DOCSLIB.ORG

Hypoxia‑Inducible Factor 1Α and ROCK1 Regulate Proliferation and Collagen Synthesis in Hepatic Stellate Cells Under Hypoxia

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Hypoxia‑Inducible Factor 1Α and ROCK1 Regulate Proliferation and Collagen Synthesis in Hepatic Stellate Cells Under Hypoxia MOLECULAR MEDICINE REPORTS 18: 3997-4003, 2018 Hypoxia‑inducible factor 1α and ROCK1 regulate proliferation and collagen synthesis in hepatic stellate cells under hypoxia YIBING HU, DANPING HU, HUANHUAN YU, WANGWANG XU and RONGQUAN FU Department of Infectious Diseases, The Third Affiliated Hospital of Wenzhou Medical University, Wenzhou, Zhejiang 325200, P.R. China Received May 16, 2017; Accepted February 27, 2018 DOI: 10.3892/mmr.2018.9397 Abstract. Hypoxia serves a critical role in the pathogenesis of be a critical factor that regulates cell proliferation and collagen liver fibrosis. Hypoxia‑inducible factor α1 (HIF1-α) is induced synthesis in rat HSCs under hypoxia. when cells are exposed to low O2 concentrations. Recently, it has been suggested that Rho‑associated coiled‑coil‑forming Introduction kinase 1 (ROCK1) may be an important HIF1‑α regulator. In the present study, it was analyzed whether crosstalk between Hepatic fibrosis is a common consequence of chronic liver HIF1-α and ROCK1 regulates cell proliferation and collagen diseases of various etiologies, such as hepatitis virus, alcoholic synthesis in hepatic stellate cells (HSCs) under hypoxic liver disease (ALD), non‑alcoholic steatohepatitis (NASH), conditions. For this purpose, a rat hepatic HSC line (HSC‑T6) and others (1). The development of liver fibrosis encompasses was cultured under hypoxic or normoxic conditions, and activation of hepatic stellate cells (HSCs) and their crosstalk HIF1-α and ROCK1 expression was measured at different with liver parenchymal cells, such as hepatocytes and biliary time points. Additionally, HSC‑T6 cells were transfected with epithelial cells (2). HSCs become activated and differentiate HIF1-α small interfering RNA (siHIF1‑α), and measured into myofibroblasts, thus playing a critical role in the produc- protein expression and mRNA transcript levels of α‑smooth tion of collagen and excessive accumulation of extracellular muscle actin, collagen 1A1 and ROCK1. Collagen 3A1 matrix (ECM), which are two main features of hepatic fibro- secretion was also measured by ELISA. Cell proliferation was genesis (3). Liver fibrosis is a form of chronic liver injury that assessed by the MTT assay under these hypoxic conditions. is strongly associated with hypoxia. Moreover, exposure of The results indicated that a specific ROCK inhibitor, Y‑27632, HSCs to hypoxia affects development of hepatic fibrosis (4,5). increased HIF1‑α and ROCK1 expression over time in During the past decades, increasing evidence has suggested HSC‑T6 cells in response to hypoxia. In addition, knockdown that overexpression of hypoxia‑inducible factor 1‑α (HIF1-α) of HIF1-α inhibited HSC‑T6 proliferation, suppressed is closely associated with liver fibrogenesis (6-8). Indeed, over- collagen 1A1 expression, decreased collagen 3A1 secretion expression of HIF1-α in vitro results in HSC activation and and attenuated ROCK1 expression. Notably, ROCK1 inhibition upregulation of genes associated with matrix deposition (9). caused HSC‑T6 quiescence, suppressed collagen secretion and HIF1-α and collagen 1A1 (Col1A1) expression increase in downregulated HIF1‑α expression. Collectively, these findings HSCs under hypoxia (4,10). Transfection with short inter- indicated that the interplay between HIF1‑α and ROCK1 may fering RNA against HIF1-α (siHIF1-α) prevents human HSC migration (7), while upregulation of HIF1‑α is associated with viral hepatitis‑derived fibrosis, HSC activation, and mitogen activated protein kinase (MAPK) activity (11). Additionally, Correspondence to: Dr Rongquan Fu, Department of Infectious expression of fibrogenic mediators is decreased in bile duct Diseases, The Third Affiliated Hospital of