Nuclear peroxisome proliferator-activated receptors alpha and gamma have opposing effects on monocyte chemotaxis in endometriosis.

The peroxisome proliferator-activated receptors (PPARs) alpha and gamma are nuclear receptors that play important roles in inflammatory diseases like ulcerative colitis and arthritis. In this study, we examined the possible role of PPARs in macrophage attraction into the peritoneal cavity of patients with endometriosis. We identified PPAR-alpha and -gamma messenger RNA by RT-PCR and protein by immunoblotting of lysates of peritoneal macrophages and monocytic U937 cells. Using immunocytochemistry, we localized PPAR-alpha and -gamma within the nuclei of both cell types. Monocyte chemotactic activity of peritoneal fluid from patients with endometriosis was quantified in Boyden chambers. Migration of U937 cells was increased by WY 14643 and reduced by rosiglitazone. Peritoneal fluid from patients with endometriosis activated U937 cells transiently transfected with a PPAR-alpha/GAL4 luciferase reporter. By contrast, peritoneal fluid did not cause significant activation of PPAR-gamma/GAL4 constructs. The U937 cells transiently transfected with a PPAR response element luciferase reporter showed disease stage-dependent up-regulation when treated with peritoneal fluid from patients with endometriosis. Treatment with peritoneal fluid from healthy controls down-regulated PPAR response element transactivation. We conclude that peritoneal fluid of endometriosis patients contains activators of PPAR-alpha that stimulate macrophage chemotaxis. Inhibitors of PPAR-alpha or activators of PPAR-gamma could be developed for the treatment of inflammation associated with endometriosis.

Albumin regulates induction of peroxisome proliferator-activated receptor-gamma (PPARgamma) by 15-deoxy-delta(12-14)-prostaglandin J(2) in vitro and may be an important regulator of PPARgamma function in vivo.

We observed that serum contains a factor(s) that inhibits the induction of peroxisome proliferator-activated receptor-gamma (PPARgamma) by 15-deoxy-Delta(12,14)-PGJ(2) (15dJ(2)). Ten percent FBS reduces 15dJ(2) induction of PPARgamma from over 150-fold to less than 15-fold in EP-JEG cells, a stably transfected choriocarcinoma cell line that expresses endogenous PPARgamma. By contrast, rosiglitazone, an unrelated pharmacological agonist of PPARgamma, is not inhibited by serum in this cell line. We have identified the inhibitory principal in serum as albumin. Serum albumin binds 15dJ(2) with a dissociation constant of 870 +/- 70 nM, effectively reducing the concentration of 15dJ(2) available to PPARgamma. Heat treatment of serum abolishes the inhibition, providing a way to test eicosanoid compounds independently of albumin's inhibitory effect. It is reasonable to assume that 15dJ(2) or structurally similar compounds or metabolites are the endogenous activators of PPARgamma. Therefore, albumin may be an important regulator of PPARgamma function in vivo.

Placental peroxisome proliferator-activated receptor-gamma is up-regulated by pregnancy serum.

Lipid metabolism plays an important role in normal pregnancy adaptation and in pathological pregnancy (e.g. preeclampsia). In the current studies we examined the expression of peroxisome proliferator-activated receptor-gamma (PPARgamma) in tissues and cells relevant to human pregnancy. We found that PPARgamma is expressed in placental cytotrophoblasts in vivo and in trophoblasts (primary and choriocarcinoma cells) and fetal endothelial cells in vitro. We characterized primary cytotrophoblasts and two cell lines with which to study PPARgamma regulation in human pregnancy. Like primary cytotrophoblasts, the choriocarcinoma cell line JEG-3 has endogenous PPARgamma expression. Normal positive and negative PPARgamma regulation was observed in the latter cells. We also created a new JEG-3-derived cell line (EP-JEG) by stable insertion of a PPAR response element-luciferase reporter gene construct. Together, these cell lines are useful for studying PPARgamma expression and activation in human trophoblasts. We examined PPARgamma regulation in these cells by human serum and found that PPARgamma protein expression and activation are dramatically increased by sera from pregnant women. Preliminary characterization of the regulatory principle(s) is consistent with a prostanoid or fatty acid derivative. The results suggest that increased activation of PPARgamma may play an important role in maternal metabolism during human pregnancy.

Pili binding to asialo-GM1 on epithelial cells can mediate cytotoxicity or bacterial internalization by Pseudomonas aeruginosa.

