Publications by authors named "Jeffrey L Wrana"

142 Publications

The hippo pathway: A master regulator of liver metabolism, regeneration, and disease.

FASEB J 2021 May;35(5):e21570

Department of Medicine, Multi-Organ Transplant Program, Toronto General Hospital, Toronto, ON, Canada.

The liver is the only visceral organ in the body with a tremendous capacity to regenerate in response to insults that induce inflammation, cell death, and injury. Liver regeneration is a complicated process involving a well-orchestrated activation of non-parenchymal cells in the injured area and proliferation of undamaged hepatocytes. Furthermore, the liver has a Hepatostat, defined as adjustment of its volume to that required for homeostasis. Understanding the mechanisms that control different steps of liver regeneration is critical to informing therapies for liver repair, to help patients with liver disease. The Hippo signaling pathway is well known for playing an essential role in the control and regulation of liver size, regeneration, stem cell self-renewal, and liver cancer. Thus, the Hippo pathway regulates dynamic cell fates in liver, and in absence of its downstream effectors YAP and TAZ, liver regeneration is severely impaired, and the proliferative expansion of liver cells blocked. We will mainly review upstream mechanisms activating the Hippo signaling pathway following partial hepatectomy in mouse model and patients, its roles during different steps of liver regeneration, metabolism, and cancer. We will also discuss how targeting the Hippo signaling cascade might improve liver regeneration and suppress liver tumorigenesis.
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http://dx.doi.org/10.1096/fj.202002284RRDOI Listing
May 2021

A multiplexed, next generation sequencing platform for high-throughput detection of SARS-CoV-2.

Nat Commun 2021 03 3;12(1):1405. Epub 2021 Mar 3.

Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, M5G 1X5, ON, Canada.

Population scale sweeps of viral pathogens, such as SARS-CoV-2, require high intensity testing for effective management. Here, we describe "Systematic Parallel Analysis of RNA coupled to Sequencing for Covid-19 screening" (C19-SPAR-Seq), a multiplexed, scalable, readily automated platform for SARS-CoV-2 detection that is capable of analyzing tens of thousands of patient samples in a single run. To address strict requirements for control of assay parameters and output demanded by clinical diagnostics, we employ a control-based Precision-Recall and Receiver Operator Characteristics (coPR) analysis to assign run-specific quality control metrics. C19-SPAR-Seq coupled to coPR on a trial cohort of several hundred patients performs with a specificity of 100% and sensitivity of 91% on samples with low viral loads, and a sensitivity of >95% on high viral loads associated with disease onset and peak transmissibility. This study establishes the feasibility of employing C19-SPAR-Seq for the large-scale monitoring of SARS-CoV-2 and other pathogens.
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http://dx.doi.org/10.1038/s41467-021-21653-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7930244PMC
March 2021

A novel negative regulatory mechanism of Smurf2 in BMP/Smad signaling in bone.

Bone Res 2020 Nov 23;8(1):41. Epub 2020 Nov 23.

Department of Molecular Medicine for Pathogenesis, Ehime University Graduate School of Medicine, 454 Shitsukawa, Toon, Ehime, 791-0295, Japan.

Transforming growth factor-β (TGF-β) and bone morphogenetic protein (BMP) play important roles in bone metabolism. Smad ubiquitination regulatory factors (Smurfs) regulate TGF-β/BMP signaling via ubiquitination, resulting in degradation of signaling molecules to prevent excessive activation of TGF-β/BMP signaling. Though Smurf2 has been shown to negatively regulate TGF-β/Smad signaling, its involvement in BMP/Smad signaling in bone metabolism has not been thoroughly investigated. In the present study, we sought to evaluate the role of Smurf2 in BMP/Smad signaling in bone metabolism. Absorbable collagen sponges containing 3 μg of recombinant human BMP2 (rhBMP2) were implanted in the dorsal muscle pouches of wild type (WT) and Smurf2 mice. The rhBMP2-induced ectopic bone in Smurf2 mice showed greater bone mass, higher mineral apposition and bone formation rates, and greater osteoblast numbers than the ectopic bone in WT mice. In WT mice, the ectopic bone consisted of a thin discontinuous outer cortical shell and scant inner trabecular bone. In contrast, in Smurf2 mice, the induced bone consisted of a thick, continuous outer cortical shell and abundant inner trabecular bone. Additionally, rhBMP2-stimulated bone marrow stromal cells (BMSCs) from Smurf2 mice showed increased osteogenic differentiation. Smurf2 induced the ubiquitination of Smad1/5. BMP/Smad signaling was enhanced in Smurf2 BMSCs stimulated with rhBMP2, and the inhibition of BMP/Smad signaling suppressed osteogenic differentiation of these BMSCs. These findings demonstrate that Smurf2 negatively regulates BMP/Smad signaling, thereby identifying a new regulatory mechanism in bone metabolism.
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http://dx.doi.org/10.1038/s41413-020-00115-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7680794PMC
November 2020

An endogenous PI3K interactome promoting astrocyte mediated neuroprotection identifies a novel association with RNA binding protein ZC3H14.

J Biol Chem 2020 Nov 24. Epub 2020 Nov 24.

Vision Sciences, Krembil Research Institute, University Health Network, Canada.

