Amitava Mukherjee

An Autophagy-Enhancing Drug Promotes Degradation of Mutant α1-Antitrypsin Z and Reduces Hepatic Fibrosis

Correcting a Liver Problem The classical form of α1-antitrypsin (AT) deficiency is caused by a point mutation that alters the folding and causes intracellular aggregation of AT—an abundant liver-derived plasma glycoprotein. AT deficiency is the most common genetic cause of liver disease in childhood and can also lead to cirrhosis and/or hepatocellular carcinoma in adulthood. Carbamazepine is a drug known to be well tolerated in humans that enhances the intracellular degradation process known as autophagy. Now, Hidvegi et al. (p. 229, published online June 3; see the Perspective by Sifers) show that carbamazepine can reduce the severity of liver disease in a mouse model of AT deficiency by enhancing the degradation of misfolded accumulated AT. A mouse model of a human liver disease can be treated using a drug known to be well tolerated. In the classical form of α1-antitrypsin (AT) deficiency, a point mutation in AT alters the folding of a liver-derived secretory glycoprotein and renders it aggregation-prone. In addition to decreased serum concentrations of AT, the disorder is characterized by accumulation of the mutant α1-antitrypsin Z (ATZ) variant inside cells, causing hepatic fibrosis and/or carcinogenesis by a gain–of–toxic function mechanism. The proteasomal and autophagic pathways are known to mediate degradation of ATZ. Here we show that the autophagy-enhancing drug carbamazepine (CBZ) decreased the hepatic load of ATZ and hepatic fibrosis in a mouse model of AT deficiency–associated liver disease. These results provide a basis for testing CBZ, which has an extensive clinical safety profile, in patients with AT deficiency and also provide a proof of principle for therapeutic use of autophagy enhancers.

EphA2 Mediates Ligand-Dependent Inhibition and Ligand-Independent Promotion of Cell Migration and Invasion via a Reciprocal Regulatory Loop with Akt

Both pro- and antioncogenic properties have been attributed to EphA2 kinase. We report that a possible cause for this apparent paradox is diametrically opposite roles of EphA2 in regulating cell migration and invasion. While activation of EphA2 with its ligand ephrin-A1 inhibited chemotactic migration of glioma and prostate cancer cells, EphA2 overexpression promoted migration in a ligand-independent manner. Surprisingly, the latter effects required phosphorylation of EphA2 on serine 897 by Akt, and S897A mutation abolished ligand-independent promotion of cell motility. Ephrin-A1 stimulation of EphA2 negated Akt activation by growth factors and caused EphA2 dephosphorylation on S897. In human astrocytoma, S897 phosphorylation was correlated with tumor grades and Akt activation, suggesting that the Akt-EphA2 crosstalk may contribute to brain tumor progression.

The Coxsackievirus B 3Cpro Protease Cleaves MAVS and TRIF to Attenuate Host Type I Interferon and Apoptotic Signaling

The host innate immune response to viral infections often involves the activation of parallel pattern recognition receptor (PRR) pathways that converge on the induction of type I interferons (IFNs). Several viruses have evolved sophisticated mechanisms to attenuate antiviral host signaling by directly interfering with the activation and/or downstream signaling events associated with PRR signal propagation. Here we show that the 3Cpro cysteine protease of coxsackievirus B3 (CVB3) cleaves the innate immune adaptor molecules mitochondrial antiviral signaling protein (MAVS) and Toll/IL-1 receptor domain-containing adaptor inducing interferon-beta (TRIF) as a mechanism to escape host immunity. We found that MAVS and TRIF were cleaved in CVB3-infected cells in culture. CVB3-induced cleavage of MAVS and TRIF required the cysteine protease activity of 3Cpro, occurred at specific sites and within specialized domains of each molecule, and inhibited both the type I IFN and apoptotic signaling downstream of these adaptors. 3Cpro-mediated MAVS cleavage occurred within its proline-rich region, led to its relocalization from the mitochondrial membrane, and ablated its downstream signaling. We further show that 3Cpro cleaves both the N- and C-terminal domains of TRIF and localizes with TRIF to signalosome complexes within the cytoplasm. Taken together, these data show that CVB3 has evolved a mechanism to suppress host antiviral signal propagation by directly cleaving two key adaptor molecules associated with innate immune recognition.

Comparative RNAi Screening Reveals Host Factors Involved in Enterovirus Infection of Polarized Endothelial Monolayers

Enteroviruses, including coxsackievirus B (CVB) and poliovirus (PV), can access the CNS through the blood brain barrier (BBB) endothelium to cause aseptic meningitis. To identify cellular components required for CVB and PV infection of human brain microvascular endothelial cells, an inxa0vitro BBB model, we performed comparative RNAi screens and identified 117 genes that influenced infection. Whereas a large proportion of genes whose depletion enhanced infection (17 of 22) were broadly antienteroviral, only 46 of the 95 genes whose depletion inhibited infection were required by both CVB and PV and included components of cell signaling pathways such as adenylate cyclases. Downregulation of genes including Rab GTPases, Src tyrosine kinases, and tyrosine phosphatases displayed specificity in their requirement for either CVB or PV infection. These findings highlight the pathways hijacked by enteroviruses for entry and replication in the BBB endothelium, a specialized and clinically relevant cell type for these viruses.

