Mechanisms of mitotic inhibition in corneal endothelium: contact inhibition and TGF-beta2.
Mechanisms of mitotic inhibition in corneal endothelium: contact inhibition and TGF-beta2.
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发表时间：
2002-07
影响因子：
4.4
通讯作者：
N. Joyce;D. Harris;David M Mello
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文献类型：
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作者：
N. Joyce;D. Harris;David M Mello
目的接触抑制被认为是发育和成熟角膜内皮的重要抗增殖机制。尽管外源性TGF-β2和房水中的TGF-β2抑制培养的大鼠角膜内皮细胞进入S期，但尚不清楚TGF-β2是否有助于体内内皮发育过程中发生的有丝分裂抑制。 TGF-β 受体 I、II 和 III 必须共表达才能传递 TGF-β2 诱导的细胞内信号。目前的研究旨在通过研究这些受体何时在新生大鼠的内皮细胞中共表达，来确定 TGF-β2 是否有助于内皮发育过程中的有丝分裂抑制。细胞周期蛋白依赖性激酶抑制剂 (CKI)，例如 p27kip1 和 p15INK4b，有助于介导其他细胞类型的有丝分裂抑制。首先通过测定新生大鼠角膜内皮中 p27kip1 表达的动力学来检查 CKI 在抑制角膜内皮增殖中的作用。然后将研究扩展到培养细胞，以更直接地比较 TGF-β2 和细胞间接触对 CKI、p27kip1 和 p15INK4b 的相对蛋白和 mRNA 表达的影响。方法 免疫细胞化学 (ICC) 检测出生后第 1、10 和 21 天以及成年（3 个月大）大鼠角膜内皮细胞中的 TGF-β 受体 I、II 和 III（分别为 RI、RII、RIII）。 p27kip1 的 ICC 在出生后第 1、7、14 和 21 天进行。在出生后第 7、14 和 21 天从成年大鼠中采集样本进行 p27kip1 RT-PCR。在 10% 血清 +/- 5 ng/mL TGF-β2 中孵育 24 小时的 G(0) 期同步亚汇合大鼠角膜内皮细胞中测定 TGF-β2 对 p27kip1 和 p15INK4b 表达的影响。还在完全汇合的培养物中检查了 CKI 表达。 RT-PCR 和 Western blot 分析分别检测 p27kip1 和 p15INK4b mRNA 和蛋白表达。在使用和不使用 2.0 mg/mL EDTA 二钠处理 1 小时的汇合培养物中研究释放细胞从细胞-细胞接触中释放对增殖和 p27kip1 蛋白表达的影响，然后在 10% 血清中维持 24 小时。然后将培养物固定用于 Ki67（活跃循环细胞的标记）的 ICC，或提取用于 p27kip1 蛋白的蛋白质印迹测定。结果 出生后第 10 天检测到 TGF-β RIII 阳性染色，出生后第 21 天检测到 RI 和 RII 染色。出生后第 1 天及之后内皮细胞 p27kip1 染色呈阳性，并且在最早测试时间点（出生后第 7 天）可检测到 p27kip1 PCR 产物。在培养细胞中，TGF-β2 和细胞间接触对 p27kip1 或 p15INK4b mRNA 表达的影响相对较小。 TGF-β2 降低了两种蛋白的水平，但 p27kip1 的水平仍高于 p15INK4b。在汇合培养物中，p15INK4b 蛋白减少；然而，p27kip1 蛋白水平增加了 20 倍。与未处理的对照相比，在 EDTA 处理的汇合细胞中检测到 Ki67 的阳性染色，p27kip1 蛋白水平显着降低。结论 该实验室之前的研究表明，新生大鼠的角膜内皮细胞增殖在出生后第 13 天停止。这一时间与稳定的细胞-细胞接触的形成相关，表明接触抑制是内皮发育过程中生长停滞的重要机制。目前的研究表明，TGF-β RI、RII 和 RIII 的共表达发生得太晚，以至于 TGF-β2 在内皮发育过程中无法发挥重要的抑制增殖作用。对培养细胞的研究表明，p27kip1 介导对 TGF-β2 诱导的增殖的抑制，尽管对该细胞因子的反应相对较弱。新生儿内皮细胞中 p27kip1 的 ICC 和 RT-PCR 以及培养细胞中的 RT-PCR 和蛋白质印迹研究表明，接触抑制在很大程度上是通过 p27kip1 的活性介导的。这些结果与该实验室先前的数据一起强烈表明接触抑制是负责在角膜内皮发育过程中诱导细胞周期停滞以及维持成熟单层处于非增殖状态的重要机制。在这两种情况下，接触诱导的抑制至少部分是由 p27kip1 介导的。 TGF-β2似乎不会诱导发育中的内皮细胞有丝分裂停滞，但如果单层细胞与细胞的接触丧失，则可能起到维持成熟内皮细胞处于非复制状态的作用。
PURPOSE Contact inhibition has been implicated as an important antiproliferative mechanism in developing and mature corneal endothelium. Although exogenous TGF-beta2 and TGF-beta2 in aqueous humor suppress S-phase entry in cultured rat corneal endothelial cells, it is not known whether TGF-beta2 contributes to the mitotic inhibition that occurs during in vivo endothelial development. TGF-beta receptors I, II, and III must be coexpressed for a TGF-beta2-induced intracellular signal to be transmitted. The current study was conducted to determine whether TGF-beta2 contributes to mitotic inhibition during endothelial development, by investigating when these receptors become coexpressed in the endothelium of neonatal rats. Cyclin-dependent kinase inhibitors (CKIs), such as p27kip1 and p15INK4b, help mediate mitotic inhibition in other cell types. The role of CKIs in inhibiting proliferation in corneal endothelium was examined by first determining the kinetics of p27kip1 expression in neonatal rat corneal endothelium. Studies were then extended to cultured cells to more directly compare the effects of TGF-beta2 and cell-cell contact on the relative protein and mRNA expression of the CKIs, p27kip1, and p15INK4b. METHODS