Astrocytes can support neuronal survival through a range of secreted signals that protect against neurotoxicity, oxidative stress, and apoptotic cascades. Thus, analyzing the effects of the astrocyte secretome may provide valuable insight into these neuroprotective mechanisms. Previously, we characterized a potent neuroprotective activity mediated by retinal astrocyte conditioned media (ACM) on retinal and cortical neurons in metabolic stress models. However, the molecular mechanism underlying this complex activity in neuronal cells has remained unclear. Here, a chemical genetics screen of kinase inhibitors revealed phosphoinositide 3-kinase (PI3K) as a central player transducing ACM-mediated neuroprotection. To identify additional proteins contributing to the protective cascade, endogenous PI3K was immunoprecipitated from neuronal cells exposed to ACM or control media, followed by MS/MS proteomic analyses. These data pointed toward a relatively small number of proteins that co-immunoprecipitated with PI3K, and surprisingly only five were regulated by the ACM signal. These hits included expected PI3K interactors, such as the platelet-derived growth factor receptor A (PDGFRA), as well as novel RNA binding protein (RBP) interactors ZC3H14 (zinc finger CCCH-type containing 14) and THOC1 (THO complex protein 1). In particular, ZC3H14 has recently emerged as an important RBP with multiple roles in post-transcriptional regulation. In validation studies, we show that PI3K recruitment of ZC3H14 is necessary for PDGF-induced neuroprotection, and that this interaction is present in primary retinal ganglion cells. Thus, we identified a novel non-cell autonomous neuroprotective signaling cascade mediated through PI3K that requires recruitment of ZC3H14 and may present a promising strategy to promote astrocyte-secreted pro-survival signals.
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http://dx.doi.org/10.1074/jbc.RA120.015389DOI Listing
November 2020

Persistence of serum and saliva antibody responses to SARS-CoV-2 spike antigens in COVID-19 patients.

Sci Immunol 2020 10;5(52)

Lunenfeld-Tanenbaum Research Institute at Mount Sinai Hospital, Sinai Health System, Toronto, ON, Canada.

While the antibody response to SARS-CoV-2 has been extensively studied in blood, relatively little is known about the antibody response in saliva and its relationship to systemic antibody levels. Here, we profiled by enzyme-linked immunosorbent assays (ELISAs) IgG, IgA and IgM responses to the SARS-CoV-2 spike protein (full length trimer) and its receptor-binding domain (RBD) in serum and saliva of acute and convalescent patients with laboratory-diagnosed COVID-19 ranging from 3-115 days post-symptom onset (PSO), compared to negative controls. Anti-SARS-CoV-2 antibody responses were readily detected in serum and saliva, with peak IgG levels attained by 16-30 days PSO. Longitudinal analysis revealed that anti-SARS-CoV-2 IgA and IgM antibodies rapidly decayed, while IgG antibodies remained relatively stable up to 105 days PSO in both biofluids. Lastly, IgG, IgM and to a lesser extent IgA responses to spike and RBD in the serum positively correlated with matched saliva samples. This study confirms that serum and saliva IgG antibodies to SARS-CoV-2 are maintained in the majority of COVID-19 patients for at least 3 months PSO. IgG responses in saliva may serve as a surrogate measure of systemic immunity to SARS-CoV-2 based on their correlation with serum IgG responses.
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http://dx.doi.org/10.1126/sciimmunol.abe5511DOI Listing
October 2020

Overexpression of the Severe Acute Respiratory Syndrome Coronavirus-2 Receptor, Angiotensin-Converting Enzyme 2, in Diabetic Kidney Disease: Implications for Kidney Injury in Novel Coronavirus Disease 2019.

Can J Diabetes 2021 Mar 18;45(2):162-166.e1. Epub 2020 Jul 18.

Keenan Research Centre for Biomedical Science, Li Ka Shing Knowledge Institute, Unity Health Toronto, St. Michael's Hospital, Toronto, Ontario, Canada.

Objectives: Diabetes is associated with adverse outcomes, including death, after coronavirus disease 19 (COVID-19) infection. Beyond the lungs, Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2), the etiologic agent of the COVID-19 pandemic, can infect a range of other tissues, including the kidney, potentially contributing to acute kidney injury in those with severe disease. We hypothesized that the renal abundance of angiotensin-converting enzyme (ACE) 2, the cell surface receptor for SARS-CoV-2, may be modulated by diabetes and agents that block the renin-angiotensin-aldosterone system (RAAS).

Methods: The expression of ACE 2 was examined in 49 archival kidney biopsies from patients with diabetic kidney disease and from 12 healthy, potential living allograft donors using next-generation sequencing technology (RNA Seq).

Results: Mean ACE 2 messenger RNA was increased approximately 2-fold in diabetes when compared with healthy control subjects (mean ± SD, 13.2±7.9 vs 7.7±3.6 reads per million reads, respectively; p=0.001). No difference in transcript abundance was noted between recipients and nonrecipients of agents that block the RAAS (12.2±6.7 vs 16.2±10.7 reads per million reads, respectively; p=0.25).

