Edited by: T. John Wu, Uniformed Services University of the Health Sciences, United States
Reviewed by: Wilson C. J. Chung, Kent State University, United States; Darwin Omar Larco, Affinivax, Inc, United States
This article was submitted to Experimental Endocrinology, a section of the journal Frontiers in Endocrinology
†These authors have contributed equally to this work
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The GnRH neurons in the hypothalamus secrete gonadotropin releasing hormone, which control the production and release of the gonadotropin-luteinizing hormone (LH) and follicle-stimulating hormone (FSH). LH and FSH stimulate gametogenesis and sex steroid production in the gonads (
CHH is a disorder with great heterogeneity in genetics and phenotype (
In this study, by using GN11 immature GnRH neuronal cell line, we demonstrated that RNF216 regulates the GnRH neuron migration by suppressing Beclin1-mediated autophagy.
To study the effect of RNF216 on the proliferation and migration of GnRH neurons, we utilized the GN11 immature GnRH neuron cell line (
We first down-regulated the RNF216 expression in GN11 cells using small interfering RNAs (siRNAs). As shown in Figure
Depletion of RNF216 inhibited GN11 cells migration.
We further used a trans-well assay to study the effect of RNF216 knockdown on the GN11 cells migration. Cells transfected with a control siRNA or siRNAs targeting RNF216 were loaded onto the upper chamber, and cells migrated to the bottom layer of the chamber were stained with DAPI and counted. As shown in Figures
As an E3 ubiquitin-protein ligase, RNF216 mediates ubiquitination of multiple molecules, such as TLR4, RIP1, TRAF3, Arc, and Beclin1 (
RNF216 was also able to mediate the ubiquitination and degradation of autophagy-related protein Beclin1, inhibiting the occurrence of autophagy (
We first measured the Beclin1 protein level after knockdown of RNF216 in GN11 cells. As shown in Figures
RNF216 regulated GN11 cells migration via Beclin1.
Beclin1 plays an essential role in autophagy induction (
The LC3 antibody used in this study can only detect LC3-II in the GN11 cells, but can detect both LC3-I and LC3-II in 293T cell (Figure
RNF216 regulated GN11 cells migration through autophagy.
To see the involvement of Beclin1 in the autophagy induced by RNF216-depletion, we measured the protein levels of LC3 in GN11 cells transfected with siRNAs targeting RNF216 and Beclin1. As shown in Figure
Autophagy plays an important role in regulating the physiological function of cells, including cell migration (
To further investigate if increased autophagy flux is sufficient to halt the GN11 cells migration, we treated GN11 cells with an autophagy activator rapamycin for 30 h and the cell migration was monitored with a trans-well assay. The promotion of autophagy was confirmed by immunoblotting (Figure
Upregulation of autophagy inhibited GN11 cells migration.
RNF216, an E3 ubiquitin ligase, can regulate the ubiquitination level of many substrates to participate in various physiological activities. In this study, we demonstrated that depletion of RNF216 disrupted the migration, but had no effect on the proliferation of GnRH neuronal cell line. This effect seems to be mediated by the Beclin1-regulated autophagy. We further showed that increasing autophagy
The CHH-associated genes are involved in the genesis, migration of GnRH neurons and/or synthesis, secretion and signaling of GnRH (
Autophagy is a biological process that maintains cell homeostasis and physiological functions of cells (
Autophagy initiation is coordinated by two kinases, unc-51 like kinase 1 (ULK1, also known as autophagy-related (ATG)-1) and vacuolar protein sorting-34 (VPS34, also known as PIK3C3). Activation of both ULK1 and VPS34 drives the recruitment of additional ATG proteins to phagophore membranes and promotes autophagosomal maturation. VPS34 complex is mainly composed of VPS34, Beclin1, and ATG14. Beclin1 governs the autophagic process by regulating PI3KC3-dependent generation of PI3P and the subsequent recruitment of additional ATG proteins that orchestrate autophagosome formation (
In this study, we found that autophagy, elicited by Beclin1 (RNF216 depletion) or mTOR inhibition (rapamycin treatment), suppressed the migration of GnRH neuronal cell line. It will be intriguing to understand the molecular mechanisms underlying the inhibitory effect of autophagy on GnRH cell migration. Our results also imply that increased autophagy by genetic mutations and/or environmental factors may contribute to the pathogenesis of hypogonadotropic hypogonadism. Genetic studies in large CHH cohorts, combined with cell/animal model studies, may identify more autophagy-related genes to be associated with CHH.
