Role of actin cytoskeleton in prostaglandin-induced protection against ethanol in an intestinal epithelial cell line

Prostaglandins (PGs) protect a variety of gastrointestinal cells against injury induced by ethanol and other noxious agents. This investigation attempted to discern the mechanism of cytoprotection as it relates to the relationship between actin and PGs in IEC-6 cells (a rat intestinal epithelial cell line). IEC-6 cells were incubated in Dulbecco's modified Eagle's medium +/- 16,16-dimethyl prostaglandin E(2) (dmPG, 2.6 microM) for 15 min and subsequently incubated in medium containing 1, 2.5, 5, 7.5, and 10% ethanol (EtOH). Cells were then processed for immunocytochemistry using FITC-phalloidin in order to stain the actin cytoskeleton, and cell viability was determined by trypan blue exclusion. Quantitative Western immunoblotting of fractioned G-actin (nonpolymerized; S1) and F-actin (polymerized; S2) was also carried out. EtOH concentrations equal to and greater than 5% led to the collapse of the actin cytoskeleton as depicted by extensive disorganization and fragmentation. In addition, these same EtOH concentrations significantly decreased the S2 fraction and increased the S1 pool of actin. Preincubation with dmPG prevented collapse of the actin cytoskeleton, significantly increased the S2 polymerized fraction as determined by quantitative immunoblotting, and increased cell viability in EtOH-treated cultures. Prior incubation with cytochalasin D, an actin disruptive agent, not only reduced cell viability but also prevented the cytoprotective effects of dmPG. Phalloidin, an actin stabilizing agent, had effects similar to that of dmPG as demonstrated by stability of the actin cytoskeleton and increased cellular viability. Such findings indicate that PGs are important in the organization and stability of actin under in vitro conditions. These effects on actin may play an essential role in the mechanism of PG-induced cytoprotection.     

Characterization of an immortalized human vaginal epithelial cell line

PURPOSE: Adherence of type 1 piliated Escherichia coli to vaginal mucosa plays a major role in the pathogenesis of ascending urinary tract infections (UTIs) in women. Progress in understanding the mechanism of adherence to the vaginal surface could be enhanced by the utilization of well-characterized vaginal epithelial cells. The objective of this study was to immortalize vaginal epithelial cells and study their bacterial adherence properties. MATERIALS AND METHODS: Primary vaginal cells were obtained from a normal post-menopausal woman, immortalized by infection with E6/E7 genes from human papillomavirus 16 (HPV 16) and cultured in serum free keratinocyte growth factor medium. RESULTS: Positive immunostaining with a pool of antibodies to cytokeratins 1, 5, 10 and 14 (K1, K5, K10 and K14) and to K13 confirmed the epithelial origin of these cells. The immortalized cells showed binding of type 1 piliated E. coli in a pili specific and mannose sensitive manner. CONCLUSION: This model system should facilitate studies on the interaction of pathogens with vaginal mucosal cells, an essential step in the progression of ascending UTIs in women.     

Alphavbeta integrins play an essential role in BMP-2 induction of osteoblast differentiation.

