Use of cDNA arrays to generate differential expression profiles for inflammatory genes in human gestational membranes delivered at term and preterm.

Inflammatory processes are implicated in preterm labour (PTL). To identify potential novel markers for PTL, we have used commercial cDNA arrays to generate profiles of differential expression of inflammation-associated genes in gestational membranes with term and PTL. RNA for cDNA probe synthesis was isolated from reflected human amnion and choriodecidua membranes delivered following Caesarean section at term before the onset of labour (TNL, n = 4), spontaneous labour at term (TSL, n = 4), and PTL with and without chorioamnionitis (PTL(+INF) and PTL(-INF) respectively, n = 4 each). Profiles were displayed relative to TNL and statistical comparisons of TSL versus TNL and PTL(+INF) versus PTL(-INF) were performed. Elevated expression of chemokines macrophage inflammatory protein 1beta(MIP-1beta) and pulmonary and activation-regulated chemokine (PARC) was observed in PTL(+INF) compared to PTL(-INF) amnion and choriodecidua respectively (P = 0.03). Likewise, the cytokines oncostatin-M and pre-B cell enhancing factor (PBEF) were more highly expressed in PTL(+INF) compared with PTL(-INF) and in TSL compared with TNL respectively (P = 0.03). Conversely, inhibin A, tissue inhibitors of matrix metalloproteinase (TIMP)-3 and TIMP-4 were all significantly elevated in PTL(-INF) compared with PTL(+INF) (P = 0.03). Furthermore, differential expression patterns of classes of genes, grouped according to function (e.g. chemokines), were noted. The cDNA array approach holds promise for identification of new candidate markers or combinations thereof for prediction or diagnosis of PTL, as well as for increasing our understanding of the particular aetiologies involved.

Transcriptional regulation of prostaglandin-H-synthase-1 in the amnion-derived AV3 cell line.

Prostaglandin-H-synthase-1 (PGHS-1), while constitutively expressed in most tissues, increases in abundance in human gestational membranes at term. This suggests that PGHS-1 may be up-regulated in preparation for labor, and thus might be a key determinant in timing labor onset. We conducted transient transfection experiments in amnion-derived AV3 cells utilizing pPGHS1CAT to identify substances that might regulate PGHS-1 expression in amnion. Transforming growth factor-beta (1 ng/ml) and 15-deoxy-delta(12,14) prostaglandin J2 (1 microM) significantly (P < 0.05) (33% and 44% respectively) increased PGHS-1 promoter activity. The activity decreased significantly (P < 0.05) in response to interleukin-1 (IL-1)beta (1 ng/ml) (45%), tumor necrosis factor (TNF)-alpha (50 ng/ml) (34%), epidermal growth factor (10 ng/ml) (54%), phorbol myristate acetate (10 nM) (70%), IL-4 (10 ng/ml) (50%), IL-8 (100 ng/ml) (72%) and Activin A (25 ng/ml) (32%). Whether this degree of change in promoter activity leads to physiologically relevant alterations in the amounts of PGHS-1 present in cells remains to be determined.

The 15-deoxy-delta(12,14)-prostaglandin J(2)receptor, peroxisome proliferator activated receptor-gamma (PPARgamma) is expressed in human gestational tissues and is functionally active in JEG3 choriocarcinoma cells.

RNA was extracted from human gestational membranes and villous placental tissue following spontaneous delivery (n = 15) or elective caesarean section (n = 15) at term. The samples were subjected to Northern analysis, using a 2 kb cDNA probe for peroxisome proliferator activated receptor (PPAR)-gamma. The mRNA was detectable in all choriodecidual and villous placental samples, irrespective of mode of delivery, but was only rarely detectable in the amnion. The JEG3 choriocarcinoma cell line also expressed PPARgamma. In order to evaluate PPAR mediated transcriptional activation in JEG3 cells, the cells were transfected with pTK-PPREx3-luc, a PPAR response element (PPRE) containing luciferase reporter construct. Subsequent treatment with 10 microm 15-deoxy-delta(12,14)prostaglandin J(2)(15dPGJ(2)) resulted in an eight-fold stimulation of luciferase production relative to controls transfected with the same construct lacking the PPRE. This stimulation was concentration-dependent. These results suggest roles for PPARgamma and its ligand in lipid, steroid and inflammatory mediator homeostasis and in remodelling of gestational tissues.

