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UV-Induced Wnt7a in the Human Skin Microenvironment Specifies the Fate of Neural Crest-Like Cells via Suppression of Notch.

Fukunaga-Kalabis M, Hristova DM, Wang JX, Li L, Heppt MV, Wei Z, Gyurdieva A, Webster MR, Oka M, Weeraratna AT, Herlyn M - J. Invest. Dermatol. (2015)

Bottom Line: Inhibition of Notch signaling reduced the proliferation of NCSC-like cells, induced cell death, and downregulated noncanonical Wnt5a, suggesting that the Notch pathway contributes to the maintenance and motility of these stem cells.This differentiation was triggered by the endogenous Notch inhibitor Numb, which is upregulated in the stem cells by Wnt7a derived from UV-irradiated keratinocytes.Together, these data reveal a cross talk between the two conserved developmental pathways in postnatal human skin, and highlight the role of the skin microenvironment in specifying the fate of stem cells.

View Article: PubMed Central - PubMed

Affiliation: Molecular and Cellular Oncogenesis Program, The Wistar Institute, Philadelphia, Pennsylvania, USA.

ABSTRACT
Multipotent stem cells with neural crest-like properties have been identified in the dermis of human skin. These neural crest stem cell (NCSC)-like cells display self-renewal capacity and differentiate into neural crest derivatives, including epidermal pigment-producing melanocytes. NCSC-like cells share many properties with aggressive melanoma cells, such as high migratory capabilities and expression of the neural crest markers. However, little is known about which intrinsic or extrinsic signals determine the proliferation or differentiation of these neural crest-like stem cells. Here we show that, in NCSC-like cells, Notch signaling is highly activated, similar to melanoma cells. Inhibition of Notch signaling reduced the proliferation of NCSC-like cells, induced cell death, and downregulated noncanonical Wnt5a, suggesting that the Notch pathway contributes to the maintenance and motility of these stem cells. In three-dimensional skin reconstructs, canonical Wnt signaling promoted the differentiation of NCSC-like cells into melanocytes. This differentiation was triggered by the endogenous Notch inhibitor Numb, which is upregulated in the stem cells by Wnt7a derived from UV-irradiated keratinocytes. Together, these data reveal a cross talk between the two conserved developmental pathways in postnatal human skin, and highlight the role of the skin microenvironment in specifying the fate of stem cells.

