Hidetoshi Yamashita

Cloning of a TGFβ type I receptor that forms a heteromeric complex with the TGFβ 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.

Hyaluronan Fragments Induce Endothelial Cell Differentiation in a CD44- and CXCL1/GRO1-dependent Manner

Hyaluronan is a glycosaminoglycan of the extracellular matrix. In tumors and during chronic inflammatory diseases, hyaluronan is degraded to smaller fragments, which are known to stimulate endothelial cell differentiation. In this study, we have compared the molecular mechanisms through which hyaluronan dodecasaccharides (HA12), and the known angiogenic factor, fibroblast growth factor 2 (FGF-2), induce capillary endothelial cell sprouting in a three-dimensional collagen gel. The gene expression profiles of unstimulated and HA12- or FGF-2-stimulated endothelial cells were compared using a microarray analysis approach. The data revealed that both FGF-2 and HA12 promoted endothelial cell morphogenesis in a process depending on the expression of ornithine decarboxylase (Odc) and ornithine decarboxylase antizyme inhibitor (Oazi) genes. Among the genes selectively up-regulated in response to HA12 was the chemokine CXCL1/GRO1 gene. The notion that the induction of CXCL1/GRO1 is of importance for HA12-induced endothelial cell sprouting was supported by the fact that morphogenesis was inhibited by antibodies specifically neutralizing the CXCL1/GRO1 protein product. HA12-stimulated endothelial cell differentiation was exerted via binding to CD44 since it was inhibited by antibodies blocking CD44 function. Our data show that hyaluronan fragments and FGF-2 affect endothelial cell morphogenesis by the induction of overlapping but also by distinct sets of genes.

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.

Effects of transforming growth factor β on corneal epithelial and stromal cell function in a rat wound healing model after excimer laser keratectomy

Abstractu2002· Background: Transforming growth factor β (TGF-β) regulates extracellular matrix deposition, cell proliferation, and migration, and is expressed in cornea. TGF-β is thought to be involved in the corneal wound healing process. · Methods: The central corneal area (3 mm in diameter) of Lewis rats was ablated using PTK mode excimer laser and the wound healing process was observed at 12 and 24 h and 2, 5, 10, and 30 days after treatment. The expression of TGF-β1, -β2 and -β3, TGF-β type I and type II receptors, α3, α5, β4 integrin subunits, laminin and fibronectin was studied immunohistochemically. Antibody neutralizing TGF-β1, -β2 and -β3 was administered intraperitoneally, 50 µg daily, for 5 days after the laser treatment to investigate the effects of TGF-β function blockade. · Results: At the leading edge of the regenerating epithelium, no TGF-β type I and type II receptors and β4 integrin subunits were expressed after 24u2005h. Regenerating epithelium covered the ablated area after 2 days. An abnormal fibrotic layer was formed in the subepithelial area. This layer contained round-shaped cells in the stroma in the early stage (2–5 days after laser ablation) and spindle-shaped fibroblast-like keratocytes after 10 days. Laminin and fibronectin expression increased in the fibrotic layer. The increased stromal cells expressed TGF-β isoforms and TGF-β receptors. Neutralizing TGF-β inhibited the stromal cell increase in the laser ablated area after 5 days. · Conclusion: TGF-β may be involved in epithelial cell migration and stromal cell reaction during the corneal wound healing process after excimer laser ablation in rat models.

Three-dimensional organization of collagen fibrils during corneal stromal wound healing after excimer laser keratectomy

Purpose: To investigate the structural changes in corneal stromal collagen fibrils after excimer laser keratectomy in relation to the degree of corneal haze. Setting: University of Tokyo Hospital, Tokyo, Japan. Methods: Corneal haze was quantitatively measured by analyzing the light scattering in Scheimpflug images of the corneas of white rabbits after excimer laser keratectomy. Collagen fibril structure was examined using scanning electron microscopy after chemical digestion with sodium hydroxide solution; the same specimens were examined by transmission electron microcopy after re‐embedding. Results: Corneal haze reached a peak 4 weeks after excimer laser keratectomy and then gradually decreased, The collagen fibrils of the normal cornea were regularly arranged parallel to the surface of the cornea, with small interfibrillar distances. After excimer laser keratectomy, the arrangement was highly disordered, with increased interfibrillar distances. These structural changes were most prominent 4 weeks after excimer laser keratectomy. Conclusion: The structural changes in the collagen fibrils of the corneal stroma, especially the increase in interfibrillar distances and the disordered arrangement, were associated with corneal haze after excimer laser keratectomy.

EXPRESSION OF TGF-BETA TYPE I AND TYPE II RECEPTORS IN RAT EYES

Transforming growth factor beta (TGF-beta) transduces signals through mediation of type I and type II serine/threonine kinase receptors. The expression of TGF-beta type I (T beta R-I) and II (T beta R-II) receptors in rat eyes was investigated immunohistochemically. T beta R-I and T beta R-II immunoreactivity was detected in corneal and conjunctival epithelial cells, corneal endothelial cells, ciliary epithelial cells, lens epithelial cells, retinal pigment epithelial cells, and choroidal vessels. This co-expression of T beta R-I and T beta R-II indicates that the above cells respond to TGF-beta and, because TGF-beta is reported to be produced in ocular tissues, that it may have important autocrine and/or paracrine roles in the growth and metabolism of ocular tissues in situ.

