Insulin‐like growth factor‐I increases bone sialoprotein (BSP) expression through fibroblast growth factor‐2 response element and homeodomain protein‐binding site in the proximal promoter of the BSP gene

Abstract

Insulin-like growth factor-I (IGF-I) promotes bone formation by stimulating proliferation and differentiation of osteoblasts. Bone sialoprotein (BSP), is thought to function in the initial mineralization of bone, is selectively expressed by differentiated osteoblast. To determine the molecular mechanism of IGF-I regulation of osteogenesis, we analyzed the effects of IGF-I on the expression of BSP in osteoblast-like Saos2 and in rat stromal bone marrow (RBMC-D8) cells. IGF-I (50 ng/ml) increased BSP mRNA levels at 12 h in Saos2 cells. In RBMC-D8 cells, IGF-I increased BSP mRNA levels at 3 h. From transient transfection assays, a twofold increase in transcription by IGF-I was observed at 12 h in pLUC3 construct that included the promoter sequence from -116 to +60. Effect of IGF-I was abrogated by 2-bp mutations in either the FGF2 response element (FRE) or homeodomain protein-binding site (HOX). Gel shift analyses showed that IGF-I increased binding of nuclear proteins to the FRE and HOX elements. Notably, the HOX-protein complex was supershifted by Smad1 antibody, while the FRE-protein complex was shifted by Smad1 and Cbfa1 antibodies. Dlx2 and Dlx5 antibodies disrupted the formation of the FRE- and HOX-protein complexes. The IGF-I effects on the formation of FRE-protein complexes were abolished by tyrosine kinase inhibitor herbimycin A (HA), PI3-kinase/Akt inhibitor LY249002, and MAP kinase kinase inhibitor U0126, while IGF-I effects on HOX-protein complexes were abolished by HA and LY249002. These studies demonstrate that IGF-I stimulates BSP transcription by targeting the FRE and HOX elements in the proximal promoter of BSP gene.

DLX3 regulates bone mass by targeting genes supporting osteoblast differentiation and mineral homeostasis in vivo

“…Among the genes differentially expressed in Dlx3 OCN-cKO metaphysis, DLX5 has been shown to regulate and bind to the promoter regions of Runx2, 10 Hox family members, 28 Sp7, 38 Alpl 39 and Ibsp. 40,41 These data together with our ChIP results demonstrating DLX3 binding to some of these genes lead us to speculate that DLX3 and DLX5 may have a coordinated role in the transcription of bone-related genes via molecular switches at their promoter regions.…”

Melatonin regulates human bone sialoprotein gene transcription

“…Activation of PKA may induce CREB1 phosphorylation, and phospho-CREB1 can bind firmly to CRE, thereby inducing gene transcription (51). HA, U0126 and LY294002 have been shown to inhibit the increased binding activities of transcription factors to the FRE and HOX elements in the rat BSP gene promoter (35). In our previous study, we showed that FGF2 stimulated BSP gene transcription via the tyrosine kinase and ERK1/2 pathways (33), PTH activated cAMP and phospholipase C through the PKA and tyrosine kinase pathways (31), and PGE2 increased BSP transcription via PKA, tyrosine kinase and ERK1/2, which target nuclear proteins binding to CRE and FRE in the rat BSP gene promoter (34).…”

“…In the human BSP gene promoter, an inverted TATA box (nts -28~-23) (27) and an inverted CCAAT box (nts-54~-50) maintain basal transcription (29,30), and two cAMP response elements (CRE1; -79~-72, CRE2; -674~-667) are also present (31,32). Additionally, a fibroblast growth factor 2 (FGF2) response element (FRE; nts -96~-89) (33,34), three activating protein 1 (AP1) response elements (AP1(1); -148~-142, AP1(2); -483~-477 and AP1(3); -797~-791) (31,32), and a homeobox binding site (HOX; -200~-191) (35,36) have been characterized.…”

Phosphoinositide 3-Kinase (PI3K) Activation is Differentially Regulated during Osteogenesis induced by TGF-β1 and BMP-2/BMP-7

“…Indeed, IGFs stimulate in vitro and in vivo osteoblast proliferation and differentiation through specific membrane receptors [18][19][20][21] .…”