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056 EXTRACELLULAR CALCIUM-SENSING RECEPTOR (CASR) AND WNT5A UTILIZE ORPHAN RECEPTOR ROR2 TO INCREASE CDX2 AND SUCRASE-ISOMALTASE OF INTESTINAL EPITHELIAL CELLS I Pacheco, RJ MacLeod BACKGROUND: Little is known about the extracellular determinants of homeodomain transcription factor CDX2 regulation. The Wnt family member Wnt5a may interact with Frizzled 2,4,7 or the orphan receptor Ror2 to signal in heterologous systems. Because stromal Wnt5a transcripts are present in the small intestine, the current experiments determined whether Wnt5a influenced expression of CDX2 and sucrase-isomaltase, as indices of epithelial differentiation.
GIDRU, Department of Physiology, Queen's University, Kingston, Ontario
METHODS: HT-29 and Caco-2 cells were transiently transfected with Wnt5a (or Wnt3a) and 48h later CDX2 protein and transcripts were assessed by Western blot and RT-PCR, respectively. Sucrase-isomaltase (SI) expression was measured by luciferase reporter in cells transfected with wild type Ror2, dominant negative Ror2 (Ror-GPI), with or without Wnt5a, and 48h later luciferase measured.
RESULTS: Transient transfection of HT-29 adenocarcinoma cells with Wnt5a but not Wnt3a increased CDX2 transcripts and protein. Comparable treatment of Caco-2 cells revealed substantial increases in CDX2 protein. The half-life of CDX2 protein was increased in proliferative cells (12h vs 0.5h) after CaSR activation or transfection with Wnt5a. SI-reporter activity was increased dose-responsively by Wnt5a but not Wnt3a or Wnt1 in both cell types. Overexpression of wild type Ror2 increased Wnt5a stimulation of SI-reporter (p<0.05). Dominant negative Ror2-GPI reduced Wnt5a stimulation of SI reporter. Increasing Ror2 expression increased Wnt5a stimulation of CDX2 transcript which was blocked by dominant negative Ror2. CaSR activation of both cell types increased Ror2 protein.
CONCLUSION: We conclude that Wnt5a interacts with endogenous Ror2 on epithelial cells to stimulate synthesis of CDX2 and sucrase-isomaltase. Both CaSR and Wnt5a increase CDX2 protein half-life. We speculated that CaSR/Wnt5a/Ror2 is a dynamic axis contributing to CDX2 homeostasis in the intestine.