Figure 8.
Suppression of cAMP or UVB-induced melanogenesis in human melanocytes and ex vivo human skin culture by altiratinib via CRTC3 phosphorylation. (A) Western blot analysis of CRTC3 and CREB and melanin content in normal human melanocytes (NHM) treated with FSK or TPA for 1 h. (B) mRNA level of melanogenesis-related genes in NHM within 12 h after FSK treatment. (C) Altiratinib (0.01-1 μM) dose-dependently suppressed FSK-stimulated transcriptional activity of CREB as measured by hEVX1 promoter activity. (D) The effect of 0.1 μM of altiratinib on FSK or CRTC3-stimulated transcriptional activity of CREB. (E) Expression levels and phosphorylation status of CRTC3 following 1 h of 0.01-1 μM altiratinib in Mel-Ab mouse melanocyte. (F) Expression levels and phosphorylation status of CRTC3 and CREB following 1 h of FSK treatment with or without 1 h of 0.1 μM altiratinib pretreatment. (G) Effects of 0.1 μM altiratinib, FSK, and both on the subcellular localization of CRTC3 in B16F10 cells transfected with CRTC3-EGFP (Bar = 100 μm). (H) The effect of 0.1 μM of altiratinib on FSK- or CRTC3-stimulated MITF-promoter activity. (I) mRNA level in Mel-Ab cells treated 2 h with vehicle (CTRL), 0.1 μM altiratinib, FSK or altiratinib plus FSK. (J) Melanin content of NHM treated with altiratinib (0.01-1 μM ) for 72 h. (K) The effect of altiratinib (0.01-1 μM) on cell viability of NHM by MTT assay. (L) Immunofluorescence staining using MLANA (red), CRTC3 (green) antibody with nuclear DAPI (blue) staining of human skin (Bar = 50 µm) with/without UVB treatment for 24 h. (M) Representative images of Fontana-Masson-stained paraffin-embedded sections and (N) melanin index, and (O) protein expression of MITF, Tyr, and DCT of ex vivo human skin exposed to UVB (75 mJ/ cm2) with/without 5 μM altiratinib for 96 h (Bar = 50 µm).