Akt-dependent Pp2a activity is required for epidermal barrier formation during late embryonic development

Abstract

Acquisition of epidermal barrier function occurs late in mouse gestation. Several days before birth a wave of barrier acquisition sweeps across murine fetal skin, converging on dorsal and ventral midlines. We investigated the molecular pathways active during epidermal barrier formation. Akt signaling increased as the barrier wave crossed epidermis and Jun was transiently dephosphorylated. Inhibitor experiments on embryonic explants showed that the dephosphorylation of Jun was dependent on both Akt and protein phosphatase 2A (Pp2a). Inhibition of Pp2a and Akt signaling also caused defects in epidermal barrier formation. These data are compatible with a model for developmental barrier acquisition mediated by Pp2a regulation of Jun dephosphorylation, downstream of Akt signaling. Support for this model was provided by siRNA-mediated knockdown of Ppp2r2a (Pr55alpha or B55alpha), a regulatory subunit of Pp2a expressed in an Akt-dependent manner in epidermis during barrier formation. Ppp2r2a reduction caused significant increase in Jun phosphorylation and interfered with the acquisition of barrier function, with barrier acquisition being restored by inhibition of Jun phosphorylation. Our data provide strong evidence that Ppp2r2a is a regulatory subunit of Pp2a that targets this phosphatase to Jun, and that Pp2a action is necessary for barrier formation. We therefore describe a novel Akt-dependent Pp2a activity that acts at least partly through Jun to affect initial barrier formation during late embryonic epidermal development.

Fig. 1.

Akt signaling and Jun dephosphorylation at the barrier front. (A) Typical Toluidine Blue dye penetration (permeability) assay to assess barrier in an E16.5 mouse embryo. The barrier has progressed half way around the animal. The strip indicates the region assessed for protein expression. V, ventral; D, dorsal. (B) Assessment of Akt expression and activity (pAkt) by western blots of lysates from an epidermal strip comprising the barrier front (top panel). (C) Immunohistochemical detection of active Akt (pAkt), keratin 10, filaggrin, phosphorylated Jun (pJun), Jun and Klf4 in skin sections flanking the barrier front. Arrowheads point to expression of Jun and pJun in the upper granular layer. Dashed lines indicate the dermal-epidermal junction. Scale bar: 50 μm.

Fig. 2.

Reduced Jun phosphorylation correlates with suprabasal Akt activity during development. Immunohistochemical analysis of pAkt, pJun and Jun in E15.5-19.5 dorsal mouse skin. pAkt expression increases from E16.5 in the lower suprabasal epidermis but is lost by E18.5. Upper granular layer pAkt expression starts at E17.5 and persists. Jun expression is maintained throughout the developmental series; however, pJun expression is markedly reduced in E17.5 epidermis. Arrowheads indicate examples of nuclear expression of pJun and Jun in the granular layer. The dashed lines indicate the dermal-epidermal junction. Scale bar: 50 μm.

Fig. 3.

Co-imaging of the barrier front and protein expression shows that barrier acquisition occurs within the pulse of suprabasal Akt activation and just before the pulse of Jun dephosphorylation. (A) Serial sections comparing the barrier front (arrowhead) in a Hematoxylin-stained mouse fetus with expression of pJun. 1, 2 and 3 correspond to the respective inset figures. The pJun figure is a composite image, allowing for better imaging of pJun at the same scale. Small arrowheads in the insets indicate examples of pJun expression in the upper granular layer. (B) Comparison of pAkt expression with the barrier front (arrowhead) in epidermis from a Hematoxylin-stained mouse fetus, with positions 1, 2 and 3 shown as insets. The dashed lines in A and B indicate the dermal-epidermal boundary. Note that the barrier front is moving from right to left in this figure. Scale bars: 100 μm.

Fig. 4.

Dephosphorylation of Jun and epidermal barrier function are Akt dependent. (A) Immunohistochemical analysis of embryonic explants treated with 100 μM SH-5 or vehicle (DMSO). Inhibition of Akt by SH-5 was effective, reducing levels of pAkt and expression of the downstream Akt target pmTOR. Loricrin expression was maintained in both explants, showing that the overall differentiation program remained unchanged. However, inhibition of Akt by SH-5 resulted in maintenance of Jun phosphorylation; note that Jun phosphorylation is developmentally downregulated, as expected, in the DMSO-treated control. (B) Surface view of epidermal explants after Toluidine Blue dye penetration assay. The DMSO-treated control was resistant to dye penetration, indicating barrier acquisition, whereas explants treated with SH5 had impaired barrier function. Arrowheads indicate examples of nuclear expression of Jun and pJun in the granular layer. The dashed lines indicate the dermal-epidermal junction. Scale bar: 50 μm.

