Peter Lichtlen

Embryonic lethality and liver degeneration in mice lacking the metal-responsive transcriptional activator MTF-1.

We have shown previously that the heavy metal‐responsive transcriptional activator MTF‐1 regulates the basal and heavy metal‐induced expression of metallothioneins. To investigate the physiological function of MTF‐1, we generated null mutant mice by targeted gene disruption. Embryos lacking MTF‐1 die in utero at approximately day 14 of gestation. They show impaired development of hepatocytes and, at later stages, liver decay and generalized edema. MTF‐1−/− embryos fail to transcribe metallothionein I and II genes, and also show diminished transcripts of the gene which encodes the heavy‐chain subunit of the γ‐glutamylcysteine synthetase, a key enzyme for glutathione biosynthesis. Metallothionein and glutathione are involved in heavy metal homeostasis and detoxification processes, such as scavenging reactive oxygen intermediates. Accordingly, primary mouse embryo fibroblasts lacking MTF‐1 show increased susceptibility to the cytotoxic effects of cadmium or hydrogen peroxide. Thus, MTF‐1 may help to control metal homeostasis and probably cellular redox state, especially during liver development. We also note that the MTF‐1 null mutant phenotype bears some similarity to those of two other regulators of cellular stress response, namely c‐Jun and NF‐κB (p65/RelA).

The transcription factors MTF-1 and USF1 cooperate to regulate mouse metallothionein-I expression in response to the essential metal zinc in visceral endoderm cells during early development.

During early development of the mouse embryo, expression of the metallothionein‐I (MT‐I) gene is heightened specifically in the endoderm cells of the visceral yolk sac. The mechanisms of regulation of this cell‐specific pattern of expression of metallothionein‐I are unknown. However, it has recently been shown that MTF‐1, functioning as a metalloregulatory transcription factor, activates metallothionein genes in response to the essential metal zinc. In contrast with the metallothionein genes, MTF‐1 is essential for development; null mutant embryos die due to liver degeneration. We report here that MTF‐1 is absolutely essential for upregulation of MT‐I gene expression in visceral endoderm cells and that optimal expression also involves interactions of the basic helix–loop–helix upstream stimulatory factor‐1 (USF1) with an E‐box1‐containing sequence at −223 bp in the MT‐I promoter. Expression of MT‐I in visceral endoderm cells was dependent on maternal dietary zinc. Thus, the essential metal, zinc, apparently provides the signaling ligand that activates cell‐ specific MT‐I expression in visceral endoderm cells.

Efficient Intraocular Penetration of Topical Anti–TNF-α Single-Chain Antibody (ESBA105) to Anterior and Posterior Segment without Penetration Enhancer

PURPOSE This study was designed to characterize ocular penetration pathways of ESBA105, a topically administered single-chain antibody (scFv) against tumor necrosis factor (TNF)-alpha, to the anterior and posterior segment of the eye. METHODS Fresh enucleated whole eyes and isolated corneas of rabbits mounted in perfusion chambers were used for ex vivo penetration studies. In vivo pharmacokinetics and ocular biodistribution of ESBA105 after intravitreal injection or topical administration as eye drops were investigated in rabbits. RESULTS After topical administration as eye drops, without a penetration enhancer, ESBA105 reached therapeutic levels in the anterior and posterior segment of the eye. ESBA105 migrated to aqueous humor via corneal penetration and vitreous and retina via intrascleral penetration pathways. In vivo, ESBA105 had a significantly prolonged elimination half-life in the vitreous of 25 hours compared with its serum half-life of 7 hours after i.v. administration. Therefore, based on frequency of topical dosing, a buildup of ESBA105 to distinct steady state levels in the vitreous could be achieved. CONCLUSIONS Topically administered ESBA105 quickly reaches therapeutic levels in the anterior and posterior segment without any need for a penetration enhancer. Drug penetration and ocular biodistribution patterns of ESBA105 applied as eye drops appear highly attractive for clinical use to treat TNF-alpha dependant diseases of the eye.

Metal-responsive transcription factor-1 (MTF-1) is essential for embryonic liver development and heavy metal detoxification in the adult liver

