Matrix Gla protein (MGP) is an extracellular mineral-binding protein expressed in several tissues but it only accumulates in bone and calcified cartilage under physiological conditions. Available evidence indicates that it acts as a physiological inhibitor of mineralization. Runx2 is a transcription factor essential for bone formation in mammals, affecting osteoblast and chondrocyte differentiation by regulating key genes crucial for bone and cartilage development. Being an important cartilage-associated gene, MGP is a potential target for Runx2, and thus we have investigated the possible functional interactions between them. In A6 cells, Runx2 was found to modulate MGP transcription and deletion analysis of MGP distal and proximal promoter-luciferase constructs identified cis-regulatory regions. Interestingly, we have also identified a runx2-binding site that mediates transcriptional repression of XIMGP. Mutation of this element, located between -54 and +33 bp, results in 18-fold up-regulation of transcription. Furthermore, and in addition to the previously reported Xlrunx2 types I and II, we have identified three transcripts encoding novel, truncated Xlrunx2 isoforms. Although only type I and type II could transactivate XIMGP, the truncated isoforms identified in this study, which result from alternative splicing, could be involved in negative regulation of MGP expression, as described for other RUNX2 truncated isoforms acting in other target genes. In vivo microinjection of XIMGP promoter constructs and runx2 mRNA confirmed that those promoters are targets for this transcription factor. These data also indicate that MGP is under dual regulation by runx2 through the use of various isoforms and context-dependent formation of transcriptional complexes. (C) 2009 Elsevier B.V. All rights reserved.