Regulation of adipocyte differentiation by activation of serotonin (5-HT) receptors 5-HT2AR and 5-HT2CR and involvement of microRNA-448-mediated repression of KLF5.
Retrovirus insertion-mediated random mutagenesis was applied in 3T3-L1 preadipocyte cells to better understand the molecular basis of obesity (the expansion of individual adipocytes). We found that tryptophan hydroxylase-1, a rate-limiting enzyme for the synthesis of serotonin (5-HT), is expressed in adipocytes and is required for their differentiation. A 5-HT type 2A receptor (5-HT(2A)R) antagonist, ketanserin, and a 5-HT(2c)R antagonist, SB-242084, inhibited adipocyte differentiation. Because 5-HT(2c)R mRNA levels are up-regulated during adipocyte differentiation and micro-RNA (miR)-448 is located in the fourth intron of Htr2c, we also studied the role of miR-448 in 3T3-L1 cells. Through a bioinformatics approach, Krüppel-like factor 5 (KLF5) was identified as a potential target of miR-448. Using a luciferase reporter assay, we confirmed that miR-448 targets the Klf5 3'-intranslated region. Overexpression of miR-448 reduced the expression of Klf5 and adipocyte differentiation, which was confirmed by the reduced expression of adipogenic genes and triglyceride accumulation. To examine the loss of miR-448 function, we constructed a decoy gene that had tandem complementary sequences for miR-448 in the 3'-untranslated region of a luciferase gene under the control of a cytomegalovirus promoter. When the miR-448 decoy gene was introduced into 3T3-L1 preadipocytes, KLF5 was up-regulated and triglyceride concentration was increased. In this study, we identified the regulation of adipocyte differentiation by 5-HT, 5-HT(2A)R, and 5-HT(2C)R. miR-448-mediated repression of KLF5 was identified as a negative regulator for adipocyte differentiation.
Kinoshita
Minako
M
Department of Cardiovascular Medicine, Graduate School of Medicine, Kyoto University, Sakyo-ku, Kyoto, Japan.
Ono
Koh
K
Horie
Takahiro
T
Nagao
Kazuya
K
Nishi
Hitoo
H
Kuwabara
Yasuhide
Y
Takanabe-Mori
Rieko
R
Hasegawa
Koji
K
Kita
Toru
T
Kimura
Takeshi
T
D041721:3T3-L1 Cells / Q000378:metabolism; D017667:Adipocytes / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D000631:Aminopyridines / Q000494:pharmacology; D000818:Animals; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology*; D007211:Indoles / Q000494:pharmacology; D007527:Isoenzymes / Q000378:metabolism; D007650:Ketanserin / Q000494:pharmacology; D051741:Kruppel-Like Transcription Factors / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D035683:MicroRNAs / Q000235:genetics / Q000378:metabolism*; D044402:Receptor, Serotonin, 5-HT2A / Q000235:genetics / Q000378:metabolism*; D044405:Receptor, Serotonin, 5-HT2C / Q000235:genetics / Q000378:metabolism*; D012701:Serotonin / Q000378:metabolism; D012702:Serotonin Antagonists / Q000494:pharmacology; D014365:Tryptophan Hydroxylase / Q000378:metabolism
C107820:6-chloro-5-methyl-1-((2-(2-methylpyrid-3-yloxy)pyrid-5-yl)carbamoyl)indoline; D000631:Aminopyridines; D007211:Indoles; D007527:Isoenzymes; C118391:Klf5 protein, mouse; D051741:Kruppel-Like Transcription Factors; C000610584:MIRN448 microRNA, mouse; D035683:MicroRNAs; D044402:Receptor, Serotonin, 5-HT2A; D044405:Receptor, Serotonin, 5-HT2C; D012702:Serotonin Antagonists; D012701:Serotonin; D007650:Ketanserin; D014365:Tryptophan Hydroxylase