Articles in PresS. Am J Physiol Gastrointest Liver Physiol (July 30, 2015). doi:10.1152/ajpgi.00140.2015
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Suppression of fibrogenic signaling in hepatic stellate cells by Twist1-dependent
microRNA-214 expression: Role of exosomes in horizontal transfer of Twist1
Li Chen1, Ruju Chen1, Sherri Kemper1, Alyssa Charrier1,2, and David R Brigstock1,2,3#
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43205 USA
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Columbus OH 43212 USA
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The Research Institute at Nationwide Children’s Hospital, 700 Children’s Drive, Columbus OH
Molecular, Cellular, and Developmental Biology Program, The Ohio State University,
Department of Surgery, Wexner Medical Center, The Ohio State University, Columbus, OH
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43212 USA
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David R. Brigstock, Ph.D.
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Room WA2011, Research Building 2
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Nationwide Children’s Hospital
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700 Children’s Drive
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Columbus OH 43205
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Tel 614-355-2824
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Email [email protected]
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Address Correspondence to:
Author's contributions: LC: study concept and design, acquisition of data, analysis and
interpretation of data, drafting of the manuscript; RC, SK, and AC: acquisition of data; DRB:
study concept and design, analysis and interpretation of data, critical revision of the manuscript
for important intellectual content, obtained funding, study supervision.
Running head: A Twist1-miR-214 axis regulates fibrogenic signaling in HSC
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Copyright © 2015 by the American Physiological Society.
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Abstract
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A hallmark of liver fibrosis is the activation of hepatic stellate cells (HSC) which results in their
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production of fibrotic molecules, a process that is largely regulated by connective tissue growth
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factor (CCN2). CCN2 is increasingly expressed during HSC activation due to diminished
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expression of microRNA-214 (miR-214), a product of dynamin 3 opposite strand (DNM3os) that
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directly suppresses CCN2 mRNA. We show that an E-box in the miR-214 promoter binds the
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basic helix-loop-helix transcription factor, Twist1, which drives miR-214 expression and results
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in CCN2 suppression. Twist1 expression was suppressed in HSC of fibrotic livers or in cultured
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HSC undergoing activation in vitro or after treatment with ethanol. Further, Twist1 decreasingly
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interacted with DNM3os as HSC underwent activation in vitro. Nanovesicular exosomes
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secreted by quiescent but not activated HSC contained high levels of Twist1, thus reflecting the
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suppression of cellular Twist1 during HSC activation. Exosomal Twist1 was intercellularly
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shuttled between HSC and stimulated expression of miR-214 in the recipient cells, causing
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expression of CCN2 and its downstream effectors to be suppressed. Additionally, the miR-214
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E-box in HSC was also regulated by hepatocyte-derived exosomes showing that functional
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transfer of exosomal Twist1 occurs between different cell types. Finally, the levels of Twist1,
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miR-214 or CCN2 in circulating exosomes from fibrotic mice reflected fibrosis-induced changes
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in the liver itself, highlighting the potential utility of these and other constituents in serum
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exosomes as novel circulating biomarkers for liver fibrosis. These findings reveal a unique
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function for cellular or exosomal Twist1 in CCN2-dependent fibrogenesis.
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Keywords: CTGF, CCN2, exosome, fibrosis, E-box
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Introduction
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Liver injury is characterized by a phenotypic and functional transformation of normally quiescent
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hepatic stellate cells (HSC) into alpha-smooth muscle actin (αSMA)-expressing myofibroblastic
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cells which promote wound closure and produce a collagen matrix that supports hepatocyte re-
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population (9, 11). Whereas this activated HSC phenotype is relatively short-lived in acute
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injury, it persists during chronic injury and results in unrelenting deposition of large amounts of
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collagen that is a hallmark of hepatic fibrosis - a serious pathology that compromises normal
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hepatic structure and function and is a harbinger of cirrhosis, hepatocarcinoma, and end-stage
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liver disease (10). Transforming growth factor beta (TGF-β) plays a central role in stimulating
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pathways of fibrogenesis in activated HSC but it is a challenging therapeutic target because it
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also regulates critical immune responses and has important tumor suppressive actions. On the
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other hand, the fibrogenic properties of TGF-β are mediated via connective tissue growth factor
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(CCN2, also known as CTGF), a complex matricellular molecule that is produced downstream
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of TGF-β and directly regulates many of the differentiated functions of activated HSC including
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mitogenesis, chemotaxis, adhesion, matrigenesis, and fibrogenesis (15). CCN2 is produced at
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high levels in activated HSC whereas its expression in quiescent HSC is substantially
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suppressed. We recently identified miR-214 as a direct negative regulator of CCN2 in primary
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mouse HSC or the human LX-2 HSC line (4). Via its direct binding of the CCN2 3’ untranslated
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region (UTR) in quiescent HSC, miR-214 inhibits CCN2 expression in HSC whereas in activated
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HSC, miR-214 expression is suppressed thereby allowing CCN2 to be expressed. CCN2 and
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miR-214 are thus dynamically and reciprocally expressed as a function of HSC activation (4).
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MiR-214 is located within the intron of the dynamin 3 gene and is encoded with miR-199a,
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producing a 7.9 kb non-coding DNM3 opposite strand transcript termed ‘DNM3os’ (27, 41).
