Mouse bone marrow macrophages & #THP1 transfection with #DNA using #Magnetofection

PARP9 and PARP14 cross-regulate macrophage activation via STAT1 ADP-ribosylation

Nature Communications,

Oct 2016

Iwata and al.

Abstract

Despite the global impact of macrophage activation in vascular disease, the underlying mechanisms remain obscure. Here we show, with global proteomic analysis of macrophage cell lines treated with either IFNγ or IL-4, that PARP9 and PARP14 regulate macrophage activation. In primary macrophages, PARP9 and PARP14 have opposing roles in macrophage activation. PARP14 silencing induces pro-inflammatory genes and STAT1 phosphorylation in M(IFNγ) cells, whereas it suppresses anti-inflammatory gene expression and STAT6 phosphorylation in M(IL-4) cells. PARP9 silencing suppresses pro-inflammatory genes and STAT1 phosphorylation in M(IFNγ) cells. PARP14 induces ADP-ribosylation of STAT1, which is suppressed by PARP9. Mutations at these ADP-ribosylation sites lead to increased phosphorylation. Network analysis links PARP9–PARP14 with human coronary artery disease. PARP14 deficiency in haematopoietic cells accelerates the development and inflammatory burden of acute and chronic arterial lesions in mice. These findings suggest that PARP9 and PARP14 cross-regulate macrophage activation.

Construction and enforced expression of mutant STAT1

Human pcDNA-GFP-STAT1 was purchased from Addgene (Cambridge, MA, USA). Step-wise mutations (glutamic acid, E, to glutamine, Q) was introduced at the two ribosylation sites flanking the phosphorylation at Tyr701—E657 and E705—by recombinant PCR mutagenesis. Mutated constructs were verified by DNA sequencing. Mouse bone marrow macrophages were differentiated from bone marrow stromal cells using 10 ng ml⁻¹ M-CSF for 12 days. pcDNA-GFP-STAT1 (WT) and the mutant pcDNA-GFP-STAT1 E657Q, E705Q, were transferred by Magnetofection (OzBiosciences, San Diego, CA, USA). HEK293 cells were transfected using Lipofectamine LTX (Invitrogen, USA). Twenty-four hours after transfection, cells were serum-starved (0.1% FBS) for 2 h and stimulated with IFNγ for 1 h (phospho-STAT1) or 24 h (mRNA expression of pro-inflammatory factors). The overexpressed STAT1 was immunoprecipitated using anti-GFP antibody, clone 9F9.F9 (1:1,000, ab1218, Abcam). STAT1 phosphorylation at Tyr 701 was detected by anti-phospho-STAT1 (Tyr701; 1:1,000, mAb #7649, Cell Signaling). Antibodies against STAT1 (ab3987, Abcam) and GFP (ab290, Abcam) served as loading controls. Transfection into THP-1 cells was performed using the magnetofection method described above.