Leonard H. Augenlicht

Mucus Enhances Gut Homeostasis and Oral Tolerance by Delivering Immunoregulatory Signals

Guardian of the Gut The intestine is able to tolerate continual exposure to large amounts of commensal bacteria and foreign food antigens without triggering an inappropriate inflammatory immune response. In the large intestine, this immunological tolerance is thought to occur via a physical separation between environment and host imposed by a continuous mucous layer built up from the secreted mucin protein, MUC2. However, in the small intestine, this mucous layer is porous, necessitating an additional layer of immune control. Shan et al. (p. 447, published online 26 September; see the Perspective by Belkaid and Grainger) now report that in the small intestine, MUC2 plays an active role in immunological tolerance by activating a transcription factor in resident dendritic cells, thereby selectively blocking their ability to launch an inflammatory response. This work identifies MUC2 as a central mediator of immune tolerance to maintain homeostasis in the gut and possibly at other mucosal surfaces in the body. Mucus not only forms a physical barrier in the intestine but also promotes immunological tolerance of bacteria and foods. [Also see Perspective by Belkaid and Grainger] A dense mucus layer in the large intestine prevents inflammation by shielding the underlying epithelium from luminal bacteria and food antigens. This mucus barrier is organized around the hyperglycosylated mucin MUC2. Here we show that the small intestine has a porous mucus layer, which permitted the uptake of MUC2 by antigen-sampling dendritic cells (DCs). Glycans associated with MUC2 imprinted DCs with anti-inflammatory properties by assembling a galectin-3–Dectin-1–FcγRIIB receptor complex that activated β-catenin. This transcription factor interfered with DC expression of inflammatory but not tolerogenic cytokines by inhibiting gene transcription through nuclear factor κB. MUC2 induced additional conditioning signals in intestinal epithelial cells. Thus, mucus does not merely form a nonspecific physical barrier, but also constrains the immunogenicity of gut antigens by delivering tolerogenic signals.

Western-style diet-induced colonic tumors and their modulation by calcium and vitamin D in C57Bl/6 mice: a preclinical model for human sporadic colon cancer

We reported previously that a new Western-style diet (NWD) for 18 months, consisting of elevated lipids and decreased calcium, vitamin D and methyl-donor nutrients, induced colonic tumors in normal C57Bl/6 mice [Newmark, H.L. et al. (2001) A Western-style diet induces benign and malignant neoplasms in the colon of normal C57Bl/6 mice. Carcinogenesis, 22, 1871-1875], suggesting a new mouse model for human sporadic colon cancer. Here, we have extended this study during a longer feeding period of 2 years wherein tumor formation, tumor inhibition by addition of dietary calcium and vitamin D and their effects on gene expression were determined. We also similarly tested individual supplements of methyl donor (transfer) nutrients (folic acid, choline, methionine and dietary fiber), but these had no significant effect on colonic tumor incidence or multiplicity, whereas supplementation with combined calcium and vitamin D produced significant decrease in both colon tumor incidence and multiplicity, during 2 years of feeding. No visible colonic tumors were found at 6 months, very few at 12 months, more at 18 months and significantly at 24 months. In a related study of gene changes of the mouse colonic mucosa at 6 months of feeding taken from this study, long before any tumors were visibly detectable, indicated altered profiles of gene expression linked to later risk of dietary initiation of colon tumor formation. This type of early genetic altered profile, an indication of increased risk of later colonic tumor development, may become a useful tool for prediction of colon tumor risk while the colon grossly still appears histologically and physiologically normal.

