3-Aminobenzamide | Taste Receptor

The regulation of autophagy in porcine blastocysts: Regulation of PARylation-mediated autophagy via mammalian target of rapamycin complex 1 (mTORC1) signaling

Hye Ran Lee a, Duk Hyoun Kim a, Min Gyeong Kim a, Jun Sung Lee a, Jeong Ho Hwang a, b, Hoon Taek Lee a, *

Abstract

Poly(ADP-ribosyl)ation (PARylation) acts as a modulator of selective autophagic degradation of ubiquitinated aggregates for cellular quality control, functioning in pro-survival role. It was reported previously that the inhibition of PARylation resulted in autophagy defects leading accumulation of ubiquitinated aggregates SQSTM1/p62 and apoptosis in porcine blastocysts. Thus, this study aims to investigate the mechanism between PARylation and autophagy in porcine blastocysts. In vitro produced (IVP) embryos were treated with 3-aminobenzamide (3ABA, poly (ADP-ribose) polymerase inhibitor) and/or rapamycin (RAPA, an mTORC1 inhibitor) during blastocyst formation. Then, these treated blastocysts were analyzed by real-time PCR, immunocytochemistry and TUNEL Assay. We found that the 3ABA treatment increased mTORC1 downstream target, phosphorylation of thr389 p70S6K (p-p70S6Kthr389), suggesting an increase in mTORC1 activity. Co-treatment with rapamycin (RAPA), mTORC1 inhibitor, restored the 3ABA-induced autophagy defects to those of the controls by normalizing mTORC1 activity. Moreover, autophagy induction, with only RAPA treatment, increased the rate of blastocyst development (70.05 ± 0.93 vs. 50.61 ± 3.49%), total cell number (58.48 ± 2.94 vs. 49.58 ± 2.43) and blastomere survival, but decreased the accumulation of SQSTM1/p62 aggregates. In summary, mTORC1 signaling is a key mechanism of PARylation-autophagy and its inhibition improved developmental ability and embryo quality by promoting selective autophagic degradation of ubiquitinated aggregates in porcine blastocysts. Therefore, these findings have significant implications for understanding the importance of autophagy regulation for successful in vitro production of porcine embryos.

Keywords:
Poly(ADP-ribosyl)ation
PARylation
Autophagy
Pig
Blastocyst
mTOR

1. Introduction

Considering their many similarities in genetics and physiology with humans, pigs have been used as ideal mammalian models for production of donors for bio-organs andas models of human disease.For this reason, many researchers have focused on the in vitro production (IVP) of porcine embryos, which requires high quality oocytes and embryos [1]. However, the quality of IVP embryos is not comparable with that of invivo embryos due to exposure to stressors (pH, temperature and gas environments etc.) during in vitro culture [2e4].Therefore,itisnecessarytoimprovethequalityofIVPembryos by identifying the developmental mechanisms.
Selective elimination of ubiquitinated proteins by autophagy is essential for cell survival by controlling the quality of cellular proteins [5e7]. In a previous study, we found that PARylation is involved in pro-survival autophagy, which selectively degrades ubiquitinated proteins during blastocyst formation in pigs [8]. During blastocyst formation, the inhibition of PARylation by 3aminobenzamide (3ABA, PARP inhibitor) suppresses selective autophagic degradation of ubiquitinated proteins, which contributes to apoptosis. Thus, the interaction between PARylation and autophagy influences the quality of IVP embryos in pigs [8].
Mammalian target of rapamycin (mTOR) signaling acts as sensors for regulation of metabolism, growth and survival [9]. mTOR is multi-protein complexes, mTOR complex1 (mTORC1, rapamycin sensitive complexes) and mTOR complex2 (mTORC2, rapamycin insensitive complexes). mTORC1 acts as positive regulator of protein synthesis and also a negative regulator of autophagy, which is a target of rapamycin. mTORC1 phosphorylates thr389 of p70S6K (pp70S6K-thr389), which is used as an indicator of mTORC1 activity [9]. mTORC1 is modulated by PARylation catalyzed by poly (ADPribose) polymerase-1 (PARP-1) [10]. PARylation inhibits mTORC1 activity by decreasing p70S6K phosphorylation (p-p70S6K-thr389), which subsequently induces autophagy. In contrast, PARylation inhibition suppresses pro-survival autophagy through increasing mTORC1, which contributes to apoptosis in cells [10e13]. These previous reports indicate that PARylation acts as critical player for inducer of autophagy through mTOR signaling, which contributes to survival of cultured cells. However, it has not yet been documented the involvement of mTOR signaling in the interaction of PARylation-autophagy in pre-implantation embryos.
Therefore, in this study, we examined whether mTOR signaling is involved in the interaction between PARylation and autophagy in porcine blastocysts. First, we identified the effect of PARylation inhibition on mTORC1 activity and examined the level of autophagy and development ability, as well as embryo quality, after cotreatment with inhibitors of PARylation and mTOR. In addition, we investigated the effect of autophagy induction, with mTORC1 inhibition, on development and embryo quality in pigs.
We found that mTORC1 signaling was involved in the interaction between PARylation and autophagy. Furthermore, inhibition of mTORC1 improved developmental ability and embryo quality by facilitating autophagic degradation as a pro-survival effect in porcine blastocysts. Our results indicate that the PARylationmTORC1-autophagy mechanism acts as a master regulator of cellular quality in porcine IVP embryos.

