Fluoride-Induced Autophagy via the Regulation of Phosphorylation of

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Fluoride induced Autophagy via the regulation of mTOR phosphorylation in mice Leydig cells Jianhai Zhang, Yuchen Zhu, Yan Shi, Yongli Han, Chen Liang, Zhiyuan Feng, Heping Zheng, Michelle Eng, and Jundong Wang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b03822 • Publication Date (Web): 20 Sep 2017 Downloaded from http://pubs.acs.org on September 21, 2017

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Journal of Agricultural and Food Chemistry

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Fluoride-induced Autophagy Via the Regulation of mTOR

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Phosphorylation in Mice Leydig Cells

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Jianhai Zhang†£*, Yuchen Zhu†£, Yan Shi†, Yongli Han†, Chen Liang†, Zhiyuan Feng†, Heping Zheng‡,

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Michelle Eng§, and Jundong Wang†*

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†

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Animal Science and Veterinary Medicine, Shanxi Agricultural University, Taigu, Shanxi, 030801, China;

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‡

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USA;

Shanxi Key Laboratory of Ecological Animal Science and Environmental Veterinary Medicine, College of

Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, VA 22908,

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§

Lewis Katz School of Medicine, Temple University, Philadelphia, PA 19140, USA;

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£

Jianhai Zhang and Yuchen Zhu contributed equally to this work.

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*Corresponding Author:

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Jianhai Zhang,

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Shanxi Key Laboratory of Ecological Animal Science and Environmental Veterinary Medicine, Shanxi

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Agricultural University, Taigu, Shanxi, China, 030801;

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Tel.: +86-354-6288335; E-mail: [email protected] ;[email protected] (J. Zhang).

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Jundong Wang,

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Shanxi Key Laboratory of Ecological Animal Science and Environmental Veterinary Medicine, Shanxi

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Agricultural University, Taigu, Shanxi, China, 030801;

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Tel.: +86-354-6288335; E-mail: [email protected] (J. Wang).

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ACS Paragon Plus Environment


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Fluoride-induced Autophagy Via the Regulation of mTOR

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Phosphorylation in Mice Leydig Cells

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Abstracts：：Fluoride is known to impair testicular functions and decrease testosterone levels, yet the

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underlying mechanisms remain inconclusive. The objective of this study is to investigate the roles of

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autophagy in fluoride-induced male reproductive toxicity by both in vivo and in vitro Leydig cell models.

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Using Transmission Electron Microscopy (TEM) and Monodansylcadaverine (MDC) staining, we observed

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increasing numbers of autophagosomes in testicular tissue, especially in Leydig cells of fluoride-exposed

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mice. Further study revealed that fluoride increased the mRNA and protein expressions of autophagy

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markers LC3, Beclin1 and Atg 5 in primary Leydig cells. Furthermore, fluoride inhibited the

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phosphorylation of mammalian targets of rapamycin (mTOR) and 4EBP1, which in turn resulted in a

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decrease in AKT and PI3K mRNA expressions, as well as an elevation of AMPK expression in both testes

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and primary Leydig cells. Additionally, fluoride exposure significantly changed the mRNA expression of

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the regulator genes PDK1, TSC, and Atg13 in primary Leydig cells but not in testicular cells. Taken

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together, our findings highlight the roles of autophagy in fluoride-induced testicular and Leydig cell

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damage and contribute to the elucidation of the underlying mechanisms of fluoride-induced male

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reproductive toxicity.

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Key words: Fluoride; Autophagy; Testis; Leydig cell; mTOR phosphorylation

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INTRODUCTION

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Fluoride is ubiquitously present in the environment and has pronounced reactivity in many

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physiological processes. Fluoride is one of the most significant inorganic pollutants in groundwater that

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affects human health globally, 1 especially in the densely populated regions. For example, in Asia nearly 35

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million people in China and 26 million people in India are at risk of fluoride toxicity.2-4 In addition to

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groundwater, other natural sources of fluoride include food, dental products and pesticides, which provide

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added exposure for many people. 5 For decades, fluoride attracted the interest of toxicologists due to its

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adverse effects in humans and experimental animals. In these populations fluoride can negatively impact

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various tissues, including brain,6 liver,7 kidney, 5 thyroid,8 aorta,9 ovary,10 skeletal system11,12 and male

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reproductive system. 13

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The toxicological effects of fluoride on the male reproductive system are usually evaluated based on

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the deterioration in sperm quality and the increase in infertility. Previous studies found that reproductive

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toxicity of fluoride was involved in lowering sperm quality and serum testosterone,14 as well as altering

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testicular structure,15-17 multiple reproductive hormones levels,18 the blood-testis barrier,19 and

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spermatogenic proteins.

