DISRUPTION OF THE CYTOKERATIN CYTOSKELETON AND INHIBITION OF HEPATOCYTIC AUTOPHAGY BY OKADAIC ACID

To learn whether autophagy might be dependent on any of the major cyto skeletal elements, the effect of various cytoskeleton inhibitors on au tophagy and cytoskeletal organization was studied in isolated rat hepa tocytes. Autophagy, measured as the sequestration of endogenous lactat e dehydrogenase, was completely inhibited in isolated rat hepatocytes by the protein phosphatase inhibitor okadaic acid (30 nM). Only small effects were seen with vinblastine (10 mu M) or cytochalasin D (10 mu M) Indirect immunofluorescence microscopy with antibody to a 55-kDa cy tokeratin, corresponding to human cytokeratin 8 (CK8), revealed that w hereas control cells contained a well-organized network of cytokeratin intermediate filaments, okadaic acid disrupted this network into smal l spherical aggregates. Treatment with cytochalasin D or vinblastine, which disrupt microfilaments and microtubules, respectively, had no de tectable effect on the cytokeratin filament distribution. Neither the microtubule network (detected by indirect immunofluorescence with anti bodies against alpha- and beta-tubulin) nor the actin microfilament ne twork (detected by rhodamine-palloidin) was disrupted by okadaic acid. Naringin (100 mu M), a putative protein kinase-inhibitory flavonoid, offered complete protection against the autophagy-inhibitory and cytok eratin-disruptive effects of okadaic acid. Two other flavonoids, genis tein (100 mu M) and prunin (100 mu M), as well as KN-62 (10 mu M), a s pecific inhibitor of Ca2+/calmodulin-dependent kinase II), likewise di splayed a good ability to protect against the effect of okadaic acid u pon cytokeratin organization, while no such protection was seen with H -89 (20 mu M), an inhibitor of the cyclic nucleotide-dependent protein kinases, or with H-7 (100 mu M), which in addition inhibits protein k inase C. The results suggest that the cytokeratin cytoskeleton of hepa tocytes is subject to rapid control by phosphorylation and dephosphory lation and that cytokeratin filaments may somehow be involved in the a utophagic process. (C) 1995 Academic Press, Inc.