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Tyrosinase Depletion Prevents the Maturation of Melanosomes in the Mouse Hair Follicle.

Paterson EK, Fielder TJ, MacGregor GR, Ito S, Wakamatsu K, Gillen DL, Eby V, Boissy RE, Ganesan AK - PLoS ONE (2015)

Bottom Line: Moderate depletion of TYR was sufficient to alter the appearance of the mouse coat in black, agouti, and yellow coat color backgrounds, even though TYR depletion did not significantly inhibit accumulation of melanin within the mouse hair.Ultra-structural studies revealed that the reduction of Tyr inhibited the accumulation of terminal melanosomes, and inhibited the expression of genes that regulate melanogenesis.These results indicate that color in skin and hair is determined not only by the total amount of melanin within the hair, but also by the relative accumulation of mature melanosomes.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Chemistry, University of California Irvine, Irvine, CA, United States of America.

ABSTRACT
The mechanisms that lead to variation in human skin and hair color are not fully understood. To better understand the molecular control of skin and hair color variation, we modulated the expression of Tyrosinase (Tyr), which controls the rate-limiting step of melanogenesis, by expressing a single-copy, tetracycline-inducible shRNA against Tyr in mice. Moderate depletion of TYR was sufficient to alter the appearance of the mouse coat in black, agouti, and yellow coat color backgrounds, even though TYR depletion did not significantly inhibit accumulation of melanin within the mouse hair. Ultra-structural studies revealed that the reduction of Tyr inhibited the accumulation of terminal melanosomes, and inhibited the expression of genes that regulate melanogenesis. These results indicate that color in skin and hair is determined not only by the total amount of melanin within the hair, but also by the relative accumulation of mature melanosomes.

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The partial depletion of Tyr impacts key genes involved in melanogenesis.Four-mm skin punch biopsies were acquired from anesthetized (A) control rtTA3 mice and experimental Tyr-shRNA/rtTA3 mice, (B) control Luc-shRNA mice and experimental Luc-shRNA/rtTA2 mice, and (C) control C57BL/6J wild-type and Tyrc/Tyrc albino mice and immediately stabilized in RNAlater. Extracted RNA was subjected to Nanostring analysis on the genes listed below the bar graphs. Data shown for all three Nanostring bar graphs (A-C) is mean normalized counts of mRNA for each gene (n = 3 mice as indicated by the error bars). * = p ≤0.05 or ** = p ≤ 0.01. (D) Three littermates (genotypes from left to right as shown in graphs above each figure) on continuous DOX feed were photographed at P50 (left images). After P110, DOX was removed from the diet for 60 days and the mice were photographed once more (middle images). Finally, the mice were fed a DOX-free diet for an additional 30 days and a final set of photographs were taken (right images). Pigment accumulation was measured spectrophotometrically as described in the methods. The percentage value below each mouse corresponds to the absorbance at 492 nm for that particular mouse divided by the absorbance at 492 nm for its control littermate, which is set to 100%.
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pone.0143702.g005: The partial depletion of Tyr impacts key genes involved in melanogenesis.Four-mm skin punch biopsies were acquired from anesthetized (A) control rtTA3 mice and experimental Tyr-shRNA/rtTA3 mice, (B) control Luc-shRNA mice and experimental Luc-shRNA/rtTA2 mice, and (C) control C57BL/6J wild-type and Tyrc/Tyrc albino mice and immediately stabilized in RNAlater. Extracted RNA was subjected to Nanostring analysis on the genes listed below the bar graphs. Data shown for all three Nanostring bar graphs (A-C) is mean normalized counts of mRNA for each gene (n = 3 mice as indicated by the error bars). * = p ≤0.05 or ** = p ≤ 0.01. (D) Three littermates (genotypes from left to right as shown in graphs above each figure) on continuous DOX feed were photographed at P50 (left images). After P110, DOX was removed from the diet for 60 days and the mice were photographed once more (middle images). Finally, the mice were fed a DOX-free diet for an additional 30 days and a final set of photographs were taken (right images). Pigment accumulation was measured spectrophotometrically as described in the methods. The percentage value below each mouse corresponds to the absorbance at 492 nm for that particular mouse divided by the absorbance at 492 nm for its control littermate, which is set to 100%.

Mentions: To assess whether the partial depletion of Tyr affected the expression of genes that play key roles in melanogenesis, we harvested fresh whole mouse skin using a four-mm round punch biopsy and subjected the extracted RNA to Nanostring nCounter analysis. Interestingly, knockdown of Tyr resulted in a slight decrease in the expression levels of Dct, Pmel5, and Mart1 compared to control samples (Fig 5A). Tyr depletion lead to a significant decrease in the expression levels of Mitf-M, Sox10, Tyrp1 and, as expected, Tyr (Fig 5A). These results indicate that the mechanism by which Tyr depletion impacts the deposition of melanin within the melanosome is secondary to an effect of TYR levels on the expression of other melanogenesis genes. Finally, depletion of Luc produced no significant change in expression of the same genes analyzed in Tyr-KD mice, demonstrating that the changes in expression resultant upon Tyr knockdown are specific to Tyr depletion (Fig 5B).


