Prostaglandin E2-dependent enhancement of tissue inhibitors of metalloproteinases-1 production limits dendritic cell migration through extracellular matrix.

Dendritic cell (DC) migration is crucial for the initiation of immune responses. The balance between metalloproteinases (MMP) and tissue inhibitors of metalloproteinases (TIMP) has been shown to modulate DC migration. PGE2, which is overproduced in a wide variety of human malignancies, has been implicated in MMP and TIMP regulation in various cells, including monocytes. In the present study, we hypothesized that tumor-derived PGE2 would affect DC migratory capacity through the extracellular matrix (ECM) by altering MMP and TIMP balance. Treatment of monocyte-derived immature DC with exogenous PGE2 induced TIMP-1 secretion but not MMP-9 production and was correlated with reduced DC migration through ECM. Because recombinant TIMP-1 replicated PGE2 inhibition of DC migration while anti-TIMP-1 neutralizing Ab reversed it, we conclude that PGE2-mediated induction of TIMP-1 was responsible for the reduced migration of PGE2-treated DC. Similarly, DC cultured for 48 h in supernatants from cyclooxygenase-2 overexpressing lung cancer cells that secrete high levels of PGE2, exhibited decreased migration through ECM. Finally, analysis of E prostanoid receptor expression and their selective inhibition revealed that the enhanced TIMP-1 secretion in PGE2-treated DC was mediated predominantly by the E prostanoid receptor 2. These findings indicate that PGE2-dependent enhancement of TIMP-1 production causes reduced migration of DC through ECM.

11-Deoxy,16,16-dimethyl prostaglandin E2 induces specific proteins in association with its ability to protect against oxidative stress.

Prostaglandins (PGs) act locally to maintain cellular homeostasis and stimulate stress response signaling pathways. These cellular effects are diverse and are tissue-dependent. PGE(2), and the synthetic analogue, 11-deoxy,16,16-dimethyl PGE(2) (DDM-PGE(2)), protect renal proximal tubular epithelial (LLC-PK1) cells against cellular injury induced by the potent nephrotoxic and nephrocarcinogenic metabolite of hydroquinone, 2,3,5-tris-(glutathion-S-yl)hydroquinone. Although this cytoprotective response (in LLC-PK1 cells) is mediated through a thromboxane or thromboxane-like receptor coupled to AP-1 signaling pathways, the mechanism of cytoprotection is unknown. In this study, we utilized HPLC-electrospray ionization tandem mass spectrometric (ESI MS/MS) and matrix-assisted laser desorption ionization time-of-flight mass spectrometric (MALDI TOF) analysis of proteins isolated from DDM-PGE(2)-stimulated LLC-PK1 cells to identify candidate cytoprotective proteins. DDM-PGE(2) selectively stimulated the synthesis of several proteins in LLC-PK1 cells. Peptide sequencing by ESI-MS/MS of in-gel tryptic protein digests revealed the identity of eight proteins: endothelial actin binding protein, myosin, elongation factor 2 (EF-2), elongation factor 1alpha-1 (EF-1alpha), heat shock protein 90beta (HSP90beta), glucose-regulated protein 78 (GRP 78), membrane-organizing extension spike protein, and actin. Both ESI-MS/MS and MALDI-MS analysis resulted in the same protein identification. Western analysis confirmed the temporal induction of the majority of these proteins, including EF-2, EF-1alpha, HSP90beta, GRP78, and actin. The collective expression of these proteins suggests that DDM-PGE(2)-mediated cytoprotection may involve alterations in cytoskeletal organization and/or stimulation of an endoplasmic reticulum (ER) stress response. The present studies provide insights into potential downstream targets of PG signaling.

Modulation of 3H-prostaglandin E2 binding sites in the rabbit gastric mucosa.

