Morphological and biochemical responses of cultured thyroid cells to thyrotropin

In the thyroid gland, TSH stimulates cAMP formation, exocytosis of precursor (noniodinated) thyroglobulin, endocytosis of thyroglobulin, and proteolytic processing of the thyroglobulin to form thyroid hormones. In this report we describe TSH effects on cAMP levels, microtubules, microfilaments, myosin fibrils, and the morphology of cultured thyroid follicle cells. The cells were normally cultured in the presence of 10 mU/ml TSH, and fresh TSH produced no stimulation when assayed for cAMP production in a 15-min assay. When such cells were cultured for up to 72 h in the absence of TSH and then assayed for cAMP production, the basal levels were much reduced, but fresh TSH stimulated cAMP levels half-maximally at 1 mU/ml and up to 50-fold at 20 mU/ml. Microtubules, myosin fibers, and microfilaments were demonstrated by indirect immunofluorescent staining. Fluorescent staining of fibers was observed in cells fixed before lysis and in cells lysed before fixation. In control cells grown without hormone, microtubules originated near the nucleus and extended to the cell periphery. Myosin-containing fibrils traversed the cell or radiated from foci. Microfilaments spanned the cell in a stress fiber pattern. After incubation with 20 mU/ml TSH and 4 mM isobutylmethylxanthine (IBMX) for 10-20 min, the microtubules in up to half of the cells appeared altered and more granular, and the cell periphery was scalloped. After 15-30 min with TSH and IBMX, normal myosin fibers were replaced with a fine lattice-work, peripheral staining disappeared, and the proportion of nonfibrous myosin increased. Stress fibers demonstrated with antibody to actin also disappeared, and the peripheral structures observed in normal cells became fragmented. Incubation with forskolin or TSH and IBMX for 2-3 h resulted in arborization of 30-60% of the cells that contained bundles of microtubules, myosin fibers, or microfilaments into dendrite-like processes and increased staining near the nucleus. At 5 h, more than 80% of the cells were arborized. These morphological changes were less pronounced with IBMX alone and minimal with TSH alone. The time course of cAMP levels observed basally or after TSH, forskolin, or TSH and IBMX was consistent with the relative effects of these agents on arborization. These studies are consistent with effects of cAMP on microtubules, myosin-containing fibrils, and microfilaments and may provide a basis for the morphological response to TSH.