Hyaluronic acid promotes Lgr5+ stem cell proliferation and crypt fission through TLR4 and PGE2 transactivation of EGFR.

Hyaluronic acid (HA), a glycosaminoglycan in the extracellular matrix, binds to CD44 and Toll-like receptor 4 (TLR4). We previously demonstrated that both CD44 and TLR4, but predominately TLR4, mediated HA stimulation of Lgr5+ stem cell proliferation, crypt fission, and intestinal growth in postnatal mice. Here we address the questions of which cell type expresses the relevant TLR4 in driving intestinal growth and what are the downstream events from TLR4 activation. Studies were done in 14-day-old mice: wild type (WT), mice deficient in cyclooxygenase 2 (COX2), mice deficient in myeloid cell TLR4, and mice deficient in epithelial cell epidermal growth factor receptor (EGFR). Biological end points included crypt fission and Lgr5 cell proliferation. In WT mice, treatment with NS-398 (a COX2 inhibitor), clodronate (a macrophage-depleting agent), or tyrphostin (an EGFR inhibitor) resulted in 30% reductions in crypt fission and Lgr5+ stem cell proliferation compared with control mice. Mice deficient in COX2 or myeloid TLR4 or epithelial cell EGFR all had 30% reductions in crypt fission and Lgr5+ stem cell proliferation compared with WT mice. Administration of dimethyl PGE2, a stable PGE2 analog, increased crypt fission and Lgr5+ stem cell proliferation. Administration of dimethyl PGE2 reversed the effects of NS-398, clodronate, COX2 deficiency, and myeloid TLR4 deficiency but had no effect on mice treated with tyrphostin or mice deficient in epithelial cell EGFR. We conclude that, in postnatal mice, ~30% of intestinal growth as manifested by crypt fission and Lgr5+ stem cell proliferation is driven by a novel pathway: Extracellular HA binds TLR4 on pericryptal macrophages, inducing the production of PGE2 through COX2. PGE2 transactivates EGFR in Lgr5+ epithelial stem cells, resulting in Lgr5+ stem cell proliferation and crypt fission.NEW & NOTEWORTHY This study, in newborn mice, describes a novel molecular pathway regulating Lgr5+ epithelial stem cell proliferation and normal intestinal elongation, as assessed by crypt fission. In this pathway, endogenous extracellular hyaluronic acid binds to Toll-like receptor 4 on pericryptal macrophages releasing PGE2 which binds to epidermal growth factor receptor on Lgr5+ stem cells resulting in proliferation. Lgr5+ stem cell proliferation leads to crypt fission and intestinal elongation. The demonstration that normal growth requires microbial-independent Toll-like receptor activation is novel.

Hyaluronic Acid Binding to TLR4 Promotes Proliferation and Blocks Apoptosis in Colon Cancer.

Hyaluronic acid (HA), a constituent of the extracellular matrix, promotes colorectal cancer growth. CD44 is a relevant HA receptor in this context. However, HA is also a ligand for TLR4, a receptor of significance in colorectal cancer. In this study, we examine the relative contribution of HA interactions with CD44 and TLR4 in colon tumorigenesis. Colorectal cancer models included ApcMin/+ mice, azoxymethane/dextran sodium sulfate (AOM-DSS), and CT26 tumor isografts. We used knockout mice and CT26 colorectal cancer cells with CRISPR knockdown of CD44 and TLR4. HA activity was modulated by PEP1 (a 12-mer peptide that blocks HA from binding its receptors), hyaluronidase (which promotes HA degradation), or 4-MU (HA synthesis inhibitor). Blockade of HA binding via PEP1 decreased growth in all colorectal cancer models and in cell culture. The effects were significant in WT and with CD44 deletion, but not with TLR4 deletion. In the AOM-DSS model, mice deficient in CD44 or TLR4 had fewer tumors. CD44- and TLR4-deficient CT26 isografts grew more slowly, exhibiting decreased tumor cell proliferation and increased apoptosis. In vitro, endogenous HA blocked LPS binding to TLR4 suggesting that HA is a relevant TLR4 ligand in colon cancer. Finally, PEP1 enhanced tumor radiation sensitivity in the isograft model. Together, these results indicate that HA binding to TLR4, as well as CD44, plays a key role in colon tumorigenesis. These findings also raise the possibility that an agent that blocks HA binding, such as PEP1, may be useful as an adjuvant therapy in colon cancer.

