Scientific Abstract
Proposal No. IBD-0210R2
Principal Investigator: Xiaojing Ma, Ph.D.
Applicant Organization: Weill Medical College of Cornell University (New York, New York, U.S.A.)
Project Title: The Crohn’s disease-associated NOD23020insC mutant has an acquired activity as an inhibitor of IL-10 production
Period of Award: October 1, 2007 - March 31, 2009
Nucleotide-binding oligomerization domain 2 (NOD2) is a monocyte-restricted member of a protein family, each of which contains a linked caspase activation and recruitment domain (CARD) and is critically involved in the activation of NF-κB in response to certain bacterial infection. NOD2 is thought to confer responsiveness to bacterial peptidoglycan through Toll-like receptors. Dysfunctional mutations in the NOD2 gene underline the occurrence of inflammatory bowel disease (IBD) in a substantial group of patients with Crohn’s disease (CD). CD is characterized by an exaggerated T helper 1 (Th1)-type immune response. One of the most common mutations in the NOD2 gene associated with CD is an insertion and frame shift mutation at position 3020, designated 3020insC, which results in the deletion of the terminal leucine-rich repeats (LRR) of the protein believe to be critical for intracellular sensing of bacteria. How 3020insC contributes to CD is a controversial issue. Recent human clinical studies revealed defective IL-10 production in the mononuclear cells of CD patients homozygous for 3020insC. IL-10 is a critical immunoregulatory cytokine produced by antigen-presenting cells and activated T and B lymphocytes. The chronically impaired IL-10 production in these CD patients may contribute to persistent inflammation in the intestinal mucosa. Our recent data indicate that wild type NOD2 per se is dispensable for IL-10 production by macrophages in response to several types of microbial challenges. However, in contrast to the prevalent perception, 3020insC is not simply a loss-of-function mutant. Instead, it can act as an inhibitor of IL-10 production by approximately 50%. Moreover, this acquired activity of 3020insC is unique to the human IL-10 gene and human 3020insC, it does not operate on the mouse IL-10 gene, nor does the engineered mouse counterpart of the 3020insC mutant modulate IL-10 expression. Our findings raise doubts about the validity of certain current mouse models of this human disease targeting NOD2. Furthermore, we have recently identified a target of 3020insC in this pathway as heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1), which is a novel and constitutive transcription factor for IL-10. 3020insC specifically targets its DNA-binding activity. Most importantly, we have confirmed that hnRNP A1’s binding activity is indeed impaired in NOD23020insC-CD patients who are deficient in IL-10 synthesis.
We hypothesize that 3020insC is both a loss-of-function and gain-of-function mutant. It can actively suppress IL-10 production by interfering with hnRNP A1’s phosphorylation and activation via MAP kinase signal-integrating kinases. This property of 3020insC may result in chronically impaired IL-10 levels in susceptible individuals carrying the 3020insC mutation, which, when combined with other mitigating factors over a lengthy period of time, causes homeostatic imbalance and persistent inflammation in the intestinal mucosa leading to the development and pathogenesis of CD.
We propose:
- To investigate the mechanism whereby 3020insC interferes with hnRNP A1's phosphorylation, DNA-binding, and transcriptional capacity.
- To determine if impaired IL-10 production in 3020insC-homozygous CD patients can be rescued through targeting hnRNP A1 expression.
This investigation challenges the current paradigm that 3020insC is just a loss of function mutant, and will lead to better understanding of the molecular mechanisms whereby 3020insC contributes to CD development and pathogenesis, and may reveal novel therapeutic targets with which to interfere with the CD-causing effects of 3020insC.