Conclusions: Increased ACE 2 messenger RNA in the diabetic kidney may increase the risk and/or severity of kidney infection with SARS-CoV-2 in the setting of COVID-19 disease. Further studies are needed to ascertain whether this diabetes-related overexpression is generalizable to other tissues, most notably the lungs.
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http://dx.doi.org/10.1016/j.jcjd.2020.07.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7368650PMC
March 2021

Modeling the Control of TGF-β/Smad Nuclear Accumulation by the Hippo Pathway Effectors, Taz/Yap.

iScience 2020 Aug 29;23(8):101416. Epub 2020 Jul 29.

Department of Biochemistry, University of Toronto, Toronto, ON M5S 1A8, Canada; Donnelly Centre, University of Toronto, Toronto, ON M5S 3E1, Canada. Electronic address:

Integration of transforming growth factor β (TGF-β) signals with those of other pathways allows for precise temporal and spatial control of gene expression patterns that drive development and homeostasis. The Hippo pathway nuclear effectors, Taz/Yap, interact with the TGF-β transcriptional mediators, Smads, to control Smad activity. Key to TGF-β signaling is the nuclear localization of Smads. Thus, to investigate the role of Taz/Yap in Smad nuclear accumulation, we developed mathematical models of Hippo and TGF-β cross talk. The models were based on experimental measurements of TGF-β-induced changes in Taz/Yap and Smad subcellular localization obtained using high-throughput immunofluorescence (IF) imaging in the mouse mammary epithelial cell line, EpH4. Bayesian MCMC DREAM parameter estimation was used to quantify the uncertainty in estimates of the kinetic parameters. Variation of the model parameters and statistical analysis show that our modeling predicts that Taz/Yap can alter TGF-β receptor activity and directly or indirectly act as nuclear retention factors.
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http://dx.doi.org/10.1016/j.isci.2020.101416DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7452192PMC
August 2020

Functional genomics identifies new synergistic therapies for retinoblastoma.

Oncogene 2020 07 22;39(31):5338-5357. Epub 2020 Jun 22.

Lunenfeld Tanenbaum Research Institute, Mount Sinai Hospital, Sinai Health, Toronto, ON, Canada.

Local intravitreal or intra-arterial chemotherapy has improved therapeutic success for the pediatric cancer retinoblastoma (RB), but toxicity remains a major caveat. RB initiates primarily with RB1 loss or, rarely, MYCN amplification, but the critical downstream networks are incompletely understood. We set out to uncover perturbed molecular hubs, identify synergistic drug combinations to target these vulnerabilities, and expose and overcome drug resistance. We applied dynamic transcriptomic analysis to identify network hubs perturbed in RB versus normal fetal retina, and performed in vivo RNAi screens in RB1 and RB1;MYCN orthotopic xenografts to pinpoint essential hubs. We employed in vitro and in vivo studies to validate hits, define mechanism, develop new therapeutic modalities, and understand drug resistance. We identified BRCA1 and RAD51 as essential for RB cell survival. Their oncogenic activity was independent of BRCA1 functions in centrosome, heterochromatin, or ROS regulation, and instead linked to DNA repair. RAD51 depletion or inhibition with the small molecule inhibitor, B02, killed RB cells in a Chk1/Chk2/p53-dependent manner. B02 further synergized with clinically relevant topotecan (TPT) to engage this pathway, activating p53-BAX mediated killing of RB but not human retinal progenitor cells. Paradoxically, a B02/TPT-resistant tumor exhibited more DNA damage than sensitive RB cells. Resistance reflected dominance of the p53-p21 axis, which mediated cell cycle arrest instead of death. Deleting p21 or applying the BCL2/BCL2L1 inhibitor Navitoclax re-engaged the p53-BAX axis, and synergized with B02, TPT or both to override resistance. These data expose new synergistic therapies to trigger p53-induced killing in diverse RB subtypes.
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http://dx.doi.org/10.1038/s41388-020-1372-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7391301PMC
July 2020

TNFAIP8 controls murine intestinal stem cell homeostasis and regeneration by regulating microbiome-induced Akt signaling.

Nat Commun 2020 05 22;11(1):2591. Epub 2020 May 22.

Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, 19104, USA.

The intestine is a highly dynamic environment that requires tight control of the various inputs to maintain homeostasis and allow for proper responses to injury. It was recently found that the stem cell niche and epithelium is regenerated after injury by de-differentiated adult cells, through a process that gives rise to Sca1+ fetal-like cells and is driven by a transient population of Clu revival stem cells (revSCs). However, the molecular mechanisms that regulate this dynamic process have not been fully defined. Here we show that TNFAIP8 (also known as TIPE0) is a regulator of intestinal homeostasis that is vital for proper regeneration. TIPE0 functions through inhibiting basal Akt activation by the commensal microbiota via modulating membrane phospholipid abundance. Loss of TIPE0 in mice results in injury-resistant enterocytes, that are hyperproliferative, yet have regenerative deficits and are shifted towards a de-differentiated state. Tipe0 enterocytes show basal induction of the Clu regenerative program and a fetal gene expression signature marked by Sca1, but upon injury are unable to generate Sca-1/Clu revSCs and could not regenerate the epithelium. This work demonstrates the role of TIPE0 in regulating the dynamic signaling that determines the injury response and enables intestinal epithelial cell regenerative plasticity.
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http://dx.doi.org/10.1038/s41467-020-16379-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7244529PMC
May 2020

Robust production of uniform human cerebral organoids from pluripotent stem cells.

