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While young muscle is capable of restoring the original architecture of damaged myofibers, aged muscle displays a markedly reduced regeneration. We show that expression of the “anti-aging” protein, α-Klotho, is up-regulated within young injured muscle as a result of transient Klotho promoter demethylation. However, epigenetic control of the Klotho promoter is lost with aging. Genetic inhibition of α-Klotho in vivo disrupted muscle progenitor cell (MPC) lineage progression and impaired myofiber regeneration, revealing a critical role for α-Klotho in the regenerative cascade. Genetic silencing of Klotho in young MPCs drove mitochondrial DNA (mtDNA) damage and decreased cellular bioenergetics. Conversely, supplementation with α-Klotho restored mtDNA integrity and bioenergetics of aged MPCs to youthful levels in vitro and enhanced functional regeneration of aged muscle in vivo in a temporally-dependent manner. These studies identify a role for α-Klotho in the regulation of MPC mitochondrial function and implicate α-Klotho declines as a driver of impaired muscle regeneration with age. While young muscle faithfully regenerates damaged myofibers, aged muscle is impaired. Here the authors show the “anti-aging” protein α-Klotho is upregulated in young muscle after damage via promoter demethylation and this regulation is lost in aging, resulting in mitochondrial damage and an impaired healing response.

Nuclear factor κB (NF-κB) is a transcription factor regulating expression of genes intrinsic to inflammation and cell proliferation-features of asbestos-associated diseases. In studies here, crocidolite asbestos caused protracted and dose-responsive increases in proteins binding to nuclear NF-κB-binding DNA elements in hamster tracheal epithelial (HTE) cells. This binding was modulated by cellular glutathione levels. Antibodies recognizing p65 and p50 protein members of the NF-κB family revealed these proteins in two of the DNA complexes. Transient transfection assays with a construct containing six NF-κB-binding DNA consensus sites linked to a luciferase reporter gene indicated that asbestos induced transcriptional activation of NF-κB-dependent genes, an observation that was confirmed by Northern blot analyses for c-myc mRNA levels in HTE cells. Studies suggest that NF-κB induction by asbestos is a key event in regulation of multiple genes involved in the pathogenesis of asbestos-related lung cancers.

The mechanisms of cell signaling and altered gene expression by asbestos, a potent inflammatory, fibrogenic, and carcinogenic agent, are unclear. Activation of the transcription factor, nuclear factor (NF)-kappa B, is critical in up-regulating the expression of many genes linked to inflammation and proliferation. Inhalation models of crocidolite- and chrysotile-induced inflammation and asbestosis were used to study the localization of p65, a protein subunit of the NF-kappa B transcription factor, in sham control rats and those exposed to asbestos. In addition, we investigated, using electrophoretic mobility shift analysis, whether in vitro exposure of rat lung epithelial cells and rat pleural mesothelial cells to asbestos increased binding of nuclear proteins, including p65, to the NF-kappa B DNA response element. Furthermore, translocation of p65 into the nucleus was determined by confocal microscopy. In comparison with sham animals, striking increases in p65 immunofluorescence were observed in airway epithelial cells of rats at 5 days after inhalation of asbestos. These increases were diminished by 20 days, the time period necessary for development of fibrotic lesions. In contrast, although inter-animal variability was observed, immunoreactivity for p65 was more dramatic in the interstitial compartment of asbestos-exposed rat lungs at both 5 and 20 days. Changes in p65 expression in pleural mesothelial cells exposed to asbestos in inhalation experiments were unremarkable. Exposure to asbestos also caused significant increases in nuclear protein complexes that bind the NF-kappa B consensus DNA sequence in both rat lung epithelial and rat pleural mesothelial cells. Using confocal microscopy, we observed partial nuclear translocation of p65 in rat pleural mesothelial cells exposed to asbestos. This partial response contrasted with the effects of lipopolysaccharide, which caused rapid and complete translocation of p65 from cytoplasm to nucleus. Our studies are the first to show the presence of the NF-kappa B system in lung tissue and evidence of activation in vitro and in vivo after exposure to a potent inflammatory, fibrinogenic, and carcinogenic environmental agent.

