Human familial and sporadic breast cancer: analysis of the coding regions of the 17β-hydroxysteroid dehydrogenase 2 gene (EDH17B2) using a single-strand conformation polymorphism assay

17-Hydroxysteroid dehydrogenase (17HSD) is one of the key enzymes in estrogen metabolism, catalyzing the reversible reaction between estradiol and the less active estrogen, estrone. The gene encoding this enzyme, EDH17B2, has been mapped to chromosome 17, region q12–q21, in the vicinity of BRCA1, an as yet unidentified gene that appears to be involved in familial breast cancer and in familial ovarian cancer. The possibility that EDH17B2 gene is the same as BRCA1 was tested by screening for mutations in the coding regions of EDH17B2, using a polymerase chain reaction/single-strand conformation polymorphism method. An AG transition creating a new BstUI site at exon 6 was the only frequent sequence alteration found in the coding region of the gene. This mutation also led to an amino acid substitution of serine to glycine at position 312 (312^S312^G) in the 17HSD protein. Since the nucleotide change was detected both in specimens from patients with familial or sporadic cancer and in control samples, and at similar rates, this mutation appears to be of a polymorphic nature. In addition, a rare polymorphism located at intron 5 was detected. This CT substitution creates a BbvI site and is not thought to have any effect on 17HSD activity. The results indicate that there are no major alterations in the coding areas of EDH17B2 and thus studies testing the hypothesis that EDH17B2 may be the same as BRCA1 should be extended to the promoter and regulatory elements of EDH17B2.     

Two 17beta-hydroxysteroid dehydrogenases (17HSDs) of estradiol biosynthesis: 17HSD type 1 and type 7.

Two 17β-hydroxysteroid dehydrogenases (17HSDs), type 1 and type 7, are enzymes of estradiol biosynthesis, in addition to which rodent type 1 enzymes are also able to catalyze androgens. Both of the 17HSDs are abundantly expressed in ovaries, the type 1 enzyme in granulosa cells and type 7 in luteinized cells. The expression of 17HSD7, which has also been described as a prolactin receptor-associated protein (PRAP), is particularly up-regulated in corpus luteum during the second half of rodent pregnancy. A moderate or slight signal for mouse 17HSD7/PRAP mRNA has also been demonstrated in samples of placenta and mammary gland, for example. Human, but not rodent, 17HSD1 is expressed in placenta, breast epithelium and endometrium in addition to ovaries. A cell-specific enhancer, silencer and promoter in the hHSD17B1 gene participate in the regulation of type 1 enzyme expression. The enhancer consists of several subunits, including a retinoic acid response element, the silencer has a binding motif for GATA factors, and the proximal promoter contains adjacent and competing AP-2 and Sp binding sites.     

Wnts and the female reproductive system.

Wnts are intercellular growth and differentiation factors that regulate several key developmental steps, such as gastrulation, neurulation, and organogenesis, including the development of the midbrain, central nervous system, kidney, and limbs. Wnts are also needed for a normal development of the reproductive system. Deficiency of Wnt-4, -5a, and -7a, for example, results in sex reversal, infertility, and/or malformation of the internal and external genitals. Here we focus on the importance of Wnts in the female reproductive system.     

Wnt-4 signaling is involved in the control of smooth muscle cell fate via Bmp-4 in the medullary stroma of the developing kidney

Wnt-4, a member of the Wnt family of secreted signaling molecules, is essential for nephrogenesis, but its expression in the presumptive medulla suggests additional developmental roles in kidney organogenesis. We demonstrate here that Wnt-4 signaling plays also a role in the determination of the fate of smooth muscle cells in the medullary stroma of the developing kidney, as a differentiation marker, smooth muscle alpha-actin (alpha-SMA), is markedly reduced in the absence of its signaling. Wnt-4 probably performs this function by activating the Bmp-4 gene encoding a known differentiation factor for smooth muscle cells, since Bmp-4 gene expression was lost in the absence of Wnt-4 while Wnt-4 signaling led to a rescue of Bmp-4 expression and induction of alpha-SMA-positive cells in vitro. Recombinant Bmp-4 similarly rescued the differentiation of alpha-SMA-expressing cells in cultured Wnt-4-deficient embryonic kidney. The lack of smooth muscle cell differentiation leads to an associated deficiency in the pericytes around the developing vessels of the Wnt-4-deficient kidney and apparently leads to a secondary defect in the maturation of the kidney vessels. Thus, besides being critical for regulating mesenchymal to epithelial transformation in the cortical region in nephrogenesis, Wnt-4 signaling regulates the fate of smooth muscle cells in the developing medullary region.     

