Caspase 8-mediated cleavage of plectin precedes F-actin breakdown in acinar cells during pancreatitis

Abstract

Pancreatic acinar cells depend on the integrity of the cytoskeleton for regulated secretion. Stimulation of isolated rat pancreatic acini with the secretagogue CCK serves as a model for human acute edematous pancreatitis. It induces the breakdown of the actin filament system (F-actin) with the consecutive inhibition of secretion and premature activation of digestive enzymes. However, the mechanisms that regulate F-actin breakdown are largely unknown. Plectin is a versatile cytolinker protein regulating F-actin dynamics in fibroblasts. It was recently demonstrated that plectin is a substrate of caspase 8. In pancreatic acinar cells, plectin strongly colocalizes with apical and basolateral F-actin. Supramaximal secretory stimulation of acini with CCK leads to a rapid redistribution and activation of caspase 8, followed by degradation of plectin that in turn precedes the F-actin breakdown. Inhibition of caspase 8 before CCK hyperstimulation prevents plectin cleavage, stabilizes F-actin morphology, and reverses the inhibition of secretion. Thus we propose that the caspase 8-mediated degradation of plectin represents a critical biochemical event during CCK-induced secretory blockade and cell injury.

Stabilization of Exocytosis by Dynamic F-actin Coating of Zymogen Granules in Pancreatic Acini

“…CCK receptors are known to activate signaling cascades other than Ca 2ϩ signaling (42). Such disruption of Factin coating may account for the diffuse staining of F-actin detected previously in the pancreas of animal models of acute pancreatitis by one-photon confocal microscopy (16,43).…”

“…The mixing of components of the granule and apical plasma membranes after exocytosis (31) and the lateral diffusion of an apical membrane protein and intramembrane particles into exocytic granules (32) have been demonstrated in freeze-fracture studies of the parotid gland. Candidates for plasma membrane factors that might diffuse into the granule membrane include RhoGEF (guanine nucleotide exchange factor for Rho) (33,34), phosphatidylinositol 4,5-bisphosphate (35), and plectin (16,36). Sequential exocytosis has also been proposed to be mediated by the lateral diffusion of apical membrane fusion proteins (10,37).…”

“…Vacuole formation induced by high concentrations of CCK might be due to interference with the F-actin coating of granules by the sustained increase in [Ca 2ϩ ] i (19,39), which may result in the activation of Factin-severing proteins, such as gelsolin or villin, located in the apical region of the cell (22,40,41). Dismantling of F-actin coats may result from cleavage of the scaffolding protein plectin by caspage-8 (16). Our data further demonstrated that the increases in [Ca 2ϩ ] i were not sufficient for vacuole formation since large and sustained increases in [Ca 2ϩ ] i caused by photolysis of the caged-Ca 2ϩ compound did not induce vacuole formation.…”

“…Reorganization of the actin cytoskeleton is also implicated in the pathogenesis of acute pancreatitis (13)(14)(15)(16), which is characterized at the cellular level by the appearance of vacuoles in, and disruption of the polarized secretion of digestive enzymes from, pancreatic acinar cells (17). In animal models of this condition, protracted agonist stimulation results in both actin reorganization and vacuole formation (18 -20).…”

Naringin-sensitive Phosphorylation of Plectin, a Cytoskeletal Cross-linking Protein, in Isolated Rat Hepatocytes

“…In rat hepatocytes, toxin-induced apoptosis was similarly preceded by keratin hyperphosphorylation and network disruption, in parallel with the plectin changes (3,8,21). Plectin is sensitive to endoproteolytic cleavage by calpain (88) or caspases (89) and was found to be cleaved at an early stage of receptor-mediated apoptosis in mammary carcinoma cells (89) and rat pancreatic acini (90). In the present study, however, no plectin cleavage fragments were seen on immunostained gels during the first hours after toxin treatment of hepatocytes, using an antibody capable of detecting such fragments (90).…”