EVIDENCE THAT RABBIT CULTURED GROWTH PLATE (GP) CHONDROCYTES SYNTHESIZE 40-60K and 7K PROTEINS WITH IMMUNOREACTIVE IGFI ACTIVITY (IR-IGFI A)

Abstract

The local production of IGFI like factors by cultured GP Chondrocytes was studied. IR-IGFI A in the incubation medium of prepuhertal rabbit GP Chondrocytes grown in serum-free defined medium during 20 days was evaluated by RIA. Chondrocyte phenotype was followed by specific proteoglycan and Type II collagen biosynthesis. The mean IR-IGFI A measured every 48 h from day 6 to day 16 of the primary culture was constant at 40±18 ImU/106 cells. This activity was further characterized after extraction of the culture medium at neutral and acid pH successively. At neutral pH IR-IGFI A of the eluates of G100 Sephadex column appeared as two distinct peaks:a major one migrating in the 40-60K protein region plus an additive one in the 7K region with a 40-60K:7K ratio =2:l. The 40-60K protein peak material extracted from culture medium corresponding to 10 cells, was able to specifically and reversihly bind 42±10 fmoles 1251-IGFI. At acid pH,IR-IGFI A was similarly separate? as 40-60K and 7K peaks, but their respective amount was reversed resulting in a 40-60K:7K ratio =1:2. When Chondrocytes were incubated in the presence of 35S-Meth during 20 h, only the 40-60K IR-IGFI protein peak was radiolabeled after chronatography at neutral pH. After acid extraction of the radiolabeled 40-60K peak, 60 to 80% of the radioactivity was recovered in the 7K protein peak. This radiolabeled 7K protein peak analyzed by electrophoresis on SDS 10% polyacrylamide gel pH 8.3 was cornigrating with pure 125I-IGFI. These results suggest that chondrocyte synthesize and secrete IGFI-like peptide and a pool of higher MW proteins with IGFI binding capacity.