM) protein levels in the MCC. The histological sections were also used for Alcian blue histochemistry to detect glycosaminoglycans (GAGs). Results: GO enrichment analysis revealed that the genes related to “extracellular matrix organization,” including Acan, Col1a1, Col1a2, Col2a1, Mmp3, Mmp9, and Mmp13, were most differentially expressed in the aged mandibular condyle. Among these seven genes, Mmp3 was upregulated, and the others were downregulated with aging. Histological examination showed the age-related morphological and histological changes in the MCC. Immunohistochemical investigation showed the localization of matrix metalloproteinases (MMPs)-3, -9, and -13 and their substrate proteins, aggrecan, type I collagen, and type II collagen, in the mandibular condyle at 10 and 50 weeks, indicating different localizations between the young and the aged. In the aged MCC, semi-quantitative immunohistochemistry showed a significant decrease in the aggrecan protein level, and Alcian blue histochemistry showed a decrease in GAGs. Conclusion: MMP-3, MMP-9, and MMP-13 contribute to the remodeling of the ECM of the MCC and subchondral bone during aging by degrading ECM proteins at specific times and sites under the regulation of their production and secretion.

Masseter muscle atrophy impairs bone quality of the mandibular condyle but not the alveolar process early after induction.

Masseter muscle function influences mandibular bone homeostasis. As previously reported, bone resorption markers increased in the mouse mandibular condyle two days after masseter paralysis induced with botulinum toxin type A (BoNTA), followed by local bone loss. This study aimed to evaluate the bone quality of both the mandibular condyle and alveolar process in the mandible of adult mice during the early stage of a BoNTA-induced masseter muscle atrophy, using a combined 3D histomorphometrics and shape analysis approach. Adult BALB/c mice were divided into an untreated control group and an experimental group; the latter received one single BoNTA injection in the right masseter (BoNTA-right) and saline in the left masseter (Saline-left). 3D bone microstructural changes in the mandibular condyle and alveolar process were determined with high-resolution microtomography. Additionally, landmark-based geometric morphometrics was implemented to assess external shape changes. After 2weeks, masseter mass was significantly reduced (P-value <0.001). When compared to Saline-left and untreated condyles, BoNTA-right condyles showed significant bone loss (P-value <0.001) and shape changes. No significant bone loss was observed in the alveolar processes of any of the groups (P-value >0.05). Condyle bone quality deteriorates at an early stage of BoNTA-induced masseter muscle atrophy, and before the alveolar process is affected. Since the observed bone microstructural changes resemble those in human temporomandibular joint degenerative disorders, the clinical safety of BoNTA intervention in the masticatory apparatus remains to be clarified.

Gene expression of angiogenesis markers in mouse mandibular condyle during development

Angiogenesis and osteogenesis markers are an important factor during development in relation between mandibular condyle and Meckel's cartilage. However, it has many functions in addition to the formation of mandible. We need to clearly the relationship between mandibular condyle and Meckel's cartilage to angiogenesis and osteogenesis markers during formation in mouse fetal and early postnatal stages. Therefore we try to investigate the correlation between these markers during the development of the mouse mandible. We analyzed the expression and localization of VEGF by in‐situ hybridization in the developing mouse mandibular condyle from embryonic days 12.5 to days 5. We also detected the mRNA abundance of mandible with various markers from embryonic and early postnatal stages by real‐time RT‐PCR. Our results was performed for mouse mandible in different postnatal stages and after birth and the expression of the following some mRNA markers (CD31, LYVE‐1, VEGF, collagen I, II, osteopontin, and osteocalcin). The antisense probe for VEGF was detected in mesenchymal cells surrounding around MC and bone matrices area from E17.5, and clearly localization was seen at E18.5 in the embryonic mouse mandible by in‐situ hybridization. These data suggested that angiogenesis markers were effected by development stages after or before birth in morphogenesis of mandible. VEGF also affects the other morphological markers in these specific stages terminating the differentiation in mouse mandibular condyle development.Support or Funding InformationNone

Contrast-enhanced microCT (EPIC-µCT)ex vivoapplied to the mouse and human jaw joint

