Department of Ophthalmology, Ludwig-Maximilians-University, Mathildenstrasse 8, Munich, Germany.
Biochimie. 2011 Mar;93(3):477-88. doi: 10.1016/j.biochi.2010.10.021. Epub 2010 Nov 20.
Adhesion and spreading of retinal pigment epithelial (RPE) cells on fibronectin-rich extracellular matrices is a crucial event in the pathogenesis of proliferative vitreoretinopathy (PVR). In the present study we explored the capacity of galectin-3, a β-galactoside-binding endogenous lectin, to inhibit early PVR-associated cellular events from a therapeutic perspective. We assessed the relative expression levels of galectin-3 in native RPE and dedifferentiated, cultured RPE. Galectin-3 was constitutively expressed under in vivo and in vitro conditions and was abundant in cultured cells. Treatment of human RPE cells with soluble galectin-3 disclosed no toxicity within control limits up to 250 μg/ml. When added to the medium, galectin-3 dose-dependently inhibited attachment and spreading of the cells on fibronectin by more than 75%. When coated on the plastic surface, galectin-3 alone impaired attachment and spreading of RPE cells, and reduced attachment but not spreading on fibronectin. Galectin-3 bound to the cell surface, and, as determined by the use of the competing sugar β-lactose, galectin-3-mediated effects were dependent on carbohydrate binding. To ascertain the role of the ability of galectin-3 to form pentamers, we proteolytically removed the N-terminal, cross-linking section. The remaining C-terminal carbohydrate-binding domain alone failed to bind to cells and was functionally inactive. These results emphasize the relevance of both properties, i.e., glycan-binding and cross-linking of glycan moieties, for the inhibitory activity of galectin-3. Incubation of mobilized RPE cells with galectin-3 significantly disturbed microfilament assembly and, in correlation with decreased attachment, inhibited ERK phosphorylation. Therefore, galectin-3, acting as a cross-linking lectin on the cell surface, negatively regulates attachment and spreading of RPE cells in vitro. This effect, at least in part, is attributed to an inhibition of the ERK-MAPK pathway, which prevents cytoskeletal rearrangements needed for RPE cell attachment and spreading. Further investigation at this pathway may disclose a promising nouveau perspective for treatment and prophylaxis of early PVR.
视网膜色素上皮 (RPE) 细胞在富含纤维连接蛋白的细胞外基质上的黏附和铺展是增生性玻璃体视网膜病变 (PVR) 发病机制中的关键事件。在本研究中，我们从治疗的角度探讨了半乳糖凝集素-3（一种 β-半乳糖苷结合的内源性凝集素）抑制早期与 PVR 相关的细胞事件的能力。我们评估了天然 RPE 和去分化的培养 RPE 中半乳糖凝集素-3 的相对表达水平。在体内和体外条件下，半乳糖凝集素-3 均持续表达，并且在培养细胞中含量丰富。在 250μg/ml 以内的对照限度内，可溶性半乳糖凝集素-3 处理人 RPE 细胞无毒性。当添加到培养基中时，半乳糖凝集素-3 以剂量依赖性方式抑制细胞在纤维连接蛋白上的黏附和铺展，抑制率超过 75%。当涂覆在塑料表面上时，半乳糖凝集素-3 单独即可损害 RPE 细胞的黏附和铺展，并减少细胞在纤维连接蛋白上的黏附而不影响铺展。半乳糖凝集素-3 结合到细胞表面，并且如通过使用竞争糖β-乳糖确定的那样，半乳糖凝集素-3 介导的作用依赖于碳水化合物结合。为了确定半乳糖凝集素-3 形成五聚体的能力的作用，我们使用蛋白水解法去除 N 端交联区。单独保留的 C 端碳水化合物结合结构域不能与细胞结合，并且功能失活。这些结果强调了聚糖结合和糖基部分交联的两种特性对半乳糖凝集素-3 的抑制活性的相关性。用半乳糖凝集素-3 孵育动员的 RPE 细胞可显著扰乱微丝组装，并且与附着减少相关，抑制 ERK 磷酸化。因此，半乳糖凝集素-3 在细胞表面作为交联凝集素起作用，体外抑制 RPE 细胞的黏附和铺展。该作用至少部分归因于 ERK-MAPK 途径的抑制，该途径防止了 RPE 细胞附着和铺展所需的细胞骨架重排。对该途径的进一步研究可能会揭示治疗和预防早期 PVR 的有希望的新视角。
