Scott Herdman

ERK activation drives intestinal tumorigenesis in Apc min/+ mice

Toll-like receptor (TLR) signaling is essential for intestinal tumorigenesis in Apcmin/+ mice, but the mechanisms by which Apc enhances tumor growth are unknown. Here we show that microflora-MyD88-ERK signaling in intestinal epithelial cells (IECs) promotes tumorigenesis by increasing the stability of the c-Myc oncoprotein. Activation of ERK (extracellular signal–related kinase) phosphorylates c-Myc, preventing its ubiquitination and subsequent proteasomal degradation. Accordingly, Apcmin/+/Myd88−/− mice have lower phospho-ERK (p-ERK) levels and fewer and smaller IEC tumors than Apcmin/+ mice. MyD88 (myeloid differentiation primary response gene 88)-independent activation of ERK by epidermal growth factor (EGF) increased p-ERK and c-Myc and restored the multiple intestinal neoplasia (Min) phenotype in Apcmin/+/Myd88−/− mice. Administration of an ERK inhibitor suppressed intestinal tumorigenesis in EGF-treated Apcmin/+/Myd88−/− and Apcmin/+ mice and increased their survival. Our data reveal a new facet of oncogene-environment interaction, in which microflora-induced TLR activation regulates oncogene expression and related IEC tumor growth in a susceptible host.

Cysteine proteinases from distinct cellular compartments are recruited to phagocytic vesicles by Entamoeba histolytica

Cysteine proteinases, which are encoded by at least seven genes, play a critical role in the pathogenesis of invasive amebiasis caused by Entamoeba histolytica. The study of these enzymes has been hampered by the inability to obtain significant quantities of the individual native proteinases. We have now expressed functionally active recombinant ACP1 (EhCP3) and ACP2 (EhCP2) proteinases in baculoviral expression vectors. The purified recombinant ACP1 and ACP2 proteinases exhibited similar activities for fluorogenic peptide substrates, especially in their preference for an arginine residue at the P2 position. Although ACP1 and ACP2 are structurally cathepsin L, homology modeling revealed that the aspartic acid in the S2 pocket would result in a substrate specificity for positively charged amino acids, like cathepsin B. The hydrolysis of peptide substrates was strongly inhibited by small peptidyl inhibitors specifically designed for parasitic cysteine proteinases. Confocal and immunoelectron microscopy localization of the proteinases with monoclonal and monospecific antibodies raised to the recombinant enzymes and peptides demonstrated that ACP2 was membrane-associated while ACP1 was cytoplasmic. Following phagocytosis of erythrocytes, ACP1, as well as the membrane-associated cysteine proteinase, ACP2, were incorporated into phagocytic vesicles. These studies suggest that E. histolytica has a redundancy of cysteine proteinases for intracellular digestion and that they may be recruited from different cellular compartments to the site of digestion of phagocytosed cells. The production of active proteinases in baculovirus and large scale recombinant enzymes in bacteria should further our understanding of the role of different cysteine proteinase gene products in virulence.

The ion channel TRPV1 regulates the activation and proinflammatory properties of CD4+ T cells

TRPV1 is a Ca2+-permeable channel studied mostly as a pain receptor in sensory neurons. However, its role in other cell types is poorly understood. Here we found that TRPV1 was functionally expressed in CD4+ T cells, where it acted as a non–store-operated Ca2+ channel and contributed to T cell antigen receptor (TCR)-induced Ca2+ influx, TCR signaling and T cell activation. In models of T cell–mediated colitis, TRPV1 promoted colitogenic T cell responses and intestinal inflammation. Furthermore, genetic and pharmacological inhibition of TRPV1 in human CD4+ T cells recapitulated the phenotype of mouse Trpv1−/− CD4+ T cells. Our findings suggest that inhibition of TRPV1 could represent a new therapeutic strategy for restraining proinflammatory T cell responses.

Use of recombinant Entamoeba histolytica cysteine proteinase 1 to identify a potent inhibitor of amebic invasion in a human colonic model

ABSTRACT Cysteine proteinases are key virulence factors of the protozoan parasite Entamoeba histolytica. We have shown that cysteine proteinases play a central role in tissue invasion and disruption of host defenses by digesting components of the extracellular matrix, immunoglobulins, complement, and cytokines. Analysis of the E. histolytica genome project has revealed more than 40 genes encoding cysteine proteinases. We have focused on E. histolytica cysteine proteinase 1 (EhCP1) because it is one of two cysteine proteinases unique to invasive E. histolytica and is highly expressed and released. Recombinant EhCP1 was expressed in Escherichia coli and refolded to an active enzyme with a pH optimum of 6.0. We used positional-scanning synthetic tetrapeptide combinatorial libraries to map the specificity of the P1 to P4 subsites of the active site cleft. Arginine was strongly preferred at P2, an unusual specificity among clan CA proteinases. A new vinyl sulfone inhibitor, WRR483, was synthesized based on this specificity to target EhCP1. Recombinant EhCP1 cleaved key components of the host immune system, C3, immunoglobulin G, and pro-interleukin-18, in a time- and dose-dependent manner. EhCP1 localized to large cytoplasmic vesicles, distinct from the sites of other proteinases. To gain insight into the role of secreted cysteine proteinases in amebic invasion, we tested the effect of the vinyl sulfone cysteine proteinase inhibitors K11777 and WRR483 on invasion of human colonic xenografts. The resultant dramatic inhibition of invasion by both inhibitors in this human colonic model of amebiasis strongly suggests a significant role of secreted amebic proteinases, such as EhCP1, in the pathogenesis of amebiasis.