In recent years, it has become apparent that minerals can trigger alterations in gene expression by initiating signaling events upstream of gene transactivation. These cascades may be initiated at the cell surface after interaction of minerals with the plasma membrane either through receptorlike mechanisms or integrins. Alternatively, signaling pathways may be stimulated by active oxygen species generated both during phagocytosis of minerals and by redox reactions on the mineral surface. At least two signaling cascades linked to activation of transcription factors, i.e., DNA-binding proteins involved in modulating gene expression and DNA replication, are stimulated after exposure of lung cells to asbestos fibers in vitro. These include nuclear factor κB (NFκB) and the mitogen-activated protein kinase (MAPK) cascade important in regulation of the transcription factor, activator protein-1 (AP-1). Both NFκB and AP-1 bind to specific DNA sequences within the regulatory or promoter regions of genes that are critical to cell proliferation and inflammation. Unraveling the cell signaling cascades initiated by mineral dusts and pharmacologic inhibition of these events may be important for the control and treatment of mineral-associated occupational diseases.

Examining global effects of toxic metals on gene expression can be useful for elucidating patterns of biological response, discovering underlying mechanisms of toxicity, and identifying candidate metal-specific genetic markers of exposure and response. Using a 1,200 gene nylon array, we examined changes in gene expression following low-dose, acute exposures of cadmium, chromium, arsenic, nickel, or mitomycin C (MMC) in BEAS-2B human bronchial epithelial cells. Total RNA was isolated from cells exposed to 3 μM Cd(II) (as cadmium chloride), 10 μM Cr(VI) (as sodium dichromate), 3 μ g/ cm2Ni(II) (as nickel subsulfide), 5 μM or 50 μM As(III) (as sodium arsenite), or 1 μM MMC for 4 hr. Expression changes were verified at the protein level for several genes. Only a small subset of genes was differentially expressed in response to each agent: Cd, Cr, Ni, As (5 μM), As (50 μM), and MMC each differentially altered the expression of 25, 44, 31, 110, 65, and 16 individual genes, respectively. Few genes were commonly expressed among the various treatments. Only one gene was altered in response to all four metals (hsp90), and no gene overlapped among all five treatments. We also compared low-dose (5 μM, noncytotoxic) and high-dose (50 μM, cytotoxic) arsenic treatments, which surprisingly, affected expression of almost completely nonoverlapping subsets of genes, suggesting a threshold switch from a survival-based biological response at low doses to a death response at high doses.

Environmental exposures to toxic metals result in ecological effects that are difficult to detect and quantify. There is a pressing need for biomarkers that can be used in the field and that are sensitive to the sublethal metal levels that are prevalent in the environment. Using a combination of molecular and demographic techniques, we developed a biomarker to detect arsenic stress in freshwater zooplankton. We measured changes in HSP 83 mRNA levels by RT-PCR analysis in Daphnia pulex adults and juveniles exposed to arsenite and arsenate, and we compared the HSP response to demographic effects on Daphnia survival, reproduction and development. HSP 83 appears promising as a biomarker for arsenic stress given that arsenic exposure induced an increased and sustained expression of mRNA. Both molecular and demographic measures of arsenic stress revealed differences between adult and juvenile responses and toxicity of arsenate and arsenite. The molecular responses are more sensitive than the demographic response of individuals exposed as adults but corroborate the reproductive responses for individuals exposed as neonates.[PUBLICATION ABSTRACT]

