Cryptic human growth hormone gene sequences direct gonadotroph-specific expression in transgenic mice

Our laboratory reported previously that chimeric genes encoding either rat somatostatin (SS) or human GH (hGH), but containing the identical mouse metallothionein-I (MT) promoter/enhancer sequences and hGH 3'-flanking sequences, were selectively expressed in the gonadotrophs of transgenic mice. The experiments reported here were designed to identify the DNA sequences responsible for this unexpected cell-specific expression within the anterior pituitary. We produced new transgenic mice expressing fusion genes that tested separately the requirement of the MT or 3'-hGH sequences for gonadotroph expression. A fusion gene that retained the original MT and SS sequences, with a simian virus 40 polyadenylation signal exchanged for the 3'-hGH sequences, no longer directed strong pituitary expression, but was active in the liver. In contrast, a cytomegalovirus promoter/enhancer-SS-hGH fusion gene was expressed at the same high level in the anterior pituitaries of transgenic mice as the originally studied MT-SS-hGH gene. Immunohistochemical analysis indicated that pituitary expression of the cytomegalovirus promoter/enhancer-SS-hGH fusion gene was also restricted to gonadotroph cells in adult mice. These studies indicate that sequences within the 3'-flanking region of the hGH gene can direct expression of chimeric genes to pituitary cells that do not normally produce growth hormone.     

Uptake of cadmium is diminished in transfected mouse NIH/3T3 cells enriched for metallothionein.

To determine the relationship between cellular uptake of cadmium and content of metallothionein, we measured uptake of 109Cd in cells that differed in content of metallothionein (MT). MT cells were derived from NIH/3T3 cells by transfection with a plasmid containing the genome of bovine papilloma virus and the mouse metallothionein-I gene, driven by the promotor for the glucose-regulated protein of 78 kDa. Control cells were similarly transfected with bovine papilloma virus-based plasmids with the gene for metallothionein inverted and thus separated from the promoter (TM), or deleted, along with the promoter (BPA). The number of copies of bovine papilloma virus-based plasmids was similar in MT, TM, and BPA cells, approximately 100 per cell. MT cells were more than 10 times more resistant to the lethal effect of cadmium than were the control cells. Synthesis of metallothionein was 15-fold greater in the MT cells than in the TM or BPA cells. The uptake of 109Cd by the cells enriched in metallothionein was 4-fold less than by the control cells. These data suggest that an increased content of metallothionein may protect some cells from the toxic effects of cadmium, in part, by diminishing uptake of the metal.     

Isolation of rat Schwann cell lines: use of SV40 T antigen gene regulated by synthetic metallothionein promoters

Synthetic promoter elements from the mouse metallothionein-I promoter controlling the expression of SV40 T antigen have been tested for their efficacy in cloning rat Schwann cell lines that retained the characteristic properties of these cells and could be passed in culture indefinitely. The constructed promoters contained either four (MT4) or five (MT5) copies of a metal regulatory element 5' to the CAAT and TATA elements from the HSV-1 thymidine kinase gene. Characterization of these promoters in transient expression assays and transformation assays showed that both MT5 and MT4 were approximately 10-fold and 15-fold, respectively, weaker than the wild-type MT-I promoter in the presence of heavy metal inducer. However, in the absence of inducer, the basal activity of both MT5 and MT4 was barely detectable and much lower than that of MT-I. Schwann cells were transfected with plasmids containing the SV40 T antigen gene under the control of the different metallothionein promoters and cell lines were established from each. Only with the MT5 and MT4 promoters were lines obtained that resembled secondary Schwann cells in culture in their morphology, generation time, and demonstration of contact inhibition. In the presence of zinc, the expression of T antigen in the lines derived with MT5 and MT4 was about 10-fold lower than that derived with MT-I. On removal of the inducer this level was reduced, and in one cell line T antigen was undetectable. Concomitant with the reduction in T antigen expression there was an increased expression of Po, a protein specific to myelin-forming Schwann cells, and a decreased expression of glial fibrillary acidic protein, a protein expressed only in nonmyelin-forming Schwann cells. These cell lines, therefore, closely resemble untransfected Schwann cells in culture.     

