15-keto Prostaglandin E2 Research Articles

Inhibition of 15-prostaglandin dehydrogenase attenuates acetaminophen-induced liver injury via suppression of apoptosis in liver endothelial cells

Hepatic microvascular disruption caused by injury to liver sinusoidal endothelial cells (LSECs) is an aggravating factor for drug-induced liver injury (DILI). It is suggested that prostaglandin E2 (PGE2) may be able to attenuate LSEC injury. However, it is also known that 15-keto PGE2, a metabolite of PGE2 produced by 15-prostaglandin dehydrogenase (15-PGDH) that is not a ligand of PGE2 receptors, suppresses inflammatory acute liver injury as a ligand of peroxisome proliferator-activated receptor γ. In this study, we aimed to understand whether 15-PGDH activity is essential for preventing DILI by suppressing hepatic microvascular disruption in a mouse model of acetaminophen (APAP)-induced liver injury. To inhibit 15-PGDH activity prior to APAP-induced LSEC injury, we administered the 15-PGDH inhibitor, SW033291, 1 h before and 3 h after APAP treatment. We observed that LSEC injury preceded hepatocellular injury in APAP administered mice. Hepatic endogenous PGE2 levels did not increase up till the initiation of LSEC injury but rather increased after hepatocellular injury. Moreover, hepatic 15-PGDH activity was downregulated in APAP-induced liver injury. The inhibition of 15-PGDH attenuated LSEC injury and subsequently hepatic injury by inhibiting apoptosis in APAP administered mice. Our in vitro studies also suggested that PGE2 inhibited APAP-induced apoptosis via the EP4/PI3K pathway in endothelial cells. Therefore, a decrease in 15-PGDH activity would be beneficial for preventing APAP-induced liver injury by attenuating LSEC injury.

Inhibition of 15-hydroxyprostaglandin dehydrogenase protects a mouse model of Alzheimer's disease without affecting amyloid pathology.

The 15-hydroxyprostaglandin dehydrogenase (15-PGDH) enzyme inactivates the lipid signaling molecule prostaglandin E2 (PGE2) by oxidizing it to 15-keto PGE2. We have previously shown that genetic elimination or pharmacologic inhibition of 15-PGDH (via the small molecule (+)-SW033291) protects peripheral organs by potentiating PGE2-mediated stem cell responses (1). Here, we investigate whether 15-PGDH expression in the brain is related to pathology in the 5xFAD mouse model of Alzheimer's disease, and if so then whether this might provide a novel route for therapy. Two month old 5xFAD mice and WT littermates received intraperitoneal injections of vehicle or (+)-SW033291 (5mg/kg twice daily) for 4 months. At 6 months of age, Morris water maze test was conducted to evaluate cognitive function, and then mice were sacrificed for immunohistochemical determination of neuronal cell survival in the hippocampus and amyloid plaque deposition. Blood-brain barrier structure was also examined by electron microscopy. Six-month-old 5xFAD mice display higher 15-PGDH activity in the brain than WT littermates. Treatment with (+)-SW033291 reduced 15-PGDH activity and protected 5xFAD mice from cognitive deficits in the Morris water maze test. This treatment also increased survival of adult-born young hippocampal neurons, as measured through bromodeoxyuridine labeling. Lastly, (+)-SW033291 treatment protected 5xFAD mice from developing blood-brain barrier disruption, as measured by the number of empty spaces around the vessel and astrocytic end-feet disruption. Notably, (+)-SW033291 treatment did not alter amyloid plaque formation in 5xFAD mice. An important element for discovering and developing disease-modifying therapies for Alzheimer's disease is diversification of targets, complementing and moving beyond the amyloid hypothesis. Pharmacologic inhibition of 15-PGDH in the brain, such as we have achieved here through treating mice with (+)-SW033291, may be an effective amyloid-independent therapeutic approach for patients.

Gestational long-term hypoxia induces metabolomic reprogramming and phenotypic transformations in fetal sheep pulmonary arteries.

