Amnion-derived Cells Research Articles

Amnion-Derived Cellular Cytokine Solution (ACCS) Promotes Migration of Keratinocytes and Fibroblasts

Amnion-derived Multipotent Progenitor cells appear to be useful as adjuvants in wound healing. Amnion-derived multipotent progenitor cells secrete a unique combination of cytokines and growth factors, known as amnion-derived cellular cytokine solution (ACCS). In the skin, a cytokine communication network between mesenchymal and epithelial cells tightly controls keratinocyte and fibroblast migration, proliferation and differentiation-key determinants of wound healing. To evaluate the influence of ACCS on the migratory behavior of keratinocytes and fibroblasts, cell migration was assayed quantitatively using a Boyden chamber. Chemotactic migration activity of fibroblasts or keratinocytes through the membrane determined the influence of ACCS. In the presence of ACCS, fibroblasts and keratinocytes demonstrated a statistically significant (P < 0.05) increase in migration when compared with controls. These cell types, critical to normal wound healing, may be influenced to accelerate migration in wounds, thus accelerating wound repair/healing.

Naive Rat Amnion-Derived Cell Transplantation Improved Left Ventricular Function and Reduced Myocardial Scar of Postinfarcted Heart

Stem cells contained in the amniotic membrane may be useful for cellular repair of the damaged heart. Previously, we showed that amnion-derived cells (ADCs) express embryonic stem cell surface markers and pluripotent stem cell-specific transcription factor genes. These ADCs also possess the potential for mesoderm (cardiac) lineage differentiation. In the present study we investigated whether untreated naive ADC transplantation into the injured left ventricular (LV) myocardium is beneficial as a cell-based cardiac repair strategy in a rat model. ADCs were isolated from Lewis rat embryonic day 14 amniotic membranes. FACS analysis revealed that freshly isolated ADCs contained stage-specific embryonic antigen-1 (SSEA-1), Oct-4-positive cells, and mesenchymal stromal cells, while hematopoietic stem cell marker positive cells were absent. Reverse transcription-PCR revealed that naive ADCs expressed cardiac and vascular specific genes. We injected freshly isolated ADCs (2 x 10(6) cells suspended in PBS, ADC group) into acutely infarcted LV myocardium produced by proximal left coronary ligation. PBS was injected in postinfarction controls (PBS group). Cardiac function was assessed at 2 and 6 weeks after injection. ADC treatment attenuated LV dilatation and sustained LV contractile function at 2 and 6 weeks in comparison to PBS controls (p < 0.05, ANOVA). LV peak systolic pressure and maximum dP/dt of ADC-treated heart were higher and LV end-diastolic pressure and negative dP/dt were lower than in PBS controls (p < 0.05). Histological assessment revealed that infarcted myocardium of the ADC-treated group had less fibrosis, thicker ventricular walls, and increased capillary density (p < 0.05). The fate of injected ADCs was confirmed using ADCs derived from EGFP(+) transgenic rats. Immunohistochemistry at 6 weeks revealed that EGFP(+) cells colocalized with von Willebrand factor, alpha-smooth muscle actin, or cardiac troponin-I. Our results suggest that naive ADCs are a potential cell source for cellular cardiomyoplasty.

Amnion: A Potent Graft Source for Cell Therapy in Stroke

Regenerative medicine is a new field primarily based on the concept of transplanting exogenous or stimulating endogenous stem cells to generate biological substitutes and improve tissue functions. Recently, amnion-derived cells have been reported to have multipotent differentiation ability, and these cells have attracted attention as a novel cell source for cell transplantation therapy. Cells isolated from amniotic membrane can differentiate into all three germ layers, have low immunogenicity and anti-inflammatory function, and do not require the destruction of human embryos for their isolation, thus circumventing the ethical debate commonly associated with the use of human embryonic stem cells. Accumulating evidence now suggests that the amnion, which had been discarded after parturition, is a highly potent transplant material in the field of regenerative medicine. In this report, we review the current progress on the characterization of MSCs derived from the amnion as a remarkable transplantable cell population with therapeutic potential for multiple CNS disorders, especially stroke.

