Mark Tsang

Helicobacter Infection Is Required for Inflammation and Colon Cancer in Smad3-Deficient Mice

Accumulating evidence suggests that intestinal microbial organisms may play an important role in triggering and sustaining inflammation in individuals afflicted with inflammatory bowel disease (IBD). Moreover, individuals with IBD are at increased risk for developing colorectal cancer, suggesting that chronic inflammation may initiate genetic or epigenetic changes associated with cancer development. We tested the hypothesis that bacteria may contribute to the development of colon cancer by synergizing with defective transforming growth factor-beta (TGF-beta) signaling, a pathway commonly mutated in human colon cancer. Although others have reported that mice deficient in the TGF-beta signaling molecule SMAD3 develop colon cancer, we found that SMAD3-deficient mice maintained free of the Gram-negative enterohepatic bacteria Helicobacter spp. for up to 9 months do not develop colon cancer. Furthermore, infection of SMAD3(-/-) mice with Helicobacter triggers colon cancer in 50% to 66% of the animals. Using real-time PCR, we found that Helicobacter organisms concentrate in the cecum, the preferred site of tumor development. Mucinous adenocarcinomas develop 5 to 30 weeks after infection and are preceded by an early inflammatory phase, consisting of increased proliferation of epithelial cells; increased numbers of cyclooxygenase-2-positive cells, CD4(+) T cells, macrophages; and increased MHC class II expression. Colonic tissue revealed increased transcripts for the oncogene c-myc and the proinflammatory cytokines interleukin-1alpha (IL-1alpha), IL-1beta, IL-6, IFN-gamma, and tumor necrosis factor-alpha, some of which have been implicated in colon cancer. These results suggest that bacteria may be important in triggering colorectal cancer, notably in the context of gene mutations in the TGF-beta signaling pathway, one of the most commonly affected cellular pathways in colorectal cancer in humans.

Dual Infection with Helicobacter bilis and Helicobacter hepaticus in P-Glycoprotein-Deficient mdr1a−/− Mice Results in Colitis that Progresses to Dysplasia

Patients with inflammatory bowel disease (IBD) are at increased risk for developing high-grade dysplasia and colorectal cancer. Animal IBD models that develop dysplasia and neoplasia may help elucidate the link between inflammation and colorectal cancer. Mdr1a-/- mice lack the membrane efflux pump p-glycoprotein and spontaneously develop IBD that can be modulated by infection with Helicobacter sp: H. bilis accelerates development of colitis while H. hepaticus delays disease. In this study, we determined if H. hepaticus infection could prevent H. bilis-induced colitis. Unexpectedly, a proportion of dual-infected mdr1a-/- mice showed IBD with foci of low- to high-grade dysplasia. A group of dual-infected mdr1a-/- animals were maintained long term (39 weeks) by intermittent feeding of medicated wafers to model chronic and relapsing disease. These mice showed a higher frequency of high-grade crypt dysplasia, including invasive adenocarcinoma, possibly because H. hepaticus, in delaying the development of colitis, allows time for transformation of epithelial cells. Colonic epithelial preparations from co-infected mice showed increased expression of c-myc (5- to 12-fold) and interleukin-1alpha/beta (600-fold) by real-time polymerase chain reaction relative to uninfected wild-type and mdr1a-/- animals. This animal model may have particular relevance to human IBD and colorectal cancer because certain human MDR1 polymorphisms have been linked to ulcerative colitis and increased risk for colorectal cancer.

A point mutation in the murine Hem1 gene reveals an essential role for Hematopoietic protein 1 in lymphopoiesis and innate immunity.

Hem1 (Hematopoietic protein 1) is a hematopoietic cell-specific member of the Hem family of cytoplasmic adaptor proteins. Orthologues of Hem1 in Dictyostelium discoideum, Drosophila melanogaster, and Caenorhabditis elegans are essential for cytoskeletal reorganization, embryonic cell migration, and morphogenesis. However, the in vivo functions of mammalian Hem1 are not known. Using a chemical mutagenesis strategy in mice to identify novel genes involved in immune cell functions, we positionally cloned a nonsense mutation in the Hem1 gene. Hem1 deficiency results in defective F-actin polymerization and actin capping in lymphocytes and neutrophils caused by loss of the Rac-controlled actin-regulatory WAVE protein complex. T cell development is disrupted in Hem1-deficient mice at the CD4−CD8− (double negative) to CD4+CD8+ (double positive) cell stages, whereas T cell activation and adhesion are impaired. Hem1-deficient neutrophils fail to migrate in response to chemotactic agents and are deficient in their ability to phagocytose bacteria. Remarkably, some Rac-dependent functions, such as Th1 differentiation and nuclear factor κB (NF-κB)–dependent transcription of proinflammatory cytokines proceed normally in Hem1-deficient mice, whereas the production of Th17 cells are enhanced. These results demonstrate that Hem1 is essential for hematopoietic cell development, function, and homeostasis by controlling a distinct pathway leading to cytoskeletal reorganization, whereas NF-κB–dependent transcription proceeds independently of Hem1 and F-actin polymerization.

