技术资料

Intraepithelial lymphocytes (IELs) expressing the gamma$delta$ TCR (gamma$delta$ IELs) provide continuous surveillance of the intestinal epithelium. However,the mechanisms regulating the basal motility of these cells within the epithelial compartment have not been well defined. We investigated whether IL-15 contributes to gamma$delta$ IEL localization and migratory behavior in addition to its role in IEL differentiation and survival. Using advanced live cell imaging techniques in mice,we find that compartmentalized overexpression of IL-15 in the lamina propria shifts the distribution of gamma$delta$ T cells from the epithelial compartment to the lamina propria. This mislocalization could be rescued by epithelial IL-15 overexpression,indicating that epithelial IL-15 is essential for gamma$delta$ IEL migration into the epithelium. Furthermore,in vitro analyses demonstrated that exogenous IL-15 stimulates gamma$delta$ IEL migration into cultured epithelial monolayers,and inhibition of IL-2Rbeta$ significantly attenuates the basal motility of these cells. Intravital microscopy showed that impaired IL-2Rbeta$ signaling induced gamma$delta$ IEL idling within the lateral intercellular space,which resulted in increased early pathogen invasion. Similarly,the redistribution of gamma$delta$ T cells to the lamina propria due to local IL-15 overproduction also enhanced bacterial translocation. These findings thus reveal a novel role for IL-15 in mediating gamma$delta$ T cell localization within the intestinal mucosa and regulating gamma$delta$ IEL motility and patrolling behavior as a critical component of host defense. View Publication

Long-lived multipotent stem cells (ISCs) at the base of intestinal crypts adjust their phenotypes to accommodate normal maintenance and post-injury regeneration of the epithelium. Their long life,lineage plasticity,and proliferative potential underlie the necessity for tight homeostatic regulation of the ISC compartment. In that context,the guanylate cyclase C (GUCY2C) receptor and its paracrine ligands regulate intestinal epithelial homeostasis,including proliferation,lineage commitment,and DNA damage repair. However,a role for this axis in maintaining ISCs remains unknown. Transgenic mice enabling analysis of ISCs (Lgr5-GFP) in the context of GUCY2C elimination (Gucy2c -/- ) were combined with immunodetection techniques and pharmacological treatments to define the role of the GUCY2C signaling axis in supporting ISCs. ISCs were reduced inGucy2c -/- mice,associated with loss of active Lgr5+cells but a reciprocal increase in reserve Bmi1+cells. GUCY2C was expressed in crypt base Lgr5+cells in which it mediates canonical cyclic (c) GMP-dependent signaling. Endoplasmic reticulum (ER) stress,typically absent from ISCs,was elevated throughout the crypt base inGucy2c -/- mice. The chemical chaperone tauroursodeoxycholic acid resolved this ER stress and restored the balance of ISCs,an effect mimicked by the GUCY2C effector 8Br-cGMP. Reduced ISCs inGucy2c -/- mice was associated with greater epithelial injury and impaired regeneration following sub-lethal doses of irradiation. These observations suggest that GUCY2C provides homeostatic signals that modulate ER stress and cell vulnerability as part of the machinery contributing to the integrity of ISCs. View Publication

The origins of the epithelial cells participating in the development,tissue homeostasis,and cancer of the human breast are poorly understood. However,emerging evidence suggests a role for adult tissue-specific stem cells in these processes. In a hierarchical manner,these generate the two main mammary cell lineages,producing an increasing number of cells with distinct properties. Understanding the biological characteristics of human breast stem cells and their progeny is crucial in attempts to compare the features of normal stem cells and cancer precursor cells and distinguish these from nonprecursor cells and cells from the bulk of a tumor. A historical overview of research on human breast stem cells in primary tissue and in culture reveals the progress that has been made in this area,whereas a focus on the cell-of-origin and reprogramming that occurs during neoplastic conversion provides insight into the enigmatic way in which human breast cancers are skewed toward the luminal epithelial lineage. View Publication