Life Sci Alliance 2020 05 17;3(5). Epub 2020 Apr 17.

Department of Biochemistry, University of Toronto, Toronto, Canada

Human cerebral organoid (hCO) models offer the opportunity to understand fundamental processes underlying human-specific cortical development and pathophysiology in an experimentally tractable system. Although diverse methods to generate brain organoids have been developed, a major challenge has been the production of organoids with reproducible cell type heterogeneity and macroscopic morphology. Here, we have directly addressed this problem by establishing a robust production pipeline to generate morphologically consistent hCOs and achieve a success rate of >80%. These hCOs include both a radial glial stem cell compartment and electrophysiologically competent mature neurons. Moreover, we show using immunofluorescence microscopy and single-cell profiling that individual organoids display reproducible cell type compositions that are conserved upon extended culture. We expect that application of this method will provide new insights into brain development and disease processes.
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http://dx.doi.org/10.26508/lsa.202000707DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7167289PMC
May 2020

Gaining Insights into the Function of Post-Translational Protein Modification Using Genome Engineering and Molecular Cell Biology.

J Mol Biol 2019 09 12;431(19):3920-3932. Epub 2019 Jul 12.

Institute for Molecular Health Sciences, ETH Zurich, Switzerland. Electronic address:

Modifications by kinases are a fast and reversible mechanism to diversify the function of the targeted proteins. The OCT4 transcription factor is essential for preimplantation development and pluripotency of embryonic stem cells (ESC), and its activity is tightly regulated by post-transcriptional modifications. Several phosphorylation sites have been identified by systemic approaches and their functions proposed. Here, we combined molecular and cellular biology with CRISPR/Cas9-mediated genome engineering to pinpoint the function of serine 12 of OCT4 in ESCs. Using chemical inhibitors and an antibody specific to OCT4 phosphorylated on S12, we identified cyclin-dependent kinase (CDK) 7 as upstream kinase. Surprisingly, generation of isogenic mESCs that endogenously ablate S12 revealed no effects on pluripotency and self-renewal, potentially due to compensation by other phosphorylation events. Our approach reveals that modification of distinct amino acids by precise genome engineering can help to clarify the functions of post-translational modifications on proteins encoded by essential gene in an endogenous context.
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http://dx.doi.org/10.1016/j.jmb.2019.07.015DOI Listing
September 2019

Atypical function of a centrosomal module in WNT signalling drives contextual cancer cell motility.

Nat Commun 2019 05 29;10(1):2356. Epub 2019 May 29.

Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, ON, M5G 1X5, Canada.

Centrosomes control cell motility, polarity and migration that is thought to be mediated by their microtubule-organizing capacity. Here we demonstrate that WNT signalling drives a distinct form of non-directional cell motility that requires a key centrosome module, but not microtubules or centrosomes. Upon exosome mobilization of PCP-proteins, we show that DVL2 orchestrates recruitment of a CEP192-PLK4/AURKB complex to the cell cortex where PLK4/AURKB act redundantly to drive protrusive activity and cell motility. This is mediated by coordination of formin-dependent actin remodelling through displacement of cortically localized DAAM1 for DAAM2. Furthermore, abnormal expression of PLK4, AURKB and DAAM1 is associated with poor outcomes in breast and bladder cancers. Thus, a centrosomal module plays an atypical function in WNT signalling and actin nucleation that is critical for cancer cell motility and is associated with more aggressive cancers. These studies have broad implications in how contextual signalling controls distinct modes of cell migration.
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http://dx.doi.org/10.1038/s41467-019-10241-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6541620PMC
May 2019

Single-cell transcriptomes of the regenerating intestine reveal a revival stem cell.

Nature 2019 05 24;569(7754):121-125. Epub 2019 Apr 24.

Centre for Systems Biology, Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada.

The turnover of the intestinal epithelium is driven by multipotent LGR5 crypt-base columnar cells (CBCs) located at the bottom of crypt zones. However, CBCs are lost following injury, such as irradiation, but the intestinal epithelium is nevertheless able to recover. Thus, a second population of quiescent '+4' cells, or reserve stem cells (RSCs), has previously been proposed to regenerate the damaged intestine. Although CBCs and RSCs were thought to be mutually exclusive, subsequent studies have found that LGR5 CBCs express RSC markers and that RSCs were dispensable-whereas LGR5 cells were essential-for repair of the damaged intestine. In addition, progenitors of absorptive enterocytes, secretory cells and slow cycling LGR5 cells have been shown to contribute to regeneration whereas the transcriptional regulator YAP1, which is important for intestinal regeneration, was suggested to induce a pro-survival phenotype in LGR5 cells. Thus, whether cellular plasticity or distinct cell populations are critical for intestinal regeneration remains unknown. Here we applied single-cell RNA sequencing to profile the regenerating mouse intestine and identified a distinct, damage-induced quiescent cell type that we term the revival stem cell (revSC). revSCs are marked by high clusterin expression and are extremely rare under homoeostatic conditions, yet give rise-in a temporal hierarchy-to all the major cell types of the intestine, including LGR5 CBCs. After intestinal damage by irradiation, targeted ablation of LGR5 CBCs, or treatment with dextran sodium sulfate, revSCs undergo a YAP1-dependent transient expansion, reconstitute the LGR5 CBC compartment and are required to regenerate a functional intestine. These studies thus define a unique stem cell that is mobilized by damage to revive the homoeostatic stem cell compartment and regenerate the intestinal epithelium.
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http://dx.doi.org/10.1038/s41586-019-1154-yDOI Listing
May 2019

Cell competition during reprogramming gives rise to dominant clones.

