Diacylglycerol Kinase ζ Produces 16:0‐containing Phosphatidic Acid Molecular Species during Neuroblastoma Cell Differentiation

Abstract

Phosphatidic acid (PA) is one of the phospholipids composing the plasma membrane and acts as a second messenger to regulate a wide variety of cellular functions, including cell growth, migration and differentiation. PA consists of various molecular species with different acyl chains at the sn‐1 and sn‐2 positions. However, it was unclear which PA molecular species were produced during cell stimulation. In this study, we identified the PA molecular species produced during neuroblastoma cell differentiation and the PA species production pathway using our previously developed liquid chromatography (LC)/mass spectrometry (MS) method (1).First, mouse neuroblastoma Neuro‐2a cells were differentiated by treatment with 20 μM retinoic acid (RA) for 24 hours. LC/MS analysis showed that the amount of total PA was significantly increased in RA treated Neuro‐2a cells (1.69‐fold). Notably, the amount of 30:0‐ and 32:0‐PA species were dramatically increased in RA treated Neuro‐2a cells (2.13‐, 4.67‐ and 1.85‐fold, respectively). Similar results were obtained when Neuro‐2a cells were treated with serum starvation for 24 hours. MS/MS analysis revealed that these PA species commonly contain palmitic acid (16:0). Next, we examined the production pathway of 30:0‐ and 32:0‐PA. FIPI, phospholipase D‐specific inhibitor, did not inhibit the production of these PA species. RT‐PCR analysis showed that diacylglycerol kinase (DGK) δ and DGKζ were highly expressed in Neuro‐2a cells. The silencing of DGKδ expression did not decrease the production of the PA species. However, DGKζ‐specific siRNA significantly decreased the production of 30:0‐ and 32:0‐PA. In addition, DGKζ‐specific siRNA substantially attenuated neurite outgrowth/differentiation. These results suggest that DGKζ produces 16:0‐containing 30:0‐ and 32:0‐PA species and regulates neurite outgrowth during neuroblastoma cell differentiation.Support or Funding Information