Abstract
Cyclooxygenase 2 (COX2), a key regulatory enzyme of the prostaglandin/eicosanoid pathway, is an important target for anti-inflammatory therapy. It is highly induced by pro-inflammatory cytokines in a Nuclear factor kappa B (NFκB)-dependent manner. However, the mechanisms determining the amplitude and dynamics of this important pro-inflammatory event are poorly understood. Furthermore, there is significant difference between human and mouse COX2 expression in response to the inflammatory stimulus tumor necrosis factor alpha (TNFα). Here, we report the presence of a molecular logic AND gate composed of two NFκB response elements (NREs) which controls the expression of human COX2 in a switch-like manner. Combining quantitative kinetic modeling and thermostatistical analysis followed by experimental validation in iterative cycles, we show that the human COX2 expression machinery regulated by NFκB displays features of a logic AND gate. We propose that this provides a digital, noise-filtering mechanism for a tighter control of expression in response to TNFα, such that a threshold level of NFκB activation is required before the promoter becomes active and initiates transcription. This NFκB-regulated AND gate is absent in the mouse COX2 promoter, most likely contributing to its differential graded response in promoter activity and protein expression to TNFα. Our data suggest that the NFκB-regulated AND gate acts as a novel mechanism for controlling the expression of human COX2 to TNFα, and its absence in the mouse COX2 provides the foundation for further studies on understanding species-specific differential gene regulation.Electronic supplementary materialThe online version of this article (doi:10.1007/s00018-015-1850-1) contains supplementary material, which is available to authorized users.
Highlights
Inflammation is a part of the immune system’s response to infection and injury
To quantitatively analyze and predict steady-state dynamics of the Cyclooxygenase 2 (COX2) gene expression under different modes of regulation by a transcriptional factor (e.g., NFjB), we developed three general kinetic models that describe: (1) a promoter that is regulated by a transcriptional factor through a single transcription factor (TF)-Promoter binding site; (2) a promoter regulated by a transcriptional factor through two TF-Promoter binding sites following a OR gate and (3) a promoter regulated by a transcriptional factor through two TF-Promoter binding sites following an AND gate regulation
We show that the presence of the NFjBregulated AND gate in the human COX2 promoter acts as a noise filter such that a threshold of NFjB activation is required before the promoter becomes active and initiates transcription
Summary
Inflammation is a part of the immune system’s response to infection and injury. The usual outcome of an inflammatory cellular response is its successful resolution and repair of tissue damage, whereas persistence and dysfunction of the inflammatory response has been implicated in the pathogenesis of diseases such as arthritis, cancer, neurodegenerative and cardiovascular diseases [1].Key players in the generation of the inflammatory response are the cyclooxygenase enzymes COX1 and COX2, which catalyze the conversion of arachidonic acid into pro-inflammatory prostaglandins and trigger the production of other pro-inflammatory chemokines and cytokines [2, 3]. The usual outcome of an inflammatory cellular response is its successful resolution and repair of tissue damage, whereas persistence and dysfunction of the inflammatory response has been implicated in the pathogenesis of diseases such as arthritis, cancer, neurodegenerative and cardiovascular diseases [1]. COX1 is constitutively expressed in most tissues and is involved in cellular housekeeping functions [4], while the inducible isoform COX2 is expressed in response to inflammatory stimuli such as TNFa [5]. Anti-inflammatory candidate drugs are usually developed and tested in mouse models but have very poor success rates when moved to clinical trials [7]. COX2 inhibitors, which work well in mouse, can be unfavorable in humans
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