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Biomedical subjects

K Miyazono

Publications and source records attributed to K Miyazono.

At least 163 records · Page 9Linked to original sources
PubMed1994

Structural properties of 3.0 kb and 3.2 kb transcripts encoding platelet-derived endothelial cell growth factor/thymidine phosphorylase in A431 cells.

Platelet-derived endothelial cell growth factor/thymidine phosphorylase (PD-ECGF/TP) is a 90 kDa protein consisting of two non-covalently associated subunits. In addition to the previously identified 1.8 kilobase (kb) PD-ECGF/TP mRNA, the human epidermoid carcinoma cell line A431 was found to express 3.0 kb and 3.2 kb transcripts. cDNA cloning of the larger transcripts revealed that they contain long 5' leader sequences of 1.5 kb and 1.7 kb, respectively, and the same open reading frame encoding PD-ECGF/TP as that of the 1.8 kb transcript. Comparison with the published PD-ECGF/TP genomic sequence shows that the long 5' leader sequence contain 7 of 8 copies of Sp-1 binding sites present in the transcription promoter region of the 1.8 kb transcript.

Base Sequence↗
PubMed1994

Identification of type I receptors for osteogenic protein-1 and bone morphogenetic protein-4.

Bone morphogenetic proteins (BMPs) are multifunctional proteins, structurally related to transforming growth factor-beta (TGF-beta) and activin. TGF-beta and activin exert their effects by forming heteromeric complexes of type I and type II serine/threonine kinase receptors. We have previously identified a series of type I serine/threonine kinase receptors, termed activin receptor-like kinase (ALK)-1 to -6. ALK-5 is a TGF-beta type I receptor, whereas ALK-2 and ALK-4 are activin type I receptors. Here we investigated the binding of proteins in the BMP family to ALKs. In transfected COS cells, the binding of osteogenic protein (OP)-1 and BMP-4 to certain ALKs was observed in the absence of type II receptors, and their binding was increased after co-transfection of a BMP type II receptor from Caenorhabditis elegans, DAF-4. OP-1 bound to ALK-2 and ALK-6 efficiently, and to ALK-3 less efficiently, whereas BMP-4 bound to ALK-3 and ALK-6 efficiently. Similarly, OP-1 bound to ALK-2, ALK-3, and/or ALK-6 in various nontransfected cell lines, although the binding profiles were different between different cell types. BMP-4 bound to ALK-3 in MC3T3-E1 osteoblasts and human foreskin fibroblasts. These results suggest that ALK-3 and ALK-6 are type I receptors for OP-1 and BMP-4; in addition, ALK-2 is a type I receptor shared by activin and OP-1, but not by BMP-4.

Activin Receptors↗
PubMed1994

Characterization of type I receptors for transforming growth factor-beta and activin.

Transforming growth factor-beta (TGF-beta) and activin exert their effects by binding to heteromeric complexes of type I and type II receptors. The type II receptors for TGF-beta and activin are transmembrane serine-threonine kinases; a series of related receptors, denoted activin receptor-like kinase (ALK) 1 to 5, have recently been identified, and ALK-6 is described here. ALK-5 has been shown to be a functional TGF-beta type I receptor. A systematic analysis revealed that most ALKs formed heteromeric complexes with the type II receptors for TGF-beta and activin after overexpression in COS cells; however, among the six ALKs, only ALK-5 was a functional TGF-beta type I receptor for activation of plasminogen activator inhibitor-1, and only ALK-2 and ALK-4 bound activin with high affinity.

Activin Receptors↗
PubMed1994

Characterization and autoregulation of latent transforming growth factor beta (TGF beta) complexes in osteoblast-like cell lines. Production of a latent complex lacking the latent TGF beta-binding protein.

We have previously shown that bone organ cultures produce large amounts of latent transforming growth factor beta (TGF beta), which lacks latent TGF beta-binding protein (LTBP). In this study we used the known osteoblast-like cell lines UMR-106, ROS 17/2.8, and MG63 as models to further examine latent TGF beta expression in bone. We found that the osteosarcoma cell line UMR-106 secreted latent TGF beta almost exclusively as a 100-kDa complex lacking LTBP. ROS 17/2.8 cells produced both the 100-kDa complex and also a 290-kDa complex containing the fibroblastic (190 kDa) form of LTBP. MG63 cells (like human foreskin fibroblasts) expressed almost exclusively the 290-kDa complex. To investigate the regulation of latent TGF beta complexes in bone cells we assessed the effects of TGF beta 1 treatment on expression of active and latent TGF beta. TGF beta 1 induced secretion of latent but not active TGF beta in all cell types examined. In human foreskin fibroblast cells, TGF beta 1 and LTBP mRNA were expressed concomitantly. In contrast, in osteosarcoma cell lines autoinduction of TGF beta 1 mRNA was associated with either a delayed increase or no change in LTBP mRNA. In UMR-106 cells LTBP message was virtually undetectable. We postulate that the expression of different latent TGF beta forms by osteoblast-like cells may reflect their maturation states and that different latent TGF beta complexes may have different functions, for example as secretory forms or as matrix storage forms.

