Regulation of bone sialoprotein (BSP) gene transcription by lipopolysaccharide.
Lipopolysaccharide (LPS) is a major mediator of inflammatory responses in periodontal disease that inhibits bone formation and stimulates bone resorption. To determine the molecular mechanisms involved in the suppression of bone formation, we have analyzed the effects of LPS on BSP gene expression. Bone sialoprotein (BSP) is a mineralized tissue-specific protein that appears to function in the initial mineralization of bone. Treatment of osteoblast-like ROS 17/2.8 cells with LPS (1 microg/ml) for 12 h caused a marked reduction in BSP mRNA levels. The addition of antioxidant N-acetylcysteine (NAC; 20 mM) 30 min prior to stimulation with LPS attenuated the inhibition of BSP mRNA levels. Transient transfection analyses, using chimeric constructs of the rat BSP gene promoter linked to a luciferase reporter gene, revealed that LPS (1 microg/ml) suppressed expression of luciferase construct, encompassing BSP promoter nucleotides -108 to +60, transfected into ROS17/2.8 cells. The effects of LPS were inhibited by protein kinase A (PKA) inhibitor, H89 and the tyrosine kinase inhibitor, herbimycin A (HA). Introduction of 2 bp mutations in the inverted CCAAT box (ATTGG; nts -50 and -46), a cAMP response element (CRE; nts -75 to -68), a FGF response element (FRE; nts -92 to -85), and a pituitary specific transcription factor binding element (Pit-1; nts -111 to -105) showed that the LPS effects were mediated by the CRE and FRE. Whereas the FRE and 3'-FRE DNA-protein complexes were decreased by LPS, CRE DNA-protein complex did not change after LPS treatment. These studies, therefore, show that LPS suppresses BSP gene transcription through PKA and tyrosine kinase-dependent pathways and that the LPS effects are mediated through CRE and FRE elements in the proximal BSP gene promoter.
Kato
Naoko
N
Department of Periodontology, Nihon University School of Dentistry at Matsudo, Chiba, 271-8587, Japan.
Nakayama
Youhei
Y
Nakajima
Yu
Y
Samoto
Hiroshi
H
Saito
Ryoichiro
R
Yamanouchi
Fumihiko
F
Masunaga
Hiroshi
H
Shimizu
Emi
E
Ogata
Yorimasa
Y
D000818:Animals; D000975:Antioxidants / Q000494:pharmacology; D001483:Base Sequence; D002460:Cell Line; D024202:Electrophoretic Mobility Shift Assay; D058574:Integrin-Binding Sialoprotein; D008070:Lipopolysaccharides / Q000494:pharmacology*; D008969:Molecular Sequence Data; D010012:Osteogenesis / Q000187:drug effects*; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D050436:Regulatory Elements, Transcriptional; D012795:Sialoglycoproteins / Q000235:genetics* / Q000378:metabolism*; D013489:Suppression, Genetic / Q000187:drug effects; D014158:Transcription, Genetic; D014162:Transfection
D000975:Antioxidants; C546873:Ibsp protein, rat; D058574:Integrin-Binding Sialoprotein; D008070:Lipopolysaccharides; D012333:RNA, Messenger; D012795:Sialoglycoproteins