Relationship between cyclic adenosine monophosphate and aquaporin 8 expression and distribution in human amnion-derived cells

To study the effect of cyclic adenosine monophosphate on aquaporin 8 (AQP8) expression and distribution in human amnion-derived WISH cells. Human amnion-derived WISH cells were cultured. The cells were divided into control group and study group at random. The study group was established by exposure to various concentrations of 8-Br-cAMP. Western analysis was used to quantify AQP8 expression levels. RT-PCR was used to quantify AQP8 mRNA expression levels. Immunofluorescence was used to determine the localization of AQP8 in WISH cells. With increase of cAMP, AQP8 mRNA and protein expressions significantly increased in WISH cells in vitro. When the concentration of cAMP reached 200 micromol/L, AQP8 mRNA and protein expressions were highest. Incubation with cAMP (200 micromol/L) for 2 hours resulted in a 2-fold increase in AQP8 mRNA level, and incubation for 8 hours resulted in a peak. After incubation for 16 hours AQP8 mRNA level began to descend, and after 24 hours it decreased to baseline. Incubation with cAMP for 8 hours AQP8 protein level began to increase. Incubation for 24 hours resulted in a peak in AQP8 protein level, and after incubation for 48 hours it began to decline. By immunofluorescence microscopy after incubation with cAMP (200 micromol/L) cells, AQP8 labeling in plasma membrane was enhanced and intracellular AQP8 labeling was decreased. The cAMP triggers translocation of AQP8 from cytosol to the plasma membrane via vesicle-transporting related protein instead of AQP8 itself. cAMP may upregulate the transcription of target gene protein kinase A. The cAMP may be the critical regulatory medium of AQP8 in WISH cells.

The Potential of Amniotic Membrane/Amnion-Derived Cells for Regeneration of Various Tissues

Regenerative medicine is a new field based on the use of stem cells to generate biological substitutes and improve tissue functions, restoring damaged tissue with high proliferability and differentiability. It is of interest as a potential alternative to complicated tissue/organ transplantation. Recently, amnion-derived cells have been reported to have multipotent differentiation ability, and these cells have attracted attention as a cell source for cell-transplantation therapy. The amnion possesses considerable advantageous characteristics: the isolated cells can differentiate into all three germ layers; they have low immunogenicity and anti-inflammatory functions; and they do not require the sacrifice of human embryos for their isolation, thus avoiding the current controversies associated with the use of human embryonic stem cells. Moreover, we developed human amniotic cell-sheets using a novel culture surface coated with a noncytotoxic, temperature-responsive elastic protein-based polymer. We also generated a “hyperdry-amnion”, which has already been applied clinically in the ophthalmological field. Compared to cryopreserved fresh amnion, “hyper-dry-amnion” is easy to handle and has started to bring good results to patients. These materials from the amnion are also expected to open a new field in tissue engineering. Thus, amnion, which had been discarded after parturition, has started to be appreciated as an attractive material in the field of regenerative medicine. In this review, the most recent and relevant clinical and experimental data about the use of amniotic membrane and cells derived from it are described.

Enhancement of Cortisol-Induced 11β-Hydroxysteroid dehydrogenase Type 1 Expression by Interleukin 1β in Cultured Human Chorionic Trophoblast Cells

Chorion is the most abundant site of 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) expression within intrauterine tissues. It is important to study the regulation of 11beta-HSD1 expression in the chorion in terms of local cortisol production during pregnancy. Using real-time PCR and enzyme activity assay, we found that cortisol (1 mum) and IL-1beta (10 ng/ml) for 24 h significantly increased 11beta-HSD1 mRNA expression and reductase activity in cultured human chorionic trophoblasts. A further significant increase of 11beta-HSD1 mRNA expression and reductase activity was observed with cotreatment of cortisol and IL-1beta. To explore the mechanism of induction, 11beta-HSD1 promoter was cloned into pGL3 plasmid expressing a luciferase reporter gene. By transfecting the constructed vector into WISH cells, an amnion-derived cell line, we found that cortisol (1 microM) or IL-1beta (10 ng/ml) significantly increased reporter gene expression. Likewise, an additional increase in reporter gene expression was observed with cotreatment of cortisol and IL-beta. To explore the physiological significance of 11beta-HSD1 induction in the chorion, we studied the effect of cortisol on cytosolic phospholipase A(2) and cyclooxygenase 2 expression. We found that treatment of chorionic trophoblast cells with cortisol (1 microM) induced both cytosolic phospholipase A(2) and cyclooxygenase 2 mRNA expression. We conclude that cortisol up-regulates 11beta-HSD1 expression through induction of promoter activity, and the effect was enhanced by IL-1beta, suggesting that more biologically active glucocorticoids could be generated in the fetal membranes in the presence of infection, which may consequently feed forward in up-regulation of prostaglandin synthesis.