The temporomandibular joint (TMJ) is susceptive to the development of osteoarthritis (OA). More detailed knowledge of its development is essential to improve our insight into TMJ-OA. It is imperative to have a standardized reliable three-dimensional (3D) imaging method that allows for detailed assessment of both bone and cartilage in healthy and diseased joints. We aimed to determine the applicability of a contrast-enhanced microCT (µCT) technique for ex vivo research of mouse and human TMJs. Equilibrium partitioning of an ionic contrast agent via µCT (EPIC-µCT) was previously applied for cartilage assessment in the knee joint. The method was ex vivo, applied to the mouse TMJ and adapted for the human TMJ. EPIC-µCT (30-min immersion time) was applied to mouse mandibular condyles, and 3D imaging revealed an average cartilage thickness of 110 ± 16 µm. These measurements via EPIC-µCT were similar to the histomorphometric measures (113 ± 19 µm). For human healthy OA-affected TMJ samples, the protocol was adjusted to an immersion time of 1 h. 3D imaging revealed a significant thicker cartilage layer in joints with early signs of OA compared with healthy joints (414.2 ± 122.6 and 239.7 ± 50.5 µm, respectively). A subsequent significant thinner layer was found in human joints with late signs of OA (197.4 ± 159.7 µm). The EPIC-µCT technique is effective for the ex vivo assessment of 3D cartilage morphology in the mouse as well as human TMJ and allows bone-cartilage interaction research in TMJ-OA.

An immunohistochemical study on the localization of type II collagen in the developing mouse mandibular condyle

The present chronological investigation assessed the distribution of type II collagen expression in the developing mouse mandibular condyle using immunohistochemical staining with respect to the anatomy of the anlage of the mandibular condyle, the histological characteristics of which were disclosed in our previous investigation. We analyzed fetuses, obtained by cross breeding of ICR strain mice, between 14.0 and 19.0 days post-conception (dpc) and pups on 1, 3, and 5 days post-natal (dpn) using immunohistochemical staining with 2 anti-type II collagen antibodies. The expression of type II collagen was first detected at 15.0 dpc in the lower part of the hypertrophic chondrocyte zone; thereafter, this type II collagen-positive layer was expanded and intensified (P1 layer). At 17.0 dpc, we identified a type II collagen-negative layer (N layer) around the P1 layer and we also identified another newly formed type II collagen-positive layer (P, layer) on the outer surface of the N layer. The most typical and conspicuous 3-layered distribution was observed at 1 dpn; thereafter, there was a reduction in the intensity of expression, and with it, the demarcation between the layers was weakened by 5 dpn. The P1 layer was derived from the central region of the core cell aggregate of the anlage of the mandibular condyle and participated in endochondral bone formation. The N layer was derived from the fringe of the core cell aggregate of the anlage, formed the bone collar at the side of the condyle by intramembranous bone formation, and showed a high level of proliferative activity at the vault. The P2 layer was formed from the outgrowth of the N layer, and could be considered as the secondary cartilage. The intensive expression of type II collagen from 17.0 dpc to 3 dpn was detected in the fibrous sheath covering the condylar head, which is derived from the peripheral cell aggregate of the anlage. Since its expression in the fibrous sheath was not detected in the neighboring section in the absence of hyaluronidase digestion, some changes in the extracellular matrix of the fibrous sheath appear to participate in the generation of the lower joint space. The results of the present investigation indicate that further studies are required to fully characterize the development of the mouse mandibular condyle.