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The Notch pathway is highly expressed in NCSC-like cells and is critical for their self-renewal(a) Immunoblot analysis with Notch1~4 antibodies showing that Notch1 is highly expressed in NCSC-like cells isolated from human dermis. Fom: Foreskin-derived melanocytes, NCSC-like: NCSC-like cells, WM35, WM88, WM852: human melanoma cell lines, WM35 sph: sphere-cultured WM35 melanoma cells. Blotting for β-actin serves as a loading control. (b) Activated Notch1 and the Notch target HES1 are detected in nuclear fractions of NCSC-like cells. Fom_GFP: control vector transduced melanocytes, Fom_Nic: active Notch1 transduced melanocytes (Pinnix et al., 2009). Blotting for β-actin, HSP90 and Lamin A/C serves as loading controls. (c) Immunofluorescence showing that Notch1 is localized mainly in nuclei of NCSC-like cells. Scale bar = 50 μm. (d) Immunoblot analysis showing that treatment with the γ-secretase inhibitor DAPT does not alter total expression of Notch1, but decreases total expression of Notch3 in NCSC-like cells and WM35 melanoma cells. Blotting for β-actin serves as loading controls. (e) Immunoblot analysis showing that 72 h treatment with the γ-secretase inhibitor DAPT decreases nuclear Notch1 expression in NCSC-like cells. Blotting for HSP90 and Lamin A/C serves as loading controls. (f) Left: Immunoblot analysis showing that 72 h treatment with the γ-secretase inhibitor DAPT decreases nuclear HES1 expression in NCSC-like cells. Immunofluorescence showing that the characteristic punctate staining pattern of HES1 disappears in NCSC-like cells treated with DAPT for 72 h. Scale bar = 50 μm. Right: Quantification of the nuclear HES1 positive cells. Data represent means ± SD, n = 5. *p ≤ 0.01. (g) Representative images of NCSC-like cells from control DMSO-treated and from DAPT (20 μM or 100 μM) treated cells. DAPT induces the adhesion of NCSC-like cells on the regular plastic surface and induces cell death, indicated by EthD-1 staining (red) on the low-attachment surface. Scale bar = 100 μm. (h) Left: Histogram depicting the relative growth of NCSC-like cells generated by each population. Data represent means ± SD, n = 5 for primary spheres, n=7 for secondary spheres. *p ≤ 0.01, **p ≤ 0.001, ***p ≤ 0.0001. Right: DAPT impairs the self-renewal ability of NCSC-like cells. Representative images were taken at day 13 after secondary sphere formation from single cell suspension of primary spheres. Scale bar = 100 μm.
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Figure 1: The Notch pathway is highly expressed in NCSC-like cells and is critical for their self-renewal(a) Immunoblot analysis with Notch1~4 antibodies showing that Notch1 is highly expressed in NCSC-like cells isolated from human dermis. Fom: Foreskin-derived melanocytes, NCSC-like: NCSC-like cells, WM35, WM88, WM852: human melanoma cell lines, WM35 sph: sphere-cultured WM35 melanoma cells. Blotting for β-actin serves as a loading control. (b) Activated Notch1 and the Notch target HES1 are detected in nuclear fractions of NCSC-like cells. Fom_GFP: control vector transduced melanocytes, Fom_Nic: active Notch1 transduced melanocytes (Pinnix et al., 2009). Blotting for β-actin, HSP90 and Lamin A/C serves as loading controls. (c) Immunofluorescence showing that Notch1 is localized mainly in nuclei of NCSC-like cells. Scale bar = 50 μm. (d) Immunoblot analysis showing that treatment with the γ-secretase inhibitor DAPT does not alter total expression of Notch1, but decreases total expression of Notch3 in NCSC-like cells and WM35 melanoma cells. Blotting for β-actin serves as loading controls. (e) Immunoblot analysis showing that 72 h treatment with the γ-secretase inhibitor DAPT decreases nuclear Notch1 expression in NCSC-like cells. Blotting for HSP90 and Lamin A/C serves as loading controls. (f) Left: Immunoblot analysis showing that 72 h treatment with the γ-secretase inhibitor DAPT decreases nuclear HES1 expression in NCSC-like cells. Immunofluorescence showing that the characteristic punctate staining pattern of HES1 disappears in NCSC-like cells treated with DAPT for 72 h. Scale bar = 50 μm. Right: Quantification of the nuclear HES1 positive cells. Data represent means ± SD, n = 5. *p ≤ 0.01. (g) Representative images of NCSC-like cells from control DMSO-treated and from DAPT (20 μM or 100 μM) treated cells. DAPT induces the adhesion of NCSC-like cells on the regular plastic surface and induces cell death, indicated by EthD-1 staining (red) on the low-attachment surface. Scale bar = 100 μm. (h) Left: Histogram depicting the relative growth of NCSC-like cells generated by each population. Data represent means ± SD, n = 5 for primary spheres, n=7 for secondary spheres. *p ≤ 0.01, **p ≤ 0.001, ***p ≤ 0.0001. Right: DAPT impairs the self-renewal ability of NCSC-like cells. Representative images were taken at day 13 after secondary sphere formation from single cell suspension of primary spheres. Scale bar = 100 μm.

