Purpose: The higher prevalence of metopic and sagittal suture synostosis and of non-synostotic plagiocephaly in male infants suggests an important role for circulating androgens in early craniofacial development. Previous studies in our laboratory have demonstrated the expression of androgen receptors in developing fetal dura and calvarial bone, but the influence of sex steroid hormone signaling on fetal craniofacial development has not been well investigated. The purpose of these experiments was to characterize the influence of androgen signaling on cellular proliferation and gene expression in developing fetal dura and calvarial bone. Methods: CD1 murine fetal dural cells and calvarial osteoblasts were isolated at the 18th day of gestation and grown separately in culture. Just prior to confluence, the cells were pulsed for 48 hours with 5a-dihydrotestosterone (DHT; 2-1000nM). Cell proliferation was thereafter examined utilizing a non-radioactive proliferation assay technique. Expression of genes encoding the bone matrix proteins osteopontin, osteocalcin, and type 1 collagen and of those encoding alkaline phospatase and TGF-b1 was determined by reverse transcriptase-polymerase chain reaction technique (RT-PCR). Results: Androgen stimulation at physiologic concentrations increased proliferation of fetal dural cells by 46.0% and of fetal calvarial bone cells by 20.5%. Dural expression of osteopontin, osteocalcin, and type 1 collagen was significantly enhanced by DHT in a dose-dependent fashion, as was that of TGF-b1 and alkaline phosphatase. Androgen stimulation similarly increased calvarial osteoblast expression of osteocalcin, type 1 collagen and alkaline phosphatase, but induced little change in TGF-b1 expression. Osteopontin demonstrated high baseline levels of expression in fetal calvarial osteoblasts, with increased expression after 48 hours of DHT stimulation. Conclusions: Androgen stimulation of dural cells and osteoblasts isolated from the developing fetal calvarium induces cell proliferation and osteoblastic differentiation. These results suggest that sex steroid hormone signaling may play an important role in both normal and pathologic craniofacial development.
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