title: Inhibition of oxidant-induced nuclear factor-kappaB activation and inhibitory-kappaBalpha degradation and instability of F-actin cytoskeletal dynamics and barrier function by epidermal growth factor: key role of phospholipase-gamma isoform.
abstract: Using monolayers of intestinal (Caco-2) cells as a model for studying inflammatory bowel disease (IBD), we previously showed that nuclear factor-kappaB (NF-kappaB) activation is required for oxidant-induced disruption of cytoskeletal and barrier integrity. Epidermal growth factor (EGF) stabilizes the F-actin cytoskeleton and protects against oxidant damage, but the mechanism remains unclear. We hypothesized that the mechanism involves activation of phospholipase C-gamma (PLC-gamma), which prevents NF-kappaB activation and the consequences of this activation, namely, cytoskeletal and barrier disruption. We studied wild-type and transfected cells. The latter were transfected with varying levels (1-5 microg) of cDNA to either stably overexpress PLC-gamma or to inhibit its activation. Cells were pretreated with EGF before exposure to oxidant (H(2)O(2)). Stably overexpressing PLC-gamma (+2.0-fold) or preincubating with EGF protected against oxidant injury as indicated by 1) decreases in several NF-kappaB-related variables [NF-kappaB (p50/p65 subunit) nuclear translocation, NF-kappaB subunit activity, inhibitory-kappaBalpha (I-kappaBalpha) phosphorylation and degradation]; 2) increases in F-actin and decreases in G-actin; 3) stabilization of the actin cytoskeletal architecture; and 4) enhancement of barrier function. Overexpression induced inactivation of NF-kappaB was potentiated by EGF. PLC-gamma was found mostly in membrane and cytoskeletal fractions (<9% in the cytosolic fractions), indicating its activation. Dominant negative inhibition of endogenous PLC-gamma (-99%) substantially prevented all measures of EGF protection against NF-kappaB activation. We concluded 1) EGF protects against oxidant-induced barrier disruption through PLC-gamma activation, which inactivates NF-kappaB; 2) Activation of PLC-gamma by itself is protective against NF-kappaB activation; 3) the ability to modulate the dynamics of NF-kappaB/I-kappa Balpha is a novel mechanism not previously attributed to the PLC family of isoforms in cells; and 4) development of PLC-gamma mimetics represents a possible new therapeutic strategy for IBD.
authors.affiliation: Division of Digestive Diseases, Department of Internal Medicine, Section of Gastroenterology and Nutrition, Rush University of Chicago School of Medicine, 1725 W. Harrison, Suite 206, Chicago, IL 60612, USA. ali_banan@rush.edu
authors.forename: A
authors.initials: A
authors.lastname: Banan
authors.forename: L J
authors.initials: LJ
authors.lastname: Zhang
authors.forename: M
authors.initials: M
authors.lastname: Shaikh
authors.forename: J Z
authors.initials: JZ
authors.lastname: Fields
authors.forename: A
authors.initials: A
authors.lastname: Farhadi
authors.forename: A
authors.initials: A
authors.lastname: Keshavarzian
mesh_terms: D000199:Actins / Q000378:metabolism*; D018938:Caco-2 Cells; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism; D003599:Cytoskeleton / Q000187:drug effects / Q000378:metabolism; D003600:Cytosol / Q000187:drug effects; D004795:Enzyme Stability; D004815:Epidermal Growth Factor / Q000494:pharmacology*; D006801:Humans; D020672:I-kappa B Proteins / Q000378:metabolism*; D016328:NF-kappa B / Q000378:metabolism* / Q000502:physiology; D016877:Oxidants / Q000494:pharmacology; D051966:Phospholipase C gamma; D010766:Phosphorylation; D020033:Protein Isoforms; D021122:Protein Subunits; D013347:Subcellular Fractions; D014162:Transfection; D010738:Type C Phospholipases / Q000378:metabolism*
chemical_list: D000199:Actins; D020672:I-kappa B Proteins; D016328:NF-kappa B; D016877:Oxidants; D020033:Protein Isoforms; D021122:Protein Subunits; D004815:Epidermal Growth Factor; D010738:Type C Phospholipases; D051966:Phospholipase C gamma