Hutcheon Audrey E K, Guo Xiaoqing Q, Stepp Mary Ann, Simon Kenneth J, Weinreb Paul H, Violette Shelia M, Zieske James D
Schepens Eye Research Institute and Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts 02114, USA.
Invest Ophthalmol Vis Sci. 2005 Jul;46(7):2362-8. doi: 10.1167/iovs.04-0759.
In a prior study, it was reported that both TGF-beta receptors type-I and -II are upregulated after wounding, suggesting that TGF-beta signaling may play a role in corneal epithelial repair. The Smad proteins, which translocate into the nucleus after activation of the TGF-beta receptors, are key factors in the major TGF-beta signaling pathway. The present study was undertaken to examine whether Smads 2 and 4 translocate into the nucleus during wound repair and whether the wound type affects the extent of translocation.
Either a 3-mm superficial keratectomy or epithelial debridement was performed on adult Sprague-Dawley rats. The eyes were allowed to heal from 4 hours to 2 weeks. Indirect immunofluorescence was performed with anti-Smads 2 and 4, anti-laminin, a marker of basement membrane, and anti-alphavbeta6 integrin, which has been implicated in TGF-beta activation. In addition, the effect of the p38MAPK inhibitor SB202190 on healing rates of debridement and keratectomy wounds was determined in organ culture.
In unwounded tissue, Smad 2 was cytoplasmic. By 4 hours after keratectomy, nuclear localization was visible in a few epithelial basal cells at the leading edge of the wound. The number of basal cells expressing nuclear Smad 2 in the wound area increased with time, peaking at 48 hours (95%). However, in the debridement model, Smad 2 localization remained primarily cytoplasmic. Smad 4 showed similar localization. In both wound models, p38MAPK inhibitor slowed epithelial migration, and alphavbeta6 integrin appeared to be upregulated with localization primarily observed in the basal cells migrating over the wound area.
The presence of the basement membrane appears to have an effect on the extent and duration of translocation of the Smad 2 and 4 proteins during corneal epithelial wound repair. The Smad pathway does not appear to be essential for migration; rather, it may play a role in resynthesis of the basement membrane.
在先前的一项研究中，据报道，I型和II型转化生长因子-β（TGF-β）受体在受伤后均上调，这表明TGF-β信号传导可能在角膜上皮修复中起作用。Smad蛋白在TGF-β受体激活后易位至细胞核，是主要TGF-β信号通路中的关键因子。本研究旨在检查Smad 2和4在伤口修复过程中是否易位至细胞核，以及伤口类型是否会影响易位程度。
对成年Sprague-Dawley大鼠进行3毫米的浅层角膜切除术或上皮清创术。让眼睛从4小时到2周愈合。用抗Smad 2和4、抗层粘连蛋白（基底膜标志物）和抗αvβ6整合素（与TGF-β激活有关）进行间接免疫荧光检测。此外，在器官培养中确定p38丝裂原活化蛋白激酶（p38MAPK）抑制剂SB202190对清创术和角膜切除术伤口愈合率的影响。
在未受伤组织中，Smad 2位于细胞质中。角膜切除术后4小时，在伤口前缘的一些上皮基底细胞中可见核定位。伤口区域中表达核Smad 2的基底细胞数量随时间增加，在48小时达到峰值（95%）。然而，在清创术模型中，Smad 2的定位主要仍在细胞质中。Smad 4显示出类似的定位。在两种伤口模型中，p38MAPK抑制剂均减缓上皮迁移，并且αvβ6整合素似乎上调，其定位主要在伤口区域上方迁移的基底细胞中观察到。
基底膜的存在似乎对角膜上皮伤口修复过程中Smad 2和4蛋白的易位程度和持续时间有影响。Smad途径似乎对迁移不是必需的；相反，它可能在基底膜的重新合成中起作用。
