Publications by authors named "Kostandin V Pajcini"

10 Publications

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The neurotransmitter receptor Gabbr1 regulates proliferation and function of hematopoietic stem and progenitor cells.

Blood 2021 Feb;137(6):775-787

Department of Pharmacology, University of Illinois at Chicago, Chicago, IL.

Hematopoietic and nervous systems are linked via innervation of bone marrow (BM) niche cells. Hematopoietic stem/progenitor cells (HSPCs) express neurotransmitter receptors, such as the γ-aminobutyric acid (GABA) type B receptor subunit 1 (GABBR1), suggesting that HSPCs could be directly regulated by neurotransmitters like GABA that directly bind to GABBR1. We performed imaging mass spectrometry and found that the endogenous GABA molecule is regionally localized and concentrated near the endosteum of the BM niche. To better understand the role of GABBR1 in regulating HSPCs, we generated a constitutive Gabbr1-knockout mouse model. Analysis revealed that HSPC numbers were significantly reduced in the BM compared with wild-type littermates. Moreover, Gabbr1-null hematopoietic stem cells had diminished capacity to reconstitute irradiated recipients in a competitive transplantation model. Gabbr1-null HSPCs were less proliferative under steady-state conditions and upon stress. Colony-forming unit assays demonstrated that almost all Gabbr1-null HSPCs were in a slow or noncycling state. In vitro differentiation of Gabbr1-null HSPCs in cocultures produced fewer overall cell numbers with significant defects in differentiation and expansion of the B-cell lineage. To determine whether a GABBR1 agonist could stimulate human umbilical cord blood (UCB) HSPCs, we performed brief ex vivo treatment prior to transplant into immunodeficient mice, with significant increases in long-term engraftment of HSPCs compared with GABBR1 antagonist or vehicle treatments. Our results indicate a direct role for GABBR1 in HSPC proliferation, and identify a potential target to improve HSPC engraftment in clinical transplantation.
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http://dx.doi.org/10.1182/blood.2019004415DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7885825PMC
February 2021

A Tie2-Notch1 signaling axis regulates regeneration of the endothelial bone marrow niche.

Haematologica 2019 11 28;104(11):2164-2177. Epub 2019 Mar 28.

Department of Pharmacology, The University of Illinois College of Medicine, Chicago, IL

Loss-of-function studies have determined that Notch signaling is essential for hematopoietic and endothelial development. By deleting a single allele of the Notch1 transcriptional activation domain we generated viable, post-natal mice exhibiting hypomorphic Notch signaling. These heterozygous mice, which lack only one copy of the transcriptional activation domain, appear normal and have no endothelial or hematopoietic phenotype, apart from an inherent, cell-autonomous defect in T-cell lineage development. Following chemotherapy, these hypomorphs exhibited severe pancytopenia, weight loss and morbidity. This phenotype was confirmed in an endothelial-specific, loss-of-function Notch1 model system. Ang1, secreted by hematopoietic progenitors after damage, activated endothelial Tie2 signaling, which in turn enhanced expression of Notch ligands and potentiated Notch1 receptor activation. In our heterozygous, hypomorphic model system, the mutant protein that lacks the Notch1 transcriptional activation domain accumulated in endothelial cells and interfered with optimal activity of the wildtype Notch1 transcriptional complex. Failure of the hypomorphic mutant to efficiently drive transcription of key gene targets such as and prolonged apoptosis and limited regeneration of the bone marrow niche. Thus, basal Notch1 signaling is sufficient for niche development, but robust Notch activity is required for regeneration of the bone marrow endothelial niche and hematopoietic recovery.
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http://dx.doi.org/10.3324/haematol.2018.208660DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6821596PMC
November 2019

Dlk1-Mediated Temporal Regulation of Notch Signaling Is Required for Differentiation of Alveolar Type II to Type I Cells during Repair.

