Sublethal necroptosis signaling promotes inflammation and liver cancer.

It is currently not well known how necroptosis and necroptosis responses manifest in vivo. Here, we uncovered a molecular switch facilitating reprogramming between two alternative modes of necroptosis signaling in hepatocytes, fundamentally affecting immune responses and hepatocarcinogenesis. Concomitant necrosome and NF-κB activation in hepatocytes, which physiologically express low concentrations of receptor-interacting kinase 3 (RIPK3), did not lead to immediate cell death but forced them into a prolonged "sublethal" state with leaky membranes, functioning as secretory cells that released specific chemokines including CCL20 and MCP-1. This triggered hepatic cell proliferation as well as activation of procarcinogenic monocyte-derived macrophage cell clusters, contributing to hepatocarcinogenesis. In contrast, necrosome activation in hepatocytes with inactive NF-κB-signaling caused an accelerated execution of necroptosis, limiting alarmin release, and thereby preventing inflammation and hepatocarcinogenesis. Consistently, intratumoral NF-κB-necroptosis signatures were associated with poor prognosis in human hepatocarcinogenesis. Therefore, pharmacological reprogramming between these distinct forms of necroptosis may represent a promising strategy against hepatocellular carcinoma.

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.

Intravital Dynamic and Correlative Imaging of Mouse Livers Reveals Diffusion-Dominated Canalicular and Flow-Augmented Ductular Bile Flux.

BACKGROUND AND AIMS: Small-molecule flux in tissue microdomains is essential for organ function, but knowledge of this process is scant due to the lack of suitable methods. We developed two independent techniques that allow the quantification of advection (flow) and diffusion in individual bile canaliculi and in interlobular bile ducts of intact livers in living mice, namely fluorescence loss after photoactivation and intravital arbitrary region image correlation spectroscopy. APPROACH AND RESULTS: The results challenge the prevailing "mechano-osmotic" theory of canalicular bile flow. After active transport across hepatocyte membranes, bile acids are transported in the canaliculi primarily by diffusion. Only in the interlobular ducts is diffusion augmented by regulatable advection. Photoactivation of fluorescein bis-(5-carboxymethoxy-2-nitrobenzyl)-ether in entire lobules demonstrated the establishment of diffusive gradients in the bile canalicular network and the sink function of interlobular ducts. In contrast to the bile canalicular network, vectorial transport was detected and quantified in the mesh of interlobular bile ducts. CONCLUSIONS: The liver consists of a diffusion-dominated canalicular domain, where hepatocytes secrete small molecules and generate a concentration gradient and a flow-augmented ductular domain, where regulated water influx creates unidirectional advection that augments the diffusive flux.

Kupffer Cell-Derived Tnf Triggers Cholangiocellular Tumorigenesis through JNK due to Chronic Mitochondrial Dysfunction and ROS.

Intrahepatic cholangiocarcinoma (ICC) is a highly malignant, heterogeneous cancer with poor treatment options. We found that mitochondrial dysfunction and oxidative stress trigger a niche favoring cholangiocellular overgrowth and tumorigenesis. Liver damage, reactive oxygen species (ROS) and paracrine tumor necrosis factor (Tnf) from Kupffer cells caused JNK-mediated cholangiocellular proliferation and oncogenic transformation. Anti-oxidant treatment, Kupffer cell depletion, Tnfr1 deletion, or JNK inhibition reduced cholangiocellular pre-neoplastic lesions. Liver-specific JNK1/2 deletion led to tumor reduction and enhanced survival in Akt/Notch- or p53/Kras-induced ICC models. In human ICC, high Tnf expression near ICC lesions, cholangiocellular JNK-phosphorylation, and ROS accumulation in surrounding hepatocytes are present. Thus, Kupffer cell-derived Tnf favors cholangiocellular proliferation/differentiation and carcinogenesis. Targeting the ROS/Tnf/JNK axis may provide opportunities for ICC therapy.

Lineage fate of ductular reactions in liver injury and carcinogenesis.