Lactobacillus rhamnosus GG protects the intestinal epithelium from radiation injury through release of lipoteichoic acid, macrophage activation and the migration of mesenchymal stem cells.

OBJECTIVE: Lactobacillus rhamnosus GG (LGG), a probiotic, given by gavage is radioprotective of the mouse intestine. LGG-induced radioprotection is toll-like receptor 2 (TLR2) and cyclooxygenase-2 (COX-2)-dependent and is associated with the migration of COX-2+mesenchymal stem cells (MSCs) from the lamina propria of the villus to the lamina propria near the crypt epithelial stem cells. Our goals were to define the mechanism of LGG radioprotection including identification of the TLR2 agonist, and the mechanism of the MSC migration and to determine the safety and efficacy of this approach in models relevant to clinical radiation therapy. DESIGN: Intestinal radioprotection was modelled in vitro with cell lines and enteroids as well as in vivo by assaying clinical outcomes and crypt survival. Fractionated abdominal and single dose radiation were used along with syngeneic CT26 colon tumour grafts to assess tumour radioprotection. RESULTS: LGG with a mutation in the processing of lipoteichoic acid (LTA), a TLR2 agonist, was not radioprotective, while LTA agonist and native LGG were. An agonist of CXCR4 blocked LGG-induced MSC migration and LGG-induced radioprotection. LGG given by gavage induced expression of CXCL12, a CXCR4 agonist, in pericryptal macrophages and depletion of macrophages by clodronate liposomes blocked LGG-induced MSC migration and radioprotection. LTA effectively protected the normal intestinal crypt, but not tumours in fractionated radiation regimens. CONCLUSIONS: LGG acts as a 'time-release capsule' releasing radioprotective LTA. LTA then primes the epithelial stem cell niche to protect epithelial stem cells by triggering a multicellular, adaptive immune signalling cascade involving macrophages and PGE2 secreting MSCs. TRIAL REGISTRATION NUMBER: NCT01790035; Pre-results.

TRPV4 Channel Signaling in Macrophages Promotes Gastrointestinal Motility via Direct Effects on Smooth Muscle Cells.

Intestinal macrophages are critical for gastrointestinal (GI) homeostasis, but our understanding of their role in regulating intestinal motility is incomplete. Here, we report that CX3C chemokine receptor 1-expressing muscularis macrophages (MMs) were required to maintain normal GI motility. MMs expressed the transient receptor potential vanilloid 4 (TRPV4) channel, which senses thermal, mechanical, and chemical cues. Selective pharmacologic inhibition of TRPV4 or conditional deletion of TRPV4 from macrophages decreased intestinal motility and was sufficient to reverse the GI hypermotility that is associated with chemotherapy treatment. Mechanistically, stimulation of MMs via TRPV4 promoted the release of prostaglandin E2 and elicited colon contraction in a paracrine manner via prostaglandin E receptor signaling in intestinal smooth muscle cells without input from the enteric nervous system. Collectively, our data identify TRPV4-expressing MMs as an essential component required for maintaining normal GI motility and provide potential drug targets for GI motility disorders.

CD44 and TLR4 mediate hyaluronic acid regulation of Lgr5+ stem cell proliferation, crypt fission, and intestinal growth in postnatal and adult mice.

Hyaluronic acid, a glycosaminoglycan in the extracellular matrix, binds to CD44 and Toll-like receptor 4 (TLR4). We previously addressed the role of hyaluronic acid in small intestinal and colonic growth in mice. We addressed the role of exogenous hyaluronic acid by giving hyaluronic acid intraperitoneally and the role of endogenous hyaluronic acid by giving PEP-1, a peptide that blocks hyaluronic acid binding to its receptors. Exogenous hyaluronic acid increased epithelial proliferation but had no effect on intestinal length. PEP-1 resulted in a shortened small intestine and colon and diminished epithelial proliferation. In the current study, we sought to determine whether the effects of hyaluronic acid on growth were mediated by signaling through CD44 or TLR4 by giving exogenous hyaluronic acid or PEP-1 twice a week from 3-8 wk of age to wild-type, CD44(-/-), and TLR4(-/-) mice. These studies demonstrated that signaling through both CD44 and TLR4 were important in mediating the effects of hyaluronic acid on growth in the small intestine and colon. Extending our studies to early postnatal life, we assessed the effects of exogenous hyaluronic acid and PEP-1 on Lgr5(+) stem cell proliferation and crypt fission. Administration of PEP-1 to Lgr5(+) reporter mice from postnatal day 7 to day 14 decreased Lgr5(+) cell proliferation and decreased crypt fission. These studies indicate that endogenous hyaluronic acid increases Lgr5(+) stem cell proliferation, crypt fission, and intestinal lengthening and that these effects are dependent on signaling through CD44 and TLR4.

