Hairy/enhancer-of-split related with YRPW motif protein 1 promotes osteosarcoma metastasis via matrix metallopeptidase 9 expression.

BACKGROUND: Activation of the Notch pathway has been reported in various types of cancers. However, the role of the hairy/enhancer-of-split related with YRPW motif protein 1 (HEY1) in osteosarcoma is unknown. We examined the function of HEY1 in osteosarcoma. METHODS: Expression of HEY1 was studied in human osteosarcoma. The effects of HEY1 in osteosarcoma were evaluated in vitro and in a xenograft model. Moreover, we examined the function of matrix metallopeptidase 9 (MMP9) as a downstream effector of HEY1. RESULTS: HEY1 was upregulated in human osteosarcoma. Knockdown of HEY1 inhibited the invasion of osteosarcoma cell lines. In contrast, the forced expression of HEY1 increased the invasion of mesenchymal stem cell. In addition, lung metastases were significantly inhibited by the knockdown of HEY1. We found that MMP9 was a downstream effector of HEY1 that promotes the invasion of osteosarcoma cells. Knockdown of HEY1 decreased the expression of MMP9. Addition of MMP9 rescued the invasion of osteosarcoma cells that had been rendered less invasive by knockdown of HEY1 expression. CONCLUSIONS: Our findings suggested that HEY1 augmented the metastasis of osteosarcoma via upregulation of MMP9 expression. Therefore, inhibition of HEY1 may be a novel therapeutic strategy for preventing osteosarcoma metastasis.

The N domain of Smad7 is essential for specific inhibition of transforming growth factor-beta signaling.

Inhibitory Smads (I-Smads) repress signaling by cytokines of the transforming growth factor-beta (TGF-beta) superfamily. I-Smads have conserved carboxy-terminal Mad homology 2 (MH2) domains, whereas the amino acid sequences of their amino-terminal regions (N domains) are highly divergent from those of other Smads. Of the two different I-Smads in mammals, Smad7 inhibited signaling by both TGF-beta and bone morphogenetic proteins (BMPs), whereas Smad6 was less effective in inhibiting TGF-beta signaling. Analyses using deletion mutants and chimeras of Smad6 and Smad7 revealed that the MH2 domains were responsible for the inhibition of both TGF-beta and BMP signaling by I-Smads, but the isolated MH2 domains of Smad6 and Smad7 were less potent than the full-length Smad7 in inhibiting TGF-beta signaling. The N domains of I-Smads determined the subcellular localization of these molecules. Chimeras containing the N domain of Smad7 interacted with the TGF-beta type I receptor (TbetaR-I) more efficiently, and were more potent in repressing TGF-beta signaling, than those containing the N domain of Smad6. The isolated N domain of Smad7 physically interacted with the MH2 domain of Smad7, and enhanced the inhibitory activity of the latter through facilitating interaction with TGF-beta receptors. The N domain of Smad7 thus plays an important role in the specific inhibition of TGF-beta signaling.

Characterization of a bone morphogenetic protein-responsive Smad-binding element.

Bone morphogenetic proteins (BMPs) are pleiotropic growth and differentiation factors belonging to the transforming growth factor-beta (TGF-beta) superfamily. Signals of the TGF-beta-like ligands are propagated to the nucleus through specific interaction of transmembrane serine/threonine kinase receptors and Smad proteins. GCCGnCGC has been suggested as a consensus binding sequence for Drosophila Mad regulated by a BMP-like ligand, Decapentaplegic. Smad1 is one of the mammalian Smads activated by BMPs. Here we show that Smad1 binds to this motif upon BMP stimulation in the presence of the common Smad, Smad4. The binding affinity is likely to be relatively low, because Smad1 binds to three copies of the motif weakly, but more repeats of the motif significantly enhance the binding. Heterologous reporter genes (GCCG-Lux) with multiple repeats of the motif respond to BMP stimulation but not to TGF-beta or activin. Mutational analyses reveal several bases critical for the responsiveness. A natural BMP-responsive reporter, pTlx-Lux, is activated by BMP receptors in P19 cells but not in mink lung cells. In contrast, GCCG-Lux responds to BMP stimulation in both cells, suggesting that it is a universal reporter that directly detects Smad phosphorylation by BMP receptors.

