AP-1B-mediated protein sorting regulates polarity and proliferation of intestinal epithelial cells in mice.

BACKGROUND & AIMS: In epithelial cells, protein sorting mechanisms regulate localization of plasma membrane proteins that generate and maintain cell polarity. The clathrin-adaptor protein (AP) complex AP-1B is expressed specifically in polarized epithelial cells, where it regulates basolateral sorting of membrane proteins. However, little is known about its physiological significance. METHODS: We analyzed the intestinal epithelia of mice deficient in Ap1m2 (Ap1m2(-/-) mice), which encodes the AP-1B µ1B subunit, and compared it with 129/B6/CD1 littermates (controls). Notch signaling was inhibited by intraperitoneal injection of dibenzazepine, and ß-catenin signaling was inhibited by injection of IWR1. Intestinal tissue samples were collected and analyzed by immunofluorescence analysis. RESULTS: Ap1m2(-/-) mice developed intestinal epithelial cell hyperplasia. The polarity of intestinal epithelial cells was disrupted, as indicated by the appearance of ectopic microvilli-like structures on the lateral plasma membrane and mislocalization of basolateral membrane proteins, including the low-density lipoprotein receptor and E-cadherin. The E-cadherin-ß-catenin complex therefore was disrupted at the adherens junction, resulting in nuclear translocation of ß-catenin. This resulted in up-regulation of genes regulated by ß-catenin/transcription factor 4 (Tcf4) complex, and increased the proliferation of intestinal epithelial cells. CONCLUSIONS: AP-1B is required for protein sorting and polarization of intestinal cells in mice. Loss of AP-1B in the intestinal epithelia results in mislocalization of E-cadherin, activation of ß-catenin/Tcf4 complex, proliferation, and hyperplasia.

Epithelial cell-intrinsic Notch signaling plays an essential role in the maintenance of gut immune homeostasis.

Intestinal epithelial cells (IECs) have important functions as the first line of defense against diverse microorganisms on the luminal surface. Impaired integrity of IEC has been implicated in increasing the risk for inflammatory disorders in the gut. Notch signaling plays a critical role in the maintenance of epithelial integrity by regulating the balance of secretory and absorptive cell lineages, and also by facilitating epithelial cell proliferation. We show in this article that mice harboring IEC-specific deletion of Rbpj (RBP-J(ΔIEC)), a transcription factor that mediates signaling through Notch receptors, spontaneously develop chronic colitis characterized by the accumulation of Th17 cells in colonic lamina propria. Intestinal bacteria are responsible for the development of colitis, because their depletion with antibiotics prevented the development of colitis in RBP-J(ΔIEC) mice. Furthermore, bacterial translocation was evident in the colonic mucosa of RBP-J(ΔIEC) mice before the onset of colitis, suggesting attenuated epithelial barrier functions in these mice. Indeed, RBP-J(ΔIEC) mice displayed increase in intestinal permeability after rectal administration of FITC-dextran. In addition to the defect in physical barrier, loss of Notch signaling led to arrest of epithelial cell turnover caused by downregulation of Hes1, a transcriptional repressor of p27(Kip1) and p57(Kip2). Thus, epithelial cell-intrinsic Notch signaling ensures integrity and homeostasis of IEC, and this mechanism is required for containment of intestinal inflammation.

The epithelia-specific membrane trafficking factor AP-1B controls gut immune homeostasis in mice.

