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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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Wnt7a partially rescued the emergence of gp100 positive epidermal melanocytes(a) Wnt7a partially rescued the emergence of gp100 positive epidermal melanocytes and ectopic melanocytes in the dermis in the 3D skin reconstructs treated with a porcupine inhibitor IWP2 (arrowheads). Nuclei are counterstained with DAPI (blue). Scale bars = 200 μm. (b) Quantification of the number of gp100-positive melanocytes located at the basal layer of the epidermis. The Y-axis indicates the relative ratio of marker-positive cells. Data represent means ± SD, n=4. (c) TUNEL staining (green) showed that inhibition of Wnt signaling does not affect cell death of NCSC-like cells in the 3D skin reconstructs. Nuclei are counterstained with DAPI (blue). Scale bars = 200 μm.
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Figure 5: Wnt7a partially rescued the emergence of gp100 positive epidermal melanocytes(a) Wnt7a partially rescued the emergence of gp100 positive epidermal melanocytes and ectopic melanocytes in the dermis in the 3D skin reconstructs treated with a porcupine inhibitor IWP2 (arrowheads). Nuclei are counterstained with DAPI (blue). Scale bars = 200 μm. (b) Quantification of the number of gp100-positive melanocytes located at the basal layer of the epidermis. The Y-axis indicates the relative ratio of marker-positive cells. Data represent means ± SD, n=4. (c) TUNEL staining (green) showed that inhibition of Wnt signaling does not affect cell death of NCSC-like cells in the 3D skin reconstructs. Nuclei are counterstained with DAPI (blue). Scale bars = 200 μm.

Mentions: When NCSC-like cells are embedded into the skin reconstructs, they first interact with fibroblasts and are then attracted by keratinocytes, which promote their migration and differentiation; therefore, we postulated that Wnt ligands are expressed by human keratinocytes within the epidermal layer. Among those tested, we found that human keratinocytes express high levels of Wnt7a, which induces melanocyte differentiation in mouse hair follicles (Rabbani et al., 2011; Yamada et al., 2013), (Fig. 3a, Supplementary Fig. 3a). In contrast, other cell types, such as fibroblasts and NCSC-like cells, did not express Wnt7a. Secretion of Wnt7a from keratinocytes was blocked by IWP-2 (Fig. 3b). Yamada et al. reported in mouse skin that Wnt7a expression is induced on day 1 of ultraviolet B (UVB) irradiation, and returns to the basal level on day 3 (Yamada et al., 2013). Similarly in human skin, Wnt7a was rapidly up-regulated by UVB irradiation in keratinocytes and returned to basal levels after 3 days (Fig. 3c). Since the vast majority of solar UV radiation is UVA (Seite et al., 2010), we also tested the influence of UVA on Wnt7a production (Fig. 3c). Non-lethal doses of UVA irradiation were able to induce Wnt7a expression in human keratinocytes, and this induction was enhanced towards later time points, suggesting UVA induces a gradual, but more prolonged, up-regulation of Wnt signaling compared to UVB (Fig. 3c, d). Our previous study showed that NCSC-like cells differentiate into melanocytes in adherent cultures with melanocyte differentiation media (Mel1) containing recombinant Wnt3a (Li et al., 2010). When NCSC-like cells were treated with Mel1 media, in which Wnt3a was substituted with recombinant Wnt7a, the emergence of cells positive for melanocyte markers gp100 and tyrosinase-related protein 1 (TYRP1) increased, compared to control Wnt-free Mel1 media (Fig. 3e). Mel1 media supplemented with Wnt7a induced the expression of melanocyte-specific genes such as tyrosinase and MITF in a manner similar to Mel1 media supplemented with Wnt3a (Fig. 3f, Supplementary Fig. 3b). Constitutively activated Notch1 in NCSC-like cells inhibited induction of tyrosinase by Wnt7a, suggesting that the melanocyte differentiation by Wnt7a requires down-regulation of Notch signaling (Supplementary Fig. 3c). In Mel1 media, active β-catenin was increased in NCSC-like cells shortly after treatment with Wnt7a in a manner similar to treatment with Wnt3a (Fig. 4a), along with its translocation to the nucleus (Supplementary Fig. 3d). In basal media for stem cell cultures (DMEM/F12 with GlutaMAX™), Wnt7a up-regulated phosphorylation of the JNK p46 isoform as well as β-catenin, while phosphorylation of JNK p46 was not obvious in Mel1 media (Fig. 4a). This observation suggests that Wnt7a activates both the canonical and the non-canonical Wnt pathways in a context-dependent manner. We further determined whether Wnt7a could promote melanocyte differentiation in a canonical Wnt-dependent manner using two small molecule inhibitors, XAV-939 and NSC668036 (Shan et al., 2005). Both XAV-939 and NSC668036 significantly impaired the emergence of gp100/TYRP1-positive melanocytes, which were differentiated by Mel1 media supplemented with Wnt7a (Fig. 4b, c), suggesting that Wnt7a promotes the melanocyte differentiation of NCSC-like cells in a canonical Wnt pathway-dependent manner. Exogenous Wnt7a treatment partially rescued the emergence of epidermal melanocytes in the 3D skin reconstructs treated with IWP2, while also increasing ectopic gp100 positive melanocytes in the dermis (Fig. 5a, b). To determine whether Wnt7a also induces migration of NCSC-like cells, we performed Transwell® migration assays. Wnt7a treatment did not increase the migratory potential of NCSC-like cells, suggesting that a proper gradient of Wnt7a may be necessary for melanocyte differentiation in the epidermis, but not for the migration of NCSC-like cells (Supplementary Fig. 3e). Inhibition of Wnt signaling did not affect survival of NCSC-like cells in skin reconstructs (Fig. 5c), suggesting that the appearance of gp100-positive cells is not due to selection of a subpopulation in the starting cell population.


