Regulatory mechanism of human connective tissue growth factor (CTGF/Hcs24) gene expression in a human chondrocytic cell line, HCS-2/8.
CTGF/Hcs24 is a multi-functional growth factor that potentiates either the growth or differentiation of mesenchymal cells, according to the biological conditions. Among various functional aspects of CTGF/Hcs24, it is especially notable that CTGF/Hcs24 may promote endochondral ossification in growth cartilage through all stages, and it is highly expressed in a human chondrosarcoma-derived chondrocytic cell line (HCS-2/8). In this study, to clarify the regulatory mechanism of CTGF/Hcs24 gene expression in chondrocytes, we analyzed the transcriptional activity of the CTGF/Hcs24 promoter and the effect of the CTGF/Hcs24 3'-untranslated region (3'-UTR) on gene expression in HCS-2/8 by means of an established DNA transfection and luciferase reporter gene assay system. As a result, the luciferase activity of the CTGF/Hcs24 promoter was found to be remarkably high in HCS-2/8. The 3'-UTR of the CTGF/Hcs24 gene strongly repressed the luciferase activity in HCS-2/8, when it was linked to the downstream of the luciferase reporter gene, suggesting its functionality also in chondrocytic cells. Deletion analysis of the CTGF/Hcs24 promoter clarified a major segment responsible for the enhanced CTGF/Hcs24 promoter activity in HCS-2/8. The TGF-beta response element in the DNA segment was active in HCS-2/8, and point mutations in the element moderately decreased the highly maintained promoter activity with total loss of TGF-beta responsiveness. These results indicate that the strong expression of the CTGF/Hcs24 gene in HCS-2/8 was mainly caused by high transcriptional activity of the CTGF/Hcs24 promoter, and that the TGF-beta response element is one of the critical elements that support the high transcription activity.
Eguchi
T
T
Department of Biochemistry and Molecular Dentistry, Okayama University Graduate School of Medicine and Dentistry, Okayama 700-8525, Japan.
Kubota
S
S
Kondo
S
S
Shimo
T
T
Hattori
T
T
Nakanishi
T
T
Kuboki
T
T
Yatani
H
H
Takigawa
M
M
D020413:3' Untranslated Regions / Q000235:genetics; D001483:Base Sequence; D002460:Cell Line; D019902:Chondrocytes / Q000378:metabolism*; D055513:Connective Tissue Growth Factor; D004591:Electrophoresis, Polyacrylamide Gel; D005786:Gene Expression Regulation; D006133:Growth Substances / Q000235:genetics* / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D017874:Immediate-Early Proteins / Q000235:genetics* / Q000378:metabolism*; D036341:Intercellular Signaling Peptides and Proteins; D017354:Point Mutation / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism; D020218:Response Elements / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017384:Sequence Deletion; D014158:Transcription, Genetic / Q000235:genetics; D014162:Transfection; D016212:Transforming Growth Factor beta / Q000235:genetics
D020413:3' Untranslated Regions; C526317:CCN2 protein, human; D006133:Growth Substances; D017874:Immediate-Early Proteins; D036341:Intercellular Signaling Peptides and Proteins; D012333:RNA, Messenger; D012097:Repressor Proteins; D016212:Transforming Growth Factor beta; D055513:Connective Tissue Growth Factor