Drm (Down-regulated by mos)/Gremlin is a Bone Morphogenetic Protein (BMP) antagonist, initially identified by our lab, which is expressed in a tissue-specific fashion in vivo. While many primary cells expressed the gene, most transformed cells in culture fail to express DRM/Gremlin, and its expression could be suppressed in fibroblasts by oncogene-mediated activation of the MAPK pathway. We had hypothesized that this loss of expression might be important for tumor initiation or progression, and we have continued to focus our work on the effects of Drm on transformed cells. When retroviral or plasmid clones expressing DRM/Gremlin are introduced into tumor-derived cells that express little or no Drm/Gremlin, several cell lines exhibit a reduction in their transformed phenotype. We used an ecdysone-inducible Drm/Gremlin construct to show that the p53-negative, primitive neuroectodermal-derived Daoy and the osteosarcoma-derived Saos2 cell lines exhibited slower growth in culture and reduced tumor-forming ability in vivo following Drm induction. Using p21Cip1/Waf1 promoter driven luciferase reporters and Real-time PCR, we demonstrated that growth inhibition correlated with the transcriptional induction of p21Cip1/Waf1 and an increase in the level of phosphoryalted p42/44 Mitogen Activated Protein Kinase (MAPK). The induction was independent of p53 and did not require activation of either the p38 or the p42/44 MAP kinases. Our data suggests that the BMP antagonist Drm/Gremlin can act to affect tumor cell growth through its affects on the level of p21Cip1/Waf1 and phoshorylated p42/44 MAPK. In an attempt to identify novel targets for Drm/Gremlin, we also conducted yeast-two hybrid analysis using a kidney library, in collaboration with members of Dr. Alan Perantoni's laboratory. These screens identified several novel potential interacting proteins. In particular, we confirmed that Slit 1, the soluble ligand of the Robo receptor, could interact with Drm. The Slit/Robo pathway has been shown to be involved in kidney development , axon guidance, and lymphocyte chemotaxis, and we are investigating the possible biological significance of Drm-Slit interactions in these processes.
