Muc5ac Expression Protects the Colonic Barrier in Experimental Colitis.

BACKGROUND: The mucus gel layer (MGL) lining the colon is integral to exclusion of bacteria and maintaining intestinal homeostasis in health and disease. Some MGL defects allowing bacteria to directly contact the colonic surface are commonly observed in ulcerative colitis (UC). The major macromolecular component of the colonic MGL is the secreted gel-forming mucin MUC2, whose expression is essential for homeostasis in health. In UC, another gel-forming mucin, MUC5AC, is induced. In mice, Muc5ac is protective during intestinal helminth infection. Here we tested the expression and functional role of MUC5AC/Muc5ac in UC biopsies and murine colitis. METHODS: We measured MUC5AC/Muc5ac expression in UC biopsies and in dextran sulfate sodium (DSS) colitis. We performed DSS colitis in mice deficient in Muc5ac (Muc5ac-/-) to model the potential functional role of Muc5ac in colitis. To assess MGL integrity, we quantified bacterial-epithelial interaction and translocation to mesenteric lymph nodes. Antibiotic treatment and 16S rRNA gene sequencing were performed to directly investigate the role of bacteria in murine colitis. RESULTS: Colonic MUC5AC/Muc5ac mRNA expression increased significantly in active UC and murine colitis. Muc5ac-/- mice experienced worsened injury and inflammation in DSS colitis compared with control mice. This result was associated with increased bacterial-epithelial contact and translocation to the mesenteric lymph nodes. However, no change in microbial abundance or community composition was noted. Antibiotic treatment normalized colitis severity in Muc5ac-/- mice to that of antibiotic-treated control mice. CONCLUSIONS: MUC5AC/Muc5ac induction in the acutely inflamed colon controls injury by reducing bacterial breach of the MGL.

Coordination of ENT2-dependent adenosine transport and signaling dampens mucosal inflammation.

Intestinal epithelial barrier repair is vital for remission in inflammatory bowel disease (IBD). Extracellular adenosine signaling has been implicated in promoting restoration of epithelial barrier function. Currently, no clinically approved agents target this pathway. Adenosine signaling is terminated by uptake from the extracellular space via equilibrative nucleoside transporters (ENTs). We hypothesized that ENT inhibition could dampen intestinal inflammation. Initial studies demonstrated transcriptional repression of ENT1 and ENT2 in IBD biopsies or in murine IBD models. Subsequent studies in mice with global Ent1 or Ent2 deletion revealed selective protection of Ent2-/- mice. Elevated intestinal adenosine levels in conjunction with abolished protection following pharmacologic blockade of A2B adenosine receptors implicate adenosine signaling as the mechanism of gut protection in Ent2-/- mice. Additional studies in mice with tissue-specific deletion of Ent2 uncovered epithelial Ent2 as the target. Moreover, intestinal protection provided by a selective Ent2 inhibitor was abolished in mice with epithelium-specific deletion of Ent2 or the A2B adenosine receptor. Taken together, these findings indicate that increased mucosal A2B signaling following repression or deletion of epithelial Ent2 coordinates the resolution of intestinal inflammation. This study suggests the presence of a targetable purinergic network within the intestinal epithelium designed to limit tissue inflammation.

Plasma membrane calcium ATPase 4 (PMCA4) co-ordinates calcium and nitric oxide signaling in regulating murine sperm functional activity.

Reduced sperm motility (asthenospermia) and resulting infertility arise from deletion of the Plasma Membrane Ca2+ -ATPase 4 (Pmca4) gene which encodes the highly conserved Ca2+ efflux pump, PMCA4. This is the major Ca2+ clearance protein in murine sperm. Since the mechanism underlying asthenospermia in PMCA4's absence or reduced activity is unknown, we investigated if sperm PMCA4 negatively regulates nitric oxide synthases (NOSs) and when absent NO, peroxynitrite, and oxidative stress levels are increased. Using co-immunoprecipitation (Co-IP) and Fluorescence Resonance Energy Transfer (FRET), we show an association of PMCA4 with the NOSs in elevated cytosolic [Ca2+ ] in capacitated and Ca2+ ionophore-treated sperm and with neuronal (nNOS) at basal [Ca2+ ] (ucapacitated sperm). FRET efficiencies for PMCA4-eNOS were 35% and 23% in capacitated and uncapacitated sperm, significantly (p < 0.01) different, with the molecules being <10 nm apart. For PMCA4-nNOS, this interaction was seen only for capacitated sperm where FRET efficiency was 24%, significantly (p < 0.05) higher than in uncapacitated sperm (6%). PMCA4 and the NOSs were identified as interacting partners in a quaternary complex that includes Caveolin1, which co-immunoprecipitated with eNOS in a Ca2+ -dependent manner. In Pmca4-/- sperm NOS activity was elevated twofold in capacitated/uncapacitated sperm (vs. wild-type), accompanied by a twofold increase in peroxynitrite levels and significantly (p < 0.001) increased numbers of apoptotic germ cells. The data support a quaternary complex model in which PMCA4 co-ordinates Ca2+ and NO signaling to maintain motility, with increased NO levels resulting in asthenospermia in Pmca4-/- males. They suggest the involvement of PMCA4 mutations in human asthenospermia, with diagnostic relevance.