High Wnt signaling represses the proapoptotic proteoglycan syndecan-2 in osteosarcoma cells. Osteosarcoma is characterized by frequent relapse and metastatic disease associated with resistance to chemotherapy. We previously showed that syndecan-2 is a mediator of the antioncogenic effect of chemotherapeutic drugs. The purpose of this work was to elucidate molecular mechanisms responsible for the low expression of syndecan-2 in osteosarcoma. We compared the regulatory activity of cis-acting DNA sequences of the syndecan-2 gene in osteosarcoma and osteoblastic cell lines. We identified a DNA region that negatively regulates syndecan-2 transcription in the osteosarcoma cells. T-cell factors (TCF) bind to this sequence in vivo. Wnt3a stimulation, beta-catenin activation, and TCF overexpression resulted in syndecan-2 repression, whereas Wnt inhibition using sFRP-1 increased syndecan-2 expression in U2OS cells. RhoA activation blunted the stimulatory effect of sFRP-1 on syndecan-2 transcription, whereas RhoA inhibition enhanced syndecan-2 expression. These results indicate that Wnt/beta-catenin and Wnt/RhoA signaling contribute to syndecan-2 repression. The alteration of syndecan-2 expression in osteosarcoma cell lines also seemed to be related to a higher shedding, controlled by Wnt/RhoA. Conversely, syndecan-2 was found to activate its own expression in U2OS cells through RhoA inhibition. These data identify a molecular network that may contribute to the low expression of the proapoptotic proteoglycan syndecan-2 in osteosarcoma cells. The high activity of the canonical Wnt pathway in the different osteosarcoma cells induces a constitutive repression of syndecan-2 transcription, whereas Wnt/RhoA signaling blocks the amplification loop of syndecan-2 expression. Our results identify syndecan-2 as a Wnt target and bring new insights into a possible pathologic role of Wnt signaling in osteosarcoma.Copyright 2010 AACR.