Regulatory feedback mechanisms in endothelial cells

Whereas signaling pathways mediating the activation phase of the inflammatory response have been studied in quite detail during the last decade, mechanisms that are operative during its resolution remain largely elusive. The overall aim of this project is to identify and analyze intracellular feedback mechanisms that are operative in down- regulating specific signaling pathways, transcription factors, and sets of genes during the resolution phase of the inflammatory response in endothelial cells. The hypothesis that such feedback mechanisms exist is based on the notion that already during activation, repressive mechanisms, such as the previously described inhibition of NF-B through IBa expression (Cheng et al., 1994; de Martin et al., 1993) are initiated, and function to shut down at later stages certain aspects of the inflammatory response. The impairment of such inhibitory mechanisms might be associated with pathological conditions that are associated with a switch from acute to chronic inflammation. Based on this hypothesis, we will analyze genes induced during the early activation phase of the endothelium for their repressive potential. In preliminary experiments, we have already identified approximately 150-200 genes that are induced upon IL-1 stimulation within this period (Mayer et al., 2004). Based on bioinformatics and published data, a number of candidates have been selected for more detailed analysis in regard to inhibition of individual signaling pathways and specific parameters of the inflammatory response. We will extend these studies to different stimuli and endothelial cell types in order to obtain in the initial phase of the project a broad basis for the following more specific analysis. We believe that the identification and understanding of novel negative regulatory mechanisms may, in contrast to the broad suppression through NF- B inhibition, enable a more selective shut- down of specific aspects of the inflammatory response.

We have here identified a novel function for TTP, a regulatory protein that contributes to down-regulation of inflammation. The inflammatory response represents the first line of defense of an organism against microbial and viral infection. However, this intrinsically beneficial reaction may easily turn into a destructive one in case it is exaggerated in terms of time and/or intensity, and thus become the hall-mark of several well-known chronic inflammatory diseases such as rheumatoid arthritis, inflammatory bowl disease, artheriosclerosis, and psoriasis. It is therefore of central interest from both a scientific and therapeutic view to understand the regulation of inflammation, especially aspects of how this reaction is terminated. In this regard, we have found previously that first steps for efficient down-regulation are initiated already very early upon the onset of inflammation, and occur on several levels. Interestingly, the same mechanisms that promote inflammation can, in a time-delayed manner, also evoke anti-inflammatory responses. At later stages, they act to inhibit their own activators, and are therefore called "negative feedback mechanisms". They are operative, among others, on the level of gene expression. In this project we have mainly focussed on one gene product termed TTP (tristetraprolin) that was previously described as an mRNA destabilizing protein. Surprisingly, we found that it also has another function, namely to inhibit the activity of one regulatory protein, the transcription factor NF-kB. NF-kB is a central player in inflammation, since it regulates many genes that contribute to the inflammatory phenotype. We have elucidated the mechanism and found that TTP acts to prevent the transport of NF-kB from the cells cytoplasm to the nucleus, and thereby inhibiting its activity. In addition, we have performed a detailed analysis of how TTP itself is regulated, and found that it can be phosphorylated by a specific kinase, which in turn provides a signal for further modification by ubiquitin. Together, we have gained substantial novel insight into the function and regulation of TTP as an important regulatory protein in the inflammatory reaction.