Small interfering RNA knockdown of calcium-independent phospholipases A2 β or γ inhibits the hormone-induced differentiation of 3T3-L1 preadipocytes

Xiong Su, David J. Mancuso, Perry E. Bickel, Christopher Jenkins, Richard W. Gross

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Abstract

Alterations in lipid secondary messenger generation and lipid metabolic flux are essential in promoting the differentiation of adipocytes. To determine whether specific subtypes of intracellular phospholipases A2 (PLA2s) facilitate hormone-induced differentiation of 3T3-L1 cells into adipocytes, we examined alterations in the mRNA level, protein mass, and activity of three previously characterized mammalian intracellular PLA 2s. Hormone-induced differentiation of 3T3-L1 cells resulted in 7.3 ± 0.5- and 7.4 ± 1.4-fold increases of mRNA encoding the calcium-independent phospholipases, iPLA2β and iPLA2γ, respectively. In contrast, the temporally coordinated loss of at least 90% of cPLA2α mRNA was manifest. Western analysis demonstrated the near absence of both iPLA2β and iPLA2γ protein mass in resting 3T3-L1 cells that increased dramatically during differentiation. In vitro measurement of PLA2 activities demonstrated an increase in both]PLA2β and iPLA2n γ activities that were discriminated using the chiral mechanism based inhibitors (S)- and (R)-BEL, respectively. Remarkably, treatment of 3T3-L1 cells with small interfering RNA directed against either iPLA2β or iPLA2γ prevented hormone-induced differentiation. Moreover, analysis of the temporally programmed expression of transcription factors demonstrated that the small interfering RNA knockdown of iPLA2β or iPLA2γ resulted in down-regulation of the expression of peroxisome proliferator-activated receptor γ and the CCAAT enhancer-binding protein α (C/EBPα). No alterations in the expression of the early stage transcription factors C/EBPβ and C/EBPδ were observed. Collectively, these results demonstrate prominent alterations in intracellular PLA 2s during 3T3-L1 cell differentiation into adipocytes and identify the requirement of iPLA2β and iPLA2γ for the adipogenic program that drives resting 3T3-L1 cells into adipocytes after hormone stimulation.

Original languageEnglish (US)
Pages (from-to)21740-21748
Number of pages9
JournalJournal of Biological Chemistry
Volume279
Issue number21
DOIs
Publication statusPublished - May 21 2004

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ASJC Scopus subject areas

  • Biochemistry

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