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14-3-3σ regulates keratinocyte proliferation and differentiation by modulating Yap1 cellular localization.

Sambandam SA, Kasetti RB, Xue L, Dean DC, Lu Q, Li Q - J. Invest. Dermatol. (2015)

Bottom Line: In this study, we found that Er/Er keratinocytes failed to sequester Yap1 in the cytoplasm, leading to its nuclear localization during epidermal development in vivo and under differentiation-inducing culture conditions in vitro.However, Er 14-3-3σ does not interact with Yap1, as demonstrated by coimmunoprecipitation.We conclude that Er 14-3-3σ disrupts the interaction between 14-3-3 and Yap1, and thus fails to block Yap1 nuclear transcriptional function, causing continued progenitor expansion and inhibition of differentiation in the Er/Er epidermis.

View Article: PubMed Central - PubMed

Affiliation: Departments of Ophthalmology and Visual Sciences, James Graham Brown Cancer Center, University of Louisville School of Medicine, Louisville, Kentucky, USA.

ABSTRACT
The homozygous repeated epilation (Er/Er) mouse mutant of the gene encoding 14-3-3σ displays an epidermal phenotype characterized by hyperproliferative keratinocytes and undifferentiated epidermis. Heterozygous Er/+ mice develop spontaneous skin tumors and are highly sensitive to tumor-promoting 7,12-dimethylbenzanthracene/12-O-tetradecanoyl-phorbol-13-acetate induction. The molecular mechanisms underlying 14-3-3σ regulation of epidermal proliferation, differentiation, and tumor formation have not been well elucidated. In this study, we found that Er/Er keratinocytes failed to sequester Yap1 in the cytoplasm, leading to its nuclear localization during epidermal development in vivo and under differentiation-inducing culture conditions in vitro. In addition, enhanced Yap1 nuclear localization was also evident in 7,12-dimethylbenzanthracene/12-O-tetradecanoyl-phorbol-13-acetate-induced tumors from Er/+ skin. Furthermore, short hairpin RNA (shRNA) knockdown of Yap1 expression in Er/Er keratinocytes inhibited their proliferation, suggesting that YAP1 functions as a downstream effector of 14-3-3σ controlling epidermal proliferation. We then demonstrated that keratinocytes express all seven 14-3-3 protein isoforms, some of which form heterodimers with 14-3-3σ, either full-length wild type (WT) or the mutant form found in Er/Er mice. However, Er 14-3-3σ does not interact with Yap1, as demonstrated by coimmunoprecipitation. We conclude that Er 14-3-3σ disrupts the interaction between 14-3-3 and Yap1, and thus fails to block Yap1 nuclear transcriptional function, causing continued progenitor expansion and inhibition of differentiation in the Er/Er epidermis.

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Enriched nuclear localization of Yap1 in Er/Er epidermis and induced skin tumors(a–c) Immunofluorescence staining for ΔNp63 (a), filaggrin (b) and BrdU (c) in the E18.5 Er/Er epidermis (right) compared with the WT control (left). (d) Quantification of BrdU+ cells. The number of BrdU+ cells per 500 μm of fixed length parallel to the epidermis surface was counted. Two-tail t-test: *p<0.05, N=5 embryos. n.s., not significant. (e) Immunohistochemical staining of Yap1. H&E staining (f) and Yap1 cellular localization (brown) detected by immunohistochemical staining (g) of DMBA/TPA-induced tumors derived from Er/+ mice. The scale bars represent 50 μm in (a–f) and 150 μm in (g). The arrows indicate Yap1-positive cells in (e) and (g). (h) Quantification of nuclear Yap1-positive cells. Two-tail t-test: **p<0.01, N=3 embryos.
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Figure 1: Enriched nuclear localization of Yap1 in Er/Er epidermis and induced skin tumors(a–c) Immunofluorescence staining for ΔNp63 (a), filaggrin (b) and BrdU (c) in the E18.5 Er/Er epidermis (right) compared with the WT control (left). (d) Quantification of BrdU+ cells. The number of BrdU+ cells per 500 μm of fixed length parallel to the epidermis surface was counted. Two-tail t-test: *p<0.05, N=5 embryos. n.s., not significant. (e) Immunohistochemical staining of Yap1. H&E staining (f) and Yap1 cellular localization (brown) detected by immunohistochemical staining (g) of DMBA/TPA-induced tumors derived from Er/+ mice. The scale bars represent 50 μm in (a–f) and 150 μm in (g). The arrows indicate Yap1-positive cells in (e) and (g). (h) Quantification of nuclear Yap1-positive cells. Two-tail t-test: **p<0.01, N=3 embryos.