Lack of transforming growth factor‐β type II receptor expression in human retinoblastoma cells

Retinoblastoma cells are resistant to transforming growth factor‐β (TGF‐β) activity due to the absence of TGF‐β binding. To further elucidate the mechanism of TGF‐β resistance, we studied the expression of the TGF‐β receptors and SMADs by using the Y79 and WERI‐Rb‐1 retinoblastoma cell lines. Binding of 125I‐TGF‐β1 to serine/threonine kinase receptor type II (TβR‐II) and TβR‐I was not seen in the retinoblastoma cells. TβR‐II mRNA was not expressed in these cells, but TβR‐I mRNA was detected. Mutation analysis revealed no mutation in the coding region of the TβR‐II gene, and TβR‐II mRNA could be induced after the differentiation of Y79 cells. Smad2, Smad3, and Smad4, which are involved in TGF‐β signaling, were expressed in the retinoblastoma cells. Transcriptional activation of the TGF‐β‐responsive genes was not seen by the transfection of either recep‐tor cDNA alone but could be induced by transfection of both TβR‐II and TβR‐I. These data suggest that the defect in the TGF‐β response is caused by the lack of TβR‐II in the retinoblastoma cells. In addition, TβR‐I may be functionally inactivated in these cell lines. J. Cell. Physiol. 175:305–313, 1998.

Effects of cholestanol feeding on corneal dystrophy in mice

A cholestanol-enriched diet administered for 8 months to BALB/c mice produced in 20% two kinds of corneal opacities resembling calcific band keratopathy and Schnyders crystalline dystrophy in humans. The concentrations of cholestanol in serum, liver and cornea of the corneal opacity bearing mice were 30-40-times higher than those of normal mice. On the other hand, brain cholestanol level increased only 7-times in the opacity group as compared with that of control group. There was no significant difference in the cholesterol concentrations of serum and several tissues among opacity, non-opacity and the control group. The crystal particles were observed between epithelial basement membrane and superficial stroma by the electron microscopy. Energy dispersive analysis of the particles revealed that the deposits were composed principally of calcium and phosphorus with other crystalline materials, which was presumed to be cholestanol. These results suggest that the cholestanol may deposit in the cornea from elevated serum levels. Deposition of cholestanol in cornea and related area may be a cause of corneal dystrophy in CTX.

Linkage between recycling and consumption: a case of toilet paper in Japan

This study examines consumer factors of paper recycling in Japan. The study specifically focuses on toilet paper as a paper product and attempts to reveal how individuals evaluate recycled toilet paper, how the evaluation relates to toilet paper consumption, and why people use or do not use recycled toilet paper. The study also examines what factors influence collection recycling behavior, and what people believe as necessary to achieve a society with better recycling. Responses were obtained from 1242 respondents in Japan. Four results were found. (1) People cannot judge the raw material of virgin toilet papers correctly, while people can correctly judge the raw material of recycled toilet paper. The quality and appearance of recycled toilet paper was not high enough to compete with virgin toilet paper. Furthermore, the image of recycled toilet paper also had negative impact on the willingness to use recycled toilet paper. (2) The primary criterion for purchasing recycled toilet paper was pro-environmental attitude. For the virgin toilet paper, it was brand. As expected, recycled toilet paper users had a positive evaluation and image of recycled toilet paper, while virgin toilet paper users had a negative evaluation and image of it. (3) Actual recycling behavior might not relate directly to consumption behavior of recycled paper. Rather, it was determined by the knowledge of waste collection system and payment system. (4) Most people have not realized that without the consumption of recycled products, the recycling system is not completed.

Expression and Possible Roles of Activin A in Proliferative Vitreoretinal Diseases

PURPOSEnTo examine the expression of activin A in eyes and to determine the possible functions of activin A in proliferative vitreoretinal diseases.nnnMETHODSnThe activin A concentration in vitreous specimens obtained from eyes with or without retinal ischemia was measured by a bioassay using erythroid differentiation factor effects of activin A. The expression of activin A and activin receptors in the preretinal membranes was observed by immunohistochemical analysis.nnnRESULTSnThe mean concentration of activin A in the eyes with proliferative diabetic retinopathy was 1. 50 +/- 1.27 ng/mL (mean +/- SD; n = 10), and that in the nondiabetic eyes without retinal ischemia (macular hole and epiretinal membrane) was 0.90 +/- 0.55 ng/mL (n = 5). Neither difference was significant. Activin A and its receptors were detected in the vascular endothelial cells, fibroblast-like cells and round-shaped macrophage-like cells in preretinal proliferative membranes by immunohistochemical analysis.nnnCONCLUSIONSnActivin A is involved in the proliferative membrane formation in both ischemic and nonischemic vitreoretinal proliferative diseases. Activin A, a member of TGF-beta superfamily, regulates angiogenesis and tissue fibrosis in the wound healing process.