A novel immunomodulatory function of PHLPP1: inhibition of iNOS via attenuation of STAT1 ser727 phosphorylation in mouse macrophages.
PHLPP1 is a novel tumor suppressor, but its role in the regulation of innate immune responses, which are frequently dysregulated in cancer, is unexplored. Here, we report that LPS attenuated PHLPP1 expression at mRNA and protein levels in immune cells, suggesting its involvement in immune responses. To test this, we overexpressed PHLPP1 in RAW 264.7 macrophages and observed a dramatic reduction in LPS/IFN-γ-induced iNOS expression. Conversely, silencing of PHLPP1 by siRNA or by shRNA robustly augmented LPS/IFN-γ-induced iNOS expression. qPCR and iNOS promoter reporter experiments showed that PHLPP1 inhibited iNOS transcription. Mechanistic analysis revealed that PHLPP1 suppressed LPS/IFN-γ-induced phosphorylation of ser727STAT1; however, the underlying mechanisms differed. PHLPP1 reduced IFN-γ-stimulated but not LPS-induced ERK1/2 phosphorylation, and inhibition of ERK1/2 abolished IFN-γ-induced ser727STAT1 phosphorylation and iNOS expression. In contrast, PHLPP1 knockdown augmented LPS-induced but not IFN-γ-elicited p38 phosphorylation. Blockade of p38 abolished LPS-stimulated phosphorylation of ser727STAT1 and iNOS expression. Furthermore, PHLPP1 suppressed LPS-induced phosphorylation of tyr701STAT1 by dampening p38-dependent IFN-β feedback. Collectively, our data demonstrate for the first time that PHLPP1 plays a vital role in restricting innate immune responses of macrophages, and further studies may show it to be a potential therapeutic target within the context of dysregulated macrophage activity.
Dr. Reddy's Institute of Life Sciences (DRILS), Hyderabad, India.
Neeraja P
NP
Alamuru
Dr. Reddy's Institute of Life Sciences (DRILS), Hyderabad, India.
Soma
S
Behera
Department of Biotechnology, University of Hyderabad, Hyderabad, India.
Jonathan P
JP
Butchar
Department of Biotechnology, University of Hyderabad, Hyderabad, India.
Susheela
S
Tridandapani
Department of Internal Medicine, The Ohio State University, Columbus, Ohio, USA.
Sasidhara
S
Kaimal Suraj
Department of Internal Medicine, The Ohio State University, Columbus, Ohio, USA.
P Prakash
PP
Babu
Department of Biological Sciences, Indian Institute of Technology, New Delhi, India; and.
Seyed E
SE
Hasnain
National Institute of Pathology, Safdarjung Hospital, New Delhi, India.
Nasreen Z
NZ
Ehtesham
Dr. Reddy's Institute of Life Sciences (DRILS), Hyderabad, India; kishorep@drils.org.
Kishore V L
KV
Parsa
D000818:Animals; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects / Q000235:genetics / Q000276:immunology; D056747:Immunomodulation; D007371:Interferon-gamma; D008070:Lipopolysaccharides / Q000494:pharmacology; D020935:MAP Kinase Signaling System / Q000187:drug effects / Q000235:genetics / Q000276:immunology*; D008264:Macrophages / Q000276:immunology*; D051379:Mice; D048052:Mitogen-Activated Protein Kinase 3 / Q000235:genetics / Q000276:immunology; D052247:Nitric Oxide Synthase Type II / Q000235:genetics / Q000276:immunology*; D009687:Nuclear Proteins / Q000235:genetics / Q000276:immunology*; D010749:Phosphoprotein Phosphatases / Q000235:genetics / Q000276:immunology*; D010766:Phosphorylation / Q000187:drug effects / Q000235:genetics / Q000276:immunology; D000067996:RAW 264.7 Cells; D050794:STAT1 Transcription Factor / Q000235:genetics / Q000276:immunology*; D012694:Serine / Q000276:immunology
C000611649:IFNG protein, mouse; D008070:Lipopolysaccharides; D009687:Nuclear Proteins; D050794:STAT1 Transcription Factor; C494080:Stat1 protein, mouse; D012694:Serine; D007371:Interferon-gamma; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse; D048052:Mitogen-Activated Protein Kinase 3; C519282:PHLPP1 protein, mouse; D010749:Phosphoprotein Phosphatases
ERK1/2; IFN-γ; LPS; immune responses; p38 MAPK