Sequences in intron 51 of the von Willebrand factor gene target promoter activation to a subset of lung endothelial cells in transgenic mice.

In vivo analyses of the VWF promoter previously demonstrated that a fragment spanning sequences -487 to +247 targets promoter activation to brain vascular endothelial cells, whereas a longer fragment including 2182 bp of the 5'-flanking sequences, the first exon, and the first intron activated expression in endothelial cells of the heart and muscles as well as the brain of transgenic mice. These results suggested that additional VWF gene sequences were required for expression in other vascular endothelial cells in vivo. We have now identified a region within intron 51 of the VWF gene that is DNase I-hypersensitive (HSS) specifically in non-endothelial cells and interacts with endothelial and non-endothelial specific complexes that contain YY1. We demonstrate that beta-actin is associated with YY1 specifically in the nucleus of non-endothelial cells and is a component of the nuclear protein complexes that interact with the DNase I-hypersensitive region. In vitro transfection analyses demonstrated that HSS sequences containing this YY1-binding site do not significantly affect VWF promoter activity. However, in vivo analyses demonstrated that addition of these sequences to the VWF promoter (-487 to +247) results in promoter activation in lung and brain vascular endothelial cells. These results demonstrate that the HSS sequences in intron 51 of the VWF gene contain cis-acting elements that are necessary for the VWF gene transcription in a subset of lung endothelial cells in vivo.

Three-dimensional tissue cytometer based on high-speed multiphoton microscopy.

Image cytometry technology has been extended to 3D based on high-speed multiphoton microscopy. This technique allows in situ study of tissue specimens preserving important cell-cell and cell-extracellular matrix interactions. The imaging system was based on high-speed multiphoton microscopy (HSMPM) for 3D deep tissue imaging with minimal photodamage. Using appropriate fluorescent labels and a specimen translation stage, we could quantify cellular and biochemical states of tissues in a high throughput manner. This approach could assay tissue structures with subcellular resolution down to a few hundred micrometers deep. Its throughput could be quantified by the rate of volume imaging: 1.45 mm(3)/h with high resolution. For a tissue containing tightly packed, stratified cellular layers, this rate corresponded to sampling about 200 cells/s. We characterized the performance of 3D tissue cytometer by quantifying rare cell populations in 2D and 3D specimens in vitro. The measured population ratios, which were obtained by image analysis, agreed well with the expected ratios down to the ratio of 1/10(5). This technology was also applied to the detection of rare skin structures based on endogenous fluorophores. Sebaceous glands and a cell cluster at the base of a hair follicle were identified. Finally, the 3D tissue cytometer was applied to detect rare cells that had undergone homologous mitotic recombination in a novel transgenic mouse model, where recombination events could result in the expression of enhanced yellow fluorescent protein in the cells. 3D tissue cytometry based on HSMPM demonstrated its screening capability with high sensitivity and showed the possibility of studying cellular and biochemical states in tissues in situ. This technique will significantly expand the scope of cytometric studies to the biomedical problems where spatial and chemical relationships between cells and their tissue environments are important.

Nitric oxide-induced nuclear translocation of the metal responsive transcription factor, MTF-1 is mediated by zinc release from metallothionein.

We previously showed that the major Zn-binding protein, metallothionein (MT) is a critical target for nitric oxide (NO) with resultant increases in labile Zn. We now show that NO donors also affected the activity of the metal responsive transcription factor MTF-1 that translocates from the cytosol to the nucleus in response to physiologically relevant increases in intracellular Zn and transactivates MT gene expression. Exposing mouse lung endothelial cells (MLEC) to ZnCl(2) or the NO donor, S-Nitroso-N-acetylpenicillamine (SNAP, 200 microM), caused nuclear translocation of a reporter molecule consisting of enhanced green fluorescent protein (EGFP) fused to MTF-1 (pEGFP-MTF-1). In separate experiments, NO donors induced increases in MT protein levels (Western blot). In contrast, NO did not cause nuclear translocation of EGFP-MTF-1 in MLEC from MT knockouts, demonstrating a central role for MT in mediating this response. These data suggest that S-nitrosation of Zn-thiolate clusters in MT and subsequent alterations in Zn homeostasis are participants in intracellular NO signaling pathways affecting gene expression.

Fluorescence resonance energy transfer-based assays for the real-time detection of nitric oxide signaling.

Low-molecular-weight S-nitrosothiols are found in many tissues and affect an array of signaling pathways via decomposition to *NO or exchange of their -NO function with thiol-containing proteins (transnitrosation). We used spectral laser scanning confocal imaging to visualize the effects of the membrane permeant S-nitrosothiol, S-nitrosocysteine ethyl ester (SNCEE), on a fluorescence resonance energy transfer (FRET) reporter based on the cysteine-rich heavy metal binding protein, metallothionein (FRET-MT) flanked by enhanced cyan and yellow fluorescent proteins (ECFP and EYFP, respectively). We previously showed that FRET can be used to follow metal binding and release by this construct. SNCEE (50 microM) induced a decrease in energy transfer, as shown by an increase in the peak emission intensity of the donor fluorophore (ECFP) and a decrease in that of the acceptor (EYFP). These changes in intramolecular FRET were reversed by 50 microM dithiothreitol (DTT), suggesting nitrosothiol-mediated modification of a cysteine residue in MT. Furthermore, the effects of SNCEE on the FRET-MT reporter were not affected by HbO(2), which would be expected to block any process involving (*)NO liberated by decomposition of nitrosothiol but would not necessarily affect transnitrosation. In further support of SNCEE-induced conformational changes in MT, we used live cell imaging of the zinc-sensitive fluorescent indicator FluoZin-3 to show that SNCEE also caused increases in labile Zn(2+).