Adequate availability of cellular building blocks,including lipids,is a prerequisite for cellular proliferation,but excess dietary lipids are linked to increased cancer risk. Despite these connections,specific regulatory relationships between membrane composition,intestinal stem cell (ISC) proliferation,and tumorigenesis are unclear. We reveal an unexpected link between membrane phospholipid remodeling and cholesterol biosynthesis and demonstrate that cholesterol itself acts as a mitogen for ISCs. Inhibition of the phospholipid-remodeling enzyme Lpcat3 increases membrane saturation and stimulates cholesterol biosynthesis,thereby driving ISC proliferation. Pharmacologic inhibition of cholesterol synthesis normalizes crypt hyperproliferation in Lpcat3-deficient organoids and mice. Conversely,increasing cellular cholesterol content stimulates crypt organoid growth,and providing excess dietary cholesterol or driving endogenous cholesterol synthesis through SREBP-2 expression promotes ISC proliferation in vivo. Finally,disruption of Lpcat3-dependent phospholipid and cholesterol homeostasis dramatically enhances tumor formation in Apcminmice. These findings identify a critical dietary-responsive phospholipid-cholesterol axis regulating ISC proliferation and tumorigenesis. View Publication

In recent years,the differentiation of bone marrow cells (BMCs) into myocytes has been extensively investigated,but the findings remain inconclusive. The purpose of this study was to determine the conditions necessary to induce myogenic differentiation in short-term cultures of adult BMCs,and to identify the BMC subpopulation responsible for this phenomenon. We report that high-density cultures of murine hematopoietic BMCs gave rise to spontaneous beating cell clusters in the presence of vascular endothelial and fibroblast growth factors. These clusters originated from c-kit(pos) cells. The formation of the clusters could be completely blocked by adding a c-kit/tyrosine kinase inhibitor,Gleevec (imatinib mesylate; Novartis International,Basel,Switzerland,http://www.novartis.com),to the culture. Cluster formation was also blunted in BMCs from c-kit-deficient (Kit(W)/Kit(W-v)) mice. Clustered cells expressed cardiomyocyte-specific transcription factor genes Gata-4 and Nkx2.5,sarcomeric proteins beta-MHC and MLC-2v,and ANF and connexin-43. Immunostaining revealed alpha-sarcomeric actinin expression in more than 90% of clustered cells. Under electron microscopy,the clustered cells exhibited a sarcomeric myofiber arrangement and z-bands. This study defines the microenvironment required to achieve a reproducible in vitro model of beating,myogenic cell clusters. This model could be used to examine the mechanisms responsible for the postnatal myogenic differentiation of BMCs. Our results identify c-kit(pos) bone marrow hematopoietic cells as the source of the myogenic clusters. View Publication

In models of acute lung injury,CXC chemokine receptor 2 (CXCR2) mediates migration of polymorphonuclear leukocytes (PMNs) into the lung. Since CXCR2 ligands,including CXCL1 and CXCL2/3,are chemotactic for PMNs,CXCR2 is thought to recruit PMNs by inducing chemotactic migration. In a model of PMN recruitment to the lung,aerosolized bacterial LPS inhalation induced PMN recruitment to the lung in wild-type mice,but not in littermate CXCR2-/- mice. Surprisingly,lethally irradiated wild-type mice reconstituted with CXCR2-/- BM still showed about 50% PMN recruitment into bronchoalveolar lavage fluid and into lung interstitium,but CXCR2-/- mice reconstituted with CXCR2-/- BM showed no PMN recruitment. Conversely,CXCR2-/- mice reconstituted with wild-type BM showed a surprisingly large defect in PMN recruitment,inconsistent with a role of CXCR2 on PMNs alone. Cell culture,immunohistochemistry,flow cytometry,and real-time RT-PCR were used to show expression of CXCR2 on pulmonary endothelial and bronchial epithelial cells. The LPS-induced increase in lung microvascular permeability as measured by Evans blue extravasation required CXCR2 on nonhematopoietic cells. Our data revealed what we believe to be a previously unrecognized role of endothelial and epithelial CXCR2 in LPS-induced PMN recruitment and lung injury. View Publication

