The molecular mechanisms of endogenous human skin aging

Abstract

To decipher the molecular mechanisms involved in endogenous skin aging human skin cells were maintained with GH, IGF-I, 17β-estradiol, progesterone, testosterone and DHEA at levels corresponding to average serum levels of males and females from 20 to 60 years of age. First, the expression of the corresponding receptors were identified by RT-PCR, Western blotting and immunocytochemistry. The biological activity of the skin cells under hormone treatment was evaluated by measuring the lipid synthesis, the cell proliferation and viability by means of nile red-microassay, MUH- and LDH-assay, respectively. SZ95 sebocytes incubated with the hormone mixture circulating in 60-y-old individuals showed significantly lower content of neutral lipids than cells treated with hormones of 20-y-old ones. On the other hand, proliferation of the fibroblasts was significantly affected by the hormone mixture, and fibroblasts incubated with hormones of 60-y-old individuals showed lower content of neutral and polar lipids than cells treated with hormones of 20-y-old ones. The mRNA and protein expression of two aging-associated genes- c-Myc and fibronectin- was measured via Northern and Western blotting, accordingly. Increased mRNA and protein levels of c-Myc and increased protein levels of fibronectin were detected in SZ95 sebocytes at 60-y-old hormone levels compared to those detected at 20-y-old hormone levels. Expression profiling employing a cDNA microarray composed of 15,529 cDNAs from known and novel genes identified age-related genes with altered expression levels at 20 and 60 years. The functional classification of these genes were related to several biological processes such as in mitochondrial function, oxidative damage and stress, ubiquitine-mediated proteolysis, cell cycle, immune responses, organization of the extracellular matrix, steroid biosynthesis and phospholipid degradation, which are all hallmarks of the aging process. Furthermore, the effects of the hormones were tested as single agents. In SZ95 sebocytes, while IGF-I and GH were shown to amplify neutral and polar lipid synthesis, 17β-estradiol and testosterone showed mostly an effect on polar lipid production. After treatment with progesterone and DHEA no effect was detected. Proliferation and cytotoxicity remained by all hormones tested unchanged. On the other hand, IGF-I and 17β-estradiol enhanced fibroblast proliferation and could also amplify lipid synthesis in a dose-dependent manner. In addition, in the experiments presented here, an interaction between IGF-I and estradiol signaling pathways was documented in SZ95 sebocytes and fibroblasts, which also gave evidence for a paracrine interaction between both cell types in vivo. IGF-I induced 17β-estradiol synthesis in SZ95 sebocytes and fibroblasts, while 17β-estradiol stimulated IGF-I synthesis only in fibroblasts. Moreover, IGF-IR and ERα expression in SZ95 sebocytes and IGF-IR expression in fibroblasts was shown to be influenced by 17β-estradiol and IGF-I in a time-dependent…