JNKs protect from cholestatic liver disease progression by modulating Apelin signalling.

Background & Aims: Cholestatic liver injury is associated with c-Jun N-terminal kinases (JNK) activation in distinct cell types. Its hepatocyte-specific function during cholestasis, however, has not yet been established. Therefore, in our present study, we investigated the role of JNK1/2 during cholestasis and dissected its hepatocyte-specific function. Methods: A cohort of patients with primary biliary cholangitis (n = 29) and primary sclerosing cholangitis (n = 37) was examined. Wild-type, hepatocyte-specific knockout mice for Jnk2 (Jnk2Δhepa) or Jnk1 and Jnk2 (Jnk1Δhepa/2Δhepa) were generated. Mice were subjected to bile duct ligation (BDL) or carbon tetrachloride (CCl4) treatment. Finally, Apelin signalling was blocked using a specific inhibitor. As an interventional approach, Jnk1/2 were silenced in wild-type mice using lipid nanoparticles for small interfering RNA delivery. Results: JNK activation was increased in liver specimens from patients with chronic cholestasis (primary biliary cholangitis and primary sclerosing cholangitis) and in livers of Mdr2-/- and BDL-treated animals. In Jnk1Δhepa/2Δhepa animals, serum transaminases increased after BDL, and liver histology demonstrated enhanced cell death, compensatory proliferation, hepatic fibrogenesis, and inflammation. Furthermore, microarray analysis revealed that hepatocytic Jnk1/2 ablation induces JNK-target genes involved in oxidative stress and Apelin signalling after BDL. Consequently, blocking Apelin signalling attenuated BDL-induced liver injury and fibrosis in Jnk1Δhepa/2Δhepa mice. Finally, we established an interventional small interfering RNA approach of selective Jnk1/2 targeting in hepatocytes in vivo, further demonstrating the essential protective role of Jnk1/2 during cholestasis. Conclusions: Jnk1 and Jnk2 work together to protect hepatocytes from cholestatic liver disease by controlling Apelin signalling. Dual modification of JNK signalling in hepatocytes is feasible, and enhancing its expression might be an attractive therapeutic approach for cholestatic liver disease. Impact and Implications: The cell-specific function of Jnk genes during cholestasis has not been explicitly explored. In this study, we showed that combined Jnk1/2, but not Jnk2 deficiency, in hepatocytes exacerbates liver damage and fibrosis by enhancing Apelin signalling, which contributes to cholestasis progression. Combined cell-specific Jnk targeting may be a new molecular strategy for treating cholestatic liver disease.

Hypothalamic JNK1-hepatic fatty acid synthase axis mediates a metabolic rewiring that prevents hepatic steatosis in male mice treated with olanzapine via intraperitoneal: Additional effects of PTP1B inhibition.

Olanzapine (OLA), a widely used second-generation antipsychotic (SGA), causes weight gain and metabolic alterations when administered orally to patients. Recently, we demonstrated that, contrarily to the oral treatment which induces weight gain, OLA administered via intraperitoneal (i.p.) in male mice resulted in body weight loss. This protection was due to an increase in energy expenditure (EE) through a mechanism involving the modulation of hypothalamic AMPK activation by higher OLA levels reaching this brain region compared to those of the oral treatment. Since clinical studies have shown hepatic steatosis upon chronic treatment with OLA, herein we further investigated the role of the hypothalamus-liver interactome upon OLA administration in wild-type (WT) and protein tyrosine phosphatase 1B knockout (PTP1B-KO) mice, a preclinical model protected against metabolic syndrome. WT and PTP1B-KO male mice were fed an OLA-supplemented diet or treated via i.p. Mechanistically, we found that OLA i.p. treatment induces mild oxidative stress and inflammation in the hypothalamus in a JNK1-independent and dependent manner, respectively, without features of cell dead. Hypothalamic JNK activation up-regulated lipogenic gene expression in the liver though the vagus nerve. This effect concurred with an unexpected metabolic rewiring in the liver in which ATP depletion resulted in increased AMPK/ACC phosphorylation. This starvation-like signature prevented steatosis. By contrast, intrahepatic lipid accumulation was observed in WT mice treated orally with OLA; this effect being absent in PTP1B-KO mice. We also demonstrated an additional benefit of PTP1B inhibition against hypothalamic JNK activation, oxidative stress and inflammation induced by chronic OLA i.p. treatment, thereby preventing hepatic lipogenesis. The protection conferred by PTP1B deficiency against hepatic steatosis in the oral OLA treatment or against oxidative stress and neuroinflammation in the i.p. treatment strongly suggests that targeting PTP1B might be also a therapeutic strategy to prevent metabolic comorbidities in patients under OLA treatment in a personalized manner.

