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Excess Transforming Growth Factor-? Changed the Cell Properties of Corneal Epithelium and Stroma.

ABSTRACT: Purpose:This study is to investigate the corneal anomaly caused by excess transforming growth factor-? (TGF-?) during mouse development. Methods:Bitransgenic KeraRT/TGF-? mice, generated via cross-mating tetO-TGF-? and KeraRT mice, were induced to overexpress TGF-? by doxycycline commencing at embryonic day 0 or postnatal day 0 to different developmental stages. Bitransgenic mice with doxycycline induction were defined as TGF-?ECK mice (TGF-? excess expression by corneal keratocytes). Mouse eyes were examined by hematoxylin and eosin staining, immunofluorescent staining and transmission electron microscopy. Protein and RNA from mouse cornea were subjected to western blotting and real-time quantitative polymerase chain reaction. Results:In TGF-?ECK mice, TGF-? overexpression resulted in corneal opacity. Excess TGF-? initially caused corneal epithelial hyperplasia and subsequent epithelium degeneration as the mouse developed, which was accompanied by gradually diminished K12 expression from the periphery of corneal epithelium and increased K13 expression toward the corneal center. Interestingly, K14 was detected in all layers of corneal epithelium of TGF-?ECK mice, whereas it was limited at basal layer of controls. Transmission electron microscopy showed desmosome loss between corneal epithelial cells of TGF-?ECK mice. In TGF-?ECK mice, keratocan expression was abolished; ?-SMA expression was increased while expression of Col1a1, Col1a2, and Col5a1 was diminished. Cell proliferation increased in the corneal epithelium and stroma, but not in the endothelium of TGF-?ECK mice. Conclusions:Excess TGF-? had detrimental effects on corneal morphogenesis during mouse development in that it changed the cell fate of corneal epithelial cells to assume conjunctival phenotypic expression of K13, and keratocytes to myofibroblast phenotype.

SUBMITTER: Zhang L

PROVIDER: S-EPMC7425719 | biostudies-literature | 2020 Jul

REPOSITORIES: biostudies-literature

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Excess Transforming Growth Factor-α Changed the Cell Properties of Corneal Epithelium and Stroma.

Zhang Lingling L Yuan Yong Y Yeh Lung-Kun LK Dong Fei F Zhang Jianhua J Okada Yuka Y Kao Winston W Y WWY Liu Chia-Yang CY Zhang Yujin Y

Investigative ophthalmology & visual science 20200701 8

<h4>Purpose</h4>This study is to investigate the corneal anomaly caused by excess transforming growth factor-α (TGF-α) during mouse development.<h4>Methods</h4>Bitransgenic KeraRT/TGF-α mice, generated via cross-mating tetO-TGF-α and KeraRT mice, were induced to overexpress TGF-α by doxycycline commencing at embryonic day 0 or postnatal day 0 to different developmental stages. Bitransgenic mice with doxycycline induction were defined as TGF-αECK mice (TGF-α excess expression by corneal keratocyt ...[more]

