Cloning and analysis of rat osteoclast inhibitory lectin gene promoter.
Osteoclast inhibitory lectin (OCIL) is a novel regulator of bone remodeling, however, little is known concerning how OCIL is regulated to date. In this study, approximately 4.4 kb of the 5'-flanking sequence of rat OCIL gene was cloned into the promoter-less reporter vector pGL3-basic and transiently transfected into three different cell lines. The differences in the levels of luciferase activity paralleled well with the levels of OCIL mRNA expression in these cells, suggesting that the regulation of rat OCIL gene expression occurs mainly at the transcriptional level. Additional luciferase assays using a series of constructs containing unidirectionally deleted fragments showed that the construct-1819/pGL3 (-1819 to +118) exhibited the highest luciferase activity, suggesting the presence of functional promoter in this region. The region from -4370 to -2805 might contain negative regulatory elements, while the region from -1819 to -1336 might have important positive regulatory elements that enhance OCIL transcription. Sequence analysis of the promoter revealed the absence of both TATA and CAAT boxes. However, in the proximal promoter region (-81 to +118), several potential transcription factor binding sites that may be responsible for the basal transcriptional activity of rat OCIL promoter were observed. The promoter contains several potential Sp1 binding sites, and cotransfection of a shRNA expression plasmid that knockdowns Sp1 significantly reduced OCIL promoter activity and endogenous gene expression and moreover, overexpressing Sp7, a Sp1 family member that also binds to Sp1 binding sequence, increased OCIL promoter activity and gene expression, suggesting a role of Sp1 family proteins in regulation of OCIL transcription.
Quan
Jin-Xing
JX
Key Lab of Ministry of Health for Hormone and Development, Institute of Endocrinology, Tianjin Medical University, Tianjin, PR China.
Zheng
Fang
F
Li
Xiao-Xia
XX
Hu
Li-Ling
LL
Sun
Zi-Yang
ZY
Jiao
Yan-Li
YL
Wang
Bao-Li
BL
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular / Q000379:methods; D005786:Gene Expression Regulation; D037181:Lectins, C-Type / Q000235:genetics*; D008156:Luciferases / Q000235:genetics; D008969:Molecular Sequence Data; D010010:Osteoclasts / Q000737:chemistry*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D051381:Rats; D050436:Regulatory Elements, Transcriptional; D016329:Sp1 Transcription Factor / Q000502:physiology*; D014157:Transcription Factors; D014158:Transcription, Genetic
D037181:Lectins, C-Type; D008156:Luciferases; D016329:Sp1 Transcription Factor; D014157:Transcription Factors; C541100:Clec2d protein, rat