Osteoprotegerin (OPG) and RANKL expression and distribution in developing human craniomandibular joint

During embryogenesis the bone tissue of craniomandibular joint (CMJ) is formed through two pathways: intramembranous ossification and endochondral ossification. The development process is under the control of regulatory factors.The osteoprotegerin (OPG) and the receptor activator of nuclear factor (NF)-kappaB ligand are key regulators of osteoclastogenesis. The aim of this study is the localization of OPG and RANKL mRNA and protein in the foetal CMJ by immunohistochemistry (IHC) and in situ hybridization (ISH). The main results were: OPG and RANKL mRNA and protein were co-localized in the same cell types; OPG and RANKL were specially immunolocated in osteogenic cells; immunolabeling was often seen in the nucleus and cytoplasm of otherwise negative hypertrophic chondrocytes; IHC and ISH labeling decreased from proliferative to hypertrophic chondrocytes; early osteocytes showed dual protein expression and some of the mature osteocytes were ISH-negative; periosteal osteoclasts and chondroclasts were mostly stained by IHC and variably labeled by ISH; the new bone matrix and trabecular borders showed intense immunolabeling. The co-expression of OPG and RANKL in the same bone cell types confirms their strictly coupled action in the regulation of bone metabolism in the CMJ development and their extracellular presence in the new bone matrix and trabecular borders suggests a local regulatory role.     

Immunohistochemical detection of cathepsin D, K, and L in the process of endochondral ossification in the human

Cathepsins D, K, and L were immunolocalized in tissue undergoing endochondral ossification in the human. Cathepsins D, K, and L were localized in osteoclasts and chondroclasts attached to bone matrix and cartilage matrix, respectively. Cathepsins D and L were immunostained in chondrocytes. Immunolocalization of cathepsin D was limited to hypertrophic chondrocytes adjacent to the osteochondral junction. In contrast, cathepsin L was immunolocalized in both proliferating and hypertrophic chondrocytes. In the bone marrow space, cathepsins D, K, and L were localized in multinucleated cells. Cathepsin D was diffusely detected in mononuclear bone marrow cells which were negative for cathepsins K and L. The present findings indicated that cathepsins K, D, and L were associated with the process of endochondral ossification in the human, and suggested that these cathepsins share roles in bone and cartilage turnover in the human.     

Electron microscopic observations on the fate of hypertrophic chondrocytes in condylar cartilage of rat mandible

The present study focused on the hypertrophic cell zone and the adjacent region of primary spongiosa in the mandibular condylar cartilage in growing rats (3 to 7 weeks old). In this cartilage, chondrocytes were not arranged in columns, and there was no clear distinction between longitudinal and transverse septum. The hypertrophic chondrocytes were not surrounded entirely by calcified matrix, and capillaries were in close contact with cartilage cells. The staining intensity of the pericellular matrix decreased in the lower hypertrophic cell zone in comparison with that in the upper part of the hypertrophic cell zone. Electron microscopic examinations indicated that the lowest hypertrophic cells contained lysosomes and pinocytotic vesicles. Some hypertrophic chondrocytes appeared to have been released from their lacunae and were observed in the region of the primary spongiosa. Hence it is suggested that the lowest hypertrophic chondrocytes in the rat mandibular condyle do not die but are released from their lacunae into the bone marrow. Further study is needed to determine whether or not these cells do indeed become osteoblasts and/or chondroclasts.     

The effects of three different demineralization agents on osteopontin localization in adult rat bone using immunohistochemistry