GN11 cell lines was gifted from Professor Sally Radovick and Horacio Novaira. Cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Hyclone, Logan, UT, USA) containing 15% fetal bovine serum (Gibco, Grand Island, NY, USA), and 25 mM glucose, 5 mM l-glutamine,100 mg/ml streptomycin, 100 U/ml penicillin (Gibco, Grand Island, NY, USA) in humidified air at 37°C with 5% CO2. GN11 cells were plated between fourth and tenth passages. GN11 cells were passaged when reach to 90% confluence.
GN11 cells were plated at 50% confluence and transfected with siRNAs (50 nM) specific to RNF216, BECN1, and Arc (GenePharma, Halley Road, Shanghai, P. R. China) using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. Control cells were transfected with a non-targeting siRNA (50 nM) (GenePharma, Halley Road, Shanghai, P. R. China). All treatments were performed in Reduced-Serum Medium (Opti-MEM; Gibco, Grand Island, NY, USA). The transfected cells were cultured in reduced-serum medium for 6 h, then the medium was replaced by complete medium. After 48 h, cells were harvested for immunoblotting or subsequent assays. The effective siRNA sequences are as following:
siRNF216-1: sense: 5′-GCAGACAGCAGACGAUAUUTT−3′, antisense: 5′- AAUAUCGUCUGCUGUCUGCTT-3′), siRNF216-2: sense (5′- GCUUGAAGACCAGCAGUUATT-3′), antisense(5′- UAACUGCUGGUCUUCAAGCTT-3′).
siBECN1-1: sense (5′-GGUACCGACUUGUUCCCUATT-3′), antisense(5′- UAGGGAACAAGUCGGUACCTT-3′), siBECN1-2: sense (5′- GCUCCAUGCUUUGGCCAAUTT-3′), antisense(5′- AUUGGCCAAAGCAUGGAGCTT-3′).
siArc-1: sense (5′-GCUCAGCAAUAUCAGUCUUTT-3′), antisense(5′- AAGACUGAUAUUGCUGAGCTT-3′), siArc-2: sense (5′- CCAGGAAGCUGAUGGCUAUTT-3′), antisense(5′- AUAGCCAUCAGCUUCCUGGTT-3′).
The proliferation assay used a colorimetric assay based on measuring the reduction of 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT; Sigma, St. Louis, MO, USA). GN11 cells were plated in a 96-well plate with 1,000 cells each well and cultured in complete medium. MTT assay was used to measure viable proliferating cells at 0, 24, 48, 72 and 96 h after plating. At the end of each experiment, 20 μL of the 5 mg/mL MTT solution was added to each well and incubated for 4 h at 37°C. After incubation, the medium were replaced with 100 μl DMSO (Dimethyl sulfoxide; Sigma, St. Louis, MO, USA) each well and slowly shaked for 10 min at room temperature. Absorbance was measured at 570 nm using a spectrophotometer. The experiments for each assay were performed with 8 replicates for each treatment condition and repeated 3 times using different cell passages.
When GN11 cells reach to 90% confluence, total protein lysates were harvested from cells with lysis buffer (150 mM NaCl, 50 mM PH7.5 Tris-HCl, 10% glycerol, 4% sodium dodecyl sulfate and protease inhibitor cocktail [Sigma, St. Louis, MO, USA]). Cell lysates were boiled at 100°C for 10 min and then supernatant (10 μg) was subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) after centrifugation (13,000 g for 10 min). After electrophoresis, the proteins were transferred onto polyvinylidene difluoride membranes. The membranes were blocked with 5% fat-free milk in TBS with 0.1% Tween-20 for 1 h. The primary antibodies used were as following: 1:2,000 rabbit anti-RNF216 (Sigma, St. Louis, MO, USA); 1:2,000 rabbit anti-BECN1 (Cell Signaling Technology, Danvers, MA, USA); 1:4,000 rabbit anti-P62 (Cell Signaling Technology, Danvers, MA, USA); 1:1,000 rabbit anti-LC3 (Cell Signaling Technology, Danvers, MA, USA). Primary antibodies were incubated at 4°C overnight. After washing, HRP-conjugated secondary antibodies were incubated at room temperature for 1 h. After washing, the blots were visualized with a chemiluminescent signal (Immobilon Western HRP Substrate Peroxide Solution; Sigma, St. Louis, MO, USA) and subsequently digitized (Fusion Solo; Vilber Lourmat, Paris, France) and analyzed (Image J; National Institutes of Health, Bethesda, Maryland, USA). The detection of every protein was performed at least 3 times.
