- C1orf106-negative mice exhibited defects in the intestinal epithelial cell barrier, a phenotype observed in patients with inflammatory bowel disease (IBD) that confers increased susceptibility to intestinal pathogens
- C1orf106-negative mice challenged with the murine equivalent of E. coli exhibited significantly increased bacterial loads, significantly greater histopathology and more impaired recovery from induced colitis, compared with C1orf106-positive mice
- A coding variant in C1orf106, *333F, decreases C1orf106 protein stability and thus confers increased susceptibility to IBD by compromising gut epithelial integrity through impaired turnover and degradation of cytohesin-1
- Increasing the stability of C1orf106 is worth exploring as a therapeutic strategy in IBD
Some patients with inflammatory bowel disease (IBD) have been observed to have abnormal permeability of the intestinal epithelial barrier, which is involved in nutrient absorption, protection against microbes and numerous other physiologic processes. Similar changes have been noted in healthy relatives of IBD patients, suggesting that host genetics can underlie the defects.
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In previous research, C1orf106 was identified as an IBD susceptibility gene, but the mechanism was unknown. Now, Massachusetts General Hospital researchers Vishnu Mohanan, PhD, research fellow in the Xavier Laboratory, and Ramnik J. Xavier, MD, PhD, chief of the Division of Gastroenterology and director of the Center for the Study of Inflammatory Bowel Disease, and colleagues have discovered that the *333F coding variant of C1orf106 (C1orf106 *333F) increases the risk of IBD by compromising intestinal epithelial integrity. Their findings are published in Science.
Dr. Xavier's group began by identifying cytohesin-1 and -2 as top interactors with C1orf106 in cell lines. To examine interactions between the proteins in vivo, the researchers generated C1orf106-negative mice. They found that levels of cytohesin-1 in epithelial cells from the colons and small intestines of these mice were consistently higher than in cells from C1orf106-positive mice.
The same was true when the researchers examined C1orf106-negative epithelial monolayers derived from colonic organoids.
Increasing C1orf106 expression significantly decreased levels of overexpressed or endogenous cytohesin-1, indicating that C1orf106 expression is sufficient to regulate the steady-state levels of cytohesin-1. Similar results were observed with cytohesin-2. In further experiments, the researchers determined that C1orf106 expression limits cytohesin-1 levels through ubiquitin-mediated degradation.
Epithelial Cell Function
The research team's next step was to understand how C1orf106-mediated degradation of cytohesin-1 alters epithelial cell function. It was already known that cytohesin-1 acts as a guanine exchange factor that controls the activation of ARF6, a guanosine triphosphatase involved in the control of actin remodeling. The team found that increased levels of cytohesin-1 protein in C1orf106-positive cells increased levels of ARF6 activation.
It was also known that ARF6 is key in regulating surface levels of critical adherens junction proteins and that ARF6 activation in epithelial cells increases internalization of E-cadherin. As the researchers expected, increased levels of cytohesin-1 and activated ARF6 in C1orf106-negative intestinal epithelial cells resulted in decreased surface levels of E-cadherin.
These data indicate that C1orf106 is crucial to maintaining adherens junctions by limiting ARF6 activation through regulated cytohesin degradation.
Epithelial Barrier Integrity
The researchers observed that C1orf106-negative cells demonstrated increased permeability to small solutes, compared with C1orf106-positive cells, and that changes in E-cadherin recycling delayed the ability of C1orf106-negative cells to repair epithelial junctions after injury. Cytohesin-1 was a key mediator of the barrier phenotype in C1orf106-negative cells.
Because IBD is commonly associated with increased susceptibility to microbial pathogens and dysbiosis, the researchers investigated whether the compromised epithelial barrier integrity of C1orf106-negative mice would result in increased bacterial dissemination. They challenged C1orf106-negative and C1orf106-positive mice with a murine pathogen comparable to the Escherichia coli strains associated with Crohn's disease.
C1orf106-negative mice exhibited significantly increased bacterial loads by day five, as well as significantly greater histopathology and more impaired recovery from induced colitis, compared with C1orf106-positive mice.
C1orf106 Risk Variant
Several research teams have demonstrated that a coding variant in C1orf106, *333F, increases the risk of IBD. Experiments by Dr. Xavier's group showed that:
- Expression of C1orf106 *333F was reproducibly decreased during transient transfection compared with that of wild-type C1orf106, suggesting that the risk variant is poorly expressed or unstable
- The decreased levels of C1orf106 *333F were due to ubiquitination and degradation by the proteasome
- C1orf106 *333F had a half-life of 10.2 hours, compared with almost 17 hours for wild-type C1orf106
- When C1orf106-negative organoids were transduced with either wild-type C1orf106 or C1orf106 *333F, expression of C1orf106 *333F was not sufficient to restore wild-type levels of C1orf106, mediate degradation of cytohesin-1, or increase transepithelial electrical resistance in C1orf106-negative monolayers
- Expression of C1orf106 *333F disrupted E-cadherin and actin organization and staining in monolayer-derived intestinal epithelial cells and human intestinal cells
These findings suggest that the *333F polymorphism decreases C1orf106 protein stability and thus confers increased susceptibility to IBD by compromising intestinal epithelial integrity through impaired turnover and degradation of cytohesin-1.
A "Molecular Rheostat"
The researchers conclude that C1orf106 functions as a "molecular rheostat," limiting cytohesin levels and thereby modulating intestinal epithelial barrier integrity. They find it particularly interesting that C1orf106 regulates surface levels of E-cadherin because polymorphisms in both C1orf106 and CDH1 (E-cadherin) have been linked to increased risk of ulcerative colitis.
Increasing the stability of C1orf106 is a potential therapeutic strategy in IBD, the researchers propose, to increase the integrity of the epithelial barrier.
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