Animals↗
PubMed1994

Endoglin forms a heteromeric complex with the signaling receptors for transforming growth factor-beta.

Human endoglin is a dimeric protein that binds transforming growth factor-beta (TGF-beta). A porcine cDNA clone for endoglin was obtained from a porcine uterus cDNA library. The deduced sequence of the primary translated product of endoglin consists of 643 amino acids with a high sequence identity (96%) to human endoglin in the transmembrane and intracellular domains, but with a lower sequence similarity (66%) in the extracellular domain. In contrast to human endoglin, porcine endoglin has no Arg-Gly-Asp tripeptide in its sequence. Antibodies, raised against a peptide corresponding to the intracellular domain of porcine endoglin, immunoprecipitated an 84-kDa protein under reducing condition and a 130-kDa protein under nonreducing condition in porcine aortic endothelial cells. Porcine endoglin bound TGF-beta 1 and -beta 3 efficiently, but TGF-beta 2 less efficiently. Endoglin was found to be coimmunoprecipitated with TGF-beta receptors type I and/or II by the endoglin antibodies or by TGF-beta receptor II antibodies in the presence of ligand. Thus, endoglin and TGF-beta receptors I and/or II most likely formed a heteromeric receptor complex. Endoglin was phosphorylated on serine residue(s), which did not change after stimulation by TGF-beta 1. These results revealed that endoglin is a phosphorylated protein which forms a heteromeric complex with signaling receptors for TGF-beta.

Amino Acid Sequence↗
PubMed1994

Signal transduction via serine/threonine kinase receptors.

A number of serine/threonine kinase receptors have recently been identified. Most of the members of this family are type I or type II receptors for proteins in the transforming growth factor-beta (TGF-beta) superfamily. The type I and type II receptors form heteromeric receptor complexes after binding of the ligands, and transduce intracellular signals. TGF-beta type I receptor needs type II receptor for ligand binding, and type II receptor needs type I receptor for signalling. The signal transducing molecules that interact with heteromeric serine/threonine kinase receptor complexes remain to be elucidated; cyclin-dependent kinases and the retinoblastoma protein appear to be downstream elements in the antiproliferative pathway of TGF-beta.

Animals↗
PubMed1994

Differential localization of TGF-beta-precursor isotypes in normal human skin.

Transforming growth factor-beta (TGF-beta) can act as a multi-functional regulator of both cell growth and differentiation. Three isotypes of TGF-beta s namely TGF-beta 1, TGF-beta 2 and TGF-beta 3, have been found in human tissues. Up to now, little is known about the distribution patterns of the TGF-beta isotypes in human skin. Using the TGF-beta-precursor (latency-associated peptides) specific antibodies to confirm the specificity, we studied the immunohistochemical distribution of TGF-beta 1-3 in human skin. TGF-beta 2 was found mainly in the intercellular space of all the layers of the epidermis as well as in the cytoplasm with a weak staining. In contrast, TGF-beta 3 was present in the subepidermal area of the dermis. TGF-beta 1 was observed obviously in neither epidermis nor dermis. These results showed the differential localization of TGF-beta isotypes in human skin, suggesting that the TGF-beta 2 and TGF-beta 3 may regulate the human skin function in an epithelial autocrine or mesenchymal-epithelial interaction manner.

Adolescent↗
PubMed1994

Serine/threonine kinase receptors.

A new family of transmembrane receptors that contain intracellular serine/threonine kinase domains is emerging. Ligands for this class of receptors include members of the transforming growth factor-beta (TGF-beta) superfamily, e.g. TGF-beta s and activins. TGF-beta s exert their effects on target cells via formation of heteromeric serine/threonine kinase complexes (TGF-beta type I and type II receptors). Other components, i.e. TGF-beta type III receptor and endoglin, appear to have more indirect roles, e.g. to present ligands to the signalling receptors. Given the structural similarity between members of the TGF-beta superfamily, other ligands in this family may act through structurally and functionally similar serine/threonine kinase receptors.

