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Abstract
Collagen is the major matrix protein in dermis and consists of proline and lysine, which are hydroxylated by prolyl hydroxylase (PH) and lysyl hydroxylase (LH) with cofactors such as vitamin C, oxygen, iron (Fe2+), ketoglutarate and silicon. The collagen degradation is regulated by matrix metalloproteinase-1 (MMP-1), of which is the major collagen-degrading proteinase whereas tissue inhibitors of metalloproteinase-1 (TIMP-1) bind to MMP-1 thereby inhibiting MMP-1 activity. In this study, we investigated the effects of vitamin C, silicon and iron on mRNA, protein expressions of PH, LH, MMP-1 and TIMP-1. The physiological concentrations of vitamin C (0-100 µM), silicon (0-50 µM) and iron (Fe2+:0-50 µM) were treated to human dermal fibroblast cells (HS27 cells) for 3 or 5days. The expression level of mRNA and protein was increased in not only PH but also LH when cells were incubated with vitamin C. A similar increase in LH mRNA or protein expression occurred when cells were incubated with silicon. Our results suggest that treatment of vitamin C and silicon increased mRNA and protein expression of PH and LH in human dermal fibroblast.
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Fig. 1
Effects of ascorbic acid, silicon and iron on prolyl hydroxylase (PH) mRNA expressions of cultured human dermal fibroblasts. Human dermal fibroblast cultures were treated with ascorbic acid (0, 5, 10, 50, 100 µM), silicon (0, 5, 10, 25, 50 µM) and Fe (0, 5, 10, 20, 30, 50, 100 µM) for 3 days (A) and 5 days (B). The expression levels of PH mRNA was analyzed by RT-PCR. The signal intensity were quantified the integrated area were percentized to the signal observed in control cells (% control). Results shown are means ± SEM (n = 3)(p < 0.05).
Fig. 2
Effects of ascorbic acid, silicon and iron on lysyl hydroxylase (LH) mRNA expressions of cultured human dermal fibroblasts. Human dermal fibroblast cultures were treated with ascorbic acid (0, 5, 10, 50, 100 µM), silicon (0, 5, 10, 25, 50 µM) and Fe (0, 5, 10, 20, 30, 50, 100 µM) for 3 days (A) and 5 days (B). The expression levels of LH mRNA was analyzed by RT-PCR. The signal intensity were quantified the integrated area were percentized to the signal observed in control cells (% control). Results shown are means ± SEM (n = 3)(p < 0.05).
Fig. 3
Effects of ascorbic acid, silicon and iron on prolyl hydroxylase (PH) protein expressions of cultured human dermal fibroblasts. Human dermal fibroblast cultures were treated with ascorbic acid (0, 5, 10, 50, 100 µM), silicon (0, 5, 10, 25, 50 µM) and Fe (0, 5, 10, 20, 30, 50, 100 µM) for 3 days (A) and 5 days (B). The expression levels of PH proteins was analyzed by western blotting. The signal intensity were quantified the integrated area were percentized to the signal observed in control cells (% control). Results shown are means ± SEM (n = 3)(p < 0.05).
Fig. 4
Effects of ascorbic acid, silicon and iron on Lysyl hydroxylase (LH) protein expressions of cultured human dermal fibroblasts. Human dermal fibroblast cultures were treated with ascorbic acid (0, 5, 10, 50, 100 µM), silicon (0, 5, 10, 25, 50 µM) and Fe (0, 5, 10, 20, 30, 50, 100 µM) for 3 days (A) and 5 days (B). The expression levels of LH proteins was analyzed by western blotting. The signal intensity were quantified the integrated area were percentized to the signal observed in control cells (% control). Results shown are means ± SEM (n = 3)(p < 0.05).
Fig. 5
Effects of ascorbic acid, silicon and iron on MMP-1 and TIMP-1 expressions of cultured human dermal fibroblasts. Human dermal fibroblast cultures were treated with ascorbic acid (0, 5, 10, 50, 100 µM), silicon (0, 5, 10, 25, 50 µM) and Fe (0, 5, 10, 20, 30, 50 µM) for 3 days. A: The expressions of MMP-1 protein was analysis by western blotting. B: The expressions of TIMP-1 was analyzed by western blotting. Results shown are means ± SEM.
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