Dissociation of actin polymerization and enzyme inactivation in the hormonal regulation of type II iodothyronine 5'-deiodinase activity in astrocytes.
T4 dynamically regulates the levels of type II iodothyronine 5'-deiodinase in the brain. Using an astrocyte cell culture model, we have shown that thyroxine increases inactivation of this enzyme through a mechanism using the actin cytoskeleton. In the absence of T4, the filamentous actin (F-actin) stress fibers are absent, and deiodinase inactivation is relatively slow. T4 increases inactivation of type II 5'-deiodinase by 1) restoring the F-actin stress fibers, 2) promoting the binding of the enzyme to F-actin, and 3) stimulating enzyme internalization. To determine whether inactivation of the deiodinase is due solely to the restoration of stress fibers by T4 or also involves direct thyroxine-mediated enzyme-F-actin interactions, we examined the effects of retinoids on both actin polymerization and type II 5'-deiodinase activity in cultured astrocytes, as these hormones have been shown to alter cytoskeletal organization in other tissues. In thyroid hormone-deficient astrocytes, retinoic acid increased F-actin levels, with no change in total cell actin. The F-actin content increased approximately 40% within 30 min after the addition of retinoic acid. After a plateau of 6-8 h, the F-actin content increased further to approximately 90% of the total cell actin and was associated with the reappearance of stress fibers. Only this latter retinoid-stimulated increase in F-actin content was blocked by actinomycin-D. Restoration of the F-actin stress fibers by retinoids did not increase the turnover of the type II 5'-deiodinase (t1/2, 1.99 h-1) or promote binding of the enzyme to F-actin in the absence of T4. Similarly, retinoids did not affect the rapid T4-mediated turnover (t1/2, 0.18 h-1) of type II 5'-deiodinase. These data show that an intact F-actin cytoskeleton in the absence of T4 is inadequate to alter the inactivation of type II 5'-deiodinase and that specific T4-enzyme-F-actin interactions are necessary to initiate the rapid inactivation/internalization of this enzyme.
Farwell
A P
AP
Molecular Endocrinology Laboratory, University of Massachusetts Medical School, Worcester 01655.
Leonard
J L
JL
D000199:Actins / Q000378:metabolism*; D000818:Animals; D000831:Animals, Newborn; D001253:Astrocytes / Q000201:enzymology*; D003994:Bucladesine / Q000494:pharmacology; D002478:Cells, Cultured; D003513:Cycloheximide / Q000494:pharmacology; D003609:Dactinomycin / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D005456:Fluorescent Dyes; D007453:Iodide Peroxidase / Q000378:metabolism*; D007700:Kinetics; D008856:Microscopy, Fluorescence; D051381:Rats; D013974:Thyroxine / Q000494:pharmacology; D014212:Tretinoin / Q000494:pharmacology
D000199:Actins; D005456:Fluorescent Dyes; D003609:Dactinomycin; D014212:Tretinoin; D003994:Bucladesine; D003513:Cycloheximide; D007453:Iodide Peroxidase; D013974:Thyroxine