MITOGENIC AND METABOLIC ACTIONS OF EPIDERMAL GROWTH-FACTOR ON RAT ARTICULAR CHONDROCYTES - MODULATION BY FETAL CALF SERUM, TRANSFORMING GROWTH-FACTOR-BETA, AND TYRPHOSTIN

The effects of human recombinant epidermal growth factor (EGF) on rat articular chondrocytes from humeral and femoral head cartilage of 21-d ay-old Wistar rats were analyzed. The cells were cultured under standa rd conditions as monolayers. Cell proliferation was studied by [H-3]th ymidine incorporation and determination of DNA content, proteoglycan s ynthesis by [S-35]sulfate incorporation, and collagen synthesis by [H- 3]proline incorporation, The presence of specific receptors was confir med by [I-125]-EGF binding and that of EGF and EGF-receptor (EGF-R) mR NA by reverse transcription and the polymerase chain reaction, EGF (0. 5-2.5 ng/ml) stimulated [H-3]thymidine incorporation and increased DNA content of cultures, The effect was strongest when serum concentratio n was low (less than or equal to 1%) and was lost at high (greater tha n or equal to 7.5%) serum concentrations, The EGF-induced effect on de oxynucleic acid synthesis was inhibited by transforming growth factor- beta and tyrphostin, a tyrosine kinase inhibitor that blocks the phosp horylation of tyrosine residues on EGF-R. Cultured rat articular chond rocytes possess a single class of high-affinity binding sites (Kd 0.18 nM). There were about 4.5 x 10(9) binding sites per microgram of DNA or about 37,800 binding sites per cell with 8.3 pg DNA per cell. Cultu red cells contained EGF mRNA and EGF-R mRNA. Incubation of cells with EGF for 24 h decreased the EGF mRNA transcripts and increased the EGF- R mRNA levels. These findings suggest that EGF probably takes part in the regulation of chondrocyte activity under normal and presumably pat hological conditions. (C) 1997 Academic Press.