Osmotic stress induces apoptosis in extravillous trophoblast cells. Role of TRPV-1

Biochemical and Biophysical Research Communications 514 (2019) 58e63

Contents lists available at ScienceDirect

Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc

Osmotic stress induces apoptosis in extravillous trophoblast cells. Role of TRPV-1
s Etcheverry b, Matías N. Sierra c, Alicia E. Damiano a, c, Julieta Reppetti a, Toma Mariana Farina b, Nora Martínez a, *
n, Instituto de Fisiología y Biofísica Bernardo Houssay (IFIBIO)- CONICET- Facultad de Medicina, Universidad de Laboratorio de Biología de La Reproduccio Buenos Aires. Buenos Aires, Argentina b Laboratorio de Fisiopatología Placentaria, (CEFYBO)-CONICET, Facultad de Medicina, Universidad de Buenos Aires. Buenos Aires, Argentina c

gicas, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Argentina Catedra de Biología Celular y Molecular, Departamento de Ciencias Biolo a

a r t i c l e i n f o

a b s t r a c t

Article history: Received 3 April 2019 Accepted 12 April 2019 Available online 21 April 2019

In different tissues hyperosmolarity induces cell differentiation. Nevertheless an exacerbated hyperosmolar stress alters the normal cellular development. The transient receptor potential vanilloid 1 (TRPV-1) is a non-selective cation channel that is activated by hyperosmolarity and participates in many cellular processes. TPRV-1 is expressed in human placenta at term. Here, we evaluated the expression of TRPV-1 in first trimester extravillous trophoblast cells and its participation in the survival of these cells exposed to hyperosmolar stress. Our results showed that hyperosmolar stress up-regulates the expression of TRPV-1 and induces the apoptosis in Swan 71 cells. In addition, the inhibition of TRPV-1 abrogates the apoptotic events. © 2019 Elsevier Inc. All rights reserved.

Keywords: EVT Hyperosmolarity TRPV-1 Apoptosis

1. Introduction The placenta is a transient organ that carries on critical roles during the gestation [1]. The proper growth and development of this organ requires a tight regulation of the proliferation, differentiation and turnover of the trophoblast cells. Different subtypes of trophoblast develop from a cytotrophoblast precursor. In the extravillous pathway, the trophoblast develops invasive properties, originating the extravillous trophoblasts (EVTs). EVTs are responsible for the remodeling of the maternal spiral arteries. On the other hand, cell turnover is related to apoptotic events. Apoptosis is a type of cell death that allows the continuous turnover of the trophoblasts throughout gestation. Failures in any of these processes are associated with pregnancy disorders such as intrauterine growth retardation (IUGR) and preeclampsia (PE) [2,3]. Many authors have demonstrated that changes in osmolarity induce cells differentiation [4,5]. In this regard, Liu et al. have


n, Instituto de * Corresponding author. Laboratorio de Biología de la Reproduccio Fisiología y Biofísica Bernardo Houssay (IFIBIO)- CONICET- Facultad de Medicina, Universidad de Buenos Aires, Paraguay 2155, 4º piso CP C1121ABG, Buenos Aires, Argentina. E-mail address: [email protected] (N. Martínez). https://doi.org/10.1016/j.bbrc.2019.04.091 0006-291X/© 2019 Elsevier Inc. All rights reserved.

reported that hyperosmolar stress produced the temporal and spatial trophoblast differentiation in normal gestation [6]. However, an exacerbated hyperosmolar stress may affect the normal cellular development. In addition, in other tissues, it was also reported that hyperosmolar stress induced apoptosis [7,8]. In this sense, Xie et al. have established that the peri-implantation period is a susceptible stage and hyperosmolar stress may stimulate multiple homeostatic changes that affect the normal embryo and placental development [9]. These alterations may conduce to defects in trophoblast differentiation that can lead to placental insufficiency and embryonic loss [10]. Homeostatic mechanisms that regulate cell osmolarity are critical to maintain its integrity. Cells have osmoregulation systems that sense cellular volume perturbations associated with changes in extracellular osmolarity. In this context, many ion channels play a pivotal role in cell volume regulation [11,12]. The transient receptor potential vanilloid 1 (TRPV-1) is a Ca2þ permeable non-selective cation channel. This channel is sensitive to cell volume changes, acting as an osmosensor receptor [13]. TPRV-1 is expressed in human placenta [14,15]. It is activated by many agonist and stimuli, including hyperosmolarity [16]. In other tissues, TRPV-1 participates in numerous cellular processes like apoptosis [17], cell migration [18], invasion [19], autophagy [20]

