Volume 385, Issue 7

Based on the effects of a selective experimental zinc deficiency in a rodent model we explore the use of transcriptome profiling for assessing nutrient-gene interactions in the liver at the molecular and cellular levels. Zinc deficiency caused pleiotropic alterations in mRNA/protein levels of hundreds of genes. In the context of observed metabolic alterations in hepatic metabolism, possible mechanisms are discussed for how a low zinc status may be sensed and transmitted into changes in various metabolic pathways. However, it also becomes obvious that analysis of such complex nutrient-gene interactions beyond the descriptional level is a real challenge for systems biology.

Several genes are regulated by tocopherols which can be categorized, based on their function, into five groups: genes that are involved in the uptake and degradation of tocopherols (Group 1) include α-tocopherol transfer protein (α-TTP) and cytochrome P450 (CYP3A); genes that are associated with lipid uptake and atherosclerosis (Group 2) include CD36, SRBI and SRAI/II. Genes that modulate the expression of extracellular proteins (Group 3) include tropomyosin, collagenα1, MMP-1, MMP-19 and connective tissue growth factor (CTGF). Genes that are related to inflammation, cell adhesion and platelet aggregation (Group 4) include Eselectin, ICAM-1, integrins, glycoprotein IIb, Il-2, IL-4 and IL-β. Group 5 comprises genes coding for proteins involved in cell signaling and cell cycle regulation and consists of PPARγ, cyclin D1, cyclin E, Bcl2-L1, p27 and CD95 (Apo-1/Fas ligand). The expression of P27, Bcl2, α-TTP, CYP3A, tropomyosin, Il-2, PPAR-γ, and CTGF appears to be up-regulated by one or more tocopherols whereas all other listed genes are down-regulated. Several mechanisms may underlie tocopherol-dependent gene regulation. In some cases protein kinase C has been implicated due to its deactivation by α-tocopherol and its participation in the regulation of a number of transcription factors (NFκB, AP-1). In other cases a direct involvement of PXR/ RXR has been documented. The antioxidant responsive element (ARE) appears in some cases to be involved as well as the transforming growth factor β responsive element (TGF-β-RE). This heterogeneity of mediators of tocopherol action suggests the need of a common element that could be a receptor or a co-receptor, able to interact with tocopherol and with transcription factors directed toward specific regions of promoter sequences of sensitive genes. Here we review recent results of the search for molecular mechanisms underpinning the central signaling mechanism.

The new scientific field of nutrigenomics utilizes genomic tools, like microarrays, to analyze metabolic adaptations induced by variations in nutritional status. Here we describe how transcriptional regulation patterns caused by nutritional changes can be identified using gene expression profiling. This includes technical remarks on microarray analysis and data processing, as well as giving biological meaning to statistically solid data. We highlight our recent findings of transcriptional regulation of genes representing specific signaling and metabolic pathways in mouse liver under starvation. The results show strong correlations to previously identified responses to caloric restriction, which can be linked to lifespan extension.

The zinc finger transcription factor MTF-1 (metalresponsive transcription factor-1) is conserved from insects to vertebrates. Its major role in both organisms is to control the transcription of genes involved in the homeostasis and detoxification of heavy metal ions such as Cu[2+], Zn[2+] and Cd [2+]. In mammals, MTF-1 serves at least two additional roles. First, targeted disruption of the MTF-1 gene results in death at embryonic day 14 due to liver degeneration, revealing a stagespecific developmental role. Second, under hypoxicanoxic stress, MTF-1 helps to activate the transcription of the gene placental growth factor (PlGF), an angiogenic protein. Recently we characterized dMTF-1, the Drosophila homolog of mammalian MTF-1. Here we present a series of studies to compare the metal response in mammals and insects, which reveal common features but also differences. A human MTF-1 transgene can restore to a large extent metal tolerance to flies lacking their own MTF-1 gene, both at low and high copper concentrations. Likewise, Drosophila MTF-1 can substitute for human MTF-1 in mammalian cell culture, although both the basal and the metalinduced transcript levels are lower. Finally, a clear difference was revealed in the response to mercury, a highly toxic heavy metal: metallothioneintype promoters respond poorly, if at all, to Hg[2+] in mammalian cells but strongly in Drosophila , and this response is completely dependent on dMTF-1.

The vitamin A metabolite, all-trans retinoic acid (atRA), is required for embryonic development. atRA binds to the nuclear retinoic acid receptors and regulates the transcription of specific target genes. In order to identify atRA-induced genes that play a role in neural development, a subtractive library was created from SH-SY5Y neuroblastoma cells, a human cell line that exhibits changes in cell adhesion and neurite outgrowth after exposure to the vitamin A acid. We report here the identification of 14 genes that are rapidly induced by atRA (retinoic acid induced in neurobIlastoma or RAINB), eight of which were previously not known to be atRA responsive (BTBD11, calmin, cyclin M2, ephrin B2, HOXD10, NEDD9, RAINB6 and tenascin R). mRNA regulation by atRA was confirmed in SHSY5Y cells by Northern blotting, and gene regulation was studied in additional human cell lines using the quantitative polymerase chain reaction. The majority of the atRA-responsive clones revealed in this screen are highly expressed in the nervous system of developing rat embryos. Further, the expression of several of these genes is perturbed in developing rat embryos exposed to excess atRA or conversely, deprived of sufficient retinoid during early development. We propose that a subset of these genes lie downstream of atRA and its receptors in the regulation of neurite outgrowth and cell adhesion in both neural and nonneural tissues within the developing embryo.