Mentions: Our previous study showed that forced expression of activated Notch1 dedifferentiates melanocytes to multipotent NCSC-like cells (Zabierowski et al., 2011), suggesting a role for Notch signaling in the maintenance of a neural crest-like state. NCSC-like cells expressed high levels of Notch1 and Notch3 as truncated proteins, while all 3 melanoma cell lines expressed all 4 Notch receptors (Fig. 1a). Truncated Notch1 and Notch3 were also translocated to the nuclei of NCSC-like cells (Fig. 1b, c). Of Notch target genes, the hairy and enhancer of split (HES) family was highly expressed by NCSC-like cells (Supplementary Fig. 1). Hairy/enhancer-of-split related with YRPW motif (HEY) 1 (HEY1) and HEY2 were barely expressed in NCSC-like cells compared with the 3 melanoma cell lines (Supplementary Fig. 1), suggesting that the HEY family is specifically up-regulated in malignant cells. The Notch ligand delta-like 1 gene (DLL1) was predominantly expressed by NCSC-like cells, while another Notch ligand, Jagged 1 (JAG1), was expressed predominantly by melanocytes and melanomas (Supplementary Fig. 1), indicating that the Notch pathway is activated differently between NCSC-like cells and melanoma cells. We next sought to determine whether the Notch pathway contributes to the self-renewal ability of NCSC-like cells. DAPT, a γ-secretase inhibitor, didn’t alter the total Notch1 expression, however, it mildly decreased nuclear Notch1 in NCSC-like cells after 72 h (Fig. 1d, e, Supplementary Fig. 2a). Reduction of Notch1 nuclear translocation was also observed in the NCSC-like cells treated with RO4929097, another γ-secretase inhibitor (Supplementary Fig. 2b). Notch NCSC-like cells and DAPT decreased total Notch3 expression in both bands of differently sized Notch3 (Fig. 1d), suggesting that Notch3 is directly regulated by the Notch pathway as previously reported (Zuurbier et al., 2010). Treatment with 20 μM DAPT or 5 μM RO4929097 significantly decreased the expression of Notch target molecules HES1 and HEY1 mRNA (Fig. 1e, Supplementary Fig. 2c, d), and reduced the punctate nuclear HES1 protein expression (Fig. 1f), suggesting that 20~100 μM DAPT is effective at blocking the Notch pathway in NCSC-like cells. Staining with the cell viability indicator ethidium homodimer-1 (EthD-1) revealed that DAPT and RO4929097 induced robust cell death in NCSC-like cells (Fig. 1g, Supplementary Fig. 2e). The induction of cell death was more prominent when NCSC-like cells were cultured on a low-attachment surface (Fig. 1g). Notably, DAPT induced the adhesion of NCSC-like cells on conventional culture plates. The adherent cell population was still viable, suggesting that Notch inhibition particularly decreases survival of the sphere-forming stem cells. Next, we performed serial sphere forming assays to assess the impact of Notch inhibition in the self-renewal of NCSC-like cells. The primary sphere forming capacity was impaired by DAPT in a dose-dependent manner (Fig. 1h). Furthermore, the number of secondary spheres was significantly reduced by treatment with DAPT and RO4929097 (Fig. 1h, Supplementary Fig. 2f). Together, these results showed that the activation of Notch signaling contributes to the maintenance of NCSC-like cells in vitro. Our previous study showed that NCSC-like cells are highly migratory in a collagen matrix in a similar manner to melanoma cells (Li et al., 2010). The expression of a non-canonical Wnt ligand Wnt5a is up-regulated in metastatic melanoma and has been implicated in motility in melanoma cells (Weeraratna et al., 2002). Wnt5a has also been shown to be expressed in embryonic and cancer associated fibroblasts, in addition to being a target of the Notch pathway in dermal papilla cells and endothelial progenitor cells (Hu et al., 2010; Koyanagi et al., 2007; Pourreyron et al., 2012; Sato et al., 2010). Among those tested, Wnt5a was highly expressed in NCSC-like cells (Supplementary Fig. 2c). Inhibition of Notch signaling down-regulated WNT5A, suggesting that the Notch pathway also regulates the expression of Wnt5a in NCSC-like cells (Supplementary Fig. 2c, d).


