Limits...
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.

Show MeSH

Related in: MedlinePlus

Increased nuclear localization of Yap1 in Er/Er keratinocytesYap1 cellular localization (red) was shown by immunostaining in primary cultured keratinocytes isolated from E18.5 WT (a) and Er/Er (b) skin. The keratinocytes were cultured for 48 h in SFM medium containing 0.02 mM Ca2+ (maintenance medium) or 0.5 mM Ca2+ (differentiation medium) before immunostaining. DAPI (blue) was used for nuclear counterstaining. The scale bars represent 50 μm. (c) Quantification of the number of Yap1-positive cells. The cells were immunostained with Yap1-specific antibody and calculated as percentage of total DAPI-labeled cells. The error bars represent the standard deviation from three independent experiments. The statistical significance was analyzed by two-tailed Student’s t-test. *p≤0.05 and ***p≤0.005, n=3.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4430425&req=5

Figure 2: Increased nuclear localization of Yap1 in Er/Er keratinocytesYap1 cellular localization (red) was shown by immunostaining in primary cultured keratinocytes isolated from E18.5 WT (a) and Er/Er (b) skin. The keratinocytes were cultured for 48 h in SFM medium containing 0.02 mM Ca2+ (maintenance medium) or 0.5 mM Ca2+ (differentiation medium) before immunostaining. DAPI (blue) was used for nuclear counterstaining. The scale bars represent 50 μm. (c) Quantification of the number of Yap1-positive cells. The cells were immunostained with Yap1-specific antibody and calculated as percentage of total DAPI-labeled cells. The error bars represent the standard deviation from three independent experiments. The statistical significance was analyzed by two-tailed Student’s t-test. *p≤0.05 and ***p≤0.005, n=3.

Mentions: The enhanced Yap1 nuclear localization in Er/Er epidermis led us to further analyze Yap1 expression in primary cultured keratinocytes isolated from E18.5 WT or Er/Er embryos. In primary WT control keratinocytes, Yap1 was distributed throughout both cytoplasm and nucleus but was preferentially concentrated in the nucleus in the undifferentiated cells under low-calcium culture conditions, whereas in the differentiated cells, upon induction with a high concentration of calcium, nuclear Yap1 staining signal was lost, which was accompanied by enhanced cytoplasmic staining (Fig. 2a and 2c). Compared with the Yap1 staining pattern in WT cells, Yap1 was mainly concentrated in the mutant nuclei, negligibly staining the cytoplasm, and such strong nuclear localization in the Er/Er keratinocytes remained predominant throughout the entire differentiation induction period in high-calcium culture (Fig. 2b and 2c). This data indicates that Yap1 cytoplasmic retention can’t be induced under high-calcium differentiation culture conditions in Er/Er keratinocytes that harbor the truncated 14-3-3σ mutation.


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)

Increased nuclear localization of Yap1 in Er/Er keratinocytesYap1 cellular localization (red) was shown by immunostaining in primary cultured keratinocytes isolated from E18.5 WT (a) and Er/Er (b) skin. The keratinocytes were cultured for 48 h in SFM medium containing 0.02 mM Ca2+ (maintenance medium) or 0.5 mM Ca2+ (differentiation medium) before immunostaining. DAPI (blue) was used for nuclear counterstaining. The scale bars represent 50 μm. (c) Quantification of the number of Yap1-positive cells. The cells were immunostained with Yap1-specific antibody and calculated as percentage of total DAPI-labeled cells. The error bars represent the standard deviation from three independent experiments. The statistical significance was analyzed by two-tailed Student’s t-test. *p≤0.05 and ***p≤0.005, n=3.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4430425&req=5

Figure 2: Increased nuclear localization of Yap1 in Er/Er keratinocytesYap1 cellular localization (red) was shown by immunostaining in primary cultured keratinocytes isolated from E18.5 WT (a) and Er/Er (b) skin. The keratinocytes were cultured for 48 h in SFM medium containing 0.02 mM Ca2+ (maintenance medium) or 0.5 mM Ca2+ (differentiation medium) before immunostaining. DAPI (blue) was used for nuclear counterstaining. The scale bars represent 50 μm. (c) Quantification of the number of Yap1-positive cells. The cells were immunostained with Yap1-specific antibody and calculated as percentage of total DAPI-labeled cells. The error bars represent the standard deviation from three independent experiments. The statistical significance was analyzed by two-tailed Student’s t-test. *p≤0.05 and ***p≤0.005, n=3.
Mentions: The enhanced Yap1 nuclear localization in Er/Er epidermis led us to further analyze Yap1 expression in primary cultured keratinocytes isolated from E18.5 WT or Er/Er embryos. In primary WT control keratinocytes, Yap1 was distributed throughout both cytoplasm and nucleus but was preferentially concentrated in the nucleus in the undifferentiated cells under low-calcium culture conditions, whereas in the differentiated cells, upon induction with a high concentration of calcium, nuclear Yap1 staining signal was lost, which was accompanied by enhanced cytoplasmic staining (Fig. 2a and 2c). Compared with the Yap1 staining pattern in WT cells, Yap1 was mainly concentrated in the mutant nuclei, negligibly staining the cytoplasm, and such strong nuclear localization in the Er/Er keratinocytes remained predominant throughout the entire differentiation induction period in high-calcium culture (Fig. 2b and 2c). This data indicates that Yap1 cytoplasmic retention can’t be induced under high-calcium differentiation culture conditions in Er/Er keratinocytes that harbor the truncated 14-3-3σ mutation.

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