Wenzhou Medical ligated HIF1‑α‑knockout mice compared to controls (6). University, 108 Wansong Road, Rui'an, Wenzhou, Zhejiang 325200, Altogether, this strong evidence indicates that HIF1‑α is P.R. China highly involved in hepatic fibrosis; however, the underlying E‑mail: [email protected] regulatory mechanism is still not fully understood. Previously, it was reported that the Rho/Rho‑associated Abbreviations: HSCs, hepatic stellate cells; α-SMA, coiled‑coil‑forming kinase 1 (ROCK1) pathway is involved α‑smooth muscle actin; COL1A1, collagen type 1 α1 chain; COL3A1, collagen type 3 α1 chain; RT‑qPCR, reverse in HSC activation (12). Furthermore, ROCK inhibition with transcription‑quantitative polymerase chain reaction; DMEM, Y‑27632 was demonstrated to suppress HSC proliferation Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; and type I collagen production (13). Several studies have MTT, 3‑(4,5‑dimethylthiazol‑2‑yl)‑2,5‑diphenyltetrazolium bromide clearly indicated that there is crosstalk between ROCK1 and HIF1-α (14-16). However, the relationship between HIF1‑α Key words: hypoxia inducible factor 1‑α, Rho‑associated and ROCK1 in HSCs remains unexplored. coiled‑coil‑forming kinase‑1, HSCs, hypoxia, collagen In the present study, we measured changes in HIF1‑α and ROCK1 expression in HSCs under hypoxia at different 3998 HU et al: HIF1-α AND ROCK‑1 CROSSTALK IN HYPOXIC HSC time points. In order to functionally address the role of was loaded into 8‑10% gradient SDS‑PAGE gels, and HIF1-α in HSC proliferation and collagen synthesis, and transferred to a polyvinylidene difluoride membrane the interplay between HIF1‑α and ROCK1 signaling, we (EMD Millipore, Billerica, MA, USA). Membranes were performed knockdown experiments using siRNA transfection. blocked with 5% skimmed milk for 1 h and then incubated Finally, we investigated whether ROCK1 inhibition mediates with primary antibodies (β-actin, HIF1-α, ROCK1, α-SMA, HIF1-α‑derived HSC activation, thus providing an attractive Col1A1, and Col3A1) at 4˚C overnight. Goat anti‑mouse IgG target for the treatment of hepatic fibrosis. or anti‑rabbit IgG (Jackson, USA) conjugated to horseradish peroxidase was then added for 1 h at 37˚C. Finally, antibody Materials and methods binding was developed using an Odyssey infrared scanner and Western Bright ECL reagents (Thermo Fisher Scientific, Inc.). Cell culture. The HSC‑T6 cell line was maintained in room Samples were washed three times with 1X Tris buffered saline air or in hypoxic conditions (1% O2) in an incubator at 37˚C. and 0.1% Tween‑20 (TBS/T). HSC‑T6 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS; RNA isolation and reverse transcription‑quantitative poly‑ both Gibco; Thermo Fisher Scientific, Inc. Waltham, MA, merase chain reaction (RT‑qPCR). Total cellular RNA was USA), 100 U/ml penicillin, and 100 µg/ml streptomycin. extracted with TRIzol reagent (Invitrogen; Thermo Fisher Culture medium was changed every 24 h, and the ROCK1 Scientific, Inc.), and cDNA synthesis was performed using inhibitor, Y‑27632 (100 µM) (Merck KGaA, Darmstadt, random hexamers with 1.0 mg RNA. RT‑qPCR was performed Germany ), was added. using SYBR‑Green I (Toyobo Life Science, Osaka, Japan). The reaction mixture volume was 20 µl. Primer sequences siRNA transfection. One day prior to transfection, HSC‑T6 were as follows: cells were seeded in 6‑well plates at a density of 2x105 cells per well or in 96‑well plates at a density of 1x104 cells β‑actin (131) forward, 5 ' ‑ C G T AAA GAC CTC TAT GCC AAC per well. Cells were transfected with 100 nM siHIF1‑α A‑3' and reverse, 5 ' ‑ G G A GGA GCA ATG ATC TTG ATC T ‑ 3 ' ; or mock siRNA (as a control) (Shanghai GenePharma HIF‑1α (36) forward, 5 ' ‑ G T C GGA CAG CCT CAC CAA ACA Co., Ltd., Shanghai, China) using Lipofectamine GAG C‑3' and reverse, 5 ' ‑ G T T AAC TTG ATC CAA AGC TCT 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to GAG‑3'; Col1A1 (95) forward, 5 ' ‑ G T A CAT CAG CCC AAA the manufacturer's instructions. Briefly, Lipofectamine‑siRNA