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Emodin-Mediated Cross-Linking Enhancement for Extracellular Matrix Homeostasis

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Emodin-Mediated Cross-Linking Enhancement for Extracellular Matrix Homeostasis Biochemical and Biophysical Research Communications xxx (2014) xxx–xxx Contents lists available at ScienceDirect Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc Emodin-mediated cross-linking enhancement for extracellular matrix homeostasis Lihua Jian a, Chen Zhang a, Guangfeng Chen a, Xiujuan Shi a, Yu Qiu a, Yunyun Xue a, Shuzhang Yang a, ⇑ ⇑ Lixia Lu a, Qionglan Yuan a, Guotong Xu a, Ming Ying a,b, , Xiaoqing Liu a,b, a Shanghai Tenth People’s Hospital, Tongji University School of Medicine, Shanghai, China b College of Life Sciences, Shenzhen University, Shenzhen, China article info abstract Article history: The extracellular matrix (ECM) is an essential element of mammalian organisms, and its cross-linking Received 11 March 2014 formation plays a vital role in ECM development and postnatal homeostasis. Defects in cross-link Available online xxxx formation caused by aging, genetic, or environmental factors are known to cause numerous diseases in mammals. To augment the cross-linking formation of ECM, the present study established a ZsGreen Keywords: reporter system controlled by the promoter of lysyl oxidase-like 1 gene (LOXL1), which serves as both Emodin a scaffold element and a cross-linking enzyme in the ECM. By using this system in a drug screen, we Extracellular matrix (ECM) identified emodin as a strong enhancer of LOXL1 expression that promoted cross-linking formation of Lysyl oxidase-like 1 (LOXL1) ECM in all the tested systems, including human fibroblast cells, cultured human skin tissues, and animals Cross-linking that received long-term emodin treatment. Collectively, the results suggest that emodin may serve as an effective drug or supplement for ECM homeostasis. Ó 2014 Elsevier Inc. All rights reserved. 1. Introduction in both elastic fibers and collagen bundle [14–17]. Recently, we have demonstrated that LOXL1 deficiency leads to defective elastin depo- Elastic fibers and collagens are two major components of the sition [8–10]. We and another research team have also found that extracellular matrix (ECM). Elastic fibers confer resilience and LOXL1 down-regulation is associated with aging [9,18], suggesting elasticity to lung, skin, pelvic organs, and blood vessels while that LOXL1 enhancement either by LOXL1 gene delivery or by small collagen bundles provide stiffness for tissue support [1–4]. Defects molecule supplementation may attenuate disease conditions. To in elastogenesis are known to cause numerous diseases, such as explore drugs or supplements that would be useful for elastic fiber emphysematous lung, loose skin, cardiovascular abnormalities, homeostasis, we established a LOXL1 promoter activity drug screen. and age-related macular degeneration [5–7]. In addition, we have Using this system, we found that emodin up-regulates LOXL1 gene recently found that elastic fiber dyshomeostasis confers suscepti- expression, which enhances cross-linking formation in ECM. bility to choroidal neovascularization (CNV), pelvic organ prolapse (POP) and POP-associated urinary disorders, including urinary 2. Materials and methods incontinence and retention [8–10]. Collagen defects are associated with Ehlers–Danlos syndrome and Alport syndrome [11,12]. 2.1. Drug library for screening One major process of ECM development and homeostasis is the polymerization of elastin and collagen monomers. These cross- The drug library of 31 monomeric compounds and 351 tradi- linking reactions are catalyzed by a series of enzymes, including tional Chinese medicines (TCMs) was kindly provided by Professor lysyl oxidase (LOX) family proteins and lysyl hydroxylase. Mamma- Xuexun Fang (College of Life Science, Jilin University, Changchun, lian genomes have up to five LOX members that encode prototypic China). The details for the drug library refer to the Supplementary LOX and LOX-like proteins 1 through 4 (LOXL1–LOXL4) [13]. While materials (Supplementary Tables S1 and S2). the individual roles of these LOX members remain to be fully eluci- dated, disruption of the prototypic LOX gene reduces cross-linking 2.2. Constructs and stable cells for drug screening ⇑ Corresponding authors at: Shanghai Tenth People’s Hospital, Tongji University The upstream non-coding region of the human LOXL1 gene School of Medicine, Shanghai, China. (3 kb in length), predicted to contain the LOXL1 promoter, was E-mail addresses: [email protected] (M. Ying), [email protected] (X. Liu). cloned into the pLVX-ZsGreen vector (Biovector, Beijing, China) by http://dx.doi.org/10.1016/j.bbrc.2014.03.052 0006-291X/Ó 2014 Elsevier Inc. All rights reserved. Please cite this article in press as: L. Jian et al., Emodin-mediated cross-linking