Molecular cloning of the Matrix Gla Protein gene from Xenopus laevis. Functional analysis of the promoter identifies a calcium sensitive region required for basal activity

Abstract

To analyze the regulation of Matrix Gla Protein (MGP) gene expression in Xenopus laevis, we cloned the xMGP gene and its 5¢ region, determined their molecular organization, and characterized the transcriptional properties of the core promoter. The Xenopus MGP (xMGP) gene is organized into five exons, one more as its mammalian counterparts. The first two exons in the Xenopus gene encode the DNA sequence that corresponds to the first exon in mammals whereas the last three exons show homologous organization in the Xenopus MGP gene and in the mammalian orthologs. We characterized the transcriptional regulation of the xMGP gene in transient transfections using Xenopus A6 cells. In our assay system the identified promoter was shown to be transcriptionally active, resulting in a 12-fold induction of reporter gene expression. Deletional analysis of the 5¢ end of the xMGP promoter revealed a minimal activating element in the sequence from )70 to )36 bp. Synthetic reporter constructs containing three copies of the defined regulatory element delivered 400-fold superactivation, demonstrating its potential for the recruitment of transcriptional activators. In gel mobility shift assays we demonstrate binding of X. laevis nuclear factors to an extended regulatory element from )180 to )36, the specificity of the interaction was proven in competition experiments using different fragments of the xMGP promoter. By this approach the major site of factor binding was demonstrated to be included in the minimal activating promoter fragment from )70 to )36 bp. In addition, in transient transfection experiments we could show that this element mediates calcium dependent transcription and increasing concentrations of extracellular calcium lead to a significant dose dependent activation of reporter gene expression.

Dual transcriptional regulation by runx2 of matrix Gla protein in Xenopus laevis

“…We previously reported the identification of two functional promoters for the XlMGP gene (Conceição et al, 2002;Conceição et al, 2005) and by in silico analysis identified multiple (N = 7) consensus sequence motifs homologous to runx2 binding elements found within regulatory regions of other genes expressed in osteoblasts and chondrocytes and known targets of Runx2 (Meyers et al, 1993;Ducy and Karsenty, 1995;Ducy et al, 1997;Sato et al, 1998;Jiménéz et al, 1999;Wang et al, 2004). Consistent with these findings, we show in this paper that both XlMGP gene promoters are transactivated by runx2 through three specific regions.…”

“…The plasmids −949/+33LuC, − 783/+33LuC, −464/+44LuC, − 185/+ 44LuC, − 54/+ 33LuC, − 949/− 708LuC, + 1278/ + 2083LuC, + 2123/+ 3013LuC, + 2524/+ 3013LuC, + 2733/ +3013LuC, +2843/+3013LuC and + 2818/+3013LuC were described previously (Conceição et al, 2002(Conceição et al, , 2005.…”

Development of two bone-derived cell lines from the marine teleost Sparus aurata ; evidence for extracellular matrix mineralization and cell-type-specific expression of matrix Gla protein and osteocalcin

“…VSa13 cells strongly express the MGP gene under normal culture conditions in both L-15 and D-MEM, but basal expression is higher in D-MEM. Based on recent data suggesting the direct regulation of Xenopus MGP gene expression by extracellular calcium concentrations (Conceiç¼o et al 2002), we hypothesize that the higher basal levels of MGP seen in VSa13 cells results from the higher calcium content in D-MEM (1.8 mM) than in L-15 (1.3 mM). Furthermore, this effect is at least partially mediated via a limited region of the proximal Xenopus MGP gene promoter, which is known to interact with specific protein nuclear factors, thus indicating that one of these factors may be a calcium-sensing protein (Conceiç¼o et al 2002).…”

“…Based on recent data suggesting the direct regulation of Xenopus MGP gene expression by extracellular calcium concentrations (Conceiç¼o et al 2002), we hypothesize that the higher basal levels of MGP seen in VSa13 cells results from the higher calcium content in D-MEM (1.8 mM) than in L-15 (1.3 mM). Furthermore, this effect is at least partially mediated via a limited region of the proximal Xenopus MGP gene promoter, which is known to interact with specific protein nuclear factors, thus indicating that one of these factors may be a calcium-sensing protein (Conceiç¼o et al 2002). Our efforts to correlate this calciumdependent modulation of MGP gene expression with the concomitant expression of the recently cloned S. aurata calcium-sensing receptor have not been successful, since the presence of this mRNA cannot be detected in our cells, either before or after calcium treatment (unpublished).…”

Matrix Gla protein in turbot (Scophthalmus maximus): Gene expression analysis and identification of sites of protein accumulation

“…Mgp is normally found associated with the organic matrix of cartilage and bone in vivo, and it is expressed in various vertebrate tissues such as cartilage, kidney, lung, aorta, bone and tooth Hao et al, 1995;Hashimoto et al, 2001;Hale et al, 1988). Although a wide range of cell types express Mgp, only chondrocytes, endothelial cells and VSMC (vascular smooth muscle cells) synthesize significant levels of Mgp in vivo (Conceição, et al, 2002;Luo et al, 1997;OrtizDelgado et al, 2005, b;Simes et al, 2003). After the discovery of Mgp in various soft tissues, it was proposed that this protein could act as a local inhibitor of mineralization .…”