The interaction of Pseudomonas aeruginosa type IV pili and the glycosphingolipid asialo-GM1 (aGM1) can mediate bacterial adherence to epithelial cells, but the steps subsequent to this adherence have not been elucidated. To investigate the result of the interaction of pili and aGM1, we used polarized epithelial monolayers of Madin-Darby canine kidney (MDCK) cells in culture, which contained little detectable aGM1 on their apical surface but were able to incorporate exogenous aGM1. Compared to an untreated monolayer, P. aeruginosa PA103 displayed an eightfold increase in association with and fivefold more cytotoxicity toward MDCK cells pretreated with aGM1. Cytotoxicity of either carrier-treated or aGM1-treated monolayers required the type III secreted protein ExoU. Asialo-GM1 pretreatment of MDCK monolayers likewise augmented bacterial internalization of an isogenic invasive strain approximately fourfold. These increases were not seen in monolayers treated with GM1, the sialyated form of the glycolipid, and were inhibited by treatment with an antibody to aGM1. Also, the aGM1-mediated adhesion, cytotoxicity, and internalization required intact type IV pili since nonpiliated PA103 mutants were unaffected by aGM1 pretreatment of MDCK cells. These results demonstrate that epithelial cell injury and bacterial internalization can proceed from the same adhesin-receptor interaction, and they indicate that P. aeruginosa exoproducts solely determine the steps subsequent to adhesion.

A protein dissociation step limits turnover in FLP recombinase-mediated site-specific recombination.

When two ongoing FLP-mediated recombination reactions are mixed, formation of cross-products is subject to a lag of several minutes, and the subsequent rate of cross-product formation is greatly reduced relative to normal reaction progress curves. The lag reflects the formation of a stable complex containing multiple FLP monomers and two FLP recombination target-containing DNA recombination products, a process completed within 5-10 min after addition of FLP recombinase to a reaction mixture. The reaction products are sequestered within this complex for an extended period of time, unavailable for further reaction. The length of the lag increases with increasing FLP protein concentration and is not affected by the introduction of unreacted (non FLP-bound) substrate. The results provide evidence that disassembly of FLP complexes from products occurs in a minimum of two steps. At least one FLP protein monomer is released from reaction complexes in a discrete step that leaves the reaction products sequestered. The recombination products are released in a form free to react with other FLP recombination target-containing DNA molecules only after at least one additional dissassembly step. One or both of these disassembly steps are rate limiting for reaction turnover under conditions often used to monitor FLP-mediated recombination in vitro.

Actin filament alterations in rat hepatocytes induced in vivo and in vitro by microcystin-LR, a hepatotoxin from the blue-green alga, Microcystis aeruginosa.

The morphologic effects of microcystin-LR (MCLR) were examined in vitro and in vivo to identify the specific cell type(s) affected and to characterize the actin filament changes occurring in hepatocytes. Male Sprague Dawley rats were used for all studies. For in vitro studies, hepatic cells were isolated by collagenase perfusion of liver, while parenchymal cells (hepatocytes) and nonparenchymal cells were prepared by pronase digestion and metrimazide gradient centrifugation. Cell suspensions and and primary hepatocyte monolayer cultures were treated with MCLR at doses up to 10 micrograms/ml; cultured hepatocytes were also treated with phalloidin or cytochalasin B at a dose of 10 micrograms/ml; and rats were treated intraperitoneally with MCLR at 180 mg/kg. Cultured hepatocyte preparations and frozen liver sections were stained with rhodamine-labeled phalloidin for filamentous actin. In cell suspensions, MCLR did not affect nonparenchymal cells but caused rapid, progressive, blebbing of the plasma membrane in hepatocytes. In cultured hepatocytes, MCLR caused plasma membrane blebbing as well as marked reorganization of actin microfilaments. These alterations were dose and time dependent. Cultured hepatocytes treated with phalloidin or cytochalasin B also showed extensive plasma membrane blebbing and actin filament alterations; however, actin filament changes were morphologically distinct from those induced by MCLR. In vivo, MCLR-induced hepatocyte actin alterations occurred at the same time as, or slightly preceded, histologic changes that began 30 minutes after dosing. These studies suggest that early MCLR-induced morphologic changes occurring both in vivo and in vitro are due to alterations in hepatocyte actin filaments.