BACKGROUND & AIMS: Epithelial cells that cover the intestinal mucosal surface maintain immune homeostasis and tolerance in the gastrointestinal tract. However, little is known about the molecular mechanisms that regulate epithelial immune functions. Epithelial cells are distinct in that they are highly polarized; this polarity is, at least in part, established by the epithelium-specific polarized sorting factor adaptor protein (AP)-1B. We investigated the role of AP-1B-mediated protein sorting in the maintenance of gastrointestinal immune homeostasis. METHODS: The role of AP-1B in intestinal immunity was examined in AP-1B-deficient mice (Ap1m2(-/-)) by monitoring their phenotypes, intestinal morphology, and epithelial barrier functions. AP-1B-mediated protein sorting was examined in polarized epithelial cells from AP-1B knockdown and Ap1m2(-/-) mice. RESULTS: Ap1m2(-/-) mice developed spontaneous chronic colitis, characterized by accumulation of interleukin-17A-producing, T-helper 17 cells. Deficiency of AP-1B caused epithelial immune dysfunction, such as reduced expression of antimicrobial proteins and impaired secretion of immunoglobulin A. These defects promoted intestinal dysbiosis and increased bacterial translocation within the mucosa. Importantly, AP-1B deficiency led to mistargeting of a subset of basolateral cytokine receptors to the apical plasma membrane in a polarized epithelial cell line and in colonic epithelial cells from mice. AP1M2 expression was reduced significantly in colonic epithelium samples from patients with Crohn's disease. CONCLUSIONS: AP-1B is required for proper localization of a subset of cytokine receptors in polarized epithelial cells, which allows them to respond to cytokine signals from underlying lamina propria cells. The AP-1B-mediated protein sorting machinery is required for maintenance of immune homeostasis and prevention of excessive inflammation.

Identification of TOSO/FAIM3 as an Fc receptor for IgM.

Fc receptors specifically bind to the Fc region of Igs to mediate the unique functions to each class of Igs. To identify a novel Fc receptor for IgM, we searched expressed sequence tag database for molecules containing Ig domains with homology to those of known Fc receptors for IgM, Fcalpha/muR and polymeric Ig receptor. As a result, we identified TOSO/Fas apoptotic inhibitory molecule 3 (FAIM3) as a possible Fc receptor for IgM. HeLa cells transfected with a TOSO/FAIM3-expression vector bound to IgM but not IgG and were able to internalize IgM-conjugated beads but not IgG-conjugated beads, suggesting that TOSO/FAIM3 is indeed a receptor for IgM (FcmuR). FcmuR protein was expressed predominantly on B-lineage cells; expression of the Fcmr transcripts was observed from the pre-B-cell stage and maintained thereafter during B-cell development. These results identify TOSO/FAIM3 as a receptor for IgM and suggest that FcmuR may serve as an uptake receptor for IgM-opsonized antigens by B cells.

M-Sec promotes membrane nanotube formation by interacting with Ral and the exocyst complex.

Cell-cell communication is essential for the development and homeostasis of multicellular organisms. Recently, a new type of cell-cell communication was discovered that is based on the formation of thin membranous nanotubes between remote cells. These long membrane tethers, termed tunneling nanotubes (TNTs), form an intercellular conduit and have been shown to enable the transport of various cellular components and signals. However, the molecular basis for TNT formation remains to be elucidated. Here we report that a mammalian protein, M-Sec, induces de novo formation of numerous membrane protrusions extending from the plasma membrane, some of which tether onto adjacent cells and subsequently form TNT-like structures. Depletion of M-Sec by RNA interference (RNAi) greatly reduced endogenous TNT formation as well as intercellular propagation of a calcium flux in a macrophage cell line. Furthermore, blockage of the interaction of M-Sec with Ral and the exocyst complex, which serves as a downstream effector of Ral, attenuated the formation of membrane nanotubes. Our results reveal that M-Sec functions as a key regulator of membrane nanotube formation through interaction with the Ral-exocyst pathway.

CD300 antigen like family member G: A novel Ig receptor like protein exclusively expressed on capillary endothelium.

We report the characteristics of CD300LG, a member of the CD300 antigen like family. Its genomic structure is similar in both mouse and human, and at least four isoforms exist in both species. The amino acid sequence of the immunoglobulin (Ig) V like domain of CD300LG showed approximately 35% identity to those of the polymeric Ig receptor (pIgR) and Fcalpha/muR. Interestingly, mouse CD300LG proteins were uniquely expressed on capillary endothelium. Immunoelectron microscopy revealed that mouse CD300LG is localized on both apical and basolateral plasma membranes, as well as on intracellular vesicular structures, in the capillary endothelium. Transcytosis assays using polarized MDCK epithelial cells showed that CD300LG could be transcytosed bidirectionally. Furthermore, CD300LG exogenously expressed on HeLa cells could take up IgA2 and IgM, but not IgG. These results suggest that CD300LG might play an important role in molecular traffic across the capillary endothelium.