STAT1 Regulates Human Glutaminase 1 Promoter Activity through Multiple Binding Sites in HIV-1 Infected Macrophages

Lixia Zhao,Yunlong Huang,Jialin Zheng

doi:10.1371/journal.pone.0076581

Abstract

Mononuclear phagocytes (MP, macrophages and microglia), the main targets of HIV-1 infection in the brain, play a pathogenic role in HIV-associated neurocognitive disorders (HAND) through the production and release of various soluble neurotoxic factors including glutamate. We have previously reported that glutaminase (GLS), the glutamate-generating enzyme, is upregulated in HIV-1 infected MP and in the brain tissues of HIV dementia individuals, and that HIV-1 or interferon-α (IFN-α) regulates human glutaminase 1 (GLS1) promoter through signal transducer and activator of transcription 1 (STAT1) phosphorylation in macrophages. However, there are multiple putative STAT1 binding sites in human GLS1 promoter, the exact molecular mechanism of how HIV-1 or IFN-α regulates human GLS1 promoter remains unclear. To further study the function of the putative STAT1 binding sites, we mutated the sequence of each binding site to ACTAGTCTC and found that six mutants (mut 1,3,4,5,7,8) had significantly higher promoter activity and two mutants (mut 2 and mut 6) completely lost the promoter activity compared with the wild type. To determine whether sites 2 and 6 could interfere with other inhibitory sites, particularly the nearby inhibitory sites 3 and 5, we made double mutants dmut 2/3 and dmut 5/6, and found that both the double mutants had significantly higher activity than the wild type, indicating that sites 3 and 5 are critical inhibitory elements, while sites 2 and 6 are excitatory elements. ChIP assay verified that STAT1 could bind with sites 2/3 and 5/6 within human GLS1 promoter in IFN-α stimulated or HIV-1-infected monocyte-derived macrophages. Interestingly, we found that rat Gls1 promoter was regulated through a similar way as human GLS1 promoter. Together, our data identified critical elements that regulate GLS1 promoter activity.

Highlights

HIV-associated neurocognitive disorders (HAND), the neurological complications of HIV-1 infection, remain prevalent despite the widening use of combination antiretroviral therapy
We recently found that glutamate, a neurotransmitter that is neurotoxic in high concentrations [8,9], is significantly increased in postmortem brain tissues collected from HIV-1 serum positive patients and HIV-1 associated dementia (HAD) patients [10]
As the first group to clone and characterize the human glutaminase 1 (GLS1) promoter, we found that both IFN-α and HIV-1 infection enhanced signal transducer and activator of transcription 1 (STAT1) binding with the GLS1 promoter and increased GLS1 promoter activity

Summary

Introduction

HIV-associated neurocognitive disorders (HAND), the neurological complications of HIV-1 infection, remain prevalent despite the widening use of combination antiretroviral therapy. Mononuclear phagocytes (MP, macrophages and microglia), the main targets of HIV-1 infection in the brain, play a pathogenic role in HAND through the production and release of various soluble neurotoxic factors including glutamate [5,6,7]. We recently found that glutamate, a neurotransmitter that is neurotoxic in high concentrations [8,9], is significantly increased in postmortem brain tissues collected from HIV-1 serum positive patients and HIV-1 associated dementia (HAD) patients [10]. Our previous data have demonstrated that mitochondrial glutaminase (GLS), the key enzyme that converts glutamine to glutamate in the CNS, is important for glutamate production in PLOS ONE | www.plosone.org gls Promoter Regulation by STAT1

Methods

Results

Conclusion