Fibroblast-Derived Clusterin Negatively Regulates Pigmentation Jiun Lee, Misun Kim, Tae Jun Park, Hee Young Kang  Journal of Investigative Dermatology  Volume 137, Issue 8, Pages 1812-1815 (August 2017) DOI: 10.1016/j.jid.2017.03.035 Copyright © 2017 The Authors Terms and Conditions

Figure 1 Clusterin (CLU) is expressed in the fibroblasts of the skin. (a) CLU expression was analyzed in cultured melanocytes (MC), keratinocytes (KC), and fibroblasts (FB) by real-time PCR (left panel), RT-PCR, and western blots (right panel). (b) 2 × 105 of fibroblasts were seeded and maintained for 24 or 48 hours. The media were harvested and the level of secreted CLU in a culture medium was measured by ELISA. (c) Immunohistochemical staining of CLU in normal human skin (left panel). MITF (green) and CLU (red) double-immunostained melanocytes (arrowheads) were analyzed in the basal layer of the epidermis (right panel). The inset shows a high-magnification field. (d) Receptors of CLU (LRP2, LRP8, VLDLR, and TGFβ receptors) were analyzed in cultured cells by RT-PCR. Thyrocytes (TH) and HeLa cells were used as positive controls for LRP2 and for LRP8 and VLDLR, respectively. The scale bar indicates 200 μm. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; LRP, lipoprotein receptor-related protein; MITF, microphthalmia-associated transcription factor; TGF-β, transforming growth factor-β; VLDLR, very-low-density-lipoprotein receptor. Journal of Investigative Dermatology 2017 137, 1812-1815DOI: (10.1016/j.jid.2017.03.035) Copyright © 2017 The Authors Terms and Conditions

Figure 2 Fibroblast-derived CLU negatively regulates melanogenesis. (a) Fibroblasts were infected with a control or CLU-lentivirus and normal human melanocytes were exposed to a fibroblast-derived concentrated conditioned medium (CCM). The melanin content and tyrosinase activity were then measured. (b) Expression levels of mRNA and protein of MITF and tyrosinase. (c, d) Fibroblasts were infected with a shControl (shCon) or shCLU-lentivirus (sh1, sh2) and normal human melanocytes were exposed to a fibroblast-derived CCM. Pigmentation was then analyzed by measuring the melanin content, tyrosinase activity, and expression levels of MITF and tyrosinase. (e) Ex vivo human skin was maintained in the presence of a CCM from fibroblasts infected with a CLU-lentivirus or rhCLU (10 ng/ml) for 3 days. The pigmentation was visualized by Fontana-Masson staining (left panel). The pigmented area/epidermal area (PA/EA) ratio was measured in an image analysis (right panel). The scale bar indicates 200 μm. (f) MelanoDerm was maintained with a CCM from fibroblasts infected with CLU-lentivirus or rhCLU (10 ng/ml) for 2 weeks. Macroscopic pigmentation was visualized and the total melanin content was measured. CLU, clusterin; MITF, microphthalmia-associated transcription factor; rhCLU, recombinant human CLU. Journal of Investigative Dermatology 2017 137, 1812-1815DOI: (10.1016/j.jid.2017.03.035) Copyright © 2017 The Authors Terms and Conditions