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Biomedical subjects

K Miyazono

Publications and source records attributed to K Miyazono.

At least 181 records · Page 10Linked to original sources
PubMed1993

Molecular cloning and characterization of ficolin, a multimeric protein with fibrinogen- and collagen-like domains.

We have previously identified and purified transforming growth factor-beta 1 (TGF-beta 1)-binding proteins from porcine uterus membranes (Ichijo, H., Rönnstrand, L., Miyagawa, K., Ohashi, H., Heldin, C.-H., and Miyazono, K. (1991) J. Biol. Chem. 266, 22459-22464). One of these TGF-beta 1-binding proteins, with a molecular weight of 40,000, was purified to homogeneity and subjected to amino acid sequence analysis. The amino acid sequences obtained were used to isolate two closely related cDNA clones from a porcine uterus cDNA library. The deduced amino acid sequences revealed that both cDNAs encoded proteins that were mainly composed of fibrinogen-like and collagen-like domains. Therefore, they were denoted ficolin-alpha and ficolin-beta. Expression of ficolin-alpha and -beta cDNA in mammalian cells revealed that ficolin forms dimers, trimers, and several higher order of oligomers, whose molecular weights fit well with those of the purified TGF-beta 1-binding proteins from porcine uterus. Moreover, immunoblotting analysis using a peptide anti-serum against ficolin indicated that the TGF-beta 1-binding proteins identified in porcine uterus are ficolin-alpha, -beta, and their oligomers or closely related molecules. However, recombinant ficolin-alpha and -beta did not bind TGF-beta 1, despite the similarities in molecular weights and immunoreactivity with the material from the natural source. It is possible that a specific posttranslational modification of ficolin or interaction with another component is needed for TGF-beta 1 binding. Analysis by Northern blotting revealed that the expression of ficolin-alpha mRNA is relatively restricted and most abundant in placenta and lung. On the other hand, ficolin-beta was mainly expressed in skeletal muscle. The in vivo functions of ficolin will be discussed.

Amino Acid Sequence↗
PubMed1993

Lack of the latent transforming growth factor beta binding protein in malignant, but not benign prostatic tissue.

Transforming growth factor beta (TGF-beta) is a family of proteins which act as a potent growth inhibitor for most cell types including epithelial cells. TGF-beta is synthesized as latent high molecular weight complexes, composed of TGF-beta, the NH2-terminal part of the TGF-beta precursor and the third molecule, the latent TGF-beta binding protein (LTBP). We here ascertain that TGF-beta is expressed in human prostatic cancer tissue as well as in cystectomized prostatic tissue and in materials from transurethral resections with benign prostatic hyperplasia, analyzed by immunohistochemistry. TGF-beta is observed in both epithelial cells and stromal cells. No significant correlation was obtained between TGF-beta expression in tumor cells and their degree of differentiation. However, analysis by immunohistochemistry using antibodies against LTBP revealed that specimens from histopathologically verified human prostatic cancer are mostly negative for this molecule, although it is expressed in cystectomized prostatic and benign prostatic hyperplasia tissues. These results indicate that in cystectomized prostatic and benign prostatic hyperplasia tissues, TGF-beta may be produced in a complex associated with LTBP; whereas in prostatic carcinoma, TGF-beta is produced without associating with LTBP. The biological significance of the production of TGF-beta in relation to LTBP and the possible association with prognosis are discussed.

Amino Acid Sequence↗
PubMed1993

Ca2+ binding of latent transforming growth factor-beta 1 binding protein.

Latent transforming growth factor-beta 1 binding protein (LTBP) is a constituent of the latent high molecular weight complex of TGF-beta 1 in human platelets. In the present communication, we show that LTBP binds Ca2+ in its free form as well as in the latent TGF-beta 1 complex. The binding of Ca2+ induces a structural change which protects the molecule against proteolysis and changes its elution position when analyzed by anion exchange chromatography. The in vitro activation of TGF-beta 1 is not influenced by the presence of Ca2+. The possible significance of Ca2+ binding of LTBP is discussed.

Calcium↗
PubMed1993

Immunoelectron microscopic localization of transforming growth factor beta 1 and latent transforming growth factor beta 1 binding protein in human gastrointestinal carcinomas: qualitative difference between cancer cells and stromal cells.

