Toxic Effects of TCDD on Osteogenesis through Altering IGFBP-6 Gene Expression in Osteoblasts

Abstract

Since 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) has reproductive and developmental toxicity as an estrogen antagonist, we investigated the effects of TCDD on osteogenesis in rat skeleton and the human female-responsive osteoblastic osteosarcoma cell line SaOS-2. Rat fetuses were exposed to 5, 10, or 15 m mg/kg TCDD on gestation day (GD) 10. TCDD dose-dependently induced single or multiple rat fetal skeletal development malformations in vivo. In vitro, 10 nM TCDD significantly inhibited cell proliferation in the presence of 1 m mM 17-b b-estradiol (E2) in SaOS-2 cells. Insulin-like growth factor binding protein 6 (IGFBP-6), as a crucial regulator in IGF system, plays an important role in osteogenesis and bone function. TCDD (15 m mg/kg) induced a dramatic 3-fold increase in IGFBP-6 mRNA expression in rat fetal calvaria on GD 21. On the other hand, the concurrent treatment of 10 nM TCDD and 1 m mM E2 resulted in a significant increase in IGFBP-6 mRNA and protein after 24 h in SaOS-2 cells, but TCDD and (or) E2 had no effect on the mRNA level of cytosolic aromatic hydrocarbon receptor. The functional estrogen-responsive element (ERE) [5-CCT TCA CCT G-3] (؊9 to ؉1) in the IGFBP-6 promoter region was identified in this study for the first time as the ER genomic binding site. Collectively, these results suggest that TCDD can alter the expression of IGFBP-6 gene and exerts growth-inhibitory effects on osteogenesis. In addition, TCDD exhibits an anti-estrogenic effect through its interference with the binding of activated estrogen-liganded ER to the functional ERE in IGFBP-6 gene promoter.

Transcriptional activation of insulin-like growth factor binding protein 6 by 17β-estradiol in SaOS-2 cells

“…However, the molecular mechanism of estrogen signaling through ER in IGFBP-6 transcription has not been determined. In order to understand the mechanisms of estrogen in IGF signaling, we previously investigated estrogen regulation of IGFBP-6 gene in vivo (Guo et al, 2003(Guo et al, , 2007. Herein, we present evidence that the human osteoblastic-like cell line SaOS-2 expresses IGFBP-6 and that estrogen increases the abundance of both IGFBP-6 mRNA and protein in the presence of ER.…”

“…Transfection with either ERα or ERβ causes U2OS cell sensitivity to E2 (Lima et al, 2004;Kallio et al, 2008). We previously reported that E2 (1 μM) does not positively regulate cell proliferation in MC-3T3-E1 cells (Guo et al, 2007). However, herein, SaOS-2 cells were transfected with the wild-type human ERα expression vector and E2 up-regulated cell proliferation 2 fold in SaOS-2 cells, but in dose independent manner within the range of concentration of 0.01 to 1 μM.…”

“…Estradiol differentially regulates production of IGFBP-3 and IGFBP-6 in the MCF-7 cell line (Guo et al, 2007;Martin et al, 1995). E2 (10 -8 ∼10 -10 M) has similar ability to stimulate the induction of collagenase-3 mRNA in UMR 106-01 cells (Partridge et al, 2000).…”

“…After 16 h, the cells were grown in the presence of E2 (1 μM, 0.1 μM, 0.01 μM）for 24 h before harvesting (Cesario et al, 2007, Guo et al, 2007. E2 was dissolved in 1 μM DMSO.…”

“…Nuclear extracts from SaOS-2 cells were prepared using the method of Guo et al (2007). The concentrations of nuclear extracts were determined according to the Bradford method (Bradford et al, 1976).…”