Subcellular localization of prostaglandin H synthase-2 in a human amnion cell line: implications for nuclear localized prostaglandin signaling pathways.

We have determined that prostaglandin H synthase-2 localises strongly to the nuclear membrane as well as being found in the endoplasmic reticulum in human amnion-derived WISH cells which have been stimulated with interleukin 1beta and phorbol ester. This is consistent with findings in cells of non-reproductive origin. There is strong evidence that prostaglandin J2 derivatives, which in other tissues exhibit tumour suppressing, antiproliferative and/or differentiation promoting activities, act through binding of intracellular receptors which then enter the nucleus. In addition, some arachidonic acid derivatives are clearly generated by enzymes at the nuclear envelope and localise to sites in nuclei or bind sites in nuclei. The WISH cell line will make an excellent system for studying these perinuclear intracellular prostanoid signaling mechanisms.

Amnion-derived cells express intercellular adhesion molecule-1: regulation by cytokines.

We have examined the expression of the intercellular adhesion molecule-1 (ICAM-1) mRNA in primary and established amnion-derived cell cultures and regulation of this expression by tumour necrosis factor-alpha (TNF-alpha) and interleukin (IL)-1beta. TNF-alpha (50 ng/ml) and IL-1beta (1.0 ng/ml) induced 18- and 11-fold increases respectively in expression of the ICAM-1 mRNA in WISH cells (an amnion epithelium-derived cell line). The increase was detectable within one hour of treatment and peaked by two hours. The protein synthesis inhibitor, cycloheximide (10 microg/ml) did not inhibit this induction. Increased levels of ICAM-1 protein were detected in the cells within 4 h after initiation of treatment with either cytokine. By 16 h of treatment with IL-1beta or TNF-alpha ICAM-1 reached 40 and 73 pg/microg cellular protein, representing 6- and 11-fold stimulations respectively. In primary amnion cells, basal expression of ICAM-1 mRNA was undetectable. However, TNF-alpha (50 ng/ml) induced ICAM-1 mRNA within two hours, peak expression being reached between four and eight hours after initiation of treatment. The present report demonstrates for the first time that amnion derived cells can express ICAM-1 and, further, that this expression is regulated by pro-inflammatory cytokines. This has implications for the amnion as a possible source for soluble ICAM-1, for this gene product as a marker for preterm labour, and for participation of the amnion, additional to its reported secretory role, in inflammatory processes of the fetal membranes.

Cytokines regulate prostaglandin H synthase-1 transcription in human amnion-derived cells.

We have isolated the prostaglandin H synthase-1 (PGHS-1) promoter from the human amnion cell line WISH by long template PCR. The fragment was 1124 base pairs in length and shared a 96% sequence identity with the sequence in GenBank. The putative transcription start site is located 18 bp upstream of the start codon. The sequence is TATA-less, but contains multiple Sp-1 sites and a GC box at -132. The fragment was subcloned into the promoterless reporter construct pBLCAT3 to produce the promoter reporter construct pPGHS1CAT. pPGHS1CAT expression in amnion-derived AV3 cells was inhibited by the pro-inflammatory cytokines tumor necrosis factor-alpha (TNF-alpha) and interleukin 1-beta (IL-1beta). PGHS-1 mRNA levels however, were unchanged over a 16-h time course with either treatment. These results suggest that PGHS-1 transcription is regulated in a negative manner by cytokines in human amnion-derived cells.

Methylation is co-ordinated on the putative replication origins of Physarum ribosomal DNA.

In Physarum polycephalum, the ribosomal DNA is found as 60,000 base-pair palindromes. Each rDNA has four symmetrically arranged replication origins flanked by ribosomal RNA genes. A particular sequence, the putative replication origin, is repeated at the approximate position of each origin and nowhere else in the molecule. On a typical rDNA molecule, only one origin is active per replication cycle. We show that both the level and co-ordination of methylation result in asymmetrically methylated rDNA molecules that are particularly hypomethylated at one of their four putative replication origins. This pattern of methylation on a typical rDNA molecule is consistent with a model where hypomethylation is a determinant of origin activity.