MacLeod R John, Hayes Madeline, Pacheco Ivan
Department of Physiology, Queen's University, 76 Stuart Street, Kingston, Ontario, Canada.
Am J Physiol Gastrointest Liver Physiol. 2007 Jul;293(1):G403-11. doi: 10.1152/ajpgi.00119.2007. Epub 2007 Apr 26.
To understand the role of the colonic extracellular calcium-sensing receptor (CaSR) in calcium chemoprotection against colon cancer, we activated the CaSR with 5 mM Ca(2+) on HT-29 cells, an adenocarcinoma cell line. High Ca(2+) stimulated the upregulation (as assessed by RT-PCR) and the secretion of Wnt5a (assessed by Western blot), a noncanonical Wnt family member. Inhibiting CaSR activity with a short interfering RNA (siRNA) duplex against the CaSR reduced CaSR protein and prevented the secretion of Wnt5a. Dominant negative CaSR (R185Q) or siRNA blocked the high Ca(2+)-mediated inhibition of the beta-catenin reporter TOPflash. The CaSR/Wnt5a inhibition of beta-catenin reporter was prevented by dominant negative ubiquitin ligase seven in absentia homolog 2 (Siah2). In low-calcium medium, overexpressing Wnt5a increased Siah2 amplicons and protein. Inducing the expression of full-length adenomatous polyposis coli (APC) prevented CaSRmediated increases of Siah2 and Wnt5a. Overexpressing the receptor tyrosine kinase-like orphan receptor 2 (Ror2) increased Wnt5a and CaSR-mediated inhibition of TOPflash. Conditioned medium from Wnt5a-transfected cells added to HT-29 cells in low-Ca(2+) medium inhibited the beta-catenin reporter. This inhibition was blocked dose responsively by Frizzled-8/Fc chimeric antibody. Overexpression of Ror2 in HT-29 cells in low-Ca(2+) medium increased the inhibition of beta-catenin reporter caused by recombinant Wnt5a protein compared with addition of Wnt5a protein alone. Our findings demonstrate that APC status plays a key role as a determinant of Wnt5a secretion and suggest that CaSR-mediated secretion of Wnt5a will inhibit defective Wnt signaling in APC-truncated cells in an autocrine manner.
为了解结肠细胞外钙敏感受体（CaSR）在钙对结肠癌化学保护中的作用，我们用5 mM Ca(2+) 激活了人结肠腺癌HT-29细胞系中的CaSR。高钙（Ca(2+)）刺激了非经典Wnt家族成员Wnt5a的上调（通过逆转录聚合酶链反应（RT-PCR）评估）和分泌（通过蛋白质免疫印迹法评估）。用针对CaSR的小干扰RNA（siRNA）双链体抑制CaSR活性可降低CaSR蛋白水平并阻止Wnt5a的分泌。显性负性CaSR（R185Q）或siRNA可阻断高钙（Ca(2+)）介导的β-连环蛋白报告基因TOPflash的抑制。泛素连接酶7同源物2（Siah2）显性负性突变可阻止CaSR/Wnt5a对β-连环蛋白报告基因的抑制。在低钙培养基中，过表达Wnt5a可增加Siah2扩增子和蛋白水平。诱导全长腺瘤性息肉病 coli（APC）的表达可阻止CaSR介导的Siah2和Wnt5a增加。过表达受体酪氨酸激酶样孤儿受体2（Ror2）可增加Wnt5a并增强CaSR介导的TOPflash抑制。将Wnt5a转染细胞的条件培养基添加到低钙（Ca(2+)）培养基中的HT-29细胞中可抑制β-连环蛋白报告基因。这种抑制可被卷曲蛋白8（Frizzled-8）/Fc嵌合抗体剂量依赖性阻断。与单独添加Wnt5a蛋白相比，在低钙（Ca(2+)）培养基中HT-29细胞中过表达Ror2可增强重组Wnt5a蛋白对β-连环蛋白报告基因的抑制。我们的研究结果表明，APC状态作为Wnt5a分泌的决定因素起着关键作用，并提示CaSR介导的Wnt5a分泌将以自分泌方式抑制APC截短细胞中缺陷的Wnt信号传导。