HER2-driven breast cancer suppression by the JNK signaling pathway.

The HER2+ subtype of human breast cancer is associated with the malignant transformation of luminal ductal cells of the mammary epithelium. The sequence analysis of tumor DNA identifies loss of function mutations and deletions of the MAP2K4 and MAP2K7 genes that encode direct activators of the JUN NH2-terminal kinase (JNK). We report that in vitro studies of human mammary epithelial cells with CRISPR-induced mutations in the MAPK and MAP2K components of the JNK pathway caused no change in growth in 2D culture, but these mutations promoted epithelial cell proliferation in 3D culture. Analysis of gene expression signatures in 3D culture demonstrated similar changes caused by HER2 activation and JNK pathway loss. The mechanism of signal transduction cross-talk may be mediated, in part, by JNK-suppressed expression of integrin α6ß4 that binds HER2 and amplifies HER2 signaling. These data suggest that HER2 activation and JNK pathway loss may synergize to promote breast cancer. To test this hypothesis, we performed in vivo studies using a mouse model of HER2+ breast cancer with Cre/loxP-mediated ablation of genes encoding JNK (Mapk8 and Mapk9) and the MAP2K (Map2k4 and Map2k7) that activate JNK in mammary epithelial cells. Kaplan-Meier analysis of tumor development demonstrated that JNK pathway deficiency promotes HER2+-driven breast cancer. Collectively, these data identify JNK pathway genes as potential suppressors for HER2+ breast cancer.

Phosphorylation of RXRα mediates the effect of JNK to suppress hepatic FGF21 expression and promote metabolic syndrome.

The cJun NH2-terminal kinase (JNK) signaling pathway in the liver promotes systemic changes in metabolism by regulating peroxisome proliferator-activated receptor α (PPARα)-dependent expression of the hepatokine fibroblast growth factor 21 (FGF21). Hepatocyte-specific gene ablation studies demonstrated that the Mapk9 gene (encoding JNK2) plays a key mechanistic role. Mutually exclusive inclusion of exons 7a and 7b yields expression of the isoforms JNK2α and JNK2ß. Here we demonstrate that Fgf21 gene expression and metabolic regulation are primarily regulated by the JNK2α isoform. To identify relevant substrates of JNK2α, we performed a quantitative phosphoproteomic study of livers isolated from control mice, mice with JNK deficiency in hepatocytes, and mice that express only JNK2α or JNK2ß in hepatocytes. We identified the JNK substrate retinoid X receptor α (RXRα) as a protein that exhibited JNK2α-promoted phosphorylation in vivo. RXRα functions as a heterodimeric partner of PPARα and may therefore mediate the effects of JNK2α signaling on Fgf21 expression. To test this hypothesis, we established mice with hepatocyte-specific expression of wild-type or mutated RXRα proteins. We found that the RXRα phosphorylation site Ser260 was required for suppression of Fgf21 gene expression. Collectively, these data establish a JNK-mediated signaling pathway that regulates hepatic Fgf21 expression.

Mixed lineage kinase 3 requires a functional CRIB domain for regulation of blood pressure, cardiac hypertrophy, and left ventricular function.

