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Updated: Oct 13, 2025
Direct Reprogramming of Mouse Fibroblasts into Melanocytes
Published on: August 27, 2021
The down-regulation of melanogenesis via MITF and FOXO1 signaling pathways in SIRT1 knockout cells using CRISPR/Cas9
1Department of Chemistry & Biology, Dong-Eui University, Busan 614-714, South Korea.
Abstract:
Hair graying is processed by the inactivation of tyrosinase caused by the accumulation of oxidative stress and a decrease in the number of melanocytes. Therefore, the purpose of this study was to investigate the effect of SIRT1 gene knockout using the CRISPR/Cas9 system on the protein and gene expressions related to melanogenesis. In this study, the mutation in the SIRT1 knockout(KO) gene was verified by T7EI assay and Sanger DNA sequencing. Furthermore, the expression levels of SIRT1 protein and gene in KO cells were remarkably decreased compared with normal cells. Therefore, the SIRT1 gene KO cell line was successfully established for further study. The KO cells also increased SA-β-galactosidase and decreased melanin production and the scavenging activity of hydrogen peroxide. In particular, the down-regulation of p38 and c-kit as well as the up-regulation of ERK resulted in the inactivation of MITF in the KO cells. Thus, KO cells reduced the expressions of Tyrosinase, Tyrosine hydroxylase, TRP-1 and TRP-2 through the negative modulation of MITF. Furthermore, SIRT1 gene KO cells negatively modulated antioxidant proteins such as Catalase, MnSOD, MsrA and MsrB3 through FOXO1 and Keap1. Therefore, it is suggested that SIRT1 could play a positive role in melanogenesis via MITF and FOXO1.
Insights
SIRT1 gene knockout significantly reduced melanin production and antioxidant activity, impacting hair graying. This study establishes a SIRT1 gene knockout cell line for further research into melanogenesis.
Area of Science:
- Cell Biology
- Genetics
- Dermatology
Background:
- Hair graying results from tyrosinase inactivation due to oxidative stress and reduced melanocytes.
- Understanding the regulation of melanogenesis is crucial for addressing hair graying.
Purpose of the Study:
- To investigate the impact of SIRT1 gene knockout on melanogenesis-related gene and protein expression.
- To establish a SIRT1 gene knockout (KO) cell line using CRISPR/Cas9 for further studies.
Main Methods:
- CRISPR/Cas9 system for SIRT1 gene knockout.
- T7EI assay and Sanger DNA sequencing for mutation verification.
- Analysis of SA-β-galactosidase activity, melanin production, and hydrogen peroxide scavenging activity.
Main Results:
- Successful establishment of a SIRT1 gene KO cell line with decreased SIRT1 expression.
- KO cells showed increased SA-β-galactosidase, reduced melanin production, and lower hydrogen peroxide scavenging.
- Down-regulation of p38, c-kit, and up-regulation of ERK led to MITF inactivation, reducing melanogenesis-related gene expression.
- SIRT1 KO negatively modulated antioxidant proteins via FOXO1 and Keap1 pathways.
Conclusions:
- SIRT1 plays a positive role in melanogenesis through MITF and FOXO1 pathways.
- SIRT1 gene knockout impacts key regulators of melanogenesis and antioxidant defense.
- The established SIRT1 KO cell line is a valuable tool for studying hair graying mechanisms.
