Malondialdehyde inhibits an AMPK-mediated nuclear translocation and repression activity of ALDH2 in transcription.

Aging process results from deleterious damages by reactive oxygen species, in particular, various metabolic aldehydes. Aldehyde dehydrogenase 2 (ALDH2) is one of metabolic enzymes detoxifying various aldehydes under oxidative conditions. AMP-activated protein kinase (AMPK) plays a key role in controlling metabolic process. However, little was known about the relationship of ALDH2 with AMPK under oxidative conditions. Here, we, by using MDA-specific monoclonal antibody, screened the tissues of young and old rats for MDA-modified proteins and identified an ALDH2 as a prominent MDA-modified protein band in the old rat kidney tissue. ALDH2 associates with AMPK and is phosphorylated by AMPK. In addition, AICAR, an activator of AMP-activated protein kinase, induces the nuclear translocation of ALDH2. ALDH2 in nucleus is involved in general transcription repression by association with histone deacetylases. Furthermore, MDA modification inhibited the translocation of ALDH2 and the association with AMPK, and ultimately led to de-repression of transcription in the reporter system analysis. In this study, we have demonstrated that ALDH2 acts as a transcriptional repressor in response to AMPK activation, and MDA modifies ALDH2 and inhibits repressive activity of ALDH2 in general transcription. We thus suggest that increasing amount of MDA during aging process may interrupt the nuclear function of ALDH2, modulated by AMPK.

Lysophosphatidic acid and adenylyl cyclase inhibitor increase proliferation of senescent human diploid fibroblasts by inhibiting adenosine monophosphate-activated protein kinase.

This study was designed to elucidate the molecular mechanism underlying lysophosphatidic acid (LPA) and adenylyl cyclase inhibitor SQ22536 (ACI)-induced senescent human diploid fibroblast (HDF) proliferation. Because adenosine monophosphate (AMP)-activated protein kinase (AMPK) is known to inhibit cell proliferation, we examined the phosphorylation status of AMPK and p53 and the expression level of p21(waf1/cip1) after treating HDFs with LPA and ACI. Phosphorylation of AMPKalpha on threonine-172 (p-Thr172-AMPKalpha) increases its catalytic activity but phosphorylation on serine-485/491 (p-Ser485/491-AMPKalpha) reduces the accessibility of the Thr172 phosphorylation site thereby inhibiting its catalytic activity. LPA increased p-Ser485/491-AMPKalpha, presumably by activating cAMP-dependent protein kinase (PKA). However, ACI reduced p-Thr172-AMPKalpha by inhibiting the LKB signaling. Our data demonstrated that both LPA and ACI inhibit the catalytic activity of AMPKalpha and p53 by differentially regulating phosphorylation of AMPKalpha, causing increased senescent cell proliferation. These findings suggest that the proliferation potential of senescent HDFs can be modulated through the regulation of the AMPK signaling pathway.

Caenorhabditis elegans as a screening tool for the endothelial cell-derived putative aging-related proteins detected by proteomic analysis.

Endothelial cells go through progressive pathophysiologic modification as cellular senescence progresses. In vitro, endothelial cell senescence is accompanied by failure of proliferation and by perturbations in gene and protein expressions. Moreover, this cellular senescence in culture has been proposed to reflect processes that occur in the organism in vivo and free radical theory is accepted to be the most plausible explanation for this process. We have screened proteins involved in both cellular senescence and reactive oxygen species induced condition using 2-D gel analysis and found that ubiquitin carboxyl terminal hydrolase L1, peroxyredoxin 2, peroxyredoxin 4, fatty acid binding proteins (FABPs), and 5'-AMP-activated protein kinase beta-1 subunit were candidate aging-related proteins. To evaluate in vivo function of these proteins, Caenorhabditis elegans (C. elegans) knock-down system using RNA interference was applied. Aging-specific expression of lipofucsin and the lifespan of knocked-down C. elegans were observed to assess the outcome. Interestingly, the inhibition of the genes led to short lifespan and earlier accumulation of lipofucsin with increasing age when compared with the wild type. These results suggest that the above genes may be related to cellular senescence process in determining the longevity in C. elegans and that gene inactivation renders animals susceptible to oxidative stress.

Proteomic analysis of the proteins expressed by hydrogen peroxide treated cultured human dermal microvascular endothelial cells.

