Alda Vidrich

Members of the Entamoeba histolytica transmembrane kinase family play non-redundant roles in growth and phagocytosis

Entamoeba histolytica contains a large and novel family of transmembrane kinases (TMKs). The expression patterns of the E. histolytica TMKs in individual trophozoites and the roles of the TMKs for sensing and responding to extracellular cues were incompletely characterised. Here we provide evidence that single cells express multiple TMKs and that TMK39 and TMK54 likely serve non-redundant cellular functions. Laser-capture microdissection was used in conjunction with microarray analysis to demonstrate that single trophozoites express more than one TMK gene. Anti-peptide antibodies were raised against unique regions in the extracellular domains of TMK39, TMK54 and PaTMK, and TMK expression was analysed at the protein level. Flow cytometric assays revealed that populations of trophozoites homogeneously expressed TMK39, TMK54 and PaTMK, while confocal microscopy identified different patterns of cell surface expression for TMK39 and TMK54. The functions of TMK39 and TMK54 were probed by the inducible expression of dominant-negative mutants. While TMK39 co-localised with ingested beads and expression of truncated TMK39 interfered with trophozoite phagocytosis of apoptotic lymphocytes, expression of a truncated TMK54 inhibited growth of amoebae and altered the surface expression of the heavy subunit of the E. histolytica Gal/GalNAc lectin. Overall, our data indicates that multiple members of the novel E. histolytica TMK family are utilised for non-redundant functions by the parasite.

Fibroblast growth factor receptor-3 is expressed in undifferentiated intestinal epithelial cells during murine crypt morphogenesis.

Prior studies have demonstrated that fibroblast growth factor receptor‐3 (FGFR‐3) regulates proliferation of undifferentiated intestinal epithelial cells in vitro. However, the function(s) of FGFR‐3–mediated signaling during intestinal development and epithelial differentiation in vivo remain unknown. The goal of this study was to define the temporal, regional, and cell‐specific patterns of FGFR‐3 expression and its ligands during normal intestinal ontogeny and epithelial regeneration. Both the IIIb and IIIc isoforms of FGFR‐3 mRNA, which result from differential splicing of the FGFR‐3 primary transcript, were detected in mouse small intestine as early as embryonic day 16. FGFR‐3 levels peaked in the small intestine from 7 to 21 days after birth and decreased thereafter to reach the low levels observed in adult mice. FGFR‐3 IIIb and IIIc mRNA levels were highest in the duodenum and proximal jejunum with lower levels of both seen in the distal jejunum, ileum, and colon. FGFR‐3 was expressed in a subset of proliferating undifferentiated crypt epithelial cells located in the intervillous epithelium and in the lower half of nascently forming crypts but not in differentiated epithelial cell types. FGFR‐3 IIIb was the dominant isoform expressed in both small intestinal and colonic crypts. Expression of FGF1, FGF2, and FGF9, known ligands of FGFR‐3, paralleled patterns of FGFR‐3 expression during gut development. These data suggest that signaling through FGFR‐3 plays a role in regulating morphogenic events involved in formation of intestinal crypts and/or the fate of epithelial stem cells. Developmental Dynamics 230:114–123,

Altered epithelial cell lineage allocation and global expansion of the crypt epithelial stem cell population are associated with ileitis in SAMP1/YitFc mice.

Crohns disease is characterized by cycles of mucosal injury and ulceration followed by epithelial regeneration and restoration of normal epithelial function. In this study, we examined whether ileitis in SAMP1/YitFc mice, a recombinant-inbred line that spontaneously develops ileitis resembling human Crohns disease, was associated with alterations in normal patterns of epithelial differentiation or changes in epithelial regeneration after experimental injury. Increased numbers of Paneth, goblet, and intermediate cells were present focally in the ileum of SAMP1/YitFc mice by 4 weeks of age, before any histological evidence of acute or chronic inflammation. This increase in secretory cells became more pronounced at sites of ileitis with increasing age and inflammation. Additionally, there was mispositioning of Paneth and intermediate cells along the crypt-to-villus unit. A concomitant reduction in the number of absorptive enterocytes was observed. In contrast to the ileal-specific changes in lineage allocation, crypt stem cell numbers began to increase in both the ileum and proximal jejunum at the onset of inflammation in SAMP1/YitFc mice. These data suggest that the alterations in epithelial cell differentiation and increases in the size of the crypt stem cell population observed in SAMP1/YitFc mice are regulated by distinct mechanisms. We speculate that these epithelial alterations may play a role in the pathogenesis of ileitis in this murine model of Crohns disease.