Gestational long-term hypoxia increases the risk of myriad diseases in infants including persistent pulmonary hypertension. Similar to humans, fetal lamb lung development is susceptible to long-term intrauterine hypoxia, with structural and functional changes associated with the development of pulmonary hypertension including pulmonary arterial medial wall thickening and dysregulation of arterial reactivity, which culminates in decreased right ventricular output. To further explore the mechanisms associated with hypoxia-induced aberrations in the fetal sheep lung, we examined the premise that metabolomic changes and functional phenotypic transformations occur due to intrauterine, long-term hypoxia. To address this, we performed electron microscopy, Western immunoblotting, calcium imaging, and metabolomic analyses on pulmonary arteries isolated from near-term fetal lambs that had been exposed to low- or high-altitude (3,801 m) hypoxia for the latter 110+ days of gestation. Our results demonstrate that the sarcoplasmic reticulum was swollen with high luminal width and distances to the plasma membrane in the hypoxic group. Hypoxic animals were presented with higher endoplasmic reticulum stress and suppressed calcium storage. Metabolically, hypoxia was associated with lower levels of multiple omega-3 polyunsaturated fatty acids and derived lipid mediators (e.g., eicosapentaenoic acid, docosahexaenoic acid, α-linolenic acid, 5-hydroxyeicosapentaenoic acid (5-HEPE), 12-HEPE, 15-HEPE, prostaglandin E3, and 19(20)-epoxy docosapentaenoic acid) and higher levels of some omega-6 metabolites (P < 0.02) including 15-keto prostaglandin E2 and linoleoylglycerol. Collectively, the results reveal broad evidence for long-term hypoxia-induced metabolic reprogramming and phenotypic transformations in the pulmonary arteries of fetal sheep, conditions that likely contribute to the development of persistent pulmonary hypertension.

Curcumin induces expression of 15-hydroxyprostaglandin dehydrogenase in gastric mucosal cells and mouse stomach in vivo: AP-1 as a potential target

15-Hydroxyprostaglandin dehydrogenase (15-PGDH) catalyzes the conversion of oncogenic prostaglandin E2 to non-tumerigenic 15-keto prostaglandin E2. In the present study, we found that curcumin, a yellow coloring agent present in the rhizome of Curcuma longa Linn (Zingiberaceae), induced expression of 15-PGDH at the both transcriptional and translational levels in normal rat gastric mucosal cells. By using deletion constructs of 15-PGDH promoter, we were able to demonstrate that activator protein-1 (AP-1) is the principal transcription factor responsible for regulating curcumin-induced 15-PGDH expression. Curcumin enhanced the expression of c-Jun and c-Fos that are functional subunits of AP-1, in the nuclear fraction of cells. Silencing of c-Jun suppressed curcumin-induced expression of 15-PGDH. Moreover, the chromatin immunoprecipitation assay revealed curcumin-induced binding of c-Jun to the AP-1 consensus sequence present in the 15-PGDH promoter. Curcumin increased phosphorylation of ERK1/2 and JNK, and pharmacologic inhibition of these kinases abrogated the curcumin-induced phosphorylation of c-Jun and 15-PGDH expression. In contrast, tetrahydrocurcumin which lacks the α,β-unsaturated carbonyl group failed to induce 15-PGDH expression, suggesting that the electrophilic carbonyl group of curcumin is essential for its induction of 15-PGDH expression. Curcumin restored the expression of 15-PGDH which is down-regulated by Helicobacter pylori through suppression of DNA methyltransferase 1. In addition, oral administration of curcumin increased the expression of 15-PGDH and its regulators such as p-ERK1/2, p-JNK, and c-Jun in the mouse stomach. Taken together, these findings suggest that curcumin-induced upregulation of 15-PGDH may contribute to chemopreventive effects of this phytochemical on inflammation-associated gastric carcinogenesis.

15-Keto prostaglandin E2 induces heme oxygenase-1 expression through activation of Nrf2 in human colon epithelial CCD 841 CoN cells

Prostaglandin E2 (PGE2) plays a key role in inflammation-associated carcinogenesis. NAD+-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH) catalyzes the oxidation of the 15(S)-hydroxyl group of PGE2 to generate 15-keto PGE2. 15-PGDH has been known as a tumor suppressor in various malignancies including colon cancer. However, the molecular mechanisms underlying the tumor-suppressive function of 15-PGDH remain largely unresolved. In this study, we found that 15-keto PGE2 upregulated the expression of heme oxygenase-1 (HO-1), a representative antioxidative and anti-inflammatory enzyme, at both transcriptional and translational levels, in human colon epithelial CCD 841 CoN cells. A redox-sensitive transcription factor, NF-E2-related factor (Nrf2) plays a critical role in the regulation of HO-1 and other cytoprotective proteins. 15-Keto PGE2 induced translocation of Nrf2 into the nucleus and antioxidant response element-driven luciferase activity. Furthermore, the silencing of the Nrf2 gene abolished 15-keto PGE2-induced HO-1 expression in CCD 841 CoN cells. 15-Keto PGE2 activated AKT signaling, and the pharmacological AKT inhibitor, LY294002 suppressed the 15-keto PGE2-induced HO-1 expression. 15-Keto PGE2 generates the reactive oxygen species which is suppressed by the general antioxidant N-acetyl-l-cysteine. N-acetyl-l-cysteine treatment attenuated the 15-keto PGE2-induced phosphorylation of GSK3β, transcriptional activity of Nrf2, and subsequently HO-1 expression. However, 13,14-dihydro-15-keto PGE2 lacking the α,β-unsaturated carbonyl moiety failed to induce intracellular production of reactive oxygen species, HO-1 expression and nuclear translocation of Nrf2. In conclusion, 15-keto PGE2 induces HO-1 expression through Nrf2 activation in human colon epithelial cells.

