Small molecule targeting of a myosin partner restores an intact keratin cytoskeleton despite presence of a dominant keratin mutation
Authors: Jens Bohnekamp, Thomas M. Magin
Journal: Hepatology (2015)

Potential conflict of interest: Nothing to report. See Article on Page 1858 Keratin intermediate filament (IF) proteins form the major cytoskeleton of epithelial cells and tissues. The keratin gene family encodes 28 type I and 26 type II keratins, which form isotype‐specific cytoskeletal networks in diverse epithelia where they attach to hemidesmosomes and desmosomes. By virtue of these interactions and by intrinsic biophysical and biochemical properties, they protect epithelia against mechanical force. Moreover, a subset of keratins regulates cell growth, organelle distribution, inflammation, and wound healing through ill‐defined protein interactions.1 Keratin networks underlie extensive reorganization during differentiation, wound healing, cell cycle, and tissue pathogenesis, following missense mutations in keratin genes. Keratin reorganization is regulated at the transcriptional level by expression of keratin isotypes and through posttranslational modifications which affect intracellular keratin organization and interactions with actin, microtubules, cell adhesion complexes, and additional proteins.1 The importance of keratins for epithelial tissue functions is further highlighted by numerous keratin disorders. Mutations in keratin genes result in single‐gene disorders affecting the skin and appendages and contribute to complex diseases of the liver and the intestine. In 1991, the blistering skin disorder epidermolysis bullosa simplex (EBS) was the first reported disease caused by dominant keratin mutations. Various mutations in the keratin genes KRT5 and KRT14, which are mainly expressed in the basal layer epidermis, have been identified and directly linked to the tissue fragility in EBS patients.2 Subsequently, mutations in KRT10 or KRT1, which are expressed in suprabasal cells of cornifying epithelia, were linked to epidermolytic hyperkeratosis and to pachyonychia congenita in the case of KRT6a, KRT6b, KRT16, and KRT17. While mutations in the former genes cause diseases, mutations in KRT8 and KRT18 of simple epithelia are associated with inflammatory bowel and liver disease.1KRT8 and KRT18 represent the main keratins of hepatocytes and have important mechanical and nonmechanical functions in hepatocytes including maintenance of cell integrity, protection from oxidative injury and apoptosis, regulation of mitochondrial organization/function, serving as a "phosphate sponge," and supporting protein targeting.4 Mutations in KRT8/KRT18 alter keratin functions by affecting their solubility, filament reorganization/organization, and posttranslational modifications. KRT8/KRT18 mutations have been associated with end‐stage liver disease of multiple etiologies and with liver disease progression in patients with chronic hepatitis C infection. Animal and primary hepatocyte culture studies have shown that natural and engineered mutations predispose the liver to acute or subacute injury and promote apoptosis and fibrosis. Further, KRT8/KRT18 are main components of Mallory‐Denk bodies, cytoplasmic hyaline inclusions characteristic of alcoholic liver disease.5 The notion that the engineered mutation KRT18 Arg90Cys, which is equivalent to KRT14 Arg125Cys, causing severe EBS in humans, hints toward a similarity between pathomechanisms of skin and liver diseases involving mutant keratins. In view of the dominant nature of most keratinopathies, the majority of therapeutic approaches reported so far have focused on genetic ablation/silencing of the mutant allele and on small‐molecule therapies to stabilize the IF network (Fig. 1). The use of RNA interference that specifically targets messenger RNAs encoding mutant proteins was already tested in several studies. However, there are concerns regarding potential off‐target effects of small interfering RNA. Furthermore, changes in IF expression may result in dramatic effects on tissue differentiation. Efficient delivery and long‐lasting efficacy represent major challenges connected to the above strategies. The use of small molecules to treat keratinopathies is an alternative and promising treatment option (Fig. 1). So far, small molecules either target components leading to up‐regulation of compensatory keratins, like sulforaphane‐induced up‐regulation of KRT17 to compensate for mutant KRT14, or mitogen‐activated protein kinases like c‐Jun N‐terminal kinase or extracellular signal–regulated kinases 1 and 2 that directly alter keratin phosphorylation and organization.1Figure 1: Overview of different keratinopathy treatment strategies. Abbreviations: siRNA, small interfering RNA; WT, wild type.In this issue of Hepatology, Kwan et al.6 present the multikinase inhibitor PKC412, which is a derivative of the antibiotic staurosporine, as a