Essential role of the molecular chaperone gp96 in regulating melanogenesis

Abstract

Through a process known as melanogenesis, melanocyte produces melanin in specialized organelles termed melanosomes, which regulates pigmentation of the skin, eyes, and hair. Gp96 is a constitutively expressed heat shock protein in the endoplasmic reticulum whose expression is further upregulated upon ultraviolet irradiation. However, the roles and mechanisms of this chaperone in pigmentation biology are unknown. In this study, we found that knockdown of gp96 by RNA interference significantly perturbed melanin synthesis and blocked late melanosome maturation. Gp96 knockdown did not impair the expression of tyrosinase, an essential enzyme in melanin synthesis, but compromised its catalytic activity and melanosome translocation. Further, mice with melanocyte-specific deletion of gp96 displayed decreased pigmentation. A mechanistic study revealed that the defect in melanogenesis can be rescued by activation of the canonical Wnt pathway, consistent with the critical roles of gp96 in chaperoning Wnt-coreceptor LRP6. Thus, this work uncovered the essential role of gp96 in regulating melanogenesis.

Keywords: Gp96; melanogenesis; melanosome; tyrosinase.

Fig. 1

Knockdown of gp96 in B16 cells inhibits melanin synthesis. (A) Knockdown of gp96 in B16 cells was achieved using a lentiviral shRNA system. gp96 silencing was confirmed by Western blot. (B) Gp96 knockdown B16 cell (B16-KD) pellets show decreased pigmentation compared with control vector transduced B16 cell pellets (B16-WT). (C) Quantification of melanin content in B16-KD and B16-WT cells (Mean ± SD). Shown is a representative data from three independent experiments. * P<0.05, ** P<0.01

Fig. 2

Deficiency of gp96 in melanocytes results in decreased hair pigmentation. (A) Hair on the backs of Tyr-cre/ERT2-Hsp90b1^flox/flox (Knockout, KO) and Tyr-cre-Hsp90b1^flox/wt (Wild type, WT) mice was shaved, followed by topical application of 4-OHT (10 mg/ml) or vehicle control (ethanol) on the right and left side, respectively, once a day for 4 weeks. The color of regrown hair is shown. Two independent experiments were performed with 4 mice for each experiment. (B) Epidermal melanocyte pigmentation with ethanol (left) and 4-OHT (right) treatment in KO (top) and WT (bottom) mice (400×, bars = 50 μm). (C) Lack of melanin in plucked hair shafts of KO mice compared to WT mice, 400×. Two experiments were done with similar findings.

Fig. 3

Disrupted melanosome development in B16-KD cells. Ultrastructure of B16-KD and B16-WT cells was analyzed by transmission electronic microscopy. Decreased pigmentation in melanosomes is observed in B16-KD cells. Magnification: Left, 10000×, bar=10 μm; Right, 300000×, bar=500 nm. Arrowheads indicate mature (stage IV) melanosomes. Two experiments were done with similar findings.

Fig. 4

Lack of gp96 does not interrupt expression or N-linked glycosylation of melanogenesis-associated enzymes. (A) Protein expression of Tyr, Tyrp1 and Dct in B16-WT and B16-KD cells by immunoblotting with an anti-tyrosinase monoclonal antibody (EP1577Y, EPITOMICS), an anti-Tyrp1 antibody (α-PEP1) and an anti-Dct antibody (α-PEP8’). (B,C,D) Western blotting of indicated proteins in non-digested cell lysates (-), or lysates treated with Endoglycosidase H (Endo H) or PNGase F, respectively. Data represent similar results from 2-3 experiments.

Fig. 5

Tyrosinase catalytic activity and its distribution in the late melanosome is altered in the absence of gp96. Tyrosinase activity in whole cell lysates from B16-WT and B16-KD cells was measured by spectrophotometric assay (Mean ± SD) (A) and by an in-gel colorimetric assay after electrophoretic separation (B). **P<0.01. Equal amounts of cell lysates were used, as indicated by the β-actin loading control. Three independent experiments were performed with similar findings. (C) A schematic diagram of melanocytic markers during melanosome maturation. (D) Confocal immunofluorescence analysis of subcellular localization of gp96, tyrosinase (α-PEP7) and HMB45 along with ER (Bip), Golgi (GM130) and lysosome (LAMP1/LAMP2) markers, as noted, in B16-WT and in B16-KD cells. Pictures shown are representative images from two independent experiments.

Fig. 6

Defect in melanogenesis in the absence of gp96 can be rescued by activating the Wnt/LRP6/β-catenin signaling pathway. (A) Pigmentation of cell pellets of B16-KD and B16-WT cells cultured without (UT) or with the GSK3β inhibitor TWS119 (1 μM) for 2 weeks. (B) Whole cell lysates from B16-WT and B16-KD cells treated with or without TWS119 (1 μM) were prepared, and Tyrosinase activity were measured by spectrophotometric assay (Mean ± SD). Results from three independent experiments were presented. **P<0.01. (C) In-gel colorimetric assay of tyrosinase after electrophoretic separation from the indicated cell lysates. (D) Tyrosinase expression level by immunoblot in B16-WT and B16-KD cells with or without TWS119 treatment. (E) Pigmentation of cell pellets of B16-KD and B16-WT cells cultured without (UT) or with the Wnt ligand Wnt 3a (200 ng/ml) for 2 weeks. Two experiments were done with similar findings.