Interleukin-22 drives nitric oxide-dependent DNA damage and dysplasia in a murine model of colitis-associated cancer.

The risk of colon cancer is increased in patients with Crohn's disease and ulcerative colitis. Inflammation-induced DNA damage could be an important link between inflammation and cancer, although the pathways that link inflammation and DNA damage are incompletely defined. RAG2-deficient mice infected with Helicobacter hepaticus (Hh) develop colitis that progresses to lower bowel cancer. This process depends on nitric oxide (NO), a molecule with known mutagenic potential. We have previously hypothesized that production of NO by macrophages could be essential for Hh-driven carcinogenesis, however, whether Hh infection induces DNA damage in this model and whether this depends on NO has not been determined. Here we demonstrate that Hh infection of RAG2-deficient mice rapidly induces expression of iNOS and the development of DNA double-stranded breaks (DSBs) specifically in proliferating crypt epithelial cells. Generation of DSBs depended on iNOS activity, and further, induction of iNOS, the generation of DSBs, and the subsequent development of dysplasia were inhibited by depletion of the Hh-induced cytokine IL-22. These results demonstrate a strong association between Hh-induced DNA damage and the development of dysplasia, and further suggest that IL-22-dependent induction of iNOS within crypt epithelial cells rather than macrophages is a driving force in this process.

Evidence for a specific uptake and retention mechanism for 25-hydroxyvitamin D (25OHD) in skeletal muscle cells.

Little is known about the mechanism for the prolonged residence time of 25-hydroxyvitamin D (25OHD) in blood. Several lines of evidence led us to propose that skeletal muscle could function as the site of an extravascular pool of 25OHD. In vitro studies investigated the capacity of differentiated C2 murine muscle cells to take up and release 25OHD, in comparison with other cell types and the involvement of the membrane protein megalin in these mechanisms. When C2 cells are differentiated into myotubes, the time-dependent uptake of labeled 25OHD is 2-3 times higher than in undifferentiated myoblasts or nonmuscle osteoblastic MG63 cells (P < .001). During in vitro release experiments (after 25OHD uptake), myotubes released only 32% ± 6% stored 25OHD after 4 hours, whereas this figure was 60% ± 2% for osteoblasts (P < .01). Using immunofluorescence, C2 myotubes and primary rat muscle fibers were, for the first time, shown to express megalin and cubilin, endocytotic receptors for the vitamin D binding protein (DBP), which binds nearly all 25OHD in the blood. DBP has a high affinity for actin in skeletal muscle. A time-dependent uptake of Alexafluor-488-labeled DBP into mature muscle cells was observed by confocal microscopy. Incubation of C2 myotubes (for 24 hours) with receptor-associated protein, a megalin inhibitor, led to a 40% decrease in 25OHD uptake (P < .01). These data support the proposal that 25OHD, after uptake into mature muscle cells, is held there by DBP, which has been internalized via membrane megalin and is retained by binding to actin.