Structural features of incremental line-like striations in mandibular condylar cartilage of c-src-deficient mice

Mandibular condylar cartilage is sensitive to masticatory force, while mice lacking the c-src gene (c-src-deficient mice) have osteopetrosis and tooth eruption failure. The purpose of this study was to investigate the morphology of the mandibular condyle in these mice, which were maintained with a soft-food diet for 240 days after birth. The condylar head in the c-src-deficient mice showed slight deformity in shape before weaning, but showed remarkable undergrowth after weaning. No significant morphological or histological differences were detected between the mandibular condyle in wild-type mice fed soft food and those fed hard food, indicating that osteopetrosis, as well as abnormal masticatory force, influences the morphology of the mouse mandibular condyle, and that malocclusion rather than dietary consistency may have greater influence. After 70 days, incremental line-like striations consisting of cartilaginous and non-cartilaginous layers were detected in the mandibular condyle of the c-src-deficient mice, but not in the tibial growth plate. Immunostaining of aggrecan, collagen types II and X, and osteopontin was detected in the cartilaginous layers, but not in the non-cartilaginous layers showing collagen type I immunostaining. Chondrocyte lacunae were not eroded in the cartilaginous layers, and complete circumferential mineralisation around the lacunae and impaired osteoclast (chondroclast) function can account for this phenomenon. However, repeated cessation of chondrocyte differentiation may be required to completely explain the formation of the striations. These results indicate that the mandibular condyle in the c-src-deficient mice has unique structural features, adding to its deformity.

Histochemical evidences on the chronological alterations of the hypertrophic zone of mandibular condylar cartilage

The hypertrophic chondrocytes lack the ability to proliferate, thus permitting matrix mineralization as well as vascular invasion from the bone in both the mandibular condyle and the epiphyseal cartilage. This study attempted to verify whether the histological appearance of the hypertrophic chondrocytes is in a steady state during postnatal development of the mouse mandibular condyle. Type X collagen immunohistochemistry apparently distinguished the fibrous layer described previously as the "articular zone," "articular layer," and "resting zone" from the hypertrophic zone. Interestingly, the ratio of the type X collagen-positive hypertrophic zone in the entire condyle seemed higher in the early stages but decreased in the later stages. Some apparently compacted cells in the hypertrophic zone showed proliferating cell nuclear antigen (PCNA) immunoreaction, indicating the potential for cell proliferation at the early stages. As the mice matured, in contrast, they further enlarged and assumed typical features of hypertrophic chondrocytes. Apoptotic cells were also discernible in the hypertrophic zone at the early but not later stages. Consistent with morphological configurations of hypertrophic chondrocytes, immunoreactions for alkaline phosphatase, osteopontin, and type I collagen were prominent at the later stage, but not the early stage. Cartilaginous matrices demonstrated scattered patches of mineralization at the early stage, but increased in their volume and connectivity at the later stage. Thus, the spatial and temporal occurrence of these immunoreactions as well as apoptosis likely reflect the prematurity of hypertrophying cells at the early stage, and imply a physiological relevance during the early development of the mandibular condyles.

Direct actions of leptin on bone remodeling.