enhancement for extracellular matrix homeostasis, Biochem. Biophys. Res. Commun. (2014), http://dx.doi.org/10.1016/j.bbrc.2014.03.052 2 L. Jian et al. / Biochemical and Biophysical Research Communications xxx (2014) xxx–xxx substituting its original CMV promoter, resulting in a lentiviral vec- Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA was tor pLenti-PLOXL1-ZsGreen. The 2nd generation lentiviral packaging amplified as an internal standard for quantification. PCR primers system was used to produce lentiviral particles. To generate stable for human GAPDH were (50-GCACCGTCAAGGCTGAGAAC-30) and cells containing the pLOXL1-ZsGreen component, the lentiviral par- (50-TGGTGAAGACGCCAGTGGA-30). PCR primers for mouse GAPDH ticles were used to infect a human fibroblast cell line, and then flow were (50-CTTTGGCATTGTGGAAGGGCTC-30) and (50-GCAGGGATG- cytometry was used to sort out the cells stably labeled with ZsGreen ATGTTCTGGGCAG-30). For quantitative PCR (qPCR), the reaction fluorescence. We plated 293T cells at a density of 6 Â 106 cells per was performed with a SYBRÒ Premix Ex Taq (DRR820A, TaKaRa) 100 mm dish and incubated the cell overnight before transfection and analyzed with the 7500 real-time PCR system (Applied with 10 lg pLenti-PLOXL1-ZsGreen along with 15 lg Virapower Biosystems). packaging mix using a high-efficiency transfection kit (Invitrogen). Two days after transfection, the supernatant of transfected cells was 2.6. Protein extraction and immunoblotting analysis collected and filtered through a 0.45 lm pore-size cellulose acetate filter (BD Falcon). Human fibroblasts were seeded at a density of After the human skin fibroblast cells treaded with different con- 5 8 Â 10 cells per 100-mm dish 1 day before transduction. The med- centrations of emodin dissolved in 1% DMSO or 1% DMSO as control ium was replaced with virus-containing supernatant supplemented for 24 h, the total cellular protein was extracted from human with 10 lg/ml polybrene (Nacalai Tesque) and incubated for 8 h. fibroblast cells using RIPA buffer containing protease inhibitor Two days after infection, approximately 80% of the cells exhibited (Thermo Scientific). The cellular proteins were separated by 10% ZsGreen fluorescence. ThehSF-pLenti-PLOXL1-ZsGreen cells stably SDS–polyacrylamide gel electrophoresis (SDS–PAGE) and electro- labeled with ZsGreen were collected by flow cytometry sorting transferred onto polyvinylidene difluoride (PVDF) membranes (BD FACS Aria II cell sorter) for drug screening experiments. (Millipore, Bedford, MA). The proteins were then visualized in an imager (Luminescent image analyzer, Image Quant LAS4000 mini) 2.3. Cell culture using enhanced chemiluminescence (ECL) detection reagent substrate (SuperSignal West Pico Chemiluminescent Substrate, Human skin fibroblast (hSF, Shanghai Inst Biol Sci) and human Thermo Scientific). To ensure equal protein loading, the same blot dermal fibroblast (hDF, HUM-CELL-0069, PriCells) cells were main- was subsequently developed for actin expression. The primary anti- tained in Dulbecco’s modified Eagle medium (Hyclone) supple- bodies used for Western blotting were anti-LOXL1 1:1000 (sc- mented with 1% penicillin/streptomycin and 10% fetal bovine 166632, Santa Cruz), anti-actin 1:5000 (HC201, TransGen Biotech) serum (FBS, Hyclone) at 37 °Cin5%CO2. and anti-elastin 1:500 (T3281, Epitomics). The secondary antibod- ies used were goat-anti-rabbit-HRP and goat-anti-mouse-HRP 2.4. Measurement of ZsGreen intensity and screening procedure 1:10,000 (Jackson ImmunoResearch). Human skin fibroblast (hSF-PLenti-PLOXL1-ZsGreen) cells (4000) were seeded into wells of black, clear-bottom, tissue culture 2.7. Desmosine and hydroxyproline analysis surface 96-well plate (3882, Corning) to minimize background fluo- rescence. The cells were treated with a compound for 24 h. The Desmosine and hydroxyproline levels were determined with compounds were dissolved in DMSO at the concentration of desmosine and hydroxyproline enzyme-linked immunosorbent as- 50 mM as stock solutions. Prior to use, the compounds were further say (ELISA) kits (Blue Gene). Specimens were prepared according to diluted with cell culture medium. The final concentration of test the kit instructions. For cell samples, 24 h after emodin treatment, compounds in human fibroblast cell wells was approximately washed the cells twice with ice-cold PBS. 500 ll distilled water 5 lM. DMSO at a concentration of 1% alone was added to control was added to each well and cells were collected using a scraper. wells. One day after compound treatment, the cells were then fixed The samples were then centrifuged for 5 min. The pellets were with PBS containing 4% paraformal-dehyde for 30 min at room tem- hydrolyzed with 6 M HCl aqueous solution at 110 °C for 24 h perature and then washed twice with 200 ll PBS for 5 min each [20]. Hydrolyzed samples
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