Retinoic acid-induced gene-1 (RIG-I) associates with the actin cytoskeleton via caspase activation and recruitment domain-dependent interactions

The actin cytoskeleton serves as a barrier that protects mammalian cells from environmental pathogens such as bacteria, fungi, and viruses. Several components of antimicrobial signaling pathways have been shown to associate directly with the actin cytoskeleton, indicating that the cytoskeleton may also serve as a platform for immune-associated molecules. Here we report that retinoic acid-induced gene-I (RIG-I), an important viral RNA recognition molecule, is associated with the actin cytoskeleton and localizes predominantly to actin-enriched membrane ruffles in non-polarized epithelial cells. Subcellular localization and fractionation experiments revealed that the association between RIG-I and the actin cytoskeleton was mediated by its N-terminal caspase activation and recruitment domains (CARDs), which were necessary and sufficient to induce cytoskeletal association. We also show that RIG-I plays a role in cellular migration, as ectopic expression of RIG-I enhanced cellular migration in a wound healing assay and depletion of endogenous RIG-I significantly reduced wound healing. We further show that in both cultured intestinal epithelial cells (IEC) and human colon and small intestine biopsies, RIG-I is enriched at apico-lateral cell junctions and colocalizes with markers of the tight junction. Depolymerization of the actin cytoskeleton in polarized IEC led to the rapid relocalization of RIG-I and to the induction of type I interferon signaling. These data provide evidence that RIG-I is associated with the actin cytoskeleton in non-polarized epithelial cells and with the junctional complex in polarized IECs and human intestine and colon biopsies and may point to a physiological role for RIG-I in the regulation of cellular migration.

Retinoic acid-induced gene-I (RIG-I) associates with nucleotide-binding oligomerization domain-2 (NOD2) to negatively regulate inflammatory signaling.

Cytoplasmic caspase recruiting domain (CARD)-containing molecules often function in the induction of potent antimicrobial responses in order to protect mammalian cells from invading pathogens. Retinoic acid-induced gene-I (RIG-I) and nucleotide binding oligomerization domain 2 (NOD2) serve as key factors in the detection of viral and bacterial pathogens, and in the subsequent initiation of innate immune signals to combat infection. RIG-I and NOD2 share striking similarities in their cellular localization, both localize to membrane ruffles in non-polarized epithelial cells and both exhibit a close association with the junctional complex of polarized epithelia. Here we show that RIG-I and NOD2 not only colocalize to cellular ruffles and cell-cell junctions, but that they also form a direct interaction that is mediated by the CARDs of RIG-I and multiple regions of NOD2. Moreover, we show that RIG-I negatively regulates ligand-induced nuclear factor-κB (NF-κB) signaling mediated by NOD2, and that NOD2 negatively regulates type I interferon induction by RIG-I. We also show that the three main Crohn disease-associated mutants of NOD2 (1007fs, R702W, G908R) form an interaction with RIG-I and negatively regulate its signaling to a greater extent than wild-type NOD2. Our results show that in addition to their role in innate immune recognition, RIG-I and NOD2 form a direct interaction at actin-enriched sites within cells and suggest that this interaction may impact RIG-I- and NOD2-dependent innate immune signaling.

Podocyte Injury Induces Nuclear Translocation of WTIP via Microtubule-dependent Transport

Podocyte structural and transcriptional phenotype plasticity characterizes glomerular injury. Transcriptional activity of WT1 (Wilms tumor 1) is required for normal podocyte structure and is repressed by the podocyte adherens junction protein, WTIP (WT1 interacting protein). Here we show that WTIP translocated into podocyte nuclei in lipopolysaccharide (LPS)-treated mice, a model of transient nephrotic syndrome. Cultured podocytes, which stably expressed an epitope-tagged WTIP, were treated with LPS. Imaging and cellular fractionation studies demonstrated that WTIP translocated from podocyte cell contacts into nuclei within 6 h and relocalized to cell contacts within 24 h after LPS treatment. LPS-stimulated WTIP nuclear translocation required JNK activity, which assembled a multiprotein complex of the scaffolding protein JNK-interacting protein 3 and the molecular motor dynein. Intact microtubule networks and dynein activity were necessary for LPS-stimulated WTIP translocation. Podocytes expressing sh-Wtip change morphology and demonstrate altered actin assembly in cell spreading assays. Stress signaling pathways initiate WTIP nuclear translocation, and the concomitant loss of WTIP from cell contacts changes podocyte morphology and dynamic actin assembly, suggesting a mechanism that transmits changes in podocyte morphology to the nucleus.

