Biomedical subjects

Takenobu Katagiri

Publications and source records attributed to Takenobu Katagiri.

16 recordsLinked to original sources

PubMed2006

FGF23 induces expression of two isoforms of NAB2, which are corepressors of Egr-1.

Fibroblast growth factor 23 (FGF23) is a key humoral factor in phosphate homeostasis and skeletogenesis, though the nature of its intracellular signaling is still unclear. Recently, Egr-1, a zinc-finger transcription factor, was identified as an immediate early response gene of FGF23 in the kidney. We report here, that FGF23 induces not only Egr-1 but also two isoforms of NAB2, which are specific co-repressors of Egr-1. Both isoforms of NAB2 induced by FGF23 were localized in the nucleus and suppressed the transcriptional activity of Egr-1. A negative feedback loop established by Egr-1 and NAB2 may thus be involved in mediating the physiological effects of FGF23.

Animals↗

PubMed2006

Heparin potentiates the in vivo ectopic bone formation induced by bone morphogenetic protein-2.

Although bone morphogenetic proteins (BMPs) are clinically useful for bone regeneration, large amounts are required to induce new bone formation in monkeys and humans. We found recently that heparin stimulates BMP activity in vitro (Takada, T., Katagiri, T., Ifuku, M., Morimura, N., Kobayashi, M., Hasegawa, K., Ogamo, A., and Kamijo, R. (2003) J. Biol. Chem. 278, 43229-43235). In the present study, we examined whether heparin enhances bone formation induced by BMPs in vivo and attempted to determine the molecular mechanism by which heparin stimulates BMP activity using C2C12 myoblasts. Heparin enhanced BMP-2-induced gene expression and Smad1/5/8 phosphorylation at 24 h and thereafter, although not within 12 h. Heparitinase treatment did not affect the response of cells to BMP-2. In the presence of heparin, degradation of BMP-2 was blocked, and the half-life of BMP-2 in the culture medium was prolonged by nearly 20-fold. Although noggin mRNA was induced by BMP-2 within 1 h regardless of the presence of heparin, noggin failed to inhibit BMP-2 activity in the presence of heparin. Furthermore, simultaneous administration of BMP-2 and heparin in vivo dose-dependently induced larger amounts of mineralized bone tissue compared with BMP-2 alone. These findings clearly indicate that heparin enhances BMP-induced osteoblast differentiation not only in vitro but also in vivo. This study indicates that heparin enhances BMP-induced osteoblast differentiation in vitro and in vivo by protecting BMPs from degradation and inhibition by BMP antagonists.

Alkaline Phosphatase↗

PubMed2006

Purification and identification of a BMP-like factor from bovine serum.

Myogenic differentiation is suppressed in vitro by unknown factors present in fetal bovine serum (FBS). We found that specific inhibitors of bone morphogenetic proteins (BMPs) stimulated myogenic differentiation even in the presence of 20% FBS, which in turn activated specific BMP signaling. Moreover, these specific BMP inhibitors blocked maturation of osteoblastic cells induced by FBS, indicating that BMP-like factor(s) in serum regulate both myogenic and osteoblastic differentiation. The factor identified had an apparent molecular weight (Mw) of over 100kDa on a Superdex 200 column for molecular sieving HPLC, but an apparent Mw of 33kDa on SDS-PAGE under non-reducing conditions. Analysis of a purified preparation from FBS (5L) by liquid chromatography-tandem mass spectrometry revealed the presence of an amino acid sequence conserved between mature human and murine BMP-4. This is the first study to show that BMP-4 is present in FBS as a large complex.

Amino Acid Sequence↗

PubMed2006

Establishment of primary cultures for mouse ameloblasts as a model of their lifetime.

To understand how the properties of ameloblasts are spatiotemporally regulated during amelogenesis, two primary cultures of ameloblasts in different stages of differentiation were established from mouse enamel epithelium. Mouse primary ameloblasts (MPAs) prepared from immature enamel epithelium (MPA-I) could proliferate, whereas those from mature enamel epithelium (MPA-M) could not. MPA-M but not MPA-I caused apoptosis during culture. The mRNA expression of amelogenin, a marker of immature ameloblasts, was down-regulated, and that of enamel matrix serine proteiase-1, a marker of mature ameloblasts, was induced in MPA-I during culture. Using green fluorescence protein as a reporter, a visualized reporter system was established to analyze the promoter activity of the amelogenin gene. The region between -1102bp and -261bp was required for the reporter expression in MPA-I. These results suggest that MPAs are valuable in vitro models for investigation of ameloblast biology, and that the visualized system is useful for promoter analysis in MPAs.

