Inactivation of the p19(ARF) tumor suppressor affects intestinal epithelial cell proliferation and integrity

p19(ARF) is a tumor suppressor that is frequently deleted in human cancer. It lies at chromosome 9p21 and shares exons 2 and 3 with p16(ink4a), which is also inactivated by these cancer-associated deletions. The "canonical pathway" by which p19(ARF) is thought to suppress tumorigenesis through activation of the p53 tumor suppressor. In response to hyperproliferative signals, such as expression of oncogenes, p19(ARF) is induced and binds to the MDM2 ubiquitin ligase, sequestering it in the nucleolus to allow the accumulation of p53. However, p19(ARF) also has MDM2 and p53 independent functions. In human colon cancer, p19(ARF) is only rarely deleted, but it is more frequently silenced by DNA promoter methylation. Here we show that inactivation of p19(ARF) in mice increases the number of cycling cells in the crypts of the colonic epithelium. Moreover, inactivation of p19(ARF) exacerbated the ulceration of the colonic epithelium caused by dextran sodium sulfate (DSS). These effects were similar to those observed in mice lacking myeloid translocation gene-related-1 (Mtgr1), and mice lacking both of these genes showed an even greater sensitivity to DSS. Surprisingly, inactivation of p19(ARF) restored the loss of the secretory lineage in mice deficient in Mtgr1, suggesting an additional role for p19(ARF) in the small intestinal epithelium.     

Lkb1 Deficiency Alters Goblet and Paneth Cell Differentiation in the Small Intestine

The Lkb1 tumour suppressor is a multitasking kinase participating in a range of physiological processes. We have determined the impact of Lkb1 deficiency on intestinal homeostasis, particularly focussing on secretory cell differentiation and development since we observe strong expression of Lkb1 in normal small intestine Paneth and goblet cells. We crossed mice bearing an Lkb1 allele flanked with LoxP sites with those carrying a Cyp1a1-specific inducible Cre recombinase. Lkb1 was efficiently deleted from the epithelial cells of the mouse intestine after intraperitoneal injection of the inducing agent β-naphthoflavone. Bi-allelic loss of Lkb1 led to the perturbed development of Paneth and goblet cell lineages. These changes were characterised by the lack of Delta ligand expression in Lkb1-deficient secretory cells and a significant increase in the levels of the downstream Notch signalling effector Hes5 but not Hes1. Our data show that Lkb1 is required for the normal differentiation of secretory cell lineages within the intestine, and that Lkb1 deficiency modulates Notch signalling modulation in post-mitotic cells.     

Villous B cells of the small intestine are specialized for invariant NK T cell dependence

B cells are important in mucosal microbial homeostasis through their well-known role in secretory IgA production and their emerging role in mucosal immunoregulation. Several specialized intraintestinal B cell compartments have been characterized, but the nature of conventional B cells in the lamina propria is poorly understood. In this study, we identify a B cell population predominantly composed of surface IgM(+) IgD(+) cells residing in villi of the small intestine and superficial lamina propria of the large intestine, but distinct from the intraepithelial compartment or organized intestinal lymphoid structures. Small intestinal (villous) B cells are diminished in genotypes that alter the strength of BCR signaling (Bruton tyrosine kinase(xid), Galphai2(-/-)), and in mice lacking cognate BCR specificity. They are not dependent on enteric microbial sensing, because they are abundant in mice that are germfree or genetically deficient in TLR signaling. However, villous B cells are reduced in the absence of invariant NK T cells (Jalpha18(-/-) or CD1d(-/-) mice). These findings define a distinct population of conventional B cells in small intestinal villi, and suggest an immunologic link between CD1-restricted invariant NK T cells and this B cell population.     

