Abstract
Mitochondrial dysfunction is linked to age-related senescence phenotypes. We report here the pathway increasing nucleoid remodeling and biogenesis in mitochondria during the senescence of foreskin human diploid fibroblasts (fs-HDF) and WI-38 cells. Replicative senescence in fs-HDF cells increased mitochondrial nucleoid remodeling as indicated by 5-bromo-2'-deoxyuridine (BrdU) incorporation and mitochondrial transcription factor A (TFAM) expression in enlarged and fused mitochondria. Mitochondrial nucleoid remodeling was accompanied by mitochondrial biogenesis in old cells, and the expression levels of OXPHOS complex-I, -IV and -V subunits, PGC-1α and NRF1 were greatly increased compared to young cells. Activated protein kinase C zeta (PKCζ) increased mitochondrial activity and expressed phenotypes of delayed senescence in fs-HDF cells, but not in WI-38 cells. The findings were reproduced in the doxorubicin-induced senescence of young fs-HDF and WI-38 cells via the PKCζ-LKB1-AMPK signaling pathway, which was regulated by the p53-p21WAF1 pathway when p16INK4a was silenced. The signaling enhanced PGC-1α-NRF1-TFAM axis in mitochondria, which was demonstrated by Ingenuity Pathway Analysis of young and old fs-HDF cells. Activation of the p53-p21WAF1 pathway and silencing of p16INK4a are responsible for mitochondrial reprogramming in senescent cells, which may be a compensatory mechanism to promote cell survival under senescence stress.
Highlights
Cellular senescence is involved in important biological processes, e.g., development, aging, and tumorigenesis, and is induced by activation of the p53-21WAF1 or p16INK4a-pRB axis [1]
As confirmation of mitochondrial activity in old cells, immunofluorescence microscopy showed that mitochondrial DNA (mtDNA), mtDNA polymerase γ (POL-γ), and Tom20 expression cooccurred with BrdU incorporation outside the nucleus (Supplementary Figure 1)
The amount and the activity of POL-γ did not significantly differ between the young and old cells (Supplementary Figure 2). These data are in accordance with previous reports that the activity of POL-γ is constant throughout the lifespan of cells [24], 3'-5' exonuclease activity is altered during cellular senescence [25]
Summary
Cellular senescence is involved in important biological processes, e.g., development, aging, and tumorigenesis, and is induced by activation of the p53-21WAF1 or p16INK4a-pRB axis [1]. Repression of p16INK4a expression delays senescence and regulates the replicative senescence phenotype [2]. Expression of p16INK4a is regulated by complex pathways involving lymphoid-specific helicase (Lsh), which binds to the p16INK4a promoter and creates a repressive chromatin structure by recruiting HDAC1 [3]. Mitochondrial oxidative metabolism is upregulated in senescent cells as a metabolic requirement [8, 9]. Partial uncoupling of oxidative phosphorylation in mitochondria has been reported in senescent fibroblasts [10], and BRAFV600E- and RASG12V-induced senescence upregulates the tricarboxylic acid (TCA) cycle and respiration by activating pyruvate dehydrogenase [9]. The mechanism underlying discrepant mitochondrial activity in senescent cells needs to be investigated
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