From the time of its original description almost a decade ago [1] leptin was regarded as a hormone originating in the adipocyte whose primary target was within the central nervous system. There, it regulates appetite and reproductive function. It has now become clear that leptin receptors are much more widely distributed than just the hypothalamus, and the skeleton has emerged as an important site of action of leptin. This is suggested by the demonstration of the signalling form of the leptin receptor in human osteoblasts [2–4], rat osteoblasts [5, 6], human chondrocytes [6], porcine chondrocytes [2], human mesenchymal stem cells undergoing osteogenic differentiation [7], and in osteoblastic and chondrocytic cell lines [5, 8]. Consistent with this receptor data, there is now abundant evidence indicating that leptin directly promotes the differentiation and growth of cells of the osteoblast lineage. Thomas et al. [9] found that leptin promoted differentiation of a human marrow stromal cell line into an osteoblast phenotype, and that it increased synthesis of bone matrix proteins, such as type I collagen and osteocalcin, in these cells. Similar findings have been reported by Gordeladze et al. [10] in studies of primary human osteoblasts, in which they also found that leptin reduced apoptosis and increased formation of mineralized bone nodules. Iwaniec et al. [11] have reported increased numbers of mineralized bone nodules in primary osteoblast cultures, and Reseland et al. [12] demonstrated this with both primary human osteoblast cultures and in osteosarcoma cell lines. We have found that low concentrations of leptin (>10 M) increase proliferation of isolated fetal rat osteoblasts to an extent comparable to that of IGF1 [6]. Thus, there is agreement across a range of in vitro models that leptin is anabolic to osteoblasts. There is also evidence that leptin directly regulates osteoclast development. Holloway et al. [13] have shown that leptin inhibits osteoclast generation from human peripheral blood mononuclear cells by reducing production of RANK in mononuclear cells. These findings are complemented by the report of Burguera et al. [14] that leptin increases osteoprotegerin levels and decreases RANK-ligand levels in human marrow stromal cells. Leptin (‡10 M) also inhibits osteoclastogenesis in mouse bone marrow cultures [6]. The effects of leptin on osteoclasts could be contributed to by its stimulation of the secretion of interleukin-1 receptor antagonist from monocytic and lymphocytic cells, which will reduce osteoclast recruitment [15, 16]. These in vitro findings of leptin effects on osteoclasts explain the clinical observations of an inverse relationship between serum leptin concentrations and bone resorption markers in human fetuses [17] and in postmenopausal women [18]. Thus, there is a consistent body of evidence suggesting that leptin will impact positively on bone density as a result of its dual effects on bone formation and resorption. Leptin may also impact on skeletal size since it acts on chondrocytes. We have demonstrated the presence of the leptin receptor in chondrocytes from human articular cartilage, and that proliferation of ovine and canine chondrocytes is promoted by leptin [6]. Systemic administration of leptin to adult mice increases the thickness of their growth plates [6]. Maor et al. [19] have shown specific binding sites for leptin in the chondrocyte cell population of skeletal growth centers in the mouse mandibular condyle. Leptin stimulated cell proliferation and increased the width of the chondroprogenitor zone in this model. These findings are reflected in the reduced femoral length of both the leptin-deficient mouse (Fig. 1) [2] and the leptin-resistant rat [20]. The positive effect of leptin on skeletal size suggests that this hormone may be a contributor to the increases in stature of successive generations of humans that has been observed over the last century or more [21]. As nutrition has improved, leptin concentrations may have increased with a consequent increase in growth and adult height. The final integration of the direct effects of leptin on bone cells seen in vitro occurs in at least three different ways. One is in human studies of the relationships between leptin and bone density. Circulating leptin conCorrespondence to: I. R. Reid; E-mail: i.reid@auckland.ac.nz Calcif Tissue Int (2004) 74:313–316 DOI: 10.1007/s00223-002-0015-z Calcified Tissue International

Localization and inhibitory effect of basic fibroblast growth factor on chondrogenesis in cultured mouse mandibular condyle.

The condylar cartilage, an important growth site in the mandible, shows characteristic modes of growth and differentiation, unlike the limb bud cartilage. To elucidate the mechanism of chondrogenesis at the condylar cartilage, we analyzed the effects of basic fibroblast growth factor (bFGF) on the growth and development of mouse mandibular condyle using serum-free organ culture and on the expression of genes related to the chondrogenesis. Further, we investigated the localization of bFGF in cultured condyle by immunohistochemistry. The present immunohistochemical observations showed that bFGF is localized in the extracellular matrix of the mesenchymal condylar anlage, the perichondrium and the proliferative cell zone, and that immunostaining was diminished in the metachromatically stained area. In the condyle culture with added recombinant human bFGF (rhbFGF) for 5 days, the area occupied by hypertrophic chondrocytes in the mandibular condylar cartilage was reduced. A reverse transcription-polymerase chain reaction (RT-PCR) assay also showed that the mRNA levels of aggrecan and type X collagen were reduced compared with nontreated tissues. Treatment with rhbFGF for 2 days decreased cell proliferation in the perichondrium, and bFGF downregulated the Indian hedgehog (Ihh), parathyroid hormone-related protein (PTHrP), bone morphogenetic protein 4 (BMP4), and core-binding factor alpha1 (Cbfa1) expression in the RT-PCR assay. These findings suggest that bFGF has the ability for inhibitory regulation of condylar growth, via the inhibition of proliferation and differentiation of chondrocytes, and that this inhibitory regulation is related to the downregulation of growth factors and transcription factors.

Leptin acts as a growth factor on the chondrocytes of skeletal growth centers.