Interplay of signal mediators of decapentaplegic (Dpp): molecular characterization of mothers against dpp, Medea, and daughters against dpp.

Decapentaplegic (Dpp) plays an essential role in Drosophila development, and analyses of the Dpp signaling pathway have contributed greatly to understanding of the actions of the TGF-beta superfamily. Intracellular signaling of the TGF-beta superfamily is mediated by Smad proteins, which are now grouped into three classes. Two Smads have been identified in Drosophila. Mothers against dpp (Mad) is a pathway-specific Smad, whereas Daughters against dpp (Dad) is an inhibitory Smad genetically shown to antagonize Dpp signaling. Here we report the identification of a common mediator Smad in Drosophila, which is closely related to human Smad4. Mad forms a heteromeric complex with Drosophila Smad4 (Medea) upon phosphorylation by Thick veins (Tkv), a type I receptor for Dpp. Dad stably associates with Tkv and thereby inhibits Tkv-induced Mad phosphorylation. Dad also blocks hetero-oligomerization and nuclear translocation of Mad. We also show that Mad exists as a monomer in the absence of Tkv stimulation. Tkv induces homo-oligomerization of Mad, and Dad inhibits this step. Finally, we propose a model for Dpp signaling by Drosophila Smad proteins.

A Kirschner wire as a transverse-axis guide to improve acetabular cup positioning.

PURPOSE: To compare cup-positioning accuracy in total hip arthroplasty (THA) with or without use of a Kirschner wire as a transverse-axis guide for pelvic alignment. METHODS: Records of 18 men and 73 women (mean age, 60 years) who underwent primary THA with (n=49) or without (n=42) use of a Kirschner wire as a transverse-axis guide for pelvic alignment were reviewed. A 2.4-mm Kirschner wire as a transversea-xis guide was inserted to the anterior superior iliac spine and was parallel to a line linking the left and right anterior superior iliac spine. The safe zone for cup positioning was defined as 30º to 50° abduction and 10º to 30º anteversion. Of the 5 operative surgeons, 2 were classified as experienced (total surgical volume >300) and 3 as inexperienced (total surgical volume of <50). The proportion of patients with the cup in the safe zone was compared in patients with or without use of the transverse-axis guide and in experienced and inexperienced surgeons. RESULTS: For inexperienced surgeons, the use of the transverse-axis guide significantly improved the proportion of patients with the cup in the safe zone from 90% to 100% for abduction, from 50% to 82.4% for anteversion, and from 40% to 82.4% for both. Patients with the cup inside or outside the safe zone were comparable in terms of body height, weight, BMI, subcutaneous fat thickness, incision length, and acetabular cup size. CONCLUSION: The use of the transverse-axis guide improved the accuracy of cup positioning by inexperienced surgeons.

Functional impact of human collagen alpha2(XI) gene polymorphism in pathogenesis of ossification of the posterior longitudinal ligament of the spine.