Immunohistochemical localization of osteopontin, a phosphorylated acidic glycoprotein, was compared in adult rat femur fixed in 4% paraformaldehyde at 4° C for 48 h and demineralized at 4° C in ethylenediaminetetraacetic acid (EDTA), modified Jenkin's solution, or 15% formic acid, until radiographs indicated demineralization was complete. Formic acid was also evaluated at room temperature. EDTA solution (15 days) resulted in intense staining of osteocytes, periosteal osteoclasts and osteoblastic cells in osteonal bone. Osteoblasts were negative in the periosteum. No megakaryocyte staining was present; however, occasional neutrophils in the bone marrow were non-specifically stained. Demineralization in modified Jenkin's solution (16 days) showed osteopontin localization in bone matrix, hypertrophic and articular chondrocytes, and osteocytes. In cortical bone, almost all cement lines demarcating osteons showed very dense labeling. In the bone marrow, occasional megakaryocytes were immunopositive and neutrophils were non-specifically stained. Jenkin's produced non-specific staining of skeletal muscle and connective tissue. Formic acid demineralization (14 days, 4° C) resulted in osteopontin expression in osteoblasts, osteocytes, osteoclast precursors, bone matrix, osteoid, cement lines, and chondrocytes; osteoclasts, although present in very low numbers, were also positive. More labeled osteoblasts could be identified compared to Jenkin's demineralization. Also more intense non-specific staining of the bone marrow neutrophils was obtained than with Jenkin's. Harsh, rapid demineralization with formic acid (4 days, room temperature) produced a loss in antigenicity demonstrated by a reduction in staining intensity not experienced with the 4° C protocol; however, osteopontin was still localized in bone matrix and hypertrophic zone chondrocytes. These results indicate that demineralization is compatible with retention of immunoreactive osteopontin in adult rat bone. Both EDTA and formic acid demineralization produce excellent immunostaining and are preferred over the modified Jenkin's solution to minimize background levels of non-specific staining.     

Bone morphogenetic protein (BMP) localization in developing human and rat growth plate, metaphysis, epiphysis, and articular cartilage.

We assessed the distribution and relative staining intensity of bone morphogenetic protein (BMP)-1-7 by immunohistochemistry in tibial growth plates, epiphyses, metaphyses, and articular cartilage in one 21-week and one 22-week human fetus and in five 10-week-old Sprague-Dawley rats. In the rats, articular cartilage was also examined. BMP proteins were mostly cytoplasmic, with negligible matrix staining. Highest BMP levels were seen in (a) hypertrophic and calcifying zone chondrocytes of growth plate (BMP-1-7), (b) osteoblasts and/or osteoprogenitor fibroblasts and vascular cells of the metaphyseal cortex and medulla (BMP-1-6), (c) osteoclasts of the metaphysis and epiphysis (BMP-1,-4,-5, and -6), and (d) mid to deep zone articular chondrocytes of weanling rats (BMP-1-7). BMP staining in osteoclasts, an unexpected finding, was consistently strong with BMP-4, -5, and -6 but was variable and dependent on osteoclast location with BMP-2,-3, and -7. BMP-1-7 were moderately to intensely stained in vascular canals of human fetal epiphyseal cartilage by endothelial cells and pericytes. BMP-1,-3,-5,-6, and -7 were localized in hypertrophic chondrocytes adjacent to cartilage canals. We conclude that BMP expression is associated with maturing chondrocytes of growth plate and articular cartilage, and may play a role in chondrocyte differentiation and/or apoptosis. BMP appears to be expressed by osteoclasts and might be involved in the intercellular "cross-talk" between osteoclasts and neighboring osteoprogenitor cells at sites of bone remodeling.     

Parathyroid hormone-related peptide (PTHrP)-dependent and -independent effects of transforming growth factor beta (TGF-beta) on endochondral bone formation.

Previously, we showed that expression of a dominant-negative form of the transforming growth factor beta (TGF-beta) type II receptor in skeletal tissue resulted in increased hypertrophic differentiation in growth plate and articular chondrocytes, suggesting a role for TGF-beta in limiting terminal differentiation in vivo. Parathyroid hormone-related peptide (PTHrP) has also been demonstrated to regulate chondrocyte differentiation in vivo. Mice with targeted deletion of the PTHrP gene demonstrate increased endochondral bone formation, and misexpression of PTHrP in cartilage results in delayed bone formation due to slowed conversion of proliferative chondrocytes into hypertrophic chondrocytes. Since the development of skeletal elements requires the coordination of signals from several sources, this report tests the hypothesis that TGF-beta and PTHrP act in a common signal cascade to regulate endochondral bone formation. Mouse embryonic metatarsal bone rudiments grown in organ culture were used to demonstrate that TGF-beta inhibits several stages of endochondral bone formation, including chondrocyte proliferation, hypertrophic differentiation, and matrix mineralization. Treatment with TGF-beta1 also stimulated the expression of PTHrP mRNA. PTHrP added to cultures inhibited hypertrophic differentiation and matrix mineralization but did not affect cell proliferation. Furthermore, terminal differentiation was not inhibited by TGF-beta in metatarsal rudiments from PTHrP-null embryos; however, growth and matrix mineralization were still inhibited. The data support the model that TGF-beta acts upstream of PTHrP to regulate the rate of hypertrophic differentiation and suggest that TGF-beta has both PTHrP-dependent and PTHrP-independent effects on endochondral bone formation.     

