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Elevated intracellular levels of S100A4, an S100-related calcium-binding protein, induce metastatic capability in benign mammary tumor-derived epithelial cells and in transgenic mice bearing oncogene-induced benign mammary tumors. The S100A4(p9Ka) gene in rat mammary epithelial cells expressing low levels of S100A4 yields a reduced number of fragments upon digestion with the methylation-sensitive restriction enzyme, HpaII, compared with the gene from high S100A4-expressing cells. Genomic sequencing of two potential regulatory elements in the S100A4 gene, an intronic enhancer and TATA box region, revealed that in low S100A4-expressing cells, most cytosine bases exhibited high levels of resistance to conversion to thymine by sodium bisulfite. In derivative cell lines, which express high levels of S100A4, only a small number of cytosine bases were resistant to treatment with sodium bisulfite. In contrast, cytosine bases in the DNA surrounding an upstream regulatory region, which binds inhibitory GC factor in the low-expressing cell lines, are sensitive to conversion to thymine by sodium bisulfite in both low- and high-expressing cell lines. The results suggest that the rat S100A4 gene is maintained in a different state in the low-expressing cell lines and that this state might be a consequence of the pattern of methylation in this regulated gene that does not contain a CpG island.

HLA-G, a nonclassical class I molecule, is expressed by trophoblasts, the only fetal cells in direct contact with maternal tissue. Results of previous experiments suggested that a 244-bp region located over 1 kb 5' from exon 1 is critical for extraembryonic expression of HLA-G in transgenic mice. We report here the production of HLA-G transgenic lines with a 6.0-kb HLA-G transgene that includes the 244-bp region. These lines exhibit copy number-dependent and developmentally appropriate transgene expression. One HLA-G transgenic line, G.3.2, exhibits extraembryonic HLA-G mRNA expression levels similar to those seen in human extraembryonic tissues. Studies of the cell-type-specific localization of HLA-G mRNA in placentas from the G.3.2 HLA-G transgenic mouse line show predominant localization of the HLA-G message in the spongiotrophoblast layer. This layer is in a similar anatomic location to the HLA-G-expressing human cytotrophoblast shell. The G.3.2 HLA-G transgenic mouse line should serve as an appropriate model for the study of HLA-G function at the maternal-fetal interface.

Abnormal expression of polypeptide growth factors and their receptors is closely associated with tumorigenic transformation. In this study tumor necrosis factor-alpha (TNF-alpha) mRNA and protein were analyzed in polyps and proliferative lesions of endometrium as well as in low and high grade endometrial tumors by using in situ hybridization and immunocytochemistry. All samples contained products of the TNF-alpha gene. Histochemical scores (HS), which reflect the proportion of cells positive for TNF-alpha message or protein and the intensities of the signals, were higher for epithelial than for stromal cells. Benign lesions (endometrial polyps) contained little TNF-alpha mRNA or protein, whereas specific message was abundant in proliferative lesions (hyperplasia, adenofibroma). Although neoplastic cells in both low and high grade endometrial tumors contained TNF-alpha mRNA, two major differences were observed: HS for TNF-alpha mRNA were significantly less in low grade than in high grade neoplasms, and TNF-alpha message was restricted to the nucleus in low grade adenocarcinoma cells but was abundant in the cytoplasm of high grade tumor cells. In contrast to cells in benign and proliferative lesions, TNF-alpha protein scores in endometrial tumor cells were inversely rather than positively correlated with TNF-alpha mRNA scores. Collectively, the findings in this study are consistent with the postulate that TNF-alpha is useful to endometrial tumor cells and suggest that production may increase as cells diverge from normal.

Transforming growth factor-beta 1 (TGF-beta 1) has major effects on hematopoietic cell proliferation, migration and function. In an effort to learn whether or not TGF-beta 1 might be among the cytokines that influence uterine, placental and embryonic hematopoietic cells, in situ hybridization was used to map expression of the TGF-beta 1 gene through gestation in the rat. In cycling uteri, hybridization signals with a biotinylated TGF-beta 1 antisense RNA probe were comparatively weak and were restricted to myometrium. During pregnancy, TGF-beta 1 mRNA appeared in specific cell lineages in an ordered temporal sequence. In early post-implantation tissues (g.d. 5-8), TGF-beta 1 transcripts were prominent in uterine epithelial cells, muscle and decidual cells. At g.d. 15, hybridization signals were particularly strong in uterine epithelial cells, muscle and placental trophoblast cells. At this stage, TGF-beta 1 mRNA was also present in uterine cells resembling macrophages and in natural killer-like (GMG) cells. Steady state levels of specific message in uteri and placentas declined at late stages of gestation (g.d. 18, 21). In rat embryos, specific transcripts were identified at g.d. 9 and were prominently displayed in various types of embryonic cells from mid-gestation onwards. The results of this study are consistent with the postulate that throughout pregnancy in the rat, TGF-beta 1 may, in addition to its other functions, influence the proportions, patterns of distribution and activities of maternal and fetal hematopoietic cells.