The effect of epidermal growth factor on production of vascular endothelial growth factor by amnion-derived (WISH) cells

Our objective was to clarify the physiological role of vascular endothelial growth factor (VEGF) by amnion-derived (WISH) cells. WISH cells were cultured, and the effect of epidermal growth factor (EGF), mitogen-activated protein (MAP) kinase kinase or extracellular signal-regulated kinase (ERK) kinase (MEK) inhibitors (U0126) or phosphatidylinositol (PI) 3-kinase on the production of VEGF was examined. VEGF was assayed by ELISA. The activation of MAP kinase and akt, which is phosphorylated by PI 3-kinase, were detected by Western blot analysis using anti-phosphorylated MAP kinase antibody and anti-phosphorylated akt antibody. In the time course of VEGF production following EGF treatment, VEGF production showed a significant increase only after 16 (p < 0.01)–32 h (p < 0.01). EGF increased the production of VEGF by WISH cells in a dose-dependent manner. The MAP kinase and akt activity were determined by treatment with EGF. VEGF production was significantly decreased following pretreatment with U0126 or wortmannin for two hours before treatment with EGF (p < 0.01, p < 0.01). WISH cells appeared to produce VEGF via a mechanism involving tyrosine kinase activation of EGF receptor and MAP kinase or PI 3-kinase. It is suggested that VEGF may contribute to the neovascularization and proliferation of the placenta and gestational tissue, and EGF may play an important role in regulation of VEGF production in the placenta.

Epidermal growth factor and interleukin-1beta utilize divergent signaling pathways to synergistically upregulate cyclooxygenase-2 gene expression in human amnion-derived WISH cells.

In human parturition, uterotonic prostaglandins (PGs) arise predominantly via increased expression of cyclooxygenase-2 (COX-2 [also known as prostaglandin synthase 2]) within intrauterine tissues. Interleukin-1 (IL-1) and epidermal growth factor (EGF), both inducers of COX-2 transcription, are among numerous factors that accumulate within amniotic fluid with advancing gestation. It was previously demonstrated that EGF could potentiate IL-1beta-driven PGE(2) production in amnion and amnion-derived (WISH) cells. To define the mechanism for this observation, we hypothesized that EGF and IL-1beta might exhibit synergism in regulating COX-2 gene expression. In WISH cells, combined treatment with EGF and IL-1beta resulted in a greater-than-additive increase in COX-2 mRNA relative to challenge with either agent independently. Augmentation of IL-1beta-induced transactivation by EGF was not observed in cells harboring reporter plasmids bearing nuclear factor-kappa B (NFkappaB) regulatory elements alone, but was evident when a fragment (-891/ +9) of the COX-2 gene 5'-promoter was present. Both agents transiently activated intermediates of multiple signaling pathways potentially involved in the regulation of COX-2 gene expression. The 26 S proteasome inhibitor, MG-132, selectively abrogated IL-1beta-driven NFkappaB activation and COX-2 mRNA expression. Only pharmacologic blockade of the p38 mitogen-activated protein kinase eliminated COX-2 expression following EGF stimulation. We conclude that EGF and IL-1beta appear to signal through different signaling cascades leading to COX-2 gene expression. IL-1beta employs the NFkappaB pathway predominantly, while the spectrum of EGF signaling is broader and includes p38 kinase. The synergism observed between IL-1beta and EGF does not rely on augmented NFkappaB function, but rather, occurs through differential use of independent response elements within the COX-2 promoter.

The effect of insulin-like growth factor-I on production of vascular endothelial growth factor by amnion-derived (WISH) cells

