Peroxisome proliferator-activated receptor gamma controls Muc1 transcription in trophoblasts

The nuclear receptor peroxisome proliferator-activated receptor gamma (PPARgamma) is essential for placental development. Here, we show that the mucin gene Muc1 is a PPARgamma target, whose expression is lost in PPARgamma null placentas. During differentiation of trophoblast stem cells, PPARgamma is strongly induced, and Muc1 expression is upregulated by the PPARgamma agonist rosiglitazone. Muc1 promoter is activated strongly and specifically by liganded PPARgamma but not PPARalpha or PPARdelta. A PPAR binding site (DR1) in the proximal Muc1 promoter acts as a basal silencer in the absence of PPARgamma, and its cooperation with a composite upstream enhancer element is both necessary and sufficient for PPARgamma-dependent induction of Muc1. In the placenta, MUC1 protein is localized exclusively to the apical surface of the labyrinthine trophoblast around maternal blood sinuses, resembling its luminal localization on secretory epithelia. Last, variably penetrant maternal blood sinus dilation in Muc1-deficient placentas suggests that Muc1 regulation by PPARgamma contributes to normal placental development but also that the essential functions of PPARgamma in the organ are mediated by other targets.     

Zinc-alpha2-glycoprotein, a lipid mobilizing factor, is expressed and secreted by human (SGBS) adipocytes

Zinc-alpha2-glycoprotein (ZAG), a lipid mobilizing factor, is expressed in mouse adipose tissue and is markedly upregulated in mice with cancer cachexia. We have explored whether ZAG is expressed and secreted by human adipocytes, using SGBS cells, and examined the regulation of ZAG expression. ZAG mRNA was detected by RT-PCR in mature human adipocytes and in SGBS cells post-, but not pre-, differentiation to adipocytes. Relative ZAG mRNA levels increased rapidly after differentiation of SGBS cells, peaking at day 8 post-induction. ZAG protein was evident in differentiated adipocytes (by day 3) and also detected in the culture medium (by day 6) post-induction. The PPARgamma agonist rosiglitazone induced a 3-fold increase in ZAG mRNA level, while TNF-alpha led to a 4-fold decrease. Human adipocytes express and secrete ZAG, with ZAG expression being regulated particularly through TNF-alpha and the PPARgamma nuclear receptor. ZAG is a novel adipokine, which may be involved in the local regulation of adipose tissue function.     

Phorbol ester potentiates the growth inhibitory effects of troglitazone via up-regulation of PPARgamma in A549 cells

The activation of peroxisome proliferator-activated receptor gamma (PPARgamma) has been shown to induce growth arrest and differentiation of various cancer cells. In the current study, we investigated the effect of 12-O-tetradecanoylphorbol-13-acetate (TPA) on the expression of PPARgamma and proliferation of A549 cells. TPA elicited a dose- and time-dependent increase in PPARgamma mRNA and protein levels. PPARgamma expression in response to TPA was attenuated by pretreatment with bisindolylmaleimide I, N-acetyl-L-cysteine (NAC) and PD98059. TPA-induced protein kinase C (PKC) activation was linked to the generation of reactive oxygen species (ROS), both of which were indispensable for PPARgamma expression in A549 cells. Pretreatment with bisindolylmaleimide I or NAC blocked TPA-induced phosphorylation of extracellular signal-regulated kinase (ERK), suggesting that ERK-mediated signaling is also involved in the induction of PPARgamma. Furthermore, the growth inhibitory effect of troglitazone was significantly potentiated by prolonged incubation with TPA and was attenuated in the presence of GW9662, a specific inhibitor of PPARgamma. These effects were associated with an induction of cell cycle arrest at G0/G1 phase, which was accompanied by the induction of p21Waf1/Cip1 expression and decreased cyclin D1 expression. Taken together, these observations indicate that TPA synergizes with PPARgamma ligand to inhibit cell growth through up-regulation of PPARgamma expression.     