HDAC4 Promotes Growth of Colon Cancer Cells via Repression of p21

The class II Histone deacetylase (HDAC), HDAC4, is expressed in a tissue-specific manner, and it represses differentiation of specific cell types. We demonstrate here that HDAC4 is expressed in the proliferative zone in small intestine and colon and that its expression is down-regulated during intestinal differentiation in vivo and in vitro. Subcellular localization studies demonstrated HDAC4 expression was predominantly nuclear in proliferating HCT116 cells and relocalized to the cytoplasm after cell cycle arrest. Down-regulating HDAC4 expression by small interfering RNA (siRNA) in HCT116 cells induced growth inhibition and apoptosis in vitro, reduced xenograft tumor growth, and increased p21 transcription. Conversely, overexpression of HDAC4 repressed p21 promoter activity. p21 was likely a direct target of HDAC4, because HDAC4 down-regulation increased p21 mRNA when protein synthesis was inhibited by cycloheximide. The importance of p21 repression in HDAC4-mediated growth promotion was demonstrated by the failure of HDAC4 down-regulation to induce growth arrest in HCT116 p21-null cells. HDAC4 down-regulation failed to induce p21 when Sp1 was functionally inhibited by mithramycin or siRNA-mediated down-regulation. HDAC4 expression overlapped with that of Sp1, and a physical interaction was demonstrated by coimmunoprecipitation. Chromatin immunoprecipitation (ChIP) and sequential ChIP analyses demonstrated Sp1-dependent binding of HDAC4 to the proximal p21 promoter, likely directed through the HDAC4-HDAC3-N-CoR/SMRT corepressor complex. Consistent with increased transcription, HDAC4 or SMRT down-regulation resulted in increased histone H3 acetylation at the proximal p21 promoter locus. These studies identify HDAC4 as a novel regulator of colon cell proliferation through repression of p21.

Divergent phenotypic patterns and commitment to apoptosis of Caco-2 cells during spontaneous and butyrate-induced differentiation.

Caco‐2 cells differentiate spontaneously when cultured in confluence and on exposure to the physiologically relevant short‐chain fatty acid, butyrate. This study aimed to compare the phenotype induced by these pathways and their relations to cell turnover. Caco‐2 cells were treated with butyrate at a nontoxic concentration of 2 mM for 3 days, or allowed to spontaneously differentiate for 0–21 days. Brush border hydrolase activities and carcinoembryonic antigen (CEA) expression, transepithelial resistance and dome formation, expression of components of the urokinase system, and cell turnover by flow cytometry, and the degree of DNA fragmentation were quantified. Butyrate induced increases in alkaline phosphatase activity and CEA expression but not the activities of other hydrolases, while culture alone induced progressive increases in the activities/expression of all markers. Butyrate induced a significantly greater increase in transepithelial resistance (TER) than occurred during culture alone but the densities of domes were similar. Butyrate induced a ninefold increase in urokinase receptor expression and twofold increase in urokinase activity, while culture alone induced a significantly smaller increase in receptor expression, an increase in plasminogen activator inhibitor‐1 but no change in activity. While both stimuli induced cell cycle arrest, only butyrate increased the proportion of cells undergoing apoptosis. In conclusion, differentiation of Caco‐2 cells can proceed along multiple pathways but does not necessarily lead to apoptosis. The phenotypic changes during spontaneous differentiation mimic those that occur in normal colonic epithelial cells in vivo during their migration from the crypt base to neck, while butyrate‐induced effects more closely follow those occurring when normal colonic epithelial cells migrate from crypt neck to the surface compartment. J. Cell. Physiol. 183:347–354, 2000.

The NF-κB/AKT-dependent Induction of Wnt Signaling in Colon Cancer Cells by Macrophages and IL-1β

Progression of colon cancer from microadenoma to macroscopic tumors is coupled to augmentation of canonical Wnt signaling. We recently reported that tumor associated macrophages, through interleukin 1β (IL-1β) dependent phosphorylation of GSK3β, promote Wnt signaling in colon cancer cells, demonstrating that proinflammatory cytokines can enhance TCF4/β-catenin transcriptional activity in tumor cells. Here we investigated the pathway whereby IL-1β inactivates GSK3β and promotes Wnt signaling in colon cancer cells. We showed that normal human monocytes, THP1 macrophages and IL-1 failed to induce Wnt signaling in tumor cells expressing dominant negative IκB (dnIκB), demonstrating that macrophages and IL-1 activate Wnt signaling in a NF-κB-dependent manner. NF-κB activity was required for macrophages and IL-1 to activate PDK1 and AKT in tumor cells and thereby inhibit GSK3β activity. Consistently, dominant negative AKT (dnAKT), or pharmacological inhibition of AKT in tumor cells, prevented macrophage/IL-1 mediated phosphorylation of GSK3β, activation of Wnt signaling, and induction of c-jun and c-myc, confirming that macrophages and IL-1 promote Wnt signaling in an AKT dependent manner. Finally, we showed IL-1 and macrophages failed to promote growth of colon cancer cells with impaired NF-κB or AKT signaling, confirming the requirement for NF-κB and AKT activation for the protumorigenic activity of tumor associated macrophages. Thus, we showed that IL-1 and tumor associated macrophages activate NF-κB-dependent PDK1/AKT signaling in tumor cells, and thereby inactivate GSK3β, enhance Wnt signaling and promote growth of colon cancer cells, establishing a novel molecular link between inflammation and tumor growth.

