EFFECTS OF TRIIODOTHYRONINE AND INSULIN-LIKE GROWTH FACTOR-I (IGF-I) ON ALKALINE-PHOSPHATASE ACTIVITY, [H-3] THYMIDINE INCORPORATION AND IGF-I RECEPTOR MESSENGER-RNA IN CULTURED RAT EPIPHYSEAL CHONDROCYTES

The effects of tri-iodothyronine (T3) and insulin-like growth factor-I (IGF-I) on [H-3[thymidine incorporation, alkaline phosphatase (ALP) activity and IGF-I receptor mRNA levels were studied in rat epiphyseal chondrocytes cultured in monolayer. Chondrocytes from enzymatically digested rat tibia epiphyseal growth plates were seeded in monolayer culture and precultured for 7-14 days in Ham's F-12 medium supplemented with 10% (v/v) newborn calf serum and 1% (v/v) of a serum substitute. After preculture the medium was changed to Ham's F-12 medium containing 1% (v/v) serum from hypophysectomized rats, and the effects of T3 and/or IGF-I on DNA synthesis ([H-3]thymidine incorporation), ALP activity (a late marker of differentiated epiphyseal chondrocytes) and IGF-I receptor mRNA levels were studied. ALP activity was increased by T3 in a dose-dependent manner with a maximal response at 10 mug T3/1 (678 +/- 86% compared with control culture). The increase in ALP activity was accompanied by a concomitant decrease in [H-3]thymidine incorporation (52 +/- 14% compared with control culture). Human GH (hGH; 50 mug/l) and IGF-I (25 mug/l) had no stimulatory effect on ALP activity. However IGF-I (10 mug/l) exerted an inhibition on the T3 (10 mug/l)induced increase in ALP activity (64 +/- 9% compared with T3-treated culture). T3 (3 mug/l) inhibited the increase in [H-3]thymidine incorporation caused by 25 mug IGF-I/l (51 +/- 13% compared with IGF-I-treated culture). Furthermore, IGF-I receptor mRNA levels were increased by 10 mug T3/l (137 +/- 4.2% compared with control culture) while no effect of hGH (50 mug/l) or IGF-I (25 mug/1) was demonstrated. Both T3 and IGF-I were shown to interact with epiphyseal chondrocytes and both substances seemed to affect cell proliferation and maturation and therefore longitudinal bone growth. Furthermore, the results indicated that IGF-I is important for proliferation of the cells while T3 initiates the terminal differentiation of epiphyseal chondrocytes.