Harnisha Dalwadi

Prostaglandin E2 Activates Mitogen-Activated Protein Kinase/Erk Pathway Signaling and Cell Proliferation in Non–Small Cell Lung Cancer Cells in an Epidermal Growth Factor Receptor–Independent Manner

Cyclooxygenase 2 (COX-2) overexpression is found in a wide variety of human cancers and is linked to all stages of tumorigenesis. Elevated tumor COX-2 expression is associated with increased angiogenesis, tumor invasion, suppression of host immunity and promotes tumor cell resistance to apoptosis. Previous reports have linked the COX-2 product prostaglandin E2 (PGE2) to the abnormal activation of the mitogen-activated protein kinase/Erk kinase pathway. Here we show that PGE2 is able to rapidly stimulate Erk phosphorylation in a subset of non-small cell lung cancer (NSCLC) cell lines. This effect is not evident in bronchial epithelial cells. In contrast to previous reports in colon cancer, we found that Erk activation as well as cellular proliferation induced by PGE2 was not inhibited by pretreatment of the cells with epidermal growth factor receptor (EGFR) inhibitors. Activation of the Erk pathway by PGE2 was also resistant to src kinase inhibitors but sensitive to the protein kinase C inhibition. PGE2 effects are mediated through four G protein-coupled receptors. Selective inhibition of EP receptors revealed the possible involvement of Ca2+-dependent signaling in PGE2-mediated activation of Erk. Our data indicate the presence of an EGFR-independent activation of the mitogen-activated protein kinase/Erk pathway by PGE2 in NSCLC cells. These findings provide evidence for the possible link between tumor COX-2 overexpression and elevated Erk-mediated cancer cell proliferation and migration. Importantly, these findings suggest that COX-2 overexpression may contribute to EGFR inhibitor resistance in NSCLC.

B Cell Developmental Requirement for the Gαi2 Gene

Null mutation of the Gαi2 trimeric G protein results in a discrete and profound mucosal disorder, including inflammatory bowel disease (IBD), attenuation of IL-10 expression, and immune function polarized to Th1 activity. Genetic and adoptive transfer experiments have established a role for B cells and IL-10 in mucosal immunologic homeostasis and IBD resistance. In this study, we addressed the hypothesis that Gαi2 is required for the development of IL-10-producing B cells. Gαi2−/− mice were reduced in the relative abundance of marginal zone (MZ), transitional type 2 (T2), and B-1a B cells and significantly increased in follicular mature and B-1b B cells. Reconstitution of RAG2−/− mice with Gαi2−/− bone marrow induced an IBD-like colitis and a deficiency in absolute numbers of MZ, T2, and B-1 B cells. Thus, the Gαi2−/− genotype in colitis susceptibility and B cell development involved a cis effect within the hemopoietic compartment. In vitro, the B cell population of Gαi2−/− mice was functionally deficient in LPS-induced proliferation and IL-10 production, consistent with the exclusive capacity of T2 and MZ cell subpopulations for LPS responsiveness. In vivo, Gαi2−/− mice were selectively impaired for the IgM response to T-independent type II, consistent with the relative depletion of MZ and peritoneal B-1 subpopulations. Collectively, these results reveal a selective role for Gαi2 in MZ and B-1 B cell development. Disorders of this Gαi2-dependent process in B cell development may represent a mechanism for IBD susceptibility.

Pseudomonas fluorescens Encodes the Crohn's Disease-Associated I2 Sequence and T-Cell Superantigen

ABSTRACT Commensal bacteria have emerged as an important disease factor in human Crohns disease (CD) and murine inflammatory bowel disease (IBD) models. We recently isolated I2, a novel gene segment of microbial origin that is associated with human CD and that encodes a T-cell superantigen. To identify the I2 microorganism, BLAST analysis was used to identify a microbial homologue, PA2885, a novel open reading frame (ORF) in the Pseudomonas aeruginosa genome. PCR and Southern analysis identified Pseudomonas fluorescens as the originating species of I2, with homologues detectable in 3 of 13 other Pseudomonas species. Genomic cloning disclosed a locus containing the full-length I2 gene (pfiT) and three other orthologous genes, including a homologue of the pbrA/pvdS iron response gene. CD4+ T-cell responses to recombinant proteins were potent for I2 and pfiT, but modest for PA2885. pfiT has several features of a virulence factor: association with an iron-response locus, restricted species distribution, and T-cell superantigen bioactivity. These findings suggest roles for pfiT and P. fluorescens in the pathogenesis of Crohns disease.

