The Graduate School of the University of Ulsan

The mechanisms that lead to variation in human skin and hair color are not fully understood. The skin is the body's largest organ [1], and acts as the most important barrier from Anatomically, the skin is divided into three main layers; epidermis, dermis (papillary . and reticularis) and hypodermis (subcutaneous fat layer) (Figure 1).

MITF regulation is the most crucial mode of melanocyte biology because MITF.

Materials and Methods

Animals
Cell Culture
Subcellular localization of CRTC3 and CRTC3 shRNA transfection
Antibodies
Western blot analysis
Melanin content and intracellular tyrosinase activity assay
Isolation of mouse primary melanocytes
Immunohistochemical analysis
Transmission electron microscopy (TEM)
Statistical analysis

Total protein was extracted from Mel-ab cells, washed once with cold PBS, and lysed. To separate the epidermis from the dermis, whole mouse skins were incubated in 5 mg/ml Dispase. First aid filter cDNA synthesis kit according to the manufacturer's instructions (Thermo . Scientific, Rockford, IL, USA).

Results

CRTC3 whole body K/O mice skin showed less melanin accumulation

Tyrp1 and DCT were significantly downregulated in the tail skin tissue of CRTC3 null mice (Fig. 2 E,F), indicating that CRTC3 ablation resulted in light coat color and downregulated. Data are expressed as mean ± SD of three independent experiments. K14KO-CRTC3KO CRTC3KO. RNA sequencing analysis of tail skin of WT vs. CRTC3 K/O mice. melanocyte differentiation and survival melanosomal transfer.

50 µm) The number of melanocytes in the epidermis of the tail tissue of CRTC3 WT and Null mice was counted. Electron microscopic (EM) images of epidermal cells and melanosomes in tail tissues of CRTC3 WT and Null mice. Bar = 5 µm) (D) Ratio of melanosomes in tail tissues of CRTC3 WT and Null mice based on EM images analyzed by ImageJ software.

To test whether this signal discrimination is associated with function of CRTC3, FSK. forskolin) (adenyly cyclase activator) or phorbol ester TPA (12-O-Tetradecanoylphorbol-13-. acetate) (PKC activator) were treated on mouse melanocytes Mel-Ab cells, a spontaneous. CREB to the same extent (Fig 4. C, F) but the CREB target genes only increased with FSK (Fig .. cAMP/PKA signals activate CREB target genes. Melanin content is presented as the percent change. relative to that in the vehicle -treated controls.

In B16F10 cells transfected with a plasmid containing CRTC3-EGFP, we treated with FSK and examined the TPA subcellular localization of CRTC3.

Figure 2. CRTC3 whole body K/O mice skin showed less melanin accumulation.

The level of melanogenic genes in CRTC3 O/E and K/D Mel-Ab cell

After selection of puromycin-resistant Mel-Ab cells, the efficiency of CRTC3 overexpression (O/E) (A) and knockdown (K/D) (B) was assessed by qRT-PCR. C, D) Cell viability effect in CRTC3 O/E and CRTC3 K/D Mel-Ab cells. FSK-induced melanin content and tyrosinase activity in CRTC3 O/E and K/D cells Microscopic images in Mel-Ab CRTC3 O/E (A) and K/D (D) cells after 24-, 48-, and 72- hourly treatment with FSK. Relative levels of MITF, tyrosinase, Tyrp1, and DCT mRNA in CRTC3 O/E Mel-Ab cells up to 72 h.

The protein levels of MITF, Tyrp1, DCT, tyrosinase and CRTC3 in CRTC3 O/E Mel-Ab cells were assessed by Western blotting. The protein levels of pCREB and CREB in CRTC3 K/D Mel-Ab cells for 72 hours effect of FSK treatment by Western blotting were not changed.

Figure 5. Effect on cell viability in CRTC3 K/D and O/E Mel-Ab cells.

Melanosome maturation markedly reduced in CRTC3 K/D Mel-Ab cells

Newborn mice skin tissue of CRTC3 null showed downregulation of epidermal

Instead, back skin section showed that melanosomes (assessed by . PMEL) of her unit were markedly reduced in CRTC3 Null mice but the number. Skin tissue from CRTC3 null newborn mice showed downregulation of epidermal melanogenesis-associated gene expression. Western blot (B) and qRT-PCR (n=4 per group) (C-D) analysis for melanocyte biology associated genes from CRTC3 WT and null newborn mice dorsal skin tissue.

Immunostaining was visualized with the Vectastain ABC kit, using Novared as a substrate (red color). E) Quantitative number of SOX10 in the epidermis of dorsal skin tissue of CRTC3 WT and P1 null mice based on counting multiple IHC images (Bar = 50 µm).

Figure 10. Newborn mice skin tissue of CRTC3 null showed downregulation of epidermal melanogenesis-associated gene expression

In coculture with human melanocyte and CRTC3 K/D HaCaT cells did not affect

POMC (H) in control and CRTC3 K/D HaCaT. I) POMC level of CRTC3 WT and Null mouse blood serum was assessed by ELISA.

Mouse primary melanocyte culture in WT, CRTC3 HT and K/O mice

Discussion

R Buscà, R Ballotti, Cyclic AMP a key messenger in the regulation of skin pigmentation, Pigment Cell Res. Targeted mutation of the CREB gene: compensation within the CREB/ATF family of transcription factors. Jillian C Vanover, Malinda L Spry, Laura Hamilton, Kazumasa Wakamatsu, Shosuke Ito, John A D'Orazio, Stem cell factor rescues tyrosinase expression and pigmentation at measured anatomical sites in albino mice, Pigment Cell Melanoma Res.

Brindle P, Nakajima T, Montminy M, Multiple protein kinase A-regulated events are required for transcriptional induction by cAMP, Proceedings of the National Academy of Sciences. Brenda Watt, Guillaume van Niel, Graça Raposo, Michael S Marks, PMEL: A Pigment Cell Specific Model for Functional Amyloid Formation, Pigment Cell Melanoma Res. Delineating the role of MITF isoforms in pigmentation and tissue homeostasis, Pigment Cell Melanoma Res.

마우스의 피부색 또는 털색을 결정하기 위해서는 다양한 신호 전달 경로 및 요인이 필요합니다. CREB는 멜라닌 합성 관련 유전자 전사 과정의 핵심 조절자에 해당합니다. 이러한 CREB 활성화 기능을 알기 위해서는 CREB의 co-activator인 CRTC3의 녹아웃 마우스에 대한 연구가 필요하여 CRTC3의 역할에 주목하였다.

CRTC3 KO 생쥐의 털 색깔은 야생형 생쥐에 비해 흐릿한 회색입니다. 첫째, CRTC3 KO 마우스의 꼬리 조직에서 Fontana Matheon 염색에 의해 KO 마우스에서 멜라닌 축적 정도가 감소함을 관찰하였다. 멜라닌 관련 유전자의 핵심 조절 인자인 MITF 유전자와 단백질 수치가 감소했고 대표적인 멜라닌 생성 관련 단백질인 티로시나제, Tyrp1, DCT도 감소했다.

In vitro에서 마우스 멜라노사이트 Mel-Ab 세포에서 CRTC3의 넉다운 또는 과발현을 유도하였을 때 멜라닌 관련 유전자 또는 단백질이 넉다운 시 감소하고 과발현 시 증가함을 확인하였다. 또한 마우스 멜라닌 세포의 1차 배양을 통해 CRTC3 KO 마우스 세포에서 멜라닌 생성과 관련된 유전자가 감소됨을 확인하였다. 멜라닌 세포 생존의 마커인 SOX10 단백질.