Myricetin suppresses UVB-induced wrinkle formation and MMP-9 expression by inhibiting Raf

Abstract

Chronic exposure to solar ultraviolet (UV) light causes skin photoaging. Many studies have shown that naturally occurring phytochemicals have anti-photoaging effects, but their direct target molecule(s) and mechanism(s) remain unclear. We found that myricetin, a major flavonoid in berries and red wine, inhibited wrinkle formation in mouse skin induced by chronic UVB irradiation (0.18J/cm(2), 3 days/week for 15 weeks). Myricetin treatment reduced UVB-induced epidermal thickening of mouse skin and also suppressed UVB-induced matrix metalloproteinase-9 (MMP-9) protein expression and enzyme activity. Myricetin appeared to exert its anti-aging effects by suppressing UVB-induced Raf kinase activity and subsequent attenuation of UVB-induced phosphorylation of MEK and ERK in mouse skin. In vitro and in vivo pull-down assays revealed that myricetin bound with Raf in an ATP-noncompetitive manner. Overall, these results indicate that myricetin exerts potent anti-photoaging activity by regulating MMP-9 expression through the suppression of Raf kinase activity.

Fig. 1. Effect of myricetin on UVB-induced wrinkle formation in SKH-1 hairless mice

(A) The chemical structure of myricetin (3,3′,4′,5,5′,7-hexahydroxyflavone). (B) Representative image showing the anti-photoaging effects of myricetin. Mice were topically treated with 200 μl of acetone containing 0, 1, or 5 nmol myricetin (as described in “Materials and methods”) and then irradiated with UVB light 3 times/wk for 15 wks. Images of the mouse backs were recorded using a digital camera (Samsung, Korea) before the mice were euthanized at the end of the experiment. Images shown are representative of those from 5 or 6 mice.

Fig. 2. Effect of myricetin on UVB irradiation-induced epidermal thickening in mouse skin

(A) Hematoxylin- and eosin-stained images of UVB-irradiated mouse skin. Images are representative of results from 5 tissue samples. (B) Myricetin prevents UVB induction of increased mouse epidermal thickness. After mice were treated as described for Figure 1B, the dorsal skin was excised, sectioned, mounted onto slides, and stained with hematoxylin and eosin for measurement of epidermal thickness. Bars represent the mean thickness (μm) of epidermis from 5 animals (40 measurements/section). Results are shown as means ± S.E. (n = 5). The symbol (##) indicates a significant difference (p < 0.01) between the control group and the UVB-irradiated group. Asterisks (** and ***) indicate significant differences of p < 0.01 or p < 0.001, respectively, between the myricetin-treated and non-treated groups of irradiated mice.

Fig. 3. Effect of myricetin on UVB irradiation-induced amplification of MMP-9 activity and expression

(A) Myricetin inhibits the increase in MMP-9 activity induced by UVB irradiation of mouse skin. Proteins were extracted from mouse skin samples as described in “Materials and methods”, and MMP-9 activity was determined using gelatin zymography and densitometry. Results are shown as means ± S.E. (n = 5). Symbols (# and ###) indicate a significant difference (p < 0.05 and p < 0.001, respectively) between the control group and the UVB-irradiated group. Asterisks (*, **, and ***) indicate a significant difference (p < 0.05, p < 0.01, and p < 0.001, respectively) between the myricetin-treated and non-treated groups of irradiated mice. (B) Confirmation of myricetin-mediated inhibition of UVB-induced expression of MMP-9 by Western blot analysis. Proteins were extracted from mouse skin as described in “Materials and methods”, and MMP-9 was analyzed using Western blotting and densitometry. Results are shown as means ± S.E. (n = 5). The symbol (#) indicates a significant difference (p < 0.05) between the control group and the UVB-irradiated group; the asterisk (*) indicates a significant difference (p < 0.05) between the myricetin-treated and non-treated groups of irradiated mice.

Fig. 4. Effect of myricetin on UVB-mediated signaling in mouse skin

(A) Myricetin inhibits phosphorylation of MEK, but not Raf. (B) Myricetin inhibits UVB-induced phosphorylation of ERK and p38. Mice were treated as described for Figure 1B. After euthanization, the dorsal skin tissue was removed and frozen for further study. Proteins were extracted from skin samples as described in “Materials and methods”, and phosphorylation of Raf, MEK, ERK, and p38 was determined by Western blotting. Data are representative of 3 independent experiments yielding similar results.

Fig. 5. Effect of myricetin on UVB-mediated Raf kinase activity and binding activity in mouse skin

(A) Myricetin inhibits UVB-induced Raf kinase activity. Mice were treated as described for Figure 1B. Raf kinase activity was determined using immunoprecipitation followed by a kinase assay as described in “Materials and methods”. Results are shown as means ± S.E. (n = 5). The symbol (#) indicates a significant difference (p < 0.05) between the control group and the UVB-irradiated group. Asterisks (*) indicate a significant difference (p < 0.05) between the myricetin-treated and non-treated groups of irradiated mice. (B) Myricetin directly binds Raf in mouse skin lysates. Mice were treated as described for Figure 1B. In vivo myricetin binding was confirmed by Western blot using an antibody against Raf: lane 1 (input control), whole lysate from mouse dorsal skin; lane 2 (control), mouse dorsal skin lysate precipitated with Sepharose 4B beads; and lane 3, whole-cell lysate from mouse dorsal skin precipitated by myricetin-Sepharose 4B affinity beads. (C) Myricetin binds Raf directly in an ATP-noncompetitive manner. Active Raf (2 g) was incubated with ATP at different concentrations (10 or 100 M) and 50 l of myricetin–Sepharose 4B or 50 l of Sepharose 4B (as a negative control) in reaction buffer at a final volume of 500 l. The mixtures were incubated at 4°C overnight with shaking. After washing, the pulled-down proteins were analyzed by Western blot: lane 2, negative control, Raf cannot bind with Sepharose 4B; lane 3, positive control, Raf binding with myricetin–Sepharose 4B; lanes 4 and 5, increasing amounts of ATP did not suppress myricetin binding with Raf. Each experiment was performed 3 times.

Fig. 6. Hypothetical model of the B-Raf/ATP/myricetin co-complex

Myricetin (carbon atoms shown in green) binds to the pocket adjacent to the ATP-binding pocket (carbon atoms shown in white). BAY43-9006 (yellow) is shown overlaid on the model structure, and the partially disordered activation loop is shown in white. Residues interacting with myricetin are indicated, with H-bonding depicted as a dashed line.