Fig. 5.

Dephosphorylation of Jun and epidermal barrier function are Pp2a dependent. (A) Immunohistochemical analysis of pJun and Jun in embryonic explants treated with the Pp2a inhibitors okadaic acid (10 nM), Fostriecin (20 μM) and fenvalerate (20 μM). Phosphorylation of Jun was maintained in explants in which Pp2a, but not Pp2b, was inhibited. Vertical arrowheads indicate nuclear Jun and pJun in the granular layer. (B) Immunofluorescence analysis of pAkt expression in treated explants. Expression was increased in explants treated with the Pp2a inhibitors. (C) Toluidine Blue dye penetration assay on explants treated with okadaic acid, Fostriecin or fenvalerate. Barrier function was impaired when Pp2a was inhibited, and to a lesser extent when Pp2b was inhibited. Dashed lines indicate the dermal-epidermal junction. Scale bars: 50 μm.

Fig. 6.

Ppp2r2a expression is Akt dependent and increases after barrier formation. Ppp2r2a co-localizes with, and can bind, Jun. (A) Immunofluorescence analysis for Ppp2r2a of frozen sections of E16.5 and E17.5 dorsal skin, corresponding to timepoints pre- and post-barrier acquisition. (B) Co-immunofluorescence analysis of Ppp2r2a and Jun in rat epidermal keratinocytes showing widespread co-localization. (C) Co-immunofluorescence analysis of frozen sections from drug- and vehicle-treated epidermal explants for the expression of Ppp2r2a and Pp2ac. Note that although the expression of Ppp2r2a decreased in the SH5-treated explants, the expression of Pp2ac was unchanged. Analysis of pAkt confirms that the SH-5 has inhibited Akt. (D) Immunoprecipitation (IP) western of rat keratinocyte lysate pulled down with an anti-Jun antibody or with an anti-Ppp2r2a antibody and with a respective control isotype (IgG) antibody. Scale bars: 50 μm in A,C; 10 μm in B.

Fig. 7.

siRNA knockdown of Ppp2r2a maintains Jun phosphorylation and causes defects in terminal differentiation. (A) Western blot analysis of two lines of rat epidermal keratinocytes (REKs) expressing different Ppp2r2a shRNA-containing plasmids and one expressing a scrambled control for Ppp2r2a, with actin as a loading control. siRNA2 caused substantial knockdown of Ppp2r2a. (B) Immunofluorescence analysis of Ppp2r2a in the scrambled line and siRNA2 line, confirming substantial knockdown. (C) Immunohistochemical analysis of keratin 10, involucrin and loricin in the same organotypic cultures. (D) Immunohistochemical analysis of pJun and Jun in the scrambled control and siRNA-expressing organotypic cultures. Scale bars: 50 μm.

Fig. 8.

siRNA knockdown of Ppp2r2a causes a transient barrier defect and hyperkeratosis in organotypic culture, which is restored by JNK inhibition. (A) Hematoxylin barrier assay of organotypic cultures expressing the Ppp2r2a siRNA and scrambled control, grown for 3, 5 and 10 days at the air-liquid interface. Blue nuclei indicate defective barrier function in the siRNA organotypic culture at 3 and 5 days. (B) Hematoxylin barrier assay of organotypic cultures expressing the Ppp2r2a siRNA and scrambled control and the Ppp2r2a siRNA-expressing culture grown in 50 nM SP600125 for 5 and 10 days at the air-liquid interface. (C) Hematoxylin and Eosin staining of the scrambled, siRNA-expressing and SP600125-treated siRNA-expressing cultures showing the hyperkeratotic response in the siRNA-expressing cultures, which is abrogated by addition of the JNK inhibitor SP600125. (D) pJun expression in each of the cultures, confirming reduction of Jun phosphorylation in siRNA-expressing cultures treated with SP600125. Dashed lines indicate the dermal-epidermal junction. Scale bars: 50 μm.

Fig. 9.

A model of the mechanism of Jun dephosphorylation by Akt-mediated Pp2a function. Akt activity causes upregulation of Ppp2r2a expression. Inhibition of Akt activity reduces Ppp2r2a expression and leads to barrier defects. Ppp2r2a then forms part of the Pp2a holoenzyme that specifically targets Jun for dephosphorylation, leading to barrier acquisition. Consistent with this, both siRNA knockdown of Ppp2r2a and inhibition of Pp2a activity lead to barrier defects.