Terrence E. Riehl

Cyclooxygenase 2 is induced in colonic epithelial cells in inflammatory bowel disease

BACKGROUND & AIMSnProstaglandins are synthesized by cyclooxygenases (COX)-1 and -2. The expression and cellular localization of COX-1 and COX-2 in normal human colon and inflammatory bowel disease (IBD) surgical resections were studied.nnnMETHODSnCOX-1 and COX-2 protein expression and cellular localization were assessed by Western blotting and immunohistochemistry.nnnRESULTSnCOX-1 protein was expressed at equal levels in normal, Crohns disease, and ulcerative colitis colonic epithelial cells. COX-2 protein was not detected in normal epithelial cells but was detected in Crohns disease and ulcerative colitis epithelial cells. Immunohistochemistry of normal, Crohns colitis, and ulcerative colitis tissue showed equivalent COX-1 expression in epithelial cells in the lower half of the colonic crypts. COX-2 expression was absent from normal colon, whereas in Crohns colitis and ulcerative colitis, COX-2 was observed in apical epithelial cells and in lamina propria mononuclear cells. In Crohns ileitis, COX-2 was present in the villus epithelial cells. In ulcerative colitis, colonic epithelial cells expressing COX-2 also expressed inducible nitric oxide synthase.nnnCONCLUSIONSnCOX-1 was localized in the crypt epithelium of the normal ileum and colon, and its expression was unchanged in IBD. COX-2 was undetectable in normal ileum or colon, but it was induced in apical epithelial cells of inflamed foci in IBD.

Identification of a Novel Putative Gastrointestinal Stem Cell and Adenoma Stem Cell Marker, Doublecortin and CaM Kinase‐Like‐1, Following Radiation Injury and in Adenomatous Polyposis Coli/Multiple Intestinal Neoplasia Mice

In the gut, tumorigenesis arises from intestinal or colonic crypt stem cells. Currently, no definitive markers exist that reliably identify gut stem cells. Here, we used the putative stem cell marker doublecortin and CaM kinase‐like‐1 (DCAMKL‐1) to examine radiation‐induced stem cell apoptosis and adenomatous polyposis coli (APC)/multiple intestinal neoplasia (min) mice to determine the effects of APC mutation on DCAMKL‐1 expression. Immunoreactive DCAMKL‐1 staining was demonstrated in the intestinal stem cell zone. Furthermore, we observed apoptosis of the cells negative for DCAMKL‐1 at 6 hours. We found DNA damage in all the cells in the crypt region, including the DCAMKL‐1‐positive cells. We also observed stem cell apoptosis and mitotic DCAMKL‐1‐expressing cells 24 hours after irradiation. Moreover, in APC/min mice, DCAMKL‐1‐expressing cells were negative for proliferating cell nuclear antigen and nuclear β‐catenin in normal‐appearing intestine. However, β‐catenin was nuclear in DCAMKL‐1‐positive cells in adenomas. Thus, nuclear translocation of β‐catenin distinguishes normal and adenoma stem cells. Targeting DCAMKL‐1 may represent a strategy for developing novel chemotherapeutic agents.

Myd88-dependent positioning of Ptgs2-expressing stromal cells maintains colonic epithelial proliferation during injury

We identified cellular and molecular mechanisms within the stem cell niche that control the activity of colonic epithelial progenitors (ColEPs) during injury. Here, we show that while WT mice maintained ColEP proliferation in the rectum following injury with dextran sodium sulfate, similarly treated Myd88(-/-) (TLR signaling-deficient) and prostaglandin-endoperoxide synthase 2(-/-) (Ptgs2(-/-)) mice exhibited a profound inhibition of epithelial proliferation and cellular organization within rectal crypts. Exogenous addition of 16,16-dimethyl PGE(2) (dmPGE(2)) rescued the effects of this injury in both knockout mouse strains, indicating that Myd88 signaling is upstream of Ptgs2 and PGE(2). In WT and Myd88(-/-) mice, Ptgs2 was expressed in scattered mesenchymal cells. Surprisingly, Ptgs2 expression was not regulated by injury. Rather, in WT mice, the combination of injury and Myd88 signaling led to the repositioning of a subset of the Ptgs2-expressing stromal cells from the mesenchyme surrounding the middle and upper crypts to an area surrounding the crypt base adjacent to ColEPs. These findings demonstrate that Myd88 and prostaglandin signaling pathways interact to preserve epithelial proliferation during injury using what we believe to be a previously undescribed mechanism requiring proper cellular mobilization within the crypt niche.