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Since factors that drive DNM3os transcription will enhance miR-214-dependent suppression of
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CCN2 expression and thus have potential therapeutic utility, we sought to identify the
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element(s) in the miR-214 promoter and their associated transcription factor(s) that account for
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the high levels of miR-214 expression that occur in quiescent HSC. Here we show that an E-box
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in the miR-214 promoter is a binding site for the basic helix-loop-helix (bHLH) transcription
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factor, Twist1, which drives miR-214 promoter activity and miR-214 expression, resulting in
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CCN2 suppression. Functional assays show that Twist1 decreasingly interacts with DNM3os as
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HSC undergo culture-induced activation, consistent with the finding that HSC demonstrate an
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activation-dependent suppression of Twist1 expression. Moreover, nanovesicular exosomes
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secreted by quiescent HSC or hepatocytes contain Twist1 which is intercellularly shutted to
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recipient HSC in which the E box is targeted, resulting in regulation of the miR-214-CCN2 axis.
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Finally, serum exosomes contain Twist1, miR-214 or CCN2 at levels which reflect their fibrosis-
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induced changes in the liver, suggesting that the molecular payload in circulating exosomes
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offers new possibilities in the search for non-invasive biomarkers of liver fibrosis.
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Materials and Methods
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Animal procedures
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Animal protocols were approved by the Institutional Animal Care and Use Committee of
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Nationwide Children’s Hospital (Columbus, OH). Normal male Swiss Webster mice (6–8 weeks)
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(n=10) were injected i.p. three times each week for 5 weeks with either 30 μl of vegetable oil or
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a mixture of 0.5 μl carbon tetrachloride (CCl4, Sigma-Aldrich, St Louis, MO) in 29.5 μl of
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vegetable oil. Upon sacrifice, blood was collected and individual liver lobes were tied and
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harvested either immediately and snap-frozen in liquid nitrogen for subsequent RNA or protein
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extraction, or after perfusion using PBS followed by 4% paraformaldehyde (Sigma-Aldrich) for
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histological analysis of fixed tissue. In an alternative model of liver injury, male FVB mice (6–8
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weeks) (n=10) received i.p. thioacetic acid (TAA; 100mg/kg; Sigma-Aldrich) in saline three times
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per week for 5 weeks. Control mice received i.p. saline alone. Mice were sacrificed 72 hrs after
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the last injection, livers were harvested, and RNA was isolated and processed for quantitative
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real-time polymerase chain reaction (qRT-PCR). Some livers were fixed for histological
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analysis.
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Cell Culture
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Primary HSC were isolated, essentially as we have described (5), by buoyant density
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centrifugation from normal male Swiss Webster mice (6-8 weeks) and spent medium from the
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cultured cells was replaced with fresh DMEM/F12/10%FBS medium on Day 1 and every other
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day as needed. HSC were split 1:3 every 5 days and used at passage 0-6 (P0-6). Our previous
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studies showed that when isolated from normal (non-fibrotic animals), cells studied within 24
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hours of brief culture do not exhibit characteristics typical of activated cells (eg αSMA, CCN2),
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and thereafter gradually transition to a highly activated phenotype over the ensuing 7-10 days of
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culture (5). In some experiments, triplicate wells of cells were incubated for up to 48 hours in the
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presence of 0 – 50 mM ethanol. Cells were then evaluated for Twist1, CCN2 or miR-214
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expression by qRT-PCR. Human LX-2 HSC were cultured as described (5). The AML12 mouse
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hepatocyte line was obtained from American Type Culture Collection (Manassas, VA).
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Western blotting
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Western blots of whole liver lysates (20μg), freshly isolated or cultured HSC (20μg), or
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exosomal proteins (5μg; see below) were performed using anti-Twist1 (1:300, Abcam,
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Cambridge MA), anti-CCN2 IgY (5 μg/ml; in-house (4)), or anti-CD9 (1:400; LSBio, Seattle WA),
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using our published protocols (5). Blots were stripped and incubated with anti-β-actin (1:2000;
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Abcam) to verify equal loading among samples within individual experiments (data not shown).
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Immunohistochemistry
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Fixed livers were incubated with anti-Twist1 (1:300, Abcam), anti-desmin (1:300, Abcam), anti-
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collagen α(I) (1:250, Abcam), anti-αSMA (1:1000, Dako Cytomatio, Denmark), or anti-CCN2 IgY
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(5μg/ml) (4), followed by Alexa Fluor® 488 goat-anti rabbit IgG and Alexa Fluor® 568 goat-anti
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mouse IgG, or Alexa Fluor® 647 goat-anti mouse IgG, or Alexa Fluor® 568 goat-anti-chicken
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IgG (all at 1:1000; Life Technologies, Grand Island, NY) for 1 hr at room temperature (RT). The
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slides were mounted with Vectashield Mounting Medium containing 4',6-diamidino-2-
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phenylindole (DAPI) nuclear stain (Vector Laboratories, Burlingame, CA), and examined by
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confocal microscopy.
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Cellular RNA extraction and qRT-PCR
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Total RNA from frozen liver tissues or mouse HSC or human LX-2 cells was extracted using a
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microRNeasy Plus kit (Qiagen, Valencia, CA) and was reverse transcribed using a miScript II
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RT kit (Qiagen) according to the manufacturers’ protocols. Resulting transcripts were analyzed
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by qRT-PCR as described (4, 5) with primers for Twist1, CCN2, αSMA, collagen α1(I) or miR-
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214 (Table 1). Each reaction was run in triplicate and all samples were normalized to
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glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Negative controls were a non-reverse
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transcriptase reaction or a non-sample reaction.