Metal‐responsive transcription factor‐1 (MTF‐1) activates the transcription of metallothionein genes and other target genes in response to heavy metal load and other stresses such as hypoxia and oxidative stress. It also has an essential function during embryo‐genesis: targeted disruption of Mtf1 in the mouse results in lethal liver degeneration on day 14 of gestation. Here we studied Mtf1 knockout mice at embryonic and adult stages, the latter by means of conditional knockout. Hepatocytes from Mtf1 null mutant and wild‐type embryos were taken into culture on day 12.5 of gestation. Both initially appeared normal, but mutant cells were lost within a few days. Furthermore, Mtf1 null hepatocytes were poorly, if at all, rescued by cocultivation with wild‐type rat embryo hepatocytes, indicating a cell‐autonomous defect. When the Mtf1 gene was excised by Cre recombinase after birth in liver and bone marrow and to a lesser extent in other organs, mice were viable under non‐stress conditions but highly susceptible to cadmium toxicity, in support of a role of MTF‐1 in coping with heavy metal stress. An additional MTF‐1 function was revealed upon analysis of the hematopoietic system in conditional knockout mice where leukocytes, especially lymphocytes, were found to be severely underrepresented. Together, these findings point to a critical role of MTF‐1 in embryonic liver formation, heavy metal toxicity, and hematopoiesis.— Wang, Y., Wimmer, U., Lichtlen, P., Inderbitzin, D., Stieger, B., Meier, P. J., Hunziker, L., Stallmach, T., Forrer, T., Rülicke, T., Georgiev, O., Schaffner, W. Metal‐responsive transcription factor‐1 (MTF‐1) is essential for embryonic liver development and heavy metal detoxification in the adult liver. FASEB J. 18, 1071–1079 (2004)

Pharmacokinetics and posterior segment biodistribution of ESBA105, an Anti-TNF-α single-chain antibody, upon topical administration to the rabbit eye.

PURPOSE The purpose of this study was to characterize local distribution and systemic absorption of the tumor necrosis factor (TNF)-alpha inhibitory single-chain antibody fragment (scFv) ESBA105 following topical administration to the eye in vivo. METHODS Rabbits received ESBA105 as topical eye drops in two dosing regimens. First, pharmacokinetics after the topical route of administration was compared to the intravenous (i.v.) route by means of applying the identical cumulative daily dose of ESBA105. In a second study rabbits received five eye drops daily for six consecutive days in a lower frequency topical dosing regimen. Kinetics and biodistribution of ESBA105 in ocular tissues and fluids as well as in sera were determined in all animals. RESULTS After topical administration to the eye, ESBA105 quickly reaches therapeutic concentrations in all ocular compartments. Systemic exposure after topical administration is 25,000-fold lower than exposure after i.v. injection of the identical cumulative daily dose. ESBA105 levels in vitreous humor and neuroretina are significantly higher on topical administration than after i.v. injection. Absolute and relative intraocular biodistribution of ESBA105 is different with topical and systemic delivery routes. Compared to its terminal half-life in circulation (7 hours), the vitreal half-life of ESBA105 is significantly enhanced (16-24 hours). CONCLUSIONS On topical administration, ESBA105 is efficiently absorbed and distributed to all compartments of the eye, whereby systemic drug exposure is very low. Based on its unique intraocular biodistribution and pharmacokinetics and the absolute intraocular levels reached, topical ESBA105 appears highly attractive for treatment of various ophthalmological disorders.

Relative Contribution of VEGF and TNF-α in the Cynomolgus Laser-Induced CNV Model: Comparing the Efficacy of Bevacizumab, Adalimumab, and ESBA105

PURPOSE To compare the relative contribution of VEGF and TNF-alpha in the development of laser-induced choroidal neovascularization (CNV) in monkeys and to exploit the feasibility of topical use of suitable antibody fragments for the prevention of experimental CNV. METHODS To induce experimental CNV, small high-energy laser spots were used to treat several areas of the macula in the retinas of cynomolgus monkeys according to previously published protocols. To prevent abnormalities, bevacizumab (a potent VEGF inhibitor) and adalimumab or ESBA105 (potent TNF-alpha inhibitors) were given by intravitreal injection 1 week before and 1 week and 3 weeks after laser treatment. ESBA105 was also applied topically in a separate group. Control animals were treated with either intravitreal or topical saline. Eyes were monitored by ophthalmic examination, color photography, and fluorescein angiography. RESULTS Inhibition of VEGF by bevacizumab completely blocked the formation of CNV. Both TNF-alpha inhibitors also significantly reduced laser-induced CNV abnormalities after intravitreal administration. Most important, topical use of the anti-TNF-alpha single-chain antibody fragment ESBA105 also reduced the formation of CNV. CONCLUSIONS TNF-alpha contributes to laser-induced CNV formation, and its inhibition can be a new therapeutic target for CNV. This study suggests TNF-alpha as another therapeutic target for the prevention and treatment of CNV and adds to the emerging clinical data suggesting the therapeutic value of TNF-alpha inhibitors in age-related macular degeneration (AMD). Further, this study shows that topical therapy with suitable antibody fragments has the potential of being introduced to retinal disease treatment regimens.