Childhood obesity frequently is associated with an increase in height velocity and acceleration of epiphyseal growth plate maturation despite low levels of serum growth hormone (GH). In addition, obesity is associated with higher circulating levels of leptin, a 16-kDa protein that is secreted from the adipocytes. In this study, we evaluated the direct effect of leptin on the chondrocyte population of the skeletal growth centers in the mouse mandibular condyle, a model of endochondral ossification. We found that chondrocytes in the growth centers contain specific binding sites for leptin. Leptin, at a concentration of 0.5-1.0 microg/ml, stimulated in a dose-dependent manner the width of the chondroprogenitor zone (up to 64%), whereas higher concentrations had an inhibitory effect. Leptin induction of both proliferation and differentiation activities in the mandibular condyle was confirmed by our findings of an increase in bromodeoxyuridine (BrdU) incorporation into DNA and in (acidic) Alcian blue (AB) staining of the cartilaginous matrix. Leptin also increased the abundance of the insulin-like growth factor (IGF) I receptor and IGF-I receptor messenger RNA (mRNA) within the chondrocytes and the progenitor cell population. Our results indicate that leptin acts as a skeletal growth factor with a direct peripheral effect on skeletal growth centers. Some of its effects on the growing bone may be mediated by the IGF system via regulation of IGF-I receptor expression. We speculate that the high circulating levels of leptin in obese children might contribute to their growth.

マウス下顎頭軟骨細胞コラーゲン遺伝子発現に及ぼすインターロイキン１βとヒアルロン酸の影響

Recent analyses of synovial fluid in the temporomandibular joint have confirmed the presence of various markers such as interleukin-1β(IL-1β). Hyaluronic acid (HA) has been reported to be effective effects in the management of temporomandibular joint disorders. However, the effects of IL-1β and HA on chondrocytes are not clear.This study was performed to investigate the effects of IL-1β and HA on collagen gene expression in chondrocytes of mouse mandibular condyle. Collagen gene expression was analyzed on the basis of mRNA levels by the RT-PCR procedure. We found that IL-1β significantly decreased the level of type II collagen mRNA and increased the level of type X collagen mRNA. HA significantly increased the levels of types II and X collagen mRNA. In additions, HA inhibited the IL-1β-induced decrease in type II collagen mRNA and increase in type X collagen mRNA.The results suggested that HA promotes the repair of cartilage and inhibits IL-β-induced damage of cartilage.

Genomic organization and chromosomal localization of the Itm2a gene.

Itm2A is a novel type II integral membrane protein that is involved in osteo- and chondrogenic differentiation. Itm2a cDNA was originally isolated from a cDNA library of organ cultures from prenatal mouse mandibular condyles, by subtractive hybridization and differential screening. The Itm2a gene was isolated from a BALB/c liver genomic library. In total, 9.4 kb of the gene were sequenced, of which 2649 bp are 5' flanking sequences. The Itm2a gene contains six exons and five introns. The splice sites conform to the GT/AG rule. The 5' flanking region, which contains the presumed promoter sequence, lacks the common TATAA and CCAAT sequences, but contains consensus binding sites for various transcription factors. Several of these transcription factors are known to play a role in transcriptional regulation of cartilage- or bone-specific genes (e.g. Cbfa1, Cart-1, MHox, HES-1, and CIIS1). Itm2a was mapped to mouse chromosome position XA2-XA3 by fluorescent in situ hybridization (FISH) analysis. The human homolog, ITM2A, was mapped to chromosome position Xq13.3-Xq21.2.