Transforming growth factor beta 1 (TGF-beta 1) is secreted as an inactive complex associated with latent TGF-beta 1 binding protein (LTBP). Tissue localization of these proteins has not been fully understood in human pathological conditions. We examined the immunohistochemical localization of TGF-beta 1 precursor (proTGF-beta 1) and LTBP in carcinomas and granulation tissue in the human gastrointestinal tract at the light and electron microscopic levels. In normal tissue, endothelial cells and granulocytes sporadically showed immunoreactivity for proTGF-beta 1, while epithelial cells were all negative. In cancer tissue, both cancer cells and stromal cells (fibroblasts, macrophages, and endothelial cells) were positive for proTGF-beta 1, more frequently in diffuse-type gastric carcinomas than in differentiated-type adenocarcinomas. Immunoelectron microscopy revealed that proTGF-beta 1 was localized in rough endoplasmic reticulum and perinuclear cisternae in fibroblasts, macrophages, and endothelial cells in cancer stroma and in fibrous granulation tissue. In contrast, the intracellular localization of proTGF-beta 1 in carcinoma cells was predominantly observed in the cytosol (cytoplasmic matrix). This finding suggests disarranged or blocked intracellular transportation of proTGF-beta 1 in cancer cells. The immunoreactivity for LTBP was not observed in the normal epithelial cells. It was localized in cancer stroma, not in cancer cells. Ultrastructurally, LTBP was located in the extracellular matrix around fibroblasts and smooth muscle cells. The intracellular immunoreactivity for LTBP was observed in rough endoplasmic reticulum of fibroblasts and smooth muscle cells, the same as in granulation tissue. These results suggest that gastrointestinal carcinoma cells produce no or a small amount of LTBP in vivo. Our investigation suggests that extensive fibrosis in both cancer stroma and granulation tissues may be promoted by TGF-beta 1 mainly secreted from stromal cells.

Carrier Proteins↗
PubMed1993

Different signals mediate transforming growth factor-beta 1-induced growth inhibition and extracellular matrix production in prostatic carcinoma cells.

The effects of transforming growth factor-beta 1 (TGF-beta 1) on a human prostatic carcinoma cell line PC-3, and its subclone PC-3U, were investigated. Dose-dependent inhibition of [3H]thymidine incorporation in PC-3U cells was observed by addition of TGF-beta 1, although only 50% inhibition was obtained by high concentrations (12 nM) of TGF-beta 1. The growth inhibitory effects of TGF-beta 1 on PC-3 cells was insignificant. When 0.3 ng/ml of phorbol 12-myristate 13-acetate (PMA) was added together with TGF-beta 1, TGF-beta 1 inhibited growth of PC-3 cells (about 50% inhibition), and the growth inhibitory activity of TGF-beta 1 in PC-3U cells was enhanced (more than 90% inhibition). Affinity crosslinking studies revealed that both cell lines possess all of the three described forms of TGF-beta receptors. The intensities of the crosslinked bands were weaker in the PC-3 cells than in PC-3U cells, and those were not increased by the addition of PMA. The expression of the TGF-beta type II receptor mRNA did not change after the addition of PMA or TGF-beta 1. These results suggest that the effects of PMA involved downstream components of the signal transduction pathway of TGF-beta 1. TGF-beta 1 is known to stimulate the production of extracellular matrix proteins and to induce changes in the expression of nuclear transcription factor genes. In both PC-3 and PC-3U cells, TGF-beta 1 was found to stimulate the induction of fibronectin and plasminogen activator inhibitor-1 and the expression of junB mRNA, and PMA did not affect these responses. Thus, PC-3 and PC-3U cells, which are partially resistant to the growth inhibitory activity of TGF-beta 1, could still respond to TGF-beta 1 by extracellular matrix production, independent of PMA action. These results suggest that different signalling pathways mediate TGF-beta 1-induced growth inhibition and stimulation of extracellular matrix accumulation in these cells.

Cell Division↗
PubMed1993

The mechanism of action of transforming growth factor-beta.

Transforming growth factor-beta (TGF-beta) is a family of proteins that inhibit the growth of most epithelial cells, including hepatocytes. TGF-beta also has potent effects on the accumulation of extracellular matrix components. An increased TGF-beta production has been reported after hepatectomy and in some liver diseases; TGF-beta may thus play certain roles in the regulation of growth and metabolism of liver cells and in the development of fibrosis. TGF-beta consists of latent high molecular weight complexes; knowledge about the structure and function of the latent TGF-beta s, including the activation mechanisms, will be important for the understanding of the roles of TGF-beta in vivo. TGF-beta s exert their action on target cells via binding to cell surface receptors, several types of which have been described. The TGF-beta type II receptor was recently cloned and shown to have a serine/threonine (S/T) kinase domain in the cytoplasmic part; together with the type I receptor, it is thought to be most important for signal transduction. We have purified 40, 80 and 160 kDa glycoproteins from porcine uterus, that specifically bind TGF-beta. The elucidation of the structure and function of TGF-beta receptors and TGF-beta binding proteins will make it possible to understand the mechanisms of TGF-beta action, and in addition, may give us tools to regulate its activity in vivo.