Tumor Associated Macrophages Protect Colon Cancer Cells from TRAIL-Induced Apoptosis through IL-1β- Dependent Stabilization of Snail in Tumor Cells

Background We recently reported that colon tumor cells stimulate macrophages to release IL-1β, which in turn inactivates GSK3β and enhances Wnt signaling in colon cancer cells, generating a self-amplifying loop that promotes the growth of tumor cells. Principal Findings Here we describe that macrophages protect HCT116 and Hke-3 colon cancer cells from TRAIL-induced apoptosis. Inactivation of IL-1β by neutralizing IL-1β antibody, or silencing of IL-1β in macrophages inhibited their ability to counter TRAIL-induced apoptosis. Accordingly, IL-1β was sufficient to inhibit TRAIL-induced apoptosis. TRAIL-induced collapse of the mitochondrial membrane potential (Δψ) and activation of caspases were prevented by macrophages or by recombinant IL-1β. Pharmacological inhibition of IL-1β release from macrophages by vitamin D3, a potent chemopreventive agent for colorectal cancer, restored the ability of TRAIL to induce apoptosis of tumor cells cultured with macrophages. Macrophages and IL-1β failed to inhibit TRAIL-induced apoptosis in HCT116 cells expressing dnIκB, dnAKT or dnTCF4, confirming that they oppose TRAIL-induced cell death through induction of Wnt signaling in tumor cells. We showed that macrophages and IL-1β stabilized Snail in tumor cells in an NF-κB/Wnt dependent manner and that Snail deficient tumor cells were not protected from TRAIL-induced apoptosis by macrophages or by IL-1β, demonstrating a crucial role of Snail in the resistance of tumor cells to TRAIL. Significance We have identified a positive feedback loop between tumor cells and macrophages that propagates the growth and promotes the survival of colon cancer cells: tumor cells stimulate macrophages to secrete IL-1β, which in turn, promotes Wnt signaling and stabilizes Snail in tumor cells, conferring resistance to TRAIL. Vitamin D3 halts this amplifying loop by interfering with the release of IL-1β from macrophages. Accordingly, vitamin D3 sensitizes tumor cells to TRAIL-induced apoptosis, suggesting that the therapeutic efficacy of TRAIL could be augmented by this readily available chemopreventive agent.

Expression of selenium-binding protein 1 characterizes intestinal cell maturation and predicts survival for patients with colorectal cancer

To identify candidate genes involved in the development of colorectal cancer, we used cDNA microarrays to analyze gene expression differences between human colorectal tumors and paired adjacent normal mucosa. We identified approximately 3.5-fold significant downregulation of selenium-binding protein 1 (SBP1) in colorectal tumors compared to normal mucosa (p = 0.003). Importantly, stage III colorectal cancer patients with low tumor-SBP1 expression had significantly shorter disease-free and overall survival as compared with those patients with high tumor-SBP1 expression (p = 0.04 and 0.03, respectively). We further characterized the role of SBP1 in colorectal cancer in vivo and in vitro. In normal tissue, SBP1 was maximally expressed in terminally differentiated epithelial cells on the luminal surface of crypts in the large intestine. Consistent with this in vivo localization, SBP1 was upregulated during in vitro colonic cell differentiation along the absorptive (Caco-2) and secretory (HT29 Clones 16E and 19A) cell lineages. Downregulation (approximately 50%) of SBP1 expression by small interfering RNA in colonic cancer cells was associated with reduced expression of another epithelial differentiation marker, carcinoembryonic antigen (CEA), although PCNA and p21(WAF1/cip1 )expression were not altered. These data demonstrate that higher expression of SBP1 is associated with differentiation of the normal colonic epithelia and may be a positive prognostic factor for survival in stage III colorectal carcinoma.