UV-Induced Wnt7a in the Human Skin Microenvironment Specifies the Fate of Neural Crest-Like Cells via Suppression of Notch.

Fukunaga-Kalabis M, Hristova DM, Wang JX, Li L, Heppt MV, Wei Z, Gyurdieva A, Webster MR, Oka M, Weeraratna AT, Herlyn M - J. Invest. Dermatol. (2015)

The Notch pathway is highly expressed in NCSC-like cells and is critical for their self-renewal(a) Immunoblot analysis with Notch1~4 antibodies showing that Notch1 is highly expressed in NCSC-like cells isolated from human dermis. Fom: Foreskin-derived melanocytes, NCSC-like: NCSC-like cells, WM35, WM88, WM852: human melanoma cell lines, WM35 sph: sphere-cultured WM35 melanoma cells. Blotting for β-actin serves as a loading control. (b) Activated Notch1 and the Notch target HES1 are detected in nuclear fractions of NCSC-like cells. Fom_GFP: control vector transduced melanocytes, Fom_Nic: active Notch1 transduced melanocytes (Pinnix et al., 2009). Blotting for β-actin, HSP90 and Lamin A/C serves as loading controls. (c) Immunofluorescence showing that Notch1 is localized mainly in nuclei of NCSC-like cells. Scale bar = 50 μm. (d) Immunoblot analysis showing that treatment with the γ-secretase inhibitor DAPT does not alter total expression of Notch1, but decreases total expression of Notch3 in NCSC-like cells and WM35 melanoma cells. Blotting for β-actin serves as loading controls. (e) Immunoblot analysis showing that 72 h treatment with the γ-secretase inhibitor DAPT decreases nuclear Notch1 expression in NCSC-like cells. Blotting for HSP90 and Lamin A/C serves as loading controls. (f) Left: Immunoblot analysis showing that 72 h treatment with the γ-secretase inhibitor DAPT decreases nuclear HES1 expression in NCSC-like cells. Immunofluorescence showing that the characteristic punctate staining pattern of HES1 disappears in NCSC-like cells treated with DAPT for 72 h. Scale bar = 50 μm. Right: Quantification of the nuclear HES1 positive cells. Data represent means ± SD, n = 5. *p ≤ 0.01. (g) Representative images of NCSC-like cells from control DMSO-treated and from DAPT (20 μM or 100 μM) treated cells. DAPT induces the adhesion of NCSC-like cells on the regular plastic surface and induces cell death, indicated by EthD-1 staining (red) on the low-attachment surface. Scale bar = 100 μm. (h) Left: Histogram depicting the relative growth of NCSC-like cells generated by each population. Data represent means ± SD, n = 5 for primary spheres, n=7 for secondary spheres. *p ≤ 0.01, **p ≤ 0.001, ***p ≤ 0.0001. Right: DAPT impairs the self-renewal ability of NCSC-like cells. Representative images were taken at day 13 after secondary sphere formation from single cell suspension of primary spheres. Scale bar = 100 μm.
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Figure 1: The Notch pathway is highly expressed in NCSC-like cells and is critical for their self-renewal(a) Immunoblot analysis with Notch1~4 antibodies showing that Notch1 is highly expressed in NCSC-like cells isolated from human dermis. Fom: Foreskin-derived melanocytes, NCSC-like: NCSC-like cells, WM35, WM88, WM852: human melanoma cell lines, WM35 sph: sphere-cultured WM35 melanoma cells. Blotting for β-actin serves as a loading control. (b) Activated Notch1 and the Notch target HES1 are detected in nuclear fractions of NCSC-like cells. Fom_GFP: control vector transduced melanocytes, Fom_Nic: active Notch1 transduced melanocytes (Pinnix et al., 2009). Blotting for β-actin, HSP90 and Lamin A/C serves as loading controls. (c) Immunofluorescence showing that Notch1 is localized mainly in nuclei of NCSC-like cells. Scale bar = 50 μm. (d) Immunoblot analysis showing that treatment with the γ-secretase inhibitor DAPT does not alter total expression of Notch1, but decreases total expression of Notch3 in NCSC-like cells and WM35 melanoma cells. Blotting for β-actin serves as loading controls. (e) Immunoblot analysis showing that 72 h treatment with the γ-secretase inhibitor DAPT decreases nuclear Notch1 expression in NCSC-like cells. Blotting for HSP90 and Lamin A/C serves as loading controls. (f) Left: Immunoblot analysis showing that 72 h treatment with the γ-secretase inhibitor DAPT decreases nuclear HES1 expression in NCSC-like cells. Immunofluorescence showing that the characteristic punctate staining pattern of HES1 disappears in NCSC-like cells treated with DAPT for 72 h. Scale bar = 50 μm. Right: Quantification of the nuclear HES1 positive cells. Data represent means ± SD, n = 5. *p ≤ 0.01. (g) Representative images of NCSC-like cells from control DMSO-treated and from DAPT (20 μM or 100 μM) treated cells. DAPT induces the adhesion of NCSC-like cells on the regular plastic surface and induces cell death, indicated by EthD-1 staining (red) on the low-attachment surface. Scale bar = 100 μm. (h) Left: Histogram depicting the relative growth of NCSC-like cells generated by each population. Data represent means ± SD, n = 5 for primary spheres, n=7 for secondary spheres. *p ≤ 0.01, **p ≤ 0.001, ***p ≤ 0.0001. Right: DAPT impairs the self-renewal ability of NCSC-like cells. Representative images were taken at day 13 after secondary sphere formation from single cell suspension of primary spheres. Scale bar = 100 μm.