2. Materials and methods

All reagents and chemicals were purchased from SigmaeAldrich Co. (St. Louis, MO, USA), unless otherwise specifically stated. Each experiment was conducted in at least three replicate and embryos were randomly allocated to different groups.

2.1. In vitro production of porcine embryos

Prepubertal ovaries were collected from a local slaughterhouse in 30e37 C saline. Cumulus oocyte complexes (COCs) were aspirated from follicles (3e7 mm diameter) of the ovaries, which were matured in vitro in Tissue Culture Medium 199 with Earle’s salts (TCM-199; Gibco BRL, Grand Island, NY) added to 25 mM NaHCO3, 0.57 mM cysteine, 10% (v/v) porcine follicular fluid, 10 ng/ml epidermal growth factor, 0.5 mg/ml FSH (Folltropin V; Vetrepharm, Ontario, Canada), 1 mg/ml estradiol-17b and 0.22 mg/ml sodium pyruvate, under mineral oil at 39 C in a humidified incubator containing 5% CO2 for 42e44 h. Matured oocytes were placed into fertilization medium (modified Tris-buffered medium supplemented with 1 mM caffeine sodium benzoate and 0.1% BSA). Ejaculated sperm were purchased from Darby Pig Breeding Co. (Anseong, Korea). Sperm were swim-up in Sp-TALP medium and added to the fertilization droplet to a final sperm concentration of 5 105 cells/ml. Oocytes and sperm were co-incubated for 6 h and fertilized oocytes were cultured in NCSU23 media containing 0.4% (w:v) essential fatty acid-free BSA for 7days.

2.2. Real time RT-PCR

mRNAwasisolatedfrom20oocytesorembryosusingaDynabead mRNADIRECTKit(Lifetechnology,Oslo,Norway)inaccordancewith the manufacturer’s protocol. Isolated mRNAwas reverse transcribed using a reverse transcription kit from Applied Biosystems (Applied Biosystems, Waltham, MA, USA). The level of mRNA expression was measured by real-time PCR using specific primers. The primers were designed using the Primer3 on-line software (http://bioinfo.ut.ee/ primer3-0.4.0/) or by referring to references. The sequences and references are listed in Table 1. PCR reactions were performed in accordance with the manufacturer’s instructions using a SYBR green premix Ex taq (Takara, Otsu, Japan) containing TaKaRa Ex Taq HS, SYBR Green I, dNTP Mixture, Mg2þ and TliRNase H. The PCR program consisted of the following three steps: denaturation at 95 C for 30 s, annealing at 60 or 61 C for 30 s depending on the primer pair used, and extension at 72 C for 30 s with a single fluorescence measurement. After 40 cycles, the melting curve at 65e95 C was analyzed with a heating rate of 0.5 C/s. PCR reactions were conducted using a real-time PCR machine (DNA Engine Opticon 3 fluorescence detection system; MJ Research, Waltham, MA, USA). Relative quantification of gene expression was analyzed by the 2-ddCt method. ACTB mRNAwas used as aninternalstandard. Threereplicate experiments were analyzed with 20 embryos in each group.

