TNFα-induced down-regulation of Sox18 in endothelial cells is dependent on NF-κB.

The transcription factor Sox18 plays a role in angiogenesis, including lymphangiogenesis, where it is upregulated by growth factors and directs the expression of genes encoding, e.g., guidance molecules and a matrix metalloproteinase. Conversely, we found that in human umbilical vein endothelial cells (HUVEC) Sox18 is repressed by the pro-inflammatory mediator TNFα (as well as IL-1 and LPS). Since a common feature of these mediators is the activation of the NF-κB signaling pathway, we investigated whether Sox18 downregulation is dependent on this transcription factor. Transduction of HUVEC with an adenoviral vector directing the expression of the NF-κB inhibitor IκBα prevented the downregulation of Sox18. Transient transfections of Sox18 promoter reporter genes revealed that the downregulation takes place on the level of transcription, and that the p65/RelA subunit of NF-κB was operative. Furthermore, the responsible promoter region of Sox18 is located within -1.0kb from the transcriptional start site. The repression of Sox18 and its target genes may lead to altered formation of vessels in inflamed settings.

Thrombin induces the expression of oncostatin M via AP-1 activation in human macrophages: a link between coagulation and inflammation.

Macrophages as inflammatory cells are involved in the pathogenesis of atherosclerosis that today is recognized as an inflammatory disease. Activation of coagulation leads to the late complication of atherosclerosis, namely atherothrombosis with its clinical manifestations stroke, unstable angina, myocardial infarction, and sudden cardiac death. Thus inflammation and coagulation play fundamental roles in the pathogenesis of atherosclerosis. We show that the coagulation enzyme thrombin up-regulates oncostatin M (OSM), a pleiotropic cytokine implicated in the pathophysiology of vascular disease, in human monocyte-derived macrophages (MDMs) up to 16.8-fold. A similar effect was seen in human peripheral blood monocytes and human plaque macrophages. In MDMs, the effect of thrombin on OSM was abolished by PPACK and mimicked by a PAR-1-specific peptide. Thrombin induced phosphorylation of ERK1/2 and p38 in MDMs. The ERK1/2 inhibitor PD98059 blocked the effect of thrombin on OSM production in MDMs, whereas the p38 inhibitor SB202190 had no effect. Thrombin induced translocation of c-fos and c-jun to the nucleus of MDMs. Using OSM promoter-luciferase reporter constructs transfected into MDMs, we show that a functional AP-1 site is required for promoter activation by thrombin. We present another link between coagulation and inflammation, which could impact on the pathogenesis of atherosclerosis.

In human macrophages the complement component C5a induces the expression of oncostatin M via AP-1 activation.

OBJECTIVE: Macrophages produce the cytokine oncostatin M (OSM), which beside other functions is also involved in inflammation. The complement component C5a mobilizes and activates these cells at inflammatory sites. We examined the effect of C5a on OSM production in human monocytes and in human monocyte-derived macrophages. METHODS AND RESULTS: For macrophage transformation peripheral blood monocytes were cultivated for 8 to 10 days in the presence of human serum. C5a significantly increased in these cells OSM antigen as determined by specific ELISA and mRNA as quantitated by real-time polymerase chain reaction in these cells as well as in plaque macrophages. This effect was blocked by antibodies against the receptor C5aR/CD88 and by pertussis toxin. The C5a-induced phosphorylation of p38 and JNK and the C5a-induced increase in OSM production in macrophages was abolished by 2 p38 inhibitors and by a JNK inhibitor. Furthermore C5a increased the nuclear translocation of c-fos and c-jun. Using different OSM promoter deletion mutant constructs we show that the putative AP-1 element is responsible for activation of OSM promoter activity by C5a. CONCLUSIONS: Our data establish a link between the complement system and the gp130 receptor cytokine family with possible implications for the pathology of inflammatory diseases.

A functional screening assay for the isolation of transcription factors.

Transcription factors (TFs) have been difficult to identify by bioinformatics, due to the heterogenous nature of the domains they are composed of. Therefore, we have developed a simple and generally applicable screening system for the identification of transcriptional activators, based on the presence of a functional transactivation domain (TAD). The system utilizes a retroviral vector to express a cDNA library as fusion genes with the yeast gal4 DNA-binding domain. This retroviral library is transduced into a murine NIH3T3-based reporter cell line carrying a stable integrated gal4 promoter-green fluorescent protein reporter gene. cDNA inserts encoding a functional TAD reconstitute a chimeric TF that activates the reporter gene. After fluorescence activated cell sorting (FACS) and expansion of GFP-positive cells, the responsible cDNA inserts are retrieved. From a cDNA library of cytokine-stimulated human umbilical vein endothelial cells (HUVEC), a number of known as well as potentially novel TFs were isolated, demonstrating the suitability of the system. The identification of other factors that are currently not associated with transcriptional regulation suggest additional functions for these proteins. Moreover, our results have focused attention on signaling pathways that have not been recognized previously in the context of endothelial cell biology.

The transcription factor SOX18 regulates the expression of matrix metalloproteinase 7 and guidance molecules in human endothelial cells.

BACKGROUND: 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. METHODOLOGY/PRINCIPAL FINDINGS: 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. CONCLUSIONS/SIGNIFICANCE: 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.

A novel 9-amino-acid transactivation domain in the C-terminal part of Sox18.

Sox transcription factors are members of the Sry-related protein family that play multiple roles mainly during development. Sox18 has been implicated in the development of hair follicles as well as the blood and lymphatic vasculature, due to the identification of mutations that result in the ragged phenotype in mice, and in the hypotrichosis lymphedema telangiectasia syndrome in humans. Sox18 consists of an N-terminal high-mobility group DNA binding and a central transactivation domain, followed by a C-terminal region of unknown function. We show here that this C-terminal domain consists of three blocks that are highly conserved within a subgroup of the Sox family, and that the central so-called charged block comprises an additional strong transactivating domain. This activity can be pinpointed to a recently described 9aa transactivation motif that mediates the interaction with the transcriptional cofactor TAF9. These result can explain previously controversial data on the functional consequences of Sox18 mutations in mice and humans.