Abstract
Mahogunin Ring Finger 1 (MGRN1) is an E3-ubiquitin ligase absent in dark-furred mahoganoid mice. We investigated the mechanisms of hyperpigmentation in Mgrn1-null melan-md1 melanocytes, Mgrn1-KO cells obtained by CRISPR-Cas9-mediated knockdown of Mgrn1 in melan-a6 melanocytes, and melan-a6 cells depleted of MGRN1 by siRNA treatment. Mgrn1-deficient melanocytes showed higher melanin content associated with increased melanosome abundance and higher fraction of melanosomes in highly melanized maturation stages III–IV. Expression, post-translational processing and enzymatic activity of the rate-limiting melanogenic enzyme tyrosinase measured in cell-free extracts were comparable in control and MGRN1-depleted cells. However, tyrosinase activity measured in situ in live cells and expression of genes associated with regulation of pH increased upon MGRN1 repression. Using pH-sensitive fluorescent probes, we found that downregulation of MGRN1 expression in melanocytes and melanoma cells increased the pH of acidic organelles, including melanosomes, strongly suggesting a previously unknown role of MGRN1 in the regulation of melanosomal pH. Among the pH regulatory genes upregulated by Mgrn1 knockdown, we identified those encoding several subunits of the vacuolar adenosine triphosphatase V-ATPase (mostly Atp6v0d2) and a calcium channel of the transient receptor potential channel family, Mucolipin 3 (Mcoln3). Manipulation of expression of the Mcoln3 gene showed that overexpression of Mcoln3 played a significant role in neutralization of the pH of acidic organelles and activation of tyrosinase in MGRN1-depleted cells. Therefore, lack of MGRN1 led to cell-autonomous stimulation of pigment production in melanocytes mostly by increasing tyrosinase specific activity through neutralization of the melanosomal pH in a MCOLN3-dependent manner.
Highlights
The skin and hair color of mammals is largely determined by the amount and relative proportions of two types of pigment derived from the amino acid l-tyrosine, eumelanins and pheomelanins [1]
We previously reported that Mgrn1-KO cells derived from either melan-a6 mouse melanocytes or B16 mouse melanoma cells are darker and show a higher melanin content than control, Mahogunin Ring Finger 1 (MGRN1)-expressing cells [33]
More than 50% of melanosomes in melan-a6 cells were stages I and II (Fig. 1a and c). These results must be interpreted with caution because of senescence-like features in the melan-md1 cell cultures used in this study and the less dramatic hyperpigmentation phenotype of Mgrn1-KO melan-a6 cells completely lacking MGRN1 [33]
Summary
The skin and hair color of mammals is largely determined by the amount and relative proportions of two types of pigment derived from the amino acid l-tyrosine, eumelanins and pheomelanins [1]. Active melanosomes are derived from early endosomes that receive various components required for their pigmentation, like a set of melanogenic enzymes transported to the melanosome precursors through the biosynthetic-secretory and endosomal pathways [2, 4]. These enzymes are the rate-limiting tyrosinase (TYR), a coppercontaining bifunctional enzyme catalyzing two consecutive reactions, hydroxylation of l-tyrosine to l-DOPA and. In highly pigmented stage IV melanosomes, the intraluminal structure is no longer visible due to the extensive accumulation of pigment and the melanogenic activity is reduced or absent [9]
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