Both integrins and BMP-2 exert similar effects on osteoblasts. We examined the relationship between the alphav-containing integrins (alphavbeta) and BMP-2 in osteoblast function. BMP-2 stimulates alphavbeta expression. BMP-2 receptors co-localize/overlap with alphavbeta integrins, and the intact function of alphavbeta is essential in BMP-2 activity. INTRODUCTION: Bone morphogenetic protein (BMP)-2 not only induces osteoblast differentiation and bone matrix mineralization, but also stimulates osteoblast migration on and adhesion to bone matrix proteins. The alphavbeta- and beta1- (alphabeta1) containing integrins mediate osteoblast interaction with many bone matrix proteins and play important roles in osteoblast adhesion, migration, and differentiation. Because alphavbeta integrins and BMP-2 share common effects on osteoblasts, we analyzed their relationship in osteoblast function. MATERIALS AND METHODS: The effects of BMP-2 on integrin expression were determined by surface labeling/immunoprecipitation and cell adhesion to matrix proteins. Confocal analysis of the immunostained cells and co-immunoprecipitation of cell extracts were used to study the spatial relationship between integrins and BMP-2 receptors. A function-blocking anti-alphavbeta integrin antibody (L230) was employed to investigate the roles of alphavbeta integrins in BMP-2 function. RESULTS: Human osteoblasts (HOBs) express alphabeta1, alphavbeta3, alphavbeta5, alphavbeta6, and alphavbeta8 integrins at focal adhesion sites. BMP-2 increases the levels of these integrins on osteoblast surface and enhances HOB adhesion to osteopontin and vitronectin. Immunoprecipitation and immunostaining analyses show that BMP-2 receptors co-localize or overlap with alphavbeta and alphabeta1 integrins. Incubation of HOBs with L230 abolishes the antiproliferative effect of BMP-2 and reduces the capacity of BMP-2 to stimulate alkaline phosphatase activity and the expression of osteocalcin, osteopontin, and bone sialoprotein. Furthermore, L230 prevents BMP-2 induction of matrix mineralization. Although BMP-2 retains its receptor-binding capability in the presence of L230, BMP-2 stimulation of Smad signaling is abolished by L230. CONCLUSION: BMP-2 upregulates the expression of alphavbeta integrins, and these integrins, in turn, play a critical role in BMP-2 function in osteoblasts.     

Signaling mechanisms of glucagon-like peptide 2-induced intestinal epithelial cell proliferation

BACKGROUND: Glucagon-like peptide 2 (GLP-2) stimulates intestinal epithelial growth with high potency and specificity. However, the intracellular signaling pathways responsible for the growth-stimulatory action of GLP-2 are not clearly understood. Here we report possible signaling pathways mediating GLP-2's proliferative actions in the human intestinal epithelial cell line Caco-2. MATERIALS AND METHODS: Caco-2 cells were subcultured under serum-deprived conditions in the presence or absence of GLP-2 (10 microM) and varying concentrations of inhibitors of three candidate kinases: genistein, a global tyrosine kinase inhibitor; LY294002, a phosphatidylinositide (PI) 3-kinase inhibitor; and PD 098059, a mitogen-activated/extracellular signal-regulated kinase (MEK) inhibitor. Proliferation was assessed using [(3)H]thymidine incorporation. Relative abundance of the phosphorylated forms of two specific mitogen-activated protein kinases (MAPKs), ERK1 and ERK2, was assessed by Western blotting. RESULTS: GLP-2-treated cells demonstrated a greater than 10-fold increase in proliferation. This response was inhibited by genistein, LY294002, and PD 098059 in a dose-dependent fashion. A significantly greater abundance of the phosphorylated forms of both ERK-1 and ERK-2 was present in cells within 5 min of treatment with GLP-2. CONCLUSIONS: GLP-2 stimulates the proliferation of Caco-2 cells in vitro. This increase in Caco-2 proliferation in response to GLP-2 may be due, at least in part, to the involvement of both the PI 3-kinase and the MAPK pathways.     