Ductular reactions (DRs) are observed in virtually all forms of human liver disease; however, the histogenesis and function of DRs in liver injury are not entirely understood. It is widely believed that DRs contain bipotential liver progenitor cells (LPCs) that serve as an emergency cell pool to regenerate both cholangiocytes and hepatocytes and may eventually give rise to hepatocellular carcinoma (HCC). Here, we used a murine model that allows highly efficient and specific lineage labeling of the biliary compartment to analyze the histogenesis of DRs and their potential contribution to liver regeneration and carcinogenesis. In multiple experimental and genetic liver injury models, biliary cells were the predominant precursors of DRs but lacked substantial capacity to produce new hepatocytes, even when liver injuries were prolonged up to 12 months. Genetic modulation of NOTCH and/or WNT/ß-catenin signaling within lineage-tagged DRs impaired DR expansion but failed to redirect DRs from biliary differentiation toward the hepatocyte lineage. Further, lineage-labeled DRs did not produce tumors in genetic and chemical HCC mouse models. In summary, we found no evidence in our system to support mouse biliary-derived DRs as an LPC pool to replenish hepatocytes in a quantitatively relevant way in injury or evidence that DRs give rise to HCCs.

High susceptibility to fatty liver disease in two-pore channel 2-deficient mice.

Endolysosomal organelles play a key role in trafficking, breakdown and receptor-mediated recycling of different macromolecules such as low-density lipoprotein (LDL)-cholesterol, epithelial growth factor (EGF) or transferrin. Here we examine the role of two-pore channel (TPC) 2, an endolysosomal cation channel, in these processes. Embryonic mouse fibroblasts and hepatocytes lacking TPC2 display a profound impairment of LDL-cholesterol and EGF/EGF-receptor trafficking. Mechanistically, both defects can be attributed to a dysfunction of the endolysosomal degradation pathway most likely on the level of late endosome to lysosome fusion. Importantly, endolysosomal acidification or lysosomal enzyme function are normal in TPC2-deficient cells. TPC2-deficient mice are highly susceptible to hepatic cholesterol overload and liver damage consistent with non-alcoholic fatty liver hepatitis. These findings indicate reduced metabolic reserve of hepatic cholesterol handling. Our results suggest that TPC2 plays a crucial role in trafficking in the endolysosomal degradation pathway and, thus, is potentially involved in the homoeostatic control of many macromolecules and cell metabolites.

Canonical Notch2 signaling determines biliary cell fates of embryonic hepatoblasts and adult hepatocytes independent of Hes1.

UNLABELLED: Notch signaling through the Notch2 receptor is essential for normal biliary tubulogenesis during liver development. However, the signaling events downstream of Notch2 critical for this process are less well defined. Furthermore, whether Notch signaling also underlies adult hepatic cell fate decisions is largely unknown. By implementing different genetic mouse models, we provide a comprehensive analysis that defines the role of Notch in cell fate control in the developing and adult liver. We show that cell-specific activation of Notch2 signaling by a Notch2IC (N2IC) transgene leads to rapid biliary specification of embryonic hepatoblasts, but also-when expressed in up to 6-month-old adult livers-rapidly reprograms adult hepatocytes to biliary cells with formation of tubular-cystic structures. When directed specifically to the adult biliary and facultative liver progenitor cell compartment, Notch2 is capable of inducing a ductular reaction. Furthermore, we characterized the significance of key effectors of canonical Notch signaling during normal development and in N2IC-expressing models. We demonstrate that tubule formation of intrahepatic bile ducts during embryonic development as well as N2IC-induced specification and morphogenesis of embryonic hepatoblasts and biliary conversion of adult hepatocytes all critically rely on canonical Notch signaling via recombination signal binding protein (RBP)-Jκ but do not require Hes1. CONCLUSION: Notch2 appears to be the main determinant not only of biliary commitment of embryonic hepatoblasts during development but also of biliary reprogramming of adult hepatocytes. Notch2-dictated cell fates and morphogenesis in both embryonic hepatoblasts and adult hepatocytes rely on canonical Notch signaling but do not require Hes1. Adult liver cells possess a remarkable plasticity to assume new cell fates when embryonic signaling pathways are active. (HEPATOLOGY 2013).

Constitutive activity of TRPML2 and TRPML3 channels versus activation by low extracellular sodium and small molecules.