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Twist1 overexpression or suppression in primary mouse HSC or human LX-2 cells
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Mouse Twist1 plasmids, Twist1 siRNA, or negative controls were obtained from Addgene
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(Cambridge, MA) or Santa Cruz (Santa Cruz, California). To avoid off-target effects, the Twist1
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siRNA preparation was comprised of 3 target-specific 20-25 nt siRNAs designed to knock down
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Twist1 gene expression. Primary mouse HSC or human LX-2 cells (105-106 cells) were
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transfected with 3.2 μg plasmid or 100nM siRNA by electroporation using a Nucleofector Kit
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(Lonza, Koln, Germany) and incubated for 12 hours in medium containing 10% FBS which was
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then replaced with either fresh 10% FBS-enriched or serum-free medium. Transfection
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efficiency in cells was approximately 40% in mHSC or 90% in LX-2 cells as monitored by co-
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transfection with 0.8μg of a green fluorescent protein (GFP)-expressing plasmid, pEGFP
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(Invitrogen, Carlsbad, CA). In some experiments, LX-2 cells were co-transfected for 12 hours
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with 100nM miR-214 antagomir (Qiagen). Western blot analysis of Twist1-transfected LX-2
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cells was performed using anti-Twist1 (1:300, Abcam) or anti-CCN2 IgY (5μg/ml) (4), using our
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published protocols (5).
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Transfection of mouse primary HSC with pGL 4.11[Luc2P]-DNM3os promoter plasmids
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The DNM3os promoter (647bp; Genbank SEQ ID: CM000994.2: 162217623:162225550:1) was
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amplified by PCR from primary mouse HSC genomic DNA using forward primer 5’-
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TAAAGCTTAAAGGGGGGAGCCCCAACTTATCTG-
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TACTCGAGTTCCTGCACCAGGGGCTTGT- 3’. The PCR fragment was digested with Hind III
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and Xho I, subcloned into pGL 4.11[Luc2P] Vector (Promega, Madison, WI), downstream of the
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Firefly luciferase reporter, and verified by DNA sequencing.
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containing a 6-base point mutation (CATCTG →GCGGCC) in the E-box site (nt. 162217485-
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162217490) was amplified from the wild-type mouse DNM3os promoter using forward primer 5’-
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CATTACACGAAAAGCGGCCGTACCATTTTATGC-
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GCATAAAATGGTACGGCCGCTTTTCGTGTAATG- 3’, and verified by DNA sequencing. The
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mutant DNM3os promoter fragment was released with Hind III and Xho I.
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Primary D6 mouse HSC were transfected with pGL 4.11[Luc2P]-DNM3os wild type or mutant
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plasmids, or vector alone. To control for transfection efficiency, cells were co- transfected with
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pRL-CMV vector (Promega) containing Renilla luciferase reporter gene. After 24 hrs, luciferase
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activity was measured in triplicate using an E1910 Dual Luciferase Reporter Assay System
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(Promega). Renilla luciferase activity was used for normalization, and Firefly luciferase activity
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in pGL 4.11[Luc2P]-DNM3os promoter-transfected cells was compared to that in mock-
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transfected cells.
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Electrophoretic mobility shift assay (EMSA)
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5 x 107 primary mouse HSC (D1) were lysed in EMSA lysis buffer according to the
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manufacturer’s recommendations (Thermo Scientific, Rockford IL). After centrifugation
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3’
3’
and
reverse
primer
5’-
A mutant DNM3os promoter
and
reverse
primer
5’-
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(10,000rpm, 1 min) of the cell lysate, the pellet (nuclei) was collected, resuspended in extraction
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buffer, centrifuged (14,000 rpm, 5 mins, 4oC), and the supernatant used for EMSA.
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Wild-type or mutant DNM3os promoters were labeled at their 3’-end using biotin (0.5μM;
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Thermo Scientific) and terminal deoxynucleotidyltransferase (TdT, 0.2U/μl; Thermo Scientific),
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and purified using chloroform:isoamyl alcohol (1:1). Nuclear protein (1μg) was incubated for 25
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minutes at room temperature with labeled oligonucleotides (20fmol/reaction assay) in binding
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buffer (50% glycerol, 1% NP-40, 100 mM MgCl2, 200 mM EDTA, 1X binding buffer, 1M KCl and
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1 μg/μl) Poly(dI:dC)). For some groups, (i) 0.5μg/μl anti-Twist1 antibody (Abcam) was added for
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super shift evaluation, (ii) 4pmol unlabeled oligonucleotide was used to competitively inhibit
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formation of shifted complexes, or (iii) nuclear protein was omitted to verify its requirement for
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complex formation. Samples were mixed with 5X loading buffer (Thermo Scientific) and
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electrophoresed on a 5% DNA retardation gel in 0.5x TBE buffer. Complexes were transferred
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and cross-linked to a nylon membrane prior to incubation with streptavidin-horseradish
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peroxidase conjugate (1:300; Thermo Scientific) at 37°C for 30 minutes and analysis by
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chemiluminescence (Thermo Scientific).
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Chromatin Immunoprecipitation (ChIP) Assay
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ChIP assays were performed with a EpiTect® ChIP kit (Qiagen). Briefly, 2×106 HSC were cross-
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linked with 1% formaldehyde for 10 min at room temperature after which the reaction was
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terminated with 0.125 M glycine. The cells were then isolated and sonicated on ice to generate
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DNA shear fragments of ~200-1000bp. The lysates were pelleted, pre-cleared, precipitated with
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10μg/ml Twist1 antibody (Millipore, Temecula, CA, USA) or control IgG and allowed to rotate
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overnight at 4°C with magnetic protein A beads (Qiagen). The immune complexes were
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collected and eluted using ChIP-grade proteinase K. The cross-links were destroyed by heating
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the samples at 45°C for 30min and the DNA recovered underwent ChIP-PCR using DNM3os
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primers (see above) according to the manufacturers’ instructions.
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Analysis of exosomal Twist1
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Exosomes were isolated as described from HSC conditioned medium on Days 3 or 20.