VEGF couples hypertrophic cartilage remodeling, ossification and angiogenesis during endochondral bone formation.

Hypertrophic chondrocytes in the epiphyseal growth plate express the angiogenic protein vascular endothelial growth factor (VEGF). To determine the role of VEGF in endochondral bone formation, we inactivated this factor through the systemic administration of a soluble receptor chimeric protein (Flt-(1-3)-IgG) to 24-day-old mice. Blood vessel invasion was almost completely suppressed, concomitant with impaired trabecular bone formation and expansion of hypertrophic chondrocyte zone. Recruitment and/or differentiation of chondroclasts, which express gelatinase B/matrix metalloproteinase-9, and resorption of terminal chondrocytes decreased. Although proliferation, differentiation and maturation of chondrocytes were apparently normal, resorption was inhibited. Cessation of the anti-VEGF treatment was followed by capillary invasion, restoration of bone growth, resorption of the hypertrophic cartilage and normalization of the growth plate architecture. These findings indicate that VEGF-mediated capillary invasion is an essential signal that regulates growth plate morphogenesis and triggers cartilage remodeling. Thus, VEGF is an essential coordinator of chondrocyte death, chondroclast function, extracellular matrix remodeling, angiogenesis and bone formation in the growth plate.     

Contributions of matrix metalloproteinases toward Meckel’s cartilage resorption in mice: immunohistochemical studies,including comparisons with developing endochondral bones

The middle portion of Meckel’s cartilage (one of four portions that disappear with unique fate) degrades via hypertrophy and the cell death of chondrocytes and via the resorption of cartilage by chondroclasts. We have examined the immunolocalization of matrix metalloproteinase-2 (MMP-2), MMP-9, MMP-13, and MMP-14 (members of the MMP activation cascade) and galectin-3 (an endogenous substrate for MMP-9 and an anti-apoptotic factor) during resorption of Meckel’s cartilage in embryonic mice and have compared the results with those of developing endochondral bones in hind limbs. MMP immunoreactivity, except for MMP-2, is present in nearly all chondrocytes in the middle portion of Meckel’s cartilage. On embryonic day 15 (E15), faint MMP-2-immunoreactive and intense MMP-13-immunoreactive signals occur in the periosteal bone matrix deposited by periosteal osteoblasts on the lateral surface, whereas MMP-9 and MMP-14 are immunolocalized in the peripheral chondrocytes of Meckel’s cartilage. The activation cascade of MMPs by face-to-face cross-talk between cells may thus contribute to the initiation of Meckel’s cartilage degradation. On E16, immunopositive signaling for MMP-13 is detectable in the ruffled border of chondroclasts at the resorption front, whereas immunostaining for galectin-3 is present at all stages of chondrocyte differentiation, especially in hypertrophic chondrocytes adjacent to chondroclasts. Galectin-3-positive hypertrophic chondrocytes may therefore coordinate the resorption of calcified cartilage through cell-to-cell contact with chondroclasts. In metatarsal specimens from E16, MMPs are detected in osteoblasts, young osteocytes, and the bone matrix of the periosteal envelope, whereas galectin-3 immunoreactivity is intense in young periosteal osteocytes. In addition, intense MMP-9 and MMP-14 immunostaining has been preferentially found in pre-hypertrophic chondrocytes, although galectin-3 immunoreactivity markedly decreases in hypertrophic chondrocytes. These results indicate that the degradation of Meckel’s cartilage involves an activation cascade of MMPs that differs from that in endochondral bone formation.     

Leukaemia inhibitory factor (lif) is expressed in hypertrophic chondrocytes and vascular sprouts during osteogenesis

Avascular cartilage is replaced by highly vascularized bone tissue during endochondral ossification, a process involving capillary invasion of calcified hypertrophic cartilage in association with apoptosis of hypertrophic chondrocytes, degradation of cartilage matrix and deposition of bone matrix. All of these events are closely controlled, especially by cytokines and growth factors. Leukaemia inhibitory factor (LIF), a member of the gp130 cytokine family, is involved in osteoarticular tissue metabolism and might participate in osteogenesis. Immunohistochemical staining showed that LIF is expressed in hypertrophic chondrocytes and vascular sprouts of cartilage and bone during rat and human osteogenesis. LIF is also present in osteoblasts but not in osteoclasts. Observations in a rat endochondral ossification model were confirmed by studies of human cartilage biopsies from foetuses with osteogenesis imperfecta. LIF was never detected in adult articular chondrocytes and bone-marrow mesenchymal cells. These results and other data in the literature suggest that LIF is involved in the delicate balance between the rate of formation of calcified cartilage and its vascularization for bone development.     