J. Reppetti et al. / Biochemical and Biophysical Research Communications 514 (2019) 58e63

and others. Regarding human placenta, Costa and co-workers demonstrated that TRPV-1 participates in villous trophoblast apoptosis [14]. Recently, we reported that the expression of this channel was altered in preeclamptic placentas [15], suggesting a possible participation of TRPV-1 in the pathophysiology of this syndrome. However, up to now, the expression and the role of TRPV-1 in the EVT cells were not studied. In the present work, we explored the expression of TRPV-1 in extravillous trophoblast cells, and its participation in the survival of these cells exposed to hyperosmolar stress. 2. Materials and methods 2.1. Cell culture and treatments The human extravillous trophoblast cell line Swan 71 was generously provided by Dr. Gil Mor (Yale University School of Medicine, New Haven, USA). It was generated by the introduction of human telomerase reverse transcriptase, for the immortalization of primary human cells [21]. Swan 71 cells were cultured in Dulbecco's modified Eagle medium/Nutrient Mixture F-12 (DMEM/F-12, Life Technologies, Inc. BLR, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS, Laboratorio Natocor, Cordoba, Argentina), 100 U/ml penicillin, 100 mg/ml streptomycin and 5 mM glutamine (Invitrogen, Carlsbad, CA, USA). Swan 71 cells were grown up to 80% confluence under standard conditions (20% O2, 5% CO2, 37  C) and arrested in the corresponding medium supplemented with 0.5% FBS. Then, cells were cultured for 24 h in different culture media, containing different concentrations of sucrose: 0 mM (299 ± 2 mOsM), 50 mM (341 ± 7 mOsM), 100 mM (378 ± 10 mOsM) and 150 mM (424 ± 12 mOsM) to induce osmotic stress. Osmolarity was measured using a pressure vapor osmometer VAPRO™ (Wescor Inc., Logan, UT, USA). To evaluate the participation of TRPV-1, cells were incubated 30 min at 37  C in 20% O2e 5% CO2 in the presence or absence of the TRPV-1 receptor-specific antagonist capsazepine 106 M (CPZ, Sigma-Aldrich Corp., San Luis, MO, USA).

59

by agarose gel electrophoresis and visualized by GelRed™ Nucleic Acid Gel Stain (Biotium Inc., Fremont, CA, USA). A 50 bp DNA ladder (Dongsheng Biotech, Guangzhou, Guangdong, China) was added to the electrophoresis. 2.4. Western blot As described previously [22], treated and untreated cells were collected in Lysis buffer (0.3 M NaCl, 25 mM HEPES, 1.5 mM MgCl2, 0.2 mM EDTA, 1% Triton X-100, pH 7.4) containing 0.2 mM phenylmethanesulfonyl fluoride (PMSF, Sigma-Aldrich Corp., MO, USA) and 0.01x Protease Inhibitor Cocktail Set III (Calbiochem®, EMD Millipore Corporation, Darmstadt, Germany), and protein concentration was measured using the commercial kit BCA Protein Assay kit (Thermo Scientific, Waltham, MA, USA). For immunoblotting studies, 30 mg proteins from each sample of the lysate of Swan 71 cells were loaded and resolved on a 12.5% polyacrylamide gel, and electrotransferred onto nitrocellulose membranes (Hybond ECL, Amersham Pharmacia Biotech Ltd, Pittsburgh, PA, USA). After blocking, membranes were incubated overnight with the corresponding primary antibody: anti-TRPV-1 (Alomone Labs, Jerusalem, Israel, 1:500), and anti-Bax antibody (1:1000) (Abcam, Cambridge, UK), and then with goat anti-rabbit IgG (Jackson Immuno Research Laboratories, Inc., West Grove, PA, USA; 1:10000) conjugated to peroxidase. Immunoreactivity was detected using the ECL Western Blotting Analysis System (ECL plus, (Hybond ECL, Amersham Pharmacia Biotech Ltd, Pittsburgh, PA, USA) according to the manufacturer's instructions. The densitometry of the bands was quantified using ImageJ 1.51r ® software package. Results were expressed as the relative intensity of each protein and normalized against b-actin. 2.5. Terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling assay

