Mitogenic and Metabolic Actions of Epidermal Growth Factor on Rat Articular Chondrocytes: Modulation by Fetal Calf Serum, Transforming Growth Factor-beta, and Tyrphostin

Author： Ribault D. Khatib A.-.M. Panasyuk A. Barbara A. Bouizar Z. Mitrovic R.D.

Publisher： Elsevier

ISSN： 0003-9861

Source： Archives of Biochemistry and Biophysics, Vol.337, Iss.2, 1997-01, pp. : 149-158

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Abstract

The effects of human recombinant epidermal growth factor (EGF) on rat articular chondrocytes from humeral and femoral head cartilage of 21-day-old Wistar rats were analyzed. The cells were cultured under standard conditions as monolayers. Cell proliferation was studied by [ 3 H]thymidine incorporation and determination of DNA content, proteoglycan synthesis by [ 35 S]sulfate incorporation, and collagen synthesis by [ 3 H]proline incorporation. The presence of specific receptors was confirmed by [ 125 I]-EGF binding and that of EGF and EGF-receptor (EGF-R) mRNA by reverse transcription and the polymerase chain reaction. EGF (0.5-2.5 ng/ml) stimulated [ 3 H]thymidine incorporation and increased DNA content of cultures. The effect was strongest when serum concentration was low (<1%) and was lost at high (>7.5%) serum concentrations. The EGF-induced effect on deoxynucleic acid synthesis was inhibited by transforming growth factor-beta and tyrphostin, a tyrosine kinase inhibitor that blocks the phosphorylation of tyrosine residues on EGF-R. Cultured rat articular chondrocytes possess a single class of high-affinity binding sites ( Kd 0.18 n m ). 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. Cultured 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 pathological conditions.