Group II introns are autocatalytic RNAs which selfsplice in vitro . However, in vivo additional protein factors might be involved in the splicing process. We used an affinity chromatography method called 'StreptoTag' to identify group II intron binding proteins from Saccharomyces cerevisiae . This method uses a hybrid RNA consisting of a streptomycin-binding affinity tag and the RNA of interest, which is bound to a streptomycin column and incubated with yeast protein extract. After several washing steps the bound RNPs are eluted by addition of streptomycin. The eluted RNPs are separated and the proteins identified by mass-spectrometric analysis. Using crude extract from yeast in combination with a substructure of the bI1 group II intron (domains IVVI) we were able to identify four glycolytic enzymes; glucose-6-phosphate isomerase (GPI), 3-phosphoglycerate kinase (PGK), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and triosephosphate isomerase (TPI). From these proteins GAPDH increases in vitro splicing of the bI1 group II intron by up to three times. However, in vivo GAPDH is not a group II intronsplicing factor, since it is not localised in yeast mitochondria. Therefore, the observed activity reflects an unexpected property of GAPDH. Band shift experiments and UV cross linking demonstrated the interaction of GAPDH with the group II intron RNA. This novel activity expands the reaction repertoire of GAPDH to a new RNA species.

Metalresponsive transcription factor-1 (MTF-1) is a zinc finger protein with a central role in heavy metal homeostasis/ detoxification. MTF-1 binds to DNA sequence motifs known as metal response elements (MREs) with a core consensus TGCRCNC. Since MTF-1 is also involved in other stress responses, we tested whether it is able to recognize different types of DNA sequence motifs. To this end we selected MTF-1-binding oligonucleotides from a collection of random sequences. Since MTF-1 binds to known target sequences at relatively high zinc concentrations, oligonucleotide selection was performed in a mammalian cell nuclear extract both at high and low zinc concentrations. Irrespective of zinc concentration, we find a robust representation of MRE consensus sequences, however with specific features. Selection was most efficient at 100 M zinc, yielding many oligonucleotides with two MRE motifs in divergent orientation of the sequence GTGTGCAT CACTT TGCGCAC . Oligonucleotides selected without zinc supplement contain a single high-affinity MRE with an extended flanking sequence of consensus TTT TGCGCAC GGCACTAAAT. This lowzinc MRE motif can bind MTF-1 and induce transcription in vivo , and is less dependent on zinc than the classical MREd motif from the mouse metallothioneinI promoter. At low zinc, we also found evidence for a negative role of nuclear factorI (NFI/CTFI) in MTF-1-dependent transcription. Finally, a selection in the presence of cadmium yielded no specific binding site for MTF-1, strongly supporting the concept of an indirect activation of MTF-1 by cadmium within a living cell.

Protein disulfide isomerase (PDI) is known to contain the thioredoxin box motif with a low pKa cysteine residue. To investigate the reactivity of PDI with thiol modifiers at low physiological pHs, either the reduced (PDI[red]) or oxidized form (PDI[oxid]) of PDI was exposed to various alkylating ragents. When PDI was incubated with iodoacetamide at pH 6.3 for 30 min at 38C, a remarkable inactivation (>90%) of PDI[red] was caused by iodoacetamide (IC[50]=8 M). However, PDI[oxid] was only slightly inactivated (approximately 18%) by iodoacetamide. Similarly, PDI[red] was significantly inactivated by Nethylmaleimide (NEM), but PDI[oxid] was not. When the inactivation by these alkylators was analyzed by pseudofirst order kinetics, NEM (k[3]=1.7510[-2] s[-1]; K[i]=124 M) was observed to be more potent than iodoacetamide (k[3]=9.110[-3] s[-1]; K=311 M). Interestingly, the inactivation of PDI[red] by iodoacetamide was greater at pH 6.3 than pH 7.0, in contrast to a similar inactivation potency of NEM at both pHs. Moreover, the maximal inactivation of PDI[red] or PDI[oxid] by iodoacetamide was mainly observed around pH 6.0. In addition, PDI[red] was found to be inactivated by acrolein (IC[50]=10 M) at pH 6.3, and this inactivation was also greater at pH 6.3 than at pH 7. Based on these results, we suggest that PDI[red] is susceptible to inactivation by alkylators including endogenous α,β-unsaturated aldehydes at low physiological pHs.