Doublecortin and CaM kinase-like-1 and leucine-rich-repeat-containing G-protein-coupled receptor mark quiescent and cycling intestinal stem cells, respectively.

It is thought that small intestinal epithelia (IE) undergo continuous self‐renewal primarily due to their population of undifferentiated stem cells. These stem cells give rise to transit amplifying (daughter/progenitor) cells, which can differentiate into all mature cell types required for normal gut function. Identification of stem cells in IE is paramount to fully understanding this renewal process. One major obstacle in gastrointestinal stem cell biology has been the lack of definitive markers that identify small intestinal stem cells (ISCs). Here we demonstrate that the novel putative ISC marker doublecortin and CaM kinase‐like‐1 (DCAMKL‐1) is predominantly expressed in quiescent cells in the lower two‐thirds of intestinal crypt epithelium and in occasional crypt‐based columnar cells (CBCs). In contrast, the novel putative stem cell marker leucine‐rich‐repeat‐containing G‐protein‐coupled receptor (LGR5) is observed in rapidly cycling CBCs and in occasional crypt epithelial cells. Furthermore, functionally quiescent DCAMKL‐1+ crypt epithelial cells retain bromo‐deoxyuridine in a modified label retention assay. Moreover, we demonstrate that DCAMKL‐1 is a cell surface expressing protein; DCAMKL‐1+ cells, isolated from the adult mouse small intestine by fluorescence activated cell sorting, self‐renew and ultimately form spheroids in suspension culture. These spheroids formed glandular epithelial structures in the flanks of athymic nude mice, which expressed multiple markers of gut epithelial lineage. Thus, DCAMKL‐1 is a marker of quiescent ISCs and can be distinguished from the cycling stem/progenitors (LGR5+). Moreover, DCAMKL‐1 can be used to isolate normal small intestinal stem cells and represents a novel research tool for regenerative medicine and cancer therapy. STEM CELLS 2009;27:2571–2579

Prostaglandin E2 reduces radiation-induced epithelial apoptosis through a mechanism involving AKT activation and bax translocation

Prostaglandin E2 (PGE2) synthesis modulates the response to radiation injury in the mouse intestinal epithelium through effects on crypt survival and apoptosis; however, the downstream signaling events have not been elucidated. WT mice receiving 16,16-dimethyl PGE2 (dmPGE2) had fewer apoptotic cells per crypt than untreated mice. Apoptosis in Bax(-/-) mice receiving 12 Gy was approximately 50% less than in WT mice, and the ability of dmPGE2 to attenuate apoptosis was lost in Bax(-/-) mice. Positional analysis revealed that apoptosis in the Bax(-/-) mice was diminished only in the bax-expressing cells of the lower crypts and that in WT mice, dmPGE2 decreased apoptosis only in the bax-expressing cells. The HCT-116 intestinal cell line and Bax(-/-) HCT-116 recapitulated the apoptotic response of the mouse small intestine with regard to irradiation and dmPGE2. Irradiation of HCT-116 cells resulted in phosphorylation of AKT that was enhanced by dmPGE2 through transactivation of the EGFR. Inhibition of AKT phosphorylation prevented the reduction of apoptosis by dmPGE2 following radiation. Transfection of HCT-116 cells with a constitutively active AKT reduced apoptosis in irradiated cells to the same extent as in nontransfected cells treated with dmPGE2. Treatment with dmPGE2 did not alter bax or bcl-x expression but suppressed bax translocation to the mitochondrial membrane. Our in vivo studies indicate that there are bax-dependent and bax-independent radiation-induced apoptosis in the intestine but that only the bax-dependent apoptosis is reduced by dmPGE2. The in vitro studies indicate that dmPGE2, most likely by signaling through the E prostaglandin receptor EP2, reduces radiation-induced apoptosis through transactivation of the EGFR and enhanced activation of AKT and that this results in reduced bax translocation to the mitochondria.