The effect of altered functional forces on the expression of bone-matrix proteins in developing mouse mandibular condyle

Mechanical forces are known to have an effect on bone formation, maintenance and remodelling, and there is evidence that the development of the mandibular condyle in the rat is influenced by the consistency of the diet. Here a mouse model was used to investigate the relation between food, condylar development and the expression of bone sialoprotein (BSP), osteopontin (OPN), osteocalcin (OC) and type I collagen (COL I). Twenty-four 19-day-old male mice were randomly divided into three groups. Groups 1 and 2 were fed hard pellets and soft powdered food, respectively, for 2 weeks. Group 3 mice were fed soft food for 1 week followed by a week of hard pellets. Incisors of mice in groups 2 and 3 were trimmed twice a week to reduce occlusal forces. After killing the animals, mandibular condyles were collected for RNA extraction, in situ hybridization and immunohistochemical analyses. Histological sections showed that the condyles of mice in group 2 were underdeveloped, with a thinner layer of cartilage and fewer bone trabeculae. Northern hybridization of total RNA of the condyle from mice in this soft-food group also exhibited a significant decrease in the amounts of BSP, OPN, OC and COL I, representing 79%, 75%, 77% and 79% respectively, of that from mice fed hard food. In situ hybridization of these bone-matrix proteins demonstrated signals in bone-forming cells and BSP mRNA was also seen in the hypertrophic cartilage cells in the developing condyle. Immunohistochemical study demonstrated an obvious difference in the intensity of staining, especially for BSP. Results from group 3 were similar to those from group 1. The observed decrease in bone matrix-protein expression confirms that the consistency of the diet affects the development of the mouse mandibular condyle and that a soft diet diminishes the rate of bone formation.

An ultrastructural study of cartilage resorption at the site of initial endochondral bone formation in the fetal mouse mandibular condyle.

An ultrastructural study was undertaken on cartilage resorption at the site of initial endochondral bone formation in the mouse mandibular condyle on d 16 of pregnancy. After resorbing the bone collar, the osteoclasts extended their cell processes into the cartilage matrix and made contact with hypertrophic chondrocytes. By means of cell processes or vacuolar structures, these osteoclasts entrapped the calcified cartilage matrices, cell debris, and the degraded uncalcified cartilage matrices. In particular, since the calcified cartilage matrices were sometimes seen to be disrupted within the osteoclastic vacuolar structures, they were probably disposed of by the osteoclasts. Invading endothelial cells giving rise to capillaries also directly surrounded the degraded uncalcified cartilage matrices and small deposits of cell debris. In addition, hypertrophic chondrocytes that had attached to or were in the process of attaching to the invading osteoclasts often enclosed the small calcified cartilage matrices. Other cell types that have often been reported in other regions of cartilage resorption were not seen at the site of initial endochondral bone formation in this study. Our findings in relation to cartilage resorption may therefore represent unique features of the site of initial endochondral bone formation site. We consider that the manner of cartilage resorption is likely to vary by site, age, and species.

Isolation of Markers for Chondro-osteogenic Differentiation Using cDNA Library Subtraction: MOLECULAR CLONING AND CHARACTERIZATION OF A GENE BELONGING TO A NOVEL MULTIGENE FAMILY OF INTEGRAL MEMBRANE PROTEINS

To identify novel marker molecules associated with chondro-osteogenic differentiation, we have set up a differential screening system based on a cDNA library subtraction in organ cultures of prenatal mouse mandibular condyles. Differential screening of a cDNA library constructed from in vitro cultured condyles allowed the isolation of a novel gene, named E25. Full-length E25 cDNA is predicted to encode a type II integral membrane protein of 263 amino acid residues. In situ hybridization experiments show that E25 is expressed in the outer perichondrial rim of the postnatal mandibular condyle, which contains the proliferating progenitor cells, but not in the deeper layers of the condyle containing the more differentiated chondroblasts and chondrocytes. Other cartilagenous tissues and their perichondrium were negative. Strong in situ hybridization signals were also detected on bone trabeculae of mature bone in tooth germs and in hair follicles. Northern blot analysis showed strong expression in osteogenic tissues, such as neonatal mouse calvaria, paws, tail, and in skin. This expression profile suggests that E25 could be a useful marker for chondro-osteogenic differentiation. Homology searches of DNA databanks showed that E25 belongs to a novel multigene family, containing three members both in man and mouse. The mouse E25 gene locus (Itm2) was mapped to the X chromosome.