Ameloblasts↗

PubMed2006

Establishment of a novel method for enriching osteoblast progenitors from adipose tissues using a difference in cell adhesive properties.

In the clinical field, cell-based therapies are used to treat bone defects. Adipose tissues contain many osteoblast progenitors, among other cell types. We separated mouse adipose tissue-derived stromal cells (ATSCs) according to their cell adhesive properties. Cells in a fraction adherent to the culture dishes 0.5h after inoculation (AF-0.5) had a potent ability to differentiate into both osteoblasts and adipocytes in vitro. Their differentiation pathways depended on the culture conditions. In these cells, the expression of marker genes for osteoblast differentiation was induced in osteogenic medium. Moreover, the AF-0.5 cells, which were induced to differentiate into osteoblasts in vitro, formed abundant bone tissues in vivo. These results suggest that the AF-0.5 cells have been enriched with bi-potential progenitor cells destined for either osteoblasts or adipocytes. This simple and efficient method for preparing osteoblast progenitor cells from ATSCs may be utilized for bone defect treatment clinically.

Adipose Tissue↗

PubMed2006

Bone formation enhanced by implanted octacalcium phosphate involving conversion into Ca-deficient hydroxyapatite.

The present study was designed to investigate whether hydrolysis of synthetic octacalcium phosphate (OCP) into hydroxyapatite affects bone formation. Mouse bone marrow stromal ST-2 cells and primary calvarial osteoblastic cells were cultured on the dishes pre-coated with OCP or its hydrolyzed Ca-deficient hydroxyapatite (OCP hydrolyzate; HL). The capacity of proliferation and differentiation was determined up to day 20. Granules of OCP and HL were implanted into critical-size rat calvaria defects for 4 and 12 weeks, and then bone formation was measured by histomorphometry. Structural changes of incubated and implanted OCP were determined by X-ray diffraction (XRD) and Fourier transform infrared spectroscopy (FTIR). The proliferation of both ST-2 and primary osteoblasts cultured on OCP or HL was initially inhibited, whereas their differentiation to osteoblasts was promoted at last. Implantation of OCP in bone defect more significantly enhanced bone formation than that of HL until 12 weeks. OCP tended to convert to apatite in vitro and in vivo. The conversion of the implanted OCP was ascertained to advance gradually with implantation periods. Taken together, these results suggest that OCP supports appositional bone formation and OCP-apatite conversion may be involved in this stimulatory capacity of OCP.

Animals↗

PubMed2006

Surface chemistry and biological responses to synthetic octacalcium phosphate.

Octacalcium phosphate (OCP) has been suggested as a precursor of biological apatite in bone, dentin, and cementum because its existence explains the nonstoichiometry of apatite crystals in their compositions. Synthetic inorganic calcium phosphate compounds have been used clinically to fill bone defects, and sintered hydroxyapatite (HA) and beta-tricalcium phosphate (beta-TCP), bone substitute materials, are known to be osteoconductive, with beta-TCP also being bioresorbable. Nonsintered synthetic OCP has been shown to enhance bone regeneration accompanied by conversion into hydrolyzed apatitic products in situ and biodegradation. The surfaces of the OCP implant and the converted apatite seem to be continuously exposed to biological constituents, such as extracellular matrices, inorganic biominerals, and cellular components. This article reviews the surface reaction of OCP implants and the biological responses, such as experimentally stimulated bone formation on synthetic OCP, the mechanism of OCP hydrolysis into apatite, and the adsorption of biomolecules onto OCP and the converted apatite, of particular interest in reactive bone induction with synthetic OCP implants.

Adsorption↗

PubMed2006

[Transcriptional regulation of BMP responsive genes].

Bone morphogenetic proteins (BMPs) bound to specific receptors expressed on cell membrane of target cells, then activate their specific intracellular signaling pathways. These signaling activate transcription of BMP-specific target genes. Recently, various types of genes are identified as the BMP target genes. Some critical signaling molecules are found though studies on early responsive genes of BMPs.

Animals↗

PubMed2005

Cross-talk between Wnt and bone morphogenetic protein 2 (BMP-2) signaling in differentiation pathway of C2C12 myoblasts.