Science 2019 04 21;364(6438). Epub 2019 Mar 21.

Institute of Biomaterials and Biomedical Engineering (IBBME), University of Toronto, Toronto, Ontario M5S 3E1, Canada.

The ability to generate induced pluripotent stem cells from differentiated cell types has enabled researchers to engineer cell states. Although studies have identified molecular networks that reprogram cells to pluripotency, the cellular dynamics of these processes remain poorly understood. Here, by combining cellular barcoding, mathematical modeling, and lineage tracing approaches, we demonstrate that reprogramming dynamics in heterogeneous populations are driven by dominant "elite" clones. Clones arise a priori from a population of poised mouse embryonic fibroblasts derived from Wnt1-expressing cells that may represent a neural crest-derived population. This work highlights the importance of cellular dynamics in fate programming outcomes and uncovers cell competition as a mechanism by which cells with eliteness emerge to occupy and dominate the reprogramming niche.
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http://dx.doi.org/10.1126/science.aan0925DOI Listing
April 2019

A feed forward loop enforces YAP/TAZ signaling during tumorigenesis.

Nat Commun 2018 08 29;9(1):3510. Epub 2018 Aug 29.

Department of Biochemistry, University of Toronto, Toronto, ON, M5S 1A8, Canada.

In most solid tumors, the Hippo pathway is inactivated through poorly understood mechanisms that result in the activation of the transcriptional regulators, YAP and TAZ. Here, we identify NUAK2 as a YAP/TAZ activator that directly inhibits LATS-mediated phosphorylation of YAP/TAZ and show that NUAK2 induction by YAP/TAZ and AP-1 is required for robust YAP/TAZ signaling. Pharmacological inhibition or loss of NUAK2 reduces the growth of cultured cancer cells and mammary tumors in mice. Moreover, in human patient samples, we show that NUAK2 expression is elevated in aggressive, high-grade bladder cancer and strongly correlates with a YAP/TAZ gene signature. These findings identify a positive feed forward loop in the Hippo pathway that establishes a key role for NUAK2 in enforcing the tumor-promoting activities of YAP/TAZ. Our results thus introduce a new opportunity for cancer therapeutics by delineating NUAK2 as a potential target for re-engaging the Hippo pathway.
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http://dx.doi.org/10.1038/s41467-018-05939-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6115388PMC
August 2018

Pulmonary pericytes regulate lung morphogenesis.

Nat Commun 2018 06 22;9(1):2448. Epub 2018 Jun 22.

Department of Tissue Morphogenesis, Max Planck Institute for Molecular Biomedicine, and University of Münster, Faculty of Medicine, D-48149, Münster, Germany.

Blood vessels are essential for blood circulation but also control organ growth, homeostasis, and regeneration, which has been attributed to the release of paracrine signals by endothelial cells. Endothelial tubules are associated with specialised mesenchymal cells, termed pericytes, which help to maintain vessel wall integrity. Here we identify pericytes as regulators of epithelial and endothelial morphogenesis in postnatal lung. Mice lacking expression of the Hippo pathway components YAP and TAZ in pericytes show defective alveologenesis. Mutant pericytes are present in normal numbers but display strongly reduced expression of hepatocyte growth factor leading to impaired activation of the c-Met receptor, which is expressed by alveolar epithelial cells. YAP and TAZ are also required for expression of angiopoietin-1 by pulmonary pericytes, which also controls hepatocyte growth factor expression and thereby alveologenesis in an autocrine fashion. These findings establish that pericytes have important, organ-specific signalling properties and coordinate the behavior of epithelial and vascular cells during lung morphogenesis.
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http://dx.doi.org/10.1038/s41467-018-04913-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6015030PMC
June 2018

Crucial Role of Postsynaptic Syntaxin 4 in Mediating Basal Neurotransmission and Synaptic Plasticity in Hippocampal CA1 Neurons.

Cell Rep 2018 06;23(10):2955-2966

Division of Fundamental Neurobiology, Krembil Research Institute, University Health Network, Toronto, ON M5T 2S8, Canada; Department of Physiology, Faculty of Medicine, University of Toronto, Toronto, ON M5S 1A8, Canada. Electronic address:

