Epiplakin attenuates experimental mouse liver injury by chaperoning keratin reorganization.

BACKGROUND & AIMS: Epiplakin is a member of the plakin protein family and exclusively expressed in epithelial tissues where it binds to keratins. Epiplakin-deficient (Eppk1(-/-)) mice displayed no obvious spontaneous phenotype, but their keratinocytes showed a faster keratin network breakdown in response to stress. The role of epiplakin in the stressed liver remained to be elucidated. METHODS: Wild-type (WT) and Eppk1(-/-) mice were subjected to common bile duct ligation (CBDL) or fed with a 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC)-containing diet. The importance of epiplakin during keratin reorganization was assessed in primary hepatocytes. RESULTS: Our experiments revealed that epiplakin is expressed in hepatocytes and cholangiocytes, and binds to keratin 8 (K8) and K18 via multiple domains. In several liver stress models epiplakin and K8 genes displayed identical expression patterns and transgenic K8 overexpression resulted in elevated hepatic epiplakin levels. After CBDL and DDC treatment, Eppk1(-/-) mice developed a more pronounced liver injury and their livers contained larger amounts of hepatocellular keratin granules, indicating impaired disease-induced keratin network reorganization. In line with these findings, primary Eppk1(-/-) hepatocytes showed increased formation of keratin aggregates after treatment with the phosphatase inhibitor okadaic acid, a phenotype which was rescued by the chemical chaperone trimethylamine N-oxide (TMAO). Finally, transfection experiments revealed that Eppk1(-/-) primary hepatocytes were less able to tolerate forced K8 overexpression and that TMAO treatment rescued this phenotype. CONCLUSION: Our data indicate that epiplakin plays a protective role during experimental liver injuries by chaperoning disease-induced keratin reorganization.

Stabilization of the dystroglycan complex in Cajal bands of myelinating Schwann cells through plectin-mediated anchorage to vimentin filaments.

Previous studies have unmasked plectin, a uniquely versatile intermediate filament-associated cytolinker protein, to be essential for skin and skeletal muscle integrity. Different sets of isoforms of the protein were found to stabilize cells mechanically, regulate cytoskeletal dynamics, and serve as a scaffolding platform for signaling molecules. Here, we investigated whether a similar scenario prevails in myelinating Schwann cells. Using isoform-specific antibodies, the two plectin variants predominantly expressed in the cytoplasmic compartment (Cajal bands) of Schwann cells were identified as plectin (P)1 and P1c. Coimmunoprecipitation and immunolocalization experiments revealed complex formation of Cajal band plectin with ß-dystroglycan, the core component of the dystrophin glycoprotein complex that in Schwann cells is crucial for the compartmentalization and stabilization of the myelin sheath. To study the functional implications of Schwann cell-specific plectin-ß-dystroglycan interaction, we generated conditional (Schwann cell-restricted) plectin knockout mice. Ablation of plectin in myelinating Schwann cells (SCs) was found not to affect myelin sheath formation but to abrogate the tight association of the dystroglycan complex with the intermediate filament cytoskeleton. We show that the disruption of this association leads to the destabilization of the dystroglycan complex combined with increased myelin sheath deformations observed in the peripheral nerve during ageing of the animal.

Functional and Genetic Analysis of Epiplakin in Epithelial Cells.

Epiplakin is a large member (>700 kDa) of the plakin protein family and exclusively expressed in epithelial cell types. Compared to other plakin proteins epiplakin exhibits an unusual structure as it consists entirely of a variable number of consecutive plakin repeat domains (13 in humans, 16 in mice). The only binding partners of epiplakin identified so far are keratins of simple as well as of stratified epithelia. Epiplakin-deficient mice show no obvious spontaneous phenotype. However, ex vivo studies using epiplakin-deficient primary cells indicated protective functions of epiplakin in response to stress. Recent studies using stress models for organs of the gastrointestinal tract revealed that epiplakin-deficient mice develop more pronounced pancreas and liver injuries than their wild-type littermates. In addition, impaired stress-induced keratin network reorganization was observed in the affected organs, and primary epiplakin-deficient hepatocytes showed reduced tolerance for forced keratin overexpression which could be rescued by a chemical chaperone. These findings indicate protective functions of epiplakin in chaperoning disease-induced keratin reorganization. In this review, we describe some of the methods we used to analyze epiplakin's function with the focus on biochemical and ex vivo techniques.

Epiplakin deficiency aggravates murine caerulein-induced acute pancreatitis and favors the formation of acinar keratin granules.

Epiplakin, a member of the plakin protein family, is exclusively expressed in epithelial tissues and was shown to bind to keratins. Epiplakin-deficient (EPPK-/-) mice showed no obvious spontaneous phenotype, however, EPPK-/- keratinocytes displayed faster keratin network breakdown in response to stress. The role of epiplakin in pancreas, a tissue with abundant keratin expression, was not yet known. We analyzed epiplakin's expression in healthy and inflamed pancreatic tissue and compared wild-type and EPPK-/- mice during caerulein-induced acute pancreatitis. We found that epiplakin was expressed primarily in ductal cells of the pancreas and colocalized with apicolateral keratin bundles in murine pancreatic acinar cells. Epiplakin's diffuse subcellular localization in keratin filament-free acini of K8-deficient mice indicated that its filament-associated localization in acinar cells completely depends on its binding partner keratin. During acute pancreatitis, epiplakin was upregulated in acinar cells and its redistribution closely paralleled keratin reorganization. EPPK-/- mice suffered from aggravated pancreatitis but showed no obvious regeneration phenotype. At the most severe stage of the disease, EPPK-/- acinar cells displayed more keratin aggregates than those of wild-type mice. Our data propose epiplakin to be a protective protein during acute pancreatitis, and that its loss causes impaired disease-associated keratin reorganization.