The hepatocyte IKK:NF-κB axis promotes liver steatosis by stimulating <i>de novo</i> lipogenesis and cholesterol synthesis

OBJECTIVE: Obesity-related chronic inflammation plays an important role in the development of Metabolic Associated Fatty Liver Disease (MAFLD). Although the contribution of the pro-inflammatory NF-κB signaling pathway to the progression from simple steatosis to non-alcoholic steatohepatitis (NASH) is well-established, its role as an initiator of hepatic steatosis and the underlying mechanism remains unclear. Here, we investigated the hypothesis that the hepatocytic NF-κB signaling pathway acts as a metabolic regulator, thereby promoting hepatic steatosis development. METHODS: A murine model expressing a constitutively active form of IKKβ in hepatocytes (Hep-IKKβca) was used to activate hepatocyte NF-κB. In addition, IKKβca was also expressed in hepatocyte A20-deficient mice (IKKβca;A20(LKO)). A20 is an NF-κB-target gene that inhibits the activation of the NF-κB signaling pathway upstream of IKKβ. These mouse models were fed a sucrose-rich diet for 8 weeks. Hepatic lipid levels were measured and using [1–(13)C]-acetate de novo lipogenesis and cholesterol synthesis rate were determined. Gene expression analyses and immunoblotting were used to study the lipogenesis and cholesterol synthesis pathways. RESULTS: Hepatocytic NF-κB activation by expressing IKKβca in hepatocytes resulted in hepatic steatosis without inflammation. Ablation of hepatocyte A20 in Hep-IKKβca mice (IKKβca;A20(LKO) mice) exacerbated hepatic steatosis, characterized by macrovesicular accumulation of triglycerides and cholesterol, and increased plasma cholesterol levels. Both De novo lipogenesis (DNL) and cholesterol synthesis were found elevated in IKKβca;A20(LKO) mice. Phosphorylation of AMP-activated kinase (AMPK) - a suppressor in lipogenesis and cholesterol synthesis - was decreased in IKKβca;A20(LKO) mice. This was paralleled by elevated protein levels of hydroxymethylglutaryl-CoA synthase 1 (HMGCS1) and reduced phosphorylation of HMG-CoA reductase (HMGCR) both key enzymes in the cholesterol synthesis pathway. Whereas inflammation was not observed in young IKKβca;A20(LKO) mice sustained hepatic NF-κB activation resulted in liver inflammation, together with elevated hepatic and plasma cholesterol levels in middle-aged mice. CONCLUSIONS: The hepatocytic IKK:NF-κB axis is a metabolic regulator by controlling DNL and cholesterol synthesis, independent of its central role in inflammation. The IKK:NF-κB axis controls the phosphorylation levels of AMPK and HMGCR and the protein levels of HMGCS1. Chronic IKK-mediated NF-κB activation may contribute to the initiation of hepatic steatosis and cardiovascular disease risk in MAFLD patients

T cell cholesterol efflux suppresses apoptosis and senescence and increases atherosclerosis in middle aged mice

Atherosclerosis is a chronic inflammatory disease driven by hypercholesterolemia. During aging, T cells accumulate cholesterol, potentially affecting inflammation. However, the effect of cholesterol efflux pathways mediated by ATP-binding cassette A1 and G1 (ABCA1/ABCG1) on T cell-dependent age-related inflammation and atherosclerosis remains poorly understood. In this study, we generate mice with T cell-specific Abca1/Abcg1-deficiency on the low-density-lipoprotein-receptor deficient (Ldlr-/-) background. T cell Abca1/Abcg1-deficiency decreases blood, lymph node, and splenic T cells, and increases T cell activation and apoptosis. T cell Abca1/Abcg1-deficiency induces a premature T cell aging phenotype in middle-aged (12-13 months) Ldlr-/- mice, reflected by upregulation of senescence markers. Despite T cell senescence and enhanced T cell activation, T cell Abca1/Abcg1-deficiency decreases atherosclerosis and aortic inflammation in middle-aged Ldlr-/- mice, accompanied by decreased T cells in atherosclerotic plaques. We attribute these effects to T cell apoptosis downstream of T cell activation, compromising T cell functionality. Collectively, we show that T cell cholesterol efflux pathways suppress T cell apoptosis and senescence, and induce atherosclerosis in middle-aged Ldlr-/- mice

Novel functions for atypical E2Fs, E2F7 and E2F8, in polyploidization and liver cancer

