Differentiation of HL-60 cells to granulocytes involves regulation of select diacylglycerol kinases (DGKs)

✍ Scribed by Eraldo L. Batista Jr.; Martha Warbington; John A. Badwey; Thomas E. Van Dyke

• Publisher: John Wiley and Sons
• Year: 2005
• Tongue: English
• Weight: 614 KB
• Volume: 94
• Category: Article
• ISSN: 0730-2312
• DOI: 10.1002/jcb.20356

✦ Synopsis

Abstract

Diacylglycerol Kinases (DGKs) are a family of enzymes that regulate the levels of different pools of diacylglycerol (DAG), affecting DAG‐mediated signal transduction. Since DAG is known to play several important regulatory roles in granulocyte physiology, we investigated the expression pattern of DGK isoforms throughout differentiation of HL‐60 cells to granulocytes. HL‐60 cells were incubated with 1.25% dimethyl‐sulfoxide (DMSO) to initiate differentiation and total RNA isolated at different time points. DGK expression was assessed through Northern blot, end‐point PCR, and real‐time PCR. The non‐selective inhibitors R59022 and R59949 were used to block DGK at different time points throughout differentiation. CD11b and GPI‐80, reactive oxygen species (ROS) generation, changes in the cell cycle, and apoptosis were used as markers of differentiation. Of the nine isoforms of DGK evaluated (α, δ, ε, γ, ζ, β, θ, ι, η), only five (α, δ, ε, γ, and ζ) were expressed in HL‐60 cells. DGKα was virtually absent in non‐differentiated cells, but was markedly upregulated throughout differentiation. The other isoforms (δ, ε, γ, and ζ) were expressed in undifferentiated HL‐60 cells but were substantially decreased throughout differentiation. Non‐selective blocking of DGK with R59022 and R59949 led to acceleration of differentiation, reducing the time necessary to observe upregulation of CD11b, GPI‐80 and generation of ROS by 50%. Likewise, the cell cycle was disrupted when DGK isoforms were inhibited. These results provide evidence that DGK levels are dynamically regulated throughout differentiation and that expression of DGKs play an important regulatory function during the differentiation of neutrophils. © 2004 Wiley‐Liss, Inc.