Ibuka Souji, Matsumoto Shinji, Fujii Shinsuke, Kikuchi Akira
Departments of Molecular Biology and Biochemistry, Graduate School of Medicine, Osaka University, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan Pediatric Surgery, Graduate School of Medicine, Osaka University, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan.
Departments of Molecular Biology and Biochemistry, Graduate School of Medicine, Osaka University, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan.
J Cell Sci. 2015 Jun 1;128(11):2156-68. doi: 10.1242/jcs.169060. Epub 2015 Apr 23.
Epithelial tubular structures are essential units in various organs. Here, we used rat intestinal epithelial IEC6 cells to investigate tubulogenesis and we found that tubular formation was induced by a combination of Wnt3a and EGF signaling during three-dimensional culture. Wnt3a and EGF induced the expression of the P2Y2 receptor (P2Y2R, also known as P2RY2), and knockdown of P2Y2R suppressed tubular formation. A P2Y2R mutant that lacks nucleotide responsiveness rescued the phenotypes resulting from P2Y2R knockdown, suggesting that nucleotide-dependent responses are not required for P2Y2R functions in tubular formation. The Arg-Gly-Asp (RGD) sequence of P2Y2R has been shown to interact with integrins, and a P2Y2R mutant lacking integrin-binding activity was unable to induce tubular formation. P2Y2R expression inhibited the interaction between integrins and fibronectin, and induced cell morphological changes and proliferation. Inhibition of integrin and fibronectin binding by treatment with the cyclic RGD peptide and fibronectin knockdown induced tubular formation in the presence of EGF alone, but a fibronectin coat suppressed Wnt3a- and EGF-induced tubular formation. These results suggest that Wnt3a- and EGF-induced P2Y2R expression causes tubular formation by preventing the binding of integrins and fibronectin rather than by mediating nucleotide responses.
上皮管状结构是各种器官中的基本单位。在此，我们使用大鼠肠上皮IEC6细胞来研究管形成，并且我们发现，在三维培养过程中，Wnt3a和表皮生长因子（EGF）信号的组合诱导了管状结构的形成。Wnt3a和EGF诱导P2Y2受体（P2Y2R，也称为P2RY2）的表达，并且敲低P2Y2R会抑制管状结构的形成。一种缺乏核苷酸反应性的P2Y2R突变体挽救了因P2Y2R敲低而产生的表型，这表明核苷酸依赖性反应对于P2Y2R在管形成中的功能不是必需的。已证明P2Y2R的精氨酸-甘氨酸-天冬氨酸（RGD）序列与整合素相互作用，并且一种缺乏整合素结合活性的P2Y2R突变体无法诱导管状结构的形成。P2Y2R的表达抑制了整合素与纤连蛋白之间的相互作用，并诱导了细胞形态变化和增殖。用环状RGD肽处理抑制整合素与纤连蛋白的结合以及敲低纤连蛋白，在仅存在EGF的情况下诱导了管状结构的形成，但是纤连蛋白包被抑制了Wnt3a和EGF诱导的管状结构的形成。这些结果表明，Wnt3a和EGF诱导的P2Y2R表达通过阻止整合素与纤连蛋白的结合而非通过介导核苷酸反应来导致管状结构的形成。
