Keratocan expression of murine keratocytes is maintained on amniotic membrane by down-regulating transforming growth factor-beta signaling.
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.
Kawakita
Tetsuya
T
TissueTech, Inc. and Ocular Surface Center, Miami, Florida 33173, USA.
Espana
Edgar M
EM
He
Hua
H
Hornia
Armand
A
Yeh
Lung-Kun
LK
Ouyang
Jie
J
Liu
Chia-Yang
CY
Tseng
Scheffer C G
SC
D000818:Animals; D018929:Cell Culture Techniques; D048430:Cell Shape; D002478:Cells, Cultured; D003319:Corneal Stroma / Q000166:cytology*; D004268:DNA-Binding Proteins / Q000378:metabolism; D015536:Down-Regulation; D005136:Eye Proteins / Q000032:analysis / Q000235:genetics*; D005347:Fibroblasts / Q000166:cytology*; D051379:Mice; D008815:Mice, Inbred Strains; D017346:Protein Serine-Threonine Kinases; D011509:Proteoglycans / Q000032:analysis / Q000235:genetics*; D012333:RNA, Messenger / Q000032:analysis; D000077294:Receptor, Transforming Growth Factor-beta Type II; D018125:Receptors, Transforming Growth Factor beta / Q000235:genetics; D051899:Smad2 Protein; D051901:Smad4 Protein; D015534:Trans-Activators / Q000378:metabolism; D016212:Transforming Growth Factor beta / Q000235:genetics; D053781:Transforming Growth Factor beta2
D004268:DNA-Binding Proteins; D005136:Eye Proteins; C099280:Kera protein, mouse; D011509:Proteoglycans; D012333:RNA, Messenger; D018125:Receptors, Transforming Growth Factor beta; D051899:Smad2 Protein; C497783:Smad2 protein, mouse; D051901:Smad4 Protein; C509041:TGFB2 protein, human; D015534:Trans-Activators; D016212:Transforming Growth Factor beta; D053781:Transforming Growth Factor beta2; D017346:Protein Serine-Threonine Kinases; D000077294:Receptor, Transforming Growth Factor-beta Type II