Fine structure and chemical analysis of the metathoracic scent gland of Eurygaster maura (Linnaeus, 1758) (Heteroptera: Scutelleridae)

The morphology and ultrastructure of the metathoracic scent glands (MTG) of Eurygaster maura were studied by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Also, extracts of the volatile fraction of the MTG secretion from males and females were analyzed by capillary gas chromatography-mass spectrometry (GC-MS). In SEM investigations, MTG are composed of a reservoir and a pair of lateral glands connected to the reservoir by a duct. MTG are open in between the meso- and the metacoxae. These areas, called evaporation areas, are composed of mushroom-like elements. In TEM investigations, the reservoir walls contained two types of cells. Generally, a reservoir is lined by a single layer of epithelial cells, type I cells, which have numerous organelles. Type II cells are found only in a certain area of the reservoir wall. These cells have large secretory ducts lined by a cuticular intima layer. The lateral glands are lined by secretory cells and a secretory duct found in their cytoplasm. Nuclei of secretory cells are closed to the basal region of the cells and circular-shaped. In GC-MS investigations, the MTG exhibited a typical scutellerid composition. In general, (E)-2-hexanal, (E)-2-hexenyl acetate, n-tridecane, n-hexanoic acid, octadecanoic acid, and n-dodecane compounds were present, while diisooctyl acetate and 14-Beta-H-Pregna were detected only in the male extracts of Eurygaster maura.     

NHE2X3 DKO mice exhibit gender-specific NHE8 compensation

NHE8, the newest member of the sodium/hydrogen exchanger family, is expressed in the epithelial cells of the intestine and the kidney. Intestinal expression of NHE8 is significantly higher than that of NHE2 and NHE3 at a young age, suggesting that NHE8 is an important player for intestinal sodium absorption during early development. The current study was designed to explore if NHE8 plays a compensatory role for the loss of NHE2 and NHE3 function in NHE2X3 double-knockout (NHE2X3 DKO) mice. We further explored the regulatory mechanism(s) responsible for the change in NHE8 expression in NHE2X3 DKO mice. We found that >95% of NHE2X3 DKO mice survived through weanling. However, only 60% of male NHE2X3 DKO mice and 88% of female NHE2X3 DKO mice survived to 6 wk of life. We also found that the expression of NHE8 in wild-type female mice was higher compared with wild-type male mice after puberty. In NHE2X3 KDO mice, NHE8 expression was increased in females but not in males. Using Caco-2 cells as a model of the small intestine, we showed that testosterone inhibited endogenous NHE8 expression by reducing NHE8 mRNA synthesis, whereas estrogen had no effect on NHE8 expression. Thus our data show for the first time that intestinal NHE8 has a compensatory role in NHE2X3 DKO mice and this regulation is gender-dependent.     

Rotavirus infection is not associated with small intestinal fluid secretion in the adult mouse

In contrast to humans, adult but not infant small animals are resistant to rotavirus diarrhea. The pathophysiological mechanism behind this age-restricted diarrhea is currently unresolved, and this question was investigated by studying the secretory state of the small intestines of adult mice infected with rotavirus. Immunohistochemistry and histological examinations revealed that rotavirus (strain EDIM) infects all parts of the small intestines of adult mice, with significant numbers of infected cells in the ilea at 2 and 4 days postinfection. Furthermore, quantitative PCR revealed that 100-fold more viral RNA was produced in the ilea than in the jejuna or duodena of adult mice. In vitro perfusion experiments of the small intestine did not reveal any significant changes in net fluid secretion among mice infected for 3 days or 4 days or in those that were noninfected (37 +/- 9 microl . h(-1) . cm(-1), 22 +/- 13 microl . h(-1) . cm(-1), and 33 +/- 6 microl . h(-1) . cm(-1), respectively) or in transmucosal potential difference (4.0 +/- 0.3 mV versus 3.9 +/- 0.4 mV), a marker for active chloride secretion, between control and rotavirus-infected mice. In vivo experiments also did not show any differences in potential difference between uninfected and infected small intestines. Furthermore, no significant differences in weight between infected and uninfected small intestines were found, nor were any differences in fecal output observed between infected and control mice. Altogether, these data suggest that rotavirus infection is not sufficient to stimulate chloride and water secretion from the small intestines of adult mice.     