Mentions: Our previous study showed that forced expression of activated Notch1 dedifferentiates melanocytes to multipotent NCSC-like cells (Zabierowski et al., 2011), suggesting a role for Notch signaling in the maintenance of a neural crest-like state. NCSC-like cells expressed high levels of Notch1 and Notch3 as truncated proteins, while all 3 melanoma cell lines expressed all 4 Notch receptors (Fig. 1a). Truncated Notch1 and Notch3 were also translocated to the nuclei of NCSC-like cells (Fig. 1b, c). Of Notch target genes, the hairy and enhancer of split (HES) family was highly expressed by NCSC-like cells (Supplementary Fig. 1). Hairy/enhancer-of-split related with YRPW motif (HEY) 1 (HEY1) and HEY2 were barely expressed in NCSC-like cells compared with the 3 melanoma cell lines (Supplementary Fig. 1), suggesting that the HEY family is specifically up-regulated in malignant cells. The Notch ligand delta-like 1 gene (DLL1) was predominantly expressed by NCSC-like cells, while another Notch ligand, Jagged 1 (JAG1), was expressed predominantly by melanocytes and melanomas (Supplementary Fig. 1), indicating that the Notch pathway is activated differently between NCSC-like cells and melanoma cells. We next sought to determine whether the Notch pathway contributes to the self-renewal ability of NCSC-like cells. DAPT, a γ-secretase inhibitor, didn’t alter the total Notch1 expression, however, it mildly decreased nuclear Notch1 in NCSC-like cells after 72 h (Fig. 1d, e, Supplementary Fig. 2a). Reduction of Notch1 nuclear translocation was also observed in the NCSC-like cells treated with RO4929097, another γ-secretase inhibitor (Supplementary Fig. 2b). Notch NCSC-like cells and DAPT decreased total Notch3 expression in both bands of differently sized Notch3 (Fig. 1d), suggesting that Notch3 is directly regulated by the Notch pathway as previously reported (Zuurbier et al., 2010). Treatment with 20 μM DAPT or 5 μM RO4929097 significantly decreased the expression of Notch target molecules HES1 and HEY1 mRNA (Fig. 1e, Supplementary Fig. 2c, d), and reduced the punctate nuclear HES1 protein expression (Fig. 1f), suggesting that 20~100 μM DAPT is effective at blocking the Notch pathway in NCSC-like cells. Staining with the cell viability indicator ethidium homodimer-1 (EthD-1) revealed that DAPT and RO4929097 induced robust cell death in NCSC-like cells (Fig. 1g, Supplementary Fig. 2e). The induction of cell death was more prominent when NCSC-like cells were cultured on a low-attachment surface (Fig. 1g). Notably, DAPT induced the adhesion of NCSC-like cells on conventional culture plates. The adherent cell population was still viable, suggesting that Notch inhibition particularly decreases survival of the sphere-forming stem cells. Next, we performed serial sphere forming assays to assess the impact of Notch inhibition in the self-renewal of NCSC-like cells. The primary sphere forming capacity was impaired by DAPT in a dose-dependent manner (Fig. 1h). Furthermore, the number of secondary spheres was significantly reduced by treatment with DAPT and RO4929097 (Fig. 1h, Supplementary Fig. 2f). Together, these results showed that the activation of Notch signaling contributes to the maintenance of NCSC-like cells in vitro. Our previous study showed that NCSC-like cells are highly migratory in a collagen matrix in a similar manner to melanoma cells (Li et al., 2010). The expression of a non-canonical Wnt ligand Wnt5a is up-regulated in metastatic melanoma and has been implicated in motility in melanoma cells (Weeraratna et al., 2002). Wnt5a has also been shown to be expressed in embryonic and cancer associated fibroblasts, in addition to being a target of the Notch pathway in dermal papilla cells and endothelial progenitor cells (Hu et al., 2010; Koyanagi et al., 2007; Pourreyron et al., 2012; Sato et al., 2010). Among those tested, Wnt5a was highly expressed in NCSC-like cells (Supplementary Fig. 2c). Inhibition of Notch signaling down-regulated WNT5A, suggesting that the Notch pathway also regulates the expression of Wnt5a in NCSC-like cells (Supplementary Fig. 2c, d).

