Glutathione S-transferase P1 suppresses iNOS protein stability in RAW264.7 macrophage-like cells after LPS stimulation

Abstract

Glutathione S-transferase P1 (GSTP1) is a ubiquitous expressed protein which plays an important role in the detoxification and xenobiotics metabolism. Previous studies showed that GSTP1 was upregulated by the LPS stimulation in RAW264.7 macrophage-like cells and GSTP1 overexpression downregulated lipopolysaccharide (LPS) induced inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expression. Here we show that GSTP1 physically associates with the oxygenase domain of iNOS by the G-site domain and decreases the protein level of iNOS dimer. Both overexpression and RNA interference (RNAi) experiments indicate that GSTP1 downregulates iNOS protein level and increases S-nitrosylation and ubiquitination of iNOS. The Y7F mutant type of GSTP1 physically associates with iNOS, but shows no effect on iNOS protein content, iNOS S-nitrosylation, and changes in iNOS from dimer to monomer, suggesting the importance of enzyme activity of GSTP1 in regulating iNOS S-nitrosylation and stability. GSTM1, another member of GSTs shows no significant effect on regulation of iNOS. In conclusion, our study reveals the novel role of GSTP1 in regulation of iNOS by affecting S-nitrosylation, dimerization, and stability, which provides a new insight for analyzing the regulation of iNOS and the anti-inflammatory effects of GSTP1.

Glutathione-S-Transferases as Potential Targets for Modulation of Nitric Oxide-Mediated Vasodilation

“…Subsequent studies examining activated macrophages demonstrated that silencing Mrp1 resulted in an intracellular accumulation of DNICs, while silencing Gstp1 in these cells augmented the release of iron-59 out of the cell (as DNICs) [ 8 ]. Another intriguing GSTP1-NO interaction has been suggested by studies demonstrating the binding of GSTP1 to iNOS [ 5 ]. In this later study, GSTP1 was shown to directly interact with the oxygenase domain of iNOS through the GSTP1 G-site domain [ 5 ].…”

“…While GSTs are traditionally associated with detoxification mechanisms due to their ability to conjugate glutathione (GSH) to toxins for excretion, recent advances have explored the role of GSTs in NO metabolism [ 5 , 6 , 7 , 8 , 9 , 10 ]. Studies investigating the extensive role of NO in vascular reactivity have identified GSTs as targets for the biotransformation of organic nitrates, including nitroglycerin, that results in vasodilation [ 11 , 12 , 13 , 14 ].…”

“…These relationships are mediated by: (1) the formation of DNICs [ 6 , 10 , 21 , 22 , 23 , 24 ]; (2) the direct binding of DNICs by GSTP1 to form a stable store of NO [ 8 , 9 , 15 , 16 ]; (3) the storage of DNICs by GSTP1 that then leads to a decrease in DNIC transport out of the cell by multi-drug resistance-related protein 1 (MRP1) [ 8 , 15 ]; and (4) the direct association of GSTP1 with inducible nitric oxide synthase (iNOS) to increase its degradation [ 5 ] ( Figure 1 ). Overall, GSTP1 acts to bind and store NO, but also inhibits iNOS expression to suppress NO signaling.…”

Overview of RAW264.7 for osteoclastogensis study: Phenotype and stimuli

“…21,22 Later, further studies proved RAW264.7 cells could respond to stimuli in vitro and subsequently generate multinucleated cells with the hallmark characteristics expected for fully differentiated osteoclasts (RAW-OCs). RAW264.7 cell linage is well-characterized with regard to macrophage-mediated immune, metabolic and phagocytic functions 21 and is increasingly used and accepted as a cellular model of osteoclastogenic study; [24][25][26] however, with the vastly usage of RAW-OCs for understanding the osteocalstogenesis in the past two decades, there raised considerable requirements for the extensively understanding the RAW-OCs and associated cellular biological mechanisms. RAW264.7 cell linage is well-characterized with regard to macrophage-mediated immune, metabolic and phagocytic functions 21 and is increasingly used and accepted as a cellular model of osteoclastogenic study; [24][25][26] however, with the vastly usage of RAW-OCs for understanding the osteocalstogenesis in the past two decades, there raised considerable requirements for the extensively understanding the RAW-OCs and associated cellular biological mechanisms.…”

“…23 Therefore, RAW-OCs can be used for studying osteoclastogenesis through different methods for various study purposes, including: biochemical, immunological, physiological, molecular and functional assays according to various study procedures. RAW264.7 cell linage is well-characterized with regard to macrophage-mediated immune, metabolic and phagocytic functions 21 and is increasingly used and accepted as a cellular model of osteoclastogenic study; [24][25][26] however, with the vastly usage of RAW-OCs for understanding the osteocalstogenesis in the past two decades, there raised considerable requirements for the extensively understanding the RAW-OCs and associated cellular biological mechanisms. 27,28 On the other hand, document studies reported that during the osteoclastic induction, various stimuli might lead different cellular fates during the RAW-OC induction.…”