MC3T3E1细胞分化过程中Cbfα1及相关基因的表达

目的 研究在小鼠前成骨细胞MC3T3E1诱导成骨过程中，总Cbfα1、Cbfα1两种亚型Cbfα1／P56、Cbfα1／P57及碱性磷酸酶、Ⅰ型胶原、骨钙素、骨涎蛋白和骨桥素在前成骨细胞成熟过程中的变化。明确Cbfα1、Cbfα1亚型及相关基因表达与成骨细胞分化的关系，探求与成骨细胞成熟更密切相关的Cbfα1亚型，为进一步的Cbfα1亚型研究提供基础。方法 MC3T3E1细胞在含β-甘油磷酸钠，抗坏血酸的培养基中培养22d，在细胞培养的不同时间（0，4，10，14，18，22d），用α-磷酸奈酚法测定碱性磷酸酶（ALP）活性；VanGieSon苦味酸酸性复红染色法染色细胞Ⅰ型胶原；茜素红染色观察矿化结节形成；半定量RT-PCR检测总Cbfα1mRNA及Cbfα1／P56、Cbfα1／P57mRNA和Ⅰ型胶原、骨钙素、骨涎蛋白、骨桥索mRNA的表达；用Western-blot检测Cbfα1蛋白的表达。结果 MC3T3E1细胞在β-甘油磷酸钠存在的情况下，培养第18d开始出现矿化，第22dⅠ型胶原染色及茜素红染色均观察到明显矿化结节形成。随MC3T3E1细胞的分化，Cbfα1mRNA的表达量逐渐增高，Cbfα1／P57mRNA在第18和22d的表达量明显增高，Cbfα1／P56mRNA无变化。Cbfα1蛋白随MC3T3E1细胞的分化，表达量逐渐增高。同样，随MC3T3E1细胞分化，Ⅰ型胶原、骨钙素、骨桥素和骨涎蛋白mRNA的表达量逐渐增高。ALP活性0～10d逐渐增高，10d后逐渐下降。结论 在MC3T3E1细胞的分化过程中Ⅰ型胶原、骨钙素、骨涎蛋白、骨桥素mRNA随MC3T3E1细胞的分化表达逐渐增高。Cbfα1 mRNA及Cbfα1蛋白的表达随细胞的分化逐渐增高，与其他成骨特异性基因的变化趋势一致，并以Cbfα1／P57mRNA亚型为主。Cbfα1／P57亚型的表达与成骨分化的关系更密切，在进一步的与成骨细胞分化有关的Cbfα1亚型研究应以Cbfα1／P57亚型为主。     

Toll-like receptor 4 and myeloid differentiation factor 88 are required for gastric bypass-induced metabolic effects

BackgroundToll-like receptor 4 (TLR4) has been suggested as one of the forefront cross-communicators between the intestinal bacteria and the host to regulate inflammatory signals and energy homeostasis. High-fat diet–induced inflammation is mediated by changes in gut microbiota and requires a functional TLR-4, the deficiency of which renders mice resistant to diet-induced obesity and its associated metabolic dysfunction. Furthermore, gut microbiota was suggested to play a key role in the beneficial effects of Roux-en-Y gastric bypass (RYGB), a commonly performed bariatric procedure.ObjectivesTo explore whether TLR4, myeloid differentiation factor 8 (MyD88; 1 of its key downstream signaling regulators) and gut microbiota play an integrative role in RYGB-induced metabolic outcomes.SettingAnimal- based study.MethodWe performed RYGB in TLR4 and MyD88 knock-out (KO) mice and used fecal microbiota transplant (FMT) from RYGB-operated animals to these genetic mouse models to address our questions.ResultsWe demonstrate that RYGB reduces TLR4 expression explicitly in the small and large intestine of C57Blc/6J mice. We also show that TLR4 KO mice have an attenuated glucoregulatory response to RYGB. In addition, we reveal that MyD88 KO mice fail to respond to all RYGB-induced metabolic effects. Finally, fecal microbiota transplant from RYGB-operated mice into TLR4 KO and MyD88 KO naïve recipients fails to induce a metabolic phenotype similar to that of the donors, as it does in wild-type recipients.ConclusionTLR4 and MyD88 are required for RYGB-induced metabolic response that is likely mediated by gut microbiome.     

Effects of glutamine isomers on human (Caco-2) intestinal epithelial proliferation, strain-responsiveness, and differentiation