PMID: 32668000

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Project description:Specific mutations in the transforming growth factor beta induced (TGFBI) gene are associated with lattice corneal dystrophy (LCD) type 1 and its variants. In this study, we performed an in-depth proteomic analysis of human corneal amyloid deposits associated with the heterozygous A546D mutation in TGFBI.Corneal amyloid deposits and the surrounding corneal stroma were procured by laser capture microdissection from a patient with an A546D mutation in TGFBI. Proteins in the captured corneal samples and healthy corneal stroma were identified with liquid chromatography-tandem mass spectrometry and quantified by calculating exponentially modified Protein Abundance Index values. Mass spectrometry data were further compared for identifying enriched regions of transforming growth factor beta induced protein (TGFBIp/keratoepithelin/?ig-h3) and detecting proteolytic cleavage sites in TGFBIp.A C-terminal fragment of TGFBIp containing residues Y571-R588 derived from the fourth fasciclin 1 domain (FAS1-4), serum amyloid P-component, apolipoprotein A-IV, clusterin, and serine protease HtrA1 were significantly enriched in the amyloid deposits compared to the healthy cornea. The proteolytic cleavage sites in TGFBIp from the diseased cornea are in accordance with the activity of serine protease HtrA1. We also identified small amounts of the serine protease kallikrein-14 in the amyloid deposits.Corneal amyloid caused by the A546D mutation in TGFBI involves several proteins associated with other varieties of amyloidosis. The proteomic data suggest that the sequence 571-YHIGDEILVSGGIGALVR-588 contains the amyloid core of the FAS1-4 domain of TGFBIp and point at serine protease HtrA1 as the most likely candidate responsible for the proteolytic processing of amyloidogenic and aggregated TGFBIp, which explains the accumulation of HtrA1 in the amyloid deposits. With relevance to identifying serine proteases, we also found glia-derived nexin (protease-nexin 1) in the amyloid deposits, making this serine protease inhibitor a good candidate for the physiologically relevant inhibitor of one of the amyloid-associated serine proteases in the cornea and probably in other tissues. Noteworthy, the present results are in accordance with our findings from a previous study of corneal amyloid deposits caused by the V624M mutation in TGFBI, suggesting a common mechanism for lattice corneal dystrophies (LCDs) associated with mutations in the TGFBIp FAS1-4 domain.

Project description:Radiation fibrosis of the lung is a late toxicity of thoracic irradiation. Epidermal growth factor (EGF) signaling has previously been implicated in radiation lung injury. We hypothesized that TGF-?, an EGF receptor ligand, plays a key role in radiation-induced fibrosis in lung. Mice deficient in transforming growth factor (TGF-?(-/-)) and control C57Bl/6J (C57-WT) mice were exposed to thoracic irradiation in 5 daily fractions of 6 Gy. Cohorts of mice were followed for survival (n ? 5 per group) and tissue collection (n = 3 per strain and time point). Collagen accumulation in irradiated lungs was assessed by Masson's trichrome staining and analysis of hydroxyproline content. Cytokine levels in lung tissue were assessed with ELISA. The effects of TGF-? on pneumocyte and fibroblast proliferation and collagen production were analyzed in vitro. Lysyl oxidase (LOX) expression and activity were measured in vitro and in vivo. Irradiated C57-WT mice had a median survival of 24.4 weeks compared to 48.2 weeks for irradiated TGF-?(-/-) mice (P = 0.001). At 20 weeks after irradiation, hydroxyproline content was markedly increased in C57-WT mice exposed to radiation compared to TGF-?(-/-) mice exposed to radiation or unirradiated C57-WT mice (63.0, 30.5 and 37.6 ?g/lung, respectively, P = 0.01). C57-WT mice exposed to radiation had dense foci of subpleural fibrosis at 20 weeks after exposure, whereas the lungs of irradiated TGF-? (-/-) mice were largely devoid of fibrotic foci. Lung tissue concentrations of IL-1?, IL-4, TNF-?, TGF-? and EGF at multiple time points after irradiation were similar in C57-WT and TGF-?(-/-) mice. TGF-? in lung tissue of C57-WT mice rose rapidly after irradiation and remained elevated through 20 weeks. TGF-?(-/-) mice had lower basal LOX expression than C57-WT mice. Both LOX expression and LOX activity were increased after irradiation in all mice but to a lesser degree in TGF-?(-/-) mice. Treatment of NIH-3T3 fibroblasts with TGF-? resulted in increases in proliferation, collagen production and LOX activity. These studies identify TGF-? as a critical mediator of radiation-induced lung injury and a novel therapeutic target in this setting. Further, these data implicate TGF-? as a mediator of collagen maturation through a TGF-? independent activation of lysyl oxidase.

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Excess Transforming Growth Factor-? Changed the Cell Properties of Corneal Epithelium and Stroma.

Publications

Excess Transforming Growth Factor-α Changed the Cell Properties of Corneal Epithelium and Stroma.

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