Epiplakin Is Dispensable for Skin Barrier Function and for Integrity of Keratin Network Cytoarchitecture in Simple and Stratified Epithelia

Abstract

Epiplakin, a giant epithelial protein of >700 kDa, belongs to the plakin family of cytolinker proteins. It represents an atypical family member, however, as it consists entirely of plakin repeat domains but lacks any of the other domains commonly shared by plakins. Hence, its putative function as a cytolinker protein remains to be shown. To investigate epiplakin's biological role, we generated epiplakin-deficient mice by gene targeting in embryonic stem cells. Epiplakin-deficient mice were viable and fertile, without developing any discernible phenotype. Ultrastructurally, their epidermis revealed no differences compared to wild-type littermates, and cornified envelopes isolated from skin showed no alterations in shape or stability. Furthermore, neither embryonal formation nor later function of the epithelial barrier was affected. In primary cultures of epiplakindeficient keratinocytes, the organization of actin filaments, microtubules, and keratin networks was found to be normal. Similarly, no alterations in keratin network organization were observed in simple epithelia of small intestine and liver or in primary hepatocytes. We conclude that, despite epiplakin's abundant and highly specific expression in stratified and simple epithelia, its absence in mice does not lead to severe skin dysfunctions, nor has it detectable consequences for keratin filament organization and cytoarchitecture of cells.

Stress-induced recruitment of epiplakin to keratin networks increases their resistance to hyperphosphorylation-induced disruption

“…Epiplakin was also found to colocalize with K8/18 in the human colon carcinoma cell line HT29 (our unpublished results) and in HeLa cells (Jang et al, 2005). Furthermore, the distribution of epiplakin in liver matches that of keratins Spazierer et al, 2006). Therefore, the question arises, whether the subcellular localization of epiplakin is dependent on the type of cell or on the type of keratin involved.…”

“…Nevertheless we observed no differences in keratin intermediate filament cytoarchitecture when comparing primary cultures of wild-type and epiplakin-null epidermal keratinocytes (stratified epithelium), nor of corresponding hepatocyte (simple epithelium) cultures (Spazierer et al, 2006). To assess whether epiplakin was associated with keratin filaments in such cells, primary keratinocyte and hepatocyte cell cultures from wild-type mice were subjected to double immunofluorescence microscopy using antibodies to epiplakin combined with anti-pankeratin (keratinocytes) or anti-keratin K8/18 (hepatocytes) antibodies.…”

“…The protein is expressed in epithelial tissues, with especially high levels found in skin . Its function is still unknown, because mice deficient in epiplakin did not show any discernible phenotype (Spazierer et al, 2006), except for a slight acceleration of keratinocyte migration, as reported by Goto et al (Goto et al, 2006). Surprisingly, not even when the skin of knockout mice was challenged by tape stripping or wounding, were differences to wild-type mice observed (Spazierer et al, 2006).…”

“…That this protective effect was noticed only in cells that had undergone differentiation, but not under basal conditions prompts us to speculate that epiplakin executes this function mainly in cell layers where filament systems span more cells, such as in the upper epidermis. Nevertheless, the initial analysis of mice deficient in epiplakin did not show any skin abnormalities, and even challenging the skin by tape-stripping or wounding did not reveal any discernible phenotype (Goto et al, 2006;Spazierer et al, 2006). However, this kind of mechanical stress might not have affected the keratin filament network system in the same way that phosphatase inhibitors do.…”

“…Cell culture, fractionation and immunofluorescence microscopy Primary keratinocytes were isolated from 2-to 3-day-old mice as described previously (Spazierer et al, 2006), and grown in keratinocyte growth medium (KGM; Cambrex, East Rutherford, NJ) containing 0.1 mM CaCl 2 . For differentiation of keratinocytes, KGM containing 1.3 mM CaCl 2 was used.…”