Protection against ethanol injury by prostaglandin in a human intestinal cell line: role of microtubules

Abstract

Prostaglandins have been shown to protect the gastrointestinal (GI) epithelium from injury induced by various luminal insults independent of their known acid-inhibitory effects, a process termed “cytoprotection.” The mechanism of this protective action remains unknown. The present investigation determined the role of microtubules (a major cytoskeletal component) in GI injury induced by ethanol (EtOH) and its prevention by 16,16-dimethylprostaglandin E2(dmPGE2) using cells from a human colonic cell line known as Caco-2 cells. These cells were preincubated in Eagle’s minimum essential medium with and without dmPGE2 (2.6 μM) for 15 min and subsequently incubated in media containing 1, 2.5, 5, 7.5, and 10% EtOH. The effects on cell viability and tubulin (the major protein backbone of microtubules) were then determined. EtOH concentrations ≥2.5% extensively disrupted the microtubules as demonstrated by fragmentation, kinking, and perturbation of the microtubule organizer center. EtOH treatment also led to a significant decrease in the S2 (polymerized) fraction and an increase in the S1 (monomeric) pool of tubulin. Concomitant with these effects were marked decreases in cellular viability. DmPGE2pretreatment abolished the disruption of microtubules, significantly increased the S2 fraction of tubulin, and increased cellular viability in cultures exposed to EtOH. Furthermore, pretreatment with colchicine, an inhibitor of microtubule assembly, prevented the cytoprotective action of dmPGE2. Taxol, a microtubule stabilizing agent, mimicked the effects of dmPGE2 by also enhancing microtubule integrity and increasing cellular viability in cells exposed to EtOH. Our data indicate that organization and stabilization of microtubules may play an essential role in the mechanism of prostaglandin-induced protection.

Inhibition of arachidonic acid metabolism and its implication on cell proliferation and tumour-angiogenesis

“…Sudden dramatic increases in mucosal PGs are positively correlated with disease activity of inflammatory bowel disease [21] and experimental colitis [22], whereas base-line expression of PGs generally exert a protective function against gastrointestinal injury [23] and ulcers [24] as well as acute and chronic enterocolitis [25]. It has been extensively documented that overexpression of COX-2 is implicated in various forms of human cancers such as cancer of the lung [26], breast [27], colorectal [28], prostate [29], head and neck [30] and others [31,32].…”

Key role of PLC-γ in EGF protection of epithelial barrier against iNOS upregulation and F-actin nitration and disassembly

“…At this seeding density, Caco-2 cells form confluent and differentiated monolayers in typically 7-10 days (e.g., see Refs. 14,18,33,36,42,45,58). We were therefore able to measure alterations in intestinal barrier integrity and related outcomes in differentiated and nonleaky monolayers.…”

“…Furthermore, these cells, once differentiated, are similar to native intestinal epithelial cells in that they have receptors for prostaglandins, growth factors, VIP, LDL, insulin, and specific substrates such as dipeptides, fructose, glucose, hexoses, and vitamin B 12. All experiments were performed at least 7 days postconfluence. The utility and characterization of this cell line has been extensively reported (14,45).…”

“…After staining, cells were observed with an argon laser ( ϭ 488 nm) using a ϫ63 oil-immersion plan-apochromat objective, NA 1.4 (Zeiss). The cytoskeletal elements were examined in a blinded fashion for their overall morphology, orientation, and disruption (1,2,10,11,13,14,18). The identity of the treatment groups for all slides was decoded only after examination was complete.…”

The use of Cellomics to study enterocyte cytoskeletal proteins in coeliac disease patients

“…Disruption of cytoskeletal structures can severely limit cell function, and if not reversed can adversely affect cell integrity and viability [13][14][15]. Rearrangement of the cytoskeletal proteins is thought to result in nuclear disarray and alteration of enterocyte shape, described features of the coeliac lesion [7].…”