Estrogen enhances differentiation of osteoblasts in mouse bone marrow culture

Abstract

The effects of estrogen on bone are possibly mediated by several cell types. In the present study, the effect of 17β-estradiol (E 2) on osteoblast-like cells was investigated by using mouse bone marrow cultures. Bone marrow cells were harvested from the shafts of femurs of 10-week-old NMRI mice and cultured. On day 6, confluent primary cultures were trypsinized and subcultured. Under the conditions used (Keila, S., Pitaru, S., Grosskopf, A., and Wernreb, M. Bone marrow from mechanically unloaded rat bones expresses reduced osteogenic capacity in vitro. J Bone Miner Res 9:321–327; 1994), the bone marrow cultures showed differentiation towards the osteoblastic phenotype. This was demonstrated by the appearance of osteoblastic markers such as α1(I) collagen (COL1), alkaline phosphatase (ALP), osteocalcin (OCN), osteopontin (OP), and transforming growth factor-β1 (TGF-β1), which were detected by using reverse transcriptase polymerase chain reaction (RT-PCR). Bone nodule formation, including deposition of collagen fibers and matrix mineralization, was also studied at several time points of the 3-week culture period. The effect of E 2 on the appearance of osteoblastic markers was studied by incubating cultures in the presence or absence of the hormone. The messenger ribonucleic acid (mRNA) for the estrogen receptor (ER) was found to be expressed at all time points as demonstrated by RT-PCR. When grown with E 2, the rate of cell proliferation was increased in the early phase of cultures, but not after day 6. The addition of E 2 in subcultures resulted in an increase of levels of mRNA for COL1, ALP, OCN, OP, and TGF-β1. ALP activity was also increased. Bone nodule formation, as well as calcium contents, were significantly increased in the cultures grown in the presence of E 2. All E 2 concentrations used (0.01–10 nmol/L) were effective but the maximum response was obtained with 0.1 nmol/L E 2. Addition of the antiestrogen ICI 182,780 abolished the E 2-induced stimulation of proliferation and later an increase in ALP activity. Addition of ICI 182,780 without the hormone did not cause any changes when compared to control cultures. In conclusion, our results demonstrate that E 2 stimulates sequential differentiation of osteoblasts and increases deposition and mineralization of matrix in mouse bone marrow cultures in an estrogen receptor-dependent manner.