potential new candidate for a novel therapeutic approach for keratin and possibly other IF‐associated diseases. Kwan et al. performed a high‐throughput screening of A549 lung carcinoma cells cotransduced with wild‐type KRT8 and green fluorescent protein‐tagged KRT18 Arg90Cys, using a 320‐compound kinase inhibitor library. As a readout, re‐formation of a normal keratin cytoskeleton containing KRT18 Arg90Cys was employed at different time points. This identified staurosporine and its derivate PKC412 as potential hits. After validation, staurosporine was dismissed because of ensuing cell death after 24 hours. In contrast, PKC412 reduced aggregate‐containing cells by ∼60%. Resulting aggregate‐free cells formed normal‐appearing keratin filaments. Additional experiments showed that PKC412 also protected against apoptosis in cultured cells and in mouse liver, which represents a major step to overcoming mutation‐induced predisposition to cell injury, which in the case of mutated KRT18 translates into predisposition to apoptosis.7 Protection against apoptosis was shown by decreased levels of various apoptosis markers in green fluorescent protein‐KRT18 Arg90Cys‐expressing A549 cells pretreated with PKC412, followed by apoptosis induction with interferon‐γ and Fas ligand. Moreover, mice transgenic for KRT18 Arg90Cys that were pretreated with PKC412 were protected from liver injury and parenchymal hemorrhage as measured by histopathology and serum alanine aminotransferase levels (PKC412 266 versus control 6546 U/L) after Fas‐induced injury. The protective effect appeared to depend on PKC412 pretreatment time and the extent of keratin reorganization from aggregates into filaments. The usefulness of PKC412 was further highlighted by the absence of toxic damage based on serology testing and histological assessment of livers. To gain insight into underlying mechanisms, the authors tested the hypothesis that associated proteins may be involved in keratin reorganization. In support, the cochaperone carboxy terminus of Hsc70 interacting protein (CHIP) had been reported to assist reorganization of aggregated keratins into filaments.8 By mass spectrometry they identified nonmuscle myosin heavy chain (NMHC)‐IIA in the insoluble keratin‐containing fraction of mutant KRT18 Arg90Cys‐expressing A549 cells as a likely PKC412 target. Association of NMHC‐IIA with keratins was independently confirmed by coimmunoprecipitation of mouse liver extracts using KRT18 antibodies and by double immunofluorescence using antibodies against NMHC‐IIA and KRT18 in normal mouse liver and livers of PKC412‐treated KRT18 Arg90Cys mice. Dependence of NMHC‐IIA on keratins was further corroborated by studying livers from KRT8–/– or KRT18–/– mice which lack keratin IF. In this setting, NMHC‐IIA changed from a membrane‐proximal to a diffuse pattern. It appears that dephosphorylation of NMHC‐IIA is required for an association with keratins. Pulldown experiments suggest that NMHC‐IIA preferentially binds to KRT8 but not KRT18 or KRT19. Finally, small interfering RNA against NMHC‐IIA in KRT18 Arg90Cys A549 cells confirmed that NMHC‐IIA expression is required for PKC412‐mediated keratin filament normalization. Kwan et al.6 are the first to describe an interaction of NMHC‐IIA with an IF protein and provide evidence that the phosphorylation status of NMHC‐IIA is crucial for keratin reorganization. Whether this interaction is direct and limited to KRT8 or whether NMHC‐IIA or the two additional isoforms, B and C, are also able to interact with other keratins or IF proteins remains to be addressed. The widespread expression of NMHC‐II9 qualifies it to interact with other IF proteins. Given that the KRT18 Arg90Cys mutation has not been identified in patients so far, the essential test will be to examine whether PKC412 has the potential to ameliorate severe keratinopathies including EBS. Kwan et al.6 hypothesize that NMHC‐II serves as a stabilizer of keratins and additional IF proteins. If this were true, PKC412 could represent a magic bullet that is useful to treat severe keratinopathies and other IF disorders. Unlike small interfering RNA and classic gene therapy approaches, small molecules like PKC412 can be applied systemically with ease and in the long term. The significance of the study is further emphasized by the fact that PKC412 is already used in clinical trials for the treatment of acute myeloid leukemia, lowering the hurdle for applications in additional disorders. Finally, the study emphasizes the value of basic research that identifies a framework of protein interactions and its regulation. It is their understanding that provides druggable targets that improve conditions in life‐threatening illnesses. In addition to their own models, excellent keratinocyte, Drosophila,10 and mouse models are available to further dissect NMHC‐IIA interactions with keratins. Author names in bold designate shared co‐first authorship.