Mixed lineage kinase 3 (MLK3) modulates blood pressure and left ventricular function, but the mechanisms governing these effects remain unclear. In the current study, we therefore investigated the role of the MLK3 Cdc42/Rac interactive binding (CRIB) domain in cardiovascular physiology. We examined baseline and left ventricular pressure overload responses in a MLK3 CRIB mutant (MLK3C/C) mouse, which harbors point mutations in the CRIB domain to disrupt MLK3 activation by Cdc42. Male and female MLK3C/C mice displayed increased invasively measured blood pressure compared with wild-type (MLK3+/+) littermate controls. MLK3C/C mice of both sexes also developed left and right ventricular hypertrophy but normal baseline LV function by echocardiography and invasive hemodynamics. In LV tissue from MLK3C/C mice, map3k11 mRNA, which encodes MLK3, and MLK3 protein were reduced by 74 ± 6% and 73 ± 7%, respectively. After 1-wk LV pressure overload with 25-gauge transaortic constriction (TAC), male MLK3C/C mice developed no differences in LV hypertrophy but displayed reduction in the LV systolic indices ejection fraction and dP/dt normalized to instantaneous pressure. JNK activation was also reduced in LV tissue of MLK3C/C TAC mice. TAC induced MLK3 translocation from cytosolic fraction to membrane fraction in LV tissue from MLK3+/+ but not MLK3C/C mice. These findings identify a role of the MLK3 CRIB domain in MLK3 regulation of basal blood pressure and cardiac morphology, and in promoting the compensatory LV response to pressure overload.NEW & NOTEWORTHY Here, we identified that the presence of two discrete point mutations within the Cdc42/Rac interaction and binding domain of the protein MLK3 recapitulates the effects of whole body MLK3 deletion on blood pressure, cardiac hypertrophy, and left ventricular compensation after pressure overload. These findings implicate the CRIB domain, and thus MLK3 activation by this domain, as critical for maintenance of cardiovascular homeostasis.

Cdk5-mediated JIP1 phosphorylation regulates axonal outgrowth through Notch1 inhibition.

BACKGROUND: Activated Cdk5 regulates a number of processes during nervous system formation, including neuronal differentiation, growth cone stabilization, and axonal growth. Cdk5 phosphorylates its downstream substrates located in axonal growth cones, where the highly expressed c-Jun N-terminal kinase (JNK)-interacting protein1 (JIP1) has been implicated as another important regulator of axonal growth. In addition, stringent control of the level of intracellular domain of Notch1 (Notch1-IC) plays a regulatory role in axonal outgrowth during neuronal differentiation. However, whether Cdk5-JIP1-Notch1 cooperate to regulate axonal outgrowth, and the mechanism of such joint contribution to this pathway, is presently unknown, and here we explore their potential interaction. RESULTS: Our interactome screen identified JIP1 as an interactor of p35, a Cdk5 activator, and we sought to explore the relationship between Cdk5 and JIP1 on the regulation of axonal outgrowth. We demonstrate that JIP1 phosphorylated by Cdk5 at Thr205 enhances axonal outgrowth and a phosphomimic JIP1 rescues the axonal outgrowth defects in JIP1-/- and p35-/- neurons. Axonal outgrowth defects caused by the specific increase of Notch1 in JIP1-/- neurons are rescued by Numb-mediated inhibition of Notch1. Finally, we demonstrate that Cdk5 phosphorylation of JIP1 further amplifies the phosphorylation status of yet another Cdk5 substrate E3-ubiquitin ligase Itch, resulting in increased Notch1 ubiquitination. CONCLUSIONS: Our findings identify a potentially critical signaling axis involving Cdk5-JIP1-Itch-Notch1, which plays an important role in the regulation of CNS development. Future investigation into the way this pathway integrates with additional pathways regulating axonal growth will further our knowledge of normal central nervous system development and pathological conditions.

c-JUN n-Terminal Kinase (JNK) Signaling in Autosomal Dominant Polycystic Kidney Disease.