CCC CAA G‑3' and reverse, 5 ' ‑ C G G AAC CTT CGC TTC CAT was incubated for 20 min before being added to cells. The ACT C ‑ 3 ' ; Col3A1 (88) forward, 5'‑GAC TGC CCC AAC CCA transfection medium was replaced with fresh medium after 6 h GAG ATC‑3' and reverse, 5 ' ‑ T A C CAT CAG GAA TGA CAG and cells were collected after another 48 h for the follow‑up GAG CAG‑3'; α‑SMA (77) forward, 5'‑GTG CTG TCC CTC tests. TAT GCC TCT GG‑3' and reverse, 5 ' ‑ G G C ACG TTG TGA GTC ACA CCA T C ‑ 3 ' ; ROCK1 (123) forward, 5'‑CGG TAT CTC TAC Enzyme linked immunosorbent assay (ELISA). We measured ATG GTG ATG GAG‑3' and reverse, 5 ' ‑ A T C CAA TGC AAG secreted collagen 3A1 (Col3AI) using a commercially available AAC TAC TTC T G C ‑ 3 ' ELISA kit (Uscn Life Sciences, Inc., Wuhan, China). After cells were transfected with siRNA or treated with the ROCK1 All PCR reactions were normalized to β‑actin. Relative inhibitor, supernatants were collected at 48 h. Samples were quantification of gene expression is shown using the 2‑∆∆Cq added into wells according to the manufacturers' instructions. method. The optical density (OD) values were read at 450 nm with an ELISA reader. The Col3A1 concentration was measured using MTT assay. Cells were cultured at a density of 1.0x104 cells a standard curve diagram. per well in 96‑well plates (each group with 6 duplicates) for 24 h. Then, cells were either transfected with siRNA or Immunofluorescence detection. HSC‑T6 cells were cultured treated with the ROCK1 inhibitor, Y‑27632, and incubated for in hypoxic conditions at 37℃ as described above, and then 48 h. Thereafter, 50 µl of MTT (5 mg/ml) (Nanjing KeyGen collected at 0, 12 and 48 h. The cells were fixed in 4% parafor- Biotech Co., Ltd., Nanjing, China) was added to each well and maldehyde for 15 min, permeabilized with 0.1% Triton X‑100 cells were incubated at 37˚C in an incubator for another 4 h. for 15 min and blocked with 4% bovine serum albumin (BSA) Finally, the medium was discarded and replaced with 150 µl for 1 h.
Load more

Recommended publications
  • Core Lab Brochure
    CHOOSE THE MOST TRUSTED LONG-READ TECHNOLOGY FOR YOUR CORE Sequence with Confidence The Sequel® II and IIe Systems are powered by Single Molecule, Real-Time (SMRT®) Sequencing, a technology proven to produce highly accurate long reads, known as HiFi reads, for sequencing data you and your customers can trust. SMRT SEQUENCING IS SMART BUSINESS HiFi Reads: PacBio is the only sequencing technology to offer highly accurate long reads. Because HiFi reads are extremely accurate, downstream analysis is simplified and streamlined, requiring less compute time than the error-prone long reads of other technologies. High Throughput: The Sequel II and IIe Systems have high data yields on robust, highly automated platforms to increase productivity and reduce project costs. Efficient and Easy-To-Use Workflows: Our end-to-end solutions feature library preparation in <3 hours and many push- button analysis workflows, so you can run projects quickly and easily. Support: All of our products are backed by a global team of scientists, bioinformaticians, and engineers who stand ready to provide you with outstanding service. OUTSTANDING PERFORMANCE AND RELIABILITY 99% of runs on the Sequel II System completed successfully Sequel II Systems provide reliable performance with the total bases produced by the PacBio fleet steadily increasing, and 99% of runs completed successfully. “In our experience, the Sequel II System was essentially production-ready right out of the box. We have used it for a range of applications and sample types — from human genome sequencing to metagenome and microbiome profiling to non-model plant and animal genomes — and results have been very good.” — Luke Tallon, Director of the Genomics Resource Center at Maryland Genomics pacb.com/Sequel SMRT SEQUENCING APPLICATIONS – EFFICIENT AND COST EFFECTIVE The Sequel II and IIe Systems support a wide range of applications, each adding unique value to a sequencing study.
