André Sabine, Sanchez-Ruderisch Hugo, Nakagawa Hiroaki, Buchholz Malte, Kopitz Jürgen, Forberich Pia, Kemmner Wolfgang, Böck Corina, Deguchi Kisaburo, Detjen Katharia M, Wiedenmann Bertram, von Knebel Doeberitz Magnus, Gress Thomas M, Nishimura Shin-Ichiro, Rosewicz Stefan, Gabius Hans-Joachim
Institute of Physiological Chemistry, Faculty of Veterinary Medicine, Ludwig-Maximilians-University Munich, Germany.
FEBS J. 2007 Jul;274(13):3233-56. doi: 10.1111/j.1742-4658.2007.05851.x. Epub 2007 May 29.
Expression of the tumor suppressor p16(INK4a) after stable transfection can restore the susceptibility of epithelial tumor cells to anoikis. This property is linked to increases in the expression and cell-surface presence of the fibronectin receptor. Considering its glycan chains as pivotal signals, we assumed an effect of p16(INK4a) on glycosylation. To test this hypothesis for human Capan-1 pancreatic carcinoma cells, we combined microarray for selected glycosyltransferase genes with 2D chromatographic glycan profiling and plant lectin binding. Major differences between p16-positive and control cells were detected. They concerned expression of beta1,4-galactosyltransferases (down-regulation of beta1,4-galactosyltransferases-I/V and up-regulation of beta1,4-galactosyltransferase-IV) as well as decreased alpha2,3-sialylation of O-glycans and alpha2,6-sialylation of N-glycans. The changes are compatible with increased beta(1)-integrin maturation, subunit assembly and binding activity of the alpha(5)beta(1)-integrin. Of further functional relevance in line with our hypothesis, we revealed differential reactivity towards endogenous lectins, especially galectin-1. As a result of reduced sialylation, the cells' capacity to bind galectin-1 was enhanced. In parallel, the level of transcription of the galectin-1 gene increased conspicuously in p16(INK4a)-positive cells, and even figured prominently in a microarray on 1996 tumor-associated genes and in proteomic analysis. The cells therefore gain optimal responsiveness. The correlation between genetically modulated galectin-1 levels and anoikis rates in engineered transfectants inferred functional significance. To connect these findings to the fibronectin receptor, galectin-1 was shown to be co-immunoprecipitated. We conclude that p16(INK4a) orchestrates distinct aspects of glycosylation that are relevant for integrin maturation and reactivity to an endogenous effector as well as the effector's expression. This mechanism establishes a new aspect of p16(INK4a) functionality.
稳定转染后肿瘤抑制因子p16(INK4a)的表达可恢复上皮肿瘤细胞对失巢凋亡的敏感性。这一特性与纤连蛋白受体表达增加及其在细胞表面的存在有关。鉴于其糖链为关键信号，我们推测p16(INK4a)对糖基化有影响。为了在人Capan-1胰腺癌细胞中验证这一假设，我们将选定的糖基转移酶基因微阵列与二维色谱聚糖谱分析及植物凝集素结合分析相结合。检测到p16阳性细胞与对照细胞之间存在主要差异。这些差异涉及β1,4-半乳糖基转移酶的表达（β1,4-半乳糖基转移酶-I/V下调，β1,4-半乳糖基转移酶-IV上调）以及O-聚糖的α2,3-唾液酸化减少和N-聚糖的α2,6-唾液酸化减少。这些变化与β(1)-整合素成熟、亚基组装及α(5)β(1)-整合素的结合活性增加相一致。与我们的假设一致，进一步具有功能相关性的是，我们发现细胞对内源凝集素，尤其是半乳糖凝集素-1的反应性存在差异。由于唾液酸化减少，细胞结合半乳糖凝集素-1的能力增强。同时，半乳糖凝集素-1基因的转录水平在p16(INK4a)阳性细胞中显著增加，甚至在1996个肿瘤相关基因的微阵列及蛋白质组分析中也表现突出。因此，细胞获得了最佳反应性。基因调控的半乳糖凝集素-1水平与工程转染细胞中失巢凋亡率之间存在相关性，这推断出其功能意义。为了将这些发现与纤连蛋白受体联系起来，研究表明半乳糖凝集素-1可被共免疫沉淀。我们得出结论，p16(INK4a)协调糖基化的不同方面，这些方面与整合素成熟、对内源效应物的反应性以及效应物的表达相关。这一机制确立了p16(INK4a)功能的一个新方面。