L-Maf,a downstream target of Pax6, is essential for chick lens development

Vascular endothelial growth factor (VEGF)-mediated angiogenesis is an important part of bone formation. To clarify the role of VEGF isoforms in endochondral bone formation, we examined long bone development in mice expressing exclusively the VEGF120 isoform (VEGF120/120 mice). Neonatal VEGF120/120 long bones showed a completely disturbed vascular pattern, concomitant with a 35% decrease in trabecular bone volume, reduced bone growth and a 34% enlargement of the hypertrophic chondrocyte zone of the growth plate. Surprisingly, embryonic hindlimbs at a stage preceding capillary invasion exhibited a delay in bone collar formation and hypertrophic cartilage calcification. Expression levels of marker genes of osteoblast and hypertrophic chondrocyte differentiation were significantly decreased in VEGF120/120 bones. Furthermore, inhibition of all VEGF isoforms in cultures of embryonic cartilaginous metatarsals, through the administration of a soluble receptor chimeric protein (mFlt-1/Fc), retarded the onset and progression of ossification, suggesting that osteoblast and/or hypertrophic chondrocyte development were impaired. The initial invasion by osteoclasts and endothelial cells into VEGF120/120 bones was retarded, associated with decreased expression of matrix metalloproteinase-9. Our findings indicate that expression of VEGF164 and/or VEGF188 is important for normal endochondral bone development, not only to mediate bone vascularization but also to allow normal differentiation of hypertrophic chondrocytes, osteoblasts, endothelial cells and osteoclasts.     

Role of proteoglycans in endochondral ossification: immunofluorescent localization of link protein and proteoglycan monomer in bovine fetal epiphyseal growth plate

The hypothesis is widely held that, in growth plate during endochondral ossification, proteoglycans in the extracellular matrix of the lower hypertrophic zone are degraded by proteases and removed before mineralization, and that this is the mechanism by which a noncalcifiable matrix is transformed into a calcifiable matrix. We have evaluated this hypothesis by examining the immunofluorescent localization and concentrations of proteoglycan monomer core protein and link protein, and the concentrations of glycosaminoglycans demonstrated by safranin 0 staining, in the different zones of the bovine fetal cartilage growth plate. Monospecific antibodies were prepared to proteoglycan monomer core protein and to link protein. The immunofluorescent localization of these species was examined in decalcified and undecalcified sections containing the zones of proliferating and hypertrophic chondrocytes and in sections containing the zones of proliferating and hypertrophic chondrocytes and the metaphysis, decalcified in 0.5 M EDTA, pH 7.5, in the presence of protease inhibitors. Proteoglycan monomer core protein and link protein are demonstrable without detectable loss throughout the extracellular matrix of the longitudinal septa of the hypertrophic zone and in the calcified cartilage of the metaphysis. In fact, increased staining is observed in the calcifying cartilage. Contrary to the prevailing hypothesis, our results indicate that there is no net loss of proteoglycans during mineralization and that the proteoglycans become entombed in the calcified cartilage which provides a scaffolding on which osteoid and bone are formed. Proteoglycans appear to persist unaltered in the calcified cartilage core of the trabeculae, until at last the entire trabeculae are eroded from their surfaces and removed by osteoclasts, when the primary spongiosa is replaced by the secondary spongiosa.     

Transforming growth factor-beta 1, -beta 2, and -beta 3 secreted by a human glioblastoma cell line. Identification of small and different forms of large latent complexes.

