Abstract
Lipid phosphate phosphatase 3 (LPP3), encoded by the PLPP3 gene, is an integral membrane enzyme that dephosphorylates phosphate esters of glycero- and sphingophospholipids. Cell surface LPP3 can terminate the signaling actions of bioactive lysophosphatidic acid (LPA) and sphingosine 1 phosphate, which likely explains its role in developmental angiogenesis, vascular injury responses, and cell migration. Heritable variants in the final intron PLPP3 associate with interindividual variability in coronary artery disease risk that may result from disruption of enhancer sequences that normally act in cis to increase expression of the gene. However, the mechanisms regulating PLPP3 expression are not well understood. We show that the human PLPP3 promoter contains three functional NF-κB response elements. All of these are required for maximal induction of PLPP3 promoter activity in reporter assays. The identified sequences recruit RelA and RelB components of the NF-κB transcription complex to chromatin, and these transcription factors bind to the identified target sequences in two different cell types. LPA promotes binding of Rel family transcription factors to the PLPP3 promoter and increases PLPP3 gene expression through mechanisms that are attenuated by an NF-κB inhibitor, LPA receptor antagonists, and inhibitors of phosphoinositide 3 kinase. These findings indicate that up-regulation of PLPP3 during inflammation and atherosclerosis results from canonical activation of the NF-κB signaling cascade to increase PLPP3 expression through nuclear import and binding of RelA and RelB transcription factors to the PLPP3 promoter and suggest a mechanism by which the LPP3 substrate, LPA, can regulate PLPP3 expression.
Highlights
Lipid phosphate phosphatase 3 (LPP3), encoded by the PLPP3 gene, is an integral membrane enzyme that dephosphorylates phosphate esters of glycero- and sphingophospholipids
Given the strong up-regulation of PLPP3 expression observed by others and ourselves in settings of inflammation, vascular injury, and atherosclerosis, we were interested in the presence of three distinct binding sites for cRel components of the NF-B transcription factor complex in the human PLPP3 promoter
Studies in mice and humans indicate that PLPP3 expression is tightly regulated, with dramatic increases observed in response to broadly pro-inflammatory signals, both in vitro and in vivo [17, 19]
Summary
We used online databases and data visualization tools (20 – 22), as detailed in the legend of Fig. S1, to examine predicted and experimentally validated functional elements in the vicinity of the human PLPP3 gene transcriptional initiation codon and 5Ј UTR. Overexpression of some of these proteins, p50/p65RelA and p52/RelB, significantly increased transcriptional activity of the PLPP3 promoter In separate experiments, these effects appeared to be more pronounced with the ϳ1.5-kb PLPP3 promoter/enhancer construct (Fig. 1, B and C), and this difference was confirmed in a separate experiment, suggesting that the human PLPP3 promoter–specific Rel-responsive element present in this promoter construct is active. As with HEK293 cells, we observed physical association of all three Rel-responsive element target sequences with RelB in these studies using differentiated THP-1 cells These associations were moderately strengthened by treatment of the cells with the PLPP3 substrate 18:1 LPA, suggesting that NF-B– dependent transcriptional activation of the PLPP3 promoter might be controlled by activation of cell-surface LPA receptors (Fig. 3B). To further demonstrate the functional relevance of these observations and investigate the role of NF-B in increases in PLPP3 expression, we measured LPP activity in detergent-extracted membrane proteins from differentiated THP-1 cells that were stimulated with 18:1 LPA. LPA1/2 receptors can function as activators of this pathway by initiating activation of PI3K-dependent signaling pathways that likely impact the well-described canonical activation of NF-B signaling through dissociation and degradation of the IB inhibitor [28] (Fig. 6)
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