    [Show full text]
  • The Drug Sensitivity and Resistance Testing (DSRT) Approach
    A phenotypic screening and machine learning platform eciently identifies triple negative breast cancer-selective and readily druggable targets Prson Gautam 1 Alok Jaiswal 1 Tero Aittokallio 1, 2 Hassan Al Ali 3 Krister Wennerberg 1,4 Identifying eective oncogenic targets is challenged by the complexity of genetic alterations in 1Institute for Molecular Medicine Finland (FIMM), HiLIFE, University of Helsinki, Finland cancer and their poorly understood relation to cell function and survival. There is a need for meth- Current kinome coverage of kinase inhibitors in TNBC exhibit diverse kinase dependencies MFM-223 is selectively addicted to FGFR2 2Department of Mathematics and Statistics, University of Turku, Finland 3The Miami Project to Cure Paralysis, Peggy and Harold Katz Family Drug Discovery Center, A A Sylvester Comprehensive Cancer Center, and Department of Neurological Surgery and Medicine ods that rapidly and accurately identify “pharmacologically eective” targets without the require- clinical evaluation TN Kinases MFM-223 CAL-120 MDA-MB-231 TNBC TNBC TNBC TNBC TNBC TNBC HER2+ 100 University of Miami Miller School of Medicine, Miami, FL 33136, USA. non- HER2+ FGFR1 0.97 0.00 0.00 MFM-223 BL1 BL2 M MSL IM LAR ER+, PR+ 50 ment for priori knowledge of complex signaling networks. We developed an approach that uses ma- cancerous FGFR2 56.46 0.00 0.00 CAL-120 25 4 MDA-MB-231 Biotech Research & Innovation Centre (BRIC) and Novo Nordisk Foundation Center HCC1937 CAL-85-1 CAL-120 MDA-MB-231 DU4475 CAL-148 MCF-10A SK-BR-3 BT-474 FGFR3 25.10 0.00 0.00 0 chine learning to relate results from unbiased phenotypic screening of kinase inhibitors to their bio- for Stem Cell Biology (DanStem), University of Copenhagen, Denmark HCC1599 HDQ-P1 BT-549 MDA-MB-436 MFM-223 FGFR4 0.00 0.00 0.00 MAXIS*Bk Clinical status MDA-MB-468 CAL-51 Hs578T MDA-MB-453 score chemical activity data.
    [Show full text]
  • Application of a MYC Degradation
    SCIENCE SIGNALING | RESEARCH ARTICLE CANCER Copyright © 2019 The Authors, some rights reserved; Application of a MYC degradation screen identifies exclusive licensee American Association sensitivity to CDK9 inhibitors in KRAS-mutant for the Advancement of Science. No claim pancreatic cancer to original U.S. Devon R. Blake1, Angelina V. Vaseva2, Richard G. Hodge2, McKenzie P. Kline3, Thomas S. K. Gilbert1,4, Government Works Vikas Tyagi5, Daowei Huang5, Gabrielle C. Whiten5, Jacob E. Larson5, Xiaodong Wang2,5, Kenneth H. Pearce5, Laura E. Herring1,4, Lee M. Graves1,2,4, Stephen V. Frye2,5, Michael J. Emanuele1,2, Adrienne D. Cox1,2,6, Channing J. Der1,2* Stabilization of the MYC oncoprotein by KRAS signaling critically promotes the growth of pancreatic ductal adeno- carcinoma (PDAC). Thus, understanding how MYC protein stability is regulated may lead to effective therapies. Here, we used a previously developed, flow cytometry–based assay that screened a library of >800 protein kinase inhibitors and identified compounds that promoted either the stability or degradation of MYC in a KRAS-mutant PDAC cell line. We validated compounds that stabilized or destabilized MYC and then focused on one compound, Downloaded from UNC10112785, that induced the substantial loss of MYC protein in both two-dimensional (2D) and 3D cell cultures. We determined that this compound is a potent CDK9 inhibitor with a previously uncharacterized scaffold, caused MYC loss through both transcriptional and posttranslational mechanisms, and suppresses PDAC anchorage- dependent and anchorage-independent growth. We discovered that CDK9 enhanced MYC protein stability 62 through a previously unknown, KRAS-independent mechanism involving direct phosphorylation of MYC at Ser .