Immunocytochemistry (ICC) detected TGF-beta receptors I, II, and III (RI, RII, RIII, respectively) in the endothelium of rat corneas on postnatal days 1, 10, and 21, and in adult (3-month-old) rats. ICC for p27kip1 was conducted on postnatal days 1, 7, 14, and 21. Samples were taken for p27kip1 RT-PCR on postnatal days 7, 14, and 21 and from adult rats. The effect of TGF-beta2 on p27kip1 and p15INK4b expression was determined in G(0)-phase synchronized subconfluent rat corneal endothelial cells incubated for 24 hours in 10% serum +/- 5 ng/mL TGF-beta2. CKI expression was also examined in fully confluent cultures. RT-PCR and Western blot analysis detected p27kip1 and p15INK4b mRNA and protein expression, respectively. The effect of releasing cells from cell-cell contact on proliferation and p27kip1 protein expression was studied in confluent cultures treated for 1 hour with and without 2.0 mg/mL di-sodium EDTA and then maintained for 24 hours in 10% serum. Cultures were then either fixed for ICC of Ki67, a marker of actively cycling cells, or extracted for Western blot determination of p27kip1 protein. RESULTS Positive staining for TGF-beta RIII was detected on postnatal day 10, and staining for RI and RII was detected on postnatal day 21. The endothelium stained positively for p27kip1 on postnatal day 1 and thereafter, and p27kip1 PCR product was detectable at the earliest time point tested (postnatal day 7). In cultured cells, TGF-beta2 and cell-cell contact had relatively little effect on expression of p27kip1 or p15INK4b mRNA. TGF-beta2 lowered the levels of both proteins, but p27kip1 remained at a higher level than p15INK4b. In confluent cultures, p15INK4b protein was reduced; however, p27kip1 protein levels increased 20-fold. Positive staining for Ki67 was detected, and p27kip1 protein levels substantially decreased in EDTA-treated confluent cells compared with the untreated control. CONCLUSIONS Previous studies from this laboratory showed that corneal endothelial cell proliferation ceases in neonatal rat by postnatal day 13. This timing correlated with the formation of stable cell-cell contacts, implicating contact inhibition as an important mechanism of growth arrest during endothelial development. The current studies showed that coexpression of TGF-beta RI, RII, and RIII occurred too late for TGF-beta2 to have a significant role in inhibiting proliferation during endothelial development. Studies in cultured cells suggest that p27kip1 mediates inhibition of proliferation induced by TGF-beta2, although the response to this cytokine was relatively weak. ICC and RT-PCR of p27kip1 in neonatal endothelium and RT-PCR and Western blot studies in cultured cells indicate that contact inhibition is mediated, in large part, through the activity of p27kip1. These results, together with previous data from this laboratory, strongly suggest that contact inhibition is an important mechanism responsible for inducing cell cycle arrest during corneal endothelial development and for maintaining the mature monolayer in a nonproliferative state. In both cases, contact-induced inhibition is mediated, at least in part, by p27kip1. TGF-beta2 appears not to induce mitotic arrest in the developing endothelium, but may function to maintain the mature endothelium in a nonreplicative state should cell-cell contact be lost in the monolayer.