Enzymatic Activity of HPGD in Treg Cells Suppresses Tconv Cells to Maintain Adipose Tissue Homeostasis and Prevent Metabolic Dysfunction

Regulatory Tcells (Treg cells) are important for preventing autoimmunity and maintaining tissue homeostasis, but whether Treg cells can adopt tissue- or immune-context-specific suppressive mechanisms is unclear. Here, we found that the enzyme hydroxyprostaglandin dehydrogenase (HPGD), which catabolizes prostaglandin E2 (PGE2) into the metabolite 15-keto PGE2, was highly expressed in Treg cells, particularly those in visceral adipose tissue (VAT). Nuclear receptor peroxisome proliferator-activated receptor-γ (PPARγ)-induced HPGD expression in VAT Treg cells, and consequential Treg-cell-mediated generation of 15-keto PGE2 suppressed conventional Tcell activation and proliferation. Conditional deletion of Hpgd in mouse Treg cells resulted in the accumulation of functionally impaired Treg cells specifically in VAT, causing local inflammation and systemic insulin resistance. Consistent with this mechanism, humans with type 2 diabetes showed decreased HPGD expression in Treg cells. These data indicate that HPGD-mediated suppression is a tissue- and context-dependent suppressive mechanism used by Treg cells to maintain adipose tissue homeostasis.

15-Keto prostaglandin E2 suppresses STAT3 signaling and inhibits breast cancer cell growth and progression

Overproduction of prostaglandin E2 (PGE2) has been linked to enhanced tumor cell proliferation, invasiveness and metastasis as well as resistance to apoptosis. 15-Keto prostaglandin E2 (15-keto PGE2), a product formed from 15-hydroxyprostaglandin dehydrogenase-catalyzed oxidation of PGE2, has recently been shown to have anti-inflammatory and anticarcinogenic activities. In this study, we observed that 15-keto PGE2 suppressed the phosphorylation, dimerization and nuclear translocation of signal transducer and activator of transcription 3 (STAT3) in human mammary epithelial cells transfected with H-ras (MCF10A-ras). 15-Keto PGE2 inhibited the migration and clonogenicity of MCF10A-ras cells. In addition, subcutaneous injection of 15-keto PGE2 attenuated xenograft tumor growth and phosphorylation of STAT3 induced by breast cancer MDA-MB-231 cells. However, a non-electrophilic analogue, 13,14-dihydro-15-keto PGE2 failed to inhibit STAT3 signaling and was unable to suppress the growth and transformation of MCF10A-ras cells. These findings suggest that the α,β-unsaturated carbonyl moiety of 15-keto PGE2 is essential for its suppression of STAT3 signaling. We observed that the thiol reducing agent, dithiothreitol abrogated 15-keto PGE2-induced STAT3 inactivation and disrupted the direct interaction between 15-keto PGE2 and STAT3. Furthermore, a molecular docking analysis suggested that Cys251 and Cys259 residues of STAT3 could be preferential binding sites for this lipid mediator. Mass spectral analysis revealed the covalent modification of recombinant STAT3 by 15-keto PGE2 at Cys259. Taken together, thiol modification of STAT3 by 15-keto PGE2 inactivates STAT3 which may account for its suppression of breast cancer cell proliferation and progression.