Organization and Regulatory Aspects of the Human Intestinal Mucin Gene (MUC2) Locus

The human MUC2 gene maps to chromosome 11p15, where three additional mucin genes have been located, and encodes the most abundant gastrointestinal mucin normally expressed in the intestinal goblet cell lineage. However, in pathological conditions, including colorectal cancer, MUC2 can be abnormally expressed. Therefore, it is of considerable interest to understand the regulation of the MUC2 gene and how the mechanism is altered in colon cancer. Toward this goal, we have isolated a group of overlapping clones (contig) spanning 85 kilobases harboring the entire MUC2 locus, including sequences located upstream of the gene. Detection of two DNase I-hypersensitive sites in the 5′ region of the MUC2 gene suggests the presence of DNA regulatory elements. To better characterize this region, we have sequenced 12 kilobases of the upstream region and analyzed it for functional activity by cloning portions of it into a luciferase reporter vector and assaying for promoter/enhancer activity using a transient transfection assay. A fragment from the AUG translational initiation codon +1 to −848 confers maximal transcriptional activity in several intestinal cell lines. Elements located further upstream exert a negative effect on the expression of the reporter gene when tested in conjunction with homologous or heterologous promoters. The same pattern of expression is observed when the MUC2/luciferase constructs are transfected into HeLa cells, which do not express the endogenous MUC2 gene. However, the level of activity in HeLa cells is at least an order of magnitude higher, suggesting that additional sequences singularly or in combination are responsible for the tissue- and cell lineage-specific expression of MUC2 Finally, we have identified an additional mucin-like gene (MUCX), located upstream of MUC2 We show that this MUCX gene, that is transcribed in opposite orientation to that of MUC2, is expressed with a pattern distinct from that of MUC2, yet similar to that of MUC5B and MUC6, two additional mucin genes located at chromosome 11p15. Recent information on the order of the mucin genes at chromosome 11p15 suggests that MUCX may be MUC6, one of the already identified mucin genes, or a novel one, yet to be fully characterized.

KLF4 regulation in intestinal epithelial cell maturation

The Krüppel-like factor 4 (KLF4) transcription factor suppresses tumorigenesis in gastrointestinal epithelium. Thus, its expression is decreased in gastric and colon cancers. Moreover, KLF4 regulates both differentiation and growth that is likely fundamental to its tumor suppressor activity. We dissected the expression of Klf4 in the normal mouse intestinal epithelium along the crypt-villus and cephalo-caudal axes. Klf4 reached its highest level in differentiated cells of the villus, with levels in the duodenum>jejunum>ileum, in inverse relation to the representation of goblet cells in these regions, the lineage previously linked to KLF4. In parallel, in vitro studies using HT29cl.16E and Caco2 colon cancer cell lines clarified that KLF4 increased coincident with differentiation along both the goblet and absorptive cell lineages, respectively, and that KLF4 levels also increased during differentiation induced by the short chain fatty acid butyrate, independently of cell fate. Moreover, we determined that lower levels of KLF4 expression in the proliferative compartment of the intestinal epithelium are regulated by the transcription factors TCF4 and SOX9, an effector and a target, respectively, of beta-catenin/Tcf signaling, and independently of CDX2. Thus, reduced levels of KLF4 tumor suppressor activity in colon tumors may be driven by elevated beta-catenin/Tcf signaling.

Cell type‐ and promoter‐dependent modulation of the Wnt signaling pathway by sodium butyrate

The Wnt signaling pathway modulates the transcription of genes linked to proliferation, differentiation and tumor progression. β‐Catenin‐Tcf (BCT)‐dependent Wnt signaling is influenced by the short‐chain fatty acid sodium butyrate, which induces growth arrest and/or maturation of colonic carcinoma cells. We have compared the effects of sodium butyrate on BCT‐dependent signaling in 2 colon carcinoma cell lines that differ in their physiologic response to butyrate, with SW620 cells responding to butyrate by undergoing terminal differentiation and apoptosis, and HCT‐116 cells undergoing reversible growth arrest, but no significant apoptotic cell death. Furthermore, these colon carcinoma cell lines differ in their mechanism of Wnt pathway activation, with adenomatous polyposis coli (APC) mutant SW620 cells having high levels of BCT complexes and APC wild‐type HCT‐116 cells having mutant β‐catenin, low levels of BCT complexes and correspondingly higher levels of free Tcf. We have demonstrated that in SW620 cells, butyrate downregulates BCT‐dependent expression of the Tcf‐TK, matrilysin and cyclin D1 promoters, whereas in HCT‐116 cells, butyrate upregulates expression of these promoters. Cotransfection with expression vectors that interfere with the Wnt pathway suggests that butyrate enhances BCT complex‐DNA binding. Butyrate reduces the expression of Tcf4 in HCT‐116 cells, consistent with the induction by butyrate of Tcf‐repressible promoters in these cells. These findings indicate that sodium butyrate modulates the Wnt pathway in SW620 and HCT‐116 cells in a different manner and that these differences have consequences for promoter activity that may influence the physiologic response to butyrate.