Growth Hormone in Vivo Potentiates the Stimulatory Effect of Insulin-Like Growth Factor-1 in Vitro on Colony Formation of Epiphyseal Chondrocytes Isolated from Hypophysectomized Rats*

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Abstract
The effect of GH pretreatment in vivo on the colony formation of epiphyseal chondrocytes from hypophysectomized rats and the subsequent responsiveness to insulin-like growth factor (IGF-I) was studied in vitro. Chondrocytes from epiphyseal growth plates of the proximal tibia of 36-day-old hypophysectomized rats were enzymatically isolated and cultured in suspension, stabilized with agarose (0.5%) in Ham''s F-12 medium and serum supplement. After 14 days the cultures were terminated and screened for cloning efficiency (number of colonies with a diameter > 56 .mu.m/100 0 seeded cells) and for distribution of cloning efficiency as a function of colony size. Pretreatment with human GH in vivo for 24 h (10 .mu.g .times. 3) increased the cloning efficiency during the subsequent culture period (control, 1.5 .+-. 0.1; human GH, 4.4 .+-. 0.3). Addition of IGF-I to the chondrocyte cultures from control rats caused a slight increase in cloning efficiency (control, 1.5 .+-. 0.1; IGF-I, 2.2 .+-. 0.3) but caused a marked increase in chondrocyte cultures from GH-pretreated rats (control, 4.4 .+-. 0.4; IGF-I, 8.2 .+-. 0.9). The cloning efficiency was increased 12 and 24 h, but not 4 h, after start of GH-treatment in vivo. The increased responsiveness to IGF-I in vivo showed a similar course after GH pretreatment. The distribution of cloning efficiency was altered in cultures of chondrocytes isolated from the GH-pretreated rats; large colonies were overrepresented in the GH-treated group. Colonies with a diameter exceeding 180 .mu.m were only seen in cultures of chondrocytes isolated from the GH-pretreated animals. Addition of IGF-I in vitro did not alter the distribution of cloning efficiency, but increased the mean colony size of all colonies. Pretreatment of the rats with two different doses of IGF-I in vivo for 24 h (5 .mu.g .times. 3 or 50 .mu.g .times. 3) had a slight stimulatory effect on subsequent colony formation, but no potentiation of IGF-I in vitro was demonstrated. The results of the present study show that pretreatment of hypophysectomized rats with GH, but not with IGF-I, promotes the formation of chondrocyte colonies and make the chondrocytes susceptible to IGF-I in vitro. The results suggest that GH induces colony formation by IGF-I-independent mechanisms and that IGF-I is a second effector in GH action as previously shown for cultured 3T3-preadipose cells. The present study supports the dual effector theory of GH action and suggests that this theory applies to the mechanism of action of GH on longitudinal bone growth.