Hendrika M. A. VanDongen

Hedgehog signaling regulates epithelial-mesenchymal transition during biliary fibrosis in rodents and humans

Epithelial-mesenchymal transitions (EMTs) play an important role in tissue construction during embryogenesis, and evidence suggests that this process may also help to remodel some adult tissues after injury. Activation of the hedgehog (Hh) signaling pathway regulates EMT during development. This pathway is also induced by chronic biliary injury, a condition in which EMT has been suggested to have a role. We evaluated the hypothesis that Hh signaling promotes EMT in adult bile ductular cells (cholangiocytes). In liver sections from patients with chronic biliary injury and in primary cholangiocytes isolated from rats that had undergone bile duct ligation (BDL), an experimental model of biliary fibrosis, EMT was localized to cholangiocytes with Hh pathway activity. Relief of ductal obstruction in BDL rats reduced Hh pathway activity, EMT, and biliary fibrosis. In mouse cholangiocytes, coculture with myofibroblastic hepatic stellate cells, a source of soluble Hh ligands, promoted EMT and cell migration. Addition of Hh-neutralizing antibodies to cocultures blocked these effects. Finally, we found that EMT responses to BDL were enhanced in patched-deficient mice, which display excessive activation of the Hh pathway. Together, these data suggest that activation of Hh signaling promotes EMT and contributes to the evolution of biliary fibrosis during chronic cholestasis.

Fate-Mapping Evidence that Hepatic Stellate Cells are Epithelial Progenitors in Adult Mouse Livers

Liver injury activates quiescent hepatic stellate cells (Q‐HSC) to proliferative myofibroblasts. Accumulation of myofibroblastic hepatic stellate cells (MF‐HSC) sometimes causes cirrhosis and liver failure. However, MF‐HSC also promote liver regeneration by producing growth factors for oval cells, bipotent progenitors of hepatocytes and cholangiocytes. Genes that are expressed by primary hepatic stellate cell (HSC) isolates overlap those expressed by oval cells, and hepatocytic and ductular cells emerge when HSC are cultured under certain conditions. We evaluated the hypothesis that HSC are a type of oval cell and, thus, capable of generating hepatocytes to regenerate injured livers. Because Q‐HSC express glial fibrillary acidic protein (GFAP), we crossed mice in which GFAP promoter elements regulated Cre‐recombinase with ROSA‐loxP‐stop‐loxP‐green fluorescent protein (GFP) mice to generate GFAP‐Cre/GFP double‐transgenic mice. These mice were fed methionine choline‐deficient, ethionine‐supplemented diets to activate and expand HSC and oval cell populations. GFP(+) progeny of GFAP‐expressing precursors were characterized by immunohistochemistry. Basal expression of mesenchymal markers was negligible in GFAP(+)Q‐HSC. When activated by liver injury or culture, HSC downregulated expression of GFAP but remained GFP(+); they became highly proliferative and began to coexpress markers of mesenchyme and oval cells. These transitional cells disappeared as GFP‐expressing hepatocytes emerged, began to express albumin, and eventually repopulated large areas of the hepatic parenchyma. Ductular cells also expressed GFAP and GFP, but their proliferative activity did not increase in this model. These findings suggest that HSC are a type of oval cell that transitions through a mesenchymal phase before differentiating into hepatocytes during liver regeneration.

Activation-Dependent Subconductance Levels in the drk1 K Channel Suggest a Subunit Basis for Ion Permeation and Gating

Ion permeation and channel opening are two fundamental properties of ion channels, the molecular bases of which are poorly understood. Channels can exist in two permeability states, open and closed. The relative amount of time a channel spends in the open conformation depends on the state of activation. In voltage-gated ion channels, activation involves movement of a charged voltage sensor, which is required for channel opening. Single-channel recordings of drk1 K channels expressed in Xenopus oocytes suggested that intermediate current levels (sublevels) may be associated with transitions between the closed and open states. Because K channels are formed by four identical subunits, each contributing to the lining of the pore, it was hypothesized that these sublevels resulted from heteromeric pore conformations. A formal model based on this hypothesis predicted that sublevels should be more frequently observed in partially activated channels, in which some but not all subunits have undergone voltage-dependent conformational changes required for channel opening. Experiments using the drk1 K channel, as well as drk1 channels with mutations in the pore and in the voltage sensor, showed that the probability of visiting a sublevel correlated with voltage- and time-dependent changes in activation. A subunit basis is proposed for channel opening and permeation in which these processes are coupled.

