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

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 A(2) (PLA(2)s) 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(2)s. 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, iPLA(2)beta and iPLA(2)gamma, respectively. In contrast, the temporally coordinated loss of at least 90% of cPLA(2)alpha mRNA was manifest. Western analysis demonstrated the near absence of both iPLA(2)beta and iPLA(2)gamma protein mass in resting 3T3-L1 cells that increased dramatically during differentiation. In vitro measurement of PLA(2) activities demonstrated an increase in both iPLA(2)beta and iPLA(2)gamma 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 iPLA(2)beta or iPLA(2)gamma prevented hormone-induced differentiation. Moreover, analysis of the temporally programmed expression of transcription factors demonstrated that the small interfering RNA knockdown of iPLA(2)beta or iPLA(2)gamma resulted in down-regulation of the expression of peroxisome proliferator-activated receptor gamma and the CCAAT enhancer-binding protein alpha (C/EBPalpha). No alterations in the expression of the early stage transcription factors C/EBPbeta and C/EBPdelta were observed. Collectively, these results demonstrate prominent alterations in intracellular PLA(2)s during 3T3-L1 cell differentiation into adipocytes and identify the requirement of iPLA(2)beta and iPLA(2)gamma for the adipogenic program that drives resting 3T3-L1 cells into adipocytes after hormone stimulation.