Abstract 2231: 15-Keto prostaglandin E2 inhibits STAT3 signaling in H-Ras transformed mammary epithelial cells

Abstract Prostaglandin E2 (PGE2) is a potent lipid mediator that plays a key role in inflammation and carcinogenesis. The intracellular level of PGE2 is regulated by NAD+-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH). This enzyme catalyzes the oxidation of the 15(S)-hydroxyl group of PGE2 to generate 15-keto PGE2. Down-regulation of 15-PGDH was observed in various malignancies and overexpression of 15-PGDH has been known to suppress the carcinogenic processes. However, mechanisms underlying anti-carcinogenic effects and tumor suppressive functions of 15-keto PGE2 are poorly understood in various cancer. H-Ras transformed mammary epithelial MCF10A-ras cells show enhanced expression of cyclooxygenase-2 and activation of signal transducer and activator of transcription 3 (STAT3) and down-regulation of 15-PGDH compared to normal mammary epithelial MCF10A cells. STAT 3 plays a key role in growth of cancer cells as well as inflammation-associated carcinogenesis. This prompted us to investigate the effect of 15-keto PGE2 on STAT3 activation in MCF10A-ras cells. 15-Keto PGE2 suppressed the phosphorylation of STAT3 in MCF10A-ras cells in concentration- and time-dependent manners as determined by Western blot and immunocytochemical analyses. 15-Keto PGE2 also inhibited the dimerization of STAT3 which led to suppression of nuclear translocation and transcriptional activity of STAT3 as determined by Western blot analysis and the luciferase assay, respectively. 15-keto PGE2 suppressed the expression of cyclin D1. In addition, 15-keto PGE2 inhibited the growth of MCF10A-ras cells as determined by anchorage-independent growth and colony forming assays. However, a non-electrophilic analogue 13,14-dihydro-15-keto PGE2 which lacks the α,β-unsaturated carbonyl moiety failed to inhibit the phosphorylation, dimerization, and nuclear translocation of STAT3. Moreover, 13,14-dihydro-15-keto PGE2 did not affect the growth of MCF10A ras cells. These findings suggest that the α,β-unsaturated carbonyl moiety of 15-keto PGE2 is essential for its suppression of STAT3 signaling. STAT3 has several cysteine residues in its activation sites. We observed that reducing agents N-acetyl-L-cysteine and dithiothreitol abrogated the suppressive effect of 15-keto PGE2 on STAT3 phosphorylation, suggesting that the involvement of the thiol modification. Biotinylated 15-keto PGE2 covalently bound to STAT3 as determined by immunoprecipitation with STAT3 followed by immunoblotted with streptavidin. Molecular docking analysis predicted Cys251 and Cys259 residues of STAT3 as putative binding sites of 15-keto PGE2. In conclusion, 15-keto PGE2 inhibits STAT3 signaling through cysteine thiol modification, thereby suppressing MCF10A-ras cell growth and proliferation. Thus, tumor suppressor function of 15-PGDH is attributable, at least in part, to inactivation of oncogenic STAT3 signaling by its catalytic product 15-keto PGE2. Citation Format: Eun-Ji Lee, Young-Il Hahn, Su-Jung Kim, Young-Joon Surh, Hye-Kyung Na. 15-Keto prostaglandin E2 inhibits STAT3 signaling in H-Ras transformed mammary epithelial cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2231. doi:10.1158/1538-7445.AM2017-2231

Prostaglandin E2 Regulates Its Own Inactivating Enzyme, 15-PGDH, by EP2 Receptor-Mediated Cervical Cell-Specific Mechanisms

Prostaglandins play important roles in parturition and have been used to induce cervical ripening and labor. Prior to cervical ripening at term, 15-hydroxyprostaglandin dehydrogenase (15-PGDH) is highly expressed in the cervix and metabolizes cyclooxygenase-2-mediated increases in active prostaglandin E2 (PGE2) to inactive 15-keto PGE2. At term, 15-PGDH gene expression decreases and PGE2 accumulates, leading to cervical ripening and labor. Previously, we found that the cervical isoform of microphthalmia-associated transcription factor (MiTF-CX) serves as a progestational transcription factor that represses IL-8 and hypoxia-mediated increases in cyclooxygenase-2. We tested the hypothesis that PGE2 regulates its own inactivation through MiTF-CX. We used human cervical stromal cells to investigate the regulation of 15-PGDH. This was a laboratory-based study using cells from clinical tissue samples. We evaluated the mechanisms by which PGE2 regulates 15-PGDH in human cervical stromal cells. PGE2 repressed MiTF-CX and 15-PGDH, whereas ectopic overexpression of MiTF-CX induced 15-PGDH expression levels. Stabilization of HIF-1α by deferoxamine resulted in concomitant down-regulation of MiTF-CX and 15-PGDH. Ectopic overexpression of MiTF-CX abrogated PGE2- and deferoxamine-mediated loss of MiTF-CX and 15-PGDH. PGE2-induced loss of MiTF-CX and 15-PGDH was mediated through prostaglandin E2 receptor (EP2) receptors (PTGER2), but not cAMP. The 15-PGDH gene is a MiTF-CX target gene in cervical stromal cells and is down-regulated by PGE2 through EP2 receptors. The findings suggest that EP2 receptor-specific antagonists may be used as an adjunct to present clinical management for the prevention of preterm cervical ripening and preterm labor.