Polycystic kidney disease is an inherited degenerative disease in which the uriniferous tubules are replaced by expanding fluid-filled cysts that ultimately destroy organ function. Autosomal dominant polycystic kidney disease (ADPKD) is the most common form, afflicting approximately 1 in 1,000 people and is caused by mutations in the transmembrane proteins polycystin-1 (Pkd1) and polycystin-2 (Pkd2). The mechanisms by which polycystin mutations induce cyst formation are not well understood, however pro-proliferative signaling must be involved for tubule epithelial cell number to increase over time. We recently found that the stress-activated mitogen-activated protein kinase (MAPK) pathway c-Jun N-terminal kinase (JNK) pathway is activated in cystic disease and genetically removing JNK reduces cyst growth driven by a loss of Pkd2. This review covers the current state of knowledge of signaling in ADPKD with an emphasis on the JNK pathway.

Aberrant Ca2+ signaling by IP3Rs in adipocytes links inflammation to metabolic dysregulation in obesity.

Chronic metabolic inflammation is a key feature of obesity, insulin resistance, and diabetes. Here, we showed that altered regulation of the Ca2+ channel inositol trisphosphate receptor (IP3R) was an adipocyte-intrinsic event involved in the emergence and propagation of inflammatory signaling and the resulting insulin resistance. Inflammation induced by cytokine exposure in vitro or by obesity in vivo led to increases in the abundance and activity of IP3Rs and in the phosphorylation of the Ca2+-dependent kinase CaMKII in adipocytes in a manner dependent on the kinase JNK. In mice, adipocyte-specific loss of IP3R1/2 protected against adipose tissue inflammation and insulin resistance, despite the mice exhibiting substantial diet-induced weight gain. Thus, this work suggests that increased IP3R activity is a key link between obesity, inflammation, and insulin resistance. These data also suggest that approaches to target IP3R-mediated Ca2+ homeostasis in adipocytes may offer new therapeutic opportunities against metabolic diseases, especially because GWAS studies also implicate this locus in human obesity.

c-Jun N-terminal kinase (JNK) signaling contributes to cystic burden in polycystic kidney disease.

Polycystic kidney disease is an inherited degenerative disease in which the uriniferous tubules are replaced by expanding fluid-filled cysts that ultimately destroy organ function. Autosomal dominant polycystic kidney disease (ADPKD) is the most common form, afflicting approximately 1 in 1,000 people. It primarily is caused by mutations in the transmembrane proteins polycystin-1 (Pkd1) and polycystin-2 (Pkd2). The most proximal effects of Pkd mutations leading to cyst formation are not known, but pro-proliferative signaling must be involved for the tubule epithelial cells to increase in number over time. The c-Jun N-terminal kinase (JNK) pathway promotes proliferation and is activated in acute and chronic kidney diseases. Using a mouse model of cystic kidney disease caused by Pkd2 loss, we observe JNK activation in cystic kidneys and observe increased nuclear phospho c-Jun in cystic epithelium. Genetic removal of Jnk1 and Jnk2 suppresses the nuclear accumulation of phospho c-Jun, reduces proliferation and reduces the severity of cystic disease. While Jnk1 and Jnk2 are thought to have largely overlapping functions, we find that Jnk1 loss is nearly as effective as the double loss of Jnk1 and Jnk2. Jnk pathway inhibitors are in development for neurodegeneration, cancer, and fibrotic diseases. Our work suggests that the JNK pathway should be explored as a therapeutic target for ADPKD.

Mitogen Kinase Kinase (MKK7) Controls Cytokine Production In Vitro and In Vivo in Mice.