Fibroblast growth factor receptor-3 regulates Paneth cell lineage allocation and accrual of epithelial stem cells during murine intestinal development.

Fibroblast growth factor receptor 3 (FGFR-3) is expressed in the lower crypt epithelium, where stem cells of the intestine reside. The role of FGFR-3 signaling in regulating features of intestinal morphogenesis was examined in FGFR-3-null (FGFR-3(-/-)) mice. FGFR-3(-/-) mice had only about half the number of intestinal crypts and a marked decrease in the number of functional clonogenic stem cells, as assessed by an in vivo microcolony-forming assay, compared with wild-type littermates. A marked deficit in allocation of progenitor cells to Paneth cell differentiation was noted, although all the principal epithelial lineages were represented in FGFR-3(-/-) mice. The total cellular content and nuclear localization of beta-catenin protein were reduced in FGFR-3(-/-) mice, as was expression of cyclin D1 and matrix metalloproteinase-7, major downstream targets of beta-catenin/T cell factor-4 (Tcf-4) signaling. Activation of FGFR-3 in Caco-2 cells, an intestinal epithelial cell line, abrogated the fall in beta-catenin/Tcf-4 signaling activity that is normally observed in these cells as cultures become progressively more confluent. These findings are consistent with the hypothesis that, during intestinal development, FGFR-3 signaling regulates crypt epithelial stem cell expansion and crypt morphogenesis, as well as Paneth cell lineage specification, through beta-catenin/Tcf-4-dependent and -independent pathways.

Resistin-like molecule β (RELMβ/FIZZ2) is highly expressed in the ileum of SAMP1/YitFc mice and is associated with initiation of ileitis

SAMP1/Fc mice develop spontaneous ileitis that shares many features with human Crohn’s disease. One of the earliest features of ileitis in SAMP1/Fc mice is an increase in the number of ileal goblet and intermediate cells. Resistin-like molecule β (RELMβ) is a goblet cell-specific, cysteine-rich peptide previously shown to function as part of the innate immune response. In this study, we examined the role of expression of RELMβ in the initiation of ileal inflammation in SAMP1/Fc mice. RELMβ was highly induced in the ilea of SAMP1/Fc mice beginning at age 5 wk, coincident with the histological appearance of inflammation. RELMβ was found in ileal goblet cells and some intermediate and Paneth cells. Surprisingly, RELMβ mRNA levels were significantly increased in the ilea of 80% of germ-free SAMP1/Fc mice examined compared with specific pathogen-free AKR control mice of similar age. Ileitis was observed in germfree SAMP1/Fc mice, although it was attenuated relative to specific pathogen-free SAMP1/Fc mice. These data suggest that neither the early induction of RELMβ expression nor ileal inflammation requires the presence of viable intestinal flora. Neither was the induction of RELMβ dependent on the major Th1 or Th2 cytokines. However, RELMβ stimulated naive bone marrow-derived macrophages to secrete significant amounts of TNF-α, IL-6, and RANTES. Our data suggest that RELMβ is involved in the initiation of ileitis in SAMP1/Fc mice and may act through the induction of proinflammatory cytokines from resident immune cells within the mucosa.

Intestinal cell kinase, a MAP kinase-related kinase, regulates proliferation and G1 cell cycle progression of intestinal epithelial cells.

Intestinal cell kinase (ICK), originally cloned from the intestine and expressed in the intestinal crypt epithelium, is a highly conserved serine/threonine protein kinase that is similar to mitogen-activated protein kinases (MAPKs) in the catalytic domain and requires dual phosphorylation within a MAPK-like TDY motif for full activation. Despite these similarities to MAPKs, the biological functions of ICK remain unknown. In this study, we report that suppression of ICK expression in cultured intestinal epithelial cells by short hairpin RNA (shRNA) interference significantly impaired cellular proliferation and induced features of gene expression characteristic of colonic or enterocytic differentiation. Downregulation of ICK altered expression of cell cycle regulators (cyclin D1, c-Myc, and p21(Cip1/WAF1)) of G(1)-S transition, consistent with the G(1) cell cycle delay induced by ICK shRNA. ICK deficiency also led to a significant decrease in the expression and/or activity of p70 ribosomal protein S6 kinase (S6K1) and eukaryotic initiation factor 4E (eIF4E), concomitant with reduced expression of their upstream regulators, the mammalian target of rapamycin (mTOR) and the regulatory associated protein of mTOR (Raptor). Furthermore, ICK interacts with the mTOR/Raptor complex in vivo and phosphorylates Raptor in vitro. These results suggest that disrupting ICK function may downregulate protein translation of specific downstream targets of eIF4E and S6K1 such as cyclin D1 and c-Myc through the mTOR/Raptor signaling pathway. Taken together, our findings demonstrate an important role for ICK in proliferation and differentiation of intestinal epithelial cells.