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Cryogenic transmission electron microscopy of purified exosomes was performed as described
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(4). Exosomal Twist1 protein or mRNA were determined by, respectively, Western blot using
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anti-Twist1 (Abcam) or qRT-PCR, the latter of which was normalized to let-7a which we
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determined to be an optimal exosomal housekeeping miR for these studies (data not shown).
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Exosome uptake in P6 HSC was shown by confocal microscopy of the cells after incubation for
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12 hrs with exosomes isolated from Day 3 HSC and subsequently stained with PKH26.
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Exosomes were purified from primary HSC after transfection of Day 6 cells for 48 hrs with or
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without 100 nM Twist1 siRNA. Suppressed exosomal Twist1 levels in the exosomes from
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siRNA-treated cells was confirmed by RT-PCR using exosomal let-7a as a reference control.
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Exosomal proteins were evaluated by Western blot as described above. Control or Twist1-
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deficient exosomes were added (3 ug/ml) for 48 hrs to P4 primary HSC after which Twist1 levels
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in the cells were evaluated by RT-PCR or Western blot (see above). Cells were further analyzed
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by RT-PCR for miR-214, CCN2, αSMA or collagen α1(I).
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Exosomal regulation of DNM3os was shown by assessment of dual luciferase activity in Day 6
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HSC transfected with pGL 4.11[Luc2P]-DNM3os wild type or mutant plasmids, or vector alone,
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when co-cultured for 24hrs with Day 1 HSC or AML12 hepatocytes that had been treated for 24
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hrs with or without 100 μM GW4869, an inhibitor of nSMase2 which is required for biosynthesis
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of ceramide on which exosome production depends (3, 4).
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Circulating exosomes were harvested using PureExo Exosome Isolation Kits (101Bio, Palo Alto,
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CA) from serum of mice treated for up to 5 weeks with CCl4 as described above. Total RNA
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from exosomes in 200 μl of serum was prepared using miRNeasy mini kits (Qiagen) as
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described above. Each reaction was run in triplicate and all samples were normalized to let-7a.
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Statistical Analysis
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All experiments were performed at least three times with triplicate measurements. For controls,
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error bars were derived by setting the mean value as 1 and defining variance of replicates from
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1. Treatment groups were then expressed as fold of mean ± s.e.m. The data from qRT-PCR or
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luciferase activity assays were analyzed by student’s t-test using Sigma plot 12.0 software
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(SPSS Inc., Chicago, IL) and P values < 0.05 were considered statistically significant.
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Results
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Suppression of Twist1 expression during fibrosing liver injury or during HSC activation in vivo or
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vitro.
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Analysis of total hepatic RNA showed that hepatic Twist1 expression was high in livers
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recovered from control oil-treated mice but was significantly decreased in livers from CCl4-
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treated mice (Figure 1A). This response was associated with suppressed expression of hepatic
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miR-214 and stimulated expression of CCN2, αSMA, or collagen α1(I) (Figure 1A). Isolated
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activated HSC from this 5-week injury model showed an overall similar expression pattern in
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that Twist1 or miR214 were inhibited and CCN2, αSMA, or collagen α1(I) were enhanced
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(Figure 1A). Consistent with these findings, Western blot analysis showed that Twist1 protein
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levels were suppressed in fibrotic livers or in activated HSC recovered from fibrotic livers, and
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that CCN2 protein levels increased under the same conditions (Figure 1A). Immunostaining for
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Twist1 in liver sections showed that it was present in desmin-positive non-parenchymal cells
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(presumptive quiescent HSC) in control animals but that, after CCl4 injury, Twist1 staining was
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absent from activated HSC, which stained positively for αSMA as well as desmin (Figure 1B,
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upper panel, arrows). Some parenchymal cells also strongly stained positively for Twist1 but
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this was not reduced after CCl4 treatment (Figure 1B, upper panel). Nonetheless, since
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background hepatocyte staining might potentially confound our interpretation of Twist1 staining
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in HSC, we alternatively isolated HSC from the livers of control or fibrotic animals to verify their
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Twist1 status in vivo. As assessed by immunostaining, quiescent HSC isolated from control
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animals were positive for desmin or Twist1, but not for CCN2, αSMA or collagen α(I). In
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contrast, activated HSC isolated from animals treated with CCl4 for 5 weeks were positive for
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desmin, CCN2, αSMA or collagen α(I), but not for Twist1 (Figure 1B, lower panel). Thus, since
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HSC activation in vivo was associated with the loss of Twist1 mRNA expression or protein
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production (Figure 1A,B), this phenomenon was the focus of the studies described herein.
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In a TAA liver fibrosis model exhibiting enhanced staining in HSC for CCN2, αSMA, or collagen
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α1(I), decreased expression of hepatic Twist1 mRNA or miR-214 and increased expression of
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hepatic CCN2 mRNA was also documented (Figure 1C). Analysis of HSC isolated from normal
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livers and maintained in vitro showed that there was a large decrease in Twist1 expression
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between Days 2 and 4 of culture and then a more gradual decline in its expression up to Day 14
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of culture as the cells became progressively activated and expressed decreasing levels of miR-
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214 and increasing levels of CCN2 (Figure 1D). Treatment of Day 3 primary HSC with ethanol
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resulted in decreased Twist1 or miR-214 expression and increased CCN2 expression (Figure
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1E). Thus, the decreased Twist1 expression in HSC during culture-induced activation or in
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response to fibrosing stimulus was comparable to that observed in HSC undergoing activation in
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vivo during CCl4 injury.
