Thyroid Hormone Receptor ¦Á1 And Insulin-like Growth Factor Binding Protein 6 Interaction Into The Regulation Of Bone Cell Differentiation

Insulin-like growth factor binding protein-6(IGFBP-6) is a member of the insulin-like growth factor binding protein family, which has both IGF-dependent and independent effects on cell growth. Insulin-like growth factor binding protein-6(IGFBP-6) inhibits the tumorigenic properties of IGF-II-dependent cancer cells by directly inhibiting IGF-Ⅱ actions. However, in some cases, IGFBP-6is associated with increased cancer cell tumorigenicity, which is unlikely to be due to IGF-Ⅱ inhibition. The mechanisms underlying the contradictory actions of IGFBP-6remain unclear. In previous studies, a group and us have shown that IGFBP-6could be translocated into the cell nucleus. But the effect in the nucleus of IGFBP-6is not clear. Thyroid hormones (THs) have extensive effects on ontogeny, maintaining homeostasis, cellular proliferation and differentiation. They bind to thyroid hormone receptors (TRs), TRalpha and TRbeta, which belong to the nuclear hormone receptor superfamily. These receptors also bind to enhancer elements in the promoters of target genes, and can regulate both positive and negative transcription. Unliganded thyroid hormone receptor represses transcription through recruitment of a corepressor complex. Ligand binding alters the conformation of the thyroid hormone receptor in such a way as to release the corepressor complex and recruit a coactivator complex. T3-induced differentiation of osteoblasts comprises the sequential induction of cell cycle arrest at G0/G1and the expression of bone matrix proteins. It is disrupt on the effects of IGFBP-6on bone cell growth and osteoblastic function. We now show that IGFBP-6interacts with nuclear thyroid hormone receptor α1(TRα1) and retinoid X receptor a (RXR a), blocking TR a1:RXR a heterodimerization in vitro by GST pull-down assay. TR α1DBD, LBD interacts with IGFBP-6。IGFBP-6C terminal is the main domain in interaction with TR α1. IGFBP-6N terminal takes apart role in the interaction. Deletion of IGFBP-6LXXLL motif did not influence it. TR α1and IGFBP-6were shown to colocalize to the nuclei of HEK-293, MG-63and Hela cells by indirect cell immunocytochemistry and cofocal. IGFBP-6interacts with TR α1in whole cell lysis and nuclear extracts from cotransfected HEK-293cells were shown by co-immunoprecipitation. The FRET results confirmed the interaction between IGFBP-6and TR α1. Induction of growth hormone promoter activity by T3were significantly decreased when IGFBP-6was overexpressed in HEK-293cells which were demonstrated by transcriptional reporter assays. Furthermore, according with the increasing of IGFBP-6plasmid, inhibition increased. Over expression of IGFBP-6reduced the effects of T3in blocking cell cycle progression at G0/G1by FCAS and decreased the expression of cyclin D1, Ki67by real-time qRT-PCR. Alkaline phosphatase activity by T3was significantly decreased when IGFBP-6was overexpressed in U2OS cells. These results demonstrate that IGFBP-6can interact with TR a1to prevent TR a1:RXR a heterodimerization and suggest that IGFBP-6may attenuate the T3-induced expression of bone differentiation markers while IGFBP-6may attenuate the T3-induced expression of bone differentiation markers while having a modest effect on the T3-mediated inhibition of cell cycle progression in bone cells.