Cell viability was assessed by the 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide assay (MTT, Sigma-Aldrich Corp., San Luis, MO, USA). Swan 71 cells (7  103 per well) were loaded in a 96-well microplate until confluency in DMEM/F-12 with 10% FBS. Then CPZ and hyperosmolarity treatments were performed and the cells were arrested in DMEM-F12 0.5% for 24 h. Subsequently, MTT (5 mg/ml in PBS) was added and incubated for 2 h at 37  C. Once the incubation time has ended, the formazan was solubilized in 100 ml of absolute ethanol and the optical density was measured at 570 nm on ELISA Reader. The cell proliferation was calculated as a percentage compared to the control.

Terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling (TUNEL) staining on Swan 71 cells was performed with a fluorescein-based cell death detection kit (In Situ Cell Death Detection kit, Roche Applied Science, Mannheim, Germany) according to the manufacturer's instructions. Treated and untreated cells grown on coverslips were washed, fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton X-100, and blocked with goat serum. Nuclear counterstain was performed with Hoechst 33 342 (Sigma-Aldrich Corp., San Luis, MO, USA) fluorescent dye. Data was documented with an epifluorescent microscope (Nikon, Eclipse E:200). To quantify the number of apoptotic nuclei, several photographs were taken per sample in a total of four samples of each experimental condition, and the counting of the apoptotic nuclei marked by the ImageJ 1.51r ® program. The percentage of apoptotic nuclei in each condition was graphed [23].

2.3. RT-PCR assay

2.6. DNA fragmentation assay

Total RNA was isolated with RNAzol® RT (Molecular Research Center, Inc., Cincinnati, OH, USA). Reverse transcription was performed with M-MLV Reverse Transcriptase (Promega Co., Madison, WI, USA) for 60 min on 2 mg of total RNA according to the manufacturer's instructions. PCR (30 cycles) at 95  C for 20 s, at 60  C for 30 s and at 72  C for 20s, followed by a final extension of 10 min at 72  C, was carried out by using 0,25 mM of specific oligonucleotide primers for human TRPV-1, (forward 50 -GCTGTCTTCATCATCCTGCTGCT-30 and reverse 50 -TGTTCTTGCTCTCCTGTGCGAT-30 ). PCR products were separated

Fragmentation of DNA was analyzed by agarose gel electrophoresis. After treatments, Swan 71 cells were incubated with Lysis buffer (50 mM Trise HCl, 50 mM EDTA, 1% sodium dodecyl sulfate, 50 mM NaCl, pH 8.0) and Proteinase K (10 mg/ml; Sigma-Aldrich Corp., MO, USA) for 2 h at 55  C. Samples were centrifuged at 21000 g for 15 min. After that, 5 M NaCl was added to supernatants and then centrifuged at 21000 g for 15 min. Ice-cold absolute ethanol was added to supernatants, and samples were incubated overnight at 20  C. Finally, the samples were centrifuged at 21000 g for

2.2. Cell viability assay

60

J. Reppetti et al. / Biochemical and Biophysical Research Communications 514 (2019) 58e63

Fig. 1. Effect of hyperosmolar stress on TRPV-1 levels in Swan 71 cells. A) Detection of TRPV-1 in mRNA of Swan 71 cells (Sw71) (n ¼ 3). Control: term placenta (TP). L: 50 bp DNA ladder. NC: negative control. Immunofluorescence of TRPV-1 in Swan 71 cells showing the expression of TRPV-1 en cell membrane (arrow) and in the cytosol (head arrow). Nuclei are stained in blue (n ¼ 3). Magnification x400. B) Representative Western blot and densitometric analysis of TRPV-1 protein expression relativized to bActin in Swan 71 cell line grown in iso-osmolar or control medium (0 mM sucrose) and hyperosmolar medium (150 mM sucrose). (n ¼ 6, *p < 0.05 vs. Control). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 2. Effect of hyperosmolar stress on Swan 71 cell viability and integrity. A) The effect of hyperosmolarity (50, 100 and 150 mM sucrose) and CPZ (106 M) on Swan 71 cell viability was examined using MTT (*p < 0.05 vs. Control: 0 mM sucrose, n ¼ 15). B) Detection of Lactate Dehydrogenase (LDH) release on Swan 71 cells cultured in isoosmolar or control medium (0 mM sucrose) and hyperosmolar medium (150 mM sucrose) in the presence and/or absence of CPZ (106 M), (NS: non-significant, n ¼ 4).

