A mutation of keratin 18 within the coil 1A consensus motif causes widespread keratin aggregation but cell type-restricted lethality in mice

Mutations in genes encoding epidermal keratins cause skin disorders, while those in internal epithelial keratins, such as K8 and K18, are risk factors for liver diseases. The effect of dominant mutations in K8 or K18 during embryonic development and tissue homeostasis has not been examined so far. Here we demonstrate that the dominant mutation hK18 R89C, that is highly similar to hK14 R125C, causing EBS in humans, leads to cell type-specific lethality in mice, depending on the ratio of mutant to endogenous keratins. Mice expressing hK18 R89C in the absence of endogenous K19 and K18 died at mid-gestation from defects in trophoblast giant cells, accompanied by haematomas. A single, endogenous K18 allele rescued embryonic lethality but caused aggregation of keratins in all adult internal epithelia, surprisingly without spontaneous cell fragility. Closer analysis revealed that both filaments and aggregates coexisted in the same cell, depending on the ratio of mutant to endogenous keratins. Our results demonstrate that balanced overexpression of a wild-type keratin rescued the lethal consequences of a dominant-negative mutation. This has important implications for therapy approaches of keratinopathies, suggesting that suppressing the mutant allele is not necessary in vivo

Pairwise Assembly Determines the Intrinsic Potential for Self-Organization and Mechanical Properties of Keratin Filaments

Most type I and II keratin genes are spatially and temporally regulated in a pairwise manner in epithelial tissues, where they represent the major structural proteins. Epithelia can be partitioned into simple (single-layered) and complex (multilayered) types. We compared the structural and mechanical properties of natural keratin polymers occurring in complex (K5-K14) and simple (K8-K18) epithelia. The intrinsic properties of these distantly related keratin filaments, whether dispersed or bundled in vitro, were surprisingly similar in all respects when at high polymer concentration. When type I and II assembly partners were switched to give rise to mismatched polymers (K5-K18; K8-K14), the interfilament interactions, which determine the structural and mechanical properties of keratin polymers, were significantly altered. We also show that a K5-K16 polymer exhibits lesser elasticity than K5- K14, which may help explain the inability of K16 to fully rescue the skin blistering characteristic of K14 null mice. The property of self-interaction exhibited by keratin filaments is likely to assist their function in vivo and may account for the relative paucity of cytoplasmic and keratin-specific cross-linkers. Our findings underscore the fundamental importance of pairwise polymerization and have implications for the functional significance of keratin sequence diversity

Formation of Mallory Body-like Inclusions and Cell Death Induced by Deregulated Expression of Keratin 18

Mallory bodies (MBs) are cytoplasmic inclusions that contain keratin 8 (K8) and K18 and are present in hepatocytes of individuals with alcoholic liver disease, nonalcoholic steatohepatitis, or benign or malignant hepatocellular neoplasia. Mice fed long term with griseofulvin are an animal model of MB formation. However, the lack of a cellular model has impeded understanding of the molecular mechanism of this process. Culture of HepG2 cells with griseofulvin has now been shown to induce both the formation of intracellular aggregates containing K18 as well as an increase in the abundance of K18 mRNA. Overexpression of K18 in HepG2, HeLa, or COS-7 cells also induced the formation of intracellular aggregates that stained with antibodies to ubiquitin and with rhodamine B (characteristics of MBs formed in vivo), eventually leading to cell death. The MB-like aggregates were deposited around centrosomes and disrupted the microtubular array. Coexpression of K8 with K18 restored the normal fibrous pattern of keratin distribution and reduced the toxicity of K18. In contrast, an NH(2)-terminal deletion mutant of K8 promoted the formation of intracellular aggregates even in the absence of K18 overexpression. Deregulated expression of K18, or an imbalance between K8 and K18, may thus be an important determinant of MB formation, which compromises the function of centrosomes and the microtubule network and leads to cell death