Mentions: Given that 14-3-3 proteins are critical for excluding phosphorylated Yap1 from nuclei, we examined the cellular localization of Yap1 in Er/Er mouse skin to ask whether the Er 14-3-3σ mutation failed to retain Yap1 in the cytoplasm. E18.5 Er/Er embryos developed a multilayered, thicker epithelium lacking the cornified layer compared with well-differentiated WT skin (Li et al., 2005). Immunostaining with the antibody that specifically recognizes the C-terminus of 14-3-3σ, which was deleted in the Er mutant, showed that 14-3-3σ is mainly expressed in the differentiated suprabasal layers of the WT epidermis, but not in the Er/Er mutant epidermis (Li et al., 2005). The skin progenitor cells, as immunostained with ΔNp63α, were confined to the basal layer in the control epidermis but strongly expanded into the suprabasal layers in the mutant skin (Fig. 1a). A lack of the differentiation marker filaggrin was clearly evident in the mutant epidermis (Fig. 1b). The expansion of progenitor cells was associated with increased BrdU incorporation in the mutant suprabasal layers (Fig. 1c and 1d). Notably, Yap1 protein was clearly present in the nuclei of the mutant suprabasal layer, in contrast to the negative Yap1 nuclear staining in the WT suprabasel layer, although it was immunostained positively in the basal cell nuclei in both genotypes (Fig. 1e and 1h). Collectively, these results indicate that Yap1 is continuously localized in the nuclei from basal to suprabasal cells in the Er/Er epidermis, and this is correlated with an increase in the epidermal progenitor cell population and a lack of terminal differentiation.


14-3-3σ regulates keratinocyte proliferation and differentiation by modulating Yap1 cellular localization.

Sambandam SA, Kasetti RB, Xue L, Dean DC, Lu Q, Li Q - J. Invest. Dermatol. (2015)

Enriched nuclear localization of Yap1 in Er/Er epidermis and induced skin tumors(a–c) Immunofluorescence staining for ΔNp63 (a), filaggrin (b) and BrdU (c) in the E18.5 Er/Er epidermis (right) compared with the WT control (left). (d) Quantification of BrdU+ cells. The number of BrdU+ cells per 500 μm of fixed length parallel to the epidermis surface was counted. Two-tail t-test: *p<0.05, N=5 embryos. n.s., not significant. (e) Immunohistochemical staining of Yap1. H&E staining (f) and Yap1 cellular localization (brown) detected by immunohistochemical staining (g) of DMBA/TPA-induced tumors derived from Er/+ mice. The scale bars represent 50 μm in (a–f) and 150 μm in (g). The arrows indicate Yap1-positive cells in (e) and (g). (h) Quantification of nuclear Yap1-positive cells. Two-tail t-test: **p<0.01, N=3 embryos.
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Figure 1: Enriched nuclear localization of Yap1 in Er/Er epidermis and induced skin tumors(a–c) Immunofluorescence staining for ΔNp63 (a), filaggrin (b) and BrdU (c) in the E18.5 Er/Er epidermis (right) compared with the WT control (left). (d) Quantification of BrdU+ cells. The number of BrdU+ cells per 500 μm of fixed length parallel to the epidermis surface was counted. Two-tail t-test: *p<0.05, N=5 embryos. n.s., not significant. (e) Immunohistochemical staining of Yap1. H&E staining (f) and Yap1 cellular localization (brown) detected by immunohistochemical staining (g) of DMBA/TPA-induced tumors derived from Er/+ mice. The scale bars represent 50 μm in (a–f) and 150 μm in (g). The arrows indicate Yap1-positive cells in (e) and (g). (h) Quantification of nuclear Yap1-positive cells. Two-tail t-test: **p<0.01, N=3 embryos.
Mentions: Given that 14-3-3 proteins are critical for excluding phosphorylated Yap1 from nuclei, we examined the cellular localization of Yap1 in Er/Er mouse skin to ask whether the Er 14-3-3σ mutation failed to retain Yap1 in the cytoplasm. E18.5 Er/Er embryos developed a multilayered, thicker epithelium lacking the cornified layer compared with well-differentiated WT skin (Li et al., 2005). Immunostaining with the antibody that specifically recognizes the C-terminus of 14-3-3σ, which was deleted in the Er mutant, showed that 14-3-3σ is mainly expressed in the differentiated suprabasal layers of the WT epidermis, but not in the Er/Er mutant epidermis (Li et al., 2005). The skin progenitor cells, as immunostained with ΔNp63α, were confined to the basal layer in the control epidermis but strongly expanded into the suprabasal layers in the mutant skin (Fig. 1a). A lack of the differentiation marker filaggrin was clearly evident in the mutant epidermis (Fig. 1b). The expansion of progenitor cells was associated with increased BrdU incorporation in the mutant suprabasal layers (Fig. 1c and 1d). Notably, Yap1 protein was clearly present in the nuclei of the mutant suprabasal layer, in contrast to the negative Yap1 nuclear staining in the WT suprabasel layer, although it was immunostained positively in the basal cell nuclei in both genotypes (Fig. 1e and 1h). Collectively, these results indicate that Yap1 is continuously localized in the nuclei from basal to suprabasal cells in the Er/Er epidermis, and this is correlated with an increase in the epidermal progenitor cell population and a lack of terminal differentiation.