    [Show full text]
  • Supplementary Table 1. in Vitro Side Effect Profiling Study for LDN/OSU-0212320. Neurotransmitter Related Steroids
    Supplementary Table 1. In vitro side effect profiling study for LDN/OSU-0212320. Percent Inhibition Receptor 10 µM Neurotransmitter Related Adenosine, Non-selective 7.29% Adrenergic, Alpha 1, Non-selective 24.98% Adrenergic, Alpha 2, Non-selective 27.18% Adrenergic, Beta, Non-selective -20.94% Dopamine Transporter 8.69% Dopamine, D1 (h) 8.48% Dopamine, D2s (h) 4.06% GABA A, Agonist Site -16.15% GABA A, BDZ, alpha 1 site 12.73% GABA-B 13.60% Glutamate, AMPA Site (Ionotropic) 12.06% Glutamate, Kainate Site (Ionotropic) -1.03% Glutamate, NMDA Agonist Site (Ionotropic) 0.12% Glutamate, NMDA, Glycine (Stry-insens Site) 9.84% (Ionotropic) Glycine, Strychnine-sensitive 0.99% Histamine, H1 -5.54% Histamine, H2 16.54% Histamine, H3 4.80% Melatonin, Non-selective -5.54% Muscarinic, M1 (hr) -1.88% Muscarinic, M2 (h) 0.82% Muscarinic, Non-selective, Central 29.04% Muscarinic, Non-selective, Peripheral 0.29% Nicotinic, Neuronal (-BnTx insensitive) 7.85% Norepinephrine Transporter 2.87% Opioid, Non-selective -0.09% Opioid, Orphanin, ORL1 (h) 11.55% Serotonin Transporter -3.02% Serotonin, Non-selective 26.33% Sigma, Non-Selective 10.19% Steroids Estrogen 11.16% 1 Percent Inhibition Receptor 10 µM Testosterone (cytosolic) (h) 12.50% Ion Channels Calcium Channel, Type L (Dihydropyridine Site) 43.18% Calcium Channel, Type N 4.15% Potassium Channel, ATP-Sensitive -4.05% Potassium Channel, Ca2+ Act., VI 17.80% Potassium Channel, I(Kr) (hERG) (h) -6.44% Sodium, Site 2 -0.39% Second Messengers Nitric Oxide, NOS (Neuronal-Binding) -17.09% Prostaglandins Leukotriene,
    [Show full text]
  • GAK and PRKCD Are Positive Regulators of PRKN-Independent
    bioRxiv preprint doi: https://doi.org/10.1101/2020.11.05.369496; this version posted November 5, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 1 GAK and PRKCD are positive regulators of PRKN-independent 2 mitophagy 3 Michael J. Munson1,2*, Benan J. Mathai1,2, Laura Trachsel1,2, Matthew Yoke Wui Ng1,2, Laura 4 Rodriguez de la Ballina1,2, Sebastian W. Schultz2,3, Yahyah Aman4, Alf H. Lystad1,2, Sakshi 5 Singh1,2, Sachin Singh 2,3, Jørgen Wesche2,3, Evandro F. Fang4, Anne Simonsen1,2* 6 1Division of Biochemistry, Department of Molecular Medicine, Institute of Basic Medical Sciences, University of Oslo 7 2Centre for Cancer Cell Reprogramming, Institute of Clinical Medicine, Faculty of Medicine, University of Oslo, N-0316, Oslo, Norway. 8 3Department of Molecular Cell Biology, The Norwegian Radium Hospital Montebello, N-0379, Oslo, Norway 9 4Department of Clinical Molecular Biology, University of Oslo and Akershus University Hospital, 1478 Lørenskog, Norway 10 11 Keywords: GAK, Cyclin G Associated Kinase, PRKCD, Protein Kinase C Delta, Mitophagy, DFP, 12 DMOG, PRKN 13 14 *Corresponding Authors: 15 [email protected] 16 [email protected] 17 bioRxiv preprint doi: https://doi.org/10.1101/2020.11.05.369496; this version posted November 5, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
    [Show full text]
  • In Epha4 Signaling in <I>Xenopus Laevis</I>
    Eastern Michigan University DigitalCommons@EMU Senior Honors Theses Honors College 2008 The nI volvement of Rho-Associated Kinases (ROCKs) in EphA4 Signaling in Xenopus laevis Ashley Bate Follow this and additional works at: http://commons.emich.edu/honors Recommended Citation Bate, Ashley, "The nI volvement of Rho-Associated Kinases (ROCKs) in EphA4 Signaling in Xenopus laevis" (2008). Senior Honors Theses. 129. http://commons.emich.edu/honors/129 This Open Access Senior Honors Thesis is brought to you for free and open access by the Honors College at DigitalCommons@EMU. It has been accepted for inclusion in Senior Honors Theses by an authorized administrator of DigitalCommons@EMU. For more information, please contact lib- [email protected]. The nI volvement of Rho-Associated Kinases (ROCKs) in EphA4 Signaling in Xenopus laevis Abstract XEphA4 is a cellular receptor that functions to regulate cell and tissue interactions in amphibian embryos via a repulsive mechanism that involves actin cytoskeleton reorganization. Ectopic EphA4 signaling in Xenopus embryos results in a loss of cell-adhesion and rounded cell morphology, and this phenotype is consistent with EphA4 signaling in cultured A6 cells. How EphA4 achieves its effects on the actin cytoskeleton at the molecular level is largely unknown. One known step in the pathway is that EphA4 causes inhibition of the small GTPase RhoA. RhoA has many downstream effectors that cause cytoskeletal reorganization; the most recognized of these are the ROCK proteins (Rho-associated kinases). ROCK exists in two isoforms, ROCKI and ROCKII. We hypothesize that ROCK inhibition is one step in EphA4 signaling. To test our hypothesis we used ROCK inhibitors and mutants.
