Abstract
Regulation of expression of the intestinal epithelial actin-binding protein, villin, is poorly understood. The aim of this study was to determine whether Wnt5a stimulates Ror2 in intestinal epithelia caused transient increases in phospho-ERK1/2 (pERK1/2) and subsequently increased expression of villin transcript and protein. To demonstrate Wnt5a–Ror2 regulation of villin expression, we overexpressed wild-type, truncated, or mutant Ror2 constructs in HT29 adenocarcinoma cells and non-transformed fetally derived human intestinal epithelial cells, 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 extinguished 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 the 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. The increase in villin expression was blocked by pharmacological inhibition of MEK-1 and casein kinase 1, but not by PKC and p38 inhibitors. Neither Wnt3a nor epidermal growth factor addition caused increases in villin protein. Our findings suggest that Wnt5a/Ror2 signaling can regulate villin expression in the intestine.
Highlights
The molecular determinants of villin production in the intestine are largely unknown
Wnt5a INTERACTION WITH Ror2 STIMULATED ERK1/2 PHOSPHORYLATION IN ADENOCARCINOMA-DERIVED HT29 CELLS We first determined whether Wnt5a and Ror2 would stimulate the ERK1/2 (p44/42) MAPK cascade in adenocarcinoma-derived HT29 intestinal epithelial cells (Figure 2)
We found that Wnt5a/Ror2 signaling caused ERK1/2 activation and subsequent villin protein expression in HT29 adenocarcinoma cells and a fetally derived nontransformed human intestinal epithelial cell line
Summary
The molecular determinants of villin production in the intestine are largely unknown. Ror is expressed in murine small intestinal epithelia along the entire crypt–villus axis (Pacheco and MacLeod, 2008). CaSR activation increased Ror expression on the epithelia This paracrine Wnt5a/Ror signaling in intestinal epithelial cells led to the activation of the caudal type homeobox transcription factor 2 (CDX2), and sucrase-isomaltase (Pacheco and MacLeod, 2008) suggestive of increased epithelial differentiation. Villin is regarded a marker for differentiated epithelial cells because of its distinctive expression gradient along the villus–crypt axis (Khurana and George, 2008). It is not known if Wnt5a/Ror signaling influences villin expression
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