Ossification of the posterior longitudinal ligament (OPLL) of the spine is the leading cause of myelopathy in Japan. In earlier studies, we provided genetic linkage and allelic association evidence of distinct differences in the human collagen alpha2(XI) gene (COL11A2) that might constitute inherited predisposition to OPLL. In the present study, a strong allelic association with non-OPLL (p = 0.0003) was observed with an intron 6 polymorphism [intron 6 (-4A)], in which the intron 6 (-4A) allele is more frequently observed in non-OPLL subjects than in OPLL patients. In addition, a newly identified polymorphism in exon 6 [exon 6 (+28A)] was in linkage disequilibrium with the intron 6 (-4A). The functional impact of the polymorphisms was analyzed by comparing the differences in messenger RNA (mRNA) splicing by reverse-transcription polymerase chain reaction (RT-PCR) analysis in cultured cells from the interspinous ligament and an in vitro exon trapping study. The intron 6 (-4A) allele resulted in skipping exon 6 and retaining exon 7, while the exon 6 (+28A) allele was not associated with alteration in mRNA splicing. Similar mRNA species were observed in undifferentiated osteoblast (Ob) cells and in cells from posterior longitudinal ligament of non-OPLL subjects. The region containing exons 6-8 is an acidic subdomain presumably exposed to the surface that could interact with molecules of the extracellular matrix. Accordingly, retaining exon 7 together with removal of exon 6 observed in intron 6 (-4A) could play a protective role in the ectopic ossification process because the same pattern was observed in undifferentiated Ob cells and nonossified posterior longitudinal ligament cells.

Enhanced expression of type I receptors for bone morphogenetic proteins during bone formation.

Type I receptors for bone morphogenetic proteins (BMPs), i.e., BMPR-IA and BMPR-IB, are transmembrane serine/threonine kinases, that bind osteogenic protein-1 (OP-1, also termed BMP-7) and BMP-4. Using antibodies specific to BMPR-IA and -IB, we have studied the expression of BMP type I receptors in the bone formation process during embryonic development and fracture healing. In the mouse embryo, both BMPR-IA and -IB were expressed in condensing mesenchymal cells at 13.5 days post coitum (p.c.). At 15.5 days p.c., expression of BMPR-IB, but not of BMPR-IA, was observed in the cells in perichondrium of developing cartilage. At 17.5 and 19.5 days p.c., expression of both receptors was observed in chondrocytes and in osteoblasts. In normal rat adult bone, expression of BMPR-IA, but not of BMPR-IB, was observed in osteoblasts in the periosteum. Three days after the femoral fracture, expression of BMPR-IA and -IB was up-regulated in cells at the proliferating osteogenic layer of the periosteum. On day 7, both receptors were found in fibroblast-like spindle cells and chondrocytes in the endochondral ossification sites, and osteoblasts in the newly formed trabecular bone. Expression of BMPR-IA was higher than that BMPR-IB in osteogenic layer on day 3 and in osteoblasts in the trabecular bone on day 7. On day 14, expression of BMP type I receptors was observed at similar sites, albeit with lower expression levels than were observed on day 7. The present data suggest that expression of BMP type I receptors is up-regulated during bone formation, and that they may play important roles in bone morphogenesis.

Early effects of electrical stimulation on osteogenesis.

The mechanism by which electrical stimulation causes osteogenesis is unknown. Bone marrow of the rabbit was stimulated by direct electrical current or electromagnetic fields to clarify the mechanism of osteogenesis by electrical stimulation. A total of 105 rabbits were separated into five groups: a direct current stimulation by Kirshner wire insertion group; a pulsed electromagnetic fields stimulation (PEMF) group; a PEMF with Kirshner wire insertion group; a Kirshner wire insertion group; and an intramedullary drilling control group. Measurement of intramedullary new bone formation and determination of alkaline phosphatase activity within the bone marrow were performed. Argylophilic nuclear organizer region (AgNOR) staining was done to evaluate the change in proliferative activity of the osteoblasts during electrical stimulation. In the direct current stimulation group and the PEMF accompanied by the insertion of the Kirshner wire group, alkaline phosphatase activity in the bone marrow and AgNOR staining increased at 7 days after surgery. At 14 days after surgery, alkaline phosphatase activity and proliferative activity of osteoblast were significantly higher in these two groups than in the other groups (PEMF group, Kirshner wire insertion alone group, intramedullary drilling group). Intramedullary new bone formation was most active in the direct current stimulation group. Electromagnetic stimulation of the inserted Kirshner wire also promoted bone formation significantly. The Kirshner wire insertion alone group and the intramedullary drilling group showed bone formation, but it was significantly less. Electromagnetic stimulation without the insertion of the Kirshner wire showed little bone formation. These findings revealed that the degree of osteogenesis induced by electrical stimulation is influenced by the tissue environment, and that osteogenesis is promoted markedly when electrical stimulation is provided in the environment of inflammation and reactive cells.