Fnip1 regulates skeletal muscle fiber type specification, fatigue resistance, and susceptibility to muscular dystrophy

Significance Folliculin interacting protein-1 (Fnip1) is an intracellular protein known to interact with folliculin (a protein mutated in Birt Hogg Dube’ Syndrome) and the master metabolic sensor AMP kinase. However, the roles of Fnip1 in mammalian development and function are unclear. In this study, we used mice deficient in Fnip1 to show that Fnip1 regulates skeletal muscle fiber type specification. Mice deficient in Fnip1 were significantly enriched for highly oxidative skeletal muscle that is more resistant to fatigue than wild-type muscle. Loss of Fnip1 also decreased muscle damage in a mouse model of Duchenne muscular dystrophy. These results reveal a previously unidentified function for Fnip1 and suggest that pharmacological inhibition of Fnip1 may reduce muscle damage in patients with muscular dystrophy. Mammalian skeletal muscle is broadly characterized by the presence of two distinct categories of muscle fibers called type I “red” slow twitch and type II “white” fast twitch, which display marked differences in contraction strength, metabolic strategies, and susceptibility to fatigue. The relative representation of each fiber type can have major influences on susceptibility to obesity, diabetes, and muscular dystrophies. However, the molecular factors controlling fiber type specification remain incompletely defined. In this study, we describe the control of fiber type specification and susceptibility to metabolic disease by folliculin interacting protein-1 (Fnip1). Using Fnip1 null mice, we found that loss of Fnip1 increased the representation of type I fibers characterized by increased myoglobin, slow twitch markers [myosin heavy chain 7 (MyH7), succinate dehydrogenase, troponin I 1, troponin C1, troponin T1], capillary density, and mitochondria number. Cultured Fnip1-null muscle fibers had higher oxidative capacity, and isolated Fnip1-null skeletal muscles were more resistant to postcontraction fatigue relative to WT skeletal muscles. Biochemical analyses revealed increased activation of the metabolic sensor AMP kinase (AMPK), and increased expression of the AMPK-target and transcriptional coactivator PGC1α in Fnip1 null skeletal muscle. Genetic disruption of PGC1α rescued normal levels of type I fiber markers MyH7 and myoglobin in Fnip1-null mice. Remarkably, loss of Fnip1 profoundly mitigated muscle damage in a murine model of Duchenne muscular dystrophy. These results indicate that Fnip1 controls skeletal muscle fiber type specification and warrant further study to determine whether inhibition of Fnip1 has therapeutic potential in muscular dystrophy diseases.

Conditional Disruption of Raptor Reveals an Essential Role for mTORC1 in B Cell Development, Survival, and Metabolism

Mechanistic target of rapamycin (mTOR) is a serine-threonine kinase that coordinates nutrient and growth factor availability with cellular growth, division, and differentiation. Studies examining the roles of mTOR signaling in immune function revealed critical roles for mTOR in regulating T cell differentiation and function. However, few studies have investigated the roles of mTOR in early B cell development. In this study, we found that mTOR is highly activated during the pro- and pre-B stages of mouse B cell development. Conditional disruption of the mTOR coactivating protein Raptor in developing mouse B cells resulted in a developmental block at the pre-B cell stage, with a corresponding lack of peripheral B cells and loss of Ag-specific Ab production. Pre-B cell survival and proliferation were significantly reduced in Raptor-deficient mice. Forced expression of a transgenic BCR or a BclxL transgene on Raptor-deficient B cells failed to rescue B cell development, suggesting that pre-BCR signaling and B cell survival are impaired in a BclxL-independent manner. Raptor-deficient pre-B cells exhibited significant decreases in oxidative phosphorylation and glycolysis, indicating that loss of mTOR signaling in B cells significantly impairs cellular metabolic capacity. Treatment of mice with rapamycin, an allosteric inhibitor of mTOR, recapitulated the early B cell developmental block. Collectively, our data reveal a previously uncharacterized role for mTOR signaling in early B cell development, survival, and metabolism.