2.3. Immunocytochemistry

The embryos were fixed in 10% formaldehyde and then permeabilized by 1.0% Triton X-100 in DPBS for 30 min. The permeabilized embryos were blocked with 1% BSA in DPBS at 39 C for 1 h and then treated with a polyclonal anti-LC3 antibody (1:200 dilution; #2775, Cell Signaling Technology, Danvers, MA, USA), antiSQSTM1/p62 antibody (1:200 dilution; ab91526, Abcam, Cambridge, UK), anti-phospho p70S6K thr389 (1:200; #9205, Cell Signaling Technology, Danvers, MA, USA) and anti-p70S6K (1:200 dilution; sc230, Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 4 C overnight. FITC-conjugated anti-rabbit IgG was used to as secondary antibody (1:200 dilution; Jackson Immunoresearch, West Grove, PA, USA) and TO-PRO-3 iodide (Life Technology, Grand Island, NY, USA) was used to stain nuclei. Images from at least three independent experiments were obtained using a confocal laser microscope (Carl Zeiss, Oberkochen, Germany) and analyzed using the ImageJ software (National Institutes of Health, Bethesda, MD, USA).

2.4. TUNEL assays

Embryos were fixed with 10% formaldehyde in DPBS and permeabilized using 1.0% Triton X-100 and 0.1% sodium citrate in DPBS for 1 h. Apoptotic cells were stained with TdT and fluorescein dUTP for 1 h at 39 C (In situ Cell Death Detection Kit; Roche, Indianapolis, IN, USA), and nuclei were stained with 5 mg/ml Hoechst 33342. The slides were examined under a fluorescence microscope (Nikon Coolpix 990; Nikon Corporation, Tokyo, Japan). Embryos were analyzed according to three nuclear morphologies as previously described [14]: (i) healthy nuclei were uniformly stained with Hoechst 33342, but without TUNEL signal (F-T-); (ii) fragmented nuclei with no TUNEL signal (F þ T-); (iii) fragmented nuclei with TUNEL signal (F þ Tþ). Embryos containing only fragmented nuclei (F þ T-) were classified as ‘fragmented’. Fragmented, TUNEL and apoptotic indices were calculated for each of the embryos as follows: fragmented index (no. of F þ T-nuclei/no. of total nuclei) 100; TUNEL index (no. of FþTþ nuclei/no. of total nuclei) 100; total apoptotic index (no. of FþTþ and F þ T-nuclei/ no. of total nuclei) 100. Experiments were performed in four replicates using 20e25 embryos from each group.

2.5. Treatment of porcine embryos with 3ABA and/or RAPA

3ABA and RAPA were dissolved in DMSO. IVP embryos were cultured in NCSU23 medium supplemented with or without 3ABA, RAPA, and 3ABA þ RAPA from the morula to blastocysts. The diluents of the chemical inhibitors were used as negative controls. The chemical inhibitors were used at a fixed concentration of 5 mM (3ABA) or 1 nM (RAPA) because previous studies demonstrated these concentrations to be effective in inhibiting the PARylation and mTORC1 activity of porcine embryos [8,15,16].

2.6. Statistical analyses

Data were analyzed by one-way ANOVA using SPSS statistics (SPSS, Chicago, IL, USA). P values less than 0.05 were considered to indicate a significant difference.

3. Results

3.1. Effect of PARylation inhibition on mTORC1 activity

To determine whether mTORC1 signaling is related to the PARylation-autophagy interaction in porcine blastocysts, we examined the expression of total p70S6K and p-p70S6K-thr389 in blastocysts following 3ABA treatment. Total p70S6K showed primarily diffuse cytoplasmic localization and occasionally a dot-like pattern, while p-p70S6K-thr389 mostly appeared as a dot-like pattern in intracellular regions of blastocysts. Treatment with 3ABA resulted in an increase in p-p70s6k-thr389 dots and dots accumulation in peri-nuclear regions, without affecting total p70S6K (Fig. 1A and B). In addition, the relative fluorescence intensity of p-p70S6K-thr389 was significantly higher in 3ABA treated-blastocysts, than in the controls (Fig. 1C and D).