Objective. Our objective was to clarify the modulation of vascular endothelial growth factor (VEGF) by amniotic cells. Design. Amnion-derived (WISH) cells were cultured, and the effect of insulin-like growth factor (IGF), mitogen-activated protein (MAP) kinase kinase and/or extracellular signal-regulated kinase (ERK) kinase (MEK) inhibitors (U0126), and phosphatidylinositol (PI) 3-kinase inhibitors (wortmannin) on the production of VEGF was examined. VEGF was assayed using ELISA. The activations of MAP kinase and akt, which is phosphorylated by PI 3-kinase, were detected by Western blot analysis using anti-phosphorylated MAP kinase antibody and anti-phosphorylated akt antibody. Results. In the time course of VEGF production following IGF-I treatment, VEGF production showed significant increases only at 16 and 32 h ( p<0.01). Also, IGF-I increased the production of VEGF by WISH cells in a dose-dependent manner. The MAP kinase and akt activities were recognized by treatment with IGF-I and suppressed by U0126 and wortmannin, respectively. When WISH cells were pretreated for 2 h with U0126 and wortmannin and then treated with IGF-I for 16 h, the production of VEGF was significantly decreased in a dose-dependent manner ( p<0.01, p<0.01, respectively). Conclusions. WISH cells appeared to produce VEGF via a mechanism involving tyrosine kinase interaction with IGF-I receptor, resulting in MAP kinase and PI 3-kinase activation. It is suggested that VEGF may contribute to the neovascularization and proliferation of the placenta and gestational tissue, and IGF-I may play an important role in the modulation of VEGF production in the placenta.

Expression of aquaporin 8 and its up-regulation by cyclic adenosine monophosphate in human WISH cells

Objective: Water absorption across the fetal chorioamniotic membranes is a critical regulatory pathway for amniotic fluid volume homeostasis. Aquaporins are cell membrane proteins that significantly enhance membrane permeability to water by acting as water channels. We recently demonstrated that aquaporin 8 is expressed in human amnion, chorion, and placenta. Thus, aquaporin 8 expression represents a molecular mechanism of amniotic water absorption through intramembranous pathways. The current study sought to determine whether aquaporin 8 is expressed in human amnion-derived cell culture and to explore its regulation by second messenger cyclic adenosine monophosphate. Study Design: Human amnion–derived WISH cells were cultured. Total RNA was isolated and reverse transcriptase-polymerase chain reaction was used to determine aquaporin 8 gene expression. To determine the effect of cyclic adenosine monophosphate on aquaporin 8 expression, WISH cells were cultured in the presence of either monobutyryl cyclic adenosine monophosphate or the cyclic adenosine monophosphate–elevating agent forskolin. Multiplex semiquantitative reverse transcriptase–polymerase chain reaction was carried out to quantify aquaporin 8 messenger RNA levels. Results: Reverse transcriptase–polymerase chain reaction detected aquaporin 8 expression in WISH cells. After forskolin treatment for 2 hours, aquaporin 8 messenger RNA expression in WISH cells increased 4-fold (P <.001). Stimulation of aquaporin 8 gene expression by colforsin was observed throughout the study period of 20 hours. Incubation of WISH cells with monobutyryl cyclic adenosine monophosphate resulted in a 2-fold increase in aquaporin 8 messenger RNA level (P <.001). However, stimulation of aquaporin 8 gene expression by monobutyryl cyclic adenosine monophosphate attenuated to baseline level after 20 hours of monobutyryl cyclic adenosine monophosphate treatment. Conclusion: The current study demonstrates the expression of aquaporin 8 water channel in human amnion–derived WISH cells and aquaporin 8 expression up-regulation by second messenger cyclic adenosine monophosphate. Aquaporin 8 messenger RNA demonstrates a relatively short biologic half-life in vitro, which renders its rapid responsiveness to regulation (Am J Obstet Gynecol 2003;188:997-1001.)

WISH Cells as a Model for the “In Vitro” Study of Amnion Pathophysiology

In the course of pregnancy amnion cells produce a number of factors which include cytokines and prostaglandins (PGs) produced in response to autocrine, paracrine and endocrine signals. Recent studies performed by several researchers contributed to elucidate the mechanism through which amnion tissue is involved in the triggering of physiological labor. However, there are other possible functions to be ascribed to amniotic cells, depending on the high number of factors that they produce as well as on the receptors that enable them to act in turn as target. For instance, it has been demonstrated that amnion cells are able to produce lecithin upon the regulation of several factors, such as glucocorticoids and epidermal growth factor, a finding that suggests a protective role of the tissue on fetal pulmonary function. As regards to triggering the uterine contractions, it is accepted that prostaglandin release by amnion cells represents a key event. It is under the control of hormones, growth factors, cytokines and probably PGs themselves. A striking analogy has been found between the mechanism of inflammation and the onset of myometrial activity in labor. In this context, it has been shown that for-Met-Leu-Phe (fMLP), the prototype of a series of formylated peptides traditionally considered chemotactic agents, is also involved in the regulation of amniotic PG release. The similitude between labor and inflammatory response is enforced by the antiprostaglandin action of some classes of antibiotics observed in amnion tissue, that enable them as effective tools against preterm labor, both in the absence and in the presence of infection. As for the mechanisms responsible for the regulation of PG synthesis, some agents act by influencing protein synthesis, while others exert their effects through the production of intracellular second messengers, mainly represented by phosphatidyl-inositol-4-5 bisphosphate and cyclic AMP. The mechanism whereby second messengers induce PG release is not clear, and a crosstalk between the two transduction pathways could be hypothesized. This interaction has extensively been analysed in "WISH" cells, a human amnion-derived cell line, which represent a model for the in vitro study of amnion functions. In the present review, we intend to report the results of the studies regarding the mechanisms through which the control of the above mentioned functions is executed.