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Twist1 regulation of miR-214 expression and downstream CCN2 production
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Transfection of Twist1 in highly activated HSC resulted in increased Twist1 mRNA or protein
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levels, causing miR-214 to be increased or CCN2 expression to be decreased (Figure 2A). On
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the other hand, Twist siRNA treatment of Day 6 HSC resulted in decreased Twist1 mRNA or
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protein levels, causing miR-214 expression to be decreased or CCN2 expression to be
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increased (Figure 2B). Since miR-214 exerts inhibitory actions on the CCN2 3’UTR (4), these
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results suggested that Twist1 transcriptionally activates miR-214 which then acts to suppress
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CCN2 mRNA levels. This was supported by the finding that the activity in Day 6 HSC of a wild
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type DNM3os luciferase reporter was blocked by Twist1 siRNA whereas the luciferase reporter
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activity of a mutant form of DNM3os lacking the E-box binding site for Twist1 was suppressed
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and insensitive to addition of Twist1 siRNA (Figure 2C). To confirm a direct binding interaction in
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HSC between Twist1 and the E-box, an EMSA was performed which showed that a wild-type
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DNM3os probe formed a shifted complex in the presence of nuclear extracts that was super-
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shifted by pre-incubation of the nuclear extract with a Twist1 antibody (Figure 2D). On the
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contrary, no complexes were formed when DNM3os containing the E-box mutation was used as
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the probe (Figure 2D). To verify that the binding between Twist1 and DNM3os occurred in living
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HSC and decreased as their level of activation was increased, a ChIP assay was performed
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which showed that Twist1-DNM3os binding was approximately 100-fold greater in Day 1
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quiescent HSC than in highly activated P7 HSC (Figure 2E). Collectively, these data show that
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that in quiescent mouse HSC, Twist1 transcriptionally activates the E-box in the miR-214
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promoter and that the resulting high levels of miR-214 are inhibitory for CCN2 production. To
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confirm that the Twist1-miR-214-CCN2 axis is evolutionarily conserved in human HSC, LX-2
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cells were analyzed for CCN2 expression downstream of Twist1. As shown in Figure 2F,
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transfection of the cells with Twist1 resulted in reduced CCN2 mRNA expression or protein
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production but this was reversed by co-transfection of the cells with a miR-214 antagomir
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showing that CCN2 inhibition by Twist1 is indirect and mediated through miR-214.
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Twist1 is shuttled between HSC in exosomes and targets the miR214-CCN2 axis in recipient
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cells.
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We have previously shown that HSC secrete nanovesicular exosomes (3, 4). In studying the
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identity of the molecular constituents of these exosomes, we found that they contained Twist1
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mRNA or protein, the concentrations of which were reduced in exosomes from Day 20 HSC as
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compared to those from Day 3 HSC (Figure 3A), consistent with the activation-associated
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decrease documented earlier for cellular Twist1 (Figure 1D,E). Since purified HSC-derived
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exosomes stained with the fluorescent marker PKH26 for visualization were taken up by P4
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HSC after incubation for 12 hours (Figure 3B), we investigated whether this process resulted in
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the delivery of endogenous exosomal Twist1 and its regulation of DNM3os in the recipient cells.
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First, since primary HSC at very early stages of culture are difficult to transfect, we used
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exosome donor cells on Day 6 of culture because they are amenable to transfection and,
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importantly, still express readily detectable levels of Twist1 (see Figure 1D). As compared to
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non-transfected controls transfection of Day 6 cells with Twist1 siRNA resulted in the production
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of exosomes that contained significantly diminished Twist1 mRNA or protein levels, whereas
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exosomal CD9 protein levels remained unchanged (Figure 3C). As compared to the effects of
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control exosomes from Day 6 HSC which contained relatively high levels of endogenous Twist1,
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treatment of P4 recipient HSC (expressing negligible levels of endogenous Twist1) with Twist1-
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depleted exosomes from Twist1 siRNA-transfected HSC resulted in reduced Twist1 mRNA or
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protein in the recipient cells, and this was accompanied by decreased miR-214 expression and
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enhanced expression of CCN2, αSMA or collagen αI(1) (Figure 3C). These data showed that
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through its epigenetic regulation of miR-214, delivery of exosomal Twist1 serves to dampen
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expression of CCN2 and its downstream effectors, αSMA or collagen αI(1) .
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When Day 6 HSC were transfected with DNM3os promoter reporters, luciferase activity of the
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wild-type promoter was stimulated in the presence of co-cultured Day 1 HSC. This response
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was blocked when exosome production by the D1 cells was blocked using GW4869 (a chemical
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inhibitor that inhibits n-sphingomyelinase 2 and downstream exosome production (2-4, 19)),
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while the involvement of Twist1 in the response was shown by the lack of activity of the mutant
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DNM3os (Figure 3D). Since some parenchymal cells were also observed to stain positively for
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Twist1 in liver sections (see Figure 1B), we also investigated the ability of hepatocytes to deliver
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exosomal Twist1 to HSC in the same co-culture system. These experiments, which were
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performed with AML12 cells showed that hepatocytes were able to regulate wild type but not
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mutant DNM3os in recipient cells and that this was GW4869-dependent (Figure 3D).
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Collectively these data showed that the E-box in DNM3os in HSC is regulated by Twist1
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delivered intercellularly within exosomes that are secreted by other HSC or by hepatocytes.
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Finally, in view of the emerging interest in using circulating exosomes for disease diagnosis or
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assessment (32), we analyzed Twist1, miR-214 or CCN2 expression in exosomes recovered
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from the circulation of mice during CCl4-induced fibrosis. As shown in Figure 3E, experimental
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fibrosis was associated with a progressive decrease in circulating exosomal Twist1 or miR-214
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and an increase in circulating exosomal CCN2, which paralleled their respective changes in the
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fibrosing livers themselves (Figure 1B, (4)).