Mitogen kinase kinase 4 (MKK4) and mitogen kinase kinase 7 (MKK7) are members of the MAP2K family that can activate downstream mitogen-activated protein kinases (MAPKs). MKK4 has been implicated in the activation of both c-Jun N-terminal kinase (JNK) and p38 MAPK, while MKK7 has been reported to activate only JNK in response to different stimuli. The stimuli, as well as the cell type determine which MAP2K member will mediate a given response. In various cell types, MKK7 contributes to the activation of downstream MAPKs, JNK, which is known to regulate essential cellular processes, such as cell death, differentiation, stress response, and cytokine secretion. Previous studies have also implicated the role of MKK7 in stress signaling pathways and cytokine production. However, little is known about the degree to which MKK4 and MKK7 contribute to innate immune responses in macrophages or during inflammation in vivo. To address this question and to elucidate the role of MKK4 and MKK7 in macrophage and in vivo, we developed MKK4- and MKK7-deficient mouse models with tamoxifen-inducible Rosa26 CreERT. This study reports that MKK7 is required for JNK activation both in vitro and in vivo. Additionally, we demonstrated that MKK7 in macrophages is necessary for lipopolysaccharide (LPS)-induced cytokine production, M1 polarization, and migration, which appear to be a major contributor to the inflammatory response in vivo. Conversely, MKK4 plays a significant, but minor role in cytokine production in vivo.

MLK3 mediates impact of PKG1α on cardiac function and controls blood pressure through separate mechanisms.

cGMP-dependent protein kinase 1α (PKG1α) promotes left ventricle (LV) compensation after pressure overload. PKG1-activating drugs improve heart failure (HF) outcomes but are limited by vasodilation-induced hypotension. Signaling molecules that mediate PKG1α cardiac therapeutic effects but do not promote PKG1α-induced hypotension could therefore represent improved therapeutic targets. We investigated roles of mixed lineage kinase 3 (MLK3) in mediating PKG1α effects on LV function after pressure overload and in regulating BP. In a transaortic constriction HF model, PKG activation with sildenafil preserved LV function in MLK3+/+ but not MLK3-/- littermates. MLK3 coimmunoprecipitated with PKG1α. MLK3-PKG1α cointeraction decreased in failing LVs. PKG1α phosphorylated MLK3 on Thr277/Ser281 sites required for kinase activation. MLK3-/- mice displayed hypertension and increased arterial stiffness, though PKG stimulation with sildenafil or the soluble guanylate cyclase (sGC) stimulator BAY41-2272 still reduced BP in MLK3-/- mice. MLK3 kinase inhibition with URMC-099 did not affect BP but induced LV dysfunction in mice. These data reveal MLK3 as a PKG1α substrate mediating PKG1α preservation of LV function but not acute PKG1α BP effects. Mechanistically, MLK3 kinase-dependent effects preserved LV function, whereas MLK3 kinase-independent signaling regulated BP. These findings suggest augmenting MLK3 kinase activity could preserve LV function in HF but avoid hypotension from PKG1α activation.

JNK signaling prevents biliary cyst formation through a CASPASE-8-dependent function of RIPK1 during aging.

The c-Jun N-terminal kinase (JNK) signaling pathway mediates adaptation to stress signals and has been associated with cell death, cell proliferation, and malignant transformation in the liver. However, up to now, its function was experimentally studied mainly in young mice. By generating mice with combined conditional ablation of Jnk1 and Jnk2 in liver parenchymal cells (LPCs) (JNK1/2LPC-KO mice; KO, knockout), we unraveled a function of the JNK pathway in the regulation of liver homeostasis during aging. Aging JNK1/2LPC-KO mice spontaneously developed large biliary cysts that originated from the biliary cell compartment. Mechanistically, we could show that cyst formation in livers of JNK1/2LPC-KO mice was dependent on receptor-interacting protein kinase 1 (RIPK1), a known regulator of cell survival, apoptosis, and necroptosis. In line with this, we showed that RIPK1 was overexpressed in the human cyst epithelium of a subset of patients with polycystic liver disease. Collectively, these data reveal a functional interaction between JNK signaling and RIPK1 in age-related progressive cyst development. Thus, they provide a functional linkage between stress adaptation and programmed cell death (PCD) in the maintenance of liver homeostasis during aging.

JUN Amino-Terminal Kinase 1 Signaling in the Proximal Tubule Causes Cell Death and Acute Renal Failure in Rat and Mouse Models of Renal Ischemia/Reperfusion Injury.

