No abstract

SummaryNon-alcoholic fatty liver disease (NAFLD) is a very common indication for liver transplantation. How fat-rich diets promote progression from fatty liver to more damaging inflammatory and fibrotic stages is poorly understood. Here, we show that disrupting phosphorylation at Ser196 (S196A) in the liver X receptor alpha (LXRα, NR1H3) retards NAFLD progression in mice on a high-fat-high-cholesterol diet. Mechanistically, this is explained by key histone acetylation (H3K27) and transcriptional changes in pro-fibrotic and pro-inflammatory genes. Furthermore, S196A-LXRα expression reveals the regulation of novel diet-specific LXRα-responsive genes, including the induction of Ces1f, implicated in the breakdown of hepatic lipids. This involves induced H3K27 acetylation and altered LXR and TBLR1 cofactor occupancy at the Ces1f gene in S196A fatty livers. Overall, impaired Ser196-LXRα phosphorylation acts as a novel nutritional molecular sensor that profoundly alters the hepatic H3K27 acetylome and transcriptome during NAFLD progression placing LXRα phosphorylation as an alternative anti-inflammatory or anti-fibrotic therapeutic target.

Macrophages are key immune cells for the initiation and development of atherosclerotic lesions. However, the macrophage regulatory nodes that determine how lesions progress in response to dietary challenges are not fully understood. Liver X receptors (LXRs) are sterol-regulated transcription factors that play a central role in atherosclerosis by integrating cholesterol homeostasis and immunity. LXR pharmacological activation elicits a robust antiatherosclerotic transcriptional program in macrophages that can be affected by LXRα S196 phosphorylation in vitro. To investigate the impact of these transcriptional changes in atherosclerosis development, we have generated mice carrying a Ser-to-Ala mutation in myeloid cells in the LDL receptor (LDLR)-deficient atherosclerotic background (M-S196A). M-S196A mice fed a high-fat diet exhibit increased atherosclerotic plaque burden and lesions with smaller necrotic cores and thinner fibrous caps. These diet-induced phenotypic changes are consistent with a reprogramed macrophage transcriptome promoted by LXRα-S196A during atherosclerosis development. Remarkably, expression of several proliferation-promoting factors, including the protooncogene FoxM1 and its targets, is induced by LXRα-S196A. This is consistent with increased proliferation of plaque-resident cells in M-S196A mice. Moreover, disrupted LXRα phosphorylation increases expression of phagocytic molecules, resulting in increased apoptotic cell removal by macrophages, explaining the reduced necrotic cores. Finally, the macrophage transcriptome promoted by LXRα-S196A under dietary perturbation is markedly distinct from that revealed by LXR ligand activation, highlighting the singularity of this posttranslational modification. Overall, our findings demonstrate that LXRα phosphorylation at S196 is an important determinant of atherosclerotic plaque development through selective changes in gene transcription that affect multiple pathways.

Oncolytic viruses (OV) have been shown to activate the anti-tumor functions of specific immune cells like T cells. Here, we show OV can also reprogram TAMs to a less immunosuppressive phenotype. Syngeneic, immunocompetent mouse models of primary breast cancer were established using PyMT-TS1, 4T1 and E0771 cell lines and a metastatic model of breast cancer was established using the 4T1 cell line. Tumor growth and overall survival was assessed following intravenous administration of the OV, HSV1716 (a modified herpes simplex virus). Infiltration and function of various immune effector cells was assessed by NanoString, flow cytometry of dispersed tumors and immunofluorescence analysis of tumor sections. HSV1716 administration led to marked tumor shrinkage in primary mammary tumors and a decrease in metastases. This was associated with a significant increase in the recruitment/activation of cytotoxic T cells, a reduction in the presence of regulatory T cells and the reprograming of TAMs towards a pro-inflammatory, less immunosuppressive phenotype. These findings were supported by in vitro data demonstrating that human monocyte-derived macrophages (MDMs) host HSV1716 replication, and that this led to immunogenic macrophage lysis. These events were dependent on macrophage expression of proliferating cell nuclear antigen (PCNA). Finally, the anti-tumor effect of OV was markedly diminished when TAMs were depleted using clodronate liposomes. Together, our results show that TAMs play an essential role in support of the tumoricidal effect of the OV, HSV1716they both host viral replication via a novel, PCNA-dependent mechanism and are reprogramed to express a less immunosuppressive phenotype.

p56kk and p60c-src are closely related protein-tyrosine kinases that are activated by similar oncogenic mutations. We have used fibroblast cell lines that express p56ick from introduced DNA molecules to compare the subcellular localizations of p60c-src and p561ck and their abilities to bind polyomavirus middle T antigen (mT). p561ck is associated with the detergent-insoluble matrix, as defined by extraction with solutions containing nonionic detergents, whereas p60csrc is soluble under these conditions. p56lck is also associated with detergent-insoluble structures in a lymphoid cell line, LSTRA. p60csrC binds to mT, but P56lck does not bind detectably. In terms of both solubility and mT interactions, the nononcogenic p56lck more closely resembles oncogenically activated p60csrc mutants than it resembles p60c-src. Because published results show that an intact carboxy terminus is required for p60CsrC to bind mT and be soluble, we tested whether the different localization and mT binding properties of p561ck and p6C-srC were dictated by their different carboxy termini. A protein consisting largely of p6oc-srC sequences but carrying a p561ck carboxy terminus was soluble and bound to mT. We suggest that both the solubility and mT-binding properties of p60'src not only require sequences common to the carboxy termini of p60csrc and p56 ck, but also require sequences unique to the body of p60csrc. p561ck and p60c-src are homologous membrane-associated protein-tyrosine kinases (J. A. Cooper, in B. Kemp and P. F. Alewood, ed., Peptides and Protein Phosphorylation, in press). Both proteins have tyrosines near their carboxy termini that are normally phosphorylated (12, 37). These phosphorylations correlate with suppression of the kinase activities and transforming abilities of p60csrc and p56Ick (13,