Lipopolysaccharide is radioprotective in the mouse intestine through a prostaglandin-mediated mechanism

BACKGROUND & AIMSnThe bone marrow and the intestine are the major sites of radiation-induced injury. The cellular response to radiation injury in the intestine or bone marrow can be modulated by agents given before irradiation. Lipopolysaccharide is known to be radioprotective in the bone marrow, but its effect on the intestine is not known. We sought to determine if lipopolysaccharide is radioprotective in the intestine and, if so, to determine the mechanism of its radioprotective effects.nnnMETHODSnMice were treated with parenteral lipopolysaccharide or vehicle and then irradiated (14 Gy total body irradiation in a cesium irradiator). The number of surviving intestinal crypts was assessed 3.5 days after irradiation using a clonogenic assay.nnnRESULTSnParenteral administration of lipopolysaccharide 2-24 hours before irradiation resulted in a 2-fold increase in the number of surviving crypts 3.5 days after irradiation. The radioprotective effects of lipopolysaccharide could be eliminated by coadministration of a selective inhibitor of cyclooxygenase 2. Lipopolysaccharide was radioprotective in wild-type mice but not in mice with a disrupted cyclooxygenase 2. Parenteral administration of lipopolysaccharide resulted in increased production of prostaglandins in the intestine and in the induction of cyclooxygenase 2 expression in subepithelial fibroblasts and in villous, but not crypt, epithelial cells.nnnCONCLUSIONSnLipopolysaccharide is radioprotective in the mouse intestine through a prostaglandin-dependent pathway.

Lactobacillus probiotic protects intestinal epithelium from radiation injury in a TLR-2/cyclo-oxygenase-2-dependent manner

Background The small intestinal epithelium is highly sensitive to radiation and is a major site of injury during radiation therapy and environmental overexposure. Objective To examine probiotic bacteria as potential radioprotective agents in the intestine. Methods 8-week-old C57BL/6 wild-type or knockout mice were administered probiotic by gavage for 3u2005days before 12u2005Gy whole body radiation. The intestine was evaluated for cell-positional apoptosis (6u2005h) and crypt survival (84u2005h). Results Gavage of 5×107 Lactobacillus rhamnosus GG (LGG) improved crypt survival about twofold (p<0.01); the effect was observed when administered before, but not after, radiation. Conditioned medium (CM) from LGG improved crypt survival (1.95-fold, p<0.01), and both LGG and LGG-CM reduced epithelial apoptosis particularly at the crypt base (33% to 18%, p<0.01). LGG was detected in the distal ileal contents after the gavage cycle, but did not lead to a detectable shift in bacterial family composition. The reduction in epithelial apoptosis and improved crypt survival offered by LGG was lost in MyD88−/−, TLR-2−/− and cyclo-oxygenase-2−/− (COX-2) mice but not TLR-4−/− mice. LGG administration did not lead to increased jejunal COX-2 mRNA or prostaglandin E2 levels or a change in number of COX-2-expressing cells. However, a location shift was observed in constitutively COX-2-expressing cells of the lamina propria from the villi to a position near the crypt base (villi to crypt ratio 80:20 for control and 62:38 for LGG; p<0.001). Co-staining revealed these COX-2-expressing small intestinal lamina propria cells to be mesenchymal stem cells. Conclusions LGG or its CM reduce radiation-induced epithelial injury and improve crypt survival. A TLR-2/MyD88 signalling mechanism leading to repositioning of constitutive COX-2-expressing mesenchymal stem cells to the crypt base is invoked.

Regulation of colonic epithelial repair in mice by Toll-like receptors and hyaluronic acid.

