AKT-dependent HspB1 (Hsp27) Activity in Epidermal Differentiation

Abstract

AKT activity has been reported in the epidermis associated with keratinocyte survival and differentiation. We show in developing skin that Akt activity associates first with post-proliferative, para-basal keratinocytes and later with terminally differentiated keratinocytes that are forming the fetal stratum corneum. In adult epidermis the dominant Akt activity is in these highly differentiated granular keratinocytes, involved in stratum corneum assembly. Stratum corneum is crucial for protective barrier activity, and its formation involves complex and poorly understood processes such as nuclear dissolution, keratin filament aggregation, and assembly of a multiprotein cell cornified envelope. A key protein in these processes is filaggrin. We show that one target of Akt in granular keratinocytes is HspB1 (heat shock protein 27). Loss of epidermal HspB1 caused hyperkeratinization and misprocessing of filaggrin. Akt-mediated HspB1 phosphorylation promotes a transient interaction with filaggrin and intracellular redistribution of HspB1. This is the first demonstration of a specific interaction between HspB1 and a stratum corneum protein and indicates that HspB1 has chaperone activity during stratum corneum formation. This work demonstrates a new role for Akt in epidermis.The epidermis is the primary environmental barrier, protecting from infection, allergens, and damage from UV radiation. The major constituent of this epidermal barrier is the terminally differentiated, anuclear keratinocyte. This structure is bounded by a cornified envelope, an elaborate, cross-linked protein structure covalently bound to hydrophobic lipid externally and aggregated keratin internally (1). Formation of this structure is poorly understood.The complexity of keratinocyte terminal differentiation and the importance of precisely timed and compartmentalized processing is illustrated by the maturation of the stratum corneum protein filaggrin. Filaggrin is synthesized as a high molecular mass precursor comprising multiple subunits that are sequentially processed and modified in temporally and spatially regulated steps by diverse proteases and enzymes to produce mature filaggrin subunits. Mature filaggrin is thought to be important in the aggregation and collapse of the keratin network leading to flattening of the keratinocyte and the destruction of the nucleus in granular layer (terminally differentiating) keratinocytes (2). Filaggrin is also incorporated into the cornified envelope (2). Premature or aberrant filaggrin processing can be catastrophic, leading to disruption of cornified envelope integrity and skin barrier function (3-5) and is far more damaging than reduced filaggrin levels that, in contrast, lead only to mild skin defects (6).Possible regulators of protein processing and trafficking during terminal differentiation are heat shock proteins (Hsps). 2Hsps have diverse roles as cellular chaperones, anti-apoptotic factors, stress-protective proteins, and cytoskeletal stabilizers (7,8). Human HspB1 (Hsp27) (9 -11) and the mouse HspB1...

AKT1-mediated Lamin A/C degradation is required for nuclear degradation and normal epidermal terminal differentiation

“…16,17 As we demonstrated previously, AKT1 is expressed during late terminal differentiation in the mouse, 14 and this correlates with increased expression of epidermal terminal differentiation markers between E15.5 and E18.5 of mouse embryonic development. 14 Therefore, we investigated whether nuclear size change and the phosphorylation of Lamin A/C also occurs during this time. We found that nuclear size in terminally differentiated (Loricrin positive) cells was significantly reduced in the epidermis of E18.5 mice compared with E15.5 mice (Figure 3a).…”

“…The organotypic cultures were made by growing rat epidermal keratinocytes on a de-epidermalised dermal scaffold for 10 days at the air-medium interface as previously described. 14,32 Organotypic cultures were paraffin embedded prior to sectioning. E15.5 and E18.5 CD1 mouse embryo tissue was obtained from timed matings, with the skin processed for paraffin embedding.…”

“…All lesional and non-lesional AD samples demonstrated a significant increase in parakeratosis (Figure 1b), and this correlated with a loss of upper granular layer AKT activity, specifically associated with AKT1 ( Figures 1a and c). 14,15 We successfully knocked down AKT1 expression by expressing two separate shRNA plasmids (A1 and A3) in rat epidermal keratinocytes. In this experiment, rat epidermal keratinocytes were used because they differentiate in confluent culture to a greater degree than either human or mouse keratinocytes, do not require calcium switch, and readily form organotypic cultures with a cornified layer highly reminiscent of human epidermis.…”

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

“…The serine/threonine kinase Akt changes expression and activity during fetal barrier acquisition (O'Shaughnessy et al, 2007b) (Fig. 1), and Akt activity is essential for barrier formation.…”

“…Mice lacking both Akt1 and Akt2 die postnatally, probably due to lack of epidermal barrier function (Peng et al, 2003). However, although Akt1 nulls have defective cornified envelopes (O'Shaughnessy et al, 2007b), both Akt1 and Akt2 knockouts are viable, suggesting a degree of redundancy between these Akt proteins. Phosphorylated active Akt is found in two regions of the epidermis -the lower suprabasal epidermis and the upper granular layer (Janes et al, 2004;O'Shaughnessy et al, 2007b).…”