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chemotaxis23 in male reproductive cells. However, the molecular mechanisms underlying these effects are

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still not fully elucidated.

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Fluoride also play a role in capacitation,21 apoptosis,22 hyperactivation and

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Autophagy is a process in which the subcellular membrane structures undergo dynamic morphological

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changes that lead to the degradation of cellular proteins and organelles by lysosomes.24 Autophagy plays

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key roles in cellular homeostasis during embryonic development, postnatal cell survival and death.25,26 In

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the male reproductive system, autophagy has been associated with spermatogenesis,27 germ cell death,27

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spermatid differentiation,29 sperm function,30 Sertoli cell apoptosis31 and testosterone secretion.20,32 3



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Moreover, autophagy can be induced by various stress stimuli, such as oxidative stress33 and environmental

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factors.26 Fluoride, as an environmental factor, was reported to induce oxidative stress15 and endoplasmic

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reticulum stress34 in testes. Several studies have shown that fluoride could induce autophagy in the LS8

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ameloblast cell line.35-37 A recent study indicated that fluoride exposure was associated with the increasing

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number of autophagosomes in the testes of rats.38 However, the molecular mechanism of autophagy

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induced by fluoride in testes is still poorly understood.

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In the present study, we demonstrated that fluoride exposure induced an increase in the number of

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autophagosomes and changed expressions of genes and proteins described as markers of autophagy,

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including LC3, Beclin1 and Atg5 both in both in vivo and in vitro Leydig cell models. Furthermore, the

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phosphorylations of mammalian targets of rapamycin (mTOR) and target protein phospho-4EBP1 and

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phospho-p70-S6 Kinase were detected by using the mTOR inhibition model. The mRNA expressions of the

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critical molecular regulator genes including PI3K, AKT, AMPK, PDK1, TSC, ULK1 and Atg13 in both testis

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and Leydig cells were investigated to elucidate the association between fluoride-induced autophagy and the

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regulation of mTOR phosphorylation.

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MATERIALS AND METHODS

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Materials

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Sodium fluoride (NaF), dimethylsulfoxide (DMSO), monodansylcadaverine (MDC), rapamycin

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(mTOR inhibitor), and phenylmethanesulfonyl fluoride (PMSF) were obtained from Sigma-Aldrich

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Company. Dulbecco’s modified Eagle’s medium/F12 (DMEM/F12), 10% fetal bovine serum (FBS) and

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0.25% trypsin were purchased from Giboco (USA). Myllicin was purchased from Solarbio (Beijing, China).

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PrimeScript® RT Master Mix kit and SYBR@ Premix Ex TaqTM II kit were purchased from Takara (Dalian,

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China). TRIZOL and primers were purchased from Invitrogen (USA). RIPA lysis buffer and BCA protein 4


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assay kits were purchased from Beyotime Biotecnology (Shanghai, China). Anti-LC3 rabbit polyclonal

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antibody, anti-Beclin1 rabbit polyclonal antibody, anti-Atg5 rabbit polyclonal antibody, anti-β-actin rabbit

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polyclonal antibody and HRP-conjugated goat anti-rabbit secondary antibody were purchased from

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Proteintech (Wuhan, China). Phospho-mTOR (Ser2448), phospho-4E-EP1 (Thr37/46), and phosphor-p70

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S6 Kinase (Thr389) monoclonal antibodies were purchased from Cell Signaling Technology. All other

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chemicals used in this study not specifically mentioned above were analytical grade.

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Mice Treatment

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48 healthy male Kunming mice (each weighing approximately 20-25g), aged 8 weeks, were obtained

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from the Experimental Animal Center of Shanxi Medical University. Mice were maintained under a

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standard temperature (22-25℃) and a 12h light/dark cycle, with water and food ad libitum. After a

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one-week acclimatization period, all mice were randomly divided into four groups of twelve: one control

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group (drinking deionized water) and three fluoride-administered groups (exposed to 25, 50, 100 mg/L NaF

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in drinking water, respectively). After 60 days of fluoride exposure, mice were euthanized and testes were

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immediately isolated for further study. All animals were treated humanely and all handling procedures were

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approved by the Institutional Animal Care and Use Committee of Shanxi Agricultural University.

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Primary Leydig Cells Culture and Identification of Purity

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Leydig cells were isolated from the testes of 4-week-old Kunming mice. Mice were sacrificed and

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soaked in 75% ethanol for 5 min, and then testes were isolated and put in a sterile plate containing PBS.