Loss of function of the Wnt co-receptor, lipoprotein receptor-related protein 5, decreases bone formation, and a point mutation in this gene results in high bone mass, indicating the importance of this signaling pathway in bone formation. However, the exact mechanism is currently unknown. We examined a potential role for Wnt signaling and functional cross-talk of bone morphogenetic protein 2 (BMP-2) in osteoblast differentiation. To assess the contribution of Wnt, we generated C2C12 cells over-expressing Wnt3a or Wnt5a and treated these with BMP-2. We showed that expression of matrix extracellular phosphoglycoprotein was induced by BMP-2 in Wnt3a over-expressing C2C12 cells but not in Wnt5a over-expressing C2C12 cells. Over-expression of Wnt3a blocked BMP-2-induced inhibition of myotube formation in C2C12 cells when switched to low mitogen medium. In these cultures, expression of inhibitor of DNA binding/differentiation (Id) 1, a helix-loop-helix protein induced by BMP-2, decreased in stable Wnt3a- but not in Wnt5a-expressing cells. This suppression is mediated by a GC-rich region of the BMP-2-responsive element of the Id1 gene promoter, and interaction between Smad1/4 and beta-catenin is crucial for Wnt-mediated suppression of the BMP-2 response in C2C12 cells. Over-expression of the inhibitor of canonical Wnt signaling, Dickkopf, inhibits this suppression. In contrast, BMP-2 or Smad1/4 up-regulated Wnt3a or activated beta-catenin-induced lymphoid-enhancing factor 1/T cell factor-dependent transcriptional activity. These findings identify functional cross-talk of Id1 expression between Wnt and BMP signaling and demonstrate a novel mechanism for Wnt regulation of the BMP-2 response, linking Id1 expression to Wnt/beta-catenin signaling.

Animals↗

PubMed2005

Involvement of endogenous bone morphogenetic protein (BMP) 2 and BMP6 in bone formation.

Although accumulated evidence has shown the bone anabolic effects of bone morphogenetic proteins (BMPs) that were exogenously applied in vitro and in vivo, the roles of endogenous BMPs during bone formation remain to be clarified. This study initially investigated expression patterns of BMPs in the mouse long bone and found that BMP2 and BMP6 were the main subtypes expressed in hypertrophic chondrocytes that induce endochondral bone formation. We then examined the involvement of the combination of these BMPs in bone formation in vivo by generating the compound-deficient mice (Bmp2+/-;Bmp6-/-). Under physiological conditions, these mice exhibited moderate growth retardation compared with the wild-type (WT) littermates during the observation period up to 52 weeks of age. Both the fetal and adult compound-deficient mice showed a reduction in the trabecular bone volume with suppressed bone formation, but normal bone resorption, whereas the single deficient mice (Bmp2+/- or Bmp6-/-) did not. When a fracture was created at the femoral midshaft and the bone healing was analyzed, the endochondral bone formation, but not intramembranous bone formation, was impaired by the compound deficiency. In the cultures of bone marrow cells, however, there was no difference in osteogenic differentiation between WT and compound-deficient cells in the presence or absence of the exogenous BMP2. We thus concluded that endogenous BMP2 and BMP6 cooperatively play pivotal roles in bone formation under both physiological and pathological conditions.

Animals↗

PubMed2005

[Molecular mechanisms of bone formation and bone-forming factors].

Osteoblast is a specialized cell that forms bone tissue. The process of bone formation by osteoblasts is divided into several steps such as differentiation of cells, secretion of matrix proteins, mineralization of osteoid, and bone coupling. Although each step supposed to be controlled by various factors, the regulatory mechanisms of bone formation is unclear. Recently, a central control system of bone formation was identified. These findings will be useful to develop novel bone-forming drugs.

Bone Morphogenetic Proteins↗

PubMed2005

[BMP antagonists].

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Adaptor Proteins, Signal Transducing↗

PubMed2003

Sulfated polysaccharides enhance the biological activities of bone morphogenetic proteins.

Bone morphogenetic proteins (BMPs), which have been shown to be heparin-binding proteins, induce osteoblast differentiation in mesenchymal cells. In the present study, we examined the effects of heparin on the BMP activities in C2C12 myoblasts. Heparin dose dependently enhanced the osteoblast differentiation induced by not only homodimers of BMP-2 or BMP-4 but also heterodimers of BMP-2/6 or BMP-2/7. However, the osteoblast differentiation induced by the constitutively active BMPR-IA, a functional BMP type I receptor, was not affected by heparin. Heparan sulfate and dextran sulfate also enhanced the BMP-2 activity, although the chemically desulfated heparin-derivatives have lost this stimulatory capacity. Heparin dose-dependently suppressed the accumulation of BMP-2 from the culture media into the cell layer or BMPR-IA, and retained a large amount of BMP-2 in the culture media. The biological activity of BMP-2, which was evaluated using a BMP-responsive reporter gene expression, was prolonged in the presence of heparin. Taken together, these results suggest that sulfated polysaccharides enhance the biological activity of both homodimers and heterodimers of BMPs by continuously serving the ligands to their signaling receptors expressed on cell membranes.