Inhibition of Cyclooxygenase-2 Prevents Chronic and Recurrent Cystitis.

The spread of multidrug-resistant microorganisms globally has created an urgent need for novel therapeutic strategies to combat urinary tract infections (UTIs). Immunomodulatory therapy may provide benefit, as treatment of mice with dexamethasone during acute UTI improved outcome by reducing the development of chronic cystitis, which predisposes to recurrent infection. Here we discovered soluble biomarkers engaged in myeloid cell development and chemotaxis that were predictive of future UTI recurrence when elevated in the sera of young women with UTI. Translation of these findings revealed that temperance of the neutrophil response early during UTI, and specifically disruption of bladder epithelial transmigration of neutrophils by inhibition of cyclooxygenase-2, protected mice against chronic and recurrent cystitis. Further, proteomics identified bladder epithelial remodeling consequent to chronic infection that enhances sensitivity to neutrophil damage. Thus, cyclooxygenase-2 expression during acute UTI is a critical molecular trigger determining disease outcome and drugs targeting cyclooxygenase-2 could prevent recurrent UTI.

The hyaluronic acid receptor CD44 coordinates normal and metaplastic gastric epithelial progenitor cell proliferation.

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.

CD36-dependent signaling mediates fatty acid-induced gut release of secretin and cholecystokinin.

Genetic variants in the fatty acid (FA) translocase FAT/CD36 associate with abnormal postprandial lipids and influence risk for the metabolic syndrome. CD36 is abundant on apical enterocyte membranes in the proximal small intestine, where it facilitates FA uptake and FA-initiated signaling. We explored whether CD36 signaling influences FA-mediated secretion of cholecystokinin (CCK) and secretin, peptides released by enteroendocrine cells (EECs) in the duodenum/jejunum, which regulate events important for fat digestion and homeostasis. CD36 was immunodetected on apical membranes of secretin- and CCK-positive EECs and colocalized with cytosolic granules. Intragastric lipid administration to CD36 mice released less secretin (-60%) and CCK (-50%) compared with wild-type mice. Likewise, diminished secretin and CCK responses to FA were observed with CD36 intestinal segments in vitro, arguing against influence of alterations in fat absorption. Signaling mechanisms underlying peptide release were examined in STC-1 cells stably expressing human CD36 or a signaling-impaired mutant (CD36K/A). FA stimulation of cells expressing CD36 (vs. vector or CD36K/A) released more secretin (3.5- to 4-fold) and CCK (2- to 3-fold), generated more cAMP (2- to 2.5-fold), and enhanced protein kinase A activation. Protein kinase A inhibition (H-89) blunted secretin (80%) but not CCK release, which was reduced (50%) by blocking of calmodulin kinase II (KN-62). Coculture of STC-1 cells with Caco-2 cells stably expressing CD36 did not alter secretin or CCK release, consistent with a minimal effect of adjacent enterocytes. In summary, CD36 is a major mediator of FA-induced release of CCK and secretin. These peptides contribute to the role of CD36 in fat absorption and to its pleiotropic metabolic effects.

Hyaluronic acid regulates normal intestinal and colonic growth in mice.

Hyaluronic acid (HA), a component of the extracellular matrix, affects gastrointestinal epithelial proliferation in injury models, but its role in normal growth is unknown. We sought to determine the effects of exogenous HA on intestinal and colonic growth by intraperitoneal injection of HA twice a week into C57BL/6 mice from 3 to 8 wk of age. Similarly, to determine the effects of endogenous HA on intestinal and colonic growth, we administered PEP-1, a peptide that blocks the binding of HA to its receptors, on the same schedule. In mice treated with exogenous HA, villus height and crypt depth in the intestine, crypt depth in the colon, and epithelial proliferation in the intestine and colon were increased. In mice treated with PEP-1, intestinal and colonic length were markedly decreased and crypt depth and villus height in the intestine, crypt depth in the colon, and epithelial proliferation in the intestine and colon were decreased. Administration of HA was associated with increased levels of EGF (intestine) and IGF-I (colon), whereas administration of PEP-1 was associated with decreased levels of IGF-I (intestine) and epiregulin (colon). Exogenous HA increases intestinal and colonic epithelial proliferation, resulting in hyperplasia. Blocking the binding of endogenous HA to its receptors results in decreased intestinal and colonic length and a mucosal picture of hypoplasia, suggesting that endogenous HA contributes to the regulation of normal intestinal and colonic growth.