マウス下顎頭線維層における弾性線維システムの局在

マウス下顎頭線維層における弾性線維システムの局在

STT3, a novel essential gene related to the PKC1/STT1 protein kinase pathway, is involved in protein glycosylation in yeast

Mutations of genes involved in the STT1/PKC1 pathway in yeast show staurosporine and temperature sensitivities ( stt) which are suppressed by the addition of 1 M sorbitol [Yoshida et al., Mol. Gen. Genet. 242 (1994) 631-640]. Among the stt mutants, stt3-2 shares this phenotype. The STT3 gene encodes a novel 718-amino-acid protein with significant homology to potential transmembrane proteins of Caenorhabditis elegans and mouse mandibular condyle (about 80% homologous and 60% identical). Unlike the STT1/PKC1 gene, STT3 is essential for cell growth irrespective of osmotic support. Pulse-chase experiments show that the stt3 mutants are defective in protein glycosylation. The stt3 mutants are sensitive to hygromycin B and resistant to sodium orthovanadate, whose phenotypes are common to those defective in protein glycosylation.

An ultrastructural study of extracellular fibrillar components of developing mouse mandibular condyle with special reference to type VI collagen

The localization of type VI collagen was examined from birth to 8 weeks of age. Immunohistochemical staining with anti-type VI collagen antibody was strongly positive in the hypertrophic zone and moderately positive in the fibrous zone and the outer periphery of the proliferative zone, but negative in the inner area of the proliferative zone and mature zone. After ATP treatment, type VI collagen periodic fibrils with about 80-nm intervals were frequently observed but only in the fibrous zone. They occurred mainly in the superficial area of this zone, where striated collagen fibrils were sparse, while a few were noted in the inner area, where bundles of collagen fibrils were abundant. From these distributional differences of both components, a subzonation of the fibrous zone into superficial and inner areas is suggested. Moreover, with ATP treatment there were fewer type VI collagen periodic fibrils formed with increasing age (8 weeks). Testicular hyaluronidase digestion before ATP treatment facilitated the formation of periodic fibrils, in all the ages examined, in the intercellular space and around the fibroblastic cells. The interaction of type VI collagen with other components such as collagen fibrils, glycosaminoglycans or proteoglycans may play a part in maintaining the structural integrity of extracellular matrix in the mouse mandibular condyle.

Isolation of a cathepsin B-encoding cDNA from murine osteogenic cells

Cathepsin B-encoding cDNA ( CTSB) clones have been isolated from a λgt10 library of a murine osteosarcoma by differential screening during a search for genes which are typically expressed during osteogenic differentiation in mouse mandibular condyles in vitro. Sequencing of the CTSB 3' end revealed that the isolated sequence contained an 825-bp 3'-noncoding region, the polyadenylation signal and the poly(A) tail. The enhanced CTSB expression during the early stages of the enchondral ossification-like process in mandibular condyles in vitro suggests that CTSB participates in the degradation of cartilage matrix prior to the synthesis of bone matrix proteins.

C-fos expression precedes osteogenic differentiation of cartilage cells in vitro.

We have investigated the temporal pattern of expression of c-fos in cartilage cells in mouse mandibular condyles. During in vitro cultivation, the progenitor cells in this organ differentiate to osteoblasts, and hypertrophic chondrocytes start to show features indicative of osteogenic differentiation. Prior to these processes we observed two distinct patterns of c-fos expression. High, transient c-fos expression was found in the entire tissue within 30 min of culture. This type of c-fos expression appeared to result from mechanical forces applied during dissection. The second type of c-fos expression appeared in individual cells in the zone of hypertrophic chondrocytes. A varying number of formerly quiescent chondrocytes expressed high levels of c-fos mRNA after between 30 min and 10 d in culture, with a peak in the number of cells between days 1 and 3. c-fos expression in these cartilage cells was followed by DNA replication and expression of genes typifying osteoblastic differentiation. After 7 d in culture, groups of cells with the typical ultrastructural features of osteoblasts, and surrounded by an osteoid-like matrix, were observed in single chondrocyte-type lacunae, suggesting division of chondrocytes and differentiation to osteoblasts. The data suggest that c-fos may play a crucial role in the perturbation of determined pathways of skeletoblast differentiation and in the regulation of endochondral bone formation.