Isolation and analysis of the 3'-untranslated regions of the human relaxin H1 and H2 genes.

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.

Fetal membrane distention: I. Differentially expressed genes regulated by acute distention in amniotic epithelial (WISH) cells.

OBJECTIVE: This study was undertaken to determine which genes were up-regulated by acute distention in an amniotic epithelial cell line and in human fetal membranes. STUDY DESIGN: WISH cells, a human amniotic epithelial cell line, were grown on silicone elastomer sheets coated with extracellular matrix and reproducibly distended by 40% in a novel device for 4 hours. Differential gene expression was analyzed by means of suppression subtractive hybridization. Expression of the identified genes was then quantitated by Northern blot analysis in fetal membrane explants after distention in the same device for 4 hours. The effect of distention on apoptosis of the cells and tissue samples was concomitantly studied by means of the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling method. RESULTS: The genes for interleukin 8 and pre-B-cell colony-enhancing factor were found to be up-regulated in both the WISH cells and the distended fetal membranes. The apoptotic index values in both the cells and the tissue samples were unaffected by distention. CONCLUSIONS: Acute distention induces the up-regulation of interleukin 8 and pre-B-cell colony-enhancing factor in both WISH cells and human fetal membranes and does not cause apoptosis.

Genes upregulated in human fetal membranes by infection or labor.

OBJECTIVE: To determine whether suppression subtractive hybridization can detect genes in fetal membranes that are upregulated by infection, preterm premature rupture of membranes (PROM), or labor. METHODS: Using suppression subtractive hybridization, messenger RNAs from a preterm fetal membrane obtained at cesarean delivery without labor (control) were subtracted from a pool of messenger RNAs of three patients with preterm PROM and vaginal delivery. Eight candidate genes identified as upregulated were quantitated by Northern analysis in each of the tissues and in additional patient subgroups. RESULTS: Eight differentially upregulated genes were identified in preterm labor with PROM. Four of the genes are known to be involved in the response to inflammation or infection, and subsequent histologic examination showed one of the preterm PROM tissues to be infected. F-actin capping protein and chitinase precursor, not previously known to be involved in infection, were also upregulated in the infected tissue from preterm PROM. Northern blots using additional subgroups of patients showed that a regulatory G-protein signaling protein gene was significantly upregulated at term by labor in addition to significant upregulation of interleukin-8. There was a strong correlation between the gene expression for complement factor-B and duration of membrane rupture in the patients with preterm PROM. CONCLUSION: Two novel genes potentially involved in the response to inflammation or infection have been identified. A regulatory G-protein signaling protein and interleukin-8 gene expression were upregulated by labor. Complement factor-B gene expression was directly related to the duration of membrane rupture.

Suppression subtractive hybridization (SSH) identifies prolactin stimulation of p38 MAP kinase gene expression in Nb2 T lymphoma cells: molecular cloning of rat p38 MAP kinase.

Suppression Subtractive Hybridization (SSH) has been used to compare rat Nb2 cells treated with prolactin for 1 hour with untreated cells. This new method for identifying differentially expressed genes showed that the mRNAs for at least three genes were elevated by such treatment, including a p38 mitogen activated protein (MAP) kinase. The p38 MAP kinase was cloned and the full length cDNA sequence was determined.

The human Leydig insulin-like (hLEY I-L) gene is expressed in the corpus luteum and trophoblast.

A novel member of the insulin superfamily has previously been shown to be expressed only in porcine pre and postnatal Leydig cells and its human analogue demonstrated in the human testes but not in other organs and hence has been tentatively termed Leydig insulin-like peptide (Ley I-L). However, we have detected hLey I-L gene expression in the cyclic human corpus luteum and trophoblast by the reverse transcriptase-polymerase chain reaction (RT-PCR), with primers selected from the published human Ley I-L sequence. Normal and neoplastic breast tissue and fetal membranes with adhering decidua did not express the gene. The overall sequence of the trophoblast gene was in agreement with that reported with minor changes only in the putative connecting peptide, confirmed by restricted enzyme digestion. A 290 bp RT-PCR product was cloned and used as a cDNA probe in Northern analyses; hybridization was readily shown with cyclic corpora lutea but not with other tissues. The broader spectrum of the expression of this gene will warrant a new nomenclature when its biological activities are known. The different intensity of expression in the corpus luteum and trophoblast suggest endocrine and autocrine/paracrine roles respectively in these tissues in which H2 relaxin and H1/H2 relaxins coexist respectively and at similar levels of expression. Operationally the amino acid sequence homologies between the processed H1 and H2 relaxins and hLey I-L may qualify the specificity claimed for immunostaining the human relaxins in the corpus luteum and trophoblast.

Human relaxins in normal, benign and neoplastic breast tissue.

Immunoreactive relaxin is present in human breast cyst fluid and postpartum milk without concurrent detectable serum levels, suggesting that the breast is a site of relaxin synthesis. Monoclonal and polyclonal antibodies to human relaxin H2 have been used to immunolocalize relaxins in normal, benign and neoplastic breast tissues with the avidin-biotin immunostaining technique. In view of the similarities in amino acid sequence between H1 and H2 relaxins, these antibodies to H2 relaxin are likely to detect either or both relaxins present in tissue sections. Staining patterns with these antibodies were identical and showed positive diffuse cytoplasmic staining in normal, lobular and ductal epithelium and in myoepithelial cells in breast tissues from normal prepubertal, cyclic, gestational, lactational and postmenopausal females. Relaxin staining was also present in epithelial and myoepithelial cells of ducts and lobules in benign breast disease as well as in metaplastic epithelium of apocrine microcysts. All breast carcinomas (infiltrating ductal, tubular, medullary, intraductal and infiltrating lobular carcinomas) had strong uniform cytoplasmic staining within the neoplastic epithelial cells. All staining was abolished in normal and neoplastic tissues when the polyclonal antibody was preabsorbed with relaxin. It was necessary to distinguish between the possibilities of relaxins being sequestered by breast tissue and local synthesis. Therefore, the expression of the H1, H2 or both human relaxin genes in normal and neoplastic breast tissues was studied by the isolation of RNA, synthesis of first strand cDNA and amplification by PCR using primer sets which amplified either both H1 and H2, or specifically only H1 or H2 relaxin. The coamplification of both relaxin genes was verified by Southern analysis, diagnostic restriction enzyme digestion and sequencing. The primer set for H1 relaxin detected H1 gene expression in 1 out of 8 normal and 9 out of 12 neoplastic breast RNA samples. The H2 relaxin gene was found to be expressed in 3 out of 8 of the normal samples but in all 12 of the neoplastic samples, suggesting that this gene is expressed at higher copy number in the neoplastic tissues. This is the first demonstration of the cellular immunolocalization of relaxin and relaxin gene expression in normal and neoplastic breast. This should allow further exploration of relaxin's role(s) in normal breast physiology and in its tumorigenesis.