WISH Cells as a Model for the &amp;#x201C;In Vitro&amp;#x201D; Study of Amnion Pathophysiology

In the course of pregnancy amnion cells produce a number of factors which include cytokines and prostaglandins (PGs) produced in response to autocrine, paracrine and endocrine signals. Recent studies performed by several researchers contributed to elucidate the mechanism through which amnion tissue is involved in the triggering of physiological labor. However, there are other possible functions to be ascribed to amniotic cells, depending on the high number of factors that they produce as well as on the receptors that enable them to act in turn as target. For instance, it has been demonstrated that amnion cells are able to produce lecithin upon the regulation of several factors, such as glucocorticoids and epidermal growth factor, a finding that suggests a protective role of the tissue on fetal pulmonary function. As regards to triggering the uterine contractions, it is accepted that prostaglandin release by amnion cells represents a key event. It is under the control of hormones, growth factors, cytokines and probably PGs themselves. A striking analogy has been found between the mechanism of inflammation and the onset of myometrial activity in labor. In this context, it has been shown that for-Met-Leu-Phe (fMLP), the prototype of a series of formylated peptides traditionally considered chemotactic agents, is also involved in the regulation of amniotic PG release. The similitude between labor and inflammatory response is enforced by the antiprostaglandin action of some classes of antibiotics observed in amnion tissue, that enable them as effective tools against preterm labor, both in the absence and in the presence of infection. As for the mechanisms responsible for the regulation of PG synthesis, some agents act by influencing protein synthesis, while others exert their effects through the production of intracellular second messengers, mainly represented by phosphatidyl-inositol-4-5 bisphosphate and cyclic AMP. The mechanism whereby second messengers induce PG release is not clear, and a crosstalk between the two transduction pathways could be hypothesized. This interaction has extensively been analysed in “WISH” cells, a human amnion-derived cell line, which represent a model for the in vitro study of amnion functions. In the present review, we intend to report the results of the studies regarding the mechanisms through which the control of the above mentioned functions is executed.

Dominant negative MEKK1 inhibits survival of pancreatic cancer cells.

Human pancreatic cancers harbor mutations in the K-ras gene, and these mutations convert the gene oncogenic and constitutively active forms. However, in pancreatic cancer cells little is known about the activation of the downstream pathways of Ras, MEK-ERK and MEKK1-JNK, and their roles in cell survival and proliferation. An analysis of nine pancreatic cancer tissues revealed JNK activation in all tumor samples and ERK activation in three tumor samples. Colony formation assays by transfection of dominant negative mutants of Ras, ERK or MEKK1 into pancreatic cancer cell lines (BxPC-3, PANC-1, MIAPaCa-2 and AsPC-1) and an amnion-derived cell line (FL) revealed that DN-MEKK strongly inhibits the survival of colonies in pancreatic cancer cells, but not in FL cells. In vitro kinase assays and luciferase assays using the Gal4c-Jun system revealed that in pancreatic cancer cells DN-MEKK fails to inhibit JNK activation. In PANC-1 cells, c-Jun was found to be a major component of protein component binding to AP-1 site and CRE, but not in FL cells. The inhibitory effect of DN-MEKK in PANC-1 cells was thought to be the result of the inhibition of c-Jun DNA-binding. The difference of suppression in pancreatic cancer cells and non-pancreatic cancer cells suggested that the MEKK1 pathway mainly contributes to cell survival in pancreatic cancer cells and may provide an advantage for the gene therapy of pancreatic cancers using DN-MEKK expression vectors.

A polymorphism in the matrix metalloproteinase-9 promoter is associated with increased risk of preterm premature rupture of membranes in African Americans.