Trafficking of neurotransmitter receptors on postsynaptic membranes is critical for basal neurotransmission and synaptic plasticity, yet the underlying mechanisms remain elusive. Here, we investigated the role of syntaxin 4 in postsynaptic hippocampal CA1 neurons by analyzing conditional knockout (syntaxin 4 cKO) mice. We show that syntaxin 4 cKO resulted in reduction of basal neurotransmission without changes in paired-pulse ratios. Both α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and N-methyl-d-aspartic acid (NMDA) receptor-mediated charge transfers were diminished. Patch-clamp experiments revealed that amplitudes, but not frequencies, of spontaneous excitatory postsynaptic currents are reduced. Syntaxin 4 knockout (KO) caused drastic reduction in expression of surface α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and N-methyl-d-aspartic acid (NMDA) receptors in cultured hippocampal neurons. Furthermore, cKO caused defects in theta-burst stimulation induced long-term potentiation and spatial learning as assessed by a water maze task, indicating that synaptic plasticity was altered. Our data reveal a crucial role of syntaxin 4 in trafficking of ionotropic glutamate receptors that are essential for basal neurotransmission, synaptic plasticity, and spatial memory.
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http://dx.doi.org/10.1016/j.celrep.2018.05.026DOI Listing
June 2018

Exosomes Mediate Mobilization of Autocrine Wnt10b to Promote Axonal Regeneration in the Injured CNS.

Cell Rep 2017 07;20(1):99-111

Donald K. Johnson Eye Institute, Krembil Research Institute, University Health Network, Krembil Discovery Tower, KDT-8-418, 60 Leonard Street, Toronto, ON M5T 2S8, Canada; Department of Ophthalmology and Vision Science, Faculty of Medicine, University of Toronto, 340 College Street, Toronto, ON M5T 3A9, Canada; Department of Physiology, Faculty of Medicine, University of Toronto, 1 King's College Circle, Toronto, ON M5S 1A8, Canada. Electronic address:

Developing strategies that promote axonal regeneration within the injured CNS is a major therapeutic challenge, as axonal outgrowth is potently inhibited by myelin and the glial scar. Although regeneration can be achieved using the genetic deletion of PTEN, a negative regulator of the mTOR pathway, this requires inactivation prior to nerve injury, thus precluding therapeutic application. Here, we show that, remarkably, fibroblast-derived exosomes (FD exosomes) enable neurite growth on CNS inhibitory proteins. Moreover, we demonstrate that, upon treatment with FD exosomes, Wnt10b is recruited toward lipid rafts and activates mTOR via GSK3β and TSC2. Application of FD exosomes shortly after optic nerve injury promoted robust axonal regeneration, which was strongly reduced in Wnt10b-deleted animals. This work uncovers an intercellular signaling pathway whereby FD exosomes mobilize an autocrine Wnt10b-mTOR pathway, thereby awakening the intrinsic capacity of neurons for regeneration, an important step toward healing the injured CNS.
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http://dx.doi.org/10.1016/j.celrep.2017.06.009DOI Listing
July 2017

Recent advances in understanding contextual TGFβ signaling.

F1000Res 2017 26;6:749. Epub 2017 May 26.

Center for Systems Biology, Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, ON, Canada.

The appearance of the first animal species on earth coincides with the emergence of transforming growth factor β (TGFβ) pathways. The evolution of these animals into more complex organisms coincides with a progressively increased TGFβ repertoire through gene duplications and divergence, making secreted TGFβ molecules the largest family of morphogenetic proteins in humans. It is therefore not surprising that TGFβ pathways govern numerous aspects of human biology from early embryonic development to regeneration, hematopoiesis, neurogenesis, and immunity. Such heavy reliance on these pathways is reflected in the susceptibility to minor perturbations in pathway components that can lead to dysregulated signaling and a diverse range of human pathologies such as cancer, fibrosis, and developmental disorders. Attempts to comprehensively resolve these signaling cascades are complicated by the long-recognized paradoxical role the pathway plays in cell biology. Recently, several groups have probed examples of the disparate aspects of TGFβ biology in a variety of animal models and uncovered novel context-dependent regulatory mechanisms. Here, we briefly review recent advancements and discuss their overall impact in directing future TGFβ research.
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http://dx.doi.org/10.12688/f1000research.11295.1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5464225PMC
May 2017

Hippo signalling in intestinal regeneration and cancer.

Curr Opin Cell Biol 2017 10 18;48:17-25. Epub 2017 May 18.

Lunenfeld-Tanenbaum Research Institute, Sinai Health System, Toronto, Canada; Department of Molecular Genetics, University of Toronto, Canada. Electronic address:

The Hippo pathway is a unique signalling module that regulates cell-specific transcriptional responses and responds to a wide range of intrinsic and extrinsic cues. Besides its classical role in restricting tissue size during development, Hippo signalling is now recognized to control numerous processes including cell proliferation, survival, cell fate determination, epithelial-to-mesenchymal transitions and cell migration. Because of its highly dynamic nature, the intestinal epithelium has served as an exceptional model to study the complex roles of Hippo signalling. In this review, we shall present an overview of Hippo function in the mammalian intestine and discuss the various mechanisms regulating Hippo signalling and how they contribute to intestinal regeneration and cancer.
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http://dx.doi.org/10.1016/j.ceb.2017.04.005DOI Listing
October 2017

LUMIER: A Discovery Tool for Mammalian Protein Interaction Networks.

Methods Mol Biol 2017 ;1550:137-148

Center for Systems Biology, Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, 600 University Avenue, Toronto, ON, Canada, M5G 1X5.