Serum analysis of tryptophan catabolism pathway: correlation with Crohn's disease activity.

BACKGROUND: Indoleamine 2,3 dioxygenase-1 (IDO1) is a tryptophan catabolizing enzyme with immunotolerance-promoting functions. We sought to determine if increased gut expression of IDO1 in Crohn's disease (CD) would result in detectable changes in serum levels of tryptophan and the initial IDO1 pathway catabolite, kynurenine. METHODS: Individuals were prospectively enrolled through the Washington University Digestive Diseases Research Center. The Montreal Classification was used for disease phenotyping. Disease severity was categorized by the Physician's Global Assessment. Serum tryptophan and kynurenine were measured by high-pressure liquid chromatography. IDO1 immunohistochemical staining was performed on formalin-fixed tissue blocks. RESULTS: In all, 25 CD patients and 11 controls were enrolled. Eight CD patients had serum collected at two different timepoints and levels of disease activity compared. Strong IDO1 expression exists in both the lamina propria and epithelium during active CD compared to controls. Suppressed serum tryptophan levels and an elevated kynurenine/tryptophan (K/T) ratio were found in individuals with active CD as compared to those in remission or the control population. K/T ratios correlated positively with disease activity as well as with C-reactive protein and erythrocyte sedimentation rate. In the subgroup of CD patients with two serum measurements, tryptophan levels were elevated while kynurenine levels and the K/T ratio lowered as the disease activity lessened. CONCLUSIONS: IDO1 expression in CD is associated with lower serum tryptophan and an elevated K/T ratio. These levels may serve as a reasonable objective marker of gut mucosal immune activation and as a surrogate for CD activity.

Hyaluronic acid is radioprotective in the intestine through a TLR4 and COX-2-mediated mechanism.

The intestinal epithelium is sensitive to radiation injury. Damage to the intestinal epithelium is dose limiting in radiation therapy of abdominal cancers. There is a need for agents that can be given before radiation therapy to protect the intestinal epithelium. C57BL6 mice were subjected to 12 Gy of total body radiation. Some mice received intraperitoneal hyaluronic acid (HA) before radiation. Mice were killed 6 h after radiation to assess radiation-induced apoptosis in the intestine; other mice were killed at 84 h to assess crypt survival. Total body radiation (12 Gy) resulted in increased expression of HA synthases and HA in the intestine and increased plasma HA (5-fold). Intraperitoneal injection of HA (30 mg/kg) before radiation resulted in a 1.8-fold increase in intestinal crypt survival and a decrease in radiation-induced apoptosis. The radioprotective effects of HA were not seen in Toll-like receptor 4 (TLR4)- or cyclooxygenase-2 (COX-2)-deficient mice. Intraperitoneal injection of HA induced a 1.5-fold increase in intestinal COX-2 expression, a 1.5-fold increase in intestinal PGE2, and the migration of COX-2-expressing mesenchymal stem cells from the lamina propria in the villi to the lamina propria near the crypt. We conclude that 1) radiation induces increased HA expression through inducing HA synthases, 2) intraperitoneal HA given before radiation reduces radiation-induced apoptosis and increases crypt survival, and 3) these radioprotective effects are mediated through TLR4, COX-2, and the migration of COX-2-expressing mesenchymal stem cells.

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 3 days before 12 Gy whole body radiation. The intestine was evaluated for cell-positional apoptosis (6 h) and crypt survival (84 h). 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.

Growth factor regulation of prostaglandin-endoperoxide synthase 2 (Ptgs2) expression in colonic mesenchymal stem cells.