Transforming growth factor-beta 1 (TGF-beta 1) has been found to occur as latent high molecular weight complexes, with or without an associated component denoted latent TGF-beta 1-binding protein (LTBP). We show here that a human glioblastoma cell line (U-1240 MG) secretes all isoforms of TGF-beta s found in mammalian cells (TGF-beta 1, -beta 2, and -beta 3). Approximately 26% of the secreted TGF-beta is in an active form. Latent TGF-beta s were partially purified from medium conditioned by the U-1240 MG cell line using anion exchange chromatography. Analysis of the different fractions by immunoblotting using antisera against precursor parts of the different TGF-beta isoforms, and against LTBP, revealed that not only TGF-beta 1 but also other isoforms of TGF-beta may occur in high molecular weight forms containing LTBP. In addition, each one of the TGF-beta isoforms occurred in smaller forms not containing LTBP. Interestingly, each of the TGF-beta isoforms was also seen in complexes of about 210 kDa containing associated component(s) distinct from LTBP. These results indicate that each of the different isoforms of TGF-beta is synthesized and secreted by this glioblastoma cell line in several different high molecular weight latent forms; the biological importance of the various latent TGF-beta complexes is discussed.     

Osteoclasts from mice deficient in tartrate-resistant acid phosphatase have altered ruffled borders and disturbed intracellular vesicular transport

Tartrate-resistant acid phosphatase (TRAP) is an enzyme highly expressed in osteoclasts (OC) and chondroclasts. As an approach to pinpoint the function of TRAP in bone-resorbing osteoclasts, the morphological phenotypic alterations of bone and osteoclasts in mice with targeted disruption of the TRAP gene were assessed by quantitative histomorphometry and immunocytochemistry at the light microscopic and ultrastructural levels. TRAP-deficient mice display alterations in the epiphyseal growth plates as evidenced by increased height with disorganized columns of chondrocytes, in particular affecting the zone of hypertrophic chondrocytes, consistent with a disturbance of chondrocyte maturation and chondroclastic resorption at the epiphyseal/metaphyseal junction. TRAP -/- mice express an early onset osteopetrotic bone phenotype, apparent already at 4 weeks of age. The differentiation of OCs was apparently normal; however, the osteoclasts in TRAP-deficient mice were less active in terms of degradation or release of the resorption marker C-terminal type I collagen cross-linked peptide, indicative of an intrinsic defect. Ultrastructural morphometry disclosed that OCs from TRAP-deficient young mice exhibited an increased relative area of ruffled borders. Moreover, mutant OC accumulated cytoplasmic vesicles 200-500 nm in size in both ruffled border and basolateral parts of the cytoplasm, reflecting disturbed intracellular transport. The accumulated vesicles were not likely derived from the secretory pathway, since cathepsin K was detected at normal levels in the ruffled border area and matrix in TRAP -/- mice. In summary, the resorptive defect in TRAP-deficient OCs is reflected by a disturbance at the level of ruffled borders and intracellular transport vesicles. Consequently, accumulation of vesicles in the cytoplasm of mutant OCs indicates a novel function for TRAP in modulating intracellular vesicular transport in osteoclasts.     

Impaired endochondral bone development and osteopenia in Gli2-deficient mice

Mice homozygous for targeted disruption of the zinc finger domain of Gli2 (Gli2(zfd/zfd)) die at birth with developmental defects in several organ systems including the skeleton. The current studies were undertaken to define the role of Gli2 in endochondral bone development by characterizing the molecular defects in the limbs and vertebrae of Gli2(zfd/zfd) mice. The bones of mutant mice removed by cesarian section at E16.5 and E18.5 demonstrated delayed endochondral ossification. This was accompanied by an increase in the length of cartilaginous growth plates, reduced bone tissue in the femur and tibia and by failure to develop the primary ossification centre in vertebral bodies. The growth plates of tibiae and vertebrae exhibited increased numbers of proliferating and hypertrophic chondrocytes with no apparent alteration in matrix mineralisation. The changes in growth plate morphology were accompanied by an increase in expression of FGF2 in proliferating chondrocytes and decreased expression of Indian hedgehog (Ihh), patched (Ptc) and parathyroid-hormone-related protein (PTHrP) in prehypertrophic cells. Furthermore, there was a reduction in expression of angiogenic molecules in hypertrophic chondrocytes, which was accompanied by a decrease in chondroclasts at the cartilage bone interface, fewer osteoblasts lining trabecular surfaces and a reduced volume of metaphyseal bone. These results indicate that functional Gli2 is necessary for normal endochondral bone development and that its absence results in increased proliferation of immature chondrocytes and decreased resorption of mineralised cartilage and bone formation.     

