Endothelin‐1 expression in long‐term cultures of fetal rat calvarial osteoblasts: Regulation by BMP‐7

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Abstract
Endothelin-1 (ET-1) is a vasoactive peptide that modulates bone metabolism via regulatory effects on osteoblasts, chondrocytes, and osteoclasts. While ET-1 may circulate in the blood stream, tissue-specific expression of this peptide is more physiologically relevant. In the present study we measured ET-1 synthesis in sections of fetal rat calvaria (FRC) and in cultured FRC osteoblasts. Regulation of ET-1 synthesis in FRC osteoblasts by bone morphogenetic protein-7 (BMP-7) and transforming growth factor-β1 (TGF-β1) also was examined. Immunohistochemical analysis revealed ET-1 staining in calvarial osteoblasts, endothelial cells, and osteocytes. ET-1 mRNA expression was detected in cultured FRC cells and ET-1 peptide was present in conditioned media. During long-term culture of FRC cells (26 days) ET-1 peptide production rose sharply and peaked during the time of cellular proliferation (Days 0–3) then returned to baseline levels by Day 18, when mineralized nodules were forming. Treatment of FRC cells with BMP-7 enhanced ET-1 levels by three-fold on Day 3 and enhanced nodule formation by 15-fold on Day 26. To determine whether ET-1 was involved in an autocrine manner in BMP-7-induced nodule formation, cells were cultured in the presence of BMP-7 and BQ-123, an ETA receptor antagonist. BQ-123 had no effect on nodule formation in control or BMP-7-treated cells, indicating that osteoblast-derived ET-1 regulates other cell types in vivo during the bone formation process.