Activation of the JUN amino-terminal kinase (JNK) pathway is prominent in most forms of acute and progressive tubulointerstitial damage, including acute renal ischemia/reperfusion injury (IRI). Two forms of JNK, JNK1 and JNK2, are expressed in the kidney. Systemic administration of pan-JNK inhibitors suppresses renal IRI; however, the contribution of JNK1 versus JNK2, and the specific role of JNK activation in the proximal tubule in IRI, remains unknown. These questions were addressed in rat and mouse models of acute bilateral renal IRI. Administration of the JNK inhibitor, CC-930, substantially reduced the severity of renal failure, tubular damage, and inflammation at 24 hours in a rat IRI model. Additionally, Jnk1-/- mice, but not Jnk2-/- mice, were shown to be significantly protected against acute renal failure, tubular damage, and inflammation in the IRI model. Furthermore, mice with conditional Jnk1 deletion in the proximal tubule also showed considerable protection from IRI-induced renal failure, tubular damage, and inflammation. Finally, primary cultures of Jnk1-/-, but not Jnk2-/-, tubular epithelial cells were protected from oxidant-induced cell death, in association with preventing phosphorylation of proteins (receptor interacting serine/threonine kinase 3 and mixed lineage kinase domain-like pseudokinase) in the necroptosis pathway. In conclusion, JNK1, but not JNK2, plays a specific role in IRI-induced cell death in the proximal tubule, leading to acute renal failure.

A feed-forward regulatory loop in adipose tissue promotes signaling by the hepatokine FGF21.

The cJun NH2-terminal kinase (JNK) signaling pathway is activated by metabolic stress and promotes the development of metabolic syndrome, including hyperglycemia, hyperlipidemia, and insulin resistance. This integrated physiological response involves cross-talk between different organs. Here we demonstrate that JNK signaling in adipocytes causes an increased circulating concentration of the hepatokine fibroblast growth factor 21 (FGF21) that regulates systemic metabolism. The mechanism of organ crosstalk is mediated by a feed-forward regulatory loop caused by JNK-regulated FGF21 autocrine signaling in adipocytes that promotes increased expression of the adipokine adiponectin and subsequent hepatic expression of the hormone FGF21. The mechanism of organ cross-talk places circulating adiponectin downstream of autocrine FGF21 expressed by adipocytes and upstream of endocrine FGF21 expressed by hepatocytes. This regulatory loop represents a novel signaling paradigm that connects autocrine and endocrine signaling modes of the same hormone in different tissues.

Activation of the Unfolded Protein Response (UPR) Is Associated with Cholangiocellular Injury, Fibrosis and Carcinogenesis in an Experimental Model of Fibropolycystic Liver Disease.

Fibropolycystic liver disease is characterized by hyperproliferation of the biliary epithelium and the formation of multiple dilated cysts, a process associated with unfolded protein response (UPR). In the present study, we aimed to understand the mechanisms of cyst formation and UPR activation in hepatocytic c-Jun N-terminal kinase 1/2 (Jnk1/2) knockout mice. Floxed JNK1/2 (Jnkf/f) and Jnk∆hepa animals were sacrificed at different time points during progression of liver disease. Histological examination of specimens evidenced the presence of collagen fiber deposition, increased α-smooth muscle actin (αSMA), infiltration of CD45, CD11b and F4/80 cells and proinflammatory cytokines (Tnf, Tgfß1) and liver injury (e.g., ALT, apoptosis and Ki67-positive cells) in Jnk∆hepa compared with Jnkf/f livers from 32 weeks of age. This was associated with activation of effectors of the UPR, including BiP/GRP78, CHOP and spliced XBP1. Tunicamycin (TM) challenge strongly induced ER stress and fibrosis in Jnk∆hepa animals compared with Jnkf/f littermates. Finally, thioacetamide (TAA) administration to Jnk∆hepa mice induced UPR activation, peribiliary fibrosis, liver injury and markers of biliary proliferation and cholangiocarcinoma (CCA). Orthoallografts of DEN/CCl4-treated Jnk∆hepa liver tissue triggered malignant CCA. Altogether, these results suggest that activation of the UPR in conjunction with fibrogenesis might trigger hepatic cystogenesis and early stages of CCA.