Nitric oxide-induced modification of protein thiolate clusters as determined by spectral fluorescence resonance energy transfer in live endothelial cells.

Low-molecular-weight S-nitrosothiols are found in many tissues and affect a diverse array of signaling pathways via decomposition to *NO or exchange of their -NO function with thiol-containing proteins (transnitrosation). We used spectral laser scanning confocal imaging to visualize the effects of D- and L-stereoisomers of S-nitrosocysteine ethyl ester (SNCEE) on fluorescence resonance energy transfer (FRET)-based reporters that are targets for the following NO-related modifications: (a) S-nitrosation, via the cysteine-rich protein metallothionein (FRET-MT), and (b) nitrosyl-heme-Fe, via guanosine 3',5'-cyclic monophosphate (cygnet-2). Conformational changes consistent with S-nitrosation of FRET-MT were specific to l-SNCEE. In addition, they were reversed by dithiothreitol (DTT) but unaffected by exogenous oxyhemoglobin. In contrast, d- and l-SNCEE had comparable effects on cygnet-2, likely via activation of soluble guanylyl cyclase (sGC) by *NO as they were sensitive to the sGC inhibitor 1H-[1,2,4]-oxadiazolo[4,3-alpha] quinoxalin-1-one and exogenous oxyhemoglobin. These data demonstrate the utility of spectral laser scanning confocal imaging in revealing subtle aspects of NO signal transduction in live cells. Stereoselective transnitrosation of MT emphasizes the specificity of posttranslational modification as a component of NO signaling.

Spontaneous mitotic homologous recombination at an enhanced yellow fluorescent protein (EYFP) cDNA direct repeat in transgenic mice.

A transgenic mouse has been created that provides a powerful tool for revealing genetic and environmental factors that modulate mitotic homologous recombination. The fluorescent yellow direct-repeat (FYDR) mice described here carry two different copies of expression cassettes for truncated coding sequences of the enhanced yellow fluorescent protein (EYFP), arranged in tandem. Homologous recombination between these repeated elements can restore full-length EYFP coding sequence to yield a fluorescent phenotype, and the resulting fluorescent recombinant cells are rapidly quantifiable by flow cytometry. Analysis of genomic DNA from recombined FYDR cells shows that this mouse model detects gene conversions, and based on the arrangement of the integrated recombination substrate, unequal sister-chromatid exchanges and repair of collapsed replication forks are also expected to reconstitute EYFP coding sequence. The rate of spontaneous recombination in primary fibroblasts derived from adult ear tissue is 1.3 +/- 0.1 per 106 cell divisions. Interestingly, the rate is approximately 10-fold greater in fibroblasts derived from embryonic tissue. We observe an approximately 15-fold increase in the frequency of recombinant cells in cultures of ear fibroblasts when exposed to mitomycin C, which is consistent with the ability of interstrand crosslinks to induce homologous recombination. In addition to studies of recombination in cultured primary cells, the frequency of recombinant cells present in skin was also measured by direct analysis of disaggregated cells. Thus, the FYDR mouse model can be used for studies of mitotic homologous recombination both in vitro and in vivo.

Nitric oxide decreases the sensitivity of pulmonary endothelial cells to LPS-induced apoptosis in a zinc-dependent fashion.

We hypothesized that: (a) S-nitrosylation of metallothionein (MT) is a component of pulmonary endothelial cell nitric oxide (NO) signaling that is associated with an increase in labile zinc; and (b) NO mediated increases in labile zinc in turn reduce the sensitivity of pulmonary endothelium to LPS-induced apoptosis. We used microspectrofluorometric techniques to show that exposing mouse lung endothelial cells (MLEC) to the NO-donor, S-nitrosocysteine, resulted in a 45% increase in fluorescence of the Zn2+-specific fluorophore, Zinquin, that was rapidly reversed by exposure to the Zn2+ chelator, NNN'N'-tetrakis-(2-pyridylmethyl)ethylenediamine; TPEN). The absence of a NO-mediated increase in labile Zn2+ in MLEC from MT-I and -II knockout mice inferred a critical role for MT in the regulation of Zn2+ homeostasis by NO. Furthermore, we found that prior exposure of cultured endothelial cells from sheep pulmonary artery (SPAEC), to the NO-donor, S-nitroso-N-acetylpenicillamine (SNAP) reduced their sensitivity to lipopolysaccharide (LPS) induced apoptosis. The anti-apoptotic effects of NO were significantly inhibited by Zn2+ chelation with low doses of TPEN (10 microM). Collectively, these data suggest that S-nitrosylation of MT is associated with an increase in labile (TPEN chelatable) zinc and NO-mediated MT dependent zinc release is associated with reduced sensitivity to LPS-induced apoptosis in pulmonary endothelium.