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)

Wnt7a partially rescued the emergence of gp100 positive epidermal melanocytes(a) Wnt7a partially rescued the emergence of gp100 positive epidermal melanocytes and ectopic melanocytes in the dermis in the 3D skin reconstructs treated with a porcupine inhibitor IWP2 (arrowheads). Nuclei are counterstained with DAPI (blue). Scale bars = 200 μm. (b) Quantification of the number of gp100-positive melanocytes located at the basal layer of the epidermis. The Y-axis indicates the relative ratio of marker-positive cells. Data represent means ± SD, n=4. (c) TUNEL staining (green) showed that inhibition of Wnt signaling does not affect cell death of NCSC-like cells in the 3D skin reconstructs. Nuclei are counterstained with DAPI (blue). Scale bars = 200 μm.
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Figure 5: Wnt7a partially rescued the emergence of gp100 positive epidermal melanocytes(a) Wnt7a partially rescued the emergence of gp100 positive epidermal melanocytes and ectopic melanocytes in the dermis in the 3D skin reconstructs treated with a porcupine inhibitor IWP2 (arrowheads). Nuclei are counterstained with DAPI (blue). Scale bars = 200 μm. (b) Quantification of the number of gp100-positive melanocytes located at the basal layer of the epidermis. The Y-axis indicates the relative ratio of marker-positive cells. Data represent means ± SD, n=4. (c) TUNEL staining (green) showed that inhibition of Wnt signaling does not affect cell death of NCSC-like cells in the 3D skin reconstructs. Nuclei are counterstained with DAPI (blue). Scale bars = 200 μm.
Mentions: When NCSC-like cells are embedded into the skin reconstructs, they first interact with fibroblasts and are then attracted by keratinocytes, which promote their migration and differentiation; therefore, we postulated that Wnt ligands are expressed by human keratinocytes within the epidermal layer. Among those tested, we found that human keratinocytes express high levels of Wnt7a, which induces melanocyte differentiation in mouse hair follicles (Rabbani et al., 2011; Yamada et al., 2013), (Fig. 3a, Supplementary Fig. 3a). In contrast, other cell types, such as fibroblasts and NCSC-like cells, did not express Wnt7a. Secretion of Wnt7a from keratinocytes was blocked by IWP-2 (Fig. 3b). Yamada et al. reported in mouse skin that Wnt7a expression is induced on day 1 of ultraviolet B (UVB) irradiation, and returns to the basal level on day 3 (Yamada et al., 2013). Similarly in human skin, Wnt7a was rapidly up-regulated by UVB irradiation in keratinocytes and returned to basal levels after 3 days (Fig. 3c). Since the vast majority of solar UV radiation is UVA (Seite et al., 2010), we also tested the influence of UVA on Wnt7a production (Fig. 3c). Non-lethal doses of UVA irradiation were able to induce Wnt7a expression in human keratinocytes, and this induction was enhanced towards later time points, suggesting UVA