Cell Rep 2019 03;26(11):2942-2954.e5

Department of Pharmacology, The University of Illinois College of Medicine, Chicago, IL 60612, USA; The Center for Lung and Vascular Biology, The University of Illinois College of Medicine, Chicago, IL 60612, USA. Electronic address:

Lung alveolar type I cells (AT1) and alveolar type II cells (AT2) regulate the structural integrity and function of alveoli. AT1, covering ∼95% of the surface area, are responsible for gas exchange, whereas AT2 serve multiple functions, including alveolar repair through proliferation and differentiation into AT1. However, the signaling mechanisms for alveolar repair remain unclear. Here, we demonstrate, in Pseudomonas aeruginosa-induced acute lung injury in mice, that non-canonical Notch ligand Dlk1 (delta-like 1 homolog) is essential for AT2-to-AT1 differentiation. Notch signaling was activated in AT2 at the onset of repair but later suppressed by Dlk1. Deletion of Dlk1 in AT2 induced persistent Notch activation, resulting in stalled transition to AT1 and accumulation of an intermediate cell population that expressed low levels of both AT1 and AT2 markers. Thus, Dlk1 expression leads to precisely timed inhibition of Notch signaling and activates AT2-to-AT1 differentiation, leading to alveolar repair.
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http://dx.doi.org/10.1016/j.celrep.2019.02.046DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6464111PMC
March 2019

MAFB enhances oncogenic Notch signaling in T cell acute lymphoblastic leukemia.

Sci Signal 2017 11 14;10(505). Epub 2017 Nov 14.

Abramson Family Cancer Research Institute, University of Pennsylvania, Philadelphia, PA 19104, USA.

Activating mutations in the gene encoding the cell-cell contact signaling protein Notch1 are common in human T cell acute lymphoblastic leukemias (T-ALLs). However, expressing mutant alleles in mice fails to efficiently induce the development of leukemia. We performed a gain-of-function screen to identify proteins that enhanced signaling by leukemia-associated Notch1 mutants. The transcription factors MAFB and ETS2 emerged as candidates that individually enhanced Notch1 signaling, and when coexpressed, they synergistically increased signaling to an extent similar to that induced by core components of the Notch transcriptional complex. In mouse models of T-ALL, MAFB enhanced leukemogenesis by the naturally occurring Notch1 mutants, decreased disease latency, and increased disease penetrance. Decreasing MAFB abundance in mouse and human T-ALL cells reduced the expression of Notch1 target genes, including and , and sustained MAFB knockdown impaired T-ALL growth in a competitive setting. MAFB bound to ETS2 and interacted with the acetyltransferases PCAF and P300, highlighting its importance in recruiting coactivators that enhance Notch1 signaling. Together, these data identify a mechanism for enhancing the oncogenic potential of weak Notch1 mutants in leukemia models, and they reveal the MAFB-ETS2 transcriptional axis as a potential therapeutic target in T-ALL.
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http://dx.doi.org/10.1126/scisignal.aam6846DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5885022PMC
November 2017

Embryonic Stem Cell Differentiation to Functional Arterial Endothelial Cells through Sequential Activation of ETV2 and NOTCH1 Signaling by HIF1α.

Stem Cell Reports 2017 09 3;9(3):796-806. Epub 2017 Aug 3.

Department of Pharmacology and the Center for Lung and Vascular Biology, University of Illinois College of Medicine, 835 S. Wolcott, Chicago, IL 60612, USA. Electronic address:

The generation of functional arterial endothelial cells (aECs) from embryonic stem cells (ESCs) holds great promise for vascular tissue engineering. However, the mechanisms underlying their generation and the potential of aECs in revascularizing ischemic tissue are not fully understood. Here, we observed that hypoxia exposure of mouse ESCs induced an initial phase of HIF1α-mediated upregulation of the transcription factor Etv2, which in turn induced the commitment to the EC fate. However, sustained activation of HIF1α in these EC progenitors thereafter induced NOTCH1 signaling that promoted the transition to aEC fate. We observed that transplantation of aECs mediated arteriogenesis in the mouse hindlimb ischemia model. Furthermore, transplantation of aECs in mice showed engraftment in ischemic myocardium and restored cardiac function in contrast to ECs derived under normoxia. Thus, HIF1α activation of Etv2 in ESCs followed by NOTCH1 signaling is required for the generation aECs that are capable of arteriogenesis and revascularization of ischemic tissue.
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http://dx.doi.org/10.1016/j.stemcr.2017.07.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5599266PMC
September 2017

Trib2 Suppresses Tumor Initiation in Notch-Driven T-ALL.