Effects of the aqueous extract of Bryothamnion triquetrum on chemical hypoxia and aglycemia-induced damage in GT1-7 mouse hypothalamic immortalized cells

The neuroprotective ability of the aqueous crude extract of Bryothamnion triquetrum (S. G. Gmelin) Howe and its cinnamic acids was studied in GT1-7 cells exposed to the combination of chemical hypoxia (KCN 3 mM) and aglycemia conditions. These ischemia-like conditions provoked acute and delayed cytotoxicity in GT1-7 cells if extended for more than 90 min. The extract was able to protect from the cell death produced by severe (180 min) chemical hypoxia/aglycemia insult, which cannot be related to its glucose content, and also reduced the cytotoxicity and early production of free radicals produced by mild (105 min) insult. Results showed that some of these protective effects of the extract are partially related to the presence of ferulic acid. The data additionally suggest that neuroprotection exerted by the extract is related to its ability to reduce free-radical generation by mechanisms different from the direct scavenging of the radical entities.     

Induced repatterning of type XVIII collagen expression in ureter bud from kidney to lung type: association with sonic hedgehog and ectopic surfactant protein C

Epithelial-mesenchymal tissue interactions regulate the formation of signaling centers that play a role in the coordination of organogenesis, but it is not clear how their activity leads to differences in organogenesis. We report that type XVIII collagen, which contains both a frizzled and an endostatin domain, is expressed throughout the respective epithelial bud at the initiation of lung and kidney organogenesis. It becomes localized to the epithelial tips in the lung during the early stages of epithelial branching, while its expression in the kidney is confined to the epithelial stalk region and is lost from the nearly formed ureter tips, thus displaying the reverse pattern to that in the lung. In recombinants, between ureter bud and lung mesenchyme, type XVIII collagen expression pattern in the ureter bud shifts from the kidney to the lung type, accompanied by a shift in sonic hedgehog expression in the epithelium. The lung mesenchyme is also sufficient to induce ectopic lung surfactant protein C expression in the ureter bud. Moreover, the shift in type XVIII collagen expression is associated with changes in ureter development, thus resembling aspects of early lung type epigenesis in the recombinants. Respecification of collagen is necessary for the repatterning process, as type XVIII collagen antibody blocking had no effect on ureter development in the intact kidney, whereas it reduced the number of epithelial tips in the lung and completely blocked ureter development with lung mesenchyme. Type XVIII collagen antibody blocking also led to a notable reduction in the expression of Wnt2, which is expressed in the lung mesenchyme but not in that of the kidney, suggesting a regulatory interaction between this collagen and Wnt2. Respecification also occurred in a chimeric organ containing the ureter bud and both kidney and lung mesenchymes, indicating that the epithelial tips can integrate the morphogenetic signals independently. A glial cell line-derived neurotrophic factor signal induces loss of type XVIII collagen from the ureter tips and renders the ureter bud competent for repatterning by lung mesenchyme-derived signals. Our data suggest that differential organ morphogenesis is regulated by an intra-organ patterning process that involves coordination between inductive signals and matrix molecules, such as type XVIII collagen.     