Activation of the complete mouse metallothionein gene locus in the maternal deciduum

The mouse metallothionein (MT) gene family consists of four known members (MT-I through IV) clustered on chromosome 8. Studies reported herein examine the expression and regulation of the MT-III and MT-IV genes in specific cell types in the maternal reproductive tract, developing embryo, and fetus known to express the MT-I and -II genes. MT-III and MT-IV mRNAs were absent from the visceral yolk sac, placenta, and fetal liver, tissues with high levels of MT-I and MT-II mRNAs. In contrast, MT-III and MT-IV mRNAs were both abundant in the maternal deciduum, and in experimentally induced deciduoma on 7 and 8 days postcoitum (1 dpc = vaginal plug), as are MT-I and -II mRNAs. The abundance of each of these MT mRNAs increased coordinately during development of the deciduum (6–8 dpc), and in situ hybridization localized MT-I, MT-III, and MT-IV mRNAs to the secondary decidual zone of the antimesometrial region on 8 dpc, where in some regions all of the cells were apparently positive. Thus, all of the known mouse MT genes are co-expressed in at least some of the cells in the secondary decidual zone. Electrophoretic analysis of decidual MT suggested that the MT-I, -II, and -III isoforms are abundant proteins in the secondary deciduum. Bacterial endotoxin-lipopolysaccharide (LPS) and Zn are powerful inducers of MT-I and MT-II gene expression in many adult organs, whereas these agents apparently have little effect on MT-III and MT-IV gene expression. Neither of these agents significantly effected levels of decidual MT-III or MT-IV mRNAs in vivo or in primary cultures of decidual cells in vitro, and only modest effects of Zn on MT-I mRNA levels were noted. During 2 days of in vitro culture, decidual cell MT-I and MT-III mRNA levels remained elevated while MT-IV mRNA levels decreased. Thus, expression of the mouse MT gene locus in the deciduum appears to be developmentally regulated, and in this tissue, the MT genes are refractory to induction by Zn or inflammation. © 1996 Wiley-Liss, Inc.     

CT23 knockdown attenuating malignant behaviors of hepatocellular carcinoma cell is associated with upregulation of metallothionein 1

The cancer-testis antigen 23 (CT23) gene has been reported in association with the pathogenesis and progress of hepatocellular carcinoma (HCC). However, the alterations of gene expression profiling induced by CT23 knockdown in HCC cells remains largely unknown. In this study, the RNA interfering (RNAi) method was used to silence CT23 expression in BEL-7404 cells. Microarray analysis was performed on mRNA extracted from the CT23 knockdown cells and the control cells to determine the alterations of gene expression profiles. The result showed a total of 1051 genes expressed differentially (two-fold change), including 470 genes upregulated and 581 gene downregulated in the CT23 knockdown cells. A bioinformatic analysis showed that the functional differentially expressed genes (DEGs) were linked to cell proliferation, migration, and apoptosis, and metallothionein 1 (MT1) attained the maximum enrichment scores in functional annotation, classification, and pathway analysis of DEGs. Furthermore, Western blot analysis and cell behaviors assays verified that CT23 modulates cell proliferation, migration, and apoptosis by regulating MT1 expression in HCC cells and non-neoplastic hepatocytes. In summary, downregulated CT23 gene in BEL-7404 cells might change the expressions of carcinogenesis and progression related genes in HCC by upregulating MT1 expression, which would provide insight into searching for a novel therapeutic target for HCC.     

Induction of metallothionein-I mRNA in cultured cells by heavy metals and iodoacetate: evidence for gratuitous inducers.

A mouse hepatocyte cell line selected for growth in 80 microM CdSO4 (Cdr80 cells) was used to test the role of metallothioneins in heavy metal detoxification. The cadmium-resistant (Cdr80) cells have double minute chromosomes carrying amplified copies of the metallothionein-I gene and accumulate ca. 20-fold more metallothionein-I mRNA than unselected cadmium-sensitive (Cds) cells after optimal Cd stimulation. As a consequence, the amount of Cd which inhibits DNA synthesis by 50% is ca. 7.5-fold higher in Cdr80 cells than in Cds cells. Cds and Cdr80 cells were compared in terms of their resistance to other heavy metals. The results indicate that although Zn, Cu, Hg, Ag, Co, Ni, and Bi induce metallothionein-I mRNA accumulation in both Cdr80 and Cds cells, the Cdr80 cells show increased resistance to only a subset of these metals (Zn, Cu, Hg, and Bi). This suggests that not all metals which induce metallothionein mRNA are detoxified by metallothionein and argues against autoregulation of metallothionein genes. Metallothionein-I mRNA is also induced by iodoacetate, suggesting that the regulatory molecule has sensitive sulfhydryl groups.     

