Wnt5a Interaction with Intestinal Ror2 Regulates Villin Expression
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Regulation of expression of the intestinal actin-binding protein, villin, a marker of intestinal epithelial differentiation, is poorly understood. Activation of the extracellular calcium-sensing receptor (CaSR) on sub-epithelial myofibroblasts stimulated the secretion of Wnt5a, while activation of the CaSR on intestinal epithelia increased expression of Ror2, a Wnt-family co-receptor. Immunocytochemistry has localized Ror2 expression in the epithelia lining the small intestine from the crypt base to the villus tip. The aim of this study was to determine whether Wnt5a binding Ror2 in intestinal epithelia stimulated transient increases in phospho-ERK1/2 (pERK1/2) which lead to increased expression of villin transcript and protein. To examine Wnt5a-Ror2 regulation of villin expression, we transgenically overexpressed wild-type, truncated, or mutant Ror2 constructs in HT-29 adenocarcinoma cells and nontransformed fetally-derived human intestinal epithelial cells (HIECs), added conditioned media containing Wnt5a and measured changes in ERK1/2 phosphorylation, villin amplicons and protein expression by RT-PCR and Western blot techniques. Wnt5a addition caused a transient increase in pERK1/2, which was maximal at 10 min but diminished by 30 min. Transient transfection with a siRNA duplex against Ror2 diminished Ror2 amplicons and protein and reduced the extent of pERK1/2 activation. Structure-function analysis revealed that deletion of the cysteine-rich, kringle, or tyrosine kinase domain or substitution mutations of tyrosine residues in the intracellular Ser/Thr-1 region of Ror2 prevented the Wnt5a-stimulation of pERK1/2. Deletion of the intracellular proline and serine/threonine rich regions of Ror2 had no effect on Wnt5a-stimulation of pERK1/2 in HT29 cells. Western blot analysis demonstrated that villin protein was increased by over-expression of wild-type Ror2 in HT-29 cells and HIECs in the presence of Wnt5a. The increase in villin expression was blocked by pharmacological inhibition of MEK1&2 and casein kinase 1, but not by PKC and p38 inhibitors. Neither Wnt3a nor EGF addition increased villin protein. This work suggested that stromal Wnt5a will stimulate pERK1/2 via the Ror2 tyrosine kinase domain to generate increased villin protein. These findings suggested that Ror2 homeostasis and Wnt5a interaction with Ror2 are important determinants of the regulation of villin expression in the intestine.