UV radiation induces various cellular responses by regulating the activity of many UV-responsive enzymes, includingMAPKs.The ␤␥ subunit of the heterotrimeric GTP-binding protein (G␤␥) was found to mediate UV-induced p38 activation via epidermal growth factor receptor (EGFR). However, it is not known how G␤␥ mediates the UVB-induced activation of EGFR, and thus we undertook this study to elucidate the mechanism. Treatment of HaCaT-immortalized human keratinocytes with conditioned medium obtained from UVB-irradiated cells induced the phosphorylations of EGFR, p38, and ERK but not that of JNK. Blockade of heparin-binding EGF-like growth factor (HB-EGF) by neutralizing antibody or CRM197 toxin inhibited the UVB-induced activations of EGFR, p38, and ERK in normal human epidermal keratinocytes and in HaCaT cells. Treatment with HB-EGF also activated EGFR, p38, and ERK. UVB radiation stimulated the processing of pro-HB-EGF and increased the secretion of soluble HB-EGF in medium, which was quantified by immunoblotting and protein staining. In addition, treatment with CRM179 toxin blocked UV-induced apoptosis, but HB-EGF augmented this apoptosis. Moreover, UVB-induced apoptosis was reduced by inhibiting EGFR or p38. The overexpression of G␤ 1 ␥ 2 increased EGFR-activating activity and soluble HB-EGF content in conditioned medium, but the sequestration of G␤␥ by the carboxyl terminus of G protein-coupled receptor kinase 2 (GRK2ct) produced the opposite effect. The activation of Src increased UVB-induced, G␤␥-mediated HB-EGF secretion, but the inhibition of Src blocked that. Overexpression of G␤␥ increased UVB-induced apoptosis, and the overexpression of GRK2ct decreased this apoptosis. We conclude that G␤␥ mediates UVB-induced human keratinocyte apoptosis by augmenting the ectodomain shedding of HB-EGF, which sequentially activates EGFR and p38.

Vitamin E inhibits tyrosinase activity and acts as a melanogenesis inhibitor in epidermal melanocytes in vitro. However, there is no direct evidence indicating that melanosomes are degraded in lysosomes in the presence of vitamin E. To determine whether vitamin E-induced melanosome disintegration is related to the expression of endosome docking/fusion proteins in B16F10 melanoma cells, electron microscopy, reverse transcription-polymerase chain reaction (RT-PCR), and real-time PCR were used to observe the effects of tocomin (α-tocopherols and α,γ,δ-tocotrienols in palm oil) on B16F10 melanoma cells. Melanosomal integrity was lost in lysosomes of B16F10 melanoma cells when treated with tocomin, indicating that tocomin caused the degradation of melanosomes in the lysosomal compartment. RT-PCR and real-time PCR analysis demonstrated mRNA expression of tyrosinase and the endosome docking/fusion proteins (syntaxin7, Rab7, Vps11, Vps16, Vps33, Vps39, and Vps41). Expression of syntaxin7, Vps16, Vps33, and Vps41 mRNA increased significantly in cells treated with tocomin compared with that in controls. These results indicate that the tocomin-induced degradation of melanosomes in the lysosomal compartment occurs with an increase in endosome docking/fusion proteins (syntaxin7, Vps16, Vps33, and Vps41) in cultured B16F10 melanoma cells.