Hepatic accumulation of Hedgehog-reactive progenitors increases with severity of fatty liver damage in mice

Progenitors regenerate fatty livers but the mechanisms involved are uncertain. The Hedgehog pathway regulates mesendodermal progenitors and modulates mesenchymal–epithelial interactions during tissue remodeling. To determine if Hedgehog signaling increases in liver progenitors during fatty liver injury, we compared expression of Hedgehog ligands and target genes across a spectrum of injury. Leptin-deficient ob/ob mice with fatty livers and their healthy lean littermates were studied before and after exposure to the hepatotoxin, ethionine. At baseline, ob/ob mice had greater liver damage than controls. Ethionine induced liver injury in both ob/ob and lean mice, with greater injury occurring in ob/ob mice. After ethionine, the ob/ob mice developed liver atrophy and fibrosis. Liver injury increased hepatic accumulation of progenitors, including ductular cells that produced and responded to Hedgehog ligands. A dose–response relationship was demonstrated between liver injury and expansion of Hedgehog-responsive progenitors. In severely damaged, atrophic livers, nuclei in mature-appearing hepatocytes accumulated the Hedgehog-regulated mesenchymal transcription factor, Gli2 and lost expression of the liver epithelial transcription factor, hepatocyte nuclear factor 6 (HNF-6). Hepatic levels of collagen mRNA and pericellular collagen fibrils increased concomitantly. Hence, fatty liver injury increases Hedgehog activity in liver progenitors, and this might promote epithelial–mesenchymal transitions that result in liver fibrosis.

The Nonkinase Phorbol Ester Receptor α1-Chimerin Binds the NMDA Receptor NR2A Subunit and Regulates Dendritic Spine Density

Abnormalities in dendritic spines have long been associated with cognitive dysfunction and neurodevelopmental delay, whereas rapid changes in spine shape underlie synaptic plasticity. The key regulators of cytoskeletal reorganization in dendrites and spines are the Rho GTPases, which modify actin polymerization in response to synaptic signaling. Rho GTPase activity is modulated by multiple regulatory proteins, some of which have been found to associate with proteins localized to spines. Here, we show that the nonkinase phorbol ester receptor α1-chimerin is present in dendrites and spines, where it binds to the NMDA receptor NR2A subunit in a phorbol ester-dependent manner. α1-Chimerin contains a GTPase activating (GAP) domain, with activity toward the Rho family member Rac1. Overexpression of α1-chimerin in cultured hippocampal neurons inhibits formation of new spines and removes existing spines. This reduction in spine density is mediated by Rac1 inhibition, because it depends critically on the presence of a functional GAP domain. Conversely, depletion of α1-chimerin leads to an increase in spine density, indicating that a basal inhibition of Rac1 maintains the number of spines at a submaximal level. The ability of α1-chimerin to modulate spine number requires an interaction with the NMDA receptor, because an α1-chimerin mutant that binds weakly to NR2A fails to decrease spine density. Together, these results suggest that α1-chimerin is able to modulate dendritic spine morphology by binding to synaptic NMDA receptors and locally inactivating Rac1.

Arc/Arg3.1 Translation Is Controlled by Convergent N-Methyl-D-aspartate and Gs-coupled Receptor Signaling Pathways

Arc/Arg3.1 is an immediate early gene whose expression is necessary for the late-phase of long-term potentiation (LTP) and memory consolidation. Whereas pathways regulating Arc transcription have been extensively investigated, less is known about the role of post-transcriptional mechanisms in Arc expression. Fluorescence microscopy experiments in cultured hippocampal neurons revealed that Arc protein level was dramatically increased by activation of the cAMP-dependent protein kinase (PKA) pathway, which is implicated in long-term memory. A PKA-dependent increase in Arc protein level was observed after pharmacological or synaptic activation of N-methyl-d-aspartate (NMDA) receptors, which play a critical role in both LTP induction and learning. Arc protein was also up-regulated by activation of PKA through Gs-coupled dopamine and β-adrenergic receptors, which regulate the late-phase of LTP and memory. When agonists for the NMDA and Gs-coupled receptors were co-applied, they had an additive effect on Arc protein expression. Interestingly, Gs-coupled receptor stimulation was ineffective in the presence of an NMDA receptor antagonist, suggesting calcium influx through the NMDA receptor plays a gating role in this pathway. Stimulation of the cAMP/PKA pathway did not affect Arc mRNA level or protein stability, identifying translational efficacy as the main determinant of Arc protein expression level. It is concluded that efficient Arc translation requires NMDA receptor activity, whereas a further enhancement can be achieved with activation of Gs-coupled receptors. These experiments have, therefore, revealed remarkable similarities in the signaling pathways that control Arc expression and those that regulate LTP, learning, and memory.