Neutrophil infiltration regulates clock-gene expression to organize daily hepatic metabolism.

Liver metabolism follows diurnal fluctuations through the modulation of molecular clock genes. Disruption of this molecular clock can result in metabolic disease but its potential regulation by immune cells remains unexplored. Here, we demonstrated that in steady state, neutrophils infiltrated the mouse liver following a circadian pattern and regulated hepatocyte clock-genes by neutrophil elastase (NE) secretion. NE signals through c-Jun NH2-terminal kinase (JNK) inhibiting fibroblast growth factor 21 (FGF21) and activating Bmal1 expression in the hepatocyte. Interestingly, mice with neutropenia, defective neutrophil infiltration or lacking elastase were protected against steatosis correlating with lower JNK activation, reduced Bmal1 and increased FGF21 expression, together with decreased lipogenesis in the liver. Lastly, using a cohort of human samples we found a direct correlation between JNK activation, NE levels and Bmal1 expression in the liver. This study demonstrates that neutrophils contribute to the maintenance of daily hepatic homeostasis through the regulation of the NE/JNK/Bmal1 axis.

Every day, the body's biological processes work to an internal clock known as the circadian rhythm. This rhythm is controlled by 'clock genes' that are switched on or off by daily physical and environmental cues, such as changes in light levels. These daily rhythms are very finely tuned, and disturbances can lead to serious health problems, such as diabetes or high blood pressure. The ability of the body to cycle through the circadian rhythm each day is heavily influenced by the clock of one key organ: the liver. This organ plays a critical role in converting food and drink into energy. There is evidence that neutrophils ­ white blood cells that protect the body by being the first response to inflammation ­ can influence how the liver performs its role in obese people, by for example, releasing a protein called elastase. Additionally, the levels of neutrophils circulating in the blood change following a daily pattern. Crespo, González-Terán et al. wondered whether neutrophils enter the liver at specific times of the day to control liver's daily rhythm. Crespo, González-Terán et al. revealed that neutrophils visit the liver in a pattern that peaks when it gets light and dips when it gets dark by counting the number of neutrophils in the livers of mice at different times of the day. During these visits, neutrophils secreted elastase, which activated a protein called JNK in the cells of the mice's liver. This subsequently blocked the activity of another protein, FGF21, which led to the activation of the genes that allow cells to make fat molecules for storage. JNK activation also switched on the clock gene, Bmal1, ultimately causing fat to build up in the mice's liver. Crespo, González-Terán et al. also found that, in samples from human livers, the levels of elastase, the activity of JNK, and whether the Bmal1 gene was switched on were tightly linked. This suggests that neutrophils may be controlling the liver's rhythm in humans the same way they do in mice. Overall, this research shows that neutrophils can control and reset the liver's daily rhythm using a precisely co-ordinated series of molecular changes. These insights into the liver's molecular clock suggest that elastase, JNK and BmaI1 may represent new therapeutic targets for drugs or smart medicines to treat metabolic diseases such as diabetes or high blood pressure.

JNK-mediated disruption of bile acid homeostasis promotes intrahepatic cholangiocarcinoma.

Metabolic stress causes activation of the cJun NH2-terminal kinase (JNK) signal transduction pathway. It is established that one consequence of JNK activation is the development of insulin resistance and hepatic steatosis through inhibition of the transcription factor PPARα. Indeed, JNK1/2 deficiency in hepatocytes protects against the development of steatosis, suggesting that JNK inhibition represents a possible treatment for this disease. However, the long-term consequences of JNK inhibition have not been evaluated. Here we demonstrate that hepatic JNK controls bile acid production. We found that hepatic JNK deficiency alters cholesterol metabolism and bile acid synthesis, conjugation, and transport, resulting in cholestasis, increased cholangiocyte proliferation, and intrahepatic cholangiocarcinoma. Gene ablation studies confirmed that PPARα mediated these effects of JNK in hepatocytes. This analysis highlights potential consequences of long-term use of JNK inhibitors for the treatment of metabolic syndrome.