Exposure of low-passage endothelial cells in culture to nonlethal amounts of asbestos, but not refractory ceramic fiber-1, increases cell motility and gene expression. These changes may be initiated by the fibers mimicking matrix proteins as ligands for receptors on the cell surface. In the present study, 1- to 3-hr exposures of endothelial cells to 5 mg/ cm2of chrysotile asbestos caused marked cell elongation and motility. However, little morphological change was seen when chrysotile was added to cells pretreated with either mannosamine to prevent assembly of glycophosphatidylinositol (GPI)-anchored receptors or with herbimycin A to inhibit tyrosine kinase activity. Affinity purification of GPI-anchored urokinase-type plasminogen activator receptor (uPAR) from chrysotile-exposed cells demonstrated that asbestos altered the profile of proteins and phosphoproteins complexed with this receptor. Tyrosine kinase activities in the complexes were also increased by asbestos. Immunoprecipitations with selective monoclonal antibodies demonstrated that both chrysotile and crocidolite asbestos increase kinase activities associated with p60 Src or p120 focal adhesion kinase (FAK). Further, chrysotile also changed the profile of proteins and phosphoproteins associated with FAK in intact cells. These data suggest that asbestos initiates endothelial cell phenotypic change through interactions with uPAR-containing complexes and that this change is mediated through tyrosine kinase cascades.

Exposure to arsenic in drinking water increases incidence of cardiovascular diseases. However, the basic mechanisms and genetic changes that promote these diseases are unknown. This study investigated the effects of chronic arsenic exposure on vessel growth and expression of angiogenic and tissue remodeling genes in cardiac tissues. Male mice were exposed to low to moderately high levels of arsenite (AsIII) for 5, 10, or 20 wk in their drinking water. Vessel growth in Matrigel implants was tested during the last 2 wk of each exposure period. Implant vascularization increased in mice exposed to 5-500 ppb AsIII for 5 wk. Similar increases were seen following exposure to 50-250 ppb of AsIII over 20 wk, but the response to 500 ppb decreased with time. RT-PCR analysis of cardiac mRNA revealed differential expression of angiogenic or tissue remodeling genes, such as vascular endothelial cell growth factor (VEGF), VEGF receptors, plasminogen activator inhibitor-1, endothelin-1, and matrix metalloproteinase-9, which varied with time or amount of exposure. VEGF receptor mRNA and cardiac microvessel density were reduced by exposure to 500 ppb AsIII for 20 wk. These data demonstrate differential concentration and time-dependent effects of chronic arsenic exposure on cardiovascular phenotype and vascular remodeling that may explain the etiology for AsIII-induced disease.

Background: Roxarsone (3-nitro-4-hydroxy benzene arsonic acid) is an arsenic compound widely used in the poultry industry as a feed additive to prevent coccidiosis, stimulate growth, and to improve tissue pigmentation. Little is known about the potential human health effects from roxarsone released into the environment from chicken waste or from residual compound in chicken products. Objective: The growth potentiation and enhanced tissue pigmentation suggest that low levels of roxarsone exposure may have an angiogenic potential similar to that of inorganic arsenite (As). The goal of this investigation was to test the hypothesis described above using cultured human aortic and lung microvascular endothelial cells in high-content imaging tube-forming assays and begin developing a molecular level understanding of the process. Methods: We used a three-dimensional Matrigel assay for probing angiogenesis in cultured human endothelial cells, and a polymerase chain reaction (PCR) array to probe the gene changes as a function of roxarsone or As super(III) treatment. In addition, we used Western blot analysis for changes in protein concentration and activation. Results: Roxarsone was found to exhibit a higher angiogenic index than As super(III) at lower concentrations. Increased endothelial nitric oxide synthase (eNOS) activity was observed for roxarsone but not for As super(III)-induced angiogenesis. However, As super(III) caused more rapid and pronounced phosphorylation of eNOS. Quantitative PCR array on select genes revealed that the two compounds have different and often opposite effects on angiogenic gene expression. Conclusions: The results demonstrate that roxarsone and As super(III) promote angiogenic phenotype in human endothelial cells through distinctly different signaling mechanisms.