Cyclooxygenase 2-Dependent Expression of Survivin Is Critical for Apoptosis Resistance in Non-Small Cell Lung Cancer

Elevated tumor cyclooxygenase 2 (COX-2) expression is associated with increased angiogenesis, tumor invasion, and promotion of tumor cell resistance to apoptosis. In our previous studies using non-small cell lung cancer (NSCLC) cell lines constitutively expressing COX-2 cDNA in sense and antisense orientations, we demonstrated that constitutive overexpression of COX-2 leads to stabilization of the inhibitor of apoptosis protein survivin resulting in the elevated apoptosis resistance of COX-2–overexpressing cells. Genetic or pharmacologic suppression of COX-2 activity increased proteasomal degradation of survivin and cellular response to apoptosis induction. Our data show that expression of survivin in non-small cell lung cancer cells can be significantly down-regulated by RNA interference. Whereas COX-2–overexpressing NSCLC cells have significantly higher apoptosis resistance than the parental cells, inhibition of survivin expression by small interfering RNA decreases apoptosis resistance to the level of the parental non-small cell lung cancer. We conclude that COX-2-dependent expression of survivin is critical for apoptosis resistance in non-small cell lung cancer.

The Crohn's disease-associated bacterial protein I2 is a novel enteric t cell superantigen.

Abstract An aberrant T cell response to enteric bacteria is important in inflammatory bowel disease. However, the identity of relevant microbial antigens is unknown. Here, we report the presence of I2, a Crohns disease-associated microbial gene, in the murine intestine. The I2 protein induced a proliferative and IL-10 response by CD4 + T cells from unimmunized mice. The I2 response was dependent on MHC class II-mediated recognition but did not require antigen processing. Selective activation was observed for the TCR-Vβ5 subpopulation. These findings indicate that the I2 protein is a new class of T cell superantigen and suggest that colonization by the I2 microorganism in susceptible hosts may provide a superantigenic stimulus pertinent to Crohns disease pathogenesis.

Cyclooxygenase-2-Dependent Activation of Signal Transducer and Activator of Transcription 3 by Interleukin-6 in Non–Small Cell Lung Cancer

Purpose: Cyclooxygenase-2 (COX-2), phosphorylated signal transducers and activators of transcription 3 (STAT3), and interleukin-6 (IL-6) are elevated in non–small cell lung cancer (NSCLC). These molecules affect numerous cellular pathways, including angiogenesis and apoptosis resistance, and, therefore, may act in concert in NSCLC. Experimental Design: We examined IL-6 and phosphorylated STAT3 in COX-2-overexpressing [COX-2 sense-oriented (COX-2-S)] NSCLC cells and control cells. The effect of IL-6, STAT3, phosphatidylinositol 3-kinase, and mitogen-activated protein/extracellular signal-regulated kinase kinase on vascular endothelial growth factor (VEGF) production and apoptosis resistance was assessed in COX-2-overexpresing cells. Results: We report that NSCLC cells overexpressing COX-2 (COX-2-S) have increased IL-6 and phosphorylated STAT3 expression compared with control cells. IL-6 induced expression of VEGF in NSCLC cells. Moreover, blocking IL-6, mitogen-activated protein/extracellular signal-regulated kinase kinase, or phosphatidylinositol 3-kinase decreased VEGF production in COX-2-S cells. The addition of IL-6 to NSCLC cells resulted in increased apoptosis resistance. Furthermore, the inhibition of STAT3 or IL-6 induced apoptosis and reduced survivin expression, a member of the inhibitor of apoptosis protein family in COX-2-S cells. Conclusions: Overall, these findings suggest a novel pathway in which COX-2 activates STAT3 by inducing IL-6 expression. This pathway could contribute to tumor formation by promoting survivin-dependent apoptosis resistance and VEGF production. These findings provide a rationale for the future development of STAT3, IL-6, and/or COX-2-targeted therapies for the treatment of lung cancer.