In order to map mouse tumor necrosis factor-alpha (TNF) gene expression in detail and to determine whether transcription or translation of the TNF gene is regulated by uterine colony stimulating factor-1 (CSF-1), preimplantation embryos, oviducts, uteri, and uteroplacental units were studied in various strains of mice. These included homozygous osteopetrotic (op/op) female mice, which completely lack CSF-1, and heterozygous (+/op) females, which have normal levels of CSF-1. TNF mRNA was identified in all samples except preimplantation embryos by use of Northern blot hybridization or reverse transcriptase polymerase chain reaction. In situ hybridization and immunocytochemical experiments showed that the TNF gene was expressed in mouse oviduct and uterine epithelial cells, decidual cells, macrophage-like cells, placental trophoblast, and embryos. Despite an absence of CSF-1, TNF gene expression in the uteri, placentas, and embryos of op/op mothers did not differ in any major respect from expression in +/op or other strains of mice. The results of this study therefore indicate that the TNF gene is transcribed and translated in an ordered sequence through mouse gestation, and that maternal CSF-1 is not essential to expression of this cytokine gene. Collectively, these findings are consistent with a major role for TNF in mouse reproduction and development and with a potential compensatory function for this potent polypeptide factor in CSF-1 deficiency.

Although previous studies have shown that the tumor necrosis factor-alpha (TNF) gene is expressed in pregnancy decidua, it has not been determined whether this gene is transcribed or translated in the endometrium prior to implantation. To address this question and to identify cells expressing the TNF gene, samples of normal cycling human endometria were tested for TNF mRNA by in situ hybridization using a biotinylated antisense RNA probe, and the corresponding protein was localized in the same tissues by immunocytochemistry with a monoclonal antibody to TNF. Both TNF message and protein were identified in the endometrium throughout the menstrual cycle, and the same types of cells that contained transcripts also contained protein. As judged by the intensities of the hybridization signals, TNF mRNA increased during the proliferative phases, declined in the early secretory phase, and again rose during mid-to-late secretory phases, suggesting positive associations with levels of female sex hormones that show similar cyclic fluctuations. At the initiation of the cycle, transcripts were primarily localized to glandular epithelial cells whereas by the midproliferative phase, message was also present in stromal cells. Hybridization signals were consistently more intense in functionalis-region than in basalis-region stromal cells, and were frequently stronger in basalis-region glandular epithelia than in functionalis-region glands. These observations document that the TNF gene is expressed in normal cycling endometria, suggest that ovarian hormones may regulate TNF gene transcription, and identify differences in specific endometrial compartments. The findings are also consistent with the postulate that TNF is a local mediator of cellular communications in the human endometrium.

Tumor necrosis factor alpha (TNF-alpha), a polypeptide that regulates cellular growth and modulates the synthesis of various cell surface and secreted molecules, has been identified in the pregnant uterus. To determine which specific cells transcribed and translated this gene, extraembryonic fetal tissues (placenta and membranes) and uterine tissue from early and late stages of gestation were analyzed for TNF-alpha mRNA by in situ hybridization using biotinylated antisense and sense TNF-alpha probes, and for immunoreactive TNF-alpha using two monoclonal antibodies. Tumor necrosis factor-alpha transcripts and protein were identified in both extraembryonic and maternal cells. In first-trimester placental villi, TNF-alpha mRNA was present in syncytiotrophoblast but was low to absent in cytotrophoblast and villous stromal cells. Decidual and epithelial cells in maternal tissues contained TNF-alpha transcripts. In term placentas, both syncytiotrophoblast and villous stromal cells contained TNF-alpha mRNA, and transcripts were present in maternal cells in the decidua adjacent to the extraplacental membranes. In both first-trimester and term tissues, coincident expression of TNF-alpha mRNA and immunoreactive TNF-alpha was demonstrated. The results of this study show that TNF-alpha is synthesized by cells in both extraembryonic membranes and maternal tissues during human gestation and that transcription in specific types of cells is influenced by gestational age. These observations are consistent with a major role for TNF-alpha in the dynamic developmental events of human pregnancy.