Intestinal stem cells and mucosal gut development.

Purpose of review In the past year, the study of intestinal stem cell biology has realized significant progress toward understanding the mechanisms and pathways regulating crypt stem cell turnover, maintenance, and differentiation. Recent findings This review summarizes recent investigations that have contributed significantly to the elucidation of mechanisms operative during intestinal development and in the adult intestine that regulate maintenance of the stem cell niche, cell fate and lineage allocation, and establishment and maintenance of the architectural organization of the crypt-to-villus axis. Summary The relevance of the findings discussed in this review extends beyond the field of intestinal development to encompass the study of tissue remodeling and repair and intestinal neoplasia.

Elevated Expression of Paneth Cell CRS4C in Ileitis-prone SAMP1/YitFc Mice REGIONAL DISTRIBUTION, SUBCELLULAR LOCALIZATION, AND MECHANISM OF ACTION

Paneth cells at the base of small intestinal crypts of Lieberkühn secrete host defense peptides and proteins, including α-defensins, as mediators of innate immunity. Mouse Paneth cells also express α-defensin-related Defcr-rs genes that code for cysteine-rich sequence 4C (CRS4C) peptides that have a unique CPX triplet repeat motif. In ileitis-prone SAMP1/YitFc mice, Paneth cell levels of CRS4C mRNAs and peptides are induced more than a 1000-fold relative to non-prone strains as early as 4 weeks of age, with the mRNA and peptide levels highest in distal ileum and below detection in duodenum. CRS4C-1 peptides are found exclusively in Paneth cells where they occur only in dense core granules and thus are secreted to function in the intestinal lumen. CRS4C bactericidal peptide activity is membrane-disruptive in that it permeabilizes Escherichia coli and induces rapid microbial cell K+ efflux, but in a manner different from mouse α-defensin cryptdin-4. In in vitro studies, inactive pro-CRS4C-1 is converted to bactericidal CRS4C-1 peptide by matrix metalloproteinase-7 (MMP-7) proteolysis of the precursor proregion at the same residue positions that MMP-7 activates mouse pro-α-defensins. The absence of processed CRS4C in protein extracts of MMP-7-null mouse ileum demonstrates the in vivo requirement for intracellular MMP-7 in pro-CRS4C processing.

Fibroblast Growth Factor Receptor-3 (FGFR-3) Regulates Expression of Paneth Cell Lineage-specific Genes in Intestinal Epithelial Cells through both TCF4/β-Catenin-dependent and -independent Signaling Pathways

Fibroblast growth factor receptor-3 (FGFR-3) expression in the developing intestine is restricted to the undifferentiated epithelial cells within the lower portion of the crypt. We previously showed that mice lacking functional FGFR-3 have a significant decrease in the number of Paneth cells in the small intestine. Here, we used Caco2 cells to investigate whether FGFR-3 signaling can directly modulate expression of Paneth cell differentiation markers through its effects on TCF4/β-catenin or through other signaling pathways downstream of this receptor. Caco2 cells treated with FGFR-3 ligands or expressing FGFR-3K650E, a constitutively active mutant, resulted in a significantly increased expression of genes characteristic of mature Paneth cells, including human α-defensins 5 and 6 (HD5 and HD6) and Paneth cell lysozyme, whereas enterocytic differentiation markers were reduced. Activation of FGFR-3 signaling sustained high levels of β-catenin mRNA expression, leading to increased TCF4/β-catenin-regulated transcriptional activity in Caco2 cells. Sustained activity of the TCF4/β-catenin pathway was required for the induction of Paneth cell markers. Activation of the MAPK pathway by FGFR-3 is also required for the induction of Paneth cell markers in addition to and independent of the effect of FGFR-3 on TCF4/β-catenin activity. These studies suggest that coordinate activation of multiple independent signaling pathways downstream of FGFR-3 is involved in regulation of Paneth cell differentiation.

Response to Comment on “Resistin-Like Molecule β (RELM β/FIZZ2) Is Highly Expressed in the Ileum of SAMP1/YitFc Mice and Is Associated with Initiation of Ileitis”

We thank Wen et al. ([1][1]) for their comments regarding our recent article ([2][2]). Although SAMP1/YitFc mice have many features that closely resemble human Crohn’s disease including pathological and histologic features, cytokine profile activation, and beneficial response to treatments that