Mild mitochondrial uncoupling prevents premature senescence in human dermal fibroblasts.

TO THE EDITOR Mitochondrial uncoupling, an increased permeability of the inner membrane to protons not coupled to ATP synthesis, dissipates mitochondrial membrane potential. Mild mitochondrial uncoupling is believed to prolong lifespan of certain model organisms by reducing the production of reactive oxygen species (ROS) and preventing oxidant damage (Brand, 2000). Indeed, long-lived Caenorhabditis elegans strains have a lower membrane potential compared with wild-type strains (Lemire et al., 2009). Overexpression of the uncoupling protein UCP1 in murine skeletal muscle offers protection from age-related damage and disease (Gates et al., 2007). In addition, uncouplers extend the lifespan of yeast (Longo et al., 1999) and mice (Caldeira da Silva et al., 2008), suggesting that mild mitochondrial uncoupling might mitigate at least some deleterious aspects of aging. However, little is known about the effect of mitochondrial uncoupling on the aging of human skin. In this current study, we investigated the protective effect of mild mitochondrial uncoupling against oxidative stressinduced premature senescence in human dermal fibroblasts (HDFs). Oxidative stress is a pivotal mechanism leading to skin aging (Masaki, 2010). Accumulation of ROS elicits premature cellular senescence and deleterious alteration of collagen homeostasis, which can contribute to the development of characteristics of aged skin such as coarse, rough, and wrinkled appearance (Varani et al., 2006; Velarde et al., 2012). HDFs undergo cellular senescence by addition of 200mM H2O2 for 2 hours and prolonged subculture (Chen and Ames, 1994; Ido et al., 2012), which can be detected by changes in cellular morphology and senescence-associated b-galactosidase (SA-bgal) staining at 12 days after addition of H2O2 (Figure 1a). In order to determine whether mild mitochondrial uncoupling could inhibit oxidative stress–induced premature senescence, we pretreated HDFs with 60 nM carbonyl cyanide (P-trifluoromethoxy)-phenylhydrazone (FCCP) for 30 minutes before addition of H2O2. In a pilot study, the effective concentration of FCCP that would reduce the ROS production and mitochondrial membrane potential but have no discernible effect on cell viability was determined (Supplementary Figures S1 and S2 online). We found that cells pretreated with FCCP showed delayed senescence (Figure 1a), increased proliferative capacity (Figure 1b), reduced levels of oxidants (Figure 1c), and decreased expression levels of senescence-associated molecular markers, p21 and p16 (Figure 1d), compared with untreated senescent HDFs. Next, we investigated the uncoupling effect triggered by FCCP on collagen homeostasis in premature senescent HDFs. Aberrant collagen homeostasis, a prominent feature of aged human skin, Accepted article preview online 20 August 2013; published online 19 September 2013 Abbreviations: FCCP, carbonyl cyanide (p-trifluoromethoxy)-phenylhydrazone; HDF, human dermal fibroblasts; JNK, c-Jun N-terminal kinases; MMP-1, Matrix metalloproteinase-1; SA-bgal, senescenceassociated b-galactosidase SY Cho et al. Critical Role of Mitochondrial Uncoupling in Aging of Human Skin