Animals↗
PubMed1993

Role of the latent TGF-beta binding protein in the activation of latent TGF-beta by co-cultures of endothelial and smooth muscle cells.

Transforming growth factor beta (TGF-beta) is released from cells in a latent form consisting of the mature growth factor associated with an aminoterminal propeptide and latent TGF-beta binding protein (LTBP). The endogenous activation of latent TGF-beta has been described in co-cultures of endothelial and smooth muscle cells. However, the mechanism of this activation remains unknown. Antibodies to native platelet LTBP and to a peptide fragment of LTBP inhibit in a dose-dependent manner the activation of latent TGF-beta normally observed when endothelial cells are cocultured with smooth muscle cells. Inhibition of latent TGF-beta activation was also observed when cells were co-cultured in the presence of an excess of free LTBP. These data represent the first demonstration of a function for the LTBP in the extracellular regulation of TGF-beta activity and indicate that LTBP participates in the activation of latent TGF-beta, perhaps by concentrating the latent growth factor on the cell surface where activation occurs.

Animals↗
PubMed1993

Activin receptor-like kinases: a novel subclass of cell-surface receptors with predicted serine/threonine kinase activity.

Human cDNA clones encoding four novel putative transmembrane protein serine/threonine kinases, denoted activin receptor-like kinase (ALK) -1, -2, -3 and -4, were obtained using a polymerase chain reaction (PCR)-based strategy. The PCR primers were designed based upon the sequence similarity between the activin receptor type II and Daf-1. The cDNA clones for ALK-1, -2 and -3 encode complete proteins of 503, 509 and 532 amino acids respectively. The ALK-4 cDNA is incomplete and the predicted protein of 383 amino acids has a truncated extracellular domain. The ALKs share similar domain structures, comprising predicted signal sequences at the N-terminals, followed by hydrophilic cysteine-rich ligand-binding domains, single hydrophobic transmembrane regions and C-terminal intracellular portions that consist almost entirely of putative serine/threonine kinase domains. The ALKs have approximately 40% sequence identity to activin receptors type II and IIB, transforming growth factor-beta (TGF-beta) type II receptor and Daf-1 in the kinase domains. However, the sequence identities are higher (60-79%) between ALK-1, -2, -3 and -4, suggesting that they form a subfamily among the putative receptor serine/threonine kinases. The extracellular domains of ALKs show only little sequence identity to other putative receptor serine/threonine kinases, but the cysteine residues are conserved. Their structural properties suggest that ALK-1 to -4 are receptors that may bind ligands that are members of the TGF-beta superfamily. The expression of mRNA in human tissues varied for the different ALKs; ALK-2 and ALK-4 showed ubiquitous tissue expression patterns, whereas the distribution of ALK-1 and ALK-3 varied strongly between different tissues with more restricted expression patterns. These results suggest that each ALK may have different in vivo functions.

Activin Receptors↗
PubMed1993

Involvement of transforming growth factor-beta in the formation of fibrotic lesions in carcinoid heart disease.

Carcinoid heart disease is a complication of a neuroendocrine carcinoid tumor. Morphologically, it is characterized by the formation of fibrotic plaques with deposition of extracellular matrix in the subendocardium, frequently causing heart valve dysfunction and cardiac failure. Because members of the transforming growth factor-beta (TGF-beta) family are known to stimulate fibroblasts in their production of extracellular matrix, we investigated the expression of the three isoforms of TGF-beta and the binding protein for latent TGF-beta 1 (LTBP) in carcinoid plaques of the right side of the heart, as well as from control tissue, using immunohistochemistry. Tissue specimens were obtained intraoperatively from nine consecutive patients undergoing valve replacement surgery. TGF-beta 1 and TGF-beta 3 were detected in the fibroblasts of all plaques analyzed, whereas TGF-beta 2 was only rarely expressed. The localization of LTBP was partly concordant with that of TGF-beta 1, but the positive staining for LTBP was extracellular. Sections from unaffected heart tissue contained few fibroblasts in the subendocardium, showing only weak or no immunostaining for TGF-beta 1, -beta 2, and -beta 3 and no staining for LTBP. These results suggest that TGF-beta may play a role in the proliferation of fibroblasts and their matrix production in carcinoid heart lesions.

Adult↗
PubMed1992

Molecular cloning and characterization of the human and porcine transforming growth factor-beta type III receptors.

Full-length cDNAs for the transforming growth factor-beta (TGF-beta) type III receptors were isolated from porcine uterus and human placenta cDNA libraries. The human TGF-beta type III receptor coding region encodes a protein of 849 amino acids with a single transmembrane domain and a short stretch of the intracellular domain. Potential glycosaminoglycan attachment sites were found in the extracellular domain. The overall amino acid sequence identities with those of the porcine and rat TGF-beta type III receptors were 83% and 81%, respectively. A high degree of sequence conservation was observed in the transmembrane and intracellular domains, which also have sequence similarity with human endoglin. In addition, two portions with 29 and 52 amino acids in the extracellular domain were found to be substantially similar with human endoglin.