Enteral feeding with small amounts to stimulate bowel motility, and glutamine supplementation, which provides nutrients selectively used by intestinal epithelial cells, might preserve the gut mucosa during fasting. We evaluated the effects of the interaction between mechanical strain and glutamine supplementation in human Caco-2 intestinal epithelial cells, and pursued the finding of equivalent effects of L- and D-glutamine in Caco-2, HT-29, and primary malignant human colonocytes. Caco-2 cells were subjected to repetitive strain in media containing 2 mmol/L of L-glutamine and media supplemented with L- or D-glutamine. Proliferation was determined by automated cell counting. Differentiation and cellular production of L-glutamine were determined spectroscopically. Rhythmic deformation stimulated Caco-2 proliferation in a frequency-dependent manner. Maximal stimulation occurred at 10 cpm, consistent with in vivo frequencies of peristalsis and villous motility. Deformation at 10 cpm and L-glutamine supplementation from 2 to 5 mmol/L concentrations independently stimulated Caco-2 proliferation; the combination further increased proliferation. D-Glutamine supplementation yielded similar results, although with lesser potency. Furthermore, both L- and D-glutamine equivalently reduced Caco-2 dipeptidyl dipeptidase activity. The effects of each isoform were blocked by 1 to 3 mmol/L acivicin, a selective antagonist of glutamine metabolism. Indeed Caco-2 and HT-29 cells and primary malignant colonocytes each metabolized D-glutamine to L-glutamine. Glutamine supplementation in fasting patients might prove synergistic with stimulation of bowel motility by non-nutritive feeding, whereas tissue-specific variations in D-glutamine metabolism might facilitate selective nutripharmaceutical targeting of the gut mucosa. Supported in part by a VA Merit Award (Dr. Basson).     

Hyperaldosteronemia and activation of the epithelial sodium channel are not required for sodium retention in puromycin-induced nephrosis

Edema and ascites in nephrotic syndrome mainly result from increased Na+ reabsorption along connecting tubules and cortical collecting ducts (CCD). In puromycin aminonucleoside (PAN)-induced nephrosis, increased Na+ reabsorption is associated with increased activity of the epithelial sodium channel (ENaC) and Na+,K+-ATPase, two targets of aldosterone. Because plasma aldosterone increases in PAN-nephrotic rats, the aldosterone dependence of ENaC activation in PAN nephrosis was investigated. For this purpose, (1) the mechanism of ENaC activation was compared in nephrotic and sodium-depleted rats, and (2) ENaC activity in PAN-nephrotic rats was evaluated in the absence of hyperaldosteronemia. The mechanism of ENaC activation was similar in CCD from nephrotic and sodium-depleted rats, as demonstrated by (1) increased number of active ENaC evaluated by patch clamp, (2) recruitment of ENaC to the apical membrane determined by immunohistochemistry, (3) shift in the electrophoretic profile of gamma-ENaC, and (4) increased abundance of beta-ENaC mRNA. Corticosteroid clamp fully prevented all PAN-induced changes in ENaC but did not alter the development of a full-blown nephrotic syndrome with massive albuminuria, amiloride-sensitive sodium retention, induction of CCD Na+,K+-ATPase, and ascites. It is concluded that in PAN-nephrosis, (1) ENaC activation in CCD is secondary to hyperaldosteronemia, (2) sodium retention and induction of Na+,K+-ATPase in CCD are independent of hyperaldosteronemia, and (3) ENaC is not necessarily limiting for sodium reabsorption in the distal nephron.     

Mechanism of 2-chloroadenosine toxicity to PC3 cell line

BACKGROUND: 2-CADO inhibits the growth of several types of cells and causes apoptosis by a mechanism which involves adenosine receptors or cellular uptake or both. METHODS: Androgen-independent (PC3) prostate cancer cells were used in the study and proliferation, cell-cycle progression, and apoptosis analyzed. Deoxy-and ribonucleoside triphosphate pools were determined by HPLC. The molecular mechanism was examined by assessing the involvement of DNA synthesizing enzymes in the cellular response. RESULTS: 2-CADO treatment dramatically reduced the number of prostate cancer cells and permanently stopped cell-cycle progression in the S-phase. The role of 2-CADO in prostate cancer cells is uptake-mediated and followed by sequential phosphorylations to 2-Cl-ATP that irreversibly inhibits several key-enzymes for DNA biosynthesis. CONCLUSIONS: Arrest of DNA synthesis responsible for toxicity of 2-CADO to PC3 cells is due to the lack of substrates for DNA polymerization caused by irreversible inhibition of purine/pyrimidine ribo-and 2-deoxyribonucleotides salvage enzymes.