induces a gradual, but more prolonged, up-regulation of Wnt signaling compared to UVB (Fig. 3c, d). Our previous study showed that NCSC-like cells differentiate into melanocytes in adherent cultures with melanocyte differentiation media (Mel1) containing recombinant Wnt3a (Li et al., 2010). When NCSC-like cells were treated with Mel1 media, in which Wnt3a was substituted with recombinant Wnt7a, the emergence of cells positive for melanocyte markers gp100 and tyrosinase-related protein 1 (TYRP1) increased, compared to control Wnt-free Mel1 media (Fig. 3e). Mel1 media supplemented with Wnt7a induced the expression of melanocyte-specific genes such as tyrosinase and MITF in a manner similar to Mel1 media supplemented with Wnt3a (Fig. 3f, Supplementary Fig. 3b). Constitutively activated Notch1 in NCSC-like cells inhibited induction of tyrosinase by Wnt7a, suggesting that the melanocyte differentiation by Wnt7a requires down-regulation of Notch signaling (Supplementary Fig. 3c). In Mel1 media, active β-catenin was increased in NCSC-like cells shortly after treatment with Wnt7a in a manner similar to treatment with Wnt3a (Fig. 4a), along with its translocation to the nucleus (Supplementary Fig. 3d). In basal media for stem cell cultures (DMEM/F12 with GlutaMAX™), Wnt7a up-regulated phosphorylation of the JNK p46 isoform as well as β-catenin, while phosphorylation of JNK p46 was not obvious in Mel1 media (Fig. 4a). This observation suggests that Wnt7a activates both the canonical and the non-canonical Wnt pathways in a context-dependent manner. We further determined whether Wnt7a could promote melanocyte differentiation in a canonical Wnt-dependent manner using two small molecule inhibitors, XAV-939 and NSC668036 (Shan et al., 2005). Both XAV-939 and NSC668036 significantly impaired the emergence of gp100/TYRP1-positive melanocytes, which were differentiated by Mel1 media supplemented with Wnt7a (Fig. 4b, c), suggesting that Wnt7a promotes the melanocyte differentiation of NCSC-like cells in a canonical Wnt pathway-dependent manner. Exogenous Wnt7a treatment partially rescued the emergence of epidermal melanocytes in the 3D skin reconstructs treated with IWP2, while also increasing ectopic gp100 positive melanocytes in the dermis (Fig. 5a, b). To determine whether Wnt7a also induces migration of NCSC-like cells, we performed Transwell® migration assays. Wnt7a treatment did not increase the migratory potential of NCSC-like cells, suggesting that a proper gradient of Wnt7a may be necessary for melanocyte differentiation in the epidermis, but not for the migration of NCSC-like cells (Supplementary Fig. 3e). Inhibition of Wnt signaling did not affect survival of NCSC-like cells in skin reconstructs (Fig. 5c), suggesting that the appearance of gp100-positive cells is not due to selection of a subpopulation in the starting cell population.

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.

Show MeSH
Related in: MedlinePlus