3.2. Effect of co-treatment of 3ABA with RAPA on mTOR activity and autophagy

Since we observed that PARylation inhibition increased expression of p-p70S6K-thr389, an indicator of mTORC1 activity, we verified whether co-treatment with RAPA, an mTOR inhibitor, rescue mTORC1 activity. Immunocytochemistry showed that total p70S6K was not affected by 3ABA þ RAPA treatment (Fig.1B and D). However, 3ABA þ RAPA treatment restored the 3ABA-induced effects on localization and expression of p-p70S6K-thr3891, similar to the controls (Fig. 1A and C). In addition, we tested the effects of 3ABA þ RAPA on autophagy in blastocysts. Treatment with 3ABA alone significantly reduced the expression of ATGs (ATG5, BECLIN1 and LC3) and the punctate structure of LC3 protein, compared with controls (Fig. 2A, B and C). These results again confirmed our previous finding that treatment with 3ABA suppressed pro-survival autophagy in porcine blastocysts [8]. These 3ABA induced-defects of autophagy were restored by co-treatment with RAPA, with normalizing the expression level of ATGs and LC3 proteins, similar to the controls (Fig. 2A, B and C).

3.3. Effect of co-treatment with 3ABA and RAPA on SQSTM1/p62 degradation

Our previous study demonstrated that the autophagic substrate SQSTM1/p62 were excessively accumulated in 3ABA treatedblastocysts [8]. Therefore, we determined whether co-treatment with RAPA could prevent the excessive accumulation of SQSTM1/ p62 observed following 3ABA treatment. As previously reported, 3ABA treatment significantly increased the accumulation of SQSTM1/p62, as indicated by increased size and number of aggregate forms as well as relative fluorescence intensity, relative to controls (Fig. 3A and B). This excessively increased accumulation of SQSTM1/p62 was normalized by co-treatment with RAPA and did not differ from those observed in the controls.

3.4. Restoration of blastocyst development and embryo quality by 3ABA þ RAPA

We previously reported that the defect of autophagy in 3ABA treated-blastocysts is accompanied by decreases in blastocyst development and embryo quality [8]. Therefore, we examined the effect of co-treatment with RAPA on blastocyst development and embryo quality. Embryos were cultured in medium containing 3ABA alone and 3ABA þ RAPA from the morula to the blastocyst. As previously reported, treatment with 3ABA alone significantly reduced blastocyst development, especially expanded blastocysts (Fig. 4A). 3ABA treated-blastocysts showed decreased total cell number but increased apoptosis index and transcripts ratios of BAX: BCL2L1. These 3ABA-induced effects were restored to the levels of the controls by co-treatment with RAPA (Fig. 4B and Table 2).

3.5. Effect of RAPA on mTORC1 activity and autophagy

To identify the effect of RAPA on mTORC1 activity during blastocyst formation, we treated embryos with RAPA from the morula to the blastocyst and examined the expression of total p70S6K and p-p70S6K thr389. Immunocytochemistry showed that RAPA treatment did not affect the expression and localization of total p70S6K (Fig.1B and D). However, the relative fluorescence intensity of p-p70S6K-thr389 was significantly reduced in RAPA-treated blastocysts (Fig. 1C). In addition, the dot-like structures of pp70S6K-thr389 were diffusely distributed in the cytoplasm, without accumulation in peri-nuclear regions, following RAPA treatment (Fig. 1A). In addition, RAPA treatment significantly increased the expression of ATGs (ATG5, BECLIN1 and LC3) and the punctate structure of LC3 protein (Fig. 2A and B), while accumulation of SQSTM1/p62 was significantly reduced, compared with controls (Fig. 3A and B).

3.6. Improvement of blastocyst development and embryo quality by RAPA treatment

We examined the effect of RAPA during blastocyst formation on blastocyst development and quality. RAPA treatment significantly increased the development rate of blastocysts, especially expanded blastocysts, and the number of total cells of blastocysts (Fig. 4A and Table 2). In contrast, the apoptosis index and the ratio of transcripts of BAX:BCL2L1 were significantly reduced in RAPA treatedblastocysts, compared with controls (Fig. 4B and Table 2).