Loss of c-Jun N-terminal Kinase 1 and 2 Function in Liver Epithelial Cells Triggers Biliary Hyperproliferation Resembling Cholangiocarcinoma.

Targeted inhibition of the c-Jun N-terminal kinases (JNKs) has shown therapeutic potential in intrahepatic cholangiocarcinoma (CCA)-related tumorigenesis. However, the cell-type-specific role and mechanisms triggered by JNK in liver parenchymal cells during CCA remain largely unknown. Here, we aimed to investigate the relevance of JNK1 and JNK2 function in hepatocytes in two different models of experimental carcinogenesis, the dethylnitrosamine (DEN) model and in nuclear factor kappa B essential modulator (NEMO)hepatocyte-specific knockout (Δhepa) mice, focusing on liver damage, cell death, compensatory proliferation, fibrogenesis, and tumor development. Moreover, regulation of essential genes was assessed by reverse transcription polymerase chain reaction, immunoblottings, and immunostainings. Additionally, specific Jnk2 inhibition in hepatocytes of NEMOΔhepa/JNK1Δhepa mice was performed using small interfering (si) RNA (siJnk2) nanodelivery. Finally, active signaling pathways were blocked using specific inhibitors. Compound deletion of Jnk1 and Jnk2 in hepatocytes diminished hepatocellular carcinoma (HCC) in both the DEN model and in NEMOΔhepa mice but in contrast caused massive proliferation of the biliary ducts. Indeed, Jnk1/2 deficiency in hepatocytes of NEMOΔhepa (NEMOΔhepa/JNKΔhepa) animals caused elevated fibrosis, increased apoptosis, increased compensatory proliferation, and elevated inflammatory cytokines expression but reduced HCC. Furthermore, siJnk2 treatment in NEMOΔhepa/JNK1Δhepa mice recapitulated the phenotype of NEMOΔhepa/JNKΔhepa mice. Next, we sought to investigate the impact of molecular pathways in response to compound JNK deficiency in NEMOΔhepa mice. We found that NEMOΔhepa/JNKΔhepa livers exhibited overexpression of the interleukin-6/signal transducer and activator of transcription 3 pathway in addition to epidermal growth factor receptor (EGFR)-rapidly accelerated fibrosarcoma (Raf)-mitogen-activated protein kinase kinase (MEK)-extracellular signal-regulated kinase (ERK) cascade. The functional relevance was tested by administering lapatinib, which is a dual tyrosine kinase inhibitor of erythroblastic oncogene B-2 (ErbB2) and EGFR signaling, to NEMOΔhepa/JNKΔhepa mice. Lapatinib effectively inhibited cystogenesis, improved transaminases, and effectively blocked EGFR-Raf-MEK-ERK signaling. Conclusion: We define a novel function of JNK1/2 in cholangiocyte hyperproliferation. This opens new therapeutic avenues devised to inhibit pathways of cholangiocarcinogenesis.

Regulation of adipose tissue inflammation by interleukin 6.

Obesity is associated with a chronic state of low-grade inflammation and progressive tissue infiltration by immune cells and increased expression of inflammatory cytokines. It is established that interleukin 6 (IL6) regulates multiple aspects of metabolism, including glucose disposal, lipolysis, oxidative metabolism, and energy expenditure. IL6 is secreted by many tissues, but the role of individual cell types is unclear. We tested the role of specific cells using a mouse model with conditional expression of the Il6 gene. We found that IL6 derived from adipocytes increased, while IL6 derived from myeloid cells and muscle suppressed, macrophage infiltration of adipose tissue. These opposite actions were associated with a switch of IL6 signaling from a canonical mode (myeloid cells) to a noncanonical trans-signaling mode (adipocytes and muscle) with increased expression of the ADAM10/17 metalloprotease that promotes trans-signaling by the soluble IL6 receptor α. Collectively, these data demonstrate that the source of IL6 production plays a major role in the physiological regulation of metabolism.