Tristetraprolin (TTP) is a prototypic family member of CCCH-type tandem zinc-finger domain proteins that regulate mRNA destabilization in eukaryotic cells. TTP binds to AU-rich elements (AREs) in the 3′-untranslated region of certain mRNAs, including tumor necrosis factor α, granulocyte macrophage colony-stimulating factor, and immediate early response 3, thereby facilitating their ARE-mediated decay. Expression of TTP is up-regulated by a variety of agents, including inflammatory mediators such as tumor necrosis factor α, a prominent activator of the nuclear factor κB (NF-κB) family of transcription factors. Accordingly, TTP is involved in the negative feedback regulation of NF-κB through promoting mRNA degradation. We describe here a novel, ARE-mediated decay-independent function of TTP on the termination of NF-κB response: TTP suppresses the transcriptional activity of NF-κB-dependent promoters independent of its mRNA-destabilizing property. In TTP knock-out mouse embryonic fibroblasts, lack of TTP leads to enhanced nuclear p65 levels, which is associated with the up-regulation of specific, ARE-less NF-κB target genes. We find that attenuation of NF-κB activity is at least in part due to an interference of TTP with the nuclear import of the p65 subunit of the transcription factor. This novel role of TTP may synergize with its mRNA-degrading function to contribute to the efficient regulation of proinflammatory gene expression.

Symphytum officinale, commonly known as comfrey, constitutes a traditional medicinal plant with a long-standing therapeutic history, and preparations thereof have been widely used for the treatment of painful muscle and joint complaints, wound and bone healing, and inflammation. Today, its topical use is based on its analgesic and anti-inflammatory effects, which have been substantiated by modern clinical trials. However, the molecular basis of its action remained elusive. Here, we show that a hydroalcoholic extract of comfrey root impairs the development of a pro-inflammatory scenario in primary human endothelial cells in a dose-dependent manner. The extract, and especially its mucilage-depleted fraction, impair the interleukin-1 (IL-1) induced expression of pro-inflammatory markers including E-selectin, VCAM1, ICAM1, and COX-2. Both preparations inhibit the activation of NF-κB, a transcription factor of central importance for the expression of these and other pro-inflammatory genes. Furthermore, our biochemical studies provide evidence that comfrey inhibits NF-κB signaling at two stages: it dampens not only the activation of IKK1/2 and the subsequent IκBα degradation, but also interferes with NF-κB p65 nucleo-cytoplasmatic shuttling and transactivation. These results provide a first mechanistic insight into the mode of action of a century-old popular herbal medicine.

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.

Objective— Endothelial cells comprise a key component of the inflammatory response. We set out to obtain a comprehensive overview of the immediate-early to early gene expression program of interleukin-1 (IL-1)–stimulated endothelial cells and to identify novel transcription factors and regulatory elements. Methods and Results— Human umbilical vein endothelial cells (HUVECs) were stimulated with IL-1 for 0, 0.5, 1, 2.5, and 6 hours and analyzed using Affymetrix U133 microarrays. A total of 137 genes were found to be regulated >4-fold, including 18 transcription factors. The expression of selected genes was confirmed by real-time polymerase chain reaction. Cluster analysis was performed in order to group genes according to their expression profiles. To identify novel transcription factor–binding sites, the corresponding promoters were extracted from databases and analyzed for regulatory elements that were over-represented in specific clusters. Several potentially novel DNA binding sites were identified, and one was shown to specifically bind an IL-1–inducible protein from HUVEC. Conclusions— These results demonstrate that in the early phase after stimulation, IL-1 evokes a complex gene expression program that includes positive but also negative (feedback) regulators of diverse endothelial cell functions. Furthermore, the identification of a new promoter regulatory element demonstrates the feasibility of the bioinformatics-driven approach to discover novel regulatory mechanisms.

Summary: Timely termination of the inflammatory reaction is equally important as its elicitation, since a persistent or exaggerated response may lead to detrimental effects in the affected tissues and organs. Therefore, and in accordance with the complex and highly coordinated activation phase, negative regulatory mechanisms have evolved which function on multiple levels to ensure the appropriate termination of the inflammatory response. This review will focus on the mechanisms that are operative in endothelial cells to shut down the activity of specific signaling pathways and transcription factors that have been activated in response to pro-inflammatory mediators, and provide evidence that the stage for resolution is set already early in the activation phase of the inflammatory response. The elucidation of these feedback mechanisms is of importance for the understanding of acute versus chronic inflammation, and for novel strategies for therapeutic intervention.