Amino Acid Sequence↗
PubMed1994

Latent transforming growth factor-beta 1 associates to fibroblast extracellular matrix via latent TGF-beta binding protein.

The role of latent transforming growth factor-beta (TGF-beta) binding protein (LTBP) in the association of TGF-beta 1 to the extracellular matrix of cultured fibroblasts and HT-1080 fibrosarcoma cells was studied by immunochemical methods. The matrices were isolated from the cells, and the levels of LTBP and TGF-beta 1 were estimated by immunoblotting and immunoprecipitation. LTBP, TGF-beta 1, and its propeptide (latency-associated peptide, LAP) were found to associate to the extracellular matrix. Immunoblotting analysis indicated that treatment of the cells with plasmin resulted in a concomitant time and dose dependent release of both LTBP and TGF-beta 1 from the extracellular matrix to the supernatant. Comparison of molecular weights suggested that plasmin treatment resulted in the cleavage of LTBP from the high molecular weight fibroblast form to a form resembling the low molecular weight LTBP found in platelets. Pulse-chase and immunoprecipitation analysis indicated that both the free form of LTBP and LTBP complexed to latent TGF-beta were efficiently incorporated in the extracellular matrix, from where both complexes were slowly released to the culture medium. Addition of plasmin to the chase solution resulted, however, in a rapid release of LTBP from the matrix. Fibroblast derived LTBP was found to associate to the matrix of HT-1080 cells in a plasmin sensitive manner as shown by immunoprecipitation analysis. These results suggest that the latent form of TGF-beta 1 associates with the extracellular matrix via LTBP, and that the release of latent TGF-beta 1 from the matrix is a consequence of proteolytic cleavage(s) of LTBP.

Carrier Proteins↗
PubMed1994

Immunopathological features of palatine tonsil characteristic of IgA nephropathy: IgA1 localization in follicular dendritic cells.

IgA nephropathy (IgAN) is generally thought to be mediated by the glomerular deposition of circulating immune complexes containing IgA as the major antibody component. Upper respiratory infections and tonsillitis often precede IgAN, and in some cases tonsillectomy is effective for the treatment of IgAN. Thus, the tonsil seems to be a unique organ causing initial and/or progressive events to generate nephritogenic immune complexes in IgAN. In this study we focused on the analysis of immunopathological features of the palatine tonsil characteristic of IgAN patients by using an immunohistochemical technique. The IgA1 subclass was demonstrated in follicular dendritic cells (FDC) of the tonsil of IgAN patients, but not in FDC of non-IgAN controls. On the other hand, IgA2, IgG, IgM and C3 did not show any differences in distribution between the two groups. Moreover, the expression of decay-accelerating factor (DAF), an inhibitor of homologous complement activation, and transforming growth factor-beta 1 (TGF-beta 1), an inducer of antibody-producing cells to IgA class switching, in FDC and interdigitating dendritic cells of the tonsil, respectively, which was also clarified in this study for the first time, was found to be identically distributed in the two groups. These findings may support the idea that IgA1, possibly in an immune complex form, is trapped by FDC and plays an important role in the persistent activation of particular B cell repertoires responsible for the onset and/or progression of IgAN.

Adolescent↗
PubMed1994

Assignment of the gene encoding the latent TGF-beta 1-binding protein (LTBP1) to human chromosome 2, region p12-->q22.

Latent transforming growth factor beta 1-binding protein (LTBP1) is an important component of the large latent TGF-beta 1 complex. It plays a role in the assembly and secretion of the latent TGF-beta 1. In this paper we have used a cDNA probe for LTBP1 to determine the chromosomal localization of the human gene. Using a panel of well-defined human x rodent somatic cell hybrid lines, LTBP1 could be assigned to chromosome 2. Further sublocalization of the gene to 2p12-->q22 was achieved using three hybrid lines which contain partially over-lapping fragments of chromosome 2.

Animals↗
PubMed1994

Collagen type I is not under autocrine control by transforming growth factor-beta 1 in normal and scleroderma fibroblasts.