    [Show full text]
  • Mtor Inhibitors Potentially Reduce TGF-Β2-Induced Fibrogenic
    www.nature.com/scientificreports OPEN mTOR inhibitors potentially reduce TGF‑β2‑induced fbrogenic changes in trabecular meshwork cells Nozomi Igarashi, Megumi Honjo* & Makoto Aihara We examined the efects of mTOR inhibitors on the fbrotic response induced by transforming growth factor‑beta2 (TGF‑β2) in cultured human trabecular meshwork (hTM) cells. TGF‑β2‑induced expression of fbronectin, collagen type I, alpha 1 chain (COL1A1), and alpha‑smooth muscle actin (αSMA) in hTM cells was examined in the presence or absence of mTOR inhibitors using quantitative real‑time polymerase chain reaction, Western blotting, and immunohistochemistry. The migration rates of hTM cells were examined in the presence of TGF‑β2 with or without mTOR inhibitors. An in vitro study showed that the expression of fbronectin, COL1A1, and αSMA was upregulated by TGF‑β2 treatment of hTM cells; such upregulation was signifcantly suppressed by mTOR inhibitors. The inhibitors signifcantly reduced the migration rate of TGF‑β2‑stimulated hTM cells. mTOR inhibitors may usefully reduce the fbrotic response of hTM cells and we may have to explore if it is also efective in in vivo model. Glaucoma is known to be the second leading cause of blindness worldwide. Aberrant increases in intraocular pressure (IOP) characterize glaucoma, and such an increase in IOP can damage the optic nerve 1–3. IOP reduction is the only established efective therapy that suppresses visual impairment and blindness in both hypertensive and normotensive individuals. IOP elevation is reported to be principally the result of increased aqueous humor (AH) outfow resistance within the conventional outfow pathway 1,4,5. Damage to the collector channels is impli- cated as a cause of resistance within the distal pathway in patients with advanced glaucoma 6.
    [Show full text]
  • Inhibition of ERK 1/2 Kinases Prevents Tendon Matrix Breakdown Ulrich Blache1,2,3, Stefania L
    www.nature.com/scientificreports OPEN Inhibition of ERK 1/2 kinases prevents tendon matrix breakdown Ulrich Blache1,2,3, Stefania L. Wunderli1,2,3, Amro A. Hussien1,2, Tino Stauber1,2, Gabriel Flückiger1,2, Maja Bollhalder1,2, Barbara Niederöst1,2, Sandro F. Fucentese1 & Jess G. Snedeker1,2* Tendon extracellular matrix (ECM) mechanical unloading results in tissue degradation and breakdown, with niche-dependent cellular stress directing proteolytic degradation of tendon. Here, we show that the extracellular-signal regulated kinase (ERK) pathway is central in tendon degradation of load-deprived tissue explants. We show that ERK 1/2 are highly phosphorylated in mechanically unloaded tendon fascicles in a vascular niche-dependent manner. Pharmacological inhibition of ERK 1/2 abolishes the induction of ECM catabolic gene expression (MMPs) and fully prevents loss of mechanical properties. Moreover, ERK 1/2 inhibition in unloaded tendon fascicles suppresses features of pathological tissue remodeling such as collagen type 3 matrix switch and the induction of the pro-fbrotic cytokine interleukin 11. This work demonstrates ERK signaling as a central checkpoint to trigger tendon matrix degradation and remodeling using load-deprived tissue explants. Tendon is a musculoskeletal tissue that transmits muscle force to bone. To accomplish its biomechanical function, tendon tissues adopt a specialized extracellular matrix (ECM) structure1. Te load-bearing tendon compart- ment consists of highly aligned collagen-rich fascicles that are interspersed with tendon stromal cells. Tendon is a mechanosensitive tissue whereby physiological mechanical loading is vital for maintaining tendon archi- tecture and homeostasis2. Mechanical unloading of the tissue, for instance following tendon rupture or more localized micro trauma, leads to proteolytic breakdown of the tissue with severe deterioration of both structural and mechanical properties3–5.