Arsenite-inducible RNA-associated protein (AIRAP) protects cells from arsenite toxicity

Abstract Exposure of cells to arsenicals activates multiple stress pathways resulting in the induction of specific genes whose identity and role in the adaptation to arsenical-induced cellular stress are poorly understood. We report here the identification of a novel gene encoding an arsenite-inducible, cysteine- and histidine-rich RNA-associated protein, AIRAP, that is conserved among mammals, Drosophila and C elegans. Immunochemistry and cell fractionation experiments indicate that, when induced, AIRAP is present in both the nucleus and the cytoplasm, and cross-linking experiments indicate that it associates with RNA in vivo. The expression of a C elegans homologue of AIRAP, aip-1, is also induced by exposure to arsenite, and expression of an aip-1::gfp transgene is most pronounced in hypodermal cells. RNA-mediated interference (RNAi) of aip-1 lowers the resistance of nematodes to arsenite yet does not appear to affect viability under standard growth conditions. These experiments suggest a role for AIRAP/AIP-1 in protecting cells from the toxic effects of arsenite.

Cell growth selection system to detect extracellular and transmembrane protein interactions.

The interplay among extracellular and cell surface proteins, such as the interactions between ligands and receptors or between antigens and antibodies, is involved in a multitude of physiological and pathological phenomena. In the oxidizing milieu of the secretory pathway in eukaryotic cells, many extracellular proteins build disulfide bonds that significantly contribute to their correct folding and structural stability. Thus, conventional yeast two-hybrid interaction assays, which occur in the reducing intracellular environment, might not be adequate to detect extracellular protein-protein interactions. We have exploited the properties of yeast Ire1p, a type I endoplasmic reticulum (ER) membrane protein involved in the unfolded protein response (UPR) as a dimerization-activated receptor, to develop a novel system for the detection and study of interactions between extracellular and/or membrane proteins. In our system, named SCINEX-P (screening for interactions between extracellular proteins), proteins of interest were fused to truncated Ire1p so as to substitute its N-terminal lumenal domain (NLD). Specific interaction between two partners caused dimerization of the Ire1p moiety, which, through the endogenous UPR signalling pathway, led to activation of transcription of genes that permit cell growth under selective conditions.

Loss of metal transcription factor-1 suppresses tumor growth through enhanced matrix deposition

Metal transcription factor‐1 (MTF‐1) is a ubiquitous transcriptional regulator and chromatin in‐ sulator with roles in cellular stress responses and em‐ bryonic development. The studies described herein establish for the first time the involvement of MTF‐1 in tumor development. Genetically manipulated ras‐trans‐ formed mouse embryonic fibroblasts (MEFs), wild‐type (MTF‐1+/+), or nullizygous for MTF‐1 (MTF‐1‒/‒) were used to develop fibrosarcoma tumors. Loss of MTF‐1 resulted in delayed tumor growth associated with increased matrix collagen deposition and reduc‐ tions in vasculature density. Molecular consequences of MTF‐1 loss include increased expression and activation of the transforming growth factor–β1 (TGF‐β1) and tissue transglutaminase (tTG), two proteins with docu‐ mented roles in the production and stabilization of extracellular matrix (ECM). Our findings support the hypothesis that MTF‐1 enhances the ability of the developing tumor mass to evade fibrosis and scarring of the tumor, a critical step in tumor cell prolifera‐ tion.—Haroon, Z. A., Amin, K., Lichtlen, P., Sato, B., Huynh, N. T., Wang, Z., Schaffner, W., Murphy, B. J. Loss of metal transcription factor‐1 suppresses tumor growth through enhanced matrix deposition. FASEB J. 18, 1176 –1184 (2004)

Characterization of the mouse gene for the heavy metal-responsive transcription factor MTF-1

Abstract MTF-1 is a zinc finger transcription factor that mediates the cellular response to heavy metal stress; its targeted disruption in the mouse leads to liver decay and embryonic lethality at day E14. Recently, we have sequenced the entire MTF-1 gene in the compact genome of the pufferfish Fugu rubripes. Here we have defined the promoter sequences of human and mouse MTF-1 and the genomic structure of the mouse MTF-1 locus. The transcription unit of MTF-1 spans 42 kb (compared to 8.5 kb in Fugu) and is located downstream of the gene for a phosphatase (INPP5P) in mouse, human, and fish. In all of these species, the MTF promoter region has the features of a CpG island. In both mouse and human, the 5′ untranslated region harbors conserved short reading frames of unknown function. RNA mapping experiments revealed that in these two species, MTF-1 mRNA is transcribed from a cluster of multiple initiation sites from a TATA-less promoter without metal-responsive elements. Transcription from endogenous and transfected MTF-1 promoters was not affected by heavy metal load or other stressors, in support of the notion that MTF-1 activity is regulated at the posttranscriptional level. Tissue Northern blots normalized for poly A+ RNA indicate that MTF-1 is expressed at similar levels in all tissues, except in the testes, that contain more than 10-fold higher mRNA levels.