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Leydig cells were detached and cultured according to the procedure described previously. 32,39 The tunica

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albuginea was nipped with tweezers and gently removed from the testis to expose the seminiferous tubule.

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The testis was gently shaken until the PBS become turbid. Thus, the Leydig cells were gradually separated

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from the testis. Then, the PBS was collected and centrifuged at 1200 rpm for 5 min to pellet the cells. The 5



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supernatant was discarded, and after washing the Leydig cells 3 times in PBS, the cells were collected,

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resuspended with DMEM/F12 supplemented with 10% FBS and myllicin, seeded in culture flasks and

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cultured at 37℃ in an incubator with a humidified atmosphere containing 5% CO2.

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The purity of Leydig cells was assessed using the modified method described in Wang et al.40 Then,

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the Leydig cell suspension was smeared on slides, dried at room temperature, incubated at 22℃ for 90

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minutes, and then washed with deionized water. The positive cells were stained with a

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dehydroepiandrosterone solution. The purity was defined as the percentage of positive cells to total cells.

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Leydig cells with more than 90% purity were used for further experimentation.

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Leydig Cells Viability Assay and Treatments

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The effects of NaF on cell viability were determined using MTT assay. The Leydig cells were plated at

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a density of 5 × 104 cells per well in 96-well culture plates. The cells were treated with different

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concentrations of NaF for 24 hours. The cells were treated with 10 µL of 10 mg/mL MTT, and the resulting

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formazan crystals were dissolved in dimethyl sulfoxide. The absorbance was measured by a microplate

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spectrophotometer at 490 nm. Results were expressed as percentages of the controls, which were assigned

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with 100% viability. Half maximal inhibitory concentration (IC50) and 95% confidence intervals were

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calculated.

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When Leydig cells reached a confluence of 80-90%, they were detached from flasks using 0.25%

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trypsin and sub-cultured in 6-well plates for fluoride treatment. According to the MTT result, Leydig cells

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were treated for 24 hours with 0, 0.125, 0.25, 0.5mM NaF, respectively.

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MTOR Inhibition Model and Fluoride Treatment

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Leydig cells were divided into 6 groups equably (sub-cultured in 6-well plates, each well labeled as

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one group) for mTOR inhibition and fluoride treatment. Leydig cells without any treatment serve as control; 6


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Leydig cells treated with only DMSO (0.01‰, v/v) were used as solvent control. The other groups are

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0.5mM NaF

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rapamycin+0.5mM NaF treatment group. In some experiments, rapamycin dissolved in DMSO and

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prepared for the stock solution, and Leydig cells were pretreated with rapamycin (20 nM, diluted with

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medium) for 1 hour before 0.5mM NaF stimulation.41 After treatment for 24 hours, phosphorylated proteins

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from Leydig cells in all groups were extracted for phospho-4EBP1 and phospho-p70-S6 Kinase expression

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analysis.

treatment

group,

DMSO+0.5mM NaF

group,

rapamycin

treatment

group,

and

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MDC Staining for Autophagic Vacuoles

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The fluorescent dye monodansylcadaverine (MDC) was used to stain the autophagic vacuoles as a

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marker of autophagy in testicular tissue and primary Leydig cells in vivo.42,43 The testes were fixed in

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Bouin’s solution, dehydrated in a gradient of ethanol (30% 50% 70% 90% 100%), cleared in

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xylol and embedded in paraffin. The resulting sample were then sectioned serially into 4 µm slices using a

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Leica slicer (Leica, Germany) and mounted on glass slides. The sections were deparaffinized with xylene,

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rehydrated in a gradient of ethanol solutions, and incubated with a 50 µM solution of MDC dye (dissolved

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with DMSO) at 37℃ for 1 hour. In vitro, Leydig cells attached to the coverslip were washed with PBS for 3

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times (5 minutes each time), fixed with 4% paraformaldehyde for 15 minutes, washed again and incubated

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with 50 µM solution of MDC dye at 37℃ for 30 minutes. All sections were washed 3 times in PBS after

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incubation (5 minutes each time). MDC fluorescence levels were measured by a Leica inverted microscope

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(excitation wavelength 380 nm, emission wavelength 525 nm).

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Transmission Electron Microscope (TEM)

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The testes were cut into about 1mm x 1mm x 1mm pieces rapidly, fixed in a solution of 2%

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formaldehyde and 2% glutaraldehyde in 0.1 M phosphate buffer (PB) (pH 7.4) at room temperature for 2 7



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hours and then post-fixed in 2.5% osmium tetroxide in 0.1M PB. The ultrathin sections were prepared,

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mounted on copper grids after dehydration and embedding, and stained with uranyl acetate and citrate. The

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sections were then examined and photographed with a transmission electron microscope (JEM 1011,

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Japan).