Expression of the c-fms proto-oncogene and of the cytokine, CSF-1, during mouse embryogenesis

The c-fms gene encodes the cell surface receptor of the colony-stimulating factor, CSF-1. CSF-1 has recently been shown to be expressed in the maternal uterine endometrium of pregnant mice. The ontogenetic and spatial patterns of expression of the murine proto-oncogene c-fms were analyzed in the developing mouse placenta by the technique of in situ hybridization. c-fms expression was not detected in fetally derived tissues until 9.5 days postcoitum (pc) when expression first appeared in the mural trophoblast giant cells. Expression persisted at high levels in trophoblast cells throughout gestation. In the mature placenta from 13.5 days pc on, c-fms was expressed chiefly in the spongiotrophoblast layer and, to a lesser extent, in the labyrinthine trophoblast. CSF-1 expression was first detectable in the uterine epithelium at 8.5 days pc which loosely correlated with the appearance at 7.5 days of c-fms in the decidual cells around the developing egg cylinder. The time course and spatial pattern of expression of these two genes suggest a functional role for the c-fms receptor and its ligand, CSF-1, in trophoblast development and differentiation.     

小鼠胚胎干细胞向脂肪细胞分化过程中PPARγ2基因的表达模式

将PPARγ2基因启动子和报告基因荧光素酶相连接克隆于特定载体构建成表达质粒,电穿孔转染小鼠ES细胞,筛选阳性克隆.诱导ES细胞向脂肪细胞分化,通过定量检测荧光素酶活性跟踪PPARγ2基因的表达情况,以此研究脂肪细胞分化过程中该基因的表达模式.结果显示PPARγ2基因在未分化的ES细胞和EB形成的前两天中不表达,从EB形成的第3天开始表达,直至脂肪细胞分化完成.该基因在已完成分化的脂肪细胞中的表达远强于在分化中的前脂肪细胞中的表达.首次报道了从小鼠ES细胞到脂肪细胞分化过程中PPARγ2基因的表达模式,支持了PPARγ2基因为脂肪组织特异性表达基因的已有报道,并为脂肪细胞分化机理研究提供了线索.     

Cytological distribution of chorionic gonadotropin subunit and placental lactogen messenger RNA in neoplasms derived from human placenta

Normal trophoblast of the human placenta elaborates at least two major protein hormones, chorionic gonadotropin (hCG), and placental lactogen (hPL). There are several gestational trophoblastic diseases of the placenta called hydatidiform mole, invasive mole, and choriocarcinoma. Molar and choriocarcinoma tissues characteristically synthesize large amounts of hCG and small quantities of hPL. To examine the role of trophoblast differentiation in the expression of the hCG and hPL genes, we studied the cytological distribution of their messenger RNA (mRNA) in tissue sections of human hydatidiform mole and choriocarcinoma by in situ hybridization. Histologically, these tissues are in different stages of cellular differentiation. In normal placenta, hCG alpha and - beta mRNA can be localized to some cytotrophoblasts and primarily to the syncytium, whereas hPL mRNA appears only in the syncytial layer. In hydatidiform mole, which still retains placental villous morphology, the hPL gene and hCG alpha and -beta genes are expressed but are poorly localized because of the admixture of cyto- and syncytiotrophoblasts. By contrast, choriocarcinoma, which is devoid of placental villous pattern but in which the cyto- and syncytiotrophoblast-like components are distinguishable, expresses hCG alpha and -beta in the syncytial- like areas but little, if any, hPL. These results suggest that a certain level of trophoblast differentiation, such as villous formation, is associated with hPL expression, while the hCG alpha gene and the hCG beta gene can be expressed in more disorganized tissues that contain cytotrophoblastic elements.     

Expression of mRNA encoding insulin‐like growth factors I and II by uterine tissues and placenta during pregnancy in the rat

The uterus and the placenta synthesize insulin‐like growth factors (IGFs) and insulin‐like binding proteins (IGFBPs). These growth factors are implicated in processes of proliferation and differentiation that occur in the uterus. To determine the patterns of expression of IGFs during rat pregnancy we used in situ hybridization with digoxigenin labeled probes on uterus from day 7 to day 16 of pregnancy. In early gestation days (7–8) both IGF mRNAs showed similar tissue distribution with relative abundance in the stroma and circular muscle layer. On days 11 and 12 expression for IGF‐I mRNA was found in the mesometrial decidua and metrial gland and in the ectoplacental cone while clear expression of IGF‐II mRNA could only be found in the latter. On days 13 and 14, expression for IGF‐I mRNA could be detected in the mesometrial decidua and metrial gland but no expression was observed for IGF‐II mRNA. A gradient of IGF‐I mRNA expression could be observed in the placenta on day 16, with the trophoblastic cells of the basal zone expressing the signal with stronger intensity than in the labyrinthine zone. For IGF‐II mRNA the highest expression was associated with the labyrinthine zone. Endovascular trophoblast was positive for both mRNAs. The spatial and temporal patterns of expression suggests a role for IGFs in the process of decidualization as well as in the establishment, growth and differentiation of the various trophoblast cells of the placenta. Mol. Reprod. Dev. 53:294–305, 1999. © 1999 Wiley‐Liss, Inc.     