Fetal membrane rupture is associated with increased expression of matrix metalloproteinase-9 (MMP-9) and matrix degradation. We have determined the functional significance of a variable number tandem repeat and a single nucleotide polymorphism (SNP) in the MMP-9 gene on promoter activity and their association with preterm premature rupture of membranes (PPROM). The 14 CA-repeat allele was a stronger promoter than the 20 CA-repeat allele in amnion epithelial cells and WISH amnion-derived cells, but in THP-1 monocyte/macrophage cells the 14 and 20 CA-repeat alleles had similar activities. An SNP at -1562 did not significantly affect promoter activity. A case-control study of African American neonates revealed that the 14 CA-repeat allele was more common in newborns delivered of mothers who had PPROM than in those delivered at term. There was no association between the -1562 SNP and PPROM. We conclude that there are cell host-dependent differences in MMP-9 promoter activity related to CA-repeat number and that fetal carriage of the 14 CA-repeat allele is associated with PPROM in African Americans.

Regulation of cyclooxygenase-2 expression by phospholipase D in human amnion-derived WISH cells.

Prostaglandins (PGs) are known to play a key role in the initiation of labor, but the mechanisms regulating their synthesis in amnion are largely unknown. In this study, the regulatory mechanisms for PGE(2) production during phospholipase D (PLD) and p38-dependent activation of WISH cells were investigated. We found that the stimulation of WISH cells with interleukin (IL)-1 beta elicited dose-dependent synthesis of cyclooxygenase-2 (COX-2) mRNA, protein, and their products, PGE(2). Moreover, the treatment of [(3)H]myristate-labeled cells in the presence of 1-butanol caused the dose-dependent formation of [(3)H]phosphatidylbutanol (PBt), a product specific to PLD activity. Pretreating the cells with 1-butanol and Ro 31-8220 inhibited the IL-1 beta-induced COX-2 expression, but 3-butanol did not affect this response. In addition, evidence that PLD was involved in the stimulation of COX-2 expression was provided by the observations that COX-2 expression was stimulated by the dioctanoyl phosphatidic acid (PA) and that the prevention of PA dephosphorylation by 1-propranolol potentiated COX-2 expression by IL-1 beta. Moreover, IL-1 beta stimulation of the cells caused the phosphorylation of p38 and extracellular signal-regulated kinase (ERK), and IL-1 beta-induced COX-2 expression was inhibited by the pretreatment of WISH cells with a p38 inhibitor, in contrast ERK upstream inhibitor had no effect. Furthermore, Ro 31-8220 inhibited IL-1 beta-induced p38 phosphorylation but not ERK phosphorylation. The results of this study indicate that in human amnion cells, IL-1 beta might activate PLD through an upstream protein kinase C to elicit p38 and finally induce COX-2 expression.

The effect of dexamethasone on expression of mitogen-induced cyclooxygenase-2 mRNA by amnion-derived (WISH) cells

Objectives: It has been reported that prostaglandin E 2 (PGE 2) is synthesized in the amnion and that this synthesis increases during labor. The purpose of this study was to clarify the mechanism for the expression of cyclooxygenase-2 (COX-2) mRNA and the PGE 2 synthesis of amnion-derived (WISH) cells. Study design: Cells were cultured and treated by 12- O-tetradecanoylphorbol-13-acetate (TPA) and dexamethasone (DEX). PGE 2 in the culture medium was measured by ELISA. Total RNA was extracted from the cells, and COX-2 mRNA expression was analyzed by Northern blot analysis. Results: During the time course of PGE 2 production in response to TPA stimulation, the PGE 2 production could not be detected until incubation had continued for 2 h, but this production appeared to continue after 4 h of incubation. PGE 2 production was significantly increased by TPA and suppressed by treatment with TPA and DEX. During the time course of COX-2 mRNA expression in response to treatment with TPA, the COX-2 mRNA band was detected after 1.5 h. The strongest expression of COX-2 mRNA was observed at 2 h incubation. After pre-treatment with TPA for 1 h, the TPA-induced COX-2 mRNA was suppressed by treatment with DEX for 1 or 2 h incubation in a dose-dependent manner. Conclusions: These results suggest that COX-2 mRNA is induced by TPA which activate protein kinase C, and suppressed by DEX in WISH cells.

17beta-estradiol modulates prostaglandin E2 release from human amnion-derived wish cells.

