Dual transcriptional regulation by runx2 of matrix Gla protein in Xenopus laevis Cindy Fazenda a,1 , Brigite Simões a,1 , Robert N. Kelsh b , M. Leonor Cancela a , Natércia Conceição a, ⁎ a University of Algarve, CCMAR, Campus de Gambelas, 8005-139 Faro, Portugal b Department of Biology and Biochemistry, University of Bath, Bath, UK abstract article info Article history: Received 9 July 2009 Received in revised form 26 October 2009 Accepted 28 October 2009 Available online 4 November 2009 Received by A. Rynditch Keywords: Transcription factor Transfection Functional analysis Xenopus MGP Runx2 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 XlMGP. 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 XlMGP, 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 XlMGP 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. © 2009 Elsevier B.V. All rights reserved. 1. Introduction Matrix Gla protein (MGP) belongs to the vitamin K-dependent (VKD) protein family, characterized by the post-translational conver- sion of specific glutamate (Glu) into γ-carboxyglutamate (Gla) residues (Furie and Furie, 1988). MGP, through the calcium-binding property of its Gla residues, plays an essential role in controlling tissue calcification by acting as a physiological inhibitor of extracellular matrix mineralization, as demonstrated by the incidence of severe ectopic calcifications in MGP-null mice (Luo et al., 1997). In humans, Monckeberg sclerosis and Keutel syndrome, both characterized by ectopic calcifications, are consequences of naturally occurring MGP gene mutations (Ziereisen et al., 1993; Schurgers et al., 2005). Despite the importance of MGP as an inhibitor of calcification and its potential role in diseases such as atherosclerosis (O'Donnell et al., 2006), little is known about its transcriptional regulation although the human MGP gene structure was determined two decades ago (Cancela et al., 1990). Expression of the MGP gene in mammals is regulated by steroid hormones such as retinoic acid (RA) (Cancela and Price, 1992; Kirfel et al., 1997) and the hormonally active form of vitamin D3 (Cancela and Price, 1992), as well as by growth and cell proliferation factors (Cancela et al., 1997; Zhao and Warburton, 1997). All known MGP gene promoters are rich in consensus structural motifs similar to known DNA binding sites of nuclear factors including TATA and CCAAT boxes, AP-1, AP-2, metal responsive elements and possible binding sites for cAMP-dependent transcription factors and steroid hormone receptors such as RA. Despite this large array of possible regulatory elements, only a few have been confirmed to mediate transcriptional regulation of MGP. These include (i) a negative responsive element identified in human MGP and shown to function as common binding site for both the RA receptor complex and the CAAT binding protein (Kirfel et al., 1997), and conserved in the distal promoter of MGP in the marine fish Sparus aurata (Conceição et al., 2008); (ii) a polymorphism within the human MGP promoter shown to affect binding of the AP-1 complex, resulting in altered transcrip- tion (Farzaneh-Far et al., 2001); (iii) a partially-defined sequence in the 5′-flanking region of mouse MGP gene identified as sufficient for transcriptional activation by FGF2 (Stheneur et al., 2003); and (iv) Sp proteins and Runx2 mediating MGP transcription upon parathyroid hormone (PTH) treatment (Suttamanatwong et al., 2009). Gene 450 (2010) 94–102 Abbreviations: MGP, matrix Gla protein; TFSEARCH, transcriptional factor search; Runx2, Runt-related transcription factor 2; Cbfa1, core binding factor a 1. ⁎ Corresponding author. Tel.: +351 289800971; fax: +351 289800069. E-mail address: nconcei@ualg.pt (N. Conceição). 1 These authors contributed equally to this work. 0378-1119/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.gene.2009.10.007 Contents lists available at ScienceDirect Gene journal homepage: www.elsevier.com/locate/gene