Distinct and overlapping patterns of localization of bone morphogenetic protein (BMP) family members and a BMP type II receptor during fracture healing in rats.

Bone morphogenetic proteins (BMPs) and their receptors (BMPRs) are thought to play an important role in bone morphogenesis. The purpose of this study was to determine the locations of BMP-2/-4, osteogenic protein-1 (OP-1, also termed BMP-7), and BMP type II receptor (BMPR-II) during rat fracture healing by immunostaining, and thereby elucidate the possible roles of the BMPs and BMPR-II in intramembranous ossification and endochondral ossification. In the early stage of fracture repair, the expression of BMP-2/-4 and OP-1 was strongly induced in the thickened periosteum near the fracture ends, and coincided with an enhanced expression of BMPR-II. On day 7 after fracture, staining for BMP-2/-4 and OP-1 immunostaining was increased in various types of chondrocytes, and was strong in fibroblast-like spindle cells and proliferating chondrocytes in endochondral bone. On day 14 after fracture, staining with OP-1 antibody disappeared in proliferating and mature chondrocytes, while BMP-2/-4 staining continued in various types of chondrocytes until the late stage. In the newly formed trabecular bone, BMP-2/-4 and OP-1 were present at various levels. BMPR-II was actively expressed in both intramembranous ossification and endochondral ossification. Additionally, immunostaining for BMP-2/-4 and OP-1 was observed in multinucleated osteoclast-like cells on the newly formed trabecular bone, along with BMPR-II. In reference to our previous study of BMP type I receptors (BMPR-IA and BMPR-IB), BMPR-II was found to be co-localized with BMPR-IA and BMPR-IB. BMP-2/-4 and OP-1 antibodies exhibited distinct and overlapping immunostaining patterns during fracture repair. OP-1 may act predominantly in the initial phase of endochondral ossification, while BMP-2/-4 acts throughout this process. Thus, these findings suggested that BMPs acting through their BMP receptors may play major roles in modulating the sequential events leading to bone formation.

Expression and localization of bone morphogenetic proteins (BMPs) and BMP receptors in ossification of the ligamentum flavum.

To clarify the pathogenesis of ossification of the ligamentum flavum (OLF), we examined the expression and localization of bone morphogenetic proteins (BMPs) and their receptors (BMPRs) in the ligamentum flavum of the patients with OLF by immunohistochemical staining and compared them with staining patterns in control patients. The BMPRs appeared extensively in mature and immature chondrocytes around the calcified zone and in spindle-shaped cells and round cells in the remote part from ossified foci in examined tissue of OLF. The ligands for BMPRs, BMP-2/-4 and osteogenic protein-1 (OP-1)/BMP-7, colocalized in OLF patients. In the control cases, expression of BMPs and BMPRs was observed around the calcified zone at the insertion of the ligamentum flavum to the bone, and limited expression was found in the smaller range. Thus, the expression profile of BMPs and BMPRs in OLF patients was entirely different from the control patients, suggesting that BMPs may be involved in promoting endochondral ossification at ectopic ossification sites in OLF, and that ossification activity is continuous in these patients.

Intracellular signaling of the TGF-beta superfamily by Smad proteins.