was used when the values of two groups were analyzed. A p-value <0.05 was considered statistically significant. 3. Results

15 min, DNA pellets were resuspended in DNAase free water and run in 1.5% agarose gels with 1  TBE buffer (45 mM Tris-borate and 1 mM EDTA). GelRed™ Nucleic Acid Gel Stain (Biotium Inc., Fremont, CA, USA) was used to visualize the DNA fragments. The extent of DNA degradation was analyzed using the program ImageJ 1.51r ®, expressed in arbitrary densitometric units related to the control, which was assigned a value of 1. 2.7. Determination of cell integrity Integrity of Swan 71 cells was verified by determining the activity of lactate dehydrogenase (LDH) in the culture supernatant of monolayers of cells incubated in the different culture conditions. Measures were accomplished following the procedure of Ka-Ming Chang et al. [24].

3.1. Effect of hyperosmolar stress on TRPV-1 levels in Swan 71 cells First, we explored the expression of TRPV-1 in Swan 71 cell line. We found that both TRPV-1 mRNA and protein were expressed. In addition, the immunofluorescence showed the localization of TRPV-1 in the plasma membrane and in the cytoplasm of the Swan 71 cells (Fig. 1A). Then, Swan 71 cells were cultured in iso-osmolar (0 mM of sucrose) and hyperosmolar medium (150 mM of sucrose) for 24 h to generate an osmotic stress. TRPV-1 protein expression was evaluated by Western blot. We observed that TRPV-1 levels were upregulated by hyperosmolar stress (p < 0.05) (Fig. 1B). 3.2. Effect of hyperosmolar stress on Swan 71 cell viability and integrity

2.8. Statistical analysis Statistical analysis of data was performed by GraphPad Prism v5 software (GraphPad Software, Inc., La Jolla, CA, USA). Comparisons were executed using one way analysis of variance (ANOVA) followed by Tukey post hoc tests where appropriate. Student's t-test

Once established that Swan 71 cell line expresses TRPV-1 and that hyperosmolarity induces its expression, we investigated the effect of osmotic stress on cell viability. We cultured the cells in isoosmolarity (0 mM of sucrose) and hyperosmolarity (50, 100 and 150 mM of sucrose) for 24 h.

J. Reppetti et al. / Biochemical and Biophysical Research Communications 514 (2019) 58e63

Cell viability was analyzed by MTT incorporation, a measure for mitochondrial dehydrogenase enzymatic activity. We observed that, only after the addition of 150 mM of sucrose, cell viability significantly decreased (p < 0.05) compared to iso-osmolar condition (Fig. 2A). In some cases, previously to the exposure to hyperosmolar media, cells were treated for 30 min with 10 mM CPZ, a TRPV-1 inhibitor. Interestingly, in this case, we observed that cell viability was not modified (Fig. 2A). We also tested the addition of CPZ to cells cultured in iso-osmolarity and we did not observe any changes in cells viability (data not shown).

61

To determine cell integrity, we performed an LDH activity assay. LDH is an intracellular enzyme which, during necrotic death, is released into the culture media. In all cases, no differences were observed in the LDH activity in the cultured medium (Fig. 2B).

3.3. Effect of hyperosmolar stress on Swan 71 apoptosis To investigate whether the cells were dying by apoptosis, we analized different apoptotic index, including the expression of the pro-apoptotic protein Bax, DNA fragmentation and nuclear features by TUNEL assay.