Aromatase regulation and breast cancer.

Aromatase regulation and breast cancer.

Dietary linoleic acid, gastric acid, and prostaglandin secretion

Basal and pentagastrin-stimulated gastric acid secretion, fasting serum gastrin concentrations, and the gastric output of prostaglandin E and its major metabolite 13,14-dihydro 15-keto prostaglandin E2 were measured in 9 normal subjects before and after 14–20 days of dietary supplementation with linoleic acid. Mean maximal gastric acid output fell from 36.0 ± 3.3 (SEM) to 30.1 ± 2.9 mmol/h (p < 0.05), although mean basal acid output was not significantly affected (8.3 ± 2.1 and 7.2 ± 1.7 mmol/h, respectively). Mean fasting serum gastrin concentrations increased from 19.2 ± 3.1 to 30.9 ± 3.8 ng/L (p < 0.01) after linoleic acid, probably because of acid suppression. The mean output of prostaglandin E increased from 498 ± 110 to 1254 ± 465 ng/h (p < 0.05); that of its metabolite increased from 165 ± 18 to 1168 ± 645 ng/h (p < 0.01). These findings show that in normal subjects essential fatty acid weakly inhibits gastric acid secretion, but considerably increases gastric prostaglandin output.

Albumin-eicosanoid interactions. A model system to determine their attributes and inhibition.

A model system was developed to (a) reflect the chemical attributes of the microenvironment involved in albumin-eicosanoid interactions and (b) determine the effects of other ligands on these interactions. Albumin-dependent modulation of prostaglandin stability was chosen as the basis for this system. 15-Ketoprostaglandin E2 (PGE2) was evaluated as a model ligand because under special conditions it decomposes with formation of a visible chromophore. Human serum albumin, in a concentration-dependent fashion, catalyzed the dehydration of 15-keto-PGE2 with the concurrent generation of this chromophore (lambda max = 505 nm, epsilon = 35,000). Since chromophore production from 15-keto-PGE2 in albumin-free solution occurs only at pH greater than 10, the results suggest that albumin-eicosanoid interactions involve a microenvironment with alkaline attributes. The effect of other ligands on albumin-15-keto-prostaglandin E2 interactions was determined by monitoring their ability to inhibit the spectral component of these interactions. Inhibition correlated with an affinity for specific binding sites on albumin. At mole ratios of ligand/albumin below 1, only phenylbutazone, its analogs, and warfarin inhibited chromophore development. Other ligands including fatty acids, steroids, tryptophan, and drugs with an affinity for other binding sites were ineffective inhibitors.

Prostaglandin E2 binding and cyclic AMP production in isolated bone cells.

The binding of prostaglandin E2 (PGE2) to bone cells was studied to provide direct evidence for the existence of specific receptors in bone. Bone cells were isolated by collagenase digestion of fetal and newborn rat calvaria. Isolated cells were incubated with 3H-PGE2 and collected on Millipore filters. Specific binding was determined by subtracting the binding that occurred with 10(-6) M non-radioactive PGE2 and 3H-PGE2 from that with 3H-PGE2 alone. With heterogeneous cell preparations and at PGE2 concentrations from 10(-9) - 1.7 X 10(-8) M at 37 degrees C, specific binding reached steady state within 10 min. Bound 3H-PGE2 was displaced by the addition of increasing amounts of unlabeled PGE2. Inhibition of PGE2 binding was observed with PGE1 and the endoperoxide analog, U44069, but not with PGF2 alpha, a lipopolysaccharide, or 13,14-dihydro 15-keto PGE2. Studies with bone cell populations, obtained by sequential digestions, indicated that an osteoclastic population binds 30-fold more PGE2 than osteoblastic cells. Scatchard analyses revealed that the osteoclastic cells have an affinity constant for PGE2 binding similar to that obtained with heterogeneous populations. However, the PGE2 binding capacity in this osteoclastic population was fivefold greater than in the heterogeneous population. The osteoclastic population responded with an increase in cyclic AMP to lower concentrations of PGE2 than the osteoblastic populations. These studies suggest that differences in the binding capacity of PGE2 receptors exist among bone cell-types and that these differences are reflected in the cellular cyclic AMP response.