We previously found that a population of colonic stromal cells that constitutively express high levels of prostaglandin-endoperoxide synthase 2 (Ptgs2, also known as Cox-2) altered their location in the lamina propria in response to injury in a Myd88-dependent manner (Brown, S. L., Riehl, T. E., Walker, M. R., Geske, M. J., Doherty, J. M., Stenson, W. F., and Stappenbeck, T. S. (2007) J. Clin. Invest. 117, 258-269). At the time of this study, the identity of these cells and the mechanism by which they expressed high levels of Ptgs2 were unknown. Here we found that these colonic stromal cells were mesenchymal stem cells (MSCs). These colonic MSCs expressed high Ptgs2 levels not through interaction with bacterial products but instead as a consequence of mRNA stabilization downstream of Fgf9 (fibroblast growth factor 9), a growth factor that is constitutively expressed by the intestinal epithelium. This stabilization was mediated partially through a mechanism involving endogenous CUG-binding protein 2 (CUGbp2). These studies suggest that Fgf9 is an important factor in the regulation of Ptgs2 in colonic MSCs and may be a factor involved in its constitutive expression in vivo.

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

BACKGROUND & AIMS: The 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. METHODS: DSS (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. RESULTS: DSS 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. CONCLUSIONS: Endogenous 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.

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.

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 beta-catenin in normal-appearing intestine. However, beta-catenin was nuclear in DCAMKL-1-positive cells in adenomas. Thus, nuclear translocation of beta-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.

Azoxymethane protects intestinal stem cells and reduces crypt epithelial mitosis through a COX-1-dependent mechanism.

Azoxymethane (AOM) is a potent DNA-damaging agent and carcinogen that induces intestinal and colonic tumors in rodents. Evaluation of the stem cell population by colony formation assay reveals that, within 8 h after treatment, AOM (10 mg/kg) elicited a prosurvival response. In wild-type (WT) mice, AOM treatment induced a 2.5-fold increase in intestinal crypt stem cell survival. AOM treatment increased stem cell survival in cyclooxygenase (COX)-2(-/-) but not COX-1(-/-) mice, confirming a role of COX-1 in the AOM-induced increase in stem cell survival. COX-1 mRNA and protein expression as well as COX-1-derived PGE(2) synthesis were increased 8 h after AOM treatment. Immunohistochemical staining of COX-1 demonstrated expression of the enzyme in the crypt epithelial cells, especially in the columnar epithelial cells between the Paneth cells adjacent to the stem cell zone. WT mice receiving AOM exhibited increased intestinal apoptosis and a simultaneous reduction in crypt mitotic figures within 8 h of injection. There were no significant differences in baseline or AOM-induced intestinal epithelial apoptosis between WT and COX-1(-/-) mice, but there was a complete reversal of the AOM-mediated reduction in mitosis in COX-1(-/-) mice. This suggests that COX-1-derived PGE(2) may play a key role in the early phase of intestinal tumorigenesis in response to DNA damage and suggests that COX-1 may be a potential therapeutic target in this model of colon cancer.

Rapid transient absorption and biliary secretion of enantiomeric cholesterol in hamsters.

To probe the pathway and specificity of cholesterol absorption, the synthetic enantiomer of cholesterol (ent-cholesterol) and cholesterol were labeled with deuterium, gavaged into hamsters, and measured by negative ion mass spectrometry. Initial uptake of both tracers into the intestinal mucosa at 30 min was similar but cholesterol was temporarily retained there, whereas mucosal ent-cholesterol declined rapidly with concomitantly increased enrichment in both the systemic circulation and the gut lumen. In a 3 day fecal recovery study, ent-cholesterol was quantitatively recovered in the stool, whereas cholesterol absorption was 53.2%. ent-Cholesterol given by intracardiac injection was selectively secreted into bile, and the ratio of ent-cholesterol to cholesterol tracers in the gut lumen increased down the length of the small bowel, with the largest value being found in stool. ent-Cholesterol is efficiently taken up by the intestinal mucosa and undergoes transient enterohepatic recirculation, but it is quantitatively eliminated over 3 days as a result of selective secretion into bile and selective enrichment within the lumen of the intestine. These findings suggest that cholesterol absorption is structurally specific and likely to be mediated by enantiospecific cellular proteins.

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

Tumor necrosis factor (TNF)-alpha, 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) 1beta and total TNF-alpha (free plus receptor bound) compared with wild-type mice, but there were no differences in IL6 or IL10 concentrations. Furthermore, free TNF-alpha 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-alpha. 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-alpha-induced pathology, whereas TNFR2 mediates protective effects.