4. Discussion

PARylation induces pro-survival autophagy via mTORC1 signaling in various cell types [11e13]. However, it has not demonstrated the involvement of mTORC1 in the interaction between PARylation and autophagy in porcine IVP embryos. Therefore in this study, we investigated whether mTOR signaling is related to PARylation-autophagy in porcine IVP embryos, similar to other cells.
We first assessed the expression of P70S6 kinase as an indicator of mTORC1 activity. PARylation inhibition significantly increased mTORC1 activity by increasing the level of aggregated p-p70S6Kthr389 in the perinuclear region of blastocysts, but did not affect total p70S6K. This might be due to accumulation of p-p70S6Kthr389 in active ribosomes of the perinuclear region, indicating an increase in its ability to synthesize the protein [17]. These results are similar to previous reports that PARylation inhibition increases mTORC1 activity in cells [10e13]. PARylation activates AMPactivated protein kinase (AMPK), an upstream negative regulator of mTORC1, which leads to inhibition of mTORC1 activity. PARylation inhibition induces an increase in mTORC1 activity by preventing the activation of AMPK [10,11,13,18]. Thus, the function of mTORC1 inhibition by PARylation is fundamental to pro-survival autophagy. Considering these previous studies, our results suggest that mTORC1 may be involved in the PARylation-autophagy interaction in porcine blastocysts.
Rapamycin is widely used as a canonical inhibitor of mTORC1 and also acts as an inducer of autophagy [19]. Rapamycin induces the formation of complex of RAPA and intracellular receptor FK506 rapamycin binding protein (FKBP12). Rapamycin-FKBP12 complex directly binds totheFKBP12-RAPA binding(FRB)domain ofmTORC1, which inhibits downstream signaling of mTORC1, and subsequently induces autophagy [19,20]. Therefore, we determined whether cotreatment with RAPA could restore mTORC1 activated by 3ABA in porcine blastocysts. Treatment with RAPA and 3ABA restored 3ABAinduced mTORC1 activation to the levels of the controls, with normalizing the peri-nuclear localization induced by 3ABA treatment. These results are consistent with a previous report demonstrating that PARP inhibitor-induced mTORC1 activation was recovered to that of the controls by combinational treatment with RAPA [21].
In addition, we examined the effect of co-treatment with RAPA on 3ABA-induced autophagic defects in blastocysts. 3ABA-induced autophagic defects were restored, as indicated by normalized the level of ATGs, LC3 proteins, and SQSTM1/p62. The detrimental effects of 3ABA on blastocyst development and embryo quality were recovered in porcine blastocysts. Taken together, these results indicate that inhibition of 3ABA-induced mTOR activation, by cotreatment with RAPA, recovers the defects in autophagic degradation of ubiquitinated aggregates in 3ABA-treated blastocysts, without chemical toxicity.
Previous studies have demonstrated the positive effect of autophagic induction, caused by RAPA, on IVP of embryos in various species [15,16]. Our previous study has showed that inhibition of autophagy during the morula to blastocyst period has negative effects on developmental ability and embryo quality by causing accumulation of abnormal ubiquitinated proteins in porcine blastocysts [8]. However, the effect of autophagy induction during the morula to blastocyst period in pigs remains unknown. Therefore, we investigated the effect of RAPA during blastocyst formation on mTORC1 activity, autophagy, development of blastocysts and embryo quality in pigs. When embryos were treated with RAPA from the morula to the blastocyst, the expression and localization of total p70S6K was unaffected. However, dot-like structures of p-p70S6Kthr389 and the intensity were reduced, and its distribution was diffuse in the cytoplasm, without accumulation in peri-nuclear regions, in RAPA-treated blastocysts.
Furthermore, RAPA treated-blastocysts showed an increased level of autophagy and decreased SQSTM1/p62 accumulation. Moreover, treatment with only RAPA had positive effects on blastocyst development and embryo quality by increasing total cell number and decreasing apoptosis in porcine blastocysts. These results are consistent with previous reports showing that RAPA treatment inhibits mTORC1 activity by affecting p-p70S6K-thr389 but not total p70S6K, and increases selective autophagic degradation of SQSTM1/P62 aggregates in mouse model [22,23]. Considering previous studies, our observations suggest that RAPA treatment during blastocyst formation enhances autophagic activities through inhibiting mTORC1 activity, which results in a decreased level of the autophagic substrate SQSTM1/p62 in porcine blastocysts. The positive effects of RAPA on porcine blastocysts might be due to removal of ubiquitinated substrate, including aggregated or mis-folded proteins and damaged organelles, due to enhanced autophagy.
In summary, the present study demonstrated that PARylation is a key regulator of pro-survival autophagy via the mTOR pathway; the defects of autophagy following PARylation inhibition could be caused by activation of mTORC1 in porcine blastocysts. In addition, we revealed that autophagy induction during blastocyst formation by RAPA treatment effectively eliminates ubiquitinated substrates in porcine IVP embryos, which may contribute to the improved development ability and embryo quality. Therefore, these findings increase our understanding of the regulation of pro-survival autophagy during blastocyst formation, which may contribute to the high quality of IVP embryos in pigs.