Math6 expression during kidney development and altered expression in a mouse model of glomerulosclerosis

Math6 is a tissue‐restricted member of the Atonal family of basic helix–loop–helix (bHLH) transcription factors and has been implicated in specification and differentiation of cell lineages in the brain. We identify here Math6 as a podocyte‐expressed bHLH protein that was down‐regulated in human immunodeficiency virus–associated nephropathy (HIVAN); a collapsing glomerulopathy characterized by podocyte dedifferentiation. Early in metanephric development, Math6 was expressed in metanephric mesenchyme but not ureteric bud‐derived cells, with overall Math6 expression most abundant in the nephrogenic zone, including developing glomeruli. In adult kidney, Math6 expression was restricted to podocytes. In adult podocyte cell lines and kidneys from the transgenic mouse model of HIVAN, Math6 podocyte expression was reduced concurrent with previously reported reductions in Nephrin and Synaptopodin expression, suggesting a correlation between the loss of Math6 expression and typical podocyte terminal differentiation markers. These studies suggest that Math6 may participate in kidney development and may be a permissive factor for podocyte differentiation. Developmental Dynamics 235:3102–3109, 2006.

Pancreatic lipase-related protein-2 (PLRP2) can contribute to dietary fat digestion in human newborns

In newborn mice, PLRP2 is essential for fat digestion. In human infants, the role of PLRP2 in fat digestion is unclear, as it has poor activity against long-chain triglycerides in vitro. Also, many infants carry a genetic polymorphism resulting in a truncated protein, PLRP2 W340X, which may impact function significantly. We re-examined the properties of recombinant human PLRP2 and studied the impact of W340X mutation on its function. In the presence of bile salt micelles and colipase, human PLRP2 hydrolyzed long-chain tri-, di-, and monoglycerides. It hydrolyzed triolein at a level much lower than that of pancreatic triglyceride lipase, but close to that of carboxyl ester lipase, after a long lag phase, which could be eliminated by the addition of oleic acids. Human PLRP2 W340X was poorly secreted and largely retained inside the cell. The retention of the mutant protein triggered endoplasmic reticulum stress and unfolded protein responses. Our results show that earlier studies underestimated human PLRP2 activity against triolein by employing suboptimal assay conditions. In vivo, dietary fat emulsions contain fatty acids as a result of the action of gastric lipase. Consequently, PLRP2 can contribute to fat digestion during early infancy. Furthermore, infants with homozygous W340X alleles will not secrete functional PLRP2 and may have inefficient dietary fat digestion, particularly when breastfeeding is unavailable. Additionally, the aberrant folding of W340X mutant may cause chronic cellular stress and increase susceptibility of pancreatic exocrine cells to other metabolic stressors.

WT1-interacting protein (Wtip) regulates podocyte phenotype by cell-cell and cell-matrix contact reorganization.

Podocytes respond to environmental cues by remodeling their slit diaphragms and cell-matrix adhesive junctions. Wt1-interacting protein (Wtip), an Ajuba family LIM domain scaffold protein expressed in the podocyte, coordinates cell adhesion changes and transcriptional responses to regulate podocyte phenotypic plasticity. We evaluated effects of Wtip on podocyte cell-cell and cell-matrix contact organization using gain-of- and loss-of-function methods. Endogenous Wtip targeted to focal adhesions in adherent but isolated podocytes and then shifted to adherens junctions after cells made stable, homotypic contacts. Podocytes with Wtip knockdown (shWtip) adhered but failed to spread normally. Noncontacted shWtip podocytes did not assemble actin stress fibers, and their focal adhesions failed to mature. As shWtip podocytes established cell-cell contacts, stable adherens junctions failed to form and F-actin structures were disordered. In shWtip cells, cadherin and β-catenin clustered in irregularly distributed spots that failed to laterally expand. Cell surface biotinylation showed diminished plasma membrane cadherin, β-catenin, and α-catenin in shWtip podocytes, although protein expression was similar in shWtip and control cells. Since normal actin dynamics are required for organization of adherens junctions and focal adhesions, we determined whether Wtip regulates F-actin assembly. Undifferentiated podocytes did not elaborate F-actin stress fibers, but when induced to overexpress WTIP, formed abundant stress fibers, a process blocked by the RhoA inhibitor C3 toxin and a RhoA kinase inhibitor. WTIP directly interacted with Rho guanine nucleotide exchange factor (GEF) 12 (Arhgef12), a RhoA-specific GEF enriched in the glomerulus. In conclusion, stable assembly of podocyte adherens junctions and cell-matrix contacts requires Wtip, a process that may be mediated by spatiotemporal regulation of RhoA activity through appropriate targeting of Arhgef12.