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Real-time RT-PCR

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Total RNA was extracted from testes tissues and primary Leydig cells with TRIZOL reagent

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(Invitrogen, USA). The quality of total RNA was analyzed with 1% agarose gel electrophoresis and

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NanoDrop 2000 (Thermo Fisher, USA). RNA was reverse-transcribed using a PrimeScript® reverse

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transcription (RT) Master Mix kit. QRT-PCR was performed using the QuantStudio 7 Flex qRT-PCR

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system (Life Technologies, USA) and SYBR@ Premix Ex TaqTM II kit. The qRT-PCR primers were

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designed with Primer 3.0 plus Software (Applied Biosystems) according to the complete cDNA sequences

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deposited in GenBank (Table 1). The β-actin gene was used as an internal reference. The PCR amplification

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conditions were: Pre-degeneration at 95℃ for 30s, 50 cycles of polymerase chain reaction (PCR) at 95℃

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for 5s, 60℃ for 30s and 72℃ for 30s, and dissociation protocol at 95℃ for 15s, 60℃ for 1s and 95℃ for

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15s. Data were analyzed using the 2-∆∆Ct method.

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Western Blotting Analysis

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40 mg of testis tissues were homogenized in radioimmunoprecipitation assay (RIPA) lysis buffer

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containing protease inhibitor phenylmethanesulfonyl fluoride (PMSF). The homogenization solution was

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centrifuged at 12000g for 10 minutes at 4℃ and the supernatant was collected. Protein concentration was

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determined by bicinchoninic acid (BCA) protein assay kit with bovine serum albumin (BSA) as the

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standard. Total protein equivalents for each sample were mixed with loading buffer followed by

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denaturation at 100℃ for 10 minutes, and then separated on a 10-15% SDS-PAGE gel and subsequently 8


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transferred to nitrocellulose (NC) membranes. The membranes were first blocked with 5% (w/v) nonfat dry

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milk in Tris-buffered saline (TBS) containing 0.05% Tween-20 for 2 hours at room temperature. They were

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then incubated overnight at 4℃ with the primary antibodies against LC3 (dilution 1:300), Beclin1 (dilution

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1:1000), Atg5 (1:1000 dilution), phospho-mTOR (dilution 1:1000), phospho-4E-BP1(dilution 1:1000),

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phospho-p70-S6 Kinase (dilution 1:1000) and β-actin (dilution 1:1000). The membranes were washed with

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tris-buffered saline Tween (TBST) three times for 5 minutes each, followed by incubation with secondary

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antibody at room temperature for 2 hours. Protein bands on membranes were detected by enhanced

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chemiluminescence (ECL). Fluor Chem Q Imaging System and its analysis software system (Cell &

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Bioscience, USA) were used to acquire, quantify, and analyze the intensity of the protein bands.

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Statistical Analysis

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Statistical analysis was performed using GraphPad Prism 5 software (GraphPad Software Inc., San

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Diego, USA). The results were evaluated with one-way analysis of variance (ANOVA) followed by

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Dunnett’s multiple comparison test. The means and standard errors (mean ± S.E.) of the data were analyzed

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and compared. Statistical significances were considered if p-value was less than 0.05.

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RESULTS

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Detection of Autophagic Vacuoles in Mice Testicular Tissues

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Monodansylcadaverine (MDC) was preferentially used to label the autophagosomes as a stain via its

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integration into lipids in autophagic vacuoles.42 The number of autophagosomes increased significantly in

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testicular tissues, especially in Leydig cells in NaF-treated groups with increasing NaF concentrations (Fig.

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1A). The ultra-structure of testicular tissue by Transmission electron microscopy (TEM) (Fig. 1B) further

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demonstrated that the amount of autophagosomes was distinctly increased in mice testes exposed to 25, 50,

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and 100 mg/L fluoride. A typical example of a single Leydig cell with many autophagosome caused by 100 9



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mg/L fluoride is presented in higher magnification level (Fig. 1Be). These findings indicated that fluoride

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increased the number of autophagic vacuoles in Leydig cells in mice testes.

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Effects of Fluoride on Autophagy Marker Protein Expressions in Testes

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The mRNA levels of autophagy marker proteins LC3, Beclin1 and Atg5 were examined by real-time

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RT-PCR. A significant increase of LC3 mRNA expression was noted in testes of mice exposed to 50 and

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100 mg/L NaF (P

Fluoride-Induced Autophagy via the Regulation of Phosphorylation of

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