Cloning and functional characterization of 5'-upstream region of metallothionein-I gene from crucian carp (Carassius cuvieri)

Metallothioneins are low molecular weight, cysteine-rich, metal-binding proteins, which can be induced by heavy metal ions, cytokines, stress, and hormones. To investigate the roles of the main cis-acting elements involved in the inducible expression of metallothionein gene in fish, the 5'-upstream region of crucian carp (Carassius cuvieri) metallothionein-I gene had been cloned and analyzed after our previous work on metallothionein-II. In its upstream region, several putative cis-acting elements, including nine metal regulatory elements (MREs), one antioxidant response element, one E-box, and three interleukin-6 responsive elements, etc. were found. The nine metal regulatory elements are confined in less than 1000 bp from ATG start codon and organized into two clusters with different roles to the induction of the metallothionein-I expression. Deletion mutant assays demonstrated that both the distal and proximal clusters of metal regulatory elements contributed to the basal expression of the metallothionein-I, but only the proximal cluster was the chief contributor to the metal fold induction. In transient luciferase reporter assays, Zn2+ and Cd2+ served as much stronger inducers than Cu2+ to the metallothionein-I expression. H2O2 also could activate the metallothionein-I promoter about two-fold, which was mediated by the antioxidant response element (TGACAACGC, -437/-445). In conclusion, our studies demonstrate the roles of metal regulatory element and antioxidant response element in the induction of crucian carp metallothionein-I gene, and provide the regulatory mechanism for the use of fish metallothionein as a biomarker for monitoring of metal contamination in waters.     

Functional analysis of the promoter of a sea urchin metallothionein gene.

The 5'-flanking region of the metallothionein (MT) gene LpMT1 of the sea urchin Lytechinus pictus includes three copies of a conserved sequence that includes the metal-responsive element (MRE) consensus core sequence required for heavy metal induction of other MT genes, a GC box, a G box of a putative basal level enhancer element which includes another MRE core element, and a poly(C) tract. A fragment of LpMT1 DNA from nucleotides +31 to -309 fused to a chloramphenicol acetyltransferase reporter gene was inducible with cadmium after injection into L. pictus embryos. This induced activity was greatly reduced in a deletion mutant which retained only 195 base pairs of 5'-flanking sequence, including the proximal pair of MREs and the G box, but excluding the poly(C) tract, GC box, and distal MRE. A potent human hMT-IIA gene promoter is marginally functional in L. pictus embryos. In contrast, the LpMT1 promoter is active in HeLa cells and in embryos of the sea urchin Strongylocentrotus purpuratus. The hMT-IIA gene may lack a cis-acting sequence element required for expression of MT genes in L. pictus embryos. The LpMT1 promoter is a powerful, inducible, promiscuous promoter useful for driving the expression of heterologous genes in sea urchin embryos.     

Grape seed procyanidins inhibit the expression of metallothione in genes in human HepG2 cells

Procyanidins are the most abundant polyphenols in red wine and are also found in cereals, fruits, chocolate and tea. They exert many beneficial health effects, especially on the cardiovascular system (Bagchi et al. in Mutat Res 523-524:87-97, 2003; Williams et al. in Free Radic Biol Med 36:838-849, 2004; Dell'Agli et al. in Cardiovasc Res 63(4):593-602, 2004; Del Bas et al. FASEB J 19:479-480, 2005). Here, we show that oral administration of a grape seed procyanidins extract (GSPE) to healthy rats results, 5 h after treatment, in a 70% inhibition of metallothionein (MT) gene expression in the liver, as determined by oligonucleotide microarray hybridization. Similarly, in cultured human hepatocytes HepG2, GSPE downregulate the expression of MT genes at the mRNA level, as evaluated by quantitative RTPCR. Thus, mRNA levels of six functional MT genes, MT1A, 1E, 1F, 1G, 1X and MT2A, are diminished between 50 and 80% when HepG2 cells are treated during 12 h with GSPE. Only the expression of two human MT genes, MT1G and MT1E, is transiently increased during the first 2 h of treatment. GSPE-induced inhibition of MT genes expression is dose dependent, at concentrations that are not toxic for the cells. Our findings demonstrate that metallothionein genes are direct targets of procyanidins action, both in vivo and in vitro, in hepatic cells. Thus, this study will help to elucidate the mechanisms by which procyanidin exert their beneficial actions.     

Cell-specific metallothionein gene expression in mouse decidua and placentae

Oligodeoxyribonucleotide excess solution hybridization, Northern blot and in situ hybridization were used to analyze metallothionein gene expression in mouse decidua and placentae during gestation. Metallothionein (MT) -I and -II mRNA levels were constitutively elevated, 11- and 13-fold, respectively, relative to the adult liver, in the deciduum (D8), and decreased coordinately about 6-fold during the period of development when the deciduum is replaced by the developing placenta (D10-16). Coincident with this decline, levels of MT mRNA increased dramatically in the visceral yolk sac endoderm. In situ hybridization established that MT-I mRNA was present at low levels in the uterine luminal epithelium (D4), but was elevated at the site of embryo implantation exclusively in the primary decidual zone by D5, and then in the secondary decidual zone (D6-8). Although low levels of MT mRNA were detected in total placental RNA, in situ hybridization revealed constitutively high levels in the outer placental spongiotrophoblasts. Analysis of pulse-labeled proteins from decidua and placentae established that these tissues are active in the synthesis of MT. The constitutively high levels of MT mRNA in decidua were only slightly elevated following injection of cadmium (Cd) and/or zinc (Zn), whereas in placentae they increased several-fold. MT mRNA levels were equally high in decidua and experimentally induced deciduomata (D8) which establishes that decidual MT gene expression is not dependent on the presence of the embryo or some embryo-derived factor. Although the functional role of MT during development is speculative, these results establish the concept that, from the time of implantation to late in gestation, the mouse embryo is surrounded by cells, interposed between the maternal and embryonic environments, which actively express the MT genes. This suggests that MT plays an important role in the establishment and maintenance of normal pregnancy.     

