Identification of a functional peroxisome proliferator-activated receptor responsive element within the murine perilipin gene.
Perilipin, a family of phosphoproteins located around lipid droplets in adipocytes, is essential for enlargement of lipid droplets and lipolytic reaction by hormone-sensitive lipase. Thiazolidinediones, peroxisome proliferator-activated receptor (PPAR) gamma agonists, have been shown to increase perilipin expression in fully differentiated adipocytes. However, the precise mechanism of transcriptional regulation of murine perilipin gene heretofore remains unclear. We determined the transcription start site of murine perilipin gene by RNA ligase-mediated rapid amplification of the cDNA ends method. We generated luciferase reporter gene constructs containing various lengths of the 5'-flanking region of the murine perilipin gene and assayed promoter/enhancer activities using differentiated 3T3-L1 adipocytes. We identified a functional PPAR-responsive element (PPRE) in the murine perilipin promoter, and this was confirmed by gel EMSAs using nuclear extracts from differentiated 3T3-L1 adipocytes. Furthermore, point mutations of the identified functional PPRE markedly reduced both the reporter gene activity in differentiated 3T3-L1 adipocytes and PPARgamma/thiazolidinedione-induced transactivation in NIH-3T3 fibroblasts. Real-time RT-PCR revealed that thiazolidinedione up-regulates endogenous perilipin mRNA levels. We propose that PPARgamma plays a significant role in the transcriptional regulation of murine perilipin gene via the PPRE in its promoter.
Nagai
So
S
Department of Medicine II, Hokkaido University Graduate School of Medicine, Sapporo 060-8638, Japan.
Shimizu
Chikara
C
Umetsu
Masaaki
M
Taniguchi
Satoshi
S
Endo
Mikiko
M
Miyoshi
Hideaki
H
Yoshioka
Narihito
N
Kubo
Mitsumasa
M
Koike
Takao
T
D017667:Adipocytes / Q000737:chemistry; D000818:Animals; D001483:Base Sequence; D002352:Carrier Proteins; D002454:Cell Differentiation; D004252:DNA Mutational Analysis; D018076:DNA, Complementary / Q000737:chemistry; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D008156:Luciferases / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D041681:NIH 3T3 Cells; D000070778:Perilipin-1; D010750:Phosphoproteins / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics; D012333:RNA, Messenger; D018160:Receptors, Cytoplasmic and Nuclear / Q000819:agonists / Q000037:antagonists & inhibitors; D012045:Regulatory Sequences, Nucleic Acid; D020218:Response Elements / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D014157:Transcription Factors / Q000819:agonists / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D024363:Transcription Initiation Site; D014162:Transfection
D002352:Carrier Proteins; D018076:DNA, Complementary; D000070778:Perilipin-1; D010750:Phosphoproteins; D012333:RNA, Messenger; D018160:Receptors, Cytoplasmic and Nuclear; D014157:Transcription Factors; D008156:Luciferases