Fig. 3. Effect of hyperosmolar stress on Swan 71 apoptosis. A) Representative Western blot and densitometric analysis of Bax protein expression relativized to b-Actin in Swan 71 cell line grown in iso-osmolar or control medium (0 mM sucrose), hyperosmolar medium (150 mM sucrose) and hyperosmolar medium with CPZ (150 mM sucrose with CPZ 106 M) (*p < 0.05 vs. Control, n ¼ 6). B) DNA fragmentation and densitometric analysis of Swan 71 cells cultured in iso-osmolar or control medium (0 mM sucrose), hyperosmolar medium (150 mM sucrose) and hyperosmolar medium with CPZ (150 mM sucrose with CPZ 106 M); M ¼ Standard base pairs (Marker). White arrows show the apoptotic DNA fragments, (*p < 0.05 vs. Control, n ¼ 4). C) Apoptotic nuclei quantification determined by the TUNNEL Assay in Swan 71 cells cultured in iso-osmolar or control medium (0 mM sucrose), hyperosmolar medium (150 mM sucrose) and hyperosmolar medium with CPZ (150 mM sucrose with CPZ 106 M). TUNEL positive cells: red; Dapi: blue. White arrows indicate the presence of apoptotic nuclei. (**p < 0.01 vs. Control, n ¼ 4). Magnification x400. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

62

J. Reppetti et al. / Biochemical and Biophysical Research Communications 514 (2019) 58e63

Bax protein was significantly increased (p < 0.05) when Swan 71 cells were cultured under hyperosmolar stress, and the preincubation of the cells with CPZ did not produce any change compared to iso-osmolar condition (Fig. 3A). Regarding DNA fragmentation, we clearly observed the typical apoptotic ladder pattern, which indicates the degradation of DNA in Swan 71 cells cultured in hyperosmolarity. However, when cells were incubated with CPZ prior to the hyperosmolar treatment, DNA fragmentation was similar to iso-osmolar condition (Fig. 3B). Finally, we analyzed the number of apoptotic nuclei by TUNEL assay. We found that the number of apoptotic nuclei was significantly higher (p < 0.01) in cells cultured in hyperosmolarity compared with those cultured in iso-osmolarity. Additionally, when cells were pre-incubated with CPZ the number of apoptotic nuclei did not change (Fig. 3C).

We previously reported that AQPs participate in the apoptosis of the trophoblast [23], and detected an abnormal TRPV-1 and AQPs expression in preeclamptic tissues which may explain the dysregulation in trophoblast apoptosis exacerbated in this pathological condition [15,23]. However, until now, the role of TRPV-1 in the first trimester trophoblast was not studied. Accumulated evidence shows that TRPVs together with members of AQPs family cooperate in the regulation of the cellular volume involved in many processes such as apoptosis [36,37]. It is possible that TRPV-1 and AQPs may act together to modulate cell death in the trophoblast. In summary, this is the first work that shows evidence that TRPV-1 is expressed in EVT Swan 71 cells and suggests that this channel may have a role in the regulation of the cell volume and cell survival. Declaration of interest

