Biomedical subjects

Nancy A Louis

Publications and source records attributed to Nancy A Louis.

6 recordsLinked to original sources

PubMed2006

Selective induction of mucin-3 by hypoxia in intestinal epithelia.

Epithelial cells line mucosal surfaces (e.g., lung, intestine) and critically function as a semipermeable barrier to the outside world. Mucosal organs are highly vascular with extensive metabolic demands, and for this reason, are particularly susceptible to diminished blood flow and resultant tissue hypoxia. Here, we pursue the hypothesis that intestinal barrier function is regulated in a protective manner by hypoxia responsive genes. We demonstrate by PCR confirmation of microarray data and by avidin blotting of immunoprecipitated human Mucin 3 (MUC3), that surface MUC3 expression is induced in T84 intestinal epithelial cells following exposure to hypoxia. MUC3 RNA is minimally detectable while surface protein expression is absent under baseline normoxic conditions. There is a robust induction in both the mRNA (first evident by 8 h) and protein expression, first observed and maximally expressed following 24 h hypoxia. This is followed by a subsequent decline in protein expression, which remains well above baseline at 48 h of hypoxia. Further, we demonstrate that this induction of MUC3 protein is associated with a transient increase in the barrier restorative peptide, intestinal trefoil factor (ITF). ITF not only colocalizes with MUC3, by confocal microscopy, to the apical surface of T84 cells following exposure to hypoxia, but is also found, by co-immunoprecipitation, to be physically associated with MUC3, following 24 h of hypoxia. In exploration of the mechanism of hypoxic regulation of mucin 3 expression, we demonstrated by luciferase assay that the full-length promoter for mouse Mucin 3 (Muc3) is hypoxia-responsive with a 5.08 +/- 1.76-fold induction following 24 h of hypoxia. Furthermore, analysis of both the human (MUC3A) and mouse (Muc3) promoters revealed potential HIF-1 binding sites which were shown by chromatin immunoprecipitation to bind the pivotal hypoxia-regulating transcription factor HIF-1alpha. Taken together, these studies implicate the HIF-1alpha mediated hypoxic induced expression of mucin 3 and associated ITF in the maintenance of intestinal barrier function under hypoxic conditions.

Base Sequence↗

PubMed2006

Cell-cell interactions on solid matrices.

Models to study molecular, biochemical, and functional responses in vitro generally incorporate an individual cell type or group of cells organized in a random fashion. Normal physiological responses in vivo require that individual cell types be oriented in an organized fashion with three-dimensional architecture and appropriately positioned cellular interfaces. Much recent progress has been made in the development and implementation of models to study cell-cell contact using substrate grown cells. Here, we summarize the use of membrane permeable supports to study functional responses in appropriately positioned cell types. These models incorporate two or more different cells cultured in physiologically positioned locales on solid substrates. Models incorporating nonadherent cells (e.g., leukocytes) in co-culture with such models also are discussed. Such models have been used extensively to discovery both cell-bound as well as soluble mediators of physiological and pathophysiological processes.

Animals↗

PubMed2005

HIF-dependent induction of apical CD55 coordinates epithelial clearance of neutrophils.

Sites of inflammation are associated with dramatic shifts in tissue metabolism. Inflammation can result in significant tissue hypoxia, with resultant induction of hypoxia-responsive genes. Given this association, we hypothesized that neutrophil (PMN) ligands expressed on epithelial cells may be regulated by hypoxia. Initial studies confirmed earlier results that epithelial hypoxia enhances PMN transepithelial migration and promotes apical clearance of PMN from the epithelial surface. A screen of known PMN ligands revealed a surprisingly stable expression pattern in hypoxia. However, this screen identified one gene, CD55, as a highly hypoxia-inducible molecule expressed on the apical membrane of mucosal epithelia. Subsequent studies verified the induction of CD55 mRNA and protein expression by hypoxia. Overexpression of CD55 by transfection in nonhypoxic epithelia resulted in a similar pattern of apical PMN clearance, and peptide mimetics corresponding to the PMN binding site on DAF blocked such apical clearance of PMN. Studies directed at understanding molecular pathways of hypoxia inducibility revealed that a approximately 200 bp region of the CD55 gene conferred hypoxia inducibility for CD55. These studies identified a functional binding site for the transcriptional regulator hypoxia-inducible factor (HIF). Taken together, these results identify HIF-dependent induction of epithelial CD55 in the resolution of ongoing inflammation through clearance of apical PMN.

Amino Acid Sequence↗

PubMed2003

Antiadhesive role of apical decay-accelerating factor (CD55) in human neutrophil transmigration across mucosal epithelia.

