15-Deoxy-delta(12,14)-prostaglandin J(2) down-regulates CXCR4 on carcinoma cells through PPARgamma- and NFkappaB-mediated pathways

The chemokine receptor CXCR4 plays a key role in the metastasis of colorectal cancer and its growth at metastatic sites. Here, we have investigated the mechanisms by which CXCR4 on cancer cells might be regulated by eicosanoids present within the colorectal tumor microenvironment. We show that prostaglandins PGE(2), PGA(2), PGD(2), PGJ(2) and 15dPGJ(2) each down-regulates CXCR4 receptor expression on human colorectal carcinoma cells to differing degrees. The most potent of these were PGD(2) and its metabolites PGJ(2) and 15dPGJ(2). Down-regulation was most rapid with the end-product 15dPGJ(2) and was accompanied by a marked reduction in CXCR4 mRNA. 15dPGJ(2) is known to be a ligand for the nuclear receptor PPARgamma. Down-regulation of CXCR4 was also observed with the PPARgamma agonist rosiglitazone, while 15dPGJ(2)-induced CXCR4 down-regulation was substantially diminished by the PPARgamma antagonists GW9662 and T0070907. These data support the involvement of PPARgamma. However, the 15dPGJ(2) analogue CAY10410, which can act on PPARgamma but which lacks the intrinsic cyclopentenone structure found in 15dPGJ(2), down-regulated CXCR4 substantially less potently than 15dPGJ(2). The cyclopentenone grouping is known to inhibit the activity of NFkappaB. Consistent with an additional role for NFkappaB, we found that the cyclopentenone prostaglandin PGA(2) and cyclopentenone itself could also down-regulate CXCR4. Immunolocalization studies showed that the cellular context was sufficient to trigger a focal nuclear pattern of NFkappaB p50 and that 15dPGJ(2) interfered with this p50 nuclear localization. These data suggest that 15dPGJ(2) can down-regulate CXCR4 on cancer cells through both PPARgamma and NFkappaB. 15dPGJ(2), present within the tumor microenvironment, may act to down-regulate CXCR4 and impact upon the overall process of tumor expansion.     

15-deoxy-delta12,14-prostaglandin J2 regulates the functional state and the survival of microglial cells through multiple molecular mechanisms

We have previously reported that rat primary microglial cultures express the nuclear receptor peroxisome proliferator-activated receptor-gamma (PPAR-gamma) and that several functions associated with the activation of these cells, including nitric oxide (NO) and tumor necrosis factor-alpha synthesis, are down-regulated by 15-deoxy-delta12,14-prostaglandin J2 (15d-PGJ2) and ciglitazone, two specific PPAR-gamma agonists. Here we demonstrate that microglial cells not only express a functionally active PPAR-gamma, but also synthesize large amounts of 15d-PGJ2 upon stimulation with lipopolysaccharide (LPS). In addition, we show that, although 15d-PGJ2 and ciglitazone were equally effective in reducing microglial activation when used at 1-5 microm concentrations, 15d-PGJ2, but not of ciglitazone, reduced PGE2 production at low concentration (0.1 microm) and induced a time-dependent microglial impairment and apoptosis at high concentration (10 microm). Interestingly, the inhibition of PGE2 production was achieved mainly through the inhibition of cycloxygenase-2 enzymatic activity, as the expression of this enzyme and that of the microsomal isoform of PGE synthase remained unaltered. These findings suggest that 15d-PGJ2 affects the functional state and the survival of activated microglia through mechanisms only in part dependent on PPAR-gamma and that the concentration of 15d-PGJ2 is crucial in determining the particular microglial function affected.     

Combined microarray analysis uncovers self-renewal related signaling in mouse embryonic stem cells

