Isolation and biochemical characterization of the human Dkk-1 homologue, anovel inhibitor of mammalian Wnt signaling

In an effort to isolate novel growth factors, we identified a human protein , designated Sk, that co-eluted with Neuregulin during chromatographic sepa ration of conditioned medium from the SK-LMS-1 human leiomyosarcoma cell li ne. Degenerate oligonucleotides based on amino-terminal sequence analysis o f the purified protein were used to isolate the corresponding cDNA from a l ibrary generated from this cell line. Sk is a novel 266-amino acid protein that contains a signal peptide sequence and two cysteine-rich domains with no similarity to other known growth factors. A single major 2-kilobase tran script was expressed in several embryonic tissues, Transfection of mammalia n cells demonstrated that the protein was secreted and expressed as a doubl et of approximately 35 kDa. In vitro translation and endoglycosylase analys is indicated that this doublet, which was also observed in cells expressing the endogenous protein, arises from posttranslational modification. A sear ch of the GenBank(TM) data base revealed a match of Sk with Dkk-1, which is a novel secreted protein required for head induction in amphibian embryos and a potent Wnt inhibitor. When coexpressed with Wnt-2 in NIH3T3 cells, hu man Sk/Dkk-1 caused reversion of Wnt-2 induced morphological alterations an d inhibited the Wnt-2 induced increase in uncomplexed beta-catenin levels. These results provide biochemical evidence that human Sk/Dkk-1 antagonizes Wnt signaling upstream of its effect on beta-catenin regulation.