TGF-beta is a potent inhibitor of cell growth, and accumulating evidence suggests that perturbation of the TGF-beta signaling pathway leads to tumorigenesis. Smads are recently identified proteins that mediate intracellular signaling of the TGF-beta superfamily. Smads 2 and 3 are phosphorylated by the TGF-beta type I receptor. Smad4 was originally identified as a candidate tumor suppressor gene in pancreatic cancers. Smads 2 and 3 form complexes with Smad4 upon TGF-beta stimulation. The heteromeric Smad complexes translocate into the nucleus, where they activate expression of target genes. Our recent study demonstrated that Smads exist as monomers in the absence of TGF-beta. Smads 2 and 3 form homo- as well as hetero-oligomers with Smad4 upon ligand stimulation. Both homo-oligomers and hetero-oligomers directly bind to DNA, suggesting that the signaling pathway of Smads may be multiplex. Smads 2 and 3 associate with transcriptional coactivators such as p300 in a ligand-dependent manner, p300 enhances transactivation by TGF-beta, suggesting that coactivators link Smads to the basal transcriptional machinery. A missense mutation of Smad2 identified in colorectal and lung cancers was introduced to Smad3. The mutant, Smad3(DE), blocked the activation of wild-type Smad2 and Smad3. Thus, the missense mutation not only disrupts the function of the wild-type Smad but also creates a dominant-negative Smad, which could actively contribute to oncogenesis.

Immunohistochemical detection of activin A, follistatin, and activin receptors during fracture healing in the rat.

Activins are multifunctional proteins that belong to the transforming growth factor-beta superfamily and are thought to play an important role in modulating the formation of bone. Activins exert their cellular effects by way of activin type-I and type-II serine/threonine kinase receptors. Follistatin is an activin-binding protein that can suppress the biological effects of activins. In this study, the immunohistochemical expression of activin A, follistatin, and activin receptors was studied during fracture healing in the rat. Activin A was weakly detected in the periosteum near the fracture ends at an early stage but was absent in the chondrocytes around the fracture gap, where endochondral ossification took place. An antibody to follistatin stained osteogenic cells in the periosteum near the fracture ends; moderate and strong staining were observed in proliferating, mature, and hypertrophied chondrocytes at the sites of endochondral ossification. Levels of activin A and follistatin were high near the osteoblasts on the surface of the newly formed trabecular bone. In addition, an intense localization of activin A was noted where multinucleated osteoclast-like cells were present. This study suggests that the activin-follistatin system may contribute to cellular events related to the formation and remodeling of bone during fracture healing. Activin type-I and type-II receptors were co-expressed in intramembranous and endochondral ossification sites. The expression of activin type-I, type-II, and type-IIB receptors in the absence of activin A in the endochondral ossification suggests that other isoforms of activins may signal by way of these receptors.

Orthotopic ossification of the spinal ligaments of Zucker fatty rats: a possible animal model for ossification of the human posterior longitudinal ligament.

Ossification of the posterior longitudinal ligament is a human genetic disease in which pathological ectopic ossification of the spinal ligaments develops. This leads to myelopathy or radiculopathy due to compression of the spinal cord. In this study, we investigated the histological features of orthotopic ossification of the spinal ligaments of senile Zucker fatty rats. A remarkably high incidence of orthotopic ossification was observed mainly in the thoracic spinal ligaments as compared with controls. The histopathological findings were similar to those for ossification of the human posterior longitudinal ligament. Bone morphogenetic proteins and activins, which exert their effects by way of specific type-I and type-II serine/threonine kinase receptors, play important roles in the formation of bone and cartilage. In the spinal ligaments of Zucker fatty rats, bone morphogenetic protein receptors and activin receptors were immunohistochemically detected around the ossified foci in a manner similar to that previously shown for the ossified tissue from patients who had ossification of the posterior longitudinal ligament. Thus, bone morphogenetic proteins and activin receptors might play important roles in orthotopic ossification of the spinal ligaments of Zucker fatty rats as well as in ossification of the posterior longitudinal ligament of humans. In addition, bone morphogenetic protein-receptor-IA was expressed in the nonossified ligament, suggesting that the spinal ligaments, of the rats may have a predisposition to orthotopic ossification. In the controls, no expression of bone morphogenetic protein receptors or of activin receptors was observed. In conclusion, there is a great degree of similarity between orthotopic ossification of the spinal ligaments of Zucker fatty rats and ossification of the posterior longitudinal ligament of humans. Thus, the rats provide a useful animal model for the study of ossification of the human posterior longitudinal ligament.

