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Proteomic Analysis Uncovers Role For PARP9, PARP14 in Macrophage Activation

NEW YORK (GenomeWeb) – Through proteomic and other analyses, researchers from Brigham and Women's Hospital and Harvard Medical School have highlighted roles for PARP9 and PARP14 in regulating macrophage activation, a key step in the development of a number of diseases, including arterial disease.

Brigham and Women's Masanori Aikawa and his colleagues conducted a proteomic analysis of stimulated macrophage cell lines to zero in on PARP9 and PARP14 as regulators of macrophage activation, which they described in Nature Communications today. In addition, they noted these regulators appeared to have opposing effects: in one cell culture, silencing PARP14 increased macrophage activation, while silencing PARP9 decreased macrophage activation. A network analysis further linked PARP9 and PARP14 to human coronary artery disease.

"Macrophage activation plays a role in not only vascular disorders, but also various inflammatory and autoimmune diseases," Aikawa said in a statement. "These results could provide important information about the mechanisms of these diseases and help to develop much needed new therapeutics."

Using tandem mass tagging quantitative proteomics, he and his colleagues screened a mouse and a human macrophage cell line that were stimulated by either interferon-gamma (IFNγ) or IL-4. Previous work, the researchers noted, has suggested that different cytokines can promote different macrophage subpopulations.

They quantified more than 5,000 proteins in both cell lines under the three conditions of IFNγ-stimulated, IL-4 stimulated, or unstimulated. After filtering, Aikawa and his colleagues homed in on a set of 38 candidate proteins, including PARP9 and PARP14. IFNγ has been linked to pro-inflammatory activation and IL-4 has been found to counter that activation, the researchers noted.

A network analysis of PARP9 and PARP14 then linked them to autoimmune diseases and coronary artery disease in humans. The researchers further validated PARP9 and PARP14's link to macrophage activation through qPCR and western blot analyses, and found that both proteins are expressed in macrophages in human atherosclerotic lesions.

In a series of in vitro studies, Aikawa and his colleagues found that the two proteins have opposing effects. Namely, they reported that PARP9 is pro-inflammatory, while PARP14 is anti-inflammatory. For instance, they reported that PARP14 suppresses IFNγ-induced responses, while enhancing IL-4 response in macrophages. By contract, PARP9 promotes IFNγ responses.

They also reported that PARP9 and PARP14 appear to rely on different signaling pathways, regulating the activation of STAT1 and STAT6, to have their effect.

In addition, both co-immunoprecipitation and immunofluorescence analyses indicated that the two proteins interact and that PARP14, which is a mono-ADP-ribosyltransferase, mediates ADP-ribosylation of STAT1α, though that is suppressed by PARP9. This could be the mechanism through which the proteins affect IFNγ signaling, the researchers said.

In a series of mouse studies, the researchers found that PARP14 deletions are associated with increased arterial lesion development and atherogenesis. This suggests a protective role for PARP14, Aikawa and his colleagues said.

"Our findings provide insight into new mechanisms for macrophage activation that play a critical role in the pathogenesis of inflammatory arterial diseases, a global health burden," the researchers wrote in their paper.

Aikawa and his colleagues further said that these proteins might be or could point the way toward drug targets for arterial and other diseases. "Collectively, our discoveries indicate that inhibition of PARP9 and/or activation of PARP14 may attenuate macrophage-mediated vascular diseases, and also provide new insight into the development of effective therapies for other inflammatory disorders," they wrote.