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Pre-B-cell colony enhancing factor (PBEF) was first isolated from an activated peripheral blood lymphocyte cDNA library and was found to be involved in the maturation of B-cell precursors. It was subsequently identified as one of the genes upregulated by distending the human fetal membranes in vitro. Here we report on the genomic organization of this gene, which is composed of 11 exons and 10 introns, spanning 34.7 kb of genomic DNA. Neither the gene nor the protein has any homology with other cytokines in any currently available database. The use of two promoters (proximal and distal) may result in differential, tissue specific expression of the PBEF transcripts. The 5'-flanking region lacks the classical sequence motif that would place it with the hematopoietic cytokines; however, it has several putative regulatory elements, suggesting that this gene may be chemically and mechanically responsive to inducers of transcription. The three PBEF mRNA transcripts were observed in both normal and infected human fetal membranes but were significantly upregulated (P<0.05) in severe infection. The PBEF protein was immunolocalized, in both normal and infected tissues, to both the normal fetal cells of the amnion and chorion and the maternal decidua of the membranes, and to the invading neutrophils. These stained strongly and were likely to contribute to the increased expression in infection. The amniotic epithelial cell line (WISH cells) has been used as a model to study PBEF gene modulation. Lipopolysaccharide, interleukin (IL)-1beta, tumour necrosis factor (TNF)alpha and IL-6 all significantly increased the expression of PBEF in 4 h of treatment. The addition of dexamethasone to IL-1beta and TNFalpha significantly reduced the response of PBEF to these cytokines. IL-8 treatment failed to alter PBEF gene expression. Thus PBEF is a cytokine expressed in the normal fetal membranes and upregulated when they are infected. It is likely to have a central role in the mechanism of infection-induced preterm birth.

The human has two relaxins, termed H1 and H2, both of which are biologically active and co-expressed in the decidua, placenta and prostate; in the corpus luteum, the main source of circulating relaxin, only the H2 form is expressed. The reasons for this differential expression of the relaxin genes are unknown. The possibility that their 3'-untranslated regions (UTRs) contribute to this differential expression by affecting their mRNA stabilities was investigated. Thus the 3'-UTRs of both relaxin genes were isolated through a combined 3'-rapid amplification of cDNA ends-PCR (RACE-PCR) using poly (A)(+)RNA from human decidua, placenta, prostate and corpus luteum. The sequences obtained for each 3'-UTR were identical in the tissues examined, were AT-rich (72%) and showed 91% homology between relaxin H1 and H2 when maximally aligned to include several gaps, the significance of which is unknown. Relaxin H1 has two, and relaxin H2 has one, poly (A)(+) signal, in addition to one cytoplasmic polyadenylation element 30 nucleotides upstream of this. The mRNA levels of relaxin H1 and H2 in the prostate adenocarcinoma LNCaP.FGC cell line were determined by quantitative competitive RT-PCR. Relaxin H1 had a 10-fold greater number of molecules (approximately 2.5x10(7)) per microgram of total RNA than relaxin H2 (approximately 2.5x10(6)). The stability of relaxin H1 and H2 mRNAs were compared in LNCaP cells treated with the transcription inhibitor actinomycin D (10 mM) for 0, 1, 2, 4, 8, 10, 14, or 24 h. Half-lives of 3.17 days for relaxin H1 mRNA and 11. 4 h for relaxin H2 mRNA were obtained from semi-logarithmic plots. Thus both mRNAs are relatively stable; however, relaxin H1 mRNA is considerably more stable than relaxin H2, at least in LNCaP cells. This difference in their mRNA stability may partly explain the greater level of expression of relaxin H1 in these cells.

Relaxins are known endocrine and autocrine/paracrine hormones that play a major role in reproduction. In the human there are two relaxin genes, H1 and H2 which share 90% sequence homology within their coding region. The biological and evolutionary significance of two highly homologous and biologically active human relaxins is unknown. In order to achieve a better understanding of the regulatory mechanisms involved in the differential expression of these two genes and to gain insight into their role(s) in the preterm premature rupture of the membranes, we have investigated the properties of their 5'-upstream regions and mapped them both by radiation hybrid and breakpoint mapping into the same chromosome 9p24.1 locus. The 5' ends of these relaxin genes could be divided into a proximal highly homologous segment and a distal non-homologous region. Within the proximal region are contained several putative regulatory elements common to both genes, suggesting a similar regulatory mechanism. The clustering of the relaxin genes within the same chromosomal locus suggests that these genes may be under a common regulation. On the other hand, a distinct gene-specific regulation may also exist for the individual relaxin genes since cis elements specific to each gene were identified at their 5' ends. Moreover, the observed divergence at the distal region of their 5'-upstream sequences may provide the structural features that act as gene-specific transcription regulators. Since the two genes are highly homologous in both their coding and flanking regions, the divergence at the distal region of their 5' ends may be important in the regulation of these genes and in their involvement in the pathology of preterm birth.

The human placenta and decidua are intrauterine production sites for a range of polypeptide hormones. Relaxin is one of these hormones, its production having been demonstrated by immunocytochemistry and Northern analysis. There are two relaxin genes in the human genome, termed H1 and H2; only the latter is expressed in cyclic and pregnant corpus luteum. However, it has recently been shown that both genes are expressed in the decidua and placenta. It is not known whether both are translated. These hormone(s) may act in a paracrine fashion and be partly responsible for the control of enzymes and inhibitors involved in collagen remodelling in the fetal membranes in the last weeks of pregnancy. An autocrine role of decidual relaxin and a possible decidual-cell/macrophage/extracellular-matrix interaction is described; this may act as a unit in the elaboration of a range of hormones.