4. Discussion During placental development, the trophoblast differentiates into extravillous (EVT) and villous trophoblast (VT) cells. The VT cells fuse to form a syncytium and constitute the placental villi. On the other hand, the EVT cells proliferate, migrate and invade the maternal decidua and the myometrium to remodel the maternal spiral arteries [25]. The availability of early placental tissue may be ideal, but the limitation to obtain these tissues is critical. For this limitation, it is necessary to use experimental models to study the post-implantation response. In the present work, we reported the expression of TRPV-1 in Swan 71 cells, a cell line used as a model for first trimester ETV. In many cell types TRPV-1 is stored in intracellular vesicles and its mobilization to the plasma membrane is carried out by exocytosis [26,27]. Here, as in other cell types, TRPV-1 was localized in the cytoplasm and the plasma membrane of Swan 71 cells. Similar to other authors, we added a solution of sucrose to the basal medium to generate the hyperosmolar stress [28,29]. Here we found that hyperosmolarity up-regulates the expression of TRPV-1 in the Swan 71 trophoblast cells. It is also well established that hyperosmolarity promotes trophoblast cell differentiation during normal post-implantation [6,30]. However, cells have a window where a stress condition induces normal cell development. Consequently, a slight increment in osmolarity leads to senescence and apoptosis [31], and osmolarity must be tightly regulated to avoid cell death. Apoptosis and necrosis are the two main forms of cell death observed in normal and pathological conditions. Similar to Zhong and co-workers, in the present work we found that a high increase in osmolarity induces cell death. However, cell integrity was not modified, suggesting that cells were not dying by necrosis [9]. Li Y and co-workers demonstrated that the exposure of corneal epithelial cells to hyperosmolarity leads to oxidative stress, mitochondrial dysfunction and apoptosis [32]. In neuroblastoma cells the hyperosmotic stress promotes proteolysis and apoptosis via caspase-3 activation [33]. In several reports, TRPV-1 was associated with apoptosis. In glioma cells, it was observed that this channel induces the calcium flow and triggers the dissipation of the mitochondrial membrane potential which activates caspase-3 [34]. In urothelial cancer, Amantini and co-workers showed that TRPV-1 activates both the intrinsic and extrinsic pathways of the apoptosis process [35]. Regarding the placenta at term, TRPV-1 activation in cytotrophoblast induces apoptosis by a mechanism that involves the intrinsic pathway [14]. Here, we demostrated that the pre-incubation of Swan 71 with CPZ, the especific antagonist of TRPV-1, prevent the cell death when cells were exposed to hyperosmolarity, suggesting the participation of TRPV-1 in the apoptotic process of EVT.

The author declares that there is no conflict of interest that would prejudice the impartiality of this scientific work. Funding This study was supported by, ANCyPT PICT 2014-2112 (Argentina), PIP 2015-0590 (Argentina) and ANCyPT PICT 20160276 (Argentina). Acknowledgements The authors thank Natalia Beltramone for technical assistance. 7- References [1] S. Anin, G. Vince, S. Quenby, Trophoblast invasion, Hum. Fertil. 7 (2004) 169e174. [2] D. Roje, S. Zekic Tomas, I. Kuzmic Prusac, V. Capkun, I. Tadin, Trophoblast apoptosis in human term placentas from pregnancies complicated with idiopathic intrauterine growth retardation, J. Matern. Fetal Neonatal Med. 24 (2011) 745e751. [3] I.P. Crocker, S. Cooper, S.C. Ong, P.N. Baker, Differences in apoptotic susceptibility of cytotrophoblasts and syncytiotrophoblasts in normal pregnancy to those complicated with preeclampsia and intrauterine growth restriction, Am. J. Pathol. 162 (2003) 637e643. [4] L.G. Pescio, N.O. Favale, M.G. M
arquez, N.B. Sterin-Speziale, Glycosphingolipid synthesis is essential for MDCK cell differentiation, Biochim. Biophys. Acta Mol. Cell Biol. Lipids 1821 (2012) 884e894. [5] H. Li, J. Wang, F. Li, G. Chen, Q. Chen, The influence of hyperosmolarity in the intervertebral disc on the proliferation and chondrogenic differentiation of nucleus pulposus-derived mesenchymal stem cells, Cells Tissues Organs 205 (2018) 178e188. [6] J. Liu, W. Xu, T. Sun, F. Wang, E. Puscheck, D. Brigstock, Q. Wang, R. Davis, D. Rappolee, Hyperosmolar stress induces global mRNA responses in placental trophoblast stem cells that emulate early post-implantation differentiation, Placenta 30 (2009) 66e73. [7] Y.-H. Kim, T. Oh, E. Park, N.-H. Yim, K. Park, W. Cho, J. Ma, Anti-inflammatory and anti-apoptotic effects of Acer palmatum Thumb. extract, KIOM-2015EW, in a hyperosmolar-stress-induced in vitro dry eye model, Nutrients 10 (2018) 282. [8] F. Gamboni, C. Anderson, S. Mitra, J.A. Reisz, T. Nemkov, M. Dzieciatkowska, K.L. Jones, K.C. Hansen, A. D'Alessandro, A. Banerjee, Hypertonic saline primes activation of the p53ep21 signaling Axis in human small airway epithelial cells that prevents inflammation induced by pro-inflammatory cytokines, J. Proteome Res. 15 (2016) 3813e3826. [9] W. Zhong, Y. Xie, Y. Wang, J. Lewis, A. Trostinskaia, F. Wang, E.E. Puscheck, D.A. Rappolee, Use of hyperosmolar stress to measure stress-activated protein kinase activation and function in human HTR cells and mouse trophoblast stem cells, Reprod. Sci. 14 (2007) 534e547. [10] P. Bose, M. Kadyrov, R. Goldin, S. Hahn, M. Backos, L. Regan, B. Huppertz, Aberrations of early trophoblast differentiation predispose to pregnancy failure: lessons from the anti-phospholipid syndrome, Placenta 27 (2006) 869e875. [11] S.F. Pedersen, E.K. Hoffmann, I. Novak, Cell volume regulation in epithelial physiology and cancer, Front. Physiol. 4 (2013) 233. [12] M. Song, S.P. Yu, Ionic regulation of cell volume changes and cell death after ischemic stroke, Transl. Stroke Res. 5 (2014) 17e27. [13] T. Rosenbaum, S.A. Simon, TRPV1 Receptors and Signal Transduction, TRP Ion