In human amnion-derived WISH cells [(3)H]estradiol-17beta binding sites are not detectable, but they become measurable in cells exposed to cAMP elevating agents such as forskolin or Ro 20-1724. In cells unexposed to these drugs, 17beta-estradiol stimulates prostaglandin (PG)E(2) release but exerts an evident inhibitory effect in cells exposed to Ro 20-1724. Both stimulatory and inhibitory actions are inhibited by the estrogen receptor antagonist, tamoxifen, by cell pretreatment with cycloheximide, or when the hormone is bound to BSA. Our data demonstrate for the first time that 1) 17beta-estradiol modulates PGE(2) release from WISH cells, interacting with specific intracellular receptors and probably evoking new protein synthesis, and 2) WISH cell responsiveness to 17beta-estradiol seems to be modulated by cAMP, whose levels are significantly increased by the steroid hormone in the presence of Ro 20-1724. The nucleotide is presumably responsible for the enhacement of hormone receptor availability and for the inhibition of PGE(2) release observed in the presence of Ro 20-1724.

Effect of ceramide analogs on interleukin-1alpha-induced production of prostaglandin E2 by amnion-derived (WISH) cells.

Prostaglandin E2 (PGE2) is reportedly synthesized in the amnion, and its levels are increased during labor. Our objective was to measure the level of PGE2 induced by interleukin (IL)-1alpha following treatment with ceramide analogs in amnion-derived cells. Amnion-derived (WISH) cells were cultured and stimulated by IL-1alpha, IL-1 receptor antagonist (ra), C2-ceramide and C6-ceramide. The levels of PGE2 in the media were measured by ELISA. The induction of prostaglandin H synthase (PGHS)-2 mRNA was detected by reverse transcription and polymerase chain reaction (RT-PCR). Following stimulation with IL-1alpha, the production of PGE2 could not be detected until incubation had continued for 2 h, but this production appeared to continue after 4 h of incubation. The production of PGE2 was significantly increased by IL-1alpha, and was suppressed by IL-1 ra, in a dose-dependent manner. PGE2 production was significantly increased by IL-1alpha and C2-ceramide as compared with IL-1alpha alone. However, PGE2 production was not significantly increased by IL-1alpha and C6-ceramide as compared with IL-1alpha alone. PGHS-2 mRNA were induced by treatment with IL-1alpha, and were strongly induced by treatment with IL-1alpha and C2-ceramide by RT-PCR. Results suggest that IL-1alpha induce the PGHS-2 mRNA and stimulate the production of PGE2 by a mechanism that involves the sphingomyelin-ceramide system. Ceramide may be important in increasing the production of PGE2 during parturition.

Effect of ceramide analogs on interleukin- 1a-induced production of rostaglandin E2 by amnion-derived (WISH) cells

Effect of ceramide analogs on interleukin- 1a-induced production of rostaglandin E2 by amnion-derived (WISH) cells

Prostanoid stimulation of cytokine production in an amnion-derived cell line: evidence of a feed-forward mechanism with implications for term and preterm labor.

To test the hypothesis that amnion cytokine production might be regulated by prostanoids. Amnion-derived WISH cells were treated with a range of prostanoids and their effects on production of interleukin (IL)-6 and IL-8 were determined by enzyme-linked immunosorbent assay and Northern analysis. The effects of thromboxane inhibitors on cytokine production by term primary amnion explants also were examined. Prostaglandin (PG)A2, PGD2, PGF2 alpha, PGE2, PGJ2, and the PGI2 analogue carbaprostacyclin (1-1000 nmol/L) exhibited no significant effects on cytokine production. However, the thromboxane A2 (TXA2) agonist U46619 and carbocyclic (c)TXA2 both stimulated WISH cytokine production with similar potencies under basal or cytokine-stimulated conditions. Significant stimulation of IL-6 production was observed at concentrations > or = 8 nmol/L (P < .05 by analysis of variance), whereas IL-8 production was stimulated significantly but to a lesser extent. The effects of U46619 and cTXA2 were rapid; maximal stimulation of cytokine production occurred within 4 to 8 hours of treatment. U46619 augmented IL-1 beta-stimulated IL-6 and IL-8 mRNA expression within 2 hours of treatment. In amnion explants inhibitors of TX synthesis and action abrogated the stimulatory effects of IL-1 beta on cytokine production. These results are consistent with the presence of a feed-forward loop in amnion involving TXA2 and cytokines, which could play a significant role in the progression of the inflammatory response involved in the mechanism of infection-driven preterm labor.