PLoS One 2016 18;11(5):e0155408. Epub 2016 May 18.

Department of Pathology and Laboratory Medicine, Abramson Family Cancer Research Institute, Institute of Medicine and Engineering, Institute for Immunology, Center for Personalized Diagnostics, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, United States of America.

Trib2 is highly expressed in human T cell acute lymphoblastic leukemia (T-ALL) and is a direct transcriptional target of the oncogenic drivers Notch and TAL1. In human TAL1-driven T-ALL cell lines, Trib2 is proposed to function as an important survival factor, but there is limited information about the role of Trib2 in primary T-ALL. In this study, we investigated the role of Trib2 in the initiation and maintenance of Notch-dependent T-ALL. Trib2 had no effect on the growth and survival of murine T-ALL cell lines in vitro when expression was blocked by shRNAs. To test the function of Trib2 on leukemogenesis in vivo, we generated Trib2 knockout mice. Mice were born at the expected Mendelian frequencies without gross developmental anomalies. Adult mice did not develop pathology or shortened survival, and hematopoiesis, including T cell development, was unperturbed. Using a retroviral model of Notch-induced T-ALL, deletion of Trib2 unexpectedly decreased the latency and increased the penetrance of T-ALL development in vivo. Immunoblotting of primary murine T-ALL cells showed that the absence of Trib2 increased C/EBPα expression, a known regulator of cell proliferation, and did not alter AKT or ERK phosphorylation. Although Trib2 was suggested to be highly expressed in T-ALL, transcriptomic analysis of two independent T-ALL cohorts showed that low Trib2 expression correlated with the TLX1-expressing cortical mature T-ALL subtype, whereas high Trib2 expression correlated with the LYL1-expressing early immature T-ALL subtype. These data indicate that Trib2 has a complex role in the pathogenesis of Notch-driven T-ALL, which may vary between different T-ALL subtypes.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0155408PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4871414PMC
July 2017

The Notch1 transcriptional activation domain is required for development and reveals a novel role for Notch1 signaling in fetal hematopoietic stem cells.

Genes Dev 2014 Mar;28(6):576-93

Abramson Family Cancer Research Institute, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA;

Notch1 is required to generate the earliest embryonic hematopoietic stem cells (HSCs); however since Notch-deficient embryos die early in gestation, additional functions for Notch in embryonic HSC biology have not been described. We used two complementary genetic models to address this important biological question. Unlike Notch1-deficient mice, mice lacking the conserved Notch1 transcriptional activation domain (TAD) show attenuated Notch1 function in vivo and survive until late gestation, succumbing to multiple cardiac abnormalities. Notch1 TAD-deficient HSCs emerge and successfully migrate to the fetal liver but are decreased in frequency by embryonic day 14.5. In addition, TAD-deficient fetal liver HSCs fail to compete with wild-type HSCs in bone marrow transplant experiments. This phenotype is independently recapitulated by conditional knockout of Rbpj, a core Notch pathway component. In vitro analysis of Notch1 TAD-deficient cells shows that the Notch1 TAD is important to properly assemble the Notch1/Rbpj/Maml trimolecular transcription complex. Together, these studies reveal an essential role for the Notch1 TAD in fetal development and identify important cell-autonomous functions for Notch1 signaling in fetal HSC homeostasis.
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http://dx.doi.org/10.1101/gad.227496.113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3967047PMC
March 2014

Transient inactivation of Rb and ARF yields regenerative cells from postmitotic mammalian muscle.