References

[1] M.A. Gil, C. Cuello, I. Parrilla, J.M. Vazquez, J. Roca, E.A. Martinez, Advances in swine in vitro embryo production technologies, Reprod. Domest. Anim. Zuchthyg 45 (Suppl. 2) (2010) 40e48, http://dx.doi.org/10.1111/j.14390531.2010.01623.x.
[2] J.O. Gjørret, H.M. Knijn, S.J. Dieleman, B. Avery, L.-I. Larsson, P. Maddox-Hyttel, Chronology of apoptosis in bovine embryos produced in vivo and in vitro, Biol. Reprod. 69 (2003) 1193e1200, http://dx.doi.org/10.1095/ biolreprod.102.013243.
[3] H.M. Knijn, J.O. Gjørret, P.L.A.M. Vos, P.J.M. Hendriksen, B.C. van der Weijden, P. Maddox-Hyttel, et al., Consequences of in vivo development and subsequent culture on apoptosis, cell number, and blastocyst formation in bovine embryos, Biol. Reprod. 69 (2003) 1371e1378, http://dx.doi.org/10.1095/ biolreprod.103.017251.
[4] F.J.R. Pomar, K.J. Teerds, A. Kidson, B. Colenbrander, T. Tharasanit, B. Aguilar, et al., Differences in the incidence of apoptosis between in vivo and in vitro produced blastocysts of farm animal species: a comparative study, Theriogenology 63 (2005) 2254e2268, http://dx.doi.org/10.1016/j.theriogenology.2004.10.015.
[5] M. Komatsu, Y. Ichimura, Physiological significance of selective degradation of p62 by autophagy, FEBS Lett. 584 (2010) 1374e1378, http://dx.doi.org/10.1016/j.febslet.2010.02.017.
[6] M. Lippai, P. Low, The role of the selective adaptor p62 and ubiquitin-like} proteins in autophagy, BioMed Res. Int. 2014 (2014) 832704, http:// dx.doi.org/10.1155/2014/832704.
[7] J.R. Kovacs, C. Li, Q. Yang, G. Li, I.G. Garcia, S. Ju, et al., Autophagy promotes Tcell survival through degradation of proteins of the cell death machinery, Cell Death Differ. 19 (2012) 144e152, http://dx.doi.org/10.1038/cdd.2011.78.
[8] H.R. Lee, M.K. Gupta, D.H. Kim, J.H. Hwang, B. Kwon, H.T. Lee, Poly(ADPribosyl)ation is involved in pro-survival autophagy in porcine blastocysts, Mol. Reprod. Dev. 83 (2015) 37e49, http://dx.doi.org/10.1002/mrd.22588.
[9] M. Laplante, D.M. Sabatini, mTOR signaling at a glance, J. Cell Sci. 122 (2009) 3589e3594, http://dx.doi.org/10.1242/jcs.051011.
[10] L. Virag, A. Robaszkiewicz, J.M. Rodriguez-Vargas, F.J. Oliver, Poly(ADP-ribose) signaling in cell death, Mol. Asp. Med. 34 (2013) 1153e1167, http:// dx.doi.org/10.1016/j.mam.2013.01.007.
[11] J.M. Rodríguez-Vargas, M.J. Ruiz-Magana, C. Ruiz-Ruiz, J. Majuelos-Melguizo,~ A. Peralta-Leal, M.I. Rodríguez, et al., ROS-induced DNA damage and PARP-1 are required for optimal induction of starvation-induced autophagy, Cell Res. 22 (2012) 1181e1198, http://dx.doi.org/10.1038/cr.2012.70.
[12] J.A. Munoz-~ Gamez, J.M. Rodríguez-Vargas, R. Quiles-Perez, R. Aguilar-Quesada, D. Martín-Oliva, G. de Murcia, et al., PARP-1 is involved in autophagy induced by DNA damage, Autophagy 5 (2009) 61e74.
