Identification of Hsc70 as target for AGE modification in senescent human fibroblasts

Unterluggauer, Hermann; Mičutková, Lucia; Lindner, Herbert; Sarg, Bettina; Hernebring, Malin; Nyström, Thomas; Jansen‐Dürr, Pidder

doi:10.1007/s10522-008-9193-z

Cited by 16 publications

(6 citation statements)

References 36 publications

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“…Gutsmann-Conrad et al have shown that the expression of HSP70 also decreased with senescence in IMR-90 lung fibroblasts during in vitro studies of cells from young or old subjects [56] and in senescent human fibroblasts [57]. In our analyses, we found a significantly reduced expression of HSP70 protein 6.…”

Section: Discussionsupporting

confidence: 61%

Senescent vs. non-senescent cells in the human annulus in vivo: Cell harvest with laser capture microdissection and gene expression studies with microarray analysis

et al. 2010

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BackgroundSenescent cells are well-recognized in the aging/degenerating human disc. Senescent cells are viable, cannot divide, remain metabolically active and accumulate within the disc over time. Molecular analysis of senescent cells in tissue offers a special challenge since there are no cell surface markers for senescence which would let one use fluorescence-activated cell sorting as a method for separating out senescent cells.MethodsWe employed a novel laser capture microdissection (LCM) design to selectively harvest senescent and non-senescent annulus cells in paraffin-embedded tissue, and compared their gene expression with microarray analysis. LCM was used to separately harvest senescent and non-senescent cells from 11 human annulus specimens.ResultsMicroarray analysis revealed significant differences in expression levels in senescent cells vs non-senescent cells: 292 genes were upregulated, and 321 downregulated. Genes with established relationships to senescence were found to be significantly upregulated in senescent cells vs. non-senescent cells: p38 (MPAK14), RB-Associated KRAB zinc finger, Discoidin, CUB and LCCL domain, growth arrest and DNA-damage inducible beta, p28ING5, sphingosine-1-phosphate receptor 2 and somatostatin receptor 3; cyclin-dependent kinase 8 showed significant downregulation in senescent cells. Nitric oxidase synthase 1, and heat shock 70 kDa protein 6, both of which were significantly down-regulated in senescent cells, also showed significant changes. Additional genes related to cytokines, cell proliferation, and other processes were also identified.ConclusionsOur LCM-microarray analyses identified a set of genes associated with senescence which were significantly upregulated in senescent vs non-senescent cells in the human annulus. These genes include p38 MAP kinase, discoidin, inhibitor of growth family member 5, and growth arrest and DNA-damage-inducible beta. Other genes, including genes associated with cell proliferation, extracellular matrix formation, cell signaling and other cell functions also showed significant modulation in senescent vs non-senescent cells. The aging/degenerating disc undergoes a well-recognized loss of cells; understanding senescent cells is important since their presence further reduces the disc's ability to generate new cells to replace those lost to necrosis or apoptosis.

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Section: Discussionsupporting

confidence: 61%

Senescent vs. non-senescent cells in the human annulus in vivo: Cell harvest with laser capture microdissection and gene expression studies with microarray analysis

et al. 2010

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“…, 2006). In addition, it was identified as target for AGE modification in human senescent dermal fibroblasts (Unterluggauer et al. , 2009).…”

Section: Discussionmentioning

confidence: 99%

“…Formation of AGE on proteins is found in many tissues and is thought to contribute to a variety of age‐associated diseases (Reddy & Beyaz, 2006). Interestingly, Hsc 70 has been recently shown to be AGE‐modified in senescent human dermal fibroblasts (Unterluggauer et al. , 2009).…”

