Expression analysis of the entire MMP and TIMP gene families during mouse tissue development.

Matrix metalloproteinases (MMPs) and adamalysins (ADAMs) cleave many extracellular proteins, including matrix, growth factors, and receptors. We profiled the RNA levels of every MMP, several ADAMs, and inhibitors of metalloproteinases (TIMPs and RECK) in numerous mouse tissues during development and in the uterus during pregnancy. Observations include: most secreted MMPs are expressed at low to undetectable levels in tissues, whereas membrane-bound MMPs, ADAMs and inhibitors are abundant; almost every proteinase and inhibitor is present in the uterus or placenta at some time during gestation; the mouse collagenases mColA and mColB are found exclusively in the uterus and testis; and each tissue has its unique signature of proteinase and inhibitor expression.

The POU homeodomain protein OCT3 as a potential transcriptional activator for fibroblast growth factor-4 (FGF-4) in human breast cancer cells.

The POU (representing a homeodomain protein family of which the founder members are Pit-1, Oct-1/2 and Unc-86) homeodomain protein OCT3/Oct-3 (where OCT stands for octamer-binding protein) is an embryonic transcription factor expressed in oocytes, embryonic stem and embryonic carcinoma cells. We have demonstrated previously that human breast cancer cells regain the ability to express OCT3 mRNA [Jin, Branch, Zhang, Qi, Youngson and Goss (1999) Int. J. Cancer 81, 104-112]. Antibodies against human OCT3 were not available when this study was conducted. By using a human OCT3-glutathione S-transferase fusion protein to affinity purify a polyclonal antibody against the mouse Oct-3, we obtained an antibody that enabled us to detect OCT3 in human breast cancer cells by Western-blot analysis. Thus we have now confirmed that OCT3 is expressed in human breast cancer cells but not in normal human breasts and in three other organs. When breast cancer cell lines were treated with all- trans -retinoic acid, OCT3 expression was repressed, associated with decreased cell proliferation. Although another POU protein Brn-3 has been shown to be a repressor for BRCA1 (breast-cancer susceptibility gene 1), OCT3 does not repress human or mouse BRCA1/Brca-1 promoters. However, OCT3 is capable of activating a fusion promoter containing the fibroblast growth factor-4 (FGF-4) enhancer element. In addition, we documented for the first time that human breast cancer cells express FGF-4 protein, and its expression could be inhibited by all- trans -retinoic acid. Furthermore, overexpressing OCT3 stimulated endogenous FGF-4 expression in MCF7 breast cancer cell line. These observations indicate that OCT3 protein is selectively expressed in human breast cancer cells, and its expression may be implicated in mammary gland tumorigenesis via up-regulating FGF-4 expression.

Identification of an initiator-like element essential for the expression of the tissue inhibitor of metalloproteinases-4 (Timp-4) gene.

We have used real-time quantitative reverse transcriptase PCR (TaqMan) to quantify the expression of the four tissue inhibitor of metalloproteinases (Timp) genes in mouse tissues during development and in the adult. Among the four Timp genes, Timp-4 shows the most restricted pattern of expression, with highest RNA levels in brain, heart and testes. These data indicate that in the brain, Timp-4 transcripts are temporally regulated during development, becoming more abundant than those of the other Timps after birth. Cloning of the Timp-4 gene confirmed a five-exon organization resembling that of Timp-2 and Timp-3, and like all Timps, Timp-4 is located within an intron of a synapsin gene. Ribonuclease protection analysis and 5'-rapid amplification of cDNA ends PCR identified multiple transcription starts for Timp-4 from brain and heart mRNA. The promoter region of Timp-4 was functional in transient transfection analysis in mouse C3H10T1/2 fibroblasts, where it directed basal expression that was non-inducible by serum. The TATA-less promoter contains consensus motifs for Sp1 and an inverted CCAAT box upstream of an initiator-like element that is in close proximity to a transcription start site. Mutation of the CCAAT box caused a 2-fold increase in reporter expression. More significantly, mutation of the Sp1 motif or initiator-like element almost completely abolished reporter expression. This first functional characterization of the Timp-4 promoter shows it to be distinct from other members of the Timp family and provides insights into potential mechanisms controlling the tight spatio-temporal expression pattern of the gene.

Subtilisin proprotein convertase-6 expression in the mouse uterus during implantation and artificially induced decidualization.

During implantation, a balance of factors regulates the invasive properties of the embryo and the anti-invasive properties of uterine decidua. Although antiproteinases such as the metalloproteinase inhibitor TIMP-3 are thought to play critical roles in preventing the overaggressive invasion of trophoblasts, the mechanism of antiproteinase regulation is unknown. Recently, the prohormone convertase SPC-6 has been found to be co-expressed in embryo-proximal decidua in association with TIMP-3. As members of this serine proteinase family are known to activate latent TGFbeta family members which regulate decidual TIMP-3 levels, we sought to characterize the expression of SPC-6 during pregnancy and artificial decidualization. In this study, we demonstrate that the zone of SPC-6 gene expression exhibits a great degree of temporal and spatial overlap with TIMP-3 gene expression in uterine decidua from E5.5 through to E8.5. Like TIMP-3, we demonstrate that SPC-6 expression is induced during the decidual cell response using an in vivo model of artificial decidualization. Both the secreted and membrane bound forms of SPC-6 are expressed throughout the period of decidualization, suggesting that SPC-6 may play multiple roles during this developmental period. This is confirmed by our observation of the movement of SPC-6 expression to the presumptive placental region, as TIMP-3 expression regresses at the implantation site.

An improved polymerase chain reaction assay for the detection of Tritrichomonas foetus in cattle.

An improved polymerase chain reaction test has been developed to detect Tritrichomonas foetus, the causative agent of trichomoniasis in cattle. The test amplifies a region of the 5.8S ribosomal RNA gene of T. foetus, and it is simple, sensitive, and specific when compared with traditional methods to examine field samples.