Specificity of the Metallothionein-1 Response by Cadmium-Exposed Human Urothelial Cells

Occupational and environmental exposure to cadmium is associated with the development of urothelial cancer. The metallothionein (MT) family of genes encodes proteins that sequester metal ions and modulate physiological processes, including zinc homeostasis. Little is known about the selectivity of expression of the different MT isoforms. Here, we examined the effect of cadmium exposure on MT gene and isoform expression by normal human urothelial (NHU) cell cultures. Baseline and cadmium-induced MT gene expression was characterized by next generation sequencing and RT-PCR; protein expression was assessed by western blotting using isoform specific antibodies. Expression of the zinc transporter-1 (SLC30A1) gene was also assessed. NHU cells displayed transcription of MT-2A, but neither MT-3 nor MT-4 genes. Most striking was a highly inducer-specific expression of MT-1 genes, with cadmium inducing transcription of MT-1A, MT-1G, MT-1H and MT-1M. Whereas MT-1G was also induced by zinc and nickel ions and MT-1H by iron, both MT-1A and MT-1M were highly cadmium-specific, which was confirmed for protein using isoform-specific antibodies. Protein but not transcript endured post exposure, probably reflecting sequestration. SLC30A1 transcription was also affected by cadmium ion exposure, potentially reflecting perturbation of intracellular zinc homeostasis. We conclude that human urothelium displays a highly inductive profile of MT-1 gene expression, with two isoforms identified as highly specific to cadmium providing candidate transcript and long-lived protein biomarkers of cadmium exposur

VE-statin/egfl7 Expression in Endothelial Cells Is Regulated by a Distal Enhancer and a Proximal Promoter under the Direct Control of Erg and GATA-2

Angiogenesis is the process by which new blood vessels arise from existing ones by the budding out of endothelial cell capillaries from the luminal side of blood vessels. Blood vessel formation is essential for organ development during embryogenesis and is associated with several physiological and pathological processes, such as wound healing and tumor development. The VE-statin/egfl7 gene is specifically expressed in endothelial cells during embryonic development and in the adult. We studied here the regulatory mechanisms that control this tissue-specific expression. RT-qPCR analyses showed that the specificity of expression of VE-statin/egfl7 in endothelial cells is not shared with its closest neighbor genes notch1 and agpat2 on the mouse chromosome 2. Chromatin-immunoprecipitation analysis of histone modifications at the VE-statin/egfl7 locus showed that the chromatin is specifically opened in endothelial cells, but not in fibroblasts at the transcription start sites. A 13 kb genomic fragment of promoter was cloned and analyzed by gene reporter assays which showed that two conserved regions are important for the specific expression of VE-statin/egfl7 in endothelial cells; a −8409/−7563 enhancer and the −252/+38 region encompassing the exon-1b transcription start site. The latter contains essential GATA and ETS-binding sites, as assessed by linker-scanning analysis and site-directed mutagenesis. An analysis of expression of the ETS and GATA transcription factors showed that Erg, Fli-1 and GATA-2 are the most highly expressed factors in endothelial cells. Erg and GATA-2 directly control the expression of the endogenous VE-statin/egfl7 while Fli-1 probably exerts an indirect control, as assessed by RNA interference and chromatin immunoprecipitation. This first detailed analysis of the mechanisms that govern the expression of the VE-statin/egfl7 gene in endothelial cells pinpoints the specific importance of ETS and GATA factors in the specific regulation of genes in this cell lineage

Studies on the regulation of human metallothionein gene expression

Bibliography: p. 171-181

Age-Associated Increase in Thrombogenicity and Its Correlation with von Willebrand Factor

Endothelial cells that cover the lumen of all blood vessels have the inherent capacity to express both pro and anticoagulant molecules. However, under normal physiological condition, they generally function to maintain a non-thrombogenic surface for unobstructed blood flow. In response to injury, certain stimuli, or as a result of dysfunction, endothelial cells release a highly adhesive procoagulant protein, von Willebrand factor (VWF), which plays a central role in formation of platelet aggregates and thrombus generation. Since VWF expression is highly restricted to endothelial cells, regulation of its levels is among the most important functions of endothelial cells for maintaining hemostasis. However, with aging, there is a significant increase in VWF levels, which is concomitant with a significant rise in thrombotic events. It is not yet clear why and how aging results in increased VWF levels. In this review, we have aimed to discuss the age-related increase in VWF, its potential mechanisms, and associated coagulopathies as probable consequences

Endothelial cell dysfunction in response to intracellular overexpression of amyloid precursor protein

Previous reports have shown that exposure of vascular endothelial and smooth muscle cells to exogenous amyloid beta (Aβ) peptide results in cell damage and toxicity via oxidative injury. In this study we demonstrate that overexpression of the amyloid precursor protein (APP) is toxic to bovine aortic endothelial cells but not to bovine aortic smooth muscle cells. Intracellular coexpression of the free radical scavenger proteins metallothionein or MnSOD abolished the toxic effect of APP overexpression in endothelial cells. Our results demonstrate that endothelial cells are specifically susceptible to intracellular overexpression of APP and free radical generation is the likely mechanism of cell damage due to APP overexpression

Endothelial cell dysfunction in response to intracellular overexpression of amyloid precursor protein

Vascular damage as a result of amyloid protein (Aβ) accumulation is observed in brain vasculature of patients with Alzheimer\u27s disease and cerebral amyloid angiopathy (CAA). Aβ is a proteolytic product of amyloid precursor protein (APP) which is expressed in many cell types but it is not clear which cell type is the source of toxic Aβ production. Exposure of vascular endothelial and smooth muscle cells to exogenous Aβ results in toxicity in both cell types. To determine the effect of endogenous overexpression of APP in endothelial and smooth muscle cells plasmids containing wild type APP cDNA or a Dutch mutant variant (associated with CAA) were stably introduced into bovine aortic endothelial (BAE) cells and bovine smooth muscle (BSM) cells. A significant morphological change was observed in endothelial cells expressing either wild type or mutant APP cDNA These cells exhibited a large number of vacuoles followed by gradual cell death. Determination of eel! viability (MTT assay) confirmed these observations and demonstrated the toxic effect of the amyloid expression in endothelial cells. Intracellular coexpression of plasmids containing free radical scavengers cDNA (either metallothionein or manganese Superoxide dismutase), abolished the toxic effect of APP in endothelial cells. These results suggest that APP overexpression exerts its toxic effect on endothelial cells via generation of free radicals. In striking contrast the expression of the wild type or mutant APP in smooth muscle cells had no observed toxic effects, even when cells were maintained in culture as long as 3 months post transfection. Similar expression levels of the APP proteins in both transfected endothelial and smooth muscle cells was confirmed by immunohistochemistry. Thus the endothelial and smooth muscle cell components of the vasculature differentially respond to an endogenous overexpression of APP, and endothelial cells are specifically susceptible to increased endogenous APP levels