The transient receptor potential channels TRPML2 and TRPML3 (MCOLN2 and MCOLN3) are nonselective cation channels. They are widely expressed in mammals. However, little is known about their physiological function(s) and activation mechanism(s). TRPML3 can be activated or rather de-inhibited by exposing it first to sodium-free extracellular solution and subsequently to high extracellular sodium. TRPML3 can also be activated by a variety of small chemical compounds identified in a high throughput screen and is inhibited by low pH. Furthermore, it was found that TRPML3 is constitutively active in low or no sodium-containing extracellular solution. This constitutive activity is independent of the intracellular presence of sodium, and whole-cell current densities are similar with pipette solutions containing cesium, potassium, or sodium. Here, we present mutagenesis data generated based on the hypothesis that negatively charged amino acids in the extracellular loops of TRPML3 may interfere with the observed sodium inhibition. We systematically mutated negatively charged amino acids in the first and second extracellular loops and found that mutating Glu-361 in the second loop has a significant impact on the sodium-mediated block of TRPML3. We further demonstrate that the TRPML3-related cation channel TRPML2 is also activated by lowering the extracellular sodium concentration as well as by a subset of small chemical compounds that were previously identified as activators of TRPML3, thus confirming the functional activity of TRPML2 at the plasma membrane and suggesting similar gating mechanisms for both TRPML channels.

Gpr126 is essential for peripheral nerve development and myelination in mammals.

In peripheral nerves, Schwann cells form the myelin sheath that insulates axons and allows rapid propagation of action potentials. Although a number of regulators of Schwann cell development are known, the signaling pathways that control myelination are incompletely understood. In this study, we show that Gpr126 is essential for myelination and other aspects of peripheral nerve development in mammals. A mutation in Gpr126 causes a severe congenital hypomyelinating peripheral neuropathy in mice, and expression of differentiated Schwann cell markers, including Pou3f1, Egr2, myelin protein zero and myelin basic protein, is reduced. Ultrastructural studies of Gpr126-/- mice showed that axonal sorting by Schwann cells is delayed, Remak bundles (non-myelinating Schwann cells associated with small caliber axons) are not observed, and Schwann cells are ultimately arrested at the promyelinating stage. Additionally, ectopic perineurial fibroblasts form aberrant fascicles throughout the endoneurium of the mutant sciatic nerve. This analysis shows that Gpr126 is required for Schwann cell myelination in mammals, and defines new roles for Gpr126 in axonal sorting, formation of mature non-myelinating Schwann cells and organization of the perineurium.

Genetic inactivation of Trpml3 does not lead to hearing and vestibular impairment in mice.

TRPML3, a member of the transient receptor potential (TRP) family, is an inwardly rectifying, non-selective Ca2+-permeable cation channel that is regulated by extracytosolic Na+ and H+ and can be activated by a variety of small molecules. The severe auditory and vestibular phenotype of the TRPML3(A419P) varitint-waddler mutation made this protein particularly interesting for inner ear biology. To elucidate the physiological role of murine TRPML3, we conditionally inactivated Trpml3 in mice. Surprisingly, lack of functional TRPML3 did not lead to circling behavior, balance impairment or hearing loss.

Small molecule activators of TRPML3.

We conducted a high-throughput screen for small molecule activators of the TRPML3 ion channel, which, when mutated, causes deafness and pigmentation defects. Cheminformatics analyses of the 53 identified and confirmed compounds revealed nine different chemical scaffolds and 20 singletons. We found that agonists strongly potentiated TRPML3 activation with low extracytosolic [Na(+)]. This synergism revealed the existence of distinct and cooperative activation mechanisms and a wide dynamic range of TRPML3 activity. Testing compounds on TRPML3-expressing sensory hair cells revealed the absence of activator-responsive channels. Epidermal melanocytes showed only weak or no responses to the compounds. These results suggest that TRPML3 in native cells might be absent from the plasma membrane or that the protein is a subunit of heteromeric channels that are nonresponsive to the activators identified in this screen.

Life and death of sensory hair cells expressing constitutively active TRPML3.