[13] Z.-T. Chen, W. Zhao, S. Qu, L. Li, X.-D. Lu, F. Su, et al., PARP-1 promotes autophagy via the AMPK/mTOR pathway in CNE-2 human nasopharyngeal carcinoma cells following ionizing radiation, while inhibition of autophagy 3-Aminobenzamide contributes to the radiation sensitization of CNE-2 cells, Mol. Med. Rep. 12 (2015) 1868e1876, http://dx.doi.org/10.3892/mmr.2015.3604.
[14] A. Kidson, F.J. Rubio-Pomar, A. Van Knegsel, H.T.A. Van Tol, W. Hazeleger, D.W.B. Ducro-Steverink, et al., Quality of porcine blastocysts produced in vitro in the presence or absence of GH, Reprod. Camb. Engl. 127 (2004) 165e177, http://dx.doi.org/10.1530/rep.1.00086.
[15] B.-S. Song, S.-B. Yoon, J.-S. Kim, B.-W. Sim, Y.-H. Kim, J.-J. Cha, et al., Induction of autophagy promotes preattachment development of bovine embryos by reducing endoplasmic reticulum stress, Biol. Reprod. 87 (8) (2012) 1e11, http://dx.doi.org/10.1095/biolreprod.111.097949.
[16] B.-S. Song, J.-S. Kim, Y.-H. Kim, B.-W. Sim, S.-B. Yoon, J.-J. Cha, et al., Induction of autophagy during in vitro maturation improves the nuclear and cytoplasmic maturation of porcine oocytes, Reprod. Fertil. Dev. 26 (2014) 974e981, http://dx.doi.org/10.1071/RD13106.
[17] A. Avila-Flores, T. Santos, E. Rincon, I. M erida, Modulation of the mammalian target of rapamycin pathway by diacylglycerol kinase-produced phosphatidicacid, J. Biol. Chem. 280 (2005) 10091e10099, http://dx.doi.org/10.1074/ jbc.M412296200.
[18] J. Zhou, S. Ng, Q. Huang, Y.-T. Wu, Z. Li, S.Q. Yao, et al., AMPK mediates a prosurvival autophagy downstream of PARP-1 activation in response to DNA alkylating agents, FEBS Lett. 587 (2013) 170e177, http://dx.doi.org/10.1016/ j.febslet.2012.11.018.
[19] Y.C. Kim, K.-L. Guan, mTOR: a pharmacologic target for autophagy regulation, J. Clin. Invest. 125 (2015) 25e32, http://dx.doi.org/10.1172/JCI73939.
[20] C.H. Jung, S.-H. Ro, J. Cao, N.M. Otto, D.-H. Kim, mTOR regulation of autophagy, FEBS Lett. 584 (2010) 1287e1295, http://dx.doi.org/10.1016/j.febslet.2010.01.017.
[21] C. Sun, F. Zhang, T. Xiang, Q. Chen, T.K. Pandita, Y. Huang, et al., Phosphorylation of ribosomal protein S6 confers PARP inhibitor resistance in BRCA1-deficient cancers, Oncotarget 5 (2014) 3375e3385, http://dx.doi.org/ 10.18632/oncotarget.1952.
[22] S. Majumder, A. Richardson, R. Strong, S. Oddo, Inducing autophagy by rapamycin before, but not after, the formation of plaques and tangles ameliorates cognitive deficits, PloS One 6 (2011) e25416, http://dx.doi.org/ 10.1371/journal.pone.0025416.
[23] G. Bjørkøy, T. Lamark, A. Brech, H. Outzen, M. Perander, A. Overvatn, et al., p62/SQSTM1 forms protein aggregates degraded by autophagy and has a protective effect on huntingtin-induced cell death, J. Cell Biol. 171 (2005) 603e614, http://dx.doi.org/10.1083/jcb.200507002.