Protein-protein interactions (PPIs) play an essential role in all biological processes. In vivo, PPIs occur dynamically and depend on extracellular cues. To discover novel protein-protein interactions in mammalian cells, we developed a high-throughput automated technology called LUMIER (LUminescence-based Mammalian IntERactome). In this approach, we co-express a Luciferase (LUC)-tagged fusion protein along with a Flag-tagged protein in an efficiently transfectable cell line such as HEK-293T cells. The interaction between the two proteins is determined by co-immunoprecipitation using an anti-Flag antibody, and the presence of the LUC-tagged interactor in the complex is subsequently detected via its luciferase activity. LUMIER can easily detect transmembrane protein partners, interactions that are signaling- or splice isoform-dependent, as well as those that may occur only in the presence of posttranslational modifications. Using various collections of Flag-tagged proteins, we have generated protein interaction networks for several TGF-β family receptors, Wnt pathway members, and have systematically analyzed the effect of neural-specific alternative splicing on protein interaction networks. The results have provided important insights into the physiological and functional relevance of some of the novel interactions found. LUMIER is highly scalable and can be used for both low- and high-throughput strategies. LUMIER is thus a valuable tool for the identification and characterization of dynamically regulated PPIs in mammalian systems. Here, we describe a manual version of LUMIER in a 96-well format that can be easily implemented in any laboratory.
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http://dx.doi.org/10.1007/978-1-4939-6747-6_11DOI Listing
February 2018

Multilayered Control of Alternative Splicing Regulatory Networks by Transcription Factors.

Mol Cell 2017 Feb;65(3):539-553.e7

Donnelly Centre, University of Toronto, Toronto, ON M5S 3E1, Canada; Department of Molecular Genetics, University of Toronto, Toronto, ON M5S 1A8, Canada. Electronic address:

Networks of coordinated alternative splicing (AS) events play critical roles in development and disease. However, a comprehensive knowledge of the factors that regulate these networks is lacking. We describe a high-throughput system for systematically linking trans-acting factors to endogenous RNA regulatory events. Using this system, we identify hundreds of factors associated with diverse regulatory layers that positively or negatively control AS events linked to cell fate. Remarkably, more than one-third of the regulators are transcription factors. Further analyses of the zinc finger protein Zfp871 and BTB/POZ domain transcription factor Nacc1, which regulate neural and stem cell AS programs, respectively, reveal roles in controlling the expression of specific splicing regulators. Surprisingly, these proteins also appear to regulate target AS programs via binding RNA. Our results thus uncover a large "missing cache" of splicing regulators among annotated transcription factors, some of which dually regulate AS through direct and indirect mechanisms.
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http://dx.doi.org/10.1016/j.molcel.2017.01.011DOI Listing
February 2017

TGF-β Family Signaling in Embryonic and Somatic Stem-Cell Renewal and Differentiation.

Cold Spring Harb Perspect Biol 2017 Jul 5;9(7). Epub 2017 Jul 5.

Lunenfeld-Tanenbam Research Institute, Mount Sinai Hospital and Department of Molecular Genetics, University of Toronto, Toronto, Ontario M5G 1X5, Canada.

Soon after the discovery of transforming growth factor-β (TGF-β), seminal work in vertebrate and invertebrate models revealed the TGF-β family to be central regulators of tissue morphogenesis. Members of the TGF-β family direct some of the earliest cell-fate decisions in animal development, coordinate complex organogenesis, and contribute to tissue homeostasis in the adult. Here, we focus on the role of the TGF-β family in mammalian stem-cell biology and discuss its wide and varied activities both in the regulation of pluripotency and in cell-fate commitment.
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http://dx.doi.org/10.1101/cshperspect.a022186DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5495062PMC
July 2017

Reciprocal stabilization of ABL and TAZ regulates osteoblastogenesis through transcription factor RUNX2.

J Clin Invest 2016 12 31;126(12):4482-4496. Epub 2016 Oct 31.

Cellular identity in metazoan organisms is frequently established through lineage-specifying transcription factors, which control their own expression through transcriptional positive feedback, while antagonizing the developmental networks of competing lineages. Here, we have uncovered a distinct positive feedback loop that arises from the reciprocal stabilization of the tyrosine kinase ABL and the transcriptional coactivator TAZ. Moreover, we determined that this loop is required for osteoblast differentiation and embryonic skeletal formation. ABL potentiated the assembly and activation of the RUNX2-TAZ master transcription factor complex that is required for osteoblastogenesis, while antagonizing PPARγ-mediated adipogenesis. ABL also enhanced TAZ nuclear localization and the formation of the TAZ-TEAD complex that is required for osteoblast expansion. Last, we have provided genetic data showing that regulation of the ABL-TAZ amplification loop lies downstream of the adaptor protein 3BP2, which is mutated in the craniofacial dysmorphia syndrome cherubism. Our study demonstrates an interplay between ABL and TAZ that controls the mesenchymal maturation program toward the osteoblast lineage and is mechanistically distinct from the established model of lineage-specific maturation.
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http://dx.doi.org/10.1172/JCI87802DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5127668PMC
December 2016

KATapulting toward Pluripotency and Cancer.

J Mol Biol 2017 06 6;429(13):1958-1977. Epub 2016 Oct 6.

