A human keratin 14 "knockout": the absence of K14 leads to severe epidermolysis bullosa simplex and a function for an intermediate filament protein.

Abstract

Since their discovery, the hinction of intermediate filaments (IFs) has remained obscure. In skin, epidermal cells have extensive cytoskeletal architectures of IFs, composed of type I and type II keratin heterodimers. Clues to possible functions of these proteins have come from recent studies showing that several autosomal-dominant, blistering skin disorders are caused by defects in genes that encode epidermal keratins. These diseases all exhibit cell degeneration and keratin network perturbations in cells that express the particular mutant keratin gene. However, it is not clear from these studies whether cytolysis arises from the presence of large insoluble keratin aggregates that compromise cellular physiology or from the absence of an extensive keratin filament network, which jeopardizes mechanical integrity. We report here the analysis of an extremely rare case of severe recessive epidermolysis bullosa simplex (EBS), where the patient lacks a discernible keratin filament network in basal epidermal cells. Genetic analyses revealed a homozygous point mutation that yielded a premature termination codon in the major basal type I keratin gene and caused complete ablation of K14. The consanguineous parents were normal, each harboring one copy of the null K14 mutation. Analysis of cultured keratinocytes enabled us to document that the loss of K14 is not compensated for by the up-regulation of any other type I keratin. When taken together with the in vivo studies showing the presence of cell fragility generated from the lack of an extensive basal keratin network, these findings provide the first clear demonstration of loss of function associated with the absence of an IF protein in vivo.[Key Words: Intermediate filaments; epidermal keratin; epidermolysis bullosa simplex; epidermolytic hyperkeratosis]

Complete Cytolysis and Neonatal Lethality in Keratin 5 Knockout Mice Reveal Its Fundamental Role in Skin Integrity and in Epidermolysis Bullosa Simplex

“…The reported phenotype of K14 Ϫ/Ϫ mice that exhibited a generalized blistering of the skin accompanied by an increased mortality was compatible with that of severe cases of Dowling-Meara EBS; however, some mice survived the first 3 mo of life, possibly because of a partial compensation of the loss of K14 by the endogenous K15, which was shown to form poorly organized IFs with K5 (Lloyd et al, 1995). With the use of K14 Ϫ/Ϫ keratinocytes, even filaments between the unusual K5 and K17 were reported (Troy and Turksen, 1999).These findings were complemented by the rare occurrence of human EBS patients who lack K14 because of a point mutation leading to a premature stop codon (Chan et al, 1994;Rugg et al, 1994;Jonkman et al, 1996;Batta et al, 2000). As in K14 Ϫ/Ϫ mice, basal keratinocytes of these patients were found to contain "wispy" filaments consisting of K5 and K15 that did not form higher order keratin bundles.…”

Rigidity of Circulating Lymphocytes Is Primarily Conferred by Vimentin Intermediate Filaments

“…Fibroblasts derived from vimentin KO mice are more fragile than normal cells (31). Similarly, human and mouse epidermal cells with mutations in the IF protein keratin tend to rupture when mechanically stressed (32)(33)(34). Vimentin IFs are highly resistant to extraction and disassembly under a variety of conditions, suggesting that they are more stable than microtubules or microfilaments within the ionic conditions of the cytoplasm.…”

Regulation of hard α-keratin mechanics via control of intermediate filament hydration: matrix squeeze revisited

“…In many living cells, IFs form a dense network of filaments that provides passive mechanical support [5][6][7][8]. Mutations in IF genes are known to be the cause of several tissue fragility diseases, including the skin blistering disease epidermolysis bullosa simplex [6,9], which underscores the importance of IFs to maintaining the mechanical integrity of cells. Recent work on the mechanical properties of IFs in living cells suggests they are very soft, extensible and tough [10,11], in stark contrast to the relatively rigid cytoskeletal filaments F-actin and microtubules [12,13].…”