Metabolic regulator Fnip1 is crucial for iNKT lymphocyte development

Significance Identifying factors that regulate the development of cytokine-producing immunoregulatory invariant natural killer T (iNKT) cells is critical for understanding how to modulate these cells to promote cell-mediated immunity to cancer and infectious organisms or suppress excessive inflammation in autoimmune disease. Here, we identified an essential role for the metabolic regulator Folliculin-interacting protein 1 (Fnip1) in iNKT cell development and survival. Fnip1 physically interacts with AMPK, an energy-sensing enzyme that stimulates mitochondria and ATP production in response to energy deficit, while inhibiting mammalian target of rapamycin (mTOR)-mediated cell growth. Fnip1-deficient iNKT cells contain reduced mitochondrial number and hyperactive mTOR, which resulted in decreased ATP levels and increased sensitivity to apoptosis. Our findings indicate that Fnip1 is vital for maintaining metabolic balance during iNKT cell development. Folliculin-interacting protein 1 (Fnip1) is an adaptor protein that physically interacts with AMPK, an energy-sensing kinase that stimulates mitochondrial biogenesis and autophagy in response to low ATP, while turning off energy consumption mediated by mammalian target of rapamycin. Previous studies with Fnip1-null mice revealed that Fnip1 is essential for pre–B-cell development. Here we report a critical role of Fnip1 in invariant natural killer T (iNKT) cell development. Thymic iNKT development in Fnip1−/− mice was arrested at stage 2 (NK1.1−CD44+) but development of CD4, CD8, γδ T-cell, and NK cell lineages proceeded normally. Enforced expression of a Vα14Jα18 iNKT TCR transgene or loss of the proapoptotic protein Bim did not rescue iNKT cell maturation in Fnip1−/− mice. Whereas most known essential transcription factors for iNKT cell development were represented normally, Fnip1−/− iNKT cells failed to down-regulate Promyelocytic leukemia zinc finger compared with their WT counterparts. Moreover, Fnip1−/− iNKT cells contained hyperactive mTOR and reduced mitochondrial number despite lower ATP levels, resulting in increased sensitivity to apoptosis. These results indicate that Fnip1 is vital for iNKT cell development by maintaining metabolic homeostasis in response to metabolic stress.

Hematopoietic Protein-1 Regulates the Actin Membrane Skeleton and Membrane Stability in Murine Erythrocytes

Hematopoietic protein-1 (Hem-1) is a hematopoietic cell specific member of the WAVE (Wiskott-Aldrich syndrome verprolin-homologous protein) complex, which regulates filamentous actin (F-actin) polymerization in many cell types including immune cells. However, the roles of Hem-1 and the WAVE complex in erythrocyte biology are not known. In this study, we utilized mice lacking Hem-1 expression due to a non-coding point mutation in the Hem1 gene to show that absence of Hem-1 results in microcytic, hypochromic anemia characterized by abnormally shaped erythrocytes with aberrant F-actin foci and decreased lifespan. We find that Hem-1 and members of the associated WAVE complex are normally expressed in wildtype erythrocyte progenitors and mature erythrocytes. Using mass spectrometry and global proteomics, Coomassie staining, and immunoblotting, we find that the absence of Hem-1 results in decreased representation of essential erythrocyte membrane skeletal proteins including α- and β- spectrin, dematin, p55, adducin, ankyrin, tropomodulin 1, band 3, and band 4.1. Hem1−/− erythrocytes exhibit increased protein kinase C-dependent phosphorylation of adducin at Ser724, which targets adducin family members for dissociation from spectrin and actin, and subsequent proteolysis. Increased adducin Ser724 phosphorylation in Hem1−/− erythrocytes correlates with decreased protein expression of the regulatory subunit of protein phosphatase 2A (PP2A), which is required for PP2A-dependent dephosphorylation of PKC targets. These results reveal a novel, critical role for Hem-1 in the homeostasis of structural proteins required for formation and stability of the actin membrane skeleton in erythrocytes.