    [Show full text]
  • Lycorine Inhibits Cell Proliferation and Migration by Inhibiting ROCK1/Cofilin‑Induced Actin Dynamics in Hepg2 Hepatoblastoma Cells
    2298 ONCOLOGY REPORTS 40: 2298-2306, 2018 Lycorine inhibits cell proliferation and migration by inhibiting ROCK1/cofilin‑induced actin dynamics in HepG2 hepatoblastoma cells WUYI LIU*, QIAN ZHANG*, QIN TANG, CHANGPENG HU, JINGBIN HUANG, YALI LIU, YANYI LU, QING WANG, GUOBING LI and RONG ZHANG Department of Pharmacy, Xinqiao Hospital, Army Medical University, Chongqing 400037, P.R. China Received January 23, 2018; Accepted July 24, 2018 DOI: 10.3892/or.2018.6609 Abstract. Lycorine, a natural alkaloid extracted from the Introduction Amaryllidaceae plant family, has been reported to exhibit anti-cancer effects in various types of cancer cells. However, Hepatoblastoma, the most commonly diagnosed malignant the molecular mechanisms through which lycorine exhibits pediatric liver tumor, is frequently diagnosed in the first anti-hepatoblastoma activity are unclear. In the present 3 years of life. In recent years, the combination of surgery and study, the inhibitory effects of lycorine on the proliferation chemotherapy has improved the prognosis of patients with and migration of HepG2 hepatoblastoma cells were investi- hepatoblastoma (1). Furthermore, chemotherapeutic agents, gated. Lycorine inhibited the proliferation of HepG2 cells including cisplatin, have been applied in therapeutic strategies in a dose-dependent manner by inducing cell cycle arrest at for hepatoblastoma (2,3). However, conventional chemotherapy the G2/M phase, via downregulation of cyclin A, cyclin B1 agents frequently have limited clinical applications due to the and cyclin dependent kinase 1. Additionally, wound healing adverse side effects and drug resistance acquired following and Transwell assays revealed that treatment with lycorine long-term use. Consequently, it is vital to develop safe and resulted in a decrease in the migratory ability of HepG2 affordable alternative therapeutic agents for the treatment of cells.
    [Show full text]
  • P27 Controls Cytokinesis Via the Regulation of Citron Kinase Activation
    p27Kip1 controls cytokinesis via the regulation of citron kinase activation Murielle P. Serres, … , Nisar P. Malek, Arnaud Besson J Clin Invest. 2012;122(3):844-858. https://doi.org/10.1172/JCI60376. Research Article Oncology p27Kip1 (p27) acts as a tumor suppressor by inhibiting cyclin–cyclin-dependent kinase (cyclin-CDK) activity. However, mice expressing a form of p27 that is unable to bind or inhibit cyclin-CDK complexes (p27CK–) have increased incidence of tumor development as compared with wild-type and p27–/– mice, revealing an oncogenic role for p27. Here, we identified a phenotype of multinucleation and polyploidy in p27CK– mice not present in p27–/– animals, suggesting a role CK– for p27 in G2/M that is independent of cyclin-CDK regulation. Further analysis revealed that p27 expression caused a cytokinesis and abscission defect in mouse embryonic fibroblasts. We identified the Rho effector citron kinase (citron-K) as a p27-interacting protein in vitro and in vivo and found that p27 and citron-K colocalized at the contractile ring and mid- body during telophase and cytokinesis. Moreover, overexpression of the minimal p27-binding domain of citron-K was sufficient to rescue the phenotype caused by p27CK–. Conversely, expression of a mutant p27CK– unable to bind citron-K did not induce multinucleation. Finally, by binding to citron-K, p27 prevented the interaction of citron-K with its activator RhoA. Taken together, these data suggest a role for p27 during cytokinesis via the regulation of citron-K activity. Find the latest version: https://jci.me/60376/pdf Research article p27Kip1 controls cytokinesis via the regulation of citron kinase activation Murielle P.
