Nuclear factor-1 and metal transcription factor-1 synergistically activate the mouse metallothionein-1 gene in response to metal ions.

Metal activation of metallothionein (MT) gene transcription is dependent on the presence of metal regulatory elements (MREs), which are present in five non-identical copies (MREa through MREe) in the promoter of the mouse MT-1 gene and on the capacity of metal transcription factor-1 (MTF-1) to bind to the MREs in the presence of zinc. We detected a protein, distinct from MTF-1, specifically binding to the MREc region. DNA binding competition experiments using synthetic oligonucleotides and specific anti-NF1 antibodies showed that this protein binds to an NF1 site overlapping the MREc element as well as to a second site upstream of the Sp1a site and corresponds to NF1 or a related protein. Transfection experiments showed that loss of the two NF1 sites decreased metal-induced MT promoter activity by 55-70% in transiently transfected cells and almost completely abrogated metal and tert-butylhydroquinone (tBHQ) induction in stably transfected cells. Similarly, expression of an inactive NF1 protein strongly inhibited MT-1 promoter activity. Using synthetic promoters containing NF1 and MRE sites fused to a minimal MT promoter, we showed that these NF1 sites did not confer metal induction but enhanced metal-induced promoter activity. Chromatin immunoprecipitation assays confirmed that NF1 binds to the mouse MT-1 promoter in vivo and showed that NF1 binding is zinc-inducible. In addition, zinc-induced NF1 DNA binding was MTF-1-dependent. Taken together, these studies show that NF1 acts synergistically with MTF-1 to activate the mouse MT-1 promoter in response to metal ions and tert-butylhydroquinone and contributes to maximal activation of the gene.

Urease-induced alkalinization of extracellular pH and its antitumor activity in human breast and lung cancers.

Jack bean urease catalyzes the decomposition of urea into ammonia, which in turn increases the pH of the surrounding medium. Based on these two properties, we have investigated the antitumor effects of urease in vitro and in vivo on human lung and breast cancer cell lines either by the enzyme itself or in combination with other chemotherapeutic drugs. First, through the generation of toxic ammonia, urease exerted direct cytotoxicity on A549 and MDA-MB-231 tumor cells with LC50 of 0.22 and 0.45 U/ml, respectively. The cytotoxic effects could effectively be blocked using the reversible urease inhibitor acetohydroxamic acid. Complete protection was observed at dose > or = 2 mM. In addition, nude mouse xenograft models demonstrated that intratumoral urease injections (1 - 10 U/dose) inhibited A549 and MCF-7 tumor growth in vivo. Second, when combined with weak-base anticancer drugs, urease provided indirect antitumor effects via pH augmentation. Alkalinization of extracellular pH by urease (2 U/ml) and urea (> or = 2 mM) was found to enhance the antitumor efficacy of doxorubicin (50 microM) and vinblastine (100 microM) significantly.

Aldosterone stimulates epidermal growth factor receptor expression.

The steroid hormone aldosterone plays an important role during pathological tissue modifications, similar to cardiovascular or renal fibrosis. The underlying mechanisms for the pathological actions are not understood. Interaction of aldosterone with the epidermal growth factor (EGF) receptor is an attractive hypothesis to explain pathological tissue remodeling elicited by aldosterone, because (i) mineralocorticoids can sensitize cells for EGF, (ii) mineralocorticoid receptor (MR)-antagonists reduce EGFR-mRNA expression, (iii) EGFR itself supports the development of cardiovascular or renal fibrosis, and (iv) signaling elements involved in the pathological action of aldosterone (similar to ERK1/2 or NFkB) are typical downstream modules during EGF signaling. In addition, an interaction of aldosterone and EGF with respect to ERK1/2 activation has been described. Here we show that aldosterone stimulates EGFR expression in renal tissue of adrenalectomized rats and in human renal primary cell cultures. Furthermore, Chinese hamster ovary (CHO) cells normally devoid of EGFR or MR express EGFR after transfection with human MR (CHO-MR cells) but not after transfection with human glucocorticoid receptor (CHO-GR cells). In CHO-MR cells, EGFR-expression is up-regulated by aldosterone and inhibited by spironolactone. CHO-MR cells but not CHO-GR cells respond with ERK1/2 phosphorylation to EGF exposure. The responsiveness to other peptide hormones was virtually not affected. These data suggest that EGFR is an aldosterone-induced protein and is involved in the manifold (patho)biological actions of aldosterone.

Cellular context of coregulator and adaptor proteins regulates human adenovirus 5 early region 1A-dependent gene activation by the thyroid hormone receptor.

In mammalian cells, the human adenovirus type 5 early region 1A (E1A) oncoprotein functions as a thyroid hormone (TH)-dependent activator of the thyroid hormone receptor (TR). Interestingly, in the cellular context of the yeast Saccharomyces cerevisiae, E1A acts as a TR-specific constitutive coactivator that is down-regulated by TH. TH reduces the interaction of E1A with the TR in yeast but not HeLa cells. The N-terminal 82 amino acids of E1A are sufficient for coactivation in yeast and residues 4-29 are essential. In yeast, expression of the nuclear receptor corepressor (N-CoR) could down-regulate constitutive transcriptional activation of the TR by E1A, whereas expression of the glucocorticoid receptor interacting protein 1 (GRIP-1) coactivator reconstituted the E1A-induced pattern of enhanced TH-dependent gene activation by TR observed in mammalian cells. We further show that the mating type switching gene (SWI)/sucrose nonfermenting (SNF) gene chromatin remodeling complex is required for both TH/GRIP-1- and E1A-dependent coactivator function, whereas the general control nonrepressed protein (GCN5)/alteration/deficiency in activation protein (ADA2) components of the SPT, ADA, GCN5, acetylation (SAGA) transcriptional adaptor complex are required for TH/GRIP-1, but not E1A-dependent activation of the TR. Taken together, these studies demonstrate that the novel TR-specific coactivator function of E1A in yeast depends on the SWI/SNF chromatin remodeling complex and can be further influenced by changes in the cellular complement of transcriptional coregulatory proteins.