Reactive oxygen species (ROS) have been traditionally regarded as toxic by-products of aerobic metabolism. However, ROS also act as intracellular signaling molecules and can mediate phenotypes in vascular endothelial cells, which may be physiological or pathological in nature. To clarify the molecular mechanisms of ROS signaling, we examined hydrogen peroxide (H(2)O(2))-responsive proteins in cultured human dermal microvascular endothelial cells (HMVEC) using proteomic tools. Protein expression in HMVEC was studied after they had been exposed to low- and high-levels of H(2)O(2) for various times, and intracellular ROS production was examined by flow cytometer and UV spectrophotometer. Proteins obtained from dose- and time-dependent series were separated by two-dimensional gel electrophoresis and tentatively identified by matrix-assisted laser desorption-time of flight mass spectrometry, by matching the tryptic mass maps obtained with entries in the NCBI and Swiss-Prot protein sequence database. At least 163 proteins were changed by H(2)O(2), and 60 proteins were identified. Oxidative stress triggered dramatic change in the expression of proteins in primary microvessel endothelial cells, and their mapping to cellular process provided a view of the ubiquitous cellular changes elicited by H(2)O(2). These results could provide a framework for the understanding of the mechanisms of cellular redox homeostasis and H(2)O(2) metabolism in microendothelium environment in various biological processes as well as pathological conditions.

Expression of psoriasis-associated fatty acid-binding protein in senescent human dermal microvascular endothelial cells.

Aging is associated with the progressive pathophysiologic modification of endothelial cells. In vitro endothelial cell senescence is accompanied by proliferative activity failure and by perturbations in gene and protein expressions. Moreover, this cellular senescence in culture has been proposed to reflect processes that occur in aging organisms. In order to observe the changing patterns of protein expression in senescent human dermal microvascular endothelial cells (HDMECs), proteins obtained from both early- and late-passaged HDMECs were separated by two-dimensional electrophoresis, visualized by silver staining, and quantified by image processing. Proteins of interest were extracted by in-gel digestion with trypsin and quantified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), by searching the National Center for Biotechnology Information protein-sequence database. More than 2000 spots were detected by 2D electrophoresis within a linear pH range of 3-10. Twenty-two major differentially expressed spots were observed in serially passaged HDMECs and identified with high confidence by MALDI-TOF-MS. One of these spots was found to be a 14-15 kDa psoriasis-associated fatty acid-binding protein (PA-FABP) with high affinity for long-chain fatty acids. The expression of PA-FABP was confirmed to be elevated in senescent HDMECs (passage 20) by fluorescence-activated cell sorting (FACS), confocal laser microscopy, and by immunohistochemistry in aged human skin tissue. Our results suggest that the overexpression of FABP in cultured senescent HDMECs is closely related to skin aging.

Human alpha-enolase from endothelial cells as a target antigen of anti-endothelial cell antibody in Behçet's disease.

OBJECTIVE: To identify and recombine a protein of the human dermal microvascular endothelial cell (HDMEC) that specifically reacts with anti-endothelial cell antibody (AECA) in the serum of patients with Behçet's disease (BD), and to evaluate the usefulness of this protein in BD. METHODS: The proteomics technique, with 2-dimensional gel electrophoresis and matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry, was used to identify and recombine HDMEC antigen. Western blotting and enzyme-linked immunosorbent assay (ELISA) of recombinant protein isolated by gene cloning were performed on serum from healthy controls, patients with BD, and patients with other rheumatic diseases (rheumatoid arthritis, systemic lupus erythematosus, and Wegener's granulomatosis). RESULTS: Eighteen of 40 BD patients had serum IgM antibody to HDMEC antigen. The purified protein that reacted with AECA in BD patient sera was found to be alpha-enolase by 2-dimensional gel electrophoresis followed by immunoblotting and MALDI-TOF mass spectrometry. Recombinant alpha-enolase protein was isolated and refined by gene cloning. On Western blots, AECA-positive IgM from the sera of patients with active BD reacted strongly with recombinant human alpha-enolase. BD patient sera positive for anti-alpha-enolase did not react with human gamma-enolase. On dot-blotting, reactivity to human alpha-enolase was detected only in the IgM-positive group. Fifteen of the 18 AECA-positive sera that were positive for the HDMEC antigen showed reactivity to recombinant alpha-enolase IgM antibody by ELISA. CONCLUSION: The alpha-enolase protein is the target protein of serum AECA in BD patients. This is the first report of the presence of IgM antibodies to alpha-enolase in endothelial cells from the serum of BD patients. Although further studies relating this protein to the pathogenesis of BD will be necessary, alpha-enolase and its antibody may prove useful in the development of new diagnostic and treatment modalities in BD.