Differential expression of transforming growth factor-beta 1, -beta 2, and -beta 3 by glioblastoma cells, astrocytes, and microglia.

The type beta transforming growth factors (TGF) are potent regulators of the growth and functions of lymphocytes and macrophages. Recently the human glioblastoma cell line 308 was shown to produce TGF-beta 2. The relevance of this finding was evaluated further by comparing human glioblastoma cells with their nontransformed animal counterpart, astrocytes, with regard to the production of the three TGF-beta isoforms observed so far in mammals. In this report astrocytes are demonstrated to secrete also TGF-beta 2 and to express TGF-beta 1, -beta 2, and -beta 3 mRNA in vitro. In contrast, cultured murine brain macrophages release TGF-beta 1 and are positive for TGF-beta 1 mRNA only. Glia cell-derived TGF-beta 1 and -beta 2 are detected in latent form whereas both latent and active TGF-beta are identified in the supernatant of three human glioblastoma cell lines tested. These cell lines, however, show heterogeneity in regard to the isoform of TGF-beta expressed but share with astrocytes the inability to release TGF-beta 3. Provided production and activation of latent TGF-beta occur in vivo, astrocytes and microglia may then be expected to exert regulatory influences on immune mediated diseases of the central nervous system.     

Enhanced expression of TGF-beta and c-fos mRNAs in the growth plates of developing human long bones

The expression of mRNAs for type I and type II procollagens, transforming growth factor-beta (TGF-beta) and c-fos was studied in developing human long bones by Northern blotting and in situ hybridization. The cells producing bone and cartilage matrix were identified by hybridizations using cDNA probes for types I and II collagen, respectively. Northern blotting revealed that the highest levels of TGF-beta mRNA were associated with the growth plates. By in situ hybridization, this mRNA was localized predominantly in the osteoblasts and osteoclasts of the developing bone, in periosteal fibroblasts and in individual bone marrow cells. These findings are consistent with the view that TGF-beta may have a role in stimulation of type I collagen production and bone formation. Only a low level of TGF-beta mRNA was detected in cartilage where type II collagen mRNA is abundant. In Northern hybridization, the highest levels of c-fos mRNA were detected in epiphyseal cartilage. In situ hybridization revealed two cell types with high levels of c-fos expression: the chondrocytes bordering the joint space and the osteoclasts of developing bone. These differential expression patterns suggest specific roles for TGF-beta and c-fos in osseochondral development.     

A histochemical localization on Maclura pomifera lectin during osteogenesis

Mandibular condyles of 4-week-old Wistar strain rats and mandibles of ICR strain mice from 14 days gestation stage to 2 days postnatal stage were used to investigate the localization of Maclura pomifera lectin (MPA) during two modes of osteogenesis. During endochondral ossification of the mandibular condyle, MPA was only localized at the peripheral regions of calcified cartilage after the destruction of chondrocyte lacunae. Bone extracellular matrix (ECM) was not reacted with MPA. In intramembranous ossification of mice mandibles, MPA was stained intensively in the early bone ECM. The intensity of the MPA reaction decreased during bone development. In both cases of osteogenesis, chondroclasts and osteoclasts showed the strong affinity to MPA. These results indicated that the time- and position-specific changes within ECM proceeded during osteogenesis and that MPA was the useful probe to detect chondroclasts and osteoclasts.     

Isoform-specific changes in scleral transforming growth factor-beta expression and the regulation of collagen synthesis during myopia progression

The development of high myopia is associated with altered scleral extracellular matrix biochemistry. Previous studies highlight the importance of collagen turnover in this process, yet it is unclear which factors control scleral remodeling. This study used a mammalian model of myopia to investigate the capacity of TGF (transforming growth factor)-beta1, -beta2, and -beta3 to influence scleral remodeling in myopia. RT-PCR confirmed the presence of all mammalian TGF-beta isoforms in scleral tissue and scleral fibroblasts. Myopia was experimentally induced via monocular deprivation of pattern vision, and animals were allocated to two groups depending on the duration of treatment (1 or 5 days). Down-regulation of each isoform was apparent after only 1 day of myopia development (TGF-beta1, -32%; TGF-beta2, -27%; TGF-beta3, -42%). Whereas the decrease in TGF-beta1 and -beta3 expression was relatively constant between the two time points, differential down-regulation of TGF-beta2 was found between days 1 (-27%) and 5 (-50%). In vitro experiments, using primary scleral fibroblasts, demonstrated the capacity of all isoforms to increase collagen production in a dose-dependent manner. Changes in TGF-beta levels, which mimicked those during myopia induction, caused an approximately 15% reduction in collagen synthesis, which is qualitatively similar to those previously reported in vivo. These data represent the first demonstration of TGF-beta3 expression in the sclera and implicate all three TGF-beta isoforms in the control of scleral remodeling during myopia development. In addition, the early alterations in TGF-beta expression levels may reflect a role for these cytokines in mediating the retinoscleral signal that controls myopic eye growth.     