Bottom Line: Inhibition of Notch signaling reduced the proliferation of NCSC-like cells, induced cell death, and downregulated noncanonical Wnt5a, suggesting that the Notch pathway contributes to the maintenance and motility of these stem cells.This differentiation was triggered by the endogenous Notch inhibitor Numb, which is upregulated in the stem cells by Wnt7a derived from UV-irradiated keratinocytes.Together, these data reveal a cross talk between the two conserved developmental pathways in postnatal human skin, and highlight the role of the skin microenvironment in specifying the fate of stem cells.

View Article: PubMed Central - PubMed

Affiliation: Molecular and Cellular Oncogenesis Program, The Wistar Institute, Philadelphia, Pennsylvania, USA.

ABSTRACT
Multipotent stem cells with neural crest-like properties have been identified in the dermis of human skin. These neural crest stem cell (NCSC)-like cells display self-renewal capacity and differentiate into neural crest derivatives, including epidermal pigment-producing melanocytes. NCSC-like cells share many properties with aggressive melanoma cells, such as high migratory capabilities and expression of the neural crest markers. However, little is known about which intrinsic or extrinsic signals determine the proliferation or differentiation of these neural crest-like stem cells. Here we show that, in NCSC-like cells, Notch signaling is highly activated, similar to melanoma cells. Inhibition of Notch signaling reduced the proliferation of NCSC-like cells, induced cell death, and downregulated noncanonical Wnt5a, suggesting that the Notch pathway contributes to the maintenance and motility of these stem cells. In three-dimensional skin reconstructs, canonical Wnt signaling promoted the differentiation of NCSC-like cells into melanocytes. This differentiation was triggered by the endogenous Notch inhibitor Numb, which is upregulated in the stem cells by Wnt7a derived from UV-irradiated keratinocytes. Together, these data reveal a cross talk between the two conserved developmental pathways in postnatal human skin, and highlight the role of the skin microenvironment in specifying the fate of stem cells.

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Related in: MedlinePlus