Multipotential acceptance of Peyer's patches in the intestine for both thymus-derived T cells and extrathymic T cells in mice

Peyer's patches (PP) are important inductive sites for the mucosal immune response. It is well known that lymphocytes that migrate into PP are mainly of T-cell lineage from thymus-derived cells (i.e. alphabetaTCR(high) cells). In this study, we further characterized the properties of PP lymphocytes in mice using a mouse model of colitis induced by dextran sulphate sodium (DSS). Although the major site of the inflammation induced by DSS is known to be the large intestine, the small intestine was also damaged. When mice developed DSS-induced colitis, CD3+CD8+B220+ gammadelta T cells increased in PP in the small intestine. These gammadelta T cells, which are not seen in the PP of normal mice, resembled intraepithelial lymphocytes (IEL) in the small intestine in terms of their expression of CD5, CD103 and Thy1.2. In addition, the Vgamma/delta repertoire of these gammadelta T cells was similar to that of gammadelta IEL. When DSS-treated mice were injected with IEL isolated from normal mice, IEL including gammadelta T cells preferentially migrated to PP, raising the possibility that B220+ T cells seen in PP of diseased mice may derive from IEL in the small intestine. Our present study suggests that PP might be able to accept T-cell lineages from intestinal IEL as well as from thymus-derived T cells.     

The expression and function of MTG/ETO family proteins during neurogenesis

The proneural basic helix-loop-helix (bHLH) proteins promote neurogenesis by inducing changes in gene expression required for neuronal differentiation. Here we characterize one aspect of this differentiation program by analyzing a small family of putative corepressors encoded by MTG genes. We show that MTG genes are expressed sequentially during neurogenesis as cells undergo neuronal differentiation in both the chick spinal cord and in the Xenopus primary nervous system. Using in ovo electroporation, we show that misexpressing wild-type forms of MTG proteins in the developing chick spinal cord does not detectably alter neuronal differentiation. By contrast, the number of differentiated neurons is markedly reduced when a putative dominant-negative mutant of the MTG proteins is expressed in neural precursors in a manner that can be rescued by wild-type MTGR1. Together, these results suggest that MTG family members act downstream of proneural proteins, presumably as corepressors, to promote neuronal differentiation.     

Myeloid translocation gene 16b is a dual A-kinase anchoring protein that interacts selectively with plexins in a phospho-regulated manner

The myeloid translocation gene (MTG) homologue Nervy associates with PlexinA on the plasma membrane, where it functions as an A-kinase anchoring protein (AKAP) to modulate plexin-mediated semaphorin signaling in Drosophila. Mammalian MTG16b is an AKAP found in immune cells where plexin-mediated semaphorin signaling regulates immune responses. This study provides the first evidence that MTG16b is a dual AKAP capable of binding plexins. These interactions are selective (PlexinA1 and A3 bind MTG, while PlexinB1 does not) and can be regulated by PKA-phosphorylation. Collectively, these data suggest a possible mechanism for the targeting and integration of adenosine 3′,5′-cyclic monophosphate (cAMP) and semaphorin signaling in immune cells.

Structured summary

MINT-7556975: PlexinA3 (uniprotkb:P51805) physically interacts (MI:0915) with MTG 16b (uniprotkb:O75081) by anti tag coimmunoprecipitation (MI:0007)MINT-7557008: RI alpha (uniprotkb:Q9DBC7) physically interacts (MI:0915) with MTG 16b (uniprotkb:O75081) by anti bait coimmunoprecipitation (MI:0006)MINT-7556989: MTG 16b (uniprotkb:O75081) physically interacts (MI:0915) with PlexinA3 (uniprotkb:P51805) by pull down (MI:0096)