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Discussion
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Twist1 is a 21kDa bHLH transcription factor that was discovered based on its induction of cell
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differentiation and control of dorso-ventral patterning in Drosophila; Twist-null embryos are
339
mesoderm-deficient, have a twisted appearance, and subsequently die (35, 38). Although the
340
mammalian counterparts, Twist1 and Twist2, also play a central role in cell fate determination,
341
their actions are generally inhibitory during differentiation of muscle, bone, and other cells in the
342
mesenchymal lineage (13, 20, 21, 25, 42). During embryogenesis, Twist1 is expressed at high
343
levels and in a spatio-temporal manner that reflects its role in regulating genes that govern
344
mesoderm specification or differentiation as well as mesenchymal tissue development (12, 30).
345
In adults, Twist1 expression is low but present in stem cells of muscle, adipose tissue, bone
346
marrow, and other mesenchymal tissues of mesoderm origin (17, 40). Twist1 gene mutations in
347
humans result in Saethre-Chotzen syndrome (8, 14), and a similar condition occurs in Twist1-
348
deficient mice (1). Twist1 expression is also strongly associated with cancers of the breast,
349
prostate, esophagus, stomach, liver, pancreas, or bladder and, though its mechanisms of action
350
are variable, it regulates cancer cell senescence, apoptosis, resistance to chemotherapy,
351
differentiation, invasiveness, and metastasis, the latter due to its ability to drive epithelial-
352
mesenchymal transition (EMT) (33).
353
In these studies, we identified Twist1 as a product of quiescent HSC in adult mice which
354
indirectly inhibits CCN2 production through its transcriptional activation of miR-214, the latter of
355
which suppresses CCN2 via direct binding to the CCN2 3’-UTR. During HSC activation, higher
356
levels of CCN2 result, at least in part, from decreased expression of Twist1 resulting in reduced
357
miR-214 transcription. The regulation by Twist1 of the miR-214-CCN2 axis in HSC thus reveals
358
a hitherto unrecognized role for Twist1 as a suppressor of CCN2 expression and its
359
downstream fibrogenic signals (Figure 4). We further showed that this action of Twist1 is
360
critically dependent on its activation of the E-box in the miR-214 promoter (Figure 4), a
361
mechanism that is also responsible for Twist1-regulation of miR-214/miR-199a during
362
development (23). Our identification of miR-214 as a Twist1-regulated gene in HSC is
363
consistent with previous studies showing that Twist regulates DNM3os or miR-199a/214
364
expression during development (23, 26, 27, 41) or in epithelial ovarian cancer cells
365
differentiating from stem-like cells along a miR-199a/214-dependent axis (44). In the liver, miR-
366
214 suppresses stem-like traits, invasion or recurrence in human hepatocellular carcinoma (22,
367
28, 29) while miR-199a/214 is down-regulated in rodent models of alcoholic steatohepatitis (7)
368
or fibrosis (4). Forced over-expression of miR-214 in activated mouse HSC decreases
12
369
expression of inflammation- or fibrosis-associated genes including interleukin-1α or -10, integrin-
370
α3 or –β8, platelet-derived growth factor-α, matrixmelloproteinase-2,-8, or -13, tissue inhibitor of
371
metalloproteinase 1, and CCN2 (4). Conversely, expression of miR-214-5p, which comprises
372
the complementary sequence arising from the 5’ arm of the mir-214 hairpin, is enhanced in
373
fibrotic liver and activated HSC and is associated with expression of fibrosis-related genes (16).
374
We are not aware of previous reports of Twist1 in adults in mesenchymal cell types such as
375
HSC which play specialized roles in wound healing or fibrosis. There is, however, evidence that,
376
through its effects on EMT, Twist1 stimulates an epithelial contribution to fibrotic processes in
377
the kidney, lung, and oral cavity (6, 31, 36). Although we observed some Twist1-positive
378
hepatocytes in normal livers and the ability of hepatocyte-derived exosomal Twist1 to regulate
379
miR-214 in HSC, the substantial suppression of hepatic Twist1 expression during experimental
380
fibrosis coupled with emerging evidence that EMT does not contribute to hepatic fibrosis (43)
381
highlights the need for further studies in this area. Even so, a role for epithelial Twist1 in more
382
severe hepatic pathology is supported by studies of human hepatocellular carcinoma in which
383
Twist1 stimulates metastasis and angiogenesis by reducing expression of E-cadherin,
384
increasing expression of N-cadherin and vascular endothelial growth factor, and increasing cell
385
motility and invasiveness (22, 28, 29). EMT and metastasis of cholangiocarcinoma are also
386
dependent on a miR-214-Twist1 axis but in this case Twist1 appears to be a target of miR-214
387
(24).
388
Another novel aspect of our studies was the identification of Twist1 as a component of HSC- or
389
hepatocyte-derived exosomes that allowed for its intercellular shuttling to neighboring HSC.
390
Exosomes are membranous nanovesicles that arise by inward budding of multivesicular bodies
391
and are released extracellularly when these multivesicular bodies fuse internally with the plasma
392
membrane; thereafter exosomes traverse the intercellular spaces and may be taken up by
393
neighboring cells (18, 37, 39). Exosomes contain a complex mixture of miRs, mRNAs and
394
proteins and therefore their intercellular shuttling represents a communication pathway through
395
which genetic, epigenetic or proteomic information may be delivered from donor cells that
396
impact gene expression in recipient cells
397
exosomally delivered to recipient activated HSC in which it then targets the miR-214 promoter.
