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

Immediate early response gene 2 (IER2), a novel regulator of the cell cycle inhibitor p21waf1/cip1 [p21 hoch waf1/cip1]

Zsfassung in dt. SpracheImmediate early genes (IEGs) werden als Reaktion auf viele (extra)-zelluläre Reize schnell und transient induziert und sie sind oft Teil einer ersten Welle transkripioneller Regulation welche sodann Änderungen in einer Reihe von zellulären Prozessen wie Zellwachstum, Differenzierung, Angiogenese und Entzündung beeinflusst. IEGs haben Auswirkung auf die Biosynthese von Proteinen die u.a. als Transkriptionsfaktoren ihrerseits wiederum die Aktivierung aber auch die Beendigung zellulare Prozesse steuern. Falsche Regulation dieser Prozesse kann die Entstehung von Krankheiten wie Krebs fördern. In einem Mikroarray-basierenden Genexpressions-Experiment wurde zuvor das immediate early response gene 2 (IER2) in menschlichen Endothelzellen identifiziert. In weiterer Folge untersuchten wir in unserem Modellsystem, primären humanen Nabelschnur Endothelzellen (HUVEC), dieses bis dato schlecht charakterisierte IEG auf dessen Funktion.Wir konnten zeigen, dass Endothelzellproliferation und Angiogenese auslösende Wachstumsfaktoren wie der vascular endothelial growth factor (VEGF) oder basic fibroblast growth factor (bFGF) zu einer schnellen und transienten Induktion der IER2 Expression in HUVEC über die Aktivierung der Protein Kinase C (PKC) Signalkaskade führen. In weiterer Folge fanden wir in Zellkulturversuchen mittels Phorbolester (PMA) Stimulierung und RNAi Experimenten, dass IER2 hauptsächlich durch das Isoenzym PKC delta reguliert wird. Darüber hinaus konnten wir erstmals eine spezifische IER2 Protein Funktion zeigen: In der PKC Signalkaskade unterdrückt IER2 eine PMA-induzierte transkriptionell gesteuerte Expression des Zellzyklusinhibitors p21waf1/cip1. Ferner bemerkten wir, dass die IER2 vermittelte Inhibierung von p21waf1/cip1 zu einer veränderten E2F-abhängigen Transkription führt und dabei in HUVEC der PMA-induzierten Apoptose entgegenwirkt. IER2 war darüberhinaus in einigen Krebsmodellen überexpremiert, was auf eine mögliche onkogene Funktion von IER2 bei gleichzeitigem Verlust des Tumorsuppressors p21waf1/cip1 hinweist.Zusammenfassend können wir sagen, dass die IER2 Expression durch PKC Aktivierung reguliert, und durch posttranslationale Mechanismen beeinflusst wird. IER2 als neuer molekularer Regulator des Zellzyklusinhibitors p21waf1/cip1 könnte die Balance zwischen p21waf1/cip1 vermittelten Zelltod in Richtung E2F-angetriebenem Zellwachstum verschieben.Immediate early genes (IEGs) are rapidly and transiently induced in response to several (extra)-cellular stimuli. Often, they encode transcription factors, and thus represent the first transcriptional program that precedes changes in multiple cellular processes including cell growth, differentiation, angiogenesis and inflammation. IEGs influence the activity of secondary mediators, that regulate the activation but also the termination of cellular processes. Their correctly timed regulation is therefore of utmost importance since its impairment carries the potential for deregulated cellular processes, including oncogenic transformation.In a microarray-based gene expression study we previously identified immediate early response gene 2 (IER2) in the human endothelium. We consequently started to investigate the impact of this poorly described IEG with so far unknown protein function in our model system, primary human umbilical vein endothelial cells (HUVEC). We found that endothelial cell proliferation- and angiogenesis-associated growth factors such as vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) rapidly and transiently induce IER2 expression in HUVEC via the activation of the protein kinase C (PKC) signaling cascade. In addition, we discovered by using the phorbol ester PMA as a strong PKC activator and RNAi approaches that IER2 is mainly regulated by the PKC delta isozyme.Moreover, we described for the first time that IER2 in the PKC signaling cascade suppresses PMA-induced cell cycle inhibitor p21waf1/cip1 expression, acting at the level of transcription. Thereby, IER2 reverses the p21waf1/cip1-mediated block of E2F-dependent transcription thus counteracting PMA-induced apoptosis in HUVEC. We subsequently provided evidence that IER2 is accumulative expressed in some cancer models suggesting a potential oncogenic function of IER2 with concomitant loss of tumor suppressor p21waf1/cip1 expression in cancer. Together, our findings propose that IER2 expression is regulated upon PKC activation and posttranslational mechanisms. Moreover our data suggest that IER2 acts as a novel molecular regulator of the cell cycle inhibitor p21waf1/cip1 by influencing the balance between p21waf1/cip1-driven cell death towards E2F-mediated cell survival.1

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 1–3: different α-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 ß2 microglobulin expression, and are expressed as fold induction compared to control cells.</p

Top SOX18 regulated genes.

<p>*verified by real-time PCR;</p>†<p>induction by real-time PCR was 1.5-fold;</p>‡<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 ß2 microglobulin levels, and are expressed as fold induction compared to control cells.</p