Neutrophil migration across mucosal epithelium during inflammatory episodes involves the precise orchestration of a number a cell surface molecules and signaling pathways. After successful migration to the apical epithelial surface, apically localized epithelial proteins may serve to retain PMN at the lumenal surface. At present, identification of apical epithelial ligands and their PMN counter-receptors remain elusive. Therefore, to define the existence of apical epithelial cell surface proteins involved in PMN-epithelial interactions, we screened a panel of antibodies directed against epithelial plasma membranes. This strategy identified one antibody (OE-1) that both localized to the apical cell membrane and significantly inhibited PMN transmigration across epithelial monolayers. Microsequence analysis revealed that OE-1 recognized human decay-accelerating factor (DAF, CD55). DAF is a highly glycosylated, 70-80-kD, glycosyl-phosphatidyinositol-linked protein that functions predominantly as an inhibitor of autologous complement lysis. DAF suppression experiments using antisense oligonucleotides or RNA interference revealed that DAF may function as an antiadhesive molecule promoting the release of PMN from the lumenal surface after transmigration. Similarly, peptides corresponding to the antigen recognition domain of OE-1 resulted in accumulation of PMN on the apical epithelial surface. The elucidation of DAF as an apical epithelial ligand for PMN provides a target for novel anti-inflammatory therapies directed at quelling unwanted inflammatory episodes.

Amino Acid Sequence↗

PubMed2002

Hypoxia-inducible factor-1-dependent regulation of the multidrug resistance (MDR1) gene.

The microenvironment of rapidly growing tumors is associated with increased energy demand and diminished vascular supply, resulting in focal areas of prominent hypoxia. A number of hypoxia-responsive genes have been associated with growing tumors, and here we demonstrate that the multidrug resistance (MDR1) gene product P-glycoprotein, a Mr approximately 170,000 transmembrane protein associated with tumor resistance to chemotherapeutics, is induced by ambient hypoxia. Initial studies using quantitative microarray analysis of RNA revealed an approximately 7-fold increase in MDR in epithelial cells exposed to hypoxia (pO(2) 20 torr, 18 h). These findings were further confirmed at the mRNA and protein level. P-Glycoprotein function was studied by analysis of verapamil-inhibitable efflux of digoxin and rhodamine 123 in intact T84 cells and revealed that hypoxia enhances P-glycoprotein function by as much as 7 +/- 0.4-fold over normoxia. Subsequent studies confirmed hypoxia-elicited MDR1 gene induction and increased P-glycoprotein expression in nontransformed, primary cultures of human microvascular endothelial cells, and analysis of multicellular spheroids subjected to hypoxia revealed increased resistance to doxorubicin. Examination of the MDR1 gene identified a binding site for hypoxia inducible factor-1 (HIF-1), and inhibition of HIF-1 expression by antisense oligonucleotides resulted in significant inhibition of hypoxia-inducible MDR1 expression and a nearly complete loss of basal MDR1 expression. Studies using luciferase promoter constructs revealed a significant increase in activity in cells subjected to hypoxia, and such hypoxia inducibility was lost in truncated constructs lacking the HIF-1 site and in HIF-1 binding site mutants. Extensions of these studies also identified a role for Sp1 in this hypoxia response. Taken together, these data indicate that the MDR1 gene is hypoxia responsive, and such results may identify hypoxia-elicited P-glycoprotein expression as a pathway for resistance of some tumors to chemotherapeutics.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMedDate not supplied

Lipid mediator networks and leukocyte transmigration.

In intact tissues, vascular endothelial cells lie anatomically positioned as the central coordinator of inflammation. Endothelia communicate with underlying cells (e.g. smooth muscle, fibroblasts, epithelia) in ways that both coordinate leukocyte trafficking, and control the composition of the inflammatory microenvironment. Such coordination occurs through both direct communication (e.g. cell adhesion) as well as via soluble mediators liberated at sites of inflammation (e.g. chemokines, cytokines, lipids). Locally generated mediators bind to surface receptors, and mediate both physiologic and pathophysiologic functional responses. Important in this regard, both endothelial and subendothelial cell populations express enzymes capable of utilizing arachidonic acid substrates to generate bioactive lipid mediators (e.g. lipoxygenases, cyclooxygenases). Such lipid mediators can signal via autocrine or paracrine pathways and, depending on the tissue microenvironment, can convey a pro- or anti-inflammatory message. This review will highlight recent studies characterizing inflammatory responses to lipid mediators liberated at sites of inflammation, with a particular emphasis on neutrophil (polymorphonuclear leukocyte or PMN) trafficking.

Cyclooxygenase 2↗