398
Since miR-214 and CCN2 themselves are also individually and functionally delivered to HSC
399
within exosomes (3, 4), the manifestation of fibrogenic pathways in any given HSC likely reflects
400
the net action of its own cellular constituents in combination with those that are exosomally
401
received from neighboring HSC and other cell types. Finally, since the fibrosis-related
(34). In this study, we showed that Twist1 is
13
402
suppression of hepatic Twist1, CCN2, or mIR-214 was mirrored in circulating exosomes,
403
evaluation of hepatic fibrosis may be possible based upon the relative expression of a slate of
404
signature molecular components in circulating exosomes; this approach, which is non- or
405
minimally invasive and can be undertaken repeatedly in individual patients, is rapidly emerging
406
as a powerful diagnostic tool in other pathologies and diseases (32, 39).
407
In summary, through its transcriptional activation of the DNM3os E-box, cellular or exosomal
408
Twist1 drives miR-214 expression and suppresses CCN2 production and downstream
409
fibrogenic signaling. Twist1 is thus identified as a novel regulator of HSC function.
410
411
Grants: This work was supported by NIH/NIAAA grant 1RO1 AA021276 awarded to DRB.
14
412
Figure legends
413
Figure 1. Twist1 is expressed at high levels in normal liver or HSC and is suppressed
414
during fibrosis.
415
(A) Expression of Twist1, CCN2, αSMA, or collagen α1(I) mRNA or miR-214 assessed by qRT-
416
PCR and normalized to GAPDH mRNA after administration of oil or CCl4 for 5 weeks
417
determined for RNA from either liver tissue (upper panel) or HSC isolated from the livers and
418
maintained in culture for 24 hrs (lower panel) (n=5 independent experiments performed in
419
triplicate, *P<0.001 versus oil control). The insets show detection of Twist1 or CCN2 by Western
420
blotting of 20μg total protein (β-actin was detected equally in each set of paired samples; data
421
not shown). (B). Upper Panel: H and E staining or immunohistochemical detection of desmin,
422
Twist1, or αSMA in the livers of mice treated with oil or CCl4 for 5 weeks. Immunostained
423
specimens were also stained with DAPI nuclear stain (blue). In control livers, Twist1 staining
424
was associated with the desmin-positive quiescent HSC population, with some staining also in
425
hepatocytes. Fibrosis in response to CCl4 was accompanied by HSC activation as shown by
426
increased αSMA staining in HSC, and a reduction of Twist1 staining in both HSC and
427
hepatocytes. The exploded parts of the figure illustrate the differences in Twist1 staining in
428
desmin-positive HSC from normal versus fibrotic liver. Lower Panel: Immunocytochemical
429
staining for desmin, αSMA, Twist1, CCN2 or collagen α1 in HSC from control or CCl4-treated
430
mice obtained as described in (A) (Bars: 20 μm for immunostained sections; 50 μm for H&E
431
stained sections). (C) Immunohistochemical detection of αSMA, CCN2 or collagen α1 (upper
432
panel; Bar:20μm) or expression of Twist1, miR-214 or CCN2 (lower panel) in the livers of mice
433
treated with water (control) or TAA for 5 weeks to induce HSC activation and fibrosis (n=5
434
independent experiments performed in triplicate, *P<0.001, +P<0.05 versus no treatment). (D).
435
Twist1, miR-214 or CCN2 expression analyzed by qRT-PCR and normalized to GAPDH mRNA
436
in primary HSC isolated from livers of normal mice and cultured for up to 14 days (n=3
437
independent experiments performed in triplicate, *P<0.001 versus Day 2, +P<0.05 versus Day
438
2). (E). qRT-PCR of Twist1, CCN2 mRNA or miR-214, normalized to GAPDH mRNA, after Day
439
3 primary mouse HSC were incubated in 1% serum for 24 hours prior to 48-hour treatment with
440
0 or 50mM ethanol (n=3 independent experiments performed in triplicate, +P<0.05 versus ctrl).
441
Figure 2. Twist1 regulates CCN2 production via transcriptional control of miR-214 E-box
442
promoter element
15
443
Expression of Twist1 or CCN2 mRNA, or miR-214, was assessed by qRT-PCR and normalized
444
to GAPDH mRNA in (A) passage 6 mouse HSC transfected with Twist1 or (B) Day 6 primary
445
mouse HSC transfected with 100nM Twist1 siRNA (n=3 independent experiments performed in
446
triplicate, *P<0.001 versus scramble, +P<0.05 versus scramble). The insets show detection of
447
Twist1 by Western blotting of 20μg total protein (staining for β-actin confirmed equal protein
448
loading; data not shown). (C) Day 6 primary mouse HSC were transfected with parental pGL
449
4.11[Luc2P]-vector (“Vector”) or pGL 4.11[Luc2P] containing wild-type mouse DNM3os
450
promoter (“WT”) or a substitution mutation in the DNM3os promoter targeting the E-box.
