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

Research output: Contribution to journalArticle

36 Citations (Scopus)

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)-β2 and TGF-β 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 α-smooth muscle actin, even when 10 ng/ml TGF-β1 was added in a serum-free medium for up to 5 days. In parallel to such down-regulation of TGF-β 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-β. Collectively, these results indicate that down-regulation of Smad-mediated TGF-β 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-β signaling, achieved by AM stromal matrix in part via suppression of TGF-β gene transcription, can be used to expand keratocytes in culture without the use of AM in the future.

Original languageEnglish
Pages (from-to)27085-27092
Number of pages8
JournalJournal of Biological Chemistry
Volume280
Issue number29
DOIs
Publication statusPublished - 2005 Jul 22
Externally publishedYes

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Amnion
Transforming Growth Factors
Membranes
Plastics
Serum
Down-Regulation
Keratan Sulfate
Corneal Stroma
Growth Factor Receptors
Serum-Free Culture Media
Proteoglycans
Transcription
Neutralizing Antibodies
Cornea
Smooth Muscle
Muscle
Actins
Cultured Cells
Cytoplasm
Genes

ASJC Scopus subject areas

  • Biochemistry

Cite this

Keratocan expression of murine keratocytes is maintained on amniotic membrane by down-regulating transforming growth factor-β signaling. / Kawakita, Tetsuya; Espana, Edgar M.; He, Hua; Hornia, Armand; Yeh, Lung Kun; Ouyang, Jie; Liu, Chia Yang; Tseng, Scheffer C G.

In: Journal of Biological Chemistry, Vol. 280, No. 29, 22.07.2005, p. 27085-27092.

Research output: Contribution to journalArticle

Kawakita, Tetsuya ; Espana, Edgar M. ; He, Hua ; Hornia, Armand ; Yeh, Lung Kun ; Ouyang, Jie ; Liu, Chia Yang ; Tseng, Scheffer C G. / Keratocan expression of murine keratocytes is maintained on amniotic membrane by down-regulating transforming growth factor-β signaling. In: Journal of Biological Chemistry. 2005 ; Vol. 280, No. 29. pp. 27085-27092.
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AU - Kawakita, Tetsuya

AU - Espana, Edgar M.

AU - He, Hua

AU - Hornia, Armand

AU - Yeh, Lung Kun

AU - Ouyang, Jie

AU - Liu, Chia Yang

AU - Tseng, Scheffer C G

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