Keratocan expression of murine keratocytes is maintained on amniotic membrane by down-regulating transforming growth factor-β signaling
Keratocan expression of murine keratocytes is maintained on amniotic membrane by down-regulating transforming growth factor-β signaling
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DOI：
10.1074/jbc.m409567200
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发表时间：
2005-07-22
影响因子：
4.8
通讯作者：
Tseng, SCG
中科院分区：
文献类型：
--
作者：
Kawakita, T;Espana, EM;Tseng, SCG
角膜基质中的角膜细胞表达含硫酸角质素的蛋白聚糖，包括角膜特异性角膜蛋白聚糖。在塑料培养皿上，人、牛和兔角膜细胞在含血清培养基中培养时失去其特征性树突状形态和角膜蛋白聚糖表达。在此，我们证明，小鼠角膜细胞也获得了成纤维细胞的形状，并失去了角膜蛋白聚糖表达后，第一次传代培养的塑料在血清的存在下。与此相反，在人羊膜（AM）基质基质上扩增的细胞保持三维树突状形态，并在衰老前至少8代表达角蛋白聚糖mRNA和蛋白。角膜基质细胞在AM上培养时，转化生长因子（TGF）-β 2和TGF-β受体Ⅱ的启动子活性较在塑料上培养时下调。此外，即使在无血清培养基中加入10 ng/ml TGF-β 1长达5天，AM上的细胞仍持续将Smad 2和Smad 4保留在细胞质中，并且不表达α-平滑肌肌动蛋白。与TGF-β信号转导的这种下调平行，在血清存在下在AM上培养的细胞中或在用TGF-β中和抗体处理的含血清塑料上培养的细胞中观察到角膜蛋白聚糖启动子驱动的ECFP表达。总之，这些结果表明，Smad介导的TGF-β信号转导的下调是培养的角膜细胞在含血清培养基中持续扩增时维持正常表型的重要机制。这种抑制TGF-β信号传导的策略，部分地通过抑制TGF-β基因转录由AM基质基质实现，可以用于在将来不使用AM的情况下扩增培养中的角膜细胞。
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)-beta 2 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-beta 1 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.