Apoptosis following spinal cord injury in rats and preventative effect of N-methyl-D-aspartate receptor antagonist.

OBJECT: The aims of this study were to clarify the histological and histochemical changes associated with cell death in the spinal cord after acute traumatic injury and to examine the role of excitatory amino acid release mediated by N-methyl-D-aspartate (NMDA) receptors. METHODS: Following laminectomy, the spinal cord in 70 rats was injured at the T-9 level by applying extradural static weight-compression, in which a cylindrical compressor was used to induce complete and irreversible transverse spinal cord injury (SCI) with paralysis of the lower extremities. The injured rats were killed between 30 minutes and 14 days after injury, and the injured cord was removed en bloc. Rats that received NMDA receptor antagonist (MK-801) were killed at the same time points as those that received saline. The specimens were stained with hematoxylin and eosin, Nissl, and Klüver-Barrera Luxol fast blue and subjected to in situ nick-end labeling, a specific in situ method used to allow visualization of apoptosis. Thirty minutes post-SCI, a large hematoma was observed at the compressed segment. Six hours after injury, large numbers of dead cells that were not stained by in situ nick-end labeling were observed. Between 12 hours and 14 days postinjury, nuclei stained by using the in situ nick-end labeling technique were observed not only at the injury site but also in adjoining segments that had not undergone mechanical compression, suggesting that the delayed cell death was due to apoptosis. The number of cells stained by in situ nick-end labeling was maximum at 3 days postinjury. The results of electron microscopic examination were also consistent with apoptosis. In the MK-801-treated rats, the number of cells stained by in situ nick-end labeling was smaller than in nontreated rats at both 24 hours and 7 days after injury. CONCLUSIONS: These findings suggest that NMDA-receptor activation promotes delayed neuronal and glial cell death due to apoptosis.

Immunolocalization of transforming growth factor-beta s and type I and type II receptors in rat articular cartilage.

The activities of TGF-beta s and their receptors (T beta R) in the articular cartilage have not been fully elucidated as yet. Temporal and spatial expression of TGF-beta s and their receptors were examined immunohistochemically in the articular cartilage of growing rats to clarify their role in chondrogenesis. TGF-beta 1, -beta 2 and -beta 3 were strongly expressed from 6 to 50 weeks in the superficial, transitional, and least mature zones. The expression of TGF-beta s in hypertrophic chondrocyte was weak except TGF-beta 1. T beta R-I and T beta R-II were co-expressed with the ligand in the superficial, transitional, and least mature zones throughout the growth phase. This study revealed that TGF-beta s in the articular cartilage plays a major role in the modulation of endochondral ossification ensuring the growth and maintenance of the chondrocyte lineage in articular cartilage.

Immunohistochemical localization of bone morphogenetic proteins and the receptors in epiphyseal growth plate.

The expression of bone morphogenetic proteins (BMPs) and BMP receptors (BMPRs) in the epiphyseal growth plate has not been clarified. In this study, we studied immunohistochemically the spatial and temporal localization of BMP-2/4, osteogenic protein-1 (OP-1, or BMP-7), and BMP receptors (BMPR-IA, BMPR-IB, and BMPR-II) in the epiphyseal plate of growing rats. The proximal parts of tibia in growing rats were observed. At 12 weeks after birth, BMP-2/4 and OP-1 were expressed markedly in proliferating and maturing chondrocytes. BMPR-IA, IB and II were clearly co-expressed in proliferating and maturing chondrocytes, and the expression was decreased in hypertrophic chondrocytes. At 24 weeks, the expression of BMP-2/4 and OP-1 was decreased, but BMPRs were still well-expressed in proliferating chondrocytes. The temporal and spatial expression of BMP and BMPR suggests that BMP and BMP receptors play roles in the multistep cascade of enchondral ossification in the epiphyseal growth plate.