Normal stem cells and cancer stem cells (CSCs) share similar properties,in that both have the capacity to self-renew and differentiate into multiple cell types. In both the normal stem cell and cancer stem cell fields,there has been a great need for a universal marker that can effectively identify and isolate these rare populations of cells in order to characterize them and use this information for research and therapeutic purposes. Currently,it would appear that certain isoenzymes of the aldehyde dehydrogenase (ALDH) superfamily may be able to fulfill this role as a marker for both normal and cancer stem cells. ALDH has been identified as an important enzyme in the protection of normal hematopoietic stem cells,and is now also widely used as a marker to identify and isolate various types of normal stem cells and CSCs. In addition,emerging evidence suggests that ALDH1 is not only a marker for stem cells,but may also play important functional roles related to self-protection,differentiation,and expansion. This comprehensive review discusses the role that ALDH plays in normal stem cells and CSCs,with focus on ALDH1 and ALDH3A1. Discrepancies in the functional themes between cell types and future perspectives for therapeutic applications will also be discussed. View Publication

Inflammatory breast carcinoma (IBC) is characterized by exaggerated lymphovascular invasion (LVI),recapitulated in our human xenograft,MARY-X. This model exhibited lymphovascular emboli in vivo and corresponding spheroids in vitro. Owing to the morphological and gene profile resemblance of these spheroids to embryonal blastocysts,we wondered whether they might exhibit embryonic stem cell signaling. Specifically we investigated Notch and observed selective Notch 3 activation by expression profiling,reverse transcriptase- and real-time PCR,western blot and immunofluorescence in vitro,and immunohistochemistry in vivo. Notch 3 intracellular domain (N3icd) and six target genes,HES-5,HEY-1,c-Myc,Deltex-1,NRARP and PBX1,markedly increased in MARY-X. In addition,a significant percentage of MARY-X cells expressed aldehyde dehydrogenase (ALDH),a stem cell marker. Only the ALDH(+) cells were capable of secondary spheroidgenesis,tumorigenicity and self-renewal. Inhibiting Notch 3 activation in vitro with γ-secretase inhibitors (GSIs) or small interfering RNA resulted in a downregulation of Notch target genes,including CD133,and an induction of caspase 3-mediated apoptosis. Transfection of N3icd but not Notch 1 intracellular domain into normal human mammary epithelial cells resulted in increased expression of Notch target genes and induction of spheroidgenesis. GSI in vivo resulted in inhibitory but diffusion-limited effects on Notch 3 signaling,resulting in xenograft growth reduction. The lymphovascular emboli of human IBC exhibited dual N3icd and ALDH1 immunoreactivities independently of molecular subtype. This Notch 3 addiction of lymphovascular emboli might be exploited in future therapeutic strategies. View Publication

BACKGROUND: The cadmium (Cd) present in air pollutants and cigarette smoke has the potential of causing multiple adverse health outcomes involving damage to pulmonary and cardiovascular tissue. Injury to pulmonary epithelium may include alterations in tight junction (TJ) integrity,resulting in impaired epithelial barrier function and enhanced penetration of chemicals and biomolecules. Herein,we investigated mechanisms involved in the disruption of TJ integrity by Cd exposure using an in vitro human air-liquid-interface (ALI) airway tissue model derived from normal primary human bronchial epithelial cells. METHODS: ALI cultures were exposed to noncytotoxic doses of CdCl2 basolaterally and TJ integrity was measured by Trans-Epithelial Electrical Resistance (TEER) and immunofluorescence staining with TJ markers. PCR array analysis was used to identify genes involved with TJ collapse. To explore the involvement of kinase signaling pathways,cultures were treated with CdCl2 in the presence of kinase inhibitors specific for cellular Src or Protein Kinase C (PKC). RESULTS: Noncytotoxic doses of CdCl2 resulted in the collapse of barrier function,as demonstrated by TEER measurements and Zonula occludens-1 (ZO-1) and occludin staining. CdCl2 exposure altered the expression of several groups of genes encoding proteins involved in TJ homeostasis. In particular,down-regulation of select junction-interacting proteins suggested that a possible mechanism for Cd toxicity involves disruption of the peripheral junctional complexes implicated in connecting membrane-bound TJ components to the actin cytoskeleton. Inhibition of kinase signaling using inhibitors specific for cellular Src or PKC preserved the integrity of TJs,possibly by preventing occludin tyrosine hyperphosphorylation,rather than reversing the down-regulation of the junction-interacting proteins. CONCLUSIONS: Our findings indicate that acute doses of Cd likely disrupt TJ integrity in human ALI airway cultures both through occludin hyperphosphorylation via kinase activation and by direct disruption of the junction-interacting complex. View Publication