环腺苷一磷酸对人Ⅰ型17β羟类固醇脱氢酶在绒癌细胞系中表达的调节作用

由HSD17B1基因编码的人Ⅰ型17β-羟类固醇脱氢酶(17β-hydroxysteroid dehydrogenasetype 1,简称Ⅰ型17HSD)催化雌酮与雌二醇之间的转化。本文研究环腺苷一磷酸简称(cAM-P)对该酶在培养的绒癌细胞系(JAR和JEG-3)中表达的调节作用。用8-bromo-cAMP处理两种绒癌细胞后,观察到在伴随1.3 kbⅠ型17 HSDmRNA表达的同时,Ⅰ型17 HSD蛋白浓度也显著上升。标记基因分析表明,cAMP可诱导HSD 17 B1基因启动子在JAR和JEG-3细胞系中的转录活性,参与调节这一诱导作用的区域位于HSD 17 B1基因编码区上游-659至-550处。凝胶阻滞实验显示这一区域可同JAR、JEG-3、T-47 D和HeLa细胞核抽提物形成特异的DNA-蛋白复合物。本结果首次证实cAMP激活HSD 17 B1基因启动子在绒癌细胞中的转录。     

Immunological measurement of human 17 beta-hydroxysteroid dehydrogenase

Human placental 17 beta-hydroxysteroid dehydrogenase (17-HSD) was purified to apparent homogeneity using ammonium sulfate precipitation and chromatography on Red-Agarose and DEAE-Sepharose columns. Electrophoresis on polyacrylamide gels under denaturing conditions and using silver staining showed a single protein with an apparent molecular weight of 37,800. Antibodies to the purified protein were raised in rabbits and were found by immunoblotting to be specific to 17-HSD. A sensitive radioimmunoassay was established using 125I-labeled 17-HSD as a tracer, an appropriate dilution of the antibody, and a kaolin-coupled double antibody for separating the antibody-bound and free fractions. The detection limit of the assay was approximately 150 pg/tube (1.5 micrograms/l). The cytosol fraction (105,000 g) of term placental tissue contained approximately 0.7 mg of 17-HSD per gram of protein, and the concentrations of 17-HSD measured by immunoassay and enzymatic activity proved to be strictly parallel in different partly purified placental preparations. The supernatants from centrifugations of human endometrial homogenates at 800 g and 105,000 g (after detergent treatment) displayed cross-reactivity with the antibody. The mean concentration of the cross-reacting substance in the radioimmunoassay was 14.1 micrograms/g protein (range 2-62.3) in specimens taken on different days in the cycle. These concentrations showed a significant correlation with the 17-HSD activities measured in the endometrial specimens (r = 0.722, P less than 0.001, n = 21). Mean concentrations of substance were 8.3 micrograms/g protein in endometrial specimens taken during the follicular phase (days 4-12, n = 8) and 22.9 micrograms/g protein during the luteal phase (days 16-22, n = 6) were obtained using the radioimmunoassay. There was excellent parallelism between the competition curves for [125I]iodo-17-HSD with purified 17-HSD standards and placental and endometrial homogenate dilutions. These data strongly suggest that the substance measured in the endometrial specimens was 17-HSD.     

Patterning parameters associated with the branching of the ureteric bud regulated by epithelial-mesenchymal interactions

The mechanisms by which the branching of epithelial tissue occurs and is regulated to generate different organ structures are not well understood. In this work, image analyses of the organ rudiments demonstrate specific epithelial branching patterns for the early lung and kidney; the lung type typically generating several side branches, whereas kidney branching was mainly dichotomous. Parameters such as the number of epithelial tips, the angle of the first branch, the position index of the first branch (PIFB) in a module, and the percentage of epithelial module type (PMT) were analysed. The branching patterns in the cultured lung and kidney, and in homotypic tissue recombinants recapitulated their early in vivo branching patterns. The parameters were applied to heterotypic tissue recombinants between lung mesenchyme and ureteric bud, and tip number, PIFB and PMT values qualified the change in ureter morphogenesis and the reprogramming of the ureteric bud with lung mesenchyme. All the values for the heterotypic recombinant between ureteric bud and lung mesenchyme were significantly different from those for kidney samples but similar to those of the lung samples. Hence, lung mesenchyme can instruct the ureteric bud to undergo aspects of early lung-type epithelial morphogenesis. Different areas of the lung mesenchyme, except the tracheal region, were sufficient to promote ureteric bud growth and branching. In conclusion, our findings provide morphogenetic parameters for monitoring epithelial development in early embryonic lung and kidney and demonstrate the use of heterotypic tissue recombinants as a model for studying tissue-specific epithelial branching during organogenesis.