Co-ordination of TGF-beta and FGF signaling pathways in bone organ cultures

Transforming growth factor-beta (TGF-beta) is known to regulate chondrocyte proliferation and hypertrophic differentiation in embryonic bone cultures by a perichondrium dependent mechanism. To begin to determine which factors in the perichondrium mediate the effects of TGF-beta, we studied the effect of Insulin-like Growth Factor-1 (IGF-I) and Fibroblast Growth Factors-2 and -18 (FGF2, FGF18) on metatarsal organ cultures. An increase in chondrocyte proliferation and hypertrophic differentiation was observed after treatment with IGF-I. A similar effect was seen after the perichondrium was stripped from the metatarsals suggesting IGF-I acts directly on the chondrocytes. Treatment with FGF-2 or FGF-18 resulted in a decrease in bone elongation as well as hypertrophic differentiation. Treatment also resulted in a decrease in BrdU incorporation into chondrocytes and an increase in BrdU incorporation in perichondrial cells, similar to what is seen after treatment with TGF-beta1. A similar effect was seen with FGF2 after the perichondrium was stripped suggesting that, unlike TGF-beta, FGF2 acts directly on chondrocytes to regulate proliferation and hypertrophic differentiation. To test the hypothesis that TGF-beta regulates IGF or FGF signaling, activation of the receptors was characterized after treatment with TGF-beta. Activation was measured as the level of tyrosine phosphorylation on the receptor. Treatment with TGF-beta for 24h did not alter the level of IGFR-I tyrosine phosphorylation. In contrast, treatment with TGF-beta resulted in and increase in tyrosine phosphorylation on FGFR3 without alterations in total FGFR3 levels. TGF-beta also stimulated expression of FGF18 mRNA in the cultures and the effects of TGF-beta on metatarsal development were blocked or partially blocked by pretreatment with FGF signaling inhibitors. The results suggest a model in which FGF through FGFR3 mediates some of the effects of TGF-beta on embryonic bone formation.     

Indian hedgehog in the late-phase differentiation in mouse chondrogenic EC cells, ATDC5: upregulation of type X collagen and osteoprotegerin ligand mRNAs

Endochondral bone formation includes a cascade of cellular events such as proliferation, maturation, hypertrophic conversion and calcification of chondrocytes and the cartilage replacement by bone. During these processes, hypertrophic conversion and calcification of chondrocytes (the late-phase differentiation) is a crucial process of chondrogenic differentiation. Indian hedgehog (Ihh), a secreted protein expressed in early hypertrophic chondrocytes, is thought to be involved in regulation of hypertrophic conversion via a feedback loop through the perichondrium. In the present study, we showed by Northern analysis and in situ hybridization that Smoothened (Smo), a key component in hedgehog signal transduction, was expressed in chondrocytes in both adult mice and mouse embryos at 16 days post-coitum in vivo, suggesting that Ihh directly acts on chondrocytes. We previously reported that Ihh, Patched and Smo were all expressed in differentiated ATDC5 cells. Exogenously administered mouse recombinant N-terminal protein of Ihh (mrIhh-N) upregulated the gene expression of type X collagen, a phenotypic marker of hypertrophic chondrocytes, as well as osteoprotegerin ligand (OPGL), a potent stimulator of osteoclastogenesis and osteoclast activity, while it did not modulate the expression of Ihh itself, bone morphogenetic protein (BMP)-4, BMP-6, transforming growth factor (TGF)-beta1 and TGF-beta2 in differentiated ATDC5 cells. Moreover, when added to the osteoclast cultures, mrIhh-N markedly stimulated the formation of resorption pits on dentine slices. Our data support the hypothesis that Ihh stimulated the late-phase chondrogenic differentiation in differentiated ATDC5 cells and upregulated the gene expression of OPGL in these cells.