Tyrosinase Depletion Prevents the Maturation of Melanosomes in the Mouse Hair Follicle.

Paterson EK, Fielder TJ, MacGregor GR, Ito S, Wakamatsu K, Gillen DL, Eby V, Boissy RE, Ganesan AK - PLoS ONE (2015)

The partial depletion of Tyr impacts key genes involved in melanogenesis.Four-mm skin punch biopsies were acquired from anesthetized (A) control rtTA3 mice and experimental Tyr-shRNA/rtTA3 mice, (B) control Luc-shRNA mice and experimental Luc-shRNA/rtTA2 mice, and (C) control C57BL/6J wild-type and Tyrc/Tyrc albino mice and immediately stabilized in RNAlater. Extracted RNA was subjected to Nanostring analysis on the genes listed below the bar graphs. Data shown for all three Nanostring bar graphs (A-C) is mean normalized counts of mRNA for each gene (n = 3 mice as indicated by the error bars). * = p ≤0.05 or ** = p ≤ 0.01. (D) Three littermates (genotypes from left to right as shown in graphs above each figure) on continuous DOX feed were photographed at P50 (left images). After P110, DOX was removed from the diet for 60 days and the mice were photographed once more (middle images). Finally, the mice were fed a DOX-free diet for an additional 30 days and a final set of photographs were taken (right images). Pigment accumulation was measured spectrophotometrically as described in the methods. The percentage value below each mouse corresponds to the absorbance at 492 nm for that particular mouse divided by the absorbance at 492 nm for its control littermate, which is set to 100%.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4664286&req=5

pone.0143702.g005: The partial depletion of Tyr impacts key genes involved in melanogenesis.Four-mm skin punch biopsies were acquired from anesthetized (A) control rtTA3 mice and experimental Tyr-shRNA/rtTA3 mice, (B) control Luc-shRNA mice and experimental Luc-shRNA/rtTA2 mice, and (C) control C57BL/6J wild-type and Tyrc/Tyrc albino mice and immediately stabilized in RNAlater. Extracted RNA was subjected to Nanostring analysis on the genes listed below the bar graphs. Data shown for all three Nanostring bar graphs (A-C) is mean normalized counts of mRNA for each gene (n = 3 mice as indicated by the error bars). * = p ≤0.05 or ** = p ≤ 0.01. (D) Three littermates (genotypes from left to right as shown in graphs above each figure) on continuous DOX feed were photographed at P50 (left images). After P110, DOX was removed from the diet for 60 days and the mice were photographed once more (middle images). Finally, the mice were fed a DOX-free diet for an additional 30 days and a final set of photographs were taken (right images). Pigment accumulation was measured spectrophotometrically as described in the methods. The percentage value below each mouse corresponds to the absorbance at 492 nm for that particular mouse divided by the absorbance at 492 nm for its control littermate, which is set to 100%.
Mentions: To assess whether the partial depletion of Tyr affected the expression of genes that play key roles in melanogenesis, we harvested fresh whole mouse skin using a four-mm round punch biopsy and subjected the extracted RNA to Nanostring nCounter analysis. Interestingly, knockdown of Tyr resulted in a slight decrease in the expression levels of Dct, Pmel5, and Mart1 compared to control samples (Fig 5A). Tyr depletion lead to a significant decrease in the expression levels of Mitf-M, Sox10, Tyrp1 and, as expected, Tyr (Fig 5A). These results indicate that the mechanism by which Tyr depletion impacts the deposition of melanin within the melanosome is secondary to an effect of TYR levels on the expression of other melanogenesis genes. Finally, depletion of Luc produced no significant change in expression of the same genes analyzed in Tyr-KD mice, demonstrating that the changes in expression resultant upon Tyr knockdown are specific to Tyr depletion (Fig 5B).

Bottom Line: Moderate depletion of TYR was sufficient to alter the appearance of the mouse coat in black, agouti, and yellow coat color backgrounds, even though TYR depletion did not significantly inhibit accumulation of melanin within the mouse hair.Ultra-structural studies revealed that the reduction of Tyr inhibited the accumulation of terminal melanosomes, and inhibited the expression of genes that regulate melanogenesis.These results indicate that color in skin and hair is determined not only by the total amount of melanin within the hair, but also by the relative accumulation of mature melanosomes.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Chemistry, University of California Irvine, Irvine, CA, United States of America.

ABSTRACT
The mechanisms that lead to variation in human skin and hair color are not fully understood. To better understand the molecular control of skin and hair color variation, we modulated the expression of Tyrosinase (Tyr), which controls the rate-limiting step of melanogenesis, by expressing a single-copy, tetracycline-inducible shRNA against Tyr in mice. Moderate depletion of TYR was sufficient to alter the appearance of the mouse coat in black, agouti, and yellow coat color backgrounds, even though TYR depletion did not significantly inhibit accumulation of melanin within the mouse hair. Ultra-structural studies revealed that the reduction of Tyr inhibited the accumulation of terminal melanosomes, and inhibited the expression of genes that regulate melanogenesis. These results indicate that color in skin and hair is determined not only by the total amount of melanin within the hair, but also by the relative accumulation of mature melanosomes.

Show MeSH
Related in: MedlinePlus