The pregnant uterus contains TNF-alpha, a potent cytokine with pleotrophic effects. The uterus also contains numerous macrophages, which are well described sources of TNF-alpha. In order to determine if uterine TNF-alpha originated with these macrophages, patterns of macrophage tissue distribution and population densities were first established in rat uterine tissues from early, mid, and late stages of gestation by immunohistology. The potential of these and other uterine and placental cells to synthesize TNF-alpha was then tested by in situ hybridization with the use of biotinylated antisense and sense RNA probes. Although TNF-alpha mRNA was present during all stages of pregnancy, hybridization signals were highest in gestation day 15 tissues. The predominant TNF-alpha mRNA-containing cells were uterine epithelium, decidual cells, and placental trophoblast cells; these cells also contained immunoreactive TNF. Transcription of the TNF gene in the uterus and placenta was also documented by Northern blotting experiments, which showed that the transcript sizes for uterine, placental, and macrophage TNF mRNA were similar. Although stromal cells that were located in macrophage-rich uterine compartments (myometrium, metrial gland) contained TNF-alpha mRNA, the cells did not contain high levels of immunoreactive TNF-alpha. Thus, cells other than macrophages are likely to be the major contributors of TNF-alpha during uncomplicated pregnancy in the rat.

Rat M2-type pyruvate kinase mRNA was enriched from total polysomes isolated from AH-130 Yoshida ascites hepatoma cells, which contain a very high concentration of the M2-type enzyme, by immunoprecipitation with a specific antibody and Staphylococcus aureus cells. Double-stranded cDNA synthesized from the enriched mRNA was inserted into the PstI site of pBR322, and the resultant recombinant DNA molecules were used to transform Escherichia coli. Three clones containing DNA complementary to M2-type pyruvate kinase mRNA were identified by colony hybridization, hybrid-arrested translation, and hybrid-selected translation. A partial restriction map was constructed covering about 1.44 kilobase pairs. The cloned region of the M2-type mRNA showed a high degree of sequence homology with the M1-type mRNA and some homology with the L-type mRNA as determined by dot blot hybridization. The molecular size of the M2-type mRNA, which was estimated to be 2.35-2.65 kilobases on denaturing gel, was the same as that of the M1-type mRNA. The level of hepatic M2-type pyruvate kinase mRNA measured by hybridization assay using cloned cDNA as a probe was increased 2.5-fold 1 day and 3.9-fold 2 days after partial hepatectomy and then started to decrease. This induction was followed by similar changes in the enzyme activity. AH-130 hepatoma cells contained 100-150 times more M2-type isozyme mRNA than regenerating liver. These results suggest that the increased levels of M2-type isozyme in regenerating liver and hepatoma cells are primarily due to elevation of hybridizable M2-type mRNA concentration.

Rat liver L-type pyruvate kinase mRNA was enriched from total polysomes by immunoprecipitation with a specific antibody and Staphylococcus aureus cells. Double-stranded cDNA synthesized from the enriched mRNA was inserted into the PstI site of pBR322, and the resultant recombinant DNA molecules were used to transform Escherichia coli. Three clones containing DNA complementary to L-type pyruvate kinase mRNA were identified by colony hybridization, hybrid-selected translation, and dot blot hybridization. A partial restriction endonuclease map of cDNA inserts was constructed covering about 1.86 kilobase pairs. The cDNA insert of recombinant plasmid pLPK-14 was used as a hybridization probe to quantitate L-type pyruvate kinase mRNA in rat liver after various treatments. The level of hybridizable L-type enzyme mRNA was markedly increased by a high carbohydrate diet. Diabetes greatly reduced the mRNA level in the liver of rats maintained on a high carbohydrate diet, but insulin administration resulted in restoration of the mRNA level to normal within 24 h. These changes were approximately proportional to the changes in the level of translatable L-type pyruvate kinase mRNA. Thus, we conclude that nutritional and hormonal regulation of synthesis of hepatic L-type pyruvate kinase occurs at the pretranslational level.