The effect of epidermal growth factor (EGF) on clone MC3T3-El cells that have osteoblastic activity was examined by phase-contrast microscopy and electron microscopy; hydroxyproline content, collagen synthesis, collagen pattern, and alkaline phosphatase (ALP) activity were also determined. We found that EGF (0.4 ng/ml) transformed the cells from their normal polygonal shape to a spindle-like morphology by 8 h. This hormone also caused dose-related suppression of hydroxyproline content and ALP activity which was detectable 2 days and 1 day, respectively, after EGF addition. Indomethacin did not affect hydroxyproline content and ALP activity, suggesting that the effect of EGF on the cells may not be mediated by prostaglandins. Epidermal growth factor at concentrations of 2 to 50 ng/ml significantly decreased collagen synthesis in the cells, whereas protein synthesis was stimulated. Electron microscopy demonstrated that collagen fiber formation was also reduced by EGF; an immature type of fibril was observed compared with the typical cross-striated one in the controls. Moreover, the hormone treatment also resulted in the appearance of type III collagen in addition to the type I already present in the cells. These suppressive effects of EGF on MC3T3-El cells in vitro suggest that this hormone may be involved in bone remodelling in vivo as well.

We investigated the effect of epidermal growth factor (EGF) on collagen and protein synthesis in clone MC3T30-E1, a cell line which retains osteoblast-like characteristics. EGF at concentrations of 2-50 ng/ml significantly the hydroxyproline content of the cell layer. These effects were completely abolished by the addition of anti-EGF rabbit serum. The addition of indomethacin did not affect these EGF-induced effects. Collagen fiber formation was also reduced by EGF; a fine and unstriated type of fibril was detected compared to the typical cross-striated fibrils seen in control cultures. EGF at concentrations of 2-50 ng/ml significantly decreased collagen synthesis in the cells, whereas protein synthesis was rather stimulated. Thus, the proportion of collagen to protein synthesized decreased markedly with increasing concentrations of EGF. Unrelated to its effect on collagen synthesis, EGF at concentrations of 0.4-50 ng/ml significantly increased the activity of prolyl hydroxylase, an enzyme involved in the biosynthesis of collagen. Since the plasma concentration of EGF in humans is sufficiently high to cause the observed effect, osteoblasts in vivo may be responsive to this peptide in the same manner as those observed in vitro.

Biochemical and morphological studies were made on the effect of epidermal growth factor (EGF), isolated from male mouse submandibular gland, on collagen formation in clone RLC-18(4) epithelial cells from rat liver. EGF did not affect the number of these cells. EGF in the range of 0.5-500 ng/ml caused a dose-dependent increase in the content of hydroxyproline. It also increased the content of acidic glycosaminoglycans (AGAG), which are thought to be closely related to the formation of collagen fibers, and increased the activity of glutamine glucose-6-phosphate aminotransferase, an enzyme for AGAG synthesis but not that of N-acetyl-beta-glucosaminidase, an enzyme for AGAG degradation. It has no significant effect on the protein content of the cells. Studies on the effect of actinomycin D indicated that EGF may enhance de novo synthesis of hydroxyproline in liver epithelial cells and also that of glutamine glucose-6-phosphate aminotransferase, thus increasing the AGAG content of the cells. Antibody to EGF largely blocked collagen formation in the cells, even in the absence of EGF, indicating that EGF or EGF-like substances in the serum may affect collagen formation. In rat liver fibroblasts, EGF had little effect on collagen formation. These results show that EGF may act as a regulatory factor in collagen formation in liver epithelial cells, but not liver mesenchymal cells, in vitro.

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