The binding of 3H-prostaglandin E2 (PGE2) to rabbit gastric mucosa was investigated. Binding depended on incubation time, temperature and pH, and was saturable and reversible. Scatchard plot analysis revealed a single class of binding sites with a dissociation constant (Kd) of 5.33 +/- 0.21 nM and a maximum number of binding sites (Bmax) of 138.1 +/- 3.4 fmol/mg protein. PGE1 and 16,16-dimethyl PGE2 potently competed with 3H-PGE2 for the binding sites of gastric mucosa, whereas PGA2, PGF2 alpha, 6-keto PGF1 alpha and thromboxane B2 were less potent. The gastric mucosa prepared from the rabbits given indomethacin (5 mg/kg s.c. three times) showed a lower Kd (2.47 +/- 0.19 nM) for 3H-PGE2 than that from untreated one. Treatment with a PGE1 analog, misoprostol (320 micrograms/kg s.c. three times) lowered the Bmax to 74.1 +/- 2.4 fmol/mg protein without any significant effect on the Kd value. It is concluded that rabbit gastric mucosa has specific binding sites for 3H-PGE2 which may be modulated by the levels of PGs in vivo.

Cytoprotective effect of 16,16' dimethyl prostaglandin E2 (dm PGE2) on streptozotocin-induced biochemical alterations of Golgi-rich membrane fraction in comparison with morphology of rat liver Golgi apparatus in situ.

In 10-11 days after single, intraperitoneal injection of streptozotocin we found a lower (although not statistically significant) yield of Golgi-rich membrane fraction in comparison with control. In these rats similar to control specific activity of galactosyltransferase in nM Gal transferred per h and mg of protein, and statistically significant lower total activity of this enzyme expressed in nM Gal transferred per h and per total liver (t = 2.9666, 0.01 less than p less than 0.05) were found. Organization of Golgi apparatus in situ showed alterations that indicated suppression of secretory activity. Within the dm PGE2 protected animal group the obtained data equal control data.

Metabolism of prostaglandin E analogs in guinea pig and rat liver microsomes.

Tritium-labelled 16,16-dimethyl-PGE2, 9-methylene-PGE2 (9-deoxo-16,16-dimethyl-9-methylene-prostaglandin E2) and tetranor-9-methylene-PGE2 were incubated with guinea pig liver microsomes. All three compounds were converted to omega-oxidized products in yields of a few per cent. In addition, from incubations with 9-methylene-PGE2 and tetranor-9-methylene-PGE2 were also obtained metabolites with the methylene group transformed into a dihydrodiol. In a comparative study with rat liver microsomes, it was found that these converted tetranor-9-methylene-PGE2 in a 50 per cent yield to omega-oxidized products. Finally, 20.000 X G supernatants from guinea pig and rat liver were compared with respect to omega-oxidation. The rat liver 20.000 X G supernatant was found to convert the substrate to the same extent as washed microsomes. By contrast, the guinea pig liver 20.000 X G supernatant was considerably more efficient than washed microsomes.

Metabolism of prostaglandin E analogs in the guinea pig liver mitochondrial fraction.

Tritium-labeled 16,16-dimethyl-PGE2 and 9-methylene-PGE2 were incubated with the guinea pig liver mitochondrial fraction. The tetranor and dinor metabolites were obtained, a large portion of the latter contained a saturated carboxyl side chain. Also the beta-hydroxydinor metabolites, analogous to the beta oxidation of fatty acids, were identified. Such metabolites have not been reported earlier in connection with beta oxidation of prostaglandins or prostaglandin analogs. Furthermore, omega-hydroxylated analogs were incubated with the guinea pig liver mitochondrial fraction and were found to pass through beta oxidation to a certain extent.

Metabolism of 9-deoxo-16,16-dimethyl-9-methylene prostaglandin E2 in humans.

Tritium-labeled 9-deoxo-16,16-dimethyl-9-methylene prostaglandin E2 was administered by intravenous injection into humans. The initial half-life in plasma was determined to be about 3 min. About 35% of the injected radioactivity was excreted in the urine and about 55% in the feces. Twenty-three urinary metabolites were identified. The metabolites included beta-oxidized and omega-oxidized products as well as metabolites in which the 9-methylene group had been biotransformed.