Keratocan Expression of Murine Keratocytes Is Maintained on Amniotic Membrane by Down-regulating Transforming Growth Factor-β Signaling

Tetsuya Kawakita,Edgar M Espana,Hua He,Armand Hornia,Lung-Kun Yeh,Jie Ouyang,Chia-Yang Liu,Scheffer C.G Tseng

doi:10.1074/jbc.m409567200

Tetsuya Kawakita, Edgar M Espana + Show 6 more

Open Access

https://doi.org/10.1074/jbc.m409567200

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Abstract

Keratocytes in the corneal stroma express keratan sulfate-containing proteoglycans including cornea-specific keratocan. On plastic dishes, human, bovine, and rabbit keratocytes lose their characteristic dendritic morphology and keratocan expression when cultured in serum-containing media. Herein, we demonstrated that murine keratocytes also acquired a fibroblastic shape and lost keratocan expression after first passage when cultured on plastic in the presence of serum. In contrast, cells expanded on human amniotic membrane (AM) stromal matrix maintained a three-dimensional dendritic morphology and expressed keratocan mRNA and protein for at least 8 passages before senescence. When keratocytes were cultured on AM, the promoter activity of transforming growth factor (TGF)-beta2 and TGF-beta receptor II was down-regulated as compared with that on plastic. Furthermore, cells on AM continuously retained Smad 2 and Smad 4 in the cytoplasm and did not express alpha-smooth muscle actin, even when 10 ng/ml TGF-beta1 was added in a serum-free medium for up to 5 days. In parallel to such down-regulation of TGF-beta signaling, keratocan promoter-driven ECFP expression was observed in cells cultured either on AM in the presence of serum or on plastic containing serum treated with a neutralizing antibody to TGF-beta. Collectively, these results indicate that down-regulation of Smad-mediated TGF-beta signaling is an important mechanism for cultured keratocytes to maintain a normal phenotype while continuously expanded in a serum-containing medium. This strategy of suppressing TGF-beta signaling, achieved by AM stromal matrix in part via suppression of TGF-beta gene transcription, can be used to expand keratocytes in culture without the use of AM in the future.

Highlights

Keratocytes, a unique population of neural crest-derived cells embedded in the corneal stroma, play a major role in maintaining corneal transparency
Corneal epithelial cells or endothelial cells were not stained (Fig. 1D, inset). These results indicated that in vivo murine keratocytes exhibited a characteristic dendritic morphology and expressed keratocan
We have demonstrated that murine keratocytes expanded in an fetal bovine serum (FBS)-containing medium preserved a native three-dimensional dendritic morphology with extensive intercellular contacts and continuously expressed keratocan and CD34, but not fibronectin and ␣-SMA, for up to 8 passages, FIG. 8

Summary

Introduction

Keratocytes, a unique population of neural crest-derived cells embedded in the corneal stroma, play a major role in maintaining corneal transparency. We reported a culture system that can achieve effective expansion of human keratocytes by growing them on the stromal surface of the AM in an FBS-containing medium [3, 4] They maintained a dendritic morphology, continued to express corneal stroma-specific keratocan for at least 5 passages (at 1:2 split), and did not express ␣-SMA under serum-containing conditions or on addition of TGF-␤1 [4]. We report that murine keratocytes can be expanded on AM for at least 8 passages without losing their normal phenotype in a serum-containing medium and that suppression of the Smad-mediated TGF-␤ signaling pathway is pivotal in maintaining a keratocan-expressing phenotype

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