451
(“Mut.”). After 36 hours, firefly luciferase activity in cell lysates was measured and normalized to
452
that
453
*P<0.001,+P<0.05 versus “vector” group). (D). Nuclear extracts from D1 mHSC underwent
454
EMSA by incubation with oligonucleotide probes corresponding to the wild type or mutated
455
Twist1 binding site in the DNM3os promoter. Shifted complexes are indicative of binding
456
interactions with the probe; the involvement of Twist1 is indicated by a supershifted complex
457
using Twist1 antibody. Data are representative of three independent experiments. (E) ChIP-
458
PCR of DNM3os DNA in immune complexes generated using Twist1 antibody (“+”) to pull down
459
endogenous Twist1-DNM3os complexes from Day1 or P7 HSC. Control reactions were
460
performed with normal IgG (“-‘) *P<0.001. (F) qRT-PCR (left panel) of CCN2 mRNA normalized
461
to GAPDH mRNA, after LX-2 cells were transfected with Twist1 alone or together with miR-214
462
antagomir (n=3 independent experiments performed in triplicate, +P<0.05 versus non-
463
transfection). Western blot images and quantification thereof (center and right panels) show that
464
Twist1 transfection of LX-2 cells caused higher Twist1 but lower CCN2 protein levels, but that
465
CCN2 levels were restored to normal levels when Twist1-transfected cells were also transfected
466
with a miR-214 antagomir. The CCN2 Western blot shows the principal CCN2 38kDa protein
467
and its 10-20kDa proteolytic cleavage products.
468
Figure 3. Identification and intercellular transfer of exosomal Twist1.
469
(A)Twist1 mRNA assessed by qRT-PCR and normalized to let-7a (left) or protein assessed by
470
Western blot (right; 5μg total protein) in exosomes isolated from Day 3 or Day 20 mouse HSC
471
(n=3 independent experiments performed in triplicate, *P<0.001 versus Day 3). (B) P6 HSC
472
were incubated for 12 hrs with exosomes purified from P6 HSC and subsequently stained with
473
PKH-26. Cells were visualized for exosome fluorescence (red) and αSMA immunofluorescence
474
(green) by confocal microscopy; Inset: appearance of HSC-derived exosomes by cryogenic
475
transmission electron microscopy. (C) Reduced Twist1 mRNA expression (top left) or protein
of
Renilla
luciferase
(n=3
independent
16
experiments
performed
in
triplicate,
476
levels (top right) in exosomes produced over 24 hrs after Twist1 siRNA-transfection of Day 6
477
donor HSC and the effect of these exosomes on Twist1, miR-214,CCN2, αSMA, or collagen α(I)
478
expression assessed by RT-PCR or Twist1 protein levels assessed by Western blot (20μg total
479
protein, for which β-actin signals were detected equally between samples; data not shown) after
480
being added to recipient P4 HSC for 12 hrs (bottom). *P<0.001 and +P<0.05 versus control
481
exosomes from non-tranfected cells. (D) Day 6 primary mouse HSC were transfected with
482
parental pGL 4.11[Luc2P]-vector (“Vector”) or pGL 4.11[Luc2P] containing wild-type (WT) or
483
mutant (“Mut”) mouse DNM3os promoter (“WT”) (see Figure 2C) and co-cultured for 24hrs with
484
D1 HSC (left panel) or AML12 mouse hepatocytes (right panel) that had been pre-treated with
485
or without GW4869 for 24 hours. Firefly luciferase activity in cell lysates was measured and
486
normalized to that of Renilla luciferase (n=3 independent experiments performed in triplicate,
487
+P<0.05 versus “Vector”). (E). A PureExo exosome isolation kit was used to isolate circulating
488
exosomes from the serum of mice after 5-week administration of oil or CCl4 Twist1, miR-214 or
489
CCN2 expression in circulating exosomes were analyzed by qRT-PCR and normalized to let-7a
490
(n=5 independent experiments performed in triplicate) *P<0.001, +P<0.05 versus oil group.
491
Figure 4. The Twist1-miR-214-CCN2 axis in HSC. In quiescent HSC, Twist1 is expressed at
492
high levels and drives miR-214 expression through its binding of the E-box in DNM3os. One of
493
the targets of miR-214 is the 3’-UTR of CCN2, resulting in the inhibition of CCN2 in quiescent
494
cells. Twist1 (these results) or miR-214 (4) are exported from quiescent HSC (or hepatocytes
495
(see Figure 3D)) in exosomes allowing them to exert epigenetic effects on their targets (miR-
496
214 or CCN2 respectively) after being shuttled to neighboring HSC, causing fibrogenic signaling
497
to be suppressed. HSC activation is characterized by suppressed Twist1 production (the
498
mediators of which have yet to be determined) and reduced activation of the E-box (dashed
499
arrow). In turn, expression of miR-214 and its binding to the CCN2 3’-UTR are reduced, allowing
500
CCN2 expression and its downstream fibrogenic cascades to be manifested. Twist1 or miR-214
501
are incorporated into exosomes at lower levels (dashed arrows) with the result that exosomes
502
from activated HSC are less effective at suppressing the miR-214-CCN2 axis in neighboring
503
HSC.
504
17
505
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20
Table 1. Primers used for RT-PCR
Primers
GenBank
Product
Gene
accession number
Sense
Anti-sense
size (bp)
Twist1 (mouse)
NM_011658
5’ CGACGACAGCCTGAGCAACA 3’
5’ TGCAGCTCCTCGTACGACTG 3’
293
CCN2 (mouse)
NM_010217
5’ CACTCTGCCAGTGGAGTTCA 3’
5’ AAGATGTCATTGTCCCCAGG 3’
111
miR-214 (mouse)
NR_029796
Universal anti-sense
20
Collagen α1(I) (mouse)
NM_007742
5’ GCCCGAACCCCAAGGAAAAGAAGC 3’
5’ CTGGGAGGCCTCGGTGGACATTAG 3’
148
αSMA (mouse)
NM_007392
5’GGCTCTGGGCTCTGTAAGG3’
5’CTCTTGCTCTGGGCTTCATC3’
148
GAPDH (mouse)
NM_002046
5’ TGCACCACCAACTGCTTAGC 3’
5’ GGCATGGACTGTGGTCATGAG 3’
87
5’ ACAGCAGGCACAGACAGGCA 3’