7 research outputs found
The Transcription Factor SOX18 Regulates the Expression of Matrix Metalloproteinase 7 and Guidance Molecules in Human Endothelial Cells
Mutations in the transcription factor SOX18 are responsible for specific cardiovascular defects in humans and mice. In order to gain insight into the molecular basis of its action, we identified target genes of SOX18 and analyzed one, MMP7, in detail.SOX18 was expressed in HUVEC using a recombinant adenoviral vector and the altered gene expression profile was analyzed using microarrays. Expression of several regulated candidate SOX18 target genes was verified by real-time PCR. Knock-down of SOX18 using RNA interference was then used to confirm the effect of the transcription factor on selected genes that included the guidance molecules ephrin B2 and semaphorin 3G. One gene, MMP7, was chosen for further analysis, including detailed promoter studies using reporter gene assays, electrophoretic mobility shift analysis and chromatin-immunoprecipitation, revealing that it responds directly to SOX18. Immunohistochemical analysis demonstrated the co-expression of SOX18 and MMP7 in blood vessels of human skin.The identification of MMP7 as a direct SOX18 target gene as well as other potential candidates including guidance molecules provides a molecular basis for the proposed function of this transcription factor in the regulation of vessel formation
SOX18 binds to the proximal site in the <i>MMP7</i> promoter <i>in vitro</i> and <i>in vivo</i>.
<p>A, Electrophoretic mobility shift assay (EMSA). Binding of SOX18 to its proximal site in the <i>MMP7</i> promoter was analyzed using nuclear extracts from HUVEC. Specificity of binding was demonstrated by competition experiments using the same (wt), a mutated (mut) or an unrelated (unrel) oligonucleotide as indicated, the specific complex is indicated by an arrow. B, The same site was analyzed by chromatin immunoprecipitation in HUVEC. Lane 1β3: different Ξ±-Sox18 antibodies (SantaCruz, Thermo, Chemicon, respectively) as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030982#s2" target="_blank">Materials and Methods</a>, Ig: IgG control antibody; inp: input, M: 100 bp marker. The 120 bp PCR fragments obtained after amplification ofDNA precipitated by the anti-SOX18 antibodies and input DNA are marked by an arrow.</p
Real-time PCR analysis of SOX18 target genes.
<p>RNA was isolated from non-transduced (mock), control virus (AdvEGFP) and SOX18 adenovirus (AdvSOX18) transduced HUVEC and analyzed for the expression of matrix metalloproteinase 7 (<i>MMP7</i>), ephrinB2 <i>(EFNB2), EPHA7</i>, semaphorin 3G <i>(SEMA3G)</i>, interleukin 7 receptor (<i>IL-7R</i>), and cannabinoid receptor 1 (<i>CNR1</i>). Values were normalized for Γ2 microglobulin expression, and are expressed as fold induction compared to control cells.</p
Top SOX18 regulated genes.
<p>*verified by real-time PCR;</p>β <p>induction by real-time PCR was 1.5-fold;</p>β‘<p>no change in real-time PCR.</p
Knock-down of SOX18 diminishes the expression of target genes.
<p>HUVEC were transfected with siRNAs directed against <i>SOX17</i> or <i>SOX18</i>, or with a scrambled control (Scr), and expression of target genes analyzed by real-time PCR. Values were normalized for Γ2 microglobulin levels, and are expressed as fold induction compared to control cells.</p