Disruption of the Cytokeratin Cytoskeleton and Inhibition of Hepatocytic Autophagy by Okadaic Acid

Abstract

To learn whether autophagy might be dependent on any of the major cytoskeletal elements, the effect of various cytoskeleton inhibitors on autophagy and cytoskeletal organization was studied in isolated rat hepatocytes. Autophagy, measured as the sequestration of endogenous lactate dehydrogenase, was completely inhibited in isolated rat hepatocytes by the protein phosphatase inhibitor okadaic acid (30 n M). Only small effects were seen with vinblastine (10 μ M) or cytochalasin D (10 μ M). Indirect immunofluorescence microscopy with antibody to a 55-kDa cytokeratin, corresponding to human cytokeratin 8 (CK8), revealed that whereas control cells contained a well-organized network of cytokeratin intermediate filaments, okadaic acid disrupted this network into small spherical aggregates. Treatment with cytochalasin D or vinblastine, which disrupt microfilaments and microtubules, respectively, had no detectable effect on the cytokeratin filament distribution. Neither the microtubule network (detected by indirect immunofluorescence with antibodies against α- and β-tubulin) nor the actin microfilament network (detected by rhodamine-palloidin) was disrupted by okadaic acid. Naringin (100 μ M), a putative protein kinase-inhibitory flavonoid, offered complete protection against the autophagy-inhibitory and cytokeratin-disruptive effects of okadaic acid. Two other flavonoids, genistein (100 μ M) and prunin (100 μ M) as well as KN-62 (10 μ M), a specific inhibitor of Ca 2+/calmodulin-dependent kinase II), likewise displayed a good ability to protect against the effect of okadaic acid upon cytokeratin organization, while no such protection was seen with H-89 (20 μ M), an inhibitor of the cyclic nucleotide-dependent protein kinases, or with H-7 (100 μ M), which in addition inhibits protein kinase C. The results suggest that the cytokeratin cytoskeleton of hepatocytes is subject to rapid control by phosphorylation and dephosphorylation and that cytokeratin filaments may somehow be involved in the autophagic process.