Due to the limited understanding of self-renewal and pluripotency related signaling in stem cells, extracting information from genome-wide expression data is not only important but also challenging. With the combined use of two methods, we analyzed a set of microarray data at 11 time points from three mouse embryonic stem cell lines cultivated with and without leukemia inhibitory factor (LIF) for 14 days. Albeit the expression of individual genes in signaling pathways was not noticeably different between cells cultivated with and without LIF, at gene-set level the expression of ERK/MAPK (but not JAK/STAT) and cell cycle related genes was found significantly enriched in cells cultivated with LIF. This indicates that the Ras/Raf/ERK pathway, in addition to JAK/STAT, may also be a key player to carry on external LIF signal into mouse embryonic stem cells to promote self-renewal. When data at the first 7 time points were compared with data at the last 4 time points, the expression of several cell cycle related gene sets was apparently enriched in all three cell lines, indicating the active cell proliferation in the first 2 days. Compared with the slight decay of Oct4/Nanog/Sox2 during the 14 days, the expression of cell differentiation genes such as Gata4/6 underwent a drastic increase, which indicates that the upregulated expression of cell differentiation genes may better reflect the loss of self renewal than the down regulated expression of the stemness indicators Oct4, Sox2 and Nanog. Apart from differential expression and gene set enrichment analyses, a clustering algorithm was also used to classify genes into co-expression clusters. The possible regulation of two clusters, whose expression was most changed during cell culture from very low to very high, was explored. The drastic changes of these genes, including Slc39a8 which was a potential indicator of cell differentiation, in contrast the slight changes of self-renewal genes, imply that differentiation may be the default fate of stem cells and self-renewal may rely on a maintenance mechanism. When that mechanism weakens, cell differentiation begins. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Combined action of stem cell factor,leukemia inhibitory factor,and cAMP on in vitro proliferation of mouse primordial germ cells

In the present paper we investigated the effects of stem cell factor/mastocyte growth factor (SCF/MGF), leukemia inhibitory factor/differentiating inhibitory activity (LIF/DIA) (two growth factors known to affect primordial germ cell growth in vitro) and forskolin (FRSK) (an activator of adenylate cyclase in many cell types) alone or in combination on the survival and proliferation of primordial germ cells (PGCs) obtained from 8.5, 10.5, and 11.5 days post coitum (dpc) mouse embryos and cultured without pre-formed cell feeder layers. The results showed that both at 1 and 3 days of culture the addition of 100 ng/ml SCF, 20 μM FRSK, or in some instances 20 ng/ml LIF alone caused a significant increase of PGC number as compared with controls. The highest effects were obtained when SCF and/or LIF were used together with FRSK. Moreover, we found that FRSK elevated cAMP levels in purified 11.5 dpc PGCs and that this compound, but not SCF and LIF, stimulated PGC proliferation, as assessed by 5-bromo-2′-deoxyuridin (BrdU) incorporation. These results suggest a mechanism of combined action of cAMP with SCF and/or LIF in the control of proliferation of mouse PGCs in vitro. © 1993 Wiley-Liss, Inc.     

15-Deoxy-delta(12,14)-prostaglandin J(2) inhibits IL-1beta-induced IKK enzymatic activity and IkappaBalpha degradation in rat chondrocytes through a PPARgamma-independent pathway

Peroxisome proliferator-activated receptor gamma (PPARgamma) ligands have been shown to inhibit the effects of proinflammatory cytokines such as interleukin-1beta (IL-1beta). This cytokine plays a key role in articular pathophysiologies by inducing the production of inflammatory mediators such as nitric oxide (NO) and prostaglandin E(2) (PGE(2)). We previously demonstrated that 15d-PGJ(2) was more potent than troglitazone to counteract IL-1beta effects on chondrocytes. Here, we studied the action of 15d-PGJ(2) on intracellular targets in nuclear factor-kappaB (NF-kappaB) signalling pathway in IL-1beta treated rat chondrocytes. We found that 15d-PGJ(2) decreased inhibitor kappaBalpha (IkappaBalpha) degradation but not its phosphorylation by specifically inhibiting IkappaB kinase beta (IKKbeta), but not IKKalpha, enzymatic activity. We further evaluated the involvement of PPARgamma in the anti-inflammatory action of its ligands. In chondrocytes overexpressing functional PPARgamma protein, 15d-PGJ(2) pre-treatment inhibited inducible NO synthase and COX-2 mRNA expression, nitrite and PGE(2) production, p65 translocation and NF-kappaB activation. Troglitazone or rosiglitazone pre-treatment had no effect. 15d-PGJ(2) exhibited the same effect in chondrocytes overexpressing mutated PPARgamma protein. These results suggest that 15d-PGJ(2) exerts its anti-inflammatory effect in rat chondrocytes by a PPARgamma-independent mechanism, which can be conferred to a partial inhibition of IkappaBalpha degradation.     