Bottom Line: In this study, we found that Er/Er keratinocytes failed to sequester Yap1 in the cytoplasm, leading to its nuclear localization during epidermal development in vivo and under differentiation-inducing culture conditions in vitro.However, Er 14-3-3σ does not interact with Yap1, as demonstrated by coimmunoprecipitation.We conclude that Er 14-3-3σ disrupts the interaction between 14-3-3 and Yap1, and thus fails to block Yap1 nuclear transcriptional function, causing continued progenitor expansion and inhibition of differentiation in the Er/Er epidermis.

View Article: PubMed Central - PubMed

Affiliation: Departments of Ophthalmology and Visual Sciences, James Graham Brown Cancer Center, University of Louisville School of Medicine, Louisville, Kentucky, USA.

ABSTRACT
The homozygous repeated epilation (Er/Er) mouse mutant of the gene encoding 14-3-3σ displays an epidermal phenotype characterized by hyperproliferative keratinocytes and undifferentiated epidermis. Heterozygous Er/+ mice develop spontaneous skin tumors and are highly sensitive to tumor-promoting 7,12-dimethylbenzanthracene/12-O-tetradecanoyl-phorbol-13-acetate induction. The molecular mechanisms underlying 14-3-3σ regulation of epidermal proliferation, differentiation, and tumor formation have not been well elucidated. In this study, we found that Er/Er keratinocytes failed to sequester Yap1 in the cytoplasm, leading to its nuclear localization during epidermal development in vivo and under differentiation-inducing culture conditions in vitro. In addition, enhanced Yap1 nuclear localization was also evident in 7,12-dimethylbenzanthracene/12-O-tetradecanoyl-phorbol-13-acetate-induced tumors from Er/+ skin. Furthermore, short hairpin RNA (shRNA) knockdown of Yap1 expression in Er/Er keratinocytes inhibited their proliferation, suggesting that YAP1 functions as a downstream effector of 14-3-3σ controlling epidermal proliferation. We then demonstrated that keratinocytes express all seven 14-3-3 protein isoforms, some of which form heterodimers with 14-3-3σ, either full-length wild type (WT) or the mutant form found in Er/Er mice. However, Er 14-3-3σ does not interact with Yap1, as demonstrated by coimmunoprecipitation. We conclude that Er 14-3-3σ disrupts the interaction between 14-3-3 and Yap1, and thus fails to block Yap1 nuclear transcriptional function, causing continued progenitor expansion and inhibition of differentiation in the Er/Er epidermis.

Show MeSH
Related in: MedlinePlus