Signal Transducer and Activator of Transcription 3 (STAT3) Protein Suppresses Adenoma-to-carcinoma Transition in Apcmin/+ Mice via Regulation of Snail-1 (SNAI) Protein Stability

Background: STAT3 suppresses carcinogenesis of intestinal tumors in Apc min mice. Results: STAT3 suppresses expression of SNAI in intestinal epithelium by regulating GSK3β activity. Conclusion: STAT3 induces degradation of SNAI by promoting GSK3β activity and thereby suppresses adenoma-to-adenocarcinoma transition in Apc min mice. Significance: Our data provide a new insight into the role of STAT3 in colorectal cancer biology. STAT3 was recently reported to suppress tumor invasion in Apcmin/+ mice. We investigated the mechanisms by which STAT3 inhibits intestinal epithelial tumors using Apcmin/+/Stat3IEC-KO mice (intestinal epithelial cell (IEC)-specific deletion of STAT3 in the Apcmin/+ background) to determine the role of STAT3 in carcinogenesis in vivo as well as colorectal cancer cell lines in vitro. To inhibit invasion of IEC tumors, STAT3 functions as a molecular adaptor rather than a transcription factor. Accordingly, the tumors in Apcmin/+/Stat3IEC-KO mice undergo adenoma-to-carcinoma transition and acquire an invasive phenotype. Similarly, STAT3 knockdown in a colorectal cell line enhances IEC invasion. We demonstrate that STAT3 down-regulates SNAI (Snail-1) expression levels and hence suppresses epithelial-mesenchymal transition of colorectal cancer cells. Mechanistically, STAT3 facilitates glycogen synthase kinase (GSK) 3β-mediated degradation of SNAI by regulating phosphorylation of GSK3β. Our data identified a new role for STAT3 in the adenoma-to-carcinoma sequence of intestinal tumors.

Dual-specificity phosphatase 6 regulates CD4+ T-cell functions and restrains spontaneous colitis in IL-10-deficient mice.

Mitogen-activated protein kinase (MAPK) phosphatases are dual-specificity phosphatases (DUSPs) that dephosphorylate phosphothreonine and phosphotyrosine residues within MAPKs. DUSP6 preferentially dephosphorylates extracellular signal-regulated kinases 1 and 2 (ERK1/2) rendering them inactive. Here, we study the role of DUSP6 in CD4+ T-cell function, differentiation, and inflammatory profile in the colon. Upon T-cell receptor (TCR) stimulation, DUSP6 knockout (Dusp6−/−) CD4+ T cells showed increased ERK1/2 activation, proliferation, T helper 1 differentiation, and interferon-γ production, as well as a marked decrease in survival, interleukin- 17A (IL-17A) secretion, and regulatory T-cell function. To analyze the role of DUSP6 in vivo, we employed the Il10−/− model of colitis and generated Il10−/−/Dusp6−/− double-knockout mice. Il10−/−/Dusp6−/− mice suffered from accelerated and exacerbated spontaneous colitis, which was prevented by ERK1/2 inhibition. ERK1/2 inhibition also augmented regulatory T-cell differentiation in vitro and in vivo in both C57Bl/6 and Dusp6−/− mice. In summary, DUSP6 regulates CD4+ T-cell activation and differentiation by inhibiting the TCR-dependent ERK1/2 activation. DUSP6 might therefore be a potential intervention target for limiting aberrant T-cell responses in T-cell-mediated diseases, such as inflammatory bowel disease.

The TRPA1 ion channel is expressed in CD4+ T cells and restrains T-cell-mediated colitis through inhibition of TRPV1

Objective Transient receptor potential ankyrin-1 (TRPA1) and transient receptor potential vanilloid-1 (TRPV1) are calcium (Ca2+)-permeable ion channels mostly known as pain receptors in sensory neurons. However, growing evidence suggests their crucial involvement in the pathogenesis of IBD. We explored the possible contribution of TRPA1 and TRPV1 to T-cell-mediated colitis. Design We evaluated the role of Trpa1 gene deletion in two models of experimental colitis (ie, interleukin-10 knockout and T-cell-adoptive transfer models). We performed electrophysiological and Ca2+ imaging studies to analyse TRPA1 and TRPV1 functions in CD4+ T cells. We used genetic and pharmacological approaches to evaluate TRPV1 contribution to the phenotype of Trpa1−/− CD4+ T cells. We also analysed TRPA1 and TRPV1 gene expression and TRPA1+TRPV1+ T cell infiltration in colonic biopsies from patients with IBD. Results We identified a protective role for TRPA1 in T-cell-mediated colitis. We demonstrated the functional expression of TRPA1 on the plasma membrane of CD4+ T cells and identified that Trpa1−/− CD4+ T cells have increased T-cell receptor-induced Ca2+ influx, activation profile and differentiation into Th1-effector cells. This phenotype was abrogated upon genetic deletion or pharmacological inhibition of the TRPV1 channel in mouse and human CD4+ T cells. Finally, we found differential regulation of TRPA1 and TRPV1 gene expression as well as increased infiltration of TRPA1+TRPV1+ T cells in the colon of patients with IBD. Conclusions Our study indicates that TRPA1 inhibits TRPV1 channel activity in CD4+ T cells, and consequently restrains CD4+ T-cell activation and colitogenic responses. These findings may therefore have therapeutic implications for human IBD.