Prostaglandin E2 Stimulates Bone Sialoprotein (BSP) Expression through cAMP and Fibroblast Growth Factor 2 Response Elements in the Proximal Promoter of the Rat BSP Gene

Abstract

Bone sialoprotein (BSP), an early marker of osteoblast differentiation, has been implicated in the nucleation of hydroxyapatite during de novo bone formation. Prostaglandin E 2 (PGE 2 ) has anabolic effects on proliferation and differentiation of osteoblasts via diverse signal transduction systems. Because PGE 2 increases the proportion of functional osteoblasts in fetal rat calvarial cell cultures, we investigated the regulation of BSP, as an osteoblastic marker, by PGE 2 . Treatment of rat osteosarcoma UMR 106 cells with 3 M, 300 nM, and 30 nM PGE 2 increased the steady state levels of BSP mRNA about 2.7-, 2.5-, and 2.4-fold after 12 h. From transient transfection assays, the constructs including the promoter sequence of nucleotides (nt) ؊116 to ؉60 (pLUC3) were found to enhance transcriptional activity 3.8-and 2.2-fold treated with 3 M and 30 nM PGE 2 for 12 h. 2-bp mutations were made in an inverted CCAAT box (between nt ؊50 and ؊46), a cAMP response element (CRE; between nt ؊75 and ؊68), a fibroblast growth factor 2 response element (FRE; nt ؊92 to ؊85), and a pituitaryspecific transcription factor-1 motif (between nt ؊111 and ؊105) within pLUC3 and pLUC7 constructs. Transcriptional stimulation by PGE 2 was almost completed abrogated in constructs that included 2-bp mutations in either the CRE and FRE. In gel shift analyses an increased binding of nuclear extract components to double-stranded oligonucleotide probes containing CRE and FRE was observed following treatment with PGE 2 . These studies show that PGE 2 induces BSP transcription in UMR 106 cells through juxtaposed CRE and FRE elements in the proximal promoter of the BSP gene.Prostaglandins are considered important local factors that modulate bone metabolism through their effects on osteoblastic cells and osteoclasts (1, 2). Prostaglandin E 2 (PGE 2 ), 1 a major eicosanoid produced by osteoblasts, is a potent stimulator of bone resorption (3) that can stimulate the formation of osteoclast-like multinuclear cells in mouse bone marrow cultures (4, 5). The effects of PGE 2 on osteoclastogenesis are, at least in part, mediated by osteogenic cells, which express macrophage colony-stimulating factor (6) and receptor activator of nuclear factor B ligand (RANKL) (7) that promote, and osteoprotegerin, a decoy receptor for RANKL (8), that suppresses osteoclast formation. PGE 2 has been shown to stimulate RANKL and inhibit osteoprotegerin production (7, 9) and also increases production of interleukin-6, which can further enhance osteoclastogenesis (10 -12). In contrast, studies have revealed that PGE 2 also has bone-forming activity (2, 13). Treatment of male, female, and overiectomized mice with PGE 2 increases bone mass in vivo (14), whereas PGE 2 stimulates collagen and DNA synthesis and induces bone growth in calvarial organ (15) and cell cultures in vitro (16, 17). However, PGE 2 can either stimulate or inhibit cellular growth and differentiation of osteoblastic cells depending on PGE 2 concentration (15,18,19).To explain the diverse effects of PGE 2 , th...

Four and Half Lim Protein 2 (FHL2) Stimulates Osteoblast Differentiation

“…In nuclei, FHL2 is known to function as a co-activator for several transcription factors including CREB, AP-1, androgen receptor, and ␤-catenin. (16)(17)(18)(19)(20)(21)(22) Because these transcription factors and integrins play critical roles in osteoblast function and bone formation, (2,6,(46)(47)(48)(49)(50)(51) the enhancement in activities of these transcription factors and integrins by FHL2 may explain, at least in part, the osteogenic effect of FHL2. The stimulation of osteoblast specific transcription factor Osx lends additional support to the osteogenic effect of FHL2.…”

Melatonin regulates human bone sialoprotein gene transcription

“…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). PI3K/Akt is one of the critical pathways for differentiation of skeletal component cells, such as chondrocytes, osteoblasts, myoblasts and adipocytes (52).…”

“…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.…”

Transcriptional regulation of the human bone sialoprotein gene by fibroblast growth factor 2

“…FGF2 increased BSP transcription via FGF2 response element (FRE) in the proximal promoter of the rat BSP gene, which regulates both basal and FGF2-induced BSP transcription (10). We have also reported that the activator protein 1 (AP1) element is another target of FGF2 in the context of BSP transcription (11), and that in addition prostaglandin E 2 induces BSP gene expression through a cAMP response element (CRE) and FRE in the proximal promoter of the rat BSP gene (12).…”

“…These promoters include an inverted TATA box (-24 to -19) (27) and an inverted CCAAT box (-50 to -46) (28,29). Furthermore, a CRE (-75 to -68) (12,30), a FRE (-92 to -85) (10,12,31,32), a pituitary specific transcription factor-1 (Pit-1) motif (-111 to -105) (33,34), a homeodomain protein binding site (HOX; -199 to -192) (35,36), a transforming growth factor beta (TGF-β) activation element (-499 to -485) (35,37) and a glucocorticoid response element (GRE; -920 to -906) overlapping with AP1 (-921 to -915) have been characterized (14,38).…”