TY - JOUR
T1 - Small interfering RNA knockdown of calcium-independent phospholipases A2 β or γ inhibits the hormone-induced differentiation of 3T3-L1 preadipocytes
AU - Su, Xiong
AU - Mancuso, David J.
AU - Bickel, Perry E.
AU - Jenkins, Christopher
AU - Gross, Richard W.
PY - 2004/5/21
Y1 - 2004/5/21
N2 - 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.
AB - 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.
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U2 - 10.1074/jbc.M314166200
DO - 10.1074/jbc.M314166200
M3 - Article
C2 - 15024020
AN - SCOPUS:2542431970
SN - 0021-9258
VL - 279
SP - 21740
EP - 21748
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 21
ER -