Molecular cloning of a Bacteroides caccae TonB-linked outer membrane protein identified by an inflammatory bowel disease marker antibody.

ABSTRACT Commensal enteric bacteria are a required pathogenic factor in inflammatory bowel disease (IBD), but the identity of the pertinent bacterial species is unresolved. Using an IBD-associated pANCA monoclonal antibody, a 100-kDa protein was recently characterized from an IBD clinical isolate of Bacteroides caccae (p2Lc3). In this study, consensus oligonucleotides were designed from 100-kDa peptides and used to identify a single-copy gene from the p2Lc3 genome. Sequence analysis of the genomic clone revealed a 2,844-bp (948 amino acid) open reading frame encoding features typical of the TonB-linked outer membrane protein family. This gene, termed ompW,was detected by Southern analysis only in B. caccae and was absent in other species of Bacteroides and gram-negative coliforms. The closest homologues of OmpW included the outer membrane proteins SusC of Bacteroides thetaiotaomicron and RagA of Porphyromonas gingivalis. Recombinant OmpW protein was immunoreactive with the monoclonal antibody, and serum anti-OmpW immunoglobulin A levels were elevated in a Crohns disease patient subset. These findings suggest that OmpW may be a target of the IBD-associated immune response and reveal its structural relationship to a bacterial virulence factor of P. gingivalis and periodontal disease.

Resident enteric microbiota and CD8+ T cells shape the abundance of marginal zone B cells

Since enteric microbial composition is a distinctive and stable individual trait, microbial heterogeneity may confer lifelong, non‐genetic differences between individuals. Here we report that C57BL/6 mice bearing restricted flora microbiota, a distinct but diverse resident enteric microbial community, are numerically and functionally deficient in marginal zone (MZ) B cells. Surprisingly, MZ B‐cell levels are minimally affected by germ‐free conditions or null mutations of various TLR signaling molecules. In contrast, MZ B‐cell depletion is exquisitely dependent on cytolytic CD8+ T cells, and includes targeting of a cross‐reactive microbial/endogenous MHC class 1B antigen. Thus, members of certain enteric microbial communities link with CD8+ T cells as a previously unappreciated mechanism that shapes innate immunity dependent on innate‐like B cells.

Defining new pathogens and nonculturable infectious agents associated with inflammatory bowel disease.

The causes of inflammatory bowel disease remain uncertain, but increasing evidence supports roles for two pathogenic processes: genetic susceptibility in the host and an aberrant host response to enteric bacteria. Here, we review studies showing that the pathogenesis of inflammatory bowel disease involves both bacterial agents and genetic susceptibilities. We discuss the roles of known and novel commensal intestinal bacteria in the pathogenesis of inflammatory bowel disease and the various recombinant approaches used to identify these pathogens.

The Crohn's disease-associated bacterial protein, 12, is a novel enteric T cell superantigen

An aberrant T cell response to enteric bacteria is important in inflammatory bowel disease. However, the identity of relevant microbial antigens is unknown. Here, we report the presence of I2, a Crohns disease-associated microbial gene, in the murine intestine. The I2 protein induced a proliferative and IL-10 response by CD4(+) T cells from unimmunized mice. The I2 response was dependent on MHC class II-mediated recognition but did not require antigen processing. Selective activation was observed for the TCR-Vbeta5 subpopulation. These findings indicate that the I2 protein is a new class of T cell superantigen and suggest that colonization by the I2 microorganism in susceptible hosts may provide a superantigenic stimulus pertinent to Crohns disease pathogenesis.