SIN-1 is frequently used in cell culture studies as an extracellularly operating generator of peroxynitrite. However, little is known about the nature of the reactive species produced intracellulary from SIN-1. SIN-1 can easily penetrate cells as exemplified for both L-929 mouse fibroblasts and bovine aortic endothelial cells (BAECs) by utilizing capillary zone electrophoresis. In L-929 cells, SIN-1 produced nitric oxide (NO) as monitored by the fluorescent NO scavenger FNOCT-1 and by means of a NO electrode, as well as reactive nitrogenoxide species (RNOS, e.g. peroxynitrite, nitrogen dioxide, dinitrogen trioxide), as detected with the fluorescent indicator DAF-2. Laser scanning microscopy revealed that in L-929 cells SIN-1-derived species initially oxidized the major fraction of the NAD(P)H within the cytosol and the nuclei, whereas the mitochondrial NAD(P)H level was somewhat increased. In marked contrast to this, in BAECs no evidence for NO formation was found although the intracellular amount of SIN-1 was fourfold higher than in L-929 cells. In BAECs, the level of NAD(P)H was slightly decreased within the first 10 min after administration of SIN-1 in both the cytosol/nuclei and mitochondria. These observations reflect the capability of SIN-1 to generate intracellularly either almost exclusively RNOS as in BAECs, or RNOS and freely diffusing NO as in L-929 cells. Nitric oxide as well as RNOS may decisively affect cellular metabolism as indicated by the alterations in the NAD(P)H level. Hence, care should be taken when applying SIN-1 as an exclusively peroxynitrite-generating compound in cell culture systems.

Transforming growth factor-β1 is a potent mediator of fibrosis stimulating the secretion of extracellular matrix proteins and is involved in airway remodeling in chronic obstructive pulmonary disease (COPD). Signals from the TGF superfamily are mediated by the SMAD group of transcription factors. Here, the expression of the regulatory SMAD2, 3, the co-SMAD4 and the inhibitory SMAD6 and 7 was assessed in bronchial biopsies of COPD patients and controls by quantitative RTPCR. While SMAD2 was not expressed and SMAD3 and 4 displayed no change, the inhibitory SMAD6 and 7 were significantly downregulated in COPD. To reveal the molecular basis of tobacco smoke-induced airway remodeling and to test whether it may interfere with intracellular SMAD signaling, the airway epithelial cell line A549 was incubated with cigarette smoke extract (1% and 10%) for 48 hours, which led to down-regulation of SMAD6 and 7 at both concentrations tested. It can be concluded that TGF-β-mediated effects in COPD are influenced by a disturbed intracellular feedback mechanism of inhibitory SMADs. Also, the effects of non-volatile components in tobacco smoke may partly be regulated via a smoke-induced down-regulation of inhibitory SMADs.

The family of tissue inhibitors of metalloproteinases (TIMPs) exhibits diverse physiological/biological functions including the inhibition of active matrix metalloproteinases, regulation of proMMP activation, cell growth, and the modulation of angiogenesis. TIMP-1 is a secreted protein that can be detected on the cell surface through its interaction with surface proteins. The diverse biological functions of TIMP-1 are thought to lie, in part, in the kinetics of TIMP-1/MMP/surface protein interactions. Proteins anchored by glycoinositol phospholipids (GPIs), when purified and added to cells in vitro , are incorporated into their surface membranes. A GPI anchor was fused to TIMP-1 to generate a reagent that could be added directly to cell membranes and thus focus defined concentrations of TIMP-1 protein on any cell surface independent of protein-protein interaction. Unlike native TIMP-1, exogenously added GPI anchored TIMP-1 protein effectively blocked release of MMP-2 and MMP-9 from osteosarcoma cells. TIMP-1-GPI was a more effective modulator of migration and proliferation than TIMP 1. While control hTIMP-1 protein did not significantly affect migration of primary microvascular endothelial cells at the concentrations tested, the GPI-anchored TIMP-1 protein showed a pronounced suppression of endothelial cell migration in response to bFGF. In addition, TIMP-1-GPI was more effective at inducing microvascular endothelial proliferation. In contrast, fibroblast proliferation was suppressed by the agent. Reagents based on this method should assist in the dissection of the protease cascades and activities involved in TIMP biology. Membranefixed TIMP-1 may represent a more effective version of the protein for use in therapeutic expression.

Cathepsins are implicated in a multitude of physiological and pathophysiological processes. The aim of the present study was to investigate the function of cathepsin L (catL) in the proteolytic network of human lung epithelial cells and its role in the regulation of apoptosis. We found that catL-deficient A549 cells as well as lung tissue extracts of catL[-/-] mice express increased amounts of single-chain cathepsin D (catD). Degradation experiments indicate that catL specifically degrades the singlechain isoform of catD. Furthermore, we found that catLdeficient cells showed increased sensitivity to apoptosis. Finally, we demonstrate that the inhibition of catD activity by pepstatin A decreased the number of apoptotic cells in catLdeficient A549 cells after anti-Fas treatment. In conclusion, catL is involved in catD processing and the accumulation of catD isoforms in catL-deficient cells is associated with increased rates of spontaneous and anti-Fas-induced apoptosis.