Tri-iodothyronine inhibits multilayer formation of the osteoblastic cell line, MC3T3-E1, by promoting apoptosis

Cell death through apoptosis is a well-known mechanism for maintaining homo eostasis in many developmental and pathological processes. We have recently presented evidence for the occurrence of apoptosis during the formation of bone-like tissue in vitro. MC3T3-E1 osteoblast-like cells in culture devel op features of the osteoblastic phenotype and form many cell layers embedde d in extracellular matrix which can mineralise. Tri-iodothyronine (T-3), ev en though it enhances the expression of many osteoblastic features, attenua tes the multilayer formation to about two layers. The aim of this study was to investigate how T-3 prevents multilayer formation. MC3T3-E1 cells were seeded at different densities and cultured for up to 2 weeks. Thereafter we analysed proliferation rate and the distribution of the phases of the cell cycle and studied apoptosis. We found that Tg did not inhibit DNA synthesi s. Analysis of the cell cycle phases showed an increase in the number of ce lls in G0/G1 with increasing cell density, but no significant effect of T-3 treatment was found. Morphological investigations showed apoptotic feature s in both cell layers and culture supernatants. The cells exhibited typical plasma membrane blebbings, chromatin condensation, DNA fragmentation and p hagocytosed apoptotic bodies. T-3 treatment significantly increased the num ber of apoptotic cells. We conclude from our data that T-3 inhibits multila yer formation of MC3T3-E1 cells by increasing the rate of apoptosis and not by inhibition of proliferation. Because apoptosis is a fundamental regulat ory event during bone tissue differentiation, our findings emphasise the im portance of thyroid hormones in bone maintenance and development.