Cellular lifespan and senescence signaling in embryonic stem cells

Most mammalian cells when placed in culture will undergo a limited number of cell divisions before entering an unresponsive non-proliferating state termed senescence. However, several pathways that are activated singly or in concert can allow cells to bypass senescence at least for limited periods. These include the telomerase pathway required to maintain telomere ends, the p53 and Rb pathways required to direct senescence in response to DNA damage, telomere shortening and mitogenic signals, and the insulin-like growth factor--Akt pathway that may regulate lifespan and cell proliferation. In this review, we summarize recent findings related to these pathways in embryonic stem (ES) cells and suggest that ES cells are immortal because these pathways are tightly regulated.     

MFN2 silencing promotes neural differentiation of embryonic stem cells via the Akt signaling pathway

Mitofusin 2 (MFN2) is a regulatory protein participating in mitochondria dynamics, cell proliferation, death, differentiation, and so on. This study aims at revealing the functional role of MFN2 in the pluripotency maintenance and primitive differetiation of embryonic stem cell (ESCs). A dox inducible silencing and routine overexpressing approach was used to downregulate and upregulate MFN2 expression, respectively. We have compared the morphology, cell proliferation, and expression level of pluripotent genes in various groups. We also used directed differentiation methods to test the differentiation capacity of various groups. The Akt signaling pathway was explored by the western blot assay. MFN2 upregulation in ESCs exhibited a typical cell morphology and similar cell proliferation, but decreased pluripotent gene markers. In addition, MFN2 overexpression inhibited ESCs differentiation into the mesendoderm, while MFN2 silencing ESCs exhibited a normal cell morphology, slower cell proliferation and elevated pluripotency markers. For differentiation, MFN2 silencing ESCs exhibited enhanced three germs' differentiation ability. Moreover, the protein levels of phosphorylated Akt308 and Akt473 decreased in MFN2 silenced ESCs, and recovered in the neural differentiation process. When treated with the Akt inhibitor, the neural differentiation capacity of the MFN2 silenced ESCs can reverse to a normal level. Taken together, the data indicated that the appropriate level of MFN2 expression is essential for pluripotency and differentiation capacity in ESCs. The increased neural differentiation ability by MFN2 silencing is strongly related to the Akt signaling pathway.     

Epinephrine increases DNA synthesis via ERK1/2s through cAMP, Ca(2+)/PKC, and PI3K/Akt signaling pathways in mouse embryonic stem cells

Epinephrine is a catecholamine that plays important roles in regulating a wide variety of physiological systems by acting through the adrenergic receptors (ARs). The cellular responses to AR stimulation are mediated through various signaling pathways. Therefore, this study examined the effects of epinephrine on DNA synthesis and related signaling molecules in mouse embryonic stem cells (ESCs). Epinephrine increased DNA synthesis in a dose- and time-dependent manner, as determined by the level of [(3)H]-thymidine incorporation. AR subtypes (alpha1(A), alpha2(A), beta1, beta2, and beta3) were expressed in mouse ESCs and their expression levels were increased by epinephrine. In this experiment, epinephrine increased cAMP levels, intracellular Ca(2+) concentration ([Ca(2+)](i)), and translocation of protein kinase C (PKC) from the cytosol to the membrane compartment. In addition, we observed Akt phosphorylation in response to epinephrine; this was stimulated by phosphorylation of the epidermal growth factor receptor (EGFR). Epinephrine also induced phosphorylation of ERK1/2 (p44/42 MAPKs), while inhibition of PKC or Akt blocked this phosphorylation. Epinephrine increased the mRNA levels of proto-oncogenes (c-fos, c-jun, c-myc), while inhibition of ERK1/2 decreased these mRNA levels. In experiments aimed at examining the involvement of cell cycle regulatory proteins, epinephrine increased the levels of cyclin E/cyclin-dependent kinase 2 (CDK2) and cyclin D1/cyclin-dependent kinase 4 (CDK4). In conclusion, epinephrine stimulates DNA synthesis via ERK1/2 through cAMP, Ca(2+)/PKC, and PI3K/Akt signaling pathways in mouse ESCs.     

