Endothelin-1 promotes osteoprogenitor proliferation and differentiation in fetal rat calvarial cell cultures

Endothelin-1 (ET-1), a peptide produced by vascular endothelial cells, has been suggested to be one of the signaling factors between vascular and osteoblastic cells during bone growth and remodeling. The osteoinductive effects of ET-1 were tested on fetal rat calvaria which have the ability to form bone nodules in culture. ET-1 (10(-10) to 10(-6) M) dose-dependently increased cell proliferation. The effect of ET-1 (10(-8) M) on proliferation was greater than that of dexamethasone (Dex; 10(-8) M). ET-1 also increased the number of bone nodules by 146% over untreated cells, which coincided with a 3.1-fold increase in alkaline phosphatase activity. Limiting dilution assays showed that ET-1 treatment increased the number of osteoprogenitors (CFU-AP and CFU-OB) beyond what would be expected by a proliferative effect alone, indicating that ET-1 also stimulated osteoblast differentiation. Osteocalcin mRNA expression was upregulated as shown by Northern blot analysis. Using cDNA microarray analysis, ET-1 treatment resulted in an expression profile that included an upregulation of 163 genes and expressed sequence tags. Simultaneous addition of ET-1 and Dex to the medium further increased the number of bone nodules and alkaline phosphatase activity over either treatment alone. Our results show that ET-1 promotes both osteoblastic proliferation and differentiation and that the effects of ET-1 and Dex on differentiation are cooperative.