The effect of retinoic acid on a zinc finger transcription factor, AJ18, during differentiation of a rat clonal preosteoblastic cell line, ROB-C20, into osteoblasts.
OBJECTIVE
A zinc finger type transcription factor, AJ18, is thought to be a negative regulator of osteoblast differentiation, but its expression mechanism is not fully understood. Retinoic acid (RA) is a metabolite of vitamin A and involves the proliferation and differentiation in a variety of cells. To verify the effect of RA on osteoblast differentiation, AJ18 expression level was examined using a rat clonal preosteoblastic cell line, ROB-C20 (C20).


METHODS
Confluent C20 cells were treated with or without RA (10(-6)M) for several days. Northern, real time RT-PCR and Western blotting analyses were performed to examine AJ18 expression pattern in gene and protein levels. To identify the active promoter sequence of AJ18 gene, luciferase assay was designed. Furthermore, the effect of overexpressed AJ18 in C20 cells on alkaline phosphatase (ALP) mRNA expression and its activity was compared with that of RA-treated cells.


RESULTS
RA increased the expression of AJ18 mRNA from 2 to 13 days as well as its protein production. However, no significant changes of Runx2 mRNA expression and undetectable osterix mRNA expression were observed in C20 cells treated with or without RA. Luciferase assay showed increases in promoter activities in some constructs of 5'-flanking region of AJ18 gene in RA-treated C20 cells. On the other hand, RA decreases enzymatic activity and mRNA expression level of ALP, but mRNA expression levels of bone sialoprotein and osteocalcin were not altered. Interestingly, reduced ALP activity and its mRNA expression level were detected in exogenous AJ18-overexpressing C20 cells.


CONCLUSIONS
This study supports the hypothesis that RA may restrict to the differentiation of C20 cells into mature osteoblasts via inductive AJ18 expression with activation of multiple signal pathways.
Takahashi
Tomihisa
T
Department of Anatomy, Nihon University School of Dentistry, Tokyo 101-8310, Japan. takahashi-tm@dent.nihon-u.ac.jp
Kamiya
Naoko
N
Kawabata
Niki
N
Takagi
Minoru
M
D020121:5' Untranslated Regions; D000469:Alkaline Phosphatase / Q000032:analysis / Q000235:genetics; D000818:Animals; D015152:Blotting, Northern / Q000379:methods; D015153:Blotting, Western / Q000379:methods; D055415:Bone Morphogenetic Protein 4; D019485:Bone Morphogenetic Proteins / Q000235:genetics; D002454:Cell Differentiation / Q000187:drug effects; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000032:analysis / Q000235:genetics*; D015870:Gene Expression / Q000187:drug effects; D010006:Osteoblasts / Q000166:cytology* / Q000378:metabolism; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D012097:Repressor Proteins / Q000032:analysis / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D013424:Sulfanilamides; D014212:Tretinoin / Q000494:pharmacology*; D015854:Up-Regulation; D013777:Tetrazoles; D013876:Thiophenes; D000096926:Benzenesulfonamides