BACKGROUND: The ability of transforming growth factor-beta (TGF-beta) to induce synthesis of extracellular matrix proteins stimulated this study in which we address the hypothesis that TGF-beta can induce, in normal fibroblasts, the sustained, elevated collagen synthesis characteristic of the scleroderma fibroblast. EXPERIMENTAL DESIGN: Fibroblasts were studied for synthesis of and responsiveness to TGF-beta. Secreted TGF-beta levels were determined in a bioassay and at the transcriptional level in a series of scleroderma (SSc) and normal fibroblasts. The ability of cells to interact functionally with a 3-dimensional collagen matrix after TGF-beta treatment was examined. The kinetics of TGF-beta-induced fibrosis in fibroblasts was studied. RESULTS: SSc fibroblasts were not characterized by elevated TGF-beta synthesis. There was no evidence of coordinate regulation of TGF-beta and collagen over passage number. Repeated pulses of 200 pM of TGF-beta did not significantly induce sustained procollagen alpha 1(I) mRNA synthesis in normal fibroblasts, and this treatment did not significantly alter the characteristics of normal fibroblasts in a collagen gel. mRNA for both collagen and TGF-beta type II receptor was induced by TGF-beta in both SSc and control cells. SSc fibroblasts were found to have an impaired ability to activate the small latent complex of TGF-beta. CONCLUSIONS: Our data give no support to the hypothesis that TGF-beta can maintain the SSc phenotype in vitro or that it is able to induce this phenotype. The inducibility of TGF-beta receptor mRNA in SSc fibroblasts after exposure to TGF-beta suggests that the lack of sustained elevation in collagen synthesis is not due to lack of responsiveness by the fibroblasts but is rather a reflection of the transient nature of TGF-beta-induced fibrosis.

Cell Division↗
PubMed1994

Enhanced expression of transforming growth factor-beta s and transforming growth factor-beta type II receptor in the synovial tissues of patients with rheumatoid arthritis.

BACKGROUND: A growing body of evidences suggests that transforming growth factor-beta (TGF-beta) is produced in the synovial fluid of patients with rheumatoid arthritis (RA), and that TGF-beta is an important regulator in the course of the disease. Careful studies on the endogenous synthesis of TGF-beta as well as its receptors are therefore necessary to clarify the possible role of TGF-beta in RA. EXPERIMENTAL DESIGN: We examined the expressions of latent TGF-beta 1, -beta 2, and -beta 3, the latent TGF-beta 1-binding protein (LTBP) as well as TGF-beta type II receptor (TGF-beta RII) in the synovial biopsy tissues of 21 patients with RA by immunohistochemistry. Five specimens from these cases representing both active and chronic inactive stages were also examined for the corresponding mRNA by in situ hybridization. Northern blot analysis was performed on 3 synovial membranes taken from the RA patients together with a control synovium. RESULTS: Abundant LTBP, TGF-beta 1, and TGF-beta RII-positive cells as well as less intensively stained TGF-beta 2 and TGF-beta 3-positive cells were found in the synovial layer. These cells were positive for the histocompatibility antigen, HLA-DR. In lymphocyte aggregates, scattered cells positively labeled for LTBP and TGF-beta 1 were found. They stained in a reticular pattern that was similar to that demonstrated by an antibody against human dendritic cells, and also expressed HLA-DR. In situ hybridization revealed markedly increased signals for LTBP and TGF-beta RII mRNA in tissues with an active inflammatory process, when compared with tissues with less active inflammation. However, no clear differences in the levels of expression for any of the TGF-beta isoforms were found. Specimens with pronounced fibrosis, fibroblasts, and surrounding collagen fibers expressed positive immunoreactivities for all TGF-beta isoforms and LTBP. Northern blot analysis on 4 synovial tissues demonstrated positive signals for LTBP and TGF-beta 1 mRNA in all three RA patients in contrast to a normal control, which did not show any signals. An increased expression of TGF-beta RII mRNA was detected in the tissue from one of the patients. CONCLUSIONS: An abundant expression of TGF-beta 1 and LTBP, as well as TGF-beta RII was seen in most actively proliferating synovial intimal cells, and the level of the expression varied during the course of the disease. We conclude that TGF-beta is involved tightly in the regulation of the inflammatory process, and it is thus possible that the endogenous TGF-beta functions as a self-regulator that induces the remission periods.

Amino Acid Sequence↗
PubMed1994

Expression of transforming growth factor-beta 1 and its relation to endomysial fibrosis in progressive muscular dystrophy.

