Mahogunin Ring Finger 1 regulates pigmentation by controlling the pH of melanosomes in melanocytes and melanoma cells

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.

MGRN1 as a Phenotypic Determinant of Human Melanoma Cells and a Potential Biomarker

“…Mice homozygous for the md loss-of-function mutation of the Mgrn1 gene show a darker coat color similar to mice with gain-of-function mutations at the Extension locus coding Mc1r . Consistent with this finding, we showed previously that abrogation of MGRN1 expression in mouse melanocytes and B16 mouse melanoma cells by CRISPR/Cas9 led to a more dendritic and differentiated phenotype with higher melanin content [ 26 ]. Interestingly, these MGRN1-deficient cells also showed high genomic instability, increased adhesion to collagen, and decreased motility in both wound healing and invasion assays in vitro.…”

“…Since both the RHC mutations of MC1R and the MGRN1–MC1R interaction decrease MC1R-dependent activation of the cAMP pathway, we hypothesized that the level of expression and/or the activity of MGRN1 may still have poorly characterized effects on key properties of melanocytes and melanoma cells. In agreement with this hypothesis, we showed previously that MGRN1 is a key regulator of the phenotype of normal mouse melanocytes and melanoma cells, with marked effects on pigmentation, shape, and motility [ 26 , 27 ]. Moreover, the knockdown of MGRN1 augmented the burden of DNA strand breaks in mouse melanoma cells, thus indicating that loss of MGRN1 led to genomic instability [ 27 ].…”

“…In previous studies, we found that MGRN1-KO B16 mouse melanoma cells displayed a more differentiated phenotype with increased pigmentation, higher adhesion to collagen I, and low motility in 2D and 3D assays, compared to control cells. Moreover, when injected into the tail vein, MGRN1-KO B16 cells colonized the lungs of mice with much lower efficiency than control cells [ 26 , 27 ]. Therefore, it was of interest to analyze whether knockdown of MGRN1 in human melanoma cells caused a similar phenotypic switch.…”

Knockdowns of red Malphigian tubules reveal pigmentation roles in the milkweed bug

“…In mammals, several LysM‐domain genes interact with the vacuolar ATPase complex in mouse neurons and kidney cells, at least one of which is essential for lysosomal function (Castroflorio et al, 2021; Eaton et al, 2021). This precedent leads us to speculate that red and cho may indeed work in unison in the regulation of lysosome‐related organelles, and that their mutant phenotypes are explained by a failure of ommochromasomes and pterinosomes to properly acidify, similarly to the effect of pH misregulation in vertebrate melanosomes (Grant et al, 2016; Le et al, 2020; Sirés‐Campos et al, 2021). Further work will be needed to test this model in various insects.…”

The metabolism of melanin synthesis—From melanocytes to melanoma

“…Individuals with mutations in CLCN7 (aka CLC7) show hypopigmented skin and hair (Nicoli et al, 2019). The calcium channel MCOLN3 has been shown to neutralize the pH of melanosomes in MGRN1-depleted cells (Sirés-Campos et al, 2022). Mutations in the MCOLN3 gene is associated with the varitint-waddler phenotype characterized by pigmentation abnormalities (Di Palma et al, 2002).…”