Atypical E2F transcription factors, E2F7 and E2F8, function as transcriptional repressors of E2F target genes and are crucial for controlling the cell proliferation. In this thesis, we reveal that these two factors are crucial for liver cell polyploidization, embryonic development and prevention of liver cancer in mice. In chapter 2, we show that atypical E2Fs, especially E2F8, play an important role in promoting polyploidization in the liver. Loss of E2F7/8 resulted in dramatic reduction in binucleation and polyploidization of hepatocytes. In contrast, single loss of E2F1 enhanced polyploidization in hepatocytes. Interestingly, loss of E2f1 in E2f7/8-deficient liver led to partial rescue of polyploidization defect observed upon loss of E2f7/8. Moreover, we identified a transcriptional network program regulated by repressor E2F8 and activator E2F1 that controls polyploidization in liver. Together, these data suggest opposing functions for classical activator E2Fs and atypical repressor E2Fs in regulating polyploidization in liver. Surprisingly, contrary to long-term theory, we discovered that loss of polyploidization has no impact on liver differentiation and regeneration. We demonstrate in chapter 4 that E2F7 and E2F8 are required for murine embryonic development. While mice mutant for either E2F7 or E2F8 developed normally with no obvious developmental defects, global deletion of both E2F7 and 8 resulted in embryonic lethality owing to vascular and cell survival defects such as dilated blood vessels associated with multifocal hemorrhages and massive apoptosis. Interestingly, loss of either E2F1 or p53 suppressed the massive apoptosis observed in E2f7/8 double knockout embryos, however, the triple knockout embryos still carried the vascular defects and died around same embryonic age, suggesting complex mechanisms underlying the embryonic lethality. As described in chapter 5, we have revealed that E2f7/8 function as tumor suppressor genes and that they can cooperate with the known tumor suppressor gene Rb in preventing liver cancer. Using liver-specific knockout mice, we found that deletion of E2f7/8 results in spontaneous development of hepatocellular carcinoma in mice. Furthermore, additional deletion of Rb along with E2f7/8 resulted in development of tumors earlier than in E2f7/8 deficient livers, indicating that loss of Rb accelerates tumorigenesis

Necrosi cutanea ed epatica in un coniglio domestico: Un caso sospetto di reazione avversa a farmaco

The present report describes a case of cutaneous and hepatic necrosis likely related to drug administration in a rabbit. A 2 year-old male rabbit with intestinal constipation was examinated. Symptomatic treatment was initiated, consisted of subcutaneous fluid with Ringer's Lactate, meloxicam and metoclopramide. Five days later, after the development of subcutaneous oedema and bullous-necrotic skin lesion on the abdomen, the rabbit died. On necropsy, hepatic multifocal white foci were observed. Histopathologically, skin showed full thickness epidermal necrosis, necrotizing destructive mural folliculitis, dermal eosinophilic infiltrate, cell poor vasculitis, whereas liver exhibited multifocal atrophy and centrilobular and acute coagulative necrosis. Pathological findings, together with the time of the onset of clinical signs, were strongly suggestive of adverse drug reaction (ADR). Meloxicam, a nonsteroidal anti-inflammatory drug, commonly used for the control of the pain in rabbits, has never been associated with ADR in this specie, but recently reported most probably associated with cutaneous and hepatic manifestation in a dog

MyD88-dependent signaling in non-parenchymal cells promotes liver carcinogenesis

In Western countries, a rising incidence of obesity and type 2 diabetes correlates with an increase of non-alcoholic steatohepatitis (NASH)-a major risk factor for liver cirrhosis and hepatocellular carcinoma (HCC). NASH is associated with chronic liver injury, triggering hepatocyte death and enhanced translocation of intestinal bacteria, leading to persistent liver inflammation through activation of Toll-like receptors and their adapter protein myeloid differentiation factor 88 (MyD88). Therefore, we investigated the role of MyD88 during progression from NASH to HCC using a mouse model of chronic liver injury (hepatocyte-specific deletion of nuclear factor κB essential modulator, Nemo; NemoΔhepa). NemoΔhepa; NemoΔhepa/MyD88-/- and NemoΔhepa/MyD88Δhepa were generated and the impact on liver disease progression was investigated. Ubiquitous MyD88 ablation (NemoΔhepa/MyD88-/-) aggravated the degree of liver damage, accompanied by an overall decrease in inflammation, whereas infiltrating macrophages and natural killer cells were elevated. At a later stage, MyD88 deficiency impaired HCC formation. In contrast, hepatocyte-specific MyD88 deletion (NemoΔhepa/MyD88Δhepa) did not affect disease progression. These results suggest that signaling of Toll-like receptors through MyD88 in non-parenchymal liver cells is required for carcinogenesis during chronic liver injury. Hence, blocking MyD88 signaling may offer a therapeutic option to prevent HCC formation in patients with NASH

