Dickkopf-1 (Dkk-1) Is a New Agonist of the Orphan Receptor Tyrosine Kinase Ror-2 in Intestinal Epithelia.
Ivan I Pacheco, Jacqueline Kelly, R John MacLeod. Kingston General Hospital and Queen's University, ON, Canada
Background: Dickkopf-1 (Dkk-1), an evolutionary conserved glycoprotein, is a key modulator of the Wnt signaling required for multiple processes in development, in the adult and in carcinogenesis. We have previously reported that Ror-2 is expressed along the crypt to villus axis in the mouse jejunal epithelia. Furthermore, we have also shown that during the transition of adenoma to colorectal carcinoma, there is epithelial loss of the extracellular calcium sensing receptor, Wnt5a and Dkk-1. Interestingly, in the same tissue micro-array we found a marked increase of Ror-2 expression. While examining the significance of the previous finding, we discovered that overexpressing Ror-2 in colon cancer cell lines that constitutively secreted Dkk-1 resulted in activation of a β-catenin reporter. We then hypothesized that whether Ror-2 interacts with Dkk-1, this interaction should have distinct effects than its known ligand, Wnt5a.
Design: To determine if Dkk-1 and Ror-2 physically interact, we co-transfected several colonic adenocarcinoma cell lines with Dkk-1 and full lenght-Ror-2 or Ror-2 binding domain truncation constructs, and co-immunoprecipitation as well as co-immunofluorescence were performed. To determine whether Dkk1 and Wnt5a functionally interact with Ror2 in a distinct manner, we co-expressed either Dkk-1/Ror2 or Wnt5a/Ror2 in the colonic HT-29 and RKO adenocarcinoma cell lines for 48 hours. We then determine their respective effect on SGLT-1, a downstream target of the homeobox CDX2, at the transcriptional (semi-QRT-PCR; or, SGLT-1 promoter assay assays) and protein (Western Blot) levels.
Results: Co-immunoprecipitation assays showed that intact CRD or Kringle domains of Ror-2 are required for Dkk-1 binding. Furthermore, co-immunofluorescence demonstrated that the physical interaction of Dkk-1 and Ror-2 occurs at the membrane level. Dkk-1/Ror2 stimulation of CDX2 and SGLT1 occurred with an EC50 ∼ 5 ng/ml. Furthermore, Dkk-1/Ror2 stimulation of CDX2 production required Src. In contrast, Wnt5a/Ror2 stimulation of CDX2 appears faster and required JNK, CK1 and Src.
Conclusions: Dkk-1 physically interacts with Ror2 to signal in a different manner than Wnt5a interacting with Ror2. We speculate that our newly found Dkk-1/Ror2 interaction may account for some of the β-catenin independent effects of Dkk-1.
Wednesday, March 2, 2011 9:30 AM
Poster Session V # 222, Wednesday Morning