Loss of Fnip1 alters kidney developmental transcriptional program and synergizes with TSC1 loss to promote mTORC1 activation and renal cyst formation

Birt-Hogg-Dube’ Syndrome (BHDS) is a rare genetic disorder in humans characterized by skin hamartomas, lung cysts, pneumothorax, and increased risk of renal tumors. BHDS is caused by mutations in the BHD gene, which encodes for Folliculin, a cytoplasmic adapter protein that binds to Folliculin interacting proteins-1 and -2 (Fnip1, Fnip2) as well as the master energy sensor AMP kinase (AMPK). Whereas kidney-specific deletion of the Bhd gene in mice is known to result in polycystic kidney disease (PKD) and renal cell carcinoma, the roles of Fnip1 in renal cell development and function are unclear. In this study, we utilized mice with constitutive deletion of the Fnip1 gene to show that the loss of Fnip1 is sufficient to result in renal cyst formation, which was characterized by decreased AMPK activation, increased mTOR activation, and metabolic hyperactivation. Using RNAseq, we found that Fnip1 disruption resulted in many cellular and molecular changes previously implicated in the development of PKD in humans, including alterations in the expression of ion and amino acid transporters, increased cell adhesion, and increased inflammation. Loss of Fnip1 synergized with Tsc1 loss to hyperactivate mTOR, increase Erk activation, and greatly accelerate the development of PKD. Our results collectively define roles for Fnip1 in regulating kidney development and function, and provide a model for how loss of Fnip1 contributes to PKD and perhaps renal cell carcinoma.

Abstract B31: Disruption of Folliculin interacting protein-1 modulates Myc-driven metabolism, increasing cell death following metabolic stress

Folliculin interacting protein-1 (Fnip1) is a cytoplasmic protein originally discovered through its interaction with Folliculin (Flcn), a tumor suppressor mutated in the rare fibrofolliculoma disorder Birt-Hogg Dube syndrome. Fnip1 and Flcn both interact with the master metabolic regulator AMP kinase (AMPK). Our previous studies have shown that Fnip1 is required for the development of B-cells past the late pre-B stage (Park et al Immunity 2012) and for the development invariant natural killer T (iNKT) cells in the thymus (Park et al PNAS 2014). Fnip1 deficiency also prevented the development of B cell lymphoma in Eµ-Myc transgenic mice. Fnip1 deficient immune cells exhibited hyperactivation of mTORC1 concurrent with increased activation of AMPK. In this study, we examined the potential efficacy of Fnip1 disruption in cancer by disrupting Fnip1 in primary mouse and human B cell lines overexpressing the Myc oncogene. We show that constitutive depletion of Fnip1 in primary pre-B cells, conditional knockdown of Fnip1 in primary mouse mature B cells using the Cre-loxP system, or siRNA-mediated depletion of endogenous FNIP1 from a human B cell line expressing a conditional Myc allele, increase oxidative phosphorylation and Myc-induced glycolysis, which support cell division. Disruption of Fnip1 increases death of primary murine pre-B Eµ-Myc cells and human B cell lines in response to nutrient deprivation and chemotherapeutic drugs in vitro. Mass spectrometric analysis of a human B cell line depleted of Fnip1 reveals significant alterations in metabolites involved in glutaminolysis, glycolysis, antioxidant and serine biosynthesis pathways. These results suggest that inhibition of Fnip1 may provide a novel strategy to increase death of lymphoma cells in response to cytotoxic and metabolic stress. Citation Format: Julita A. Ramirez, Mark Tsang, Heon Park, Daciana Margineantu, Haiwei Gu, Daniel Raftery, David M. Hockenbery, Brian M. Iritani. Disruption of Folliculin interacting protein-1 modulates Myc-driven metabolism, increasing cell death following metabolic stress. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr B31.

Myc stimulates B lymphocyte differentiation and amplifies calcium signaling

Habib et al. 2007. J. Cell Biol. doi:10.1083/jcb.200704173 [OpenUrl][1][Abstract/FREE Full Text][2] [1]: {openurl}?query=rft_id%253Dinfo%253Adoi%252F10.1083%252Fjcb.200704173%26rft_id%253Dinfo%253Apmid%252F17998397%26rft.genre%253Darticle%26rft_val_fmt%253Dinfo%253Aofi%252Ffmt%253Akev%253Amtx%