Center for Cancer Epigenetics, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA; Department of Epigenetics and Molecular Carcinogenesis, The University of Texas MD Anderson Cancer Center, Science Park, Smithville, TX 78957, USA. Electronic address:

Development is generally regarded as a unidirectional process that results in the acquisition of specialized cell fates. During this process, cellular identity is precisely defined by signaling cues that tailor the chromatin landscape for cell-specific gene expression programs. Once established, these pathways and cell states are typically resistant to disruption. However, loss of cell identity occurs during tumor initiation and upon injury response. Moreover, terminally differentiated cells can be experimentally provoked to become pluripotent. Chromatin reorganization is key to the establishment of new gene expression signatures and thus new cell identity. Here, we explore an emerging concept that lysine acetyltransferase (KAT) enzymes drive cellular plasticity in the context of somatic cell reprogramming and tumorigenesis.
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http://dx.doi.org/10.1016/j.jmb.2016.09.023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5382138PMC
June 2017

Regulation of Rho GTPases from the lateral sides of migrating cells.

Small GTPases 2018 07 1;9(4):345-348. Epub 2016 Nov 1.

a Center for Systems Biology, Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital , Toronto , ON , Canada.

Migratory polarity and epithelial polarity share many common regulatory mechanisms. Rho GTPases play a key role in modulating cell polarity, which in migrating cells has been conventionally studied along the solitary front-rear axis. In recent work, we discovered that Prickle1 (Pk1), a core com-ponent of planar cell polarity (PCP) signaling, mediates a novel lateral signaling pathway that coordinates multi-axial protrusive activities from the lateral cortex of migrating cancer cells. We identified that Arhgap21 and 23 are essential effectors of Pk1, and that lateral signaling regulates RhoA, actomyosin and focal adhesion dynamics. Interestingly, we showed that lateral signaling coordinates shape dynamics that are critical during cell migration and function orthogonally to front-rear directional polarity.
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http://dx.doi.org/10.1080/21541248.2016.1234430DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5997147PMC
July 2018

The RNF146 and tankyrase pathway maintains the junctional Crumbs complex through regulation of angiomotin.

J Cell Sci 2016 09 12;129(18):3396-411. Epub 2016 Aug 12.

Center for Systems Biology, Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada M5G 1X5 Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada M5S 1A8

The Crumbs complex is an important determinant of epithelial apical-basal polarity that functions in regulation of tight junctions, resistance to epithelial-to-mesenchymal transitions and as a tumour suppressor. Although the functional role of the Crumbs complex is being elucidated, its regulation is poorly understood. Here, we show that suppression of RNF146, an E3 ubiquitin ligase that recognizes ADP-ribosylated substrates, and tankyrase, a poly(ADP-ribose) polymerase, disrupts the junctional Crumbs complex and disturbs the function of tight junctions. We show that RNF146 binds a number of polarity-associated proteins, in particular members of the angiomotin (AMOT) family. Accordingly, AMOT proteins are ADP-ribosylated by TNKS2, which drives ubiquitylation by RNF146 and subsequent degradation. Ablation of RNF146 or tankyrase, as well as overexpression of AMOT, led to the relocation of PALS1 (a Crumbs complex component) from the apical membrane to internal puncta, a phenotype that is rescued by AMOTL2 knockdown. We thus reveal a new function of RNF146 and tankyrase in stabilizing the Crumbs complex through downregulation of AMOT proteins at the apical membrane.
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http://dx.doi.org/10.1242/jcs.188417DOI Listing
September 2016

A critical role for NF2 and the Hippo pathway in branching morphogenesis.

Nat Commun 2016 08 2;7:12309. Epub 2016 Aug 2.

Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada M5G 1X5.

Branching morphogenesis is a complex biological process common to the development of most epithelial organs. Here we demonstrate that NF2, LATS1/2 and YAP play a critical role in branching morphogenesis in the mouse kidney. Removal of Nf2 or Lats1/2 from the ureteric bud (UB) lineage causes loss of branching morphogenesis that is rescued by loss of one copy of Yap and Taz, and phenocopied by YAP overexpression. Mosaic analysis demonstrates that cells with high YAP expression have reduced contribution to UB tips, similar to Ret(-/-) cells, and that YAP suppresses RET signalling and tip identity. Conversely, Yap/Taz UB-deletion leads to cyst-like branching and expansion of UB tip markers, suggesting a shift towards tip cell identity. Based on these data we propose that NF2 and the Hippo pathway locally repress YAP/TAZ activity in the UB to promote subsequent splitting of the tip to allow branching morphogenesis.
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http://dx.doi.org/10.1038/ncomms12309DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4974664PMC
August 2016

Multiple roles for the hippo effector yap in gut regeneration and cancer initiation.

Mol Cell Oncol 2016 May 16;3(3):e1143992. Epub 2016 Mar 16.

Centre for Systems Biology, and Lunenfeld Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada; Department of Molecular Genetics, University of Toronto, Ontario, Canada.

The Hippo signaling effector Yes-associated protein (Yap) is known for its potent control of tissue growth. Our recent work now shows that Yap promotes regeneration in the intestine by reprogramming intestinal stem cells and blocking their terminal differentiation. Similarly, in tumor-initiating cells Yap regenerative signaling synergizes with Wnt activation to drive adenoma formation.
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http://dx.doi.org/10.1080/23723556.2016.1143992DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4909447PMC
May 2016