Radioimmunoassay for 13, 14-dihydro-15-ketoprostaglandin F2 alpha and its application in normo- and anovulatory women.

Highly specific antibodies to 13, 14-dihydro-15-ketoprostaglandin F2 alpha (PGFM) were raised in rabbits. The animals were immunized with PGFM-bovine serum albumin (BSA)-conjugates. Prior to the incubation procedure PGFM was extracted by a rapid method with dichloromethane followed by column chromatography. The antisera dilution was 1:10 000 amd the cross-reactivity towards prostaglandin A2, E2, F2 alpha, 13, 14-dihydro-15-ketoprostaglandin E2 and the 15-ketoprostaglandin E2 and F2 alpha 2 was less than 1%. The limit of detection was 1.9 +/- 0.6 pg/ml plasma over the standard range 1.9--250 pg. The intra- and inter-assay variations were 3.9 and 15%, respectively. PGFM was measured throughout the menstrual cycle in female volunteers. In normal ovulatory women (n = 3) plasma levels of PGFM varied between 65.6 to 107.1 pg/ml. No significant variations of plasma PGFM were seen during the cycle. In anovulatory women (n = 4) no difference of PGFM was found during the cycle. PGFM levels in hyperprolactinaemic but ovulating women tend to be higher than in anovulatory, and normoprolactinaemic subjects. These data strongly indicate that PGFM is not correlated with other hormonal parameters tested here in the normal and anovulatory cycles.

Biosynthesis of Prostaglandin D2,15-ketoprostaglandin E2, and Hydroxy Fatty Acids by Ram Seminal Vesicle Microsomes

At high arachidonic acid concentrations (164 μM) and without exogenous cofactors, ram seminal vesicle microsomes produced prostaglandin E2 and two less polar products, identified as prostaglandin D2 and 15-ketoprostaglandin E2. The ratio of the biosynthetic products formed depended on the exogenous cofactor and on the arachidonic acid concentration. At high arachidonic acid concentrations (>150 μM), tryptophan, phenol, and glutathione stimulated prostaglandin E2 formation, but each affected the formation of the other prostaglandins differently. Ascorbic acid markedly stimulated hydroxy fatty acid formation. GLC-mass spectral analysis of the hydroxy fatty acid fraction indicated the presence of 11-hydroxy-5,8,12,14-eicosatetraenoic acid, 15-hydroxy-5,8,11,13-eicosatetraenoic acid, and 12-hydroxy-5,8,10-heptadecatrienoic acid. At low arachidonic acid concentrations (30 μM). glutathione still stimulated prostaglandin E2 biosynthesis, but the other cofactors stimulated 6-ketoprostaglandin F1α”︁ and hydroxy fatty acid formation.

Prostaglandin 15‐Hydroxy Dehydrogenase from Human Placenta

The enzyme system prostaglandin 15-hydroxy dehydrogenase, which catalyzes the inactivation of all biologically active prostaglandins, has been purified 1270-fold from human placenta. Kinetic studies on the enzyme have provided information on a well-organized control mechanism to avoid prostaglandin accumulation and for a fast prostaglandin degradation. 15-Ketoprostaglandin E2 and 13,14-dihydro-15-ketoprostaglandin E2 inhibit prostaglandin 15-hydroxy dehydrogenase non-competitively with respect to prostaglandin E2. The rate equation of enzyme reaction for two substrates was used for determination of the equilibrium constant and Michaelis constants of the enzyme. The following kinetic constants for prostaglandin 15-hydroxy dehydrogenase have been found. The equilibrium constant with repect to prostaglandin E2 is 18 muM, the Michaelis constant Km for prostaglandin E2 is 1 muM for NAD+ 44muM. The inhibition constants for 15-ketoprostaglandin E2 ar Ki(slope) = 70 muM, Ki(intercept) = 150 muM, and for 13,14-dihydro-15-ketoprostaglandin E2 Ki(slope) = 80 muM, and Ki(intercept) = 150 muM. The maximal velocity for the forward reaction is V1 = 0.45 mumol/min. These kinetic data exclude a random or ping-pong mechanism, and also a Theorell-Chance type as suggested by Braithwaite and Jarabak. We propose, therefore, a sequential ordered mechanism. The isoelectric point for prostaglandin 15-hydroxy dehydrogenase is at pH 5.35, judged by isoelectric focusing.