Section: Introductionmentioning

confidence: 99%

Protein modification and replicative senescence of WI‐38 human embryonic fibroblasts

et al. 2010

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SummaryOxidized proteins as well as proteins modified by the lipid peroxidation product 4-hydroxy-2-nonenal (HNE) and by glycation (AGE) have been shown to accumulate with aging in vivo and during replicative senescence in vitro.To better understand the mechanisms by which these damaged proteins build up and potentially affect cellular function during replicative senescence of WI-38 fibroblasts, proteins targeted by these modifications have been identified using a bidimensional gel electrophoresisbased proteomic approach coupled with immunodetection of HNE-, AGE-modified and carbonylated proteins. Thirty-seven proteins targeted for either one of these modifications were identified by mass spectrometry and are involved in different cellular functions such as protein quality control, energy metabolism and cytoskeleton. Almost half of the identified proteins were found to be mitochondrial, which reflects a preferential accumulation of damaged proteins within the mitochondria during cellular senescence. Accumulation of AGE-modified proteins could be explained by the senescence-associated decreased activity of glyoxalase-I, the major enzyme involved in the detoxification of the glycating agents methylglyoxal and glyoxal, in both cytosol and mitochondria. This finding suggests a role of detoxification systems in the age-related build-up of damaged proteins. Moreover, the oxidized protein repair system methionine sulfoxide reductase was more affected in the mitochondria than in the cytosol during cellular senescence. Finally, in contrast to the proteasome, the activity of which is decreased in senescent fibroblasts, the mitochondrial matrix ATP-stimulated Lon-like proteolytic activity is increased in senescent cells but does not seem to be sufficient to cope with the increased load of modified mitochondrial proteins.

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“…Detection of protein carbonylation was performed as described elsewhere (59). For analysis of PTEN oxidation, lysates were prepared in the presence of 10 mM N-ethylmaleimide to prevent cysteine oxidation during lysis.…”

Section: Western Blotting and Immunohistochemistrymentioning

confidence: 99%

ROS Signaling by NOX4 Drives Fibroblast-to-Myofibroblast Differentiation in the Diseased Prostatic Stroma

Sampson

Kozieł

Zenzmaier

et al. 2011

Molecular Endocrinology

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145

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Stromal remodeling, in particular fibroblast-to-myofibroblast differentiation, is a hallmark of benign prostatic hyperplasia (BPH) and solid tumors, including prostate cancer (PCa). Increased local production of TGFβ1 is considered the inducing stimulus. Given that stromal remodeling actively promotes BPH/PCa development, there is considerable interest in developing stromal-targeted therapies. Microarray and quantitative PCR analysis of primary human prostatic stromal cells induced to undergo fibroblast-to-myofibroblast differentiation with TGFβ1 revealed up-regulation of the reactive oxygen species (ROS) producer reduced nicotinamide adenine dinucleotide phosphate oxidase 4 (NOX4) and down-regulation of the selenium-containing ROS-scavenging enzymes glutathione peroxidase 3, thioredoxin reductase 1 (TXNRD1), and the selenium transporter selenoprotein P plasma 1. Consistently, NOX4 expression correlated specifically with the myofibroblast phenotype in vivo, and loss of selenoprotein P plasma 1 was observed in tumor-associated stroma of human PCa biopsies. Using lentiviral NOX4 short hairpin RNA-mediated knockdown, pharmacological inhibitors, antioxidants, and selenium, we demonstrate that TGFβ1 induction of NOX4-derived ROS is required for TGFβ1-mediated phosphorylation of c-jun N-terminal kinase, which in turn is essential for subsequent downstream cytoskeletal remodeling. Significantly, selenium supplementation inhibited differentiation by increasing ROS-scavenging selenoenzyme biosynthesis because glutathione peroxidase 3 and TXNRD1 expression and TXNRD1 enzyme activity were restored. Consistently, selenium depleted ROS levels downstream of NOX4 induction. Collectively, this work demonstrates that dysregulated redox homeostasis driven by elevated NOX4-derived ROS signaling underlies fibroblast-to-myofibroblast differentiation in the diseased prostatic stroma. Further, these data indicate the potential clinical value of selenium and/or NOX4 inhibitors in preventing the functional pathogenic changes of stromal cells in BPH and PCa.

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Identification of Hsc70 as target for AGE modification in senescent human fibroblasts

Cited by 16 publications

References 36 publications

Senescent vs. non-senescent cells in the human annulus in vivo: Cell harvest with laser capture microdissection and gene expression studies with microarray analysis

Senescent vs. non-senescent cells in the human annulus in vivo: Cell harvest with laser capture microdissection and gene expression studies with microarray analysis

Protein modification and replicative senescence of WI‐38 human embryonic fibroblasts

ROS Signaling by NOX4 Drives Fibroblast-to-Myofibroblast Differentiation in the Diseased Prostatic Stroma

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