BACKGROUND & AIMSnThe protective component of the host response to dextran sodium sulfate (DSS)-induced colitis in the mouse is mediated through the activation of Toll-like receptor (TLR) 4, the induction of cyclooxygenase (COX)-2, and prostaglandin E(2) production. TLR4 ligands include bacterial lipopolysaccharide and hyaluronic acid, a component of the extracellular matrix. Our hypothesis is that hyaluronic acid, through TLRs, plays a protective role in the host response to DSS-induced colitis.nnnMETHODSnDSS (2.5%) was administered for 7 days in wild-type and MyD88(-/-) mice. The mice also received intraperitoneal hyaluronic acid. The expression of hyaluronic acid, COX-2, and macrophage inflammatory protein (MIP)-2 was evaluated by immunohistochemistry.nnnRESULTSnDSS induced a marked increase in hyaluronic acid in the lamina propria of wild-type but not MyD88(-/-) mice. Treatment with DSS also induced the MyD88-dependent expression of hyaluronic acid synthases 2 and 3, enzymes involved in hyaluronic acid synthesis, in lamina propria macrophages. Exogenous hyaluronic acid induced the expression of tumor necrosis factor alpha, MIP-2, and COX-2 in the colon in a MyD88-dependent manner. In wild-type but not MyD88(-/-), TLR4(-/-), COX-2(-/-) mice, hyaluronic acid was protective against DSS-induced colitis. In wild-type mice, hyaluronic acid was therapeutic in established DSS-induced colitis.nnnCONCLUSIONSnEndogenous hyaluronic acid expression is markedly increased in DSS-induced colitis and preserves the epithelium through TLR activation and COX-2 expression. Furthermore, exogenous hyaluronic acid, through the activation of TLRs and the production of prostaglandin E(2) through COX-2, has protective effects in DSS-induced colitis.

Opposing effects of tumor necrosis factor receptor 1 and 2 in sepsis due to cecal ligation and puncture.

Tumor necrosis factor (TNF)-α, a cardinal molecule in the cascade of sepsis-induced host injury, binds to two distinct receptors: tumor necrosis factor receptor (TNFR) 1 and TNFR2. We used the cecal ligation and puncture model of polymicrobial sepsis to elucidate the role of these receptors in sepsis pathogenesis. Mice lacking TNFR1 had prolonged survival with less hypothermia, whereas mice lacking TNFR2−/− had shortened survival and more profound hypothermia than wild-type mice. TNFR1−/− and TNFR2−/− mice had increased serum concentrations of interleukin (IL) 1β and total TNF-α (free plus receptor bound) compared with wild-type mice, but there were no differences in IL6 or IL10 concentrations. Furthermore, free TNF-α was markedly elevated in the serum and peritoneal fluid of mice lacking TNFR2, supporting a role for this receptor in regulating the concentration of TNF-α. Lastly, apoptosis of ileal crypt epithelial cells was increased in mice lacking TNFR1, but there were no differences in lymphocyte apoptosis. These data suggest that in sepsis, TNFR1 mediates much of the TNF-α-induced pathology, whereas TNFR2 mediates protective effects.

The Hyaluronic Acid Receptor CD44 Coordinates Normal and Metaplastic Gastric Epithelial Progenitor Cell Proliferation

Background: Gastric parietal cell atrophy causes metaplasia, reactive stem cell proliferation, and increased risk for cancer. Results: Atrophy induces proliferation of CD44-positive epithelial cells that requires ERK → CD44 → STAT3 signaling. Conclusion: CD44 is a putative gastric stem cell marker that regulates normal and metaplasia-associated proliferation. Significance: Targeted pharmacological inhibition of ERK/CD44/STAT3 signaling may help block or reverse proliferation in precancerous atrophic/metaplastic lesions. The stem cell in the isthmus of gastric units continually replenishes the epithelium. Atrophy of acid-secreting parietal cells (PCs) frequently occurs during infection with Helicobacter pylori, predisposing patients to cancer. Atrophy causes increased proliferation of stem cells, yet little is known about how this process is regulated. Here we show that CD44 labels a population of small, undifferentiated cells in the gastric unit isthmus where stem cells are known to reside. Loss of CD44 in vivo results in decreased proliferation of the gastric epithelium. When we induce PC atrophy by Helicobacter infection or tamoxifen treatment, this CD44+ population expands from the isthmus toward the base of the unit. CD44 blockade during PC atrophy abrogates the expansion. We find that CD44 binds STAT3, and inhibition of either CD44 or STAT3 signaling causes decreased proliferation. Atrophy-induced CD44 expansion depends on pERK, which labels isthmal cells in mice and humans. Our studies delineate an in vivo signaling pathway, ERK → CD44 → STAT3, that regulates normal and atrophy-induced gastric stem/progenitor-cell proliferation. We further show that we can intervene pharmacologically at each signaling step in vivo to modulate proliferation.