J. Reppetti et al. / Biochemical and Biophysical Research Communications 514 (2019) 58e63

[14]

[15] [16]

[17]

[18]

[19] [20]

[21]

[22]

[23]

[24]

[25]

[26]

Channel Function in Sensory Transduction and Cellular Signaling Cascades, CRC Press, 2006, pp. 83e98. M. Costa, B. Fonseca, E. Keating, N. Teixeira, G. Correia-da-Silva, Transient receptor potential vanilloid 1 is expressed in human cytotrophoblasts: induction of cell apoptosis and impairment of syncytialization, Int. J. Biochem. Cell Biol. 57 (2014) 177e185. N. Martinez, C.E. Aban, G.F. Leguizamon, A.E. Damiano, M.G. Farina, TPRV-1 expression in human preeclamptic placenta, Placenta 40 (2016) 25e28. X. Hua, Z. Su, R. Deng, J. Lin, D.-Q. Li, S.C. Pflugfelder, Effects of L-carnitine, erythritol and betaine on pro-inflammatory markers in primary human corneal epithelial cells exposed to hyperosmotic stress, Curr. Eye Res. 40 (2015) 657e667. L. Pan, K. Song, F. Hu, W. Sun, I. Lee, Nitric oxide induces apoptosis associated with TRPV1 channel-mediated Ca2þ entry via S-nitrosylation in osteoblasts, Eur. J. Pharmacol. 715 (2013) 280e285. J. Waning, J. Vriens, G. Owsianik, L. Stüwe, S. Mally, A. Fabian, C. Frippiat, B. Nilius, A. Schwab, A novel function of capsaicin-sensitive TRPV1 channels: involvement in cell migration, Cell Calcium 42 (2007) 17e25. € dding, TRP proteins and cancer, Cell. Signal. 19 (2007) 617e624. M. Bo B. Li, Y. Yin, Y. Liu, Y. Pi, L. Guo, X. Cao, C. Gao, L. Zhang, J. Li, TRPV1 activation impedes foam cell formation by inducing autophagy in oxLDL-treated vascular smooth muscle cells, Cell Death Dis. 5 (2014) e1182. S.L. Straszewski-Chavez, V.M. Abrahams, A.B. Alvero, P.B. Aldo, Y. Ma, S. Guller, R. Romero, G. Mor, The isolation and characterization of a novel telomerase immortalized first trimester trophoblast cell line, Swan 71, Placenta 30 (2009) 939e948. R. Alejandra, S. Natalia, The blocking of aquaporin-3 (AQP3) impairs extravillous trophoblast cell migration, Biochem. Biophys. Res. Commun. 499 (2018) 227e232. N. Szpilbarg, M. Castro-Parodi, J. Reppetti, M. Repetto, B. Maskin, N. Martinez, A.E. Damiano, Placental programmed cell death: insights into the role of aquaporins, MHR: Basic Sci. Reproductive Med. 22 (2015) 46e56. F.K.-M. Chan, K. Moriwaki, M.J. De Rosa, Detection of Necrosis by Release of Lactate Dehydrogenase Activity, in: Immune Homeostasis, Springer, 2013, pp. 65e70. € fler, J. Pollheimer, Function and control of human invasive P. Velicky, M. Kno trophoblast subtypes: intrinsic vs. maternal control, Cell Adhes. Migrat. 10 (2016) 154e162. J. Meng, S.V. Ovsepian, J. Wang, M. Pickering, A. Sasse, K.R. Aoki, G.W. Lawrence, J.O. Dolly, Activation of TRPV1 mediates calcitonin generelated peptide release, which excites trigeminal sensory neurons and is attenuated by a retargeted botulinum toxin with anti-nociceptive potential, J. Neurosci. 29 (2009) 4981e4992.