Cell Stem Cell 2010 Aug;7(2):198-213

Baxter Laboratory for Stem Cell Biology, Department of Microbiology & Immunology, Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA 94305, USA.

An outstanding biological question is why tissue regeneration in mammals is limited, whereas urodele amphibians and teleost fish regenerate major structures, largely by cell cycle reentry. Upon inactivation of Rb, proliferation of postmitotic urodele skeletal muscle is induced, whereas in mammalian muscle this mechanism does not exist. We postulated that a tumor suppressor present in mammals but absent in regenerative vertebrates, the Ink4a product ARF (alternative reading frame), is a regeneration suppressor. Concomitant inactivation of Arf and Rb led to mammalian muscle cell cycle reentry, loss of differentiation properties, and upregulation of cytokinetic machinery. Single postmitotic myocytes were isolated by laser micro-dissection-catapulting, and transient suppression of Arf and Rb yielded myoblast colonies that retained the ability to differentiate and fuse into myofibers upon transplantation in vivo. These results show that differentiation of mammalian cells is reversed by inactivation of Arf and Rb and support the hypothesis that Arf evolved at the expense of regeneration.
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http://dx.doi.org/10.1016/j.stem.2010.05.022DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2919350PMC
August 2010

Myoblasts and macrophages share molecular components that contribute to cell-cell fusion.

J Cell Biol 2008 Mar;180(5):1005-19

Department of Microbiology & Immunology, Baxter Laboratory in Genetic Pharmacology, Stanford University School of Medicine, Stanford, CA 94305, USA.

Cell-cell fusion is critical to the normal development of certain tissues, yet the nature and degree of conservation of the underlying molecular components remains largely unknown. Here we show that the two guanine-nucleotide exchange factors Brag2 and Dock180 have evolutionarily conserved functions in the fusion of mammalian myoblasts. Their effects on muscle cell formation are distinct and are a result of the activation of the GTPases ARF6 and Rac, respectively. Inhibition of ARF6 activity results in a lack of physical association between paxillin and beta(1)-integrin, and disruption of paxillin transport to sites of focal adhesion. We show that fusion machinery is conserved among distinct cell types because Dock180 deficiency prevented fusion of macrophages and the formation of multinucleated giant cells. Our results are the first to demonstrate a role for a single protein in the fusion of two different cell types, and provide novel mechanistic insight into the function of GEFs in the morphological maturation of multinucleated cells.
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http://dx.doi.org/10.1083/jcb.200707191DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2265408PMC
March 2008

Exploring the mycobacteriophage metaproteome: phage genomics as an educational platform.

PLoS Genet 2006 Jun 9;2(6):e92. Epub 2006 Jun 9.

Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania, USA.

Bacteriophages are the most abundant forms of life in the biosphere and carry genomes characterized by high genetic diversity and mosaic architectures. The complete sequences of 30 mycobacteriophage genomes show them collectively to encode 101 tRNAs, three tmRNAs, and 3,357 proteins belonging to 1,536 "phamilies" of related sequences, and a statistical analysis predicts that these represent approximately 50% of the total number of phamilies in the mycobacteriophage population. These phamilies contain 2.19 proteins on average; more than half (774) of them contain just a single protein sequence. Only six phamilies have representatives in more than half of the 30 genomes, and only three-encoding tape-measure proteins, lysins, and minor tail proteins-are present in all 30 phages, although these phamilies are themselves highly modular, such that no single amino acid sequence element is present in all 30 mycobacteriophage genomes. Of the 1,536 phamilies, only 230 (15%) have amino acid sequence similarity to previously reported proteins, reflecting the enormous genetic diversity of the entire phage population. The abundance and diversity of phages, the simplicity of phage isolation, and the relatively small size of phage genomes support bacteriophage isolation and comparative genomic analysis as a highly suitable platform for discovery-based education.
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http://dx.doi.org/10.1371/journal.pgen.0020092DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1475703PMC
June 2006