Progressive muscular dystrophy is characterized by muscle fiber necrosis, regeneration, and endomysial fibrosis. Although absence of dystrophin has been known as the cause of muscle fiber degeneration, pathogenesis of interstitial fibrosis is still unknown. Transforming growth factor-beta 1 (TGF-beta 1) induces accumulation of extracellular matrix in various diseases, such as liver cirrhosis and interstitial pneumonitis. To investigate its function on the pathogenesis of progressive muscular dystrophy, it was necessary to determine the degree of TGF-beta 1 expression and the site of TGF-beta 1 immunoreactivity. In Duchenne muscular dystrophy and most of Becker muscular dystrophy, high TGF-beta 1 immunoreactivity expressed on muscle fibers and extracellular space. In other myopathies with endomysial fibrosis, however, TGF-beta 1 was seldom observed. We also examined the immunoreactivity of the latent TGF-beta binding protein, which is bound to the TGF-beta precursors. In all Duchenne muscular dystrophy and half of Becker muscular dystrophy cases, high latent TGF-beta 1 binding protein immunoreactivity was seen, but in other myopathies its immunoreactivity was seldom seen on muscle fibers or extracellular space. Therefore TGF-beta 1 may play an important role in synthesis and accumulation of extracellular matrix in progressive muscular dystrophy.

Animals↗
PubMed1993

Cloning of a TGF beta type I receptor that forms a heteromeric complex with the TGF beta type II receptor.

A cDNA clone encoding a 53 kd serine/threonine kinase receptor with an overall structure similar to that of the type II receptor for transforming growth factor beta (TGF beta) was obtained. 125I-TGF beta 1 bound to porcine endothelial cells transfected with the cDNA and formed a cross-linked complex of 70 kd, characteristic of a TGF beta type I receptor. Immunoprecipitation of the cross-linked complexes by antibodies against the cloned receptor revealed the 70 kd complex as well as a 94 kd TGF beta type II receptor complex. The immunoprecipitated novel serine/threonine kinase receptor had biochemical properties of the TGF beta type I receptor and was observed in different cell types. Transfection of the cloned cDNA into TGF beta type I receptor-deficient cells restored TGF beta-induced plasminogen activator inhibitor 1 production. These results suggest that signal transduction by TGF beta involves the formation of a heteromeric complex of two different serine/threonine kinase receptors.

Amino Acid Sequence↗
PubMed1993

Characterization of in vivo phosphorylation of activin type II receptor.

An antiserum was generated against a C-terminal peptide of the mouse activin type II receptor (ActR-II), which specifically immunoprecipitates ActR-II protein transiently expressed in COS-1 cells. Autophosphorylation activity of the ActR-II was examined in [32P]orthophosphate labeled COS-1 cells. The immunoprecipitated ActR-II protein was found to be phosphorylated on serine residues in the absence of ligand stimulation, and the phosphorylation was slightly increased after stimulation with activin A.

Activin Receptors↗
PubMed1993

Induction of transforming growth factor-beta during cardiac allograft rejection.

The polypeptides of the transforming growth factor-beta (TGF-beta) family are potent endogenous immuno-regulators. Using a rat cardiac allograft transplant model, we investigated the expression of the precursor forms of TGF-beta 1, TGF-beta 2, and TGF-beta 3 and the latent TGF-beta binding protein (LTBP) by immunohistochemistry. The activity of TGF-beta in the extracts from transplanted as well as normal hearts was measured using a bioassay, and Northern blot analysis was performed on RNA extracts. The transplanted hearts were analyzed both during acute rejection up to 6 days and during chronic rejection up to 6 mo after transplantation and compared with normal controls. The animals of the chronic rejection group received cyclosporin A for immunosuppression. The TGF-beta bioactivity dramatically increased in the transplanted allografts during the chronic rejection process compared to the normal hearts, and so did the immunostaining as well as the mRNA levels for TGF-beta 1 and, to a lesser extent, the immunostaining for TGF-beta 2. TGF-beta 3 expression remained unchanged and was only found in the myocardium in trace amounts. During the acute rejection process up to 6 days after transplantation, TGF-beta immunoreactivity increased only slightly, whereas the TGF-beta mRNA was severalfold increased. Control animals treated with cyclosporin A showed a similar pattern at day 6 with regard to TGF-beta expression. LTBP was induced simultaneously with TGF-beta 1 and occurred within interstitial spaces of the myocardium. The TGF-beta was produced by macrophage-like infiltrating lymphocytes. In conclusion, highly elevated levels of TGF-beta and LTBP were found during chronic rejection of cardiac allografts in rats. The induction of TGF-beta may counteract the rejection process and could be useful for new therapeutic approaches in the prevention of allograft rejection.

Animals↗