Amino Acid Sequence↗
PubMed1992

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.

Carrier Proteins↗
PubMed1992

Platelet-derived endothelial cell growth factor has thymidine phosphorylase activity.

Platelet-derived endothelial cell growth factor (PD-ECGF), a protein which stimulates angiogenesis in vivo, is shown to have a 39.2% amino acid sequence similarity over a 439 amino acid region with the thymidine phosphorylase of Escherichia coli (E. coli). Using recombinant human PD-ECGF, we show that PD-ECGF has thymidine phosphorylase activity. Analysis by gel chromatography revealed that recombinant human PD-ECGF occurs as a 90 kDa homodimer, similar to other thymidine phosphorylases. In addition to a possible effect on DNA synthesis, PD-ECGF was shown to affect [3H]thymidine assays in a manner which is not related to cell proliferation. The in vitro and in vivo effects of PD-ECGF may thus occur by an indirect mechanism through its enzymatic activity.

Amino Acid Sequence↗
PubMed1992

Retention of the transforming growth factor-beta 1 precursor in the Golgi complex in a latent endoglycosidase H-sensitive form.

Transforming growth factor-beta 1 (TGF-beta 1) is synthesized as a latent high molecular weight complex in a human erythroleukemia cell line, HEL, treated with phorbol 12-myristate 13-acetate. The complex is comprised of three components: mature TGF-beta 1, the TGF-beta 1 latency-associated peptide (beta 1-LAP), and the latent TGF-beta 1-binding protein (LTBP). LTBP plays an important role in the assembly and secretion of the latent TGF-beta 1 complex; if the TGF-beta 1 precursor fails to bind to LTBP, much of it remains inside the cells and may contain anomalous disulfide bond(s) between beta 1-LAP and the mature TGF-beta 1 molecule (Miyazono, K., Olofsson, A., Colosetti, P., and Heldin, C.-H. (1991) EMBO J. 10, 1091-1101). In the present work, we have investigated the subcellular localization and properties of the TGF-beta 1 precursor retained intracellularly. When the HEL cells were metabolically labeled and chased for up to 72 h, a considerable part of the TGF-beta 1 precursor was still observed intracellularly in an unprocessed form. The secreted form of the TGF-beta 1 precursor was resistant to endoglycosidase H, whereas the intracellular form of the TGF-beta 1 precursor was sensitive to endoglycosidase H, regardless of the presence or absence of swainsonine, an inhibitor of mannosidase II. Indirect immunofluorescence microscopy revealed that the TGF-beta 1 precursor co-localized with mannosidase II, a marker for the Golgi complex, but not with protein disulfide isomerase, a marker for the endoplasmic reticulum. The intracellular TGF-beta 1 precursor was prepared from phorbol 12-myristate 13-acetate-treated HEL cells and tested for TGF-beta 1 bioactivity. Half-maximal inhibition of the DNA synthesis in mink lung epithelial cells, Mv1Lu, was observed at 80 pM of the acid-treated TGF-beta 1 precursor, whereas nontreated material showed minimal growth inhibitory activity. Taken together, these results indicate that the TGF-beta 1 precursor is retained inside the cells in the Golgi complex, mainly in a latent, immature form.

Animals↗
PubMed1992

Structure, function and possible clinical application of transforming growth factor-beta.

Transforming growth factor-beta (TGF-beta) is a family of multifunctional 25 kDa proteins. TGF-beta was originally identified because of its ability to induce the growth of normal rodent fibroblasts in soft agar, but is now known as a potent growth inhibitor for many different cell types. In addition, TGF-beta is known to regulate the differentiation of cells, induce chemotaxis of cells, and to induce the accumulation of extracellular matrix proteins. In vivo, TGF-beta stimulates the repair of soft as well as hard tissues. It also acts as a potent immunosuppressant. TGF-beta is produced as latent high molecular weight complexes; since it is produced by many different cell types, and most cells have receptors for TGF-beta, the activation of latent TGF-beta is likely to be an important step in the regulation of its action. TGF-beta exerts its effects by binding to specific cell surface receptors. The type I and type II TGF-beta receptors are suggested to be the most important for signal transduction; a recent report has disclosed that the type II receptor has a serine/threonine kinase domain. Since TGF-beta is a potent growth regulator with multifunctional activity, it may be useful in the treatment of certain clinical disorders. Local application of TGF-beta is shown to accelerate wound healing. Since an increase in TGF-beta activity is often observed in various fibrotic disorders, antagonists for TGF-beta might be valuable in the treatment of such diseases.

Animals↗