EFFECTS OF TRIIODOTHYRONINE ON MORPHOLOGY, GROWTH-BEHAVIOR, AND THE ACTIN CYTOSKELETON IN MOUSE OSTEOBLASTIC CELLS (MC3T3-E1)

We investigated the effects of thyroid hormone treatment on morphology , growth behavior, and cytoskeletal structures of long-term cultured M C3T3-E1 cells. Morphological investigations were carried out on native cells by phase contrast microscopy and on epon-embedded semithin sect ions, The area covered by the cell and matrix layers (tissue-like area ), percent extracellular matrix, average height of tissue-like area, a nd length and height of single cells were measured histomorphometrical ly on the cross sections, F-actin was analyzed histochemically and qua ntitated after fluorochrome-labeled phalloidin staining using confocal microscopy and fluorometry, Significant differences between control a nd T-3-treated cells were found after confluency, but not in subconflu ent cultures, Control cells continued to proliferate forming multilaye rs, and produced increasing amounts of extracellular matrix, In contra st, T-3-treated cells stopped to proliferate forming two cell layers a t the maximum, These cells were flattened, distinctly enlarged, and po lygonal in shape, Histochemical staining for F-actin revealed three di fferent staining patterns, depending on the position of the cell withi n the multilayer of control cultures. Basal cells contained a large nu mber of thick stress fibers in parallel arrangement, Intermediate cell s exhibited only a few thick actin filament bundles located at the out ermost periphery, The superficial cells were characterized by a large number of thin, parallel-oriented microfilament bundles extending acro ss the entire cytoplasm. The actin pattern of T-3-treated cells resemb led that of the basal cell layer of the control cells, The amount of F -actin increased with the prolonged T-3 treatment. We conclude from th ese data that the known specific cellular responses to T-3 treatment a re accompanied by significant morphological alterations indicating piv otal effects of thyroid hormones on osteoblastic differentiation.