Blotting, Western↗

PubMed2002

Transcriptional regulation of Smad2 is required for enhancement of TGFbeta/Smad signaling by TGFbeta inducible early gene.

TGFbeta inducible early gene (TIEG) is a novel Krüppel-like transcriptional repressor that was recently shown to increase the activity of the TGFbeta/Smad signal transduction pathway by relieving negative feedback through repression of the inhibitory Smad7. Interestingly, while Smad7 is required for maximal enhancement of TGFbeta/Smad signaling, we observe that TIEG is still capable of increasing Smad pathway activity in the absence of Smad7. Furthermore, while Smad7 is known to block both TGFbeta and bone morphogenetic protein (BMP) signaling, we observe that TIEG specifically enhances only the TGFbeta pathway. Similarly, while both TIEG and the related Krüppel-like factor, FKLF2, repress Smad7 transcription, only TIEG is capable of enhancing Smad signaling. In order to identify additional regulatory targets of TIEG important for this enhancement of the Smad pathway activity, we performed microarray analysis and identified Smad2 as a TIEG target gene. We now show evidence that TIEG increases transcription of the Smad2 gene but not the Smad3 or Smad4 genes. Furthermore, while the TGFbeta/Smad pathway remains intact in Smad2 null cells, TIEG enhancement of Smad signaling is dramatically reduced. Thus we propose a new model whereby TIEG enhances Smad signaling by a dual mechanism involving both the repression of the inhibitory Smad7 as well as the activation of Smad2.

Animals↗

PubMed2002

Identification of a BMP-responsive element in Id1, the gene for inhibition of myogenesis.

BACKGROUND: Bone morphogenetic protein-2 (BMP-2) stimulates osteoblast differentiation, but inhibits myogenic differentiation in C2C12 myoblasts. BMP-2 induces transcription of Id1, an inhibitor for myogenesis, within 1 h in the cells. To examine the molecular mechanism of the action of BMP-2, we analysed a BMP-2-responsive element (BRE) in the 5' flanking region of the human Id1 gene. RESULTS: A GC-rich region between -985 bp and -957 bp of the human Id1 gene was identified as a BRE. The BRE containing promoter activity was stimulated by BMP-2 or by constitutively active BMP receptors (BMPR-IA and BMPR-IB). The stimulation was blocked by co-transfecting with dominant negative BMPR-IA or Smad7. A unique DNA-protein complex was induced in response to BMP-2 on the BRE. The complex induced by BMP-2 contained Smad1 and Smad4, possibly as a complex of both Smads. BMP-2 failed to stimulate the expression of Id1 mRNA in Smad4-deficient cells. Over-expression of Smad4, but not Smad1, stimulated the Id1 reporter activity and the expression of endogenous Id1 mRNA in Smad4-deficient cells. CONCLUSION: Signalling of BMP-2 to stimulate the expression of Id1 would be transduced by BMPR-IA and mediated by Smad1 and Smad4, both of which form a complex on the 29 bp GC-rich element.

Animals↗

PubMed2002

PTHrP and Indian hedgehog control differentiation of growth plate chondrocytes at multiple steps.

In developing murine growth plates, chondrocytes near the articular surface (periarticular chondrocytes) proliferate, differentiate into flat column-forming proliferating cells (columnar chondrocytes), stop dividing and finally differentiate into hypertrophic cells. Indian hedgehog (Ihh), which is predominantly expressed in prehypertrophic cells, stimulates expression of parathyroid hormone (PTH)-related peptide (PTHrP) which negatively regulates terminal chondrocyte differentiation through the PTH/PTHrP receptor (PPR). However, the roles of PTHrP and Ihh in regulating earlier steps in chondrocyte differentiation are unclear. We present novel mouse models with PPR abnormalities that help clarify these roles. In mice with chondrocyte-specific PPR ablation and mice with reduced PPR expression, chondrocyte differentiation was accelerated not only at the terminal step but also at an earlier step: periarticular to columnar differentiation. In these models, upregulation of Ihh action in the periarticular region was also observed. In the third model in which the PPR was disrupted in about 30% of columnar chondrocytes, Ihh action in the periarticular chondrocytes was upregulated because of ectopically differentiated hypertrophic chondrocytes that had lost PPR. Acceleration of periarticular to columnar differentiation was also noted in this mouse, while most of periarticular chondrocytes retained PPR signaling. These data suggest that Ihh positively controls differentiation of periarticular chondrocytes independently of PTHrP. Thus, chondrocyte differentiation is controlled at multiple steps by PTHrP and Ihh through the mutual regulation of their activities.

Animals↗