Effect of transforming growth factor-beta on fibroblasts in ossification of the posterior longitudinal ligament.

We studied the immunohistochemical localization of TGF-beta in the ligament tissue of patients with ossification of the posterior longitudinal ligament (OPLL), and the effects of TGF-beta on cultured cells derived from the spinal ligament of patients with OPLL. ALP activity in the cultured cells was also examined. TGF-beta was present in the ossified matrix and in the chondrocytes in cartilaginous areas adjacent to OPLL. ALP activity was high in the cultured cells of OPLL patients. Exogenous TGF-beta inhibited proliferation in the OPLL cells but promoted proliferation in control cells. These results suggest that the spinal ligaments of OPLL patients have an osteogenetic predisposition and that TFG-beta may play a role in the ossification.

Expression of decorin mRNA in the skin of patients with ossification of the posterior longitudinal ligament.

Ossification of the posterior longitudinal ligament (OPLL) is an intractable disease developing into severe myelopathy and often accompanied by ossification of several other spinal ligaments and articular ligaments. Therefore, it is highly probable that predisposition to systemic ossification underlies the onset of this disease. Terayama et al(1981) reported that the nuchal skin of OPLL patients tended to be tougher than that of healthy individuals. Decorin is a component of the extracellular matrix which antagonistically regulates the action of Transforming growth factor-beta (TGF-beta). Imamura et al(1995) demonstrated immunohistochemically that decorin increased in the epidermis of the nuchal skin of OPLL patients. This suggests an abnormal expression of the extracellular matrix in the skin of OPLL patients. In the present study, in situ hybridization with non-radioactive synthetic oligodeoxynucleotide probes revealed an enhanced expression of decorin mRNA in the epidermal keratinocytes of OPLL patients. The increased expression of decorin mRNA in the epidermis of OPLL patients may be interpreted as reflecting abnormalities of the matrix associated with ossification of the ligaments. Studies on the role of decorin in ossification will contribute to clarification of the pathophysiology and pathogenesis of OPLL.

Expression and distribution of transforming growth factor-beta and decorin during fracture healing.

To study interactions between TGF-beta and decorin, we experimentally induced femoral fracture in rats and immunohistologically examined changes in the distribution of TGF-beta and decorin in the femur 3, 7 and 14 days after fracture. In the areas where endochondral ossification occurred, decorin was detected during differentiation of mesenchymal cells into proliferating chondrocytes, while no TGB-beta was observed during same period. Once chondrocytes had hypertrophied, decorin was no longer observed, while the matrix around the hypertrophic chondrocytes was positively stained for TGB-beta. The disappearance of decorin, almost simultaneously accompanied by the appearance of the active type TGB-beta, suggests that decorin regulates the actions of TGB-beta.

The spatial and temporal immunolocalization of TGF-beta 1 and bone morphogenetic protein-2/-4 in phallic bone formation in inbred Sprague Dawley male rats.

In male rats and mice, the membranous bone is rapidly formed in the proximal segment (p-segment) of the os penis, and then endochondral ossification proceeds in the proximal half. The spatial and temporal patterns of expression of transforming growth factor-beta 1 (TGF-beta 1) and bone morphogentic protein-2/-4 (BMP-2/-4) were examined immunohistochemically until 14 days after birth and compared. BMP-2/-4 expression was weak in the mesenchymal condensation just at birth, and up-regulated in the membranous bone and cartilage 3 days after birth. BMP-2/-4 was strongly expressed in proliferating and mature chondrocytes 7 days after birth. At birth, TGF-beta 1 expression was diffuse and very strong in mesenchymal condensation. Thereafter, TGF-beta 1 expression was continuously strong in membranous bone and cartilage. Seven days after birth, TGF-beta 1 was actively expressed in proliferating chondrocytes. TGF-beta 1 and BMP-2/-4 seemed to act co-operatively. In particular, TGF-beta 1 appeared to strongly regulate immature stage and BMP-2/-4 may regulate the mature stage.