    [Show full text]
  • Mir‑145‑5P Inhibits Epithelial‑Mesenchymal Transition Via the JNK Signaling Pathway by Targeting MAP3K1 in Non‑Small Cell Lung Cancer Cells
    ONCOLOGY LETTERS 14: 6923-6928, 2017 miR‑145‑5p inhibits epithelial‑mesenchymal transition via the JNK signaling pathway by targeting MAP3K1 in non‑small cell lung cancer cells YONGMEI CHANG1, WENSEN YAN1, CONG SUN1, QINGFENG LIU1, JUN WANG1 and MINGZHI WANG2 Departments of 1Respiratory Medicine and 2Cardiothoracic Surgery, Guangdong No. 2 Provincial People Hospital, Guangzhou, Guangdong 510317, P.R. China Received January 8, 2016; Accepted July 3, 2017 DOI: 10.3892/ol.2017.7092 Abstract. Lung cancer is one of the most common types of has been made in lung cancer therapy, there is ~5% improve- tumors and the leading cause of cancer-associated mortality in ment in 5-year survival rates for the previous 20 years (3). the world. Additionally, non-small cell lung cancer (NSCLC) Epithelial-mesenchymal transition (EMT) is an impor- accounts for ~80% of all lung cancer cases. Epithelial- tant cell biological process, which is associated with cancer mesenchymal transition (EMT) is an important cell biological migration, metastasis, asthma and fibrosis in the lung (4). process, which is associated with cancer migration, metas- When EMT occurs, epithelial cells gradually transform into tasis, asthma and fibrosis in the lung. In the present study, it mesenchymal-like cells by losing their epithelial-associated was revealed that miR-145-5p was able to suppress EMT by functions and characteristics (5). Epigenetic downregula- inactivating the c-Jun N-terminal kinase (JNK) signaling tion of E-cadherin expression was observed in advanced pathway in NSCLC cells. Mitogen-activated protein kinase NSCLC and restoration of E-cadherin expression strongly kinase kinase 1 (MAP3K1) was predicted and confirmed to be suppressed the invasion/migration of tumor cells (6,7).
    [Show full text]
  • Rho Kinase Pathway Alterations in the Brain and Leukocytes in Huntington’Sdisease
    Mol Neurobiol (2016) 53:2132–2140 DOI 10.1007/s12035-015-9147-9 Rho Kinase Pathway Alterations in the Brain and Leukocytes in Huntington’sDisease K. Lakshmi Narayanan 1 & Vanita Chopra1 & H. Diana Rosas1,2,3 & Keith Malarick1,2,3 & Steven Hersch1 Received: 26 June 2014 /Accepted: 19 March 2015 /Published online: 5 May 2015 # The Author(s) 2015. This article is published with open access at Springerlink.com Abstract Huntington’s disease (HD) is a fatal neurodegener- ROCK1 and Profilin1 demonstrated significantly increased ative disease caused by an expanded polyglutamine tract in protein levels, which correlated with the mRNA increases. the huntingtin gene. Therapeutic approaches targeting mutant Collectively, we have shown the panel of Rho kinase pathway huntingtin (mtHtt) or its downstream toxic consequences are genes to be highly altered in human HD blood, postmortem under development, including Rho kinase pathway inhibition. brain tissue, and in R6/2 mice. These studies confirm that HD We investigated the messenger RNA (mRNA) expression of upregulates the Rho kinase pathway and identifies mRNAs Rho kinase pathway genes, including RhoA (Ras homolog that could serve as peripheral markers in HD patients and family member A), ROCK1 (Rho-associated kinase1), translational markers in HD mouse models. PRK2 (protein kinase C-related protein kinase 2), Profilin1, cofilin1, MYPT1 (myosin phosphatase target subunit 1), and Keywords Huntington’sdisease . Rho kinase pathway . LIMK1 (LIM domain kinase 1) in HD human blood leuko- mRNA expression . Biomarker cytes, postmortem brain, and in R6/2 HD mouse brain tissue using qPCR. RhoA, ROCK1, PRK2, Profilin1, cofilin1, and MYPT1 were significantly increased in HD blood compared Introduction to controls.
    [Show full text]