Crossing barriers; restoring barriers? Filaggrin protein replacement takes a bow.

In this issue of the Journal, Stout and colleagues report a novel and creative approach to replacement of genetically determined absence or deficiency of epidermal proteins. While these early data are certainly interesting, further validation work is required to determine the utility of this approach in genodermatoses.

Syncoilin is an intermediate filament protein in activated hepatic stellate cells.

Hepatic stellate cells (HSCs) play an important role in several (patho)physiologic conditions in the liver. In response to chronic injury, HSCs are activated and change from quiescent to myofibroblast-like cells with contractile properties. This shift in phenotype is accompanied by a change in expression of intermediate filament (IF) proteins. HSCs express a broad, but variable spectrum of IF proteins. In muscle, syncoilin was identified as an alpha-dystrobrevin binding protein with sequence homology to IF proteins. We investigated the expression of syncoilin in mouse and human HSCs. Syncoilin expression in isolated and cultured HSCs was studied by qPCR, Western blotting, and fluorescence immunocytochemistry. Syncoilin expression was also evaluated in other primary liver cell types and in in vivo-activated HSCs as well as total liver samples from fibrotic mice and cirrhotic patients. Syncoilin mRNA was present in human and mouse HSCs and was highly expressed in in vitro- and in vivo-activated HSCs. Syncoilin protein was strongly upregulated during in vitro activation of HSCs and undetectable in hepatocytes and liver sinusoidal endothelial cells. Syncoilin mRNA levels were elevated in both CCl4- and common bile duct ligation-treated mice. Syncoilin immunocytochemistry revealed filamentous staining in activated mouse HSCs that partially colocalized with α-smooth muscle actin, ß-actin, desmin, and α-tubulin. We show that in the liver, syncoilin is predominantly expressed by activated HSCs and displays very low-expression levels in other liver cell types, making it a good marker of activated HSCs. During in vitro activation of mouse HSCs, syncoilin is able to form filamentous structures or at least to closely interact with existing cellular filaments.

Recombinant filaggrin is internalized and processed to correct filaggrin deficiency.

This study was designed to engineer a functional filaggrin (FLG) monomer linked to a cell-penetrating peptide (RMR) and to test the ability of this peptide to penetrate epidermal tissue as a therapeutic strategy for genetically determined atopic dermatitis (AD). A single repeat of the murine filaggrin gene (Flg) was covalently linked to a RMR motif and cloned into a bacterial expression system for protein production. Purified FLG+RMR (mFLG+RMR) was applied in vitro to HEK-293T cells and a reconstructed human epidermis (RHE) tissue model. Immunochemistry demonstrated RMR-dependent cellular uptake of FLG+RMR in a dose- and time-dependent manner in HEK cells. Immunohistochemical staining of the RHE model identified penetration of FLG+RMR to the stratum granulosum, the epidermal layer at which FLG deficiency is thought to be pathologically relevant. In vivo application of FLG+RMR to FLG-deficient flaky tail (ft/ft) mice skin demonstrated internalization and processing of recombinant FLG+RMR to restore the normal phenotype. These results suggest that topically applied RMR-linked FLG monomers are able to penetrate epidermal tissue, be internalized into the appropriate cell type, and be processed to a size similar to wild-type functional barrier peptides to restore necessary barrier function, and prove to be therapeutic for patients with AD.