Cell-specific transgene expression from a widely transcribed promoter using Cre/lox in mice

Mice carrying two or more transgenes are used frequently to evaluate oncogene interactions during carcinogenesis. However, neoplastic transformation typically results in reduced expression both of differentiation-specific genes and of transgenes that use their promoters. In contrast, the more widely expressed metallothionein (MT) gene remains expressed at a high level in certain neoplasms, including those developing in pancreas. We have developed a system to maintain high-level, tissue-specific transgene expression during pancreatic carcinogenesis that uses Cre recombinase and a lox site-containing target transgene. Cre was expressed in pancreatic acinar cells under control of the elastase promoter (EL). Cre-mediated target transgene recombination placed a previously silent open-reading frame, encoding rat transforming growth factor alpha (TGFalpha), under control of the MT gene promoter. As long as DNA rearrangement does not occur in other cell types that express MT, TGFalpha expression will be restricted to acinar cells. Development of an effective target transgenic mouse required evaluation of multiple lineages to identify one with sufficient TGFalpha expression to induce pancreatic lesions after transgene rearrangement.     

Response of sugarcane to increasing concentrations of copper and cadmium and expression of metallothionein genes

Sugarcane (Saccharum spp.) offers the potential to be a phytoremediator species due to its outstanding biomass production, but its prospective metal accumulation and tolerance have not been fully characterized. Sugarcane plantlets were able to tolerate up to 100microM of copper in nutrient solution for 33 days, with no significant reduction in fresh weight, while accumulating 45mgCukg(-1) shoot dry weight. Higher levels of copper in solution (250 and 500microM) were lethal. Sugarcane displayed tolerance to 500microM Cd without symptoms of toxicity, accumulating 451mgCdkg(-1) shoot dry weight after 33 days, indicating its potential as Cd phytoremediator. DNA gel blot analyses detected 8 fragments using a metallothionein (MT) Type I probe, while 10 were revealed for the MT Type II and 8 for MT Type III. The number of genes for each type of MT in sugarcane might be similar to the ones identified in rice considering the interspecific origin of sugarcane cultivars. MT Type I gene appeared to present the highest level of constitutive expression, mainly in roots, followed by MT Type II, corroborating the expression pattern described based on large-scale expressed sequence tags sequencing. MT Type II and III genes were more expressed in shoots, where MT I was also importantly expressed. Increasing Cu concentration had little or no effect in modulating MT genes expression, while an apparent minor modulation of some of the MT genes could be detected in Cd treatments. However, the level of response was too small to explain the tolerance and/or accumulation of Cd in sugarcane tissues. Thus, cadmium tolerance and accumulation in sugarcane might derive from other mechanisms, although MT may be involved in oxidative responses to high levels of Cd. Sugarcane can be considered a potential candidate to be tested in Cd phytoremediation.     

Haem release from haemopexin by HxuA allows Haemophilus influenzae to escape host nutritional immunity

In this work we describe the identification of a copper-inducible regulon in Mycobacterium tuberculosis (Mtb). Among the regulated genes was Rv0190/MT0200, a paralogue of the copper metalloregulatory repressor CsoR. The five-locus regulon, which includes a gene that encodes the copper-protective metallothionein MymT, was highly induced in wild-type Mtb treated with copper, and highly expressed in an Rv0190/MT0200 mutant. Importantly, the Rv0190/MT0200 mutant was hyper-resistant to copper. The promoters of all five loci share a palindromic motif that was recognized by the gene product of Rv0190/MT0200. For this reason we named Rv0190/MT0200 RicR for regulated in copper repressor. Intriguingly, several of the RicR-regulated genes, including MymT, are unique to pathogenic Mycobacteria. The identification of a copper-responsive regulon specific to virulent mycobacterial species suggests copper homeostasis must be maintained during an infection. Alternatively, copper may provide a cue for the expression of genes unrelated to metal homeostasis, but nonetheless necessary for survival in a host.