The varitint-waddler mutation A419P renders TRPML3 constitutively active, resulting in cationic overload, particularly in sustained influx of Ca(2+). TRPML3 is expressed by inner ear sensory hair cells, and we were intrigued by the fact that hair cells are able to cope with expressing the TRPML3(A419P) isoform for weeks before they ultimately die. We hypothesized that the survival of varitint-waddler hair cells is linked to their ability to deal with Ca(2+) loads due to the abundance of plasma membrane calcium ATPases (PMCAs). Here, we show that PMCA2 significantly reduced [Ca(2+)](i) increase and apoptosis in HEK293 cells expressing TRPML3(A419P). The deaf-waddler isoform of PMCA2, operating at 30% efficacy, showed a significantly decreased ability to rescue the Ca(2+) loading of cells expressing TRPML3(A419P). When we combined mice heterozygous for the varitint-waddler mutant allele with mice heterozygous for the deaf-waddler mutant allele, we found severe hair bundle defects as well as increased hair cell loss compared with mice heterozygous for each mutant allele alone. Furthermore, 3-week-old double mutant mice lacked auditory brainstem responses, which were present in their respective littermates containing single mutant alleles. Likewise, heterozygous double mutant mice exhibited severe circling behavior, which was not observed in mice heterozygous for TRPML3(A419P) or PMCA2(G283S) alone. Our results provide a molecular rationale for the delayed hair cell loss in varitint-waddler mice. They also show that hair cells are able to survive for weeks with sustained Ca(2+) loading, which implies that Ca(2+) loading is an unlikely primary cause of hair cell death in ototoxic stress situations.

A helix-breaking mutation in TRPML3 leads to constitutive activity underlying deafness in the varitint-waddler mouse.

Homozygote varitint-waddler (Va) mice, expressing a mutant isoform (A419P) of TRPML3 (mucolipin 3), are profoundly deaf and display vestibular and pigmentation deficiencies, sterility, and perinatal lethality. Here we show that the varitint-waddler isoform of TRPML3 carrying an A419P mutation represents a constitutively active cation channel that can also be identified in native varitint-waddler hair cells as a distinct inwardly rectifying current. We hypothesize that the constitutive activation of TRPML3 occurs as a result of a helix-breaking proline substitution in transmembrane-spanning domain 5 (TM5). A proline substitution scan demonstrated that the inner third of TRPML3's TM5 is highly susceptible to proline-based kinks. Proline substitutions in TM5 of other TRP channels revealed that TRPML1, TRPML2, TRPV5, and TRPV6 display a similar susceptibility at comparable positions, whereas other TRP channels were not affected. We conclude that the molecular basis for deafness in the varitint-waddler mouse is the result of hair cell death caused by constitutive TRPML3 activity. To our knowledge, our study provides the first direct mechanistic link of a mutation in a TRP ion channel with mammalian hearing loss.

Receptor-induced activation of Drosophila TRP gamma by polyunsaturated fatty acids.

Cellular calcium homeostasis is regulated by hormones and neurotransmitters, resulting in the activation of a variety of proteins, in particular, channel proteins of the plasma membrane and of intracellular compartments. Such channels are, for example, TRP channels of the TRPC protein family that are activated by various mediators from receptor-stimulated signaling cascades. In Drosophila, two TRPC channels, TRP and TRPL, are involved in phototransduction. In addition, a third Drosophila TRPC channel, TRPgamma, has been identified and described as an auxiliary subunit of TRPL. Beyond it, our data show that heterologously expressed TRPgamma formed a receptor-activated, outwardly rectifying cation channel independent from TRPL co-expression. Analysis of the activation mechanism revealed that TRPgamma is activated by various polyunsaturated fatty acids generated in a phospholipase C- and phospholipase A(2)-dependent manner. The most potent activator of TRPgamma, the stable analogue of arachidonic acid, 5,8,11,14-eicosatetraynoic acid, induced currents in single channel recordings. Here we show that upon heterologous expression TRPgamma forms a homomeric channel complex that is activated by polyunsaturated fatty acids as mediators of receptor-dependent signaling pathways. Reverse transcription PCR analysis showed that TRPgamma is expressed in Drosophila heads and bodies. Its body-wide expression pattern and its activation mechanism suggest that TRPgamma forms a fly cation channel responsible for the regulation of intracellular calcium in a variety of hormonal signaling cascades.