Transwell assay was used to characterize GN11 cellular migration. After GN11 cells with siRNA treatment for 36h, the cells were harvested with 0.25% Trypsin-EDTA (Gibco, Grand Island, NY, USA), centrifuged for 5 min at 800 g, and resuspended in serum-free medium. GN11 cells (5 × 104 cells) were seeded onto the upper compartment of the well (Costar, Kennebunk, ME, USA) separated by 8 mm-pore filters with 200 μL serum-free DMEM. The lower compartment contained 500 μL complete medium. After incubation at 37°C with 5% CO2 for 24 h, cells on the upper side of the filters were then mechanically removed. GN11 cells at the lower side of the filter were then fixed in cold 100% methanol for 30 min and washed twice using PBS. Subsequently, the nuclei were stained with DAPI (Sigma, St. Louis, MO, USA) for 30 min and then washed twice using PBS. Images were acquired under a microscope (LEICA, Germany) and stained cells were counted in five fields to determine the average number of cells that had migrated. The experiments for each assay were performed with three replicates and repeated 3 times.
The efficiency of RNF216 silencing by siRNA and GnRH expression were measured in GN11 cells by qPCR. Total RNA was harvested 48 h after transfection with siRNA. Then 1 μg of RNA was reverse transcribed using the RevertAid First Strand cDNA Synthesis Kit, and the cDNA was analyzed by qPCR using Maxima SYBR Green qPCR Master Mix (Thermo Fisher Scientific, Waltham, Massachusetts, U.S.) with a 10 μM concentration of the appropriate primer pair. The primers used to amplify RNF216 were sense primer, 5′-GCCCATCCTCTAGGAGAGCTT-3′, and antisense primer, 5′-CCGTTTCTTTCACTAACAGTGGA-3′. The primers used to amplify GnRH were sense primer, 5′-AGCACTGGTCCTATGGGTTG-3′, and antisense primer, 5′-GGGGTTCTGCCATTTGATCCA-3′. The primers used to amplify Gapdh were sense primer, 5′-TGGATTTGGACGCATTGGTC-3′, and antisense primer, 5′-TTTGCACTGGTACGTGTTGAT-3′. All samples were assayed in three duplicate using the LightCycler® 96 System (Roche, Basel, Switzerland). The qPCR conditions were as follows: initial denaturation and enzyme activation at 95°C for 10 min followed by 40 cycles of denaturation (95°C, 15 s), annealing, and reading (60°C, 30 s). Melt curve analyses were conducted after each qPCR to demonstrate the presence of a single amplicon. Relative expression of genes was calculated by the 2−ΔΔCt method and normalized to the housekeeping gene Gapdh. This experiment was repeated 3 times.
The data was analyzed using Prism6 software (GraphPad Software, San Diego, CA, USA). Statistical significance were evaluated by unpaired
FL and DL contributed equally to this work. J-DL conceived and designed the study, performed data analysis and data interpretation, and wrote the manuscript. D-NC designed the study and performed data analysis. FL and DL performed all the experiments. HL provided the cells and techniques. B-BC provided technical assistance and helped to process the manuscript. FJ provided instrument for performing experiment and participated in writing the manuscript.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
We thank Drs. Sally Radovick, Horacio Novaira, and Tracey Sharp for providing GN11 cells. This project was financially supported by National Natural Science Foundation of China Grants (31371187, 81770780, and 81728013 ); the Key research and development programs from Hunan Province Grants (2018DK2010, 2018DK2013); Natural Science Foundation of Hunan Province (2018JJ3573); the Training Program Foundation for Excellent Innovation Youth of Changsha (kq1802019); the Fundamental Research Funds for the Central Universities of Central South University (1053320170804).
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