63

[27] T. Shimizu, M. Shibata, H. Toriumi, T. Iwashita, M. Funakubo, H. Sato, T. Kuroi, T. Ebine, K. Koizumi, N. Suzuki, Reduction of TRPV1 expression in the trigeminal system by botulinum neurotoxin type-A, Neurobiol. Dis. 48 (2012) 367e378. re, P.H. Watson, A.J. Watson, Mitogen-activated protein [28] C.E. Bell, N.M. Larivie kinase (MAPK) pathways mediate embryonic responses to culture medium osmolarity by regulating Aquaporin 3 and 9 expression and localization, as well as embryonic apoptosis, Hum. Reprod. 24 (2009) 1373e1386. [29] M. Hollborn, S. Vogler, A. Reichenbach, P. Wiedemann, A. Bringmann, L. Kohen, Regulation of the hyperosmotic induction of aquaporin 5 and VEGF in retinal pigment epithelial cells: involvement of NFAT5, Mol. Vis. 21 (2015) 360. [30] A. Awonuga, W. Zhong, M. Abdallah, J. Slater, S. Zhou, Y. Xie, E. Puscheck, D. Rappolee, Eomesodermin, HAND1, and CSH1 proteins are induced by cellular stress in a stress-activated protein kinase-dependent manner, Mol. Reprod. Dev. 78 (2011) 519e528. [31] E.E. Puscheck, A.O. Awonuga, Y. Yang, Z. Jiang, D.A. Rappolee, Molecular Biology of the Stress Response in the Early Embryo and its Stem Cells, Cell Signaling during Mammalian Early Embryo Development, Springer, 2015, pp. 77e128. [32] Y. Li, H. Liu, W. Zeng, J. Wei, Edaravone protects against hyperosmolarityinduced oxidative stress and apoptosis in primary human corneal epithelial cells, PLoS One 12 (2017), e0174437. [33] M. Olivera-Santa Catalina, M. Caballero-Bermejo, R. Argent, J.C. Alonso, A. Cuenda, M.J. Lorenzo, F. Centeno, Hyperosmotic stress induces tau proteolysis by caspase-3 activation in SH-SY5Y cells, J. Cell. Biochem. 117 (2016) 2781e2790. [34] C. Amantini, M. Mosca, M. Nabissi, R. Lucciarini, S. Caprodossi, A. Arcella, F. Giangaspero, G. Santoni, Capsaicin-induced apoptosis of glioma cells is mediated by TRPV1 vanilloid receptor and requires p38 MAPK activation, J. Neurochem. 102 (2007) 977e990. [35] C. Amantini, P. Ballarini, S. Caprodossi, M. Nabissi, M.B. Morelli, R. Lucciarini, M.A. Cardarelli, G. Mammana, G. Santoni, Triggering of transient receptor potential vanilloid type 1 (TRPV1) by capsaicin induces Fas/CD95-mediated apoptosis of urothelial cancer cells in an ATM-dependent manner, Carcinogenesis 30 (2009) 1320e1329. [36] X. Liu, B. Bandyopadhyay, T. Nakamoto, B. Singh, W. Liedtke, J.E. Melvin, I. Ambudkar, A role for AQP5 in activation of TRPV4 by hypotonicity concerted involvement of AQP5 and TRPV4 in regulation of cell volume recovery, J. Biol. Chem. 281 (2006) 15485e15495. [37] L. Galizia, A. Pizzoni, J. Fernandez, V. Rivarola, C. Capurro, P. Ford, Functional interaction between AQP2 and TRPV4 in renal cells, J. Cell. Biochem. 113 (2012) 580e589.