Localization and Phosphorylation of Abl-Interactor Proteins, Abi-1 and Abi-2, in the Developing Nervous System

Abl-interactor (Abi) proteins are targets of Abl-family nonreceptor tyrosine kinases and are required for Rac-dependent cytoskeletal reorganization in response to growth factor stimulation. We asked if the expression, phosphorylation, and cellular localization of Abi-1 and Abi-2 supports a role for these proteins in Abl signaling in the developing and adult mouse nervous system. In mid- to late-gestation embryos, abi-2 message is elevated in the central and peripheral nervous systems (CNS and PNS). Abi-1 mRNA is present, but not enhanced, in the CNS, and is not observed in PNS structures. Abi proteins from brain lysates undergo changes in apparent molecular weight and phosphorylation with increasing age. In the postnatal brain, abi-1 and abi-2 are expressed most prominently in cortical layers populated by projection neurons. In cultured neurons, Abi-1 and Abi-2 are concentrated in puncta throughout the cell body and processes. Both Abi and Abl proteins are present in synaptosomes and growth cone particles. Therefore, the Abi adaptors exhibit proper expression patterns and subcellular localization to participate in Abl kinase signaling in the nervous system.

Atomic distance estimates from disulfides and high-affinity metal-binding sites in a K+ channel pore.

The pore of potassium channels is lined by four identical, highly conserved hairpin loops, symmetrically arranged around a central permeation pathway. Introduction of cysteines into the external mouth of the drk1 K channel pore resulted in the formation of disulfide bonds that were incompatible with channel function. Breaking these bonds restored function and resulted in a high-affinity Cd(2+)-binding site, indicating coordinated ligation by multiple sulfhydryls. Dimeric constructs showed that these disulfide bonds formed between subunits. These results impose narrow constraints on intersubunit atomic distances in the pore that strongly support a radial pore model. The data also suggest an important functional role for the outer mouth of the pore in gating or permeation.

Activity-regulated cytoskeleton-associated protein Arc/Arg3.1 binds to spectrin and associates with nuclear promyelocytic leukemia (PML) bodies

Activity-regulated cytoskeleton-associated protein (Arc/Arg3.1) is an immediate early gene, whose expression in the central nervous system is induced by specific patterns of synaptic activity. Arc is required for the late-phase of long-term potentiation (LTP) and memory consolidation, and has been implicated in AMPA receptor trafficking. Since Arcs molecular function remains incompletely understood, we have determined its subcellular localization in cultured hippocampal neurons and HEK 293T cells. Fluorescence microscopy experiments revealed that both endogenous and exogenous Arc protein was primarily found in the nucleus, where it concentrated in puncta associated with promyelocytic leukemia (PML) bodies, proposed sites of transcriptional regulation. Arc co-localized and interacted with the betaIV spectrin splice variant betaSpIVSigma5, a nuclear spectrin isoform associated with PML bodies and the nuclear matrix. A small region of Arc containing the coiled-coil domain is also restricted to beta-spectrin-positive puncta, while the isolated spectrin homology domain is diffusely localized. Finally, Arc and betaSpIVSigma5 synergistically increased the number of PML bodies. These results suggest that Arc functions as a spectrin-binding protein, forming a complex that may provide a role at sites of transcriptional regulation within the nucleus.

An alanine residue in the M3-M4 linker lines the glycine binding pocket of the N-methyl-D-aspartate receptor.

While attempting to map a central region in the M3-M4 linker of the N-methyl-D-aspartate receptor NR1 subunit, we found that mutation of a single position, Ala-714, greatly reduced the apparent affinity for glycine. Proximal N-glycosylation localized this region to the extracellular space. Glycine affinities of additional Ala-714 mutations correlated with side chain volume. Substitution of alanine 714 with cysteine did not alter glycine sensitivity, although this mutant was rapidly inhibited by dithionitrobenzoate. Glycine protected the A714C mutant from modification by dithionitrobenzoate, whereas the co-agonist L-glutamate was ineffective. These experiments place Ala-714 in the glycine binding pocket of the N-methyl-D-aspartate receptor, a determination not predicted by previous structural models based on bacterial periplasmic binding protein homology.