Acute systemic loss of Mad2 leads to intestinal atrophy in adult mice

Chromosomal instability (CIN) is a hallmark of cancer, leading to aneuploid cells. To study the role that CIN plays in tumor evolution, several mouse models have been engineered over the last 2 decades. These models have unequivocally shown that systemic high-grade CIN is embryonic lethal. We and others have previously shown that embryonic lethality can be circumvented by provoking CIN in a tissue-specific fashion. In this study, we provoke systemic high-grade CIN in adult mice as an alternative to circumvent embryonic lethality. For this, we disrupt the spindle assembly checkpoint (SAC) by alleviating Mad2 or truncating Mps1, both essential genes for SAC functioning, with or without p53 inactivation. We find that disruption of the SAC leads to rapid villous atrophy, atypia and apoptosis of the epithelia of the jejunum and ileum, substantial weight loss, and death within 2–3 weeks after the start of the CIN insult. Despite this severe intestinal phenotype, most other tissues are unaffected, except for minor abnormalities in spleen, presumably due to the lower proliferation rate in these tissues. We conclude that high-grade CIN in vivo in adult mice is most toxic to the high cell turnover intestinal epithelia

E2F7 is a potent inhibitor of liver tumor growth in adult mice

Background and Aims: Up-regulation of the E2F-dependent transcriptional network has been identified in nearly every human malignancy and is an important driver of tumorigenesis. Two members of the E2F family, E2F7 and E2F8, are potent repressors of E2F-dependent transcription. They are atypical in that they do not bind to dimerization partner proteins and are not controlled by retinoblastoma protein. The physiological relevance of E2F7 and E2F8 remains incompletely understood, largely because tools to manipulate their activity in vivo have been lacking. Approach and Results: Here, we generated transgenic mice with doxycycline-controlled transcriptional activation of E2f7 and E2f8 and induced their expression during postnatal development, in adulthood, and in the context of cancer. Systemic induction of E2f7 and, to lesser extent, E2f8 transgenes in juvenile mice impaired cell proliferation, caused replication stress, DNA damage, and apoptosis, and inhibited animal growth. In adult mice, however, E2F7 and E2F8 induction was well tolerated, yet profoundly interfered with DNA replication, DNA integrity, and cell proliferation in diethylnitrosamine-induced liver tumors. Conclusion: Collectively, our findings demonstrate that atypical E2Fs can override cell-cycle entry and progression governed by other E2F family members and suggest that this property can be exploited to inhibit proliferation of neoplastic hepatocytes when growth and development have subsided during adulthood

Papillary meningioma with multifocal leptomeningeal spread in a dog

An 11-year-old, English cocker spaniel was presented with subacute progressive signs of vestibular ataxia, tetraparesis, left-sided proprioceptive deficits, positional ventrolateral strabismus of the right eye and a right-sided menace deficit. Magnetic resonance imaging of the cranium and cranial cervical spinal cord revealed multifocal T2 hyper-/T1-hypointense intradural lesions with dural tail signs and intra-axial and intramedullary extension. Medical treatment resulted in initial improvement before deterioration was noticed. Cytological examination results of computed tomography-guided fine-needle aspiration biopsy of the C1–C2 lesion were consistent with mesenchymal neoplasia. Three days later, after progressive clinical deterioration, euthanasia was performed. Postmortem examination and subsequent histological examination of the brainstem and spinal cord revealed multifocal, strongly infiltrative growth of neoplastic cells with areas of pseudo-rosette formation by cylindrical neoplastic cells with moderately large, oval nuclei in addition to areas of spindle-shaped neoplastic cells with meningothelial whorls. The final diagnosis was a papillary (grade III) meningioma with multifocal leptomeningeal spread

Atypical E2Fs either Counteract or Cooperate with RB during Tumorigenesis Depending on Tissue Context

E2F-transcription factors activate many genes involved in cell cycle progression, DNA repair, and apoptosis. Hence, E2F-dependent transcription must be tightly regulated to prevent tumorigenesis, and therefore metazoan cells possess multiple E2F regulation mechanisms. The best-known is the Retinoblastoma protein (RB), which is mutated in many cancers. Atypical E2Fs (E2F7 and -8) can repress E2F-target gene expression independently of RB and are rarely mutated in cancer. Therefore, they may act as emergency brakes in RB-mutated cells to suppress tumor growth. Currently, it is unknown if and how RB and atypical E2Fs functionally interact in vivo. Here, we demonstrate that mice with liver-specific combinatorial deletion of Rb and E2f7/8 have reduced life-spans compared to E2f7/8 or Rb deletion alone. This was associated with increased proliferation and enhanced malignant progression of liver tumors. Hence, atypical repressor E2Fs and RB cooperatively act as tumor suppressors in hepatocytes. In contrast, loss of either E2f7 or E2f8 largely prevented the formation of pituitary tumors in Rb+/- mice. To test whether atypical E2Fs can also function as oncogenes independent of RB loss, we induced long-term overexpression of E2f7 or E2f8 in mice. E2F7 and -8 overexpression increased the incidence of tumors in the lungs, but not in other tissues. Collectively, these data show that atypical E2Fs can promote but also inhibit tumorigenesis depending on tissue type and RB status. We propose that the complex interactions between atypical E2Fs and RB on maintenance of genetic stability underlie this context-dependency