A regulatory role of Wnt signaling pathway in the hematopoietic differentiation of murine embryonic stem cells

One of the most important issues in stem cell research is to understand the regulatory mechanisms responsible for their differentiation. An extensive understanding of mechanism underlying the process of differentiation is crucial in order to prompt stem cells to perform a particular function after differentiation. To elucidate the molecular mechanisms responsible for the hematopoietic differentiation of embryonic stem cells (ESCs), we investigated murine ES cells for the presence of hematopoietic lineage markers as well as Wnt signaling pathway during treatments with different cytokines alone or in combination with another. Here we report that Wnt/beta-catenin signaling is down-regulated in hematopoietic differentiation of murine ES cells. We also found that differentiation induced by the interleukin-3, interleukin-6, and erythropoietin combinations resulted in high expression of CD3e, CD11b, CD45R/B220, Ly-6G, and TER-119 in differentiated ES cells. A high expression of beta-catenin was observed in two undifferentiated ES cell lines. Gene and protein expression analysis revealed that the members downstream of Wnt in this signaling pathway including beta-catenin, GSK-3beta, Axin, and TCF4 were significantly down-regulated as ES cells differentiated into hematopoietic progenitors. Our results show that the Wnt/beta-catenin signaling pathway plays a role in the hematopoietic differentiation of murine ESCs and also may support beta-catenin as a crucial factor in the maintenance of ES cells in their undifferentiated state.     

Extrinsic factors derived from mouse embryonal carcinoma cell lines maintain pluripotency of mouse embryonic stem cells through a novel signal pathway

Embryonic carcinoma (EC) cells, which are malignant stem cells of teratocarcinoma, have numerous morphological and biochemical properties in common with pluripotent stem cells such as embryonic stem (ES) cells. However, three EC cell lines (F9, P19 and PCC3) show different developmental potential and self‐renewal capacity from those of ES cells. All three EC cell lines maintain self‐renewal capacity in serum containing medium without Leukemia Inhibitory factor (LIF) or feeder layer, and show limited differentiation capacity into restricted lineage and cell types. To reveal the underlying mechanism of these characteristics, we took the approach of characterizing extrinsic factors derived from EC cells on the self‐renewal capacity and pluripotency of mouse ES cells. Here we demonstrate that EC cell lines F9 and P19 produce factor(s) maintaining the undifferentiated state of mouse ES cells via an unidentified signal pathway, while P19 and PCC3 cells produce self‐renewal factors of ES cells other than LIF that were able to activate the STAT3 signal; however, inhibition of STAT3 activation with Janus kinase inhibitor shows only partial impairment on the maintenance of the undifferentiated state of ES cells. Thus, these factors present in EC cells‐derived conditioned medium may be responsible for the self‐renewal capacity of EC and ES cells independently of LIF signaling.     

Quantitative characterization of 15-deoxy-delta(12,14)-prostaglandin J2 in regulating EGFPSmad2 translocation in CHO cells through PPARgamma/TGFbeta/Smad2 pathway.

Smad2 is an important factor in TGFbeta/Smad2 signal transduction pathway with ability for signal propagation, it could translocate from cytoplasm to nucleus after the TGFbeta receptor-mediated phosphorylation. 15-deoxy-delta(12,14)-prostaglandin J2 (15d-PGJ2), a natural agonist of the peroxisome proliferator-activated receptor gamma (PPARgamma), is found recently to be able to function in the regulation of Smad2 activity. However, no quantification data have been yet reported, and it still keeps suspenseful whether or not 15d-PGJ2 could regulate Smad2 activity by depending on PPARgamma through PPAR gamma/TGFbeta/ Smad2 pathway. In this work, by analyzing the EGFP-Smad2 location in CHO cells according to the Nucleus Trafficking Analysis Module based on IN Cell Analyzer 1000 platform, TGFbeta stimulated EGFP-Smad2 translocation regulated by 15d-PGJ2 was quantitatively investigated. The results showed that TGFbeta could induce EGFP-Smad2 translocation from cytoplasm to nucleus by EC50 of 8.83 pM, and 15d-PGJ2 could impede the TGFbeta-stimulated Smad2 translocation by IC50 of 0.68 microM. Moreover, GW9662, a PPARgamma antagonist, could attenuate such a 15d-PGJ2 inhibitory activity by almost one order of magnitude. This result thereby implies that 15d-PGJ2 might inhibit Smad2 translocation through PPARgamma/TGFbeta/Smad2 pathway. Further investigation discovered that different from the case for 15d-PGJ2, rosiglitazone, another PPARgamma agonist, could enhance Smad2 translocation to nucleus, suggesting that rosiglitazone and 15d-PGJ(2) might take different modes in the activation of PPARgamma within the signaling pathway.