Notch1 and the activated NOTCH1 intracellular domain are expressed in differentiated trophoblast cells

The Notch signalling pathway regulates proliferation, cell death and cell type specification that is critical for organogenesis. Mouse models carrying mutations in the Notch signalling pathway display defects in development of the placenta, suggesting that this pathway is required for placental development. In particular, Notch1 mutant embryos exhibit abnormal placental morphogenesis and arrest early in development. However, expression of Notch1 gene has not been detected during placental development. Trophoblast stem cells are derived from the precursor of the placenta and express Notch1. We report that Notch1 is also expressed in differentiated trophoblast cells. Under standard differentiation conditions, Notch1 expression ceases by day 6. Furthermore, the activated NOTCH1 intracellular domain is enriched at the nucleolus of trophoblast stem cells and differentiated trophoblast cells. Our results suggest that NOTCH1 is active in both trophoblast stem cells and differentiated trophoblast cells.     

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.     

Cloning and localization of hFP(S): a six-transmembrane mRNA splice variant of the human FP prostanoid receptor

FP prostanoid receptors are G-protein coupled receptors that mediate the actions of prostaglandin F2alpha. Two isoforms, designated FP(A) and FP(B), have been previously described. We now report the cloning of a FP receptor mRNA alternative splice variant from human heart and placenta cDNA, named hFP(S). The cDNA encoding hFP(S) has a 71 bp insert that produces a frame shift resulting in a truncated receptor lacking transmembrane-7 and the intracellular carboxyl tail. This 71 bp sequence has been identified as a distinct exon localized in the human FP receptor gene on chromosome one. Northern blot analysis suggests that hFPs is expressed in skeletal muscle as well as human heart and placenta. Immunohistochemical microscopy showed positive immunoreactivity on vascular endothelial, trophoblast, and decidual cells from human placenta. hFPs represents the first confirmed alternative splice variant of the human FP prostanoid receptor gene, however, its function is presently unknown.     

An angiotensin II AT1 receptor antagonist, telmisartan augments glucose uptake and GLUT4 protein expression in 3T3-L1 adipocytes

Evidence has accumulated that some of the angiotensin II AT1 receptor antagonists have insulin-sensitizing property. We thus examined the effect of telmisartan on insulin action using 3T3-L1 adipocytes. With standard differentiation inducers, a higher dose of telmisartan effectively facilitated differentiation of 3T3-L1 preadipocytes. Treatment of both differentiating adipocytes and fully differentiated adipocytes with telmisartan caused a dose-dependent increase in mRNA levels for PPARgamma target genes such as aP2 and adiponectin. By contrast, telmisartan attenuated 11beta-hydroxysteroid dehydrogenase type 1 mRNA level in differentiated adipocytes. Of note, we demonstrated for the first time that telmisartan augmented GLUT4 protein expression and 2-deoxy glucose uptake both in basal and insulin-stimulated state of adipocytes, which may contribute, at least partly, to its insulin-sensitizing ability.     

PPAR gamma is required for placental, cardiac, and adipose tissue development.

The nuclear hormone receptor PPAR gamma promotes adipogenesis and macrophage differentiation and is a primary pharmacological target in the treatment of type II diabetes. Here, we show that PPAR gamma gene knockout results in two independent lethal phases. Initially, PPAR gamma deficiency interferes with terminal differentiation of the trophoblast and placental vascularization, leading to severe myocardial thinning and death by E10.0. Supplementing PPAR gamma null embryos with wild-type placentas via aggregation with tetraploid embryos corrects the cardiac defect, implicating a previously unrecognized dependence of the developing heart on a functional placenta. A tetraploid-rescued mutant surviving to term exhibited another lethal combination of pathologies, including lipodystrophy and multiple hemorrhages. These findings both confirm and expand the current known spectrum of physiological functions regulated by PPAR gamma.