Tyrphostin AG-1478

Activation of Protein Kinase Cδ Contributes to the Induction of Src/EGF Receptor/ERK Signaling in Ammonia-treated Astrocytes

Guizhi Jia 1 • Rui Wang2 • Yi Yue2 • Hongliang Dai3

Previously, we showed that Src-mediated EGF receptor transactivation/ERK activation mediates ammonia-induced astrocyte swelling, which represents a major component of brain edema in hyperammonemic disorders. Here, we tested the role of PKC in the induction of this signaling pathway and its involvement in ammonia-mediated cell swelling. We found that incubating astrocytes with bisindolylmaleimide (BIM, an inhibitor of classical and novel PKC isoforms) or rottlerin, a PKCδ-specific inhibitor, attenuated the ammonia-induced phosphorylation of EGFR, while GF109203X had no effect on this pathway. We further found that BIM or rottlerin pretreatment inhibited the ammonia-induced phosphorylation of Src and that ammonia significantly increased the level of PKCδ pulled down by a Src antibody. AG1478, a specific EGFR kinase activity inhibitor, effectively inhibited phosphorylation at Tyr1068 but had no discernable effect on phosphorylation at Tyr845. Moreover, BIM or rottlerin abrogated ammonia-induced ERK phosphorylation. BIM-, rottlerin-, or GF109203X-treated astrocytes showed a sig- nificant reduction in cell swelling compared to that observed after treatment with ammonia alone. Finally, it was found that AG1478 attenuated ammonia-induced PKCα translocation to the particulate fraction. Taken together, our results indicate that PKCδ mediates ammonia-induced astrocyte swelling by activating Src and downstream EGF receptor/ERK signaling, which may contribute to the pathogenesis of neuropsychiatric disorders associated with hyperammonemia.
Keywords Ammonia . Astrocyte . EGFR . Src . PKC . Hepatic encephalopathy

Hepatic encephalopathy (HE) is a common complication ac- companying hepatic diseases (Marti-Carvajal et al. 2019). Between 50% and 75% of cases of liver failure show neuro- logical symptoms to varying degrees (Jayakumar et al. 2006). According to the characteristics of liver diseases, there are three types in which HE can be observed. Of these types, the acute form of HE, i.e., type A, usually presents with an abrupt onset of delirium, seizures, and coma and is associated with an extremely poor prognosis (Dai et al. 2017a). This poor prognosis is related to its ability to induce brain edema, in- crease intracranial pressure, and induce brain herniation. Increasing evidence has demonstrated that astroglial cell swelling is the key pathophysiological mechanism behind HE-related brain edema. Many agents can result in astrocyte swelling in HE, and ammonia seems to be the main etiological factor. Multiple mechanisms regarding how ammonia induces astrocyte swelling, including oxidative stress, the activation of signaling kinases and transcriptional factors, and inflammato- ry response, have been proposed (Heidari 2018). In addition, our more recent studies demonstrated that epidermal growth factor (EGF) receptor transactivation contributes to the path- ogenesis of ammonia-induced astrocyte swelling (Dai et al. 2017a, 2013).
The ErbB family belongs to subclass I of receptor tyrosine kinases (RTKs). To date, four members of the ErbB family, i.e., ErbB1/HER1/EGFR, ErbB1/Neu/HER2, ErbB3/HER3, and ErbB3/HER3, have been identified; ErbB1/HER1/ EGFR is the most prominent member and modulates multiple biological processes (Amos et al. 2005). All ErbBs share a modular structure composed of an extracellular ligand- binding domain, a signal hydrophobic transmembrane region, and an intracellular tyrosine kinase domain. A variety of EGF- related peptide growth factors can activate ErbBs through spe- cific ligand-receptor binding, which causes receptor dimeriza- tion, the induction of the intracellular intrinsic tyrosine kinase activity of the receptor, and the subsequent autophosphoryla- tion of specific residues within the cytoplasmic domain. These phosphorylated residues act as docking sites for downstream signaling molecules to coordinately regulate cell responses (Cattaneo et al. 2011). In addition to being activated through the classical activation process by its cognate ligand as men- tioned above, EGFR can also be activated by other stimuli, such as cytokines, oxidative stress, and heat stress, through a process that is now generally termed “EGFR transactivation” (Atef and Anand-Srivastava 2016; Chen et al. 2017; Dai et al. 2017a, b). Notably, it has been shown that ammonia-induced astrocyte swelling is an EGFR transactivation/ERK activation-dependent process. Moreover, it has been further demonstrated that this EGFR transactivation is Src-mediated and ligand release–independent (Dai et al. 2017a, 2013).
PKC is a member of the phospholipid-dependent serine/threonine kinase family, and activated PKC is crit- ically involved in a variety of cellular responses, includ- ing cell growth and differentiation, hormone secretion, and apoptosis. The PKC family is composed of at least 11 isoforms and their isoforms are commonly divided into three subgroups (i.e., conventional, novel, and atyp- ical PKCs) based on their diversity in molecular struc- ture, cellular distribution, biological function, and cofac- tor dependence. Intriguingly, PKCs have been frequently reported to induce EGFR transactivation via the phos- phorylation of Src (Hsieh et al. 2008; Huang et al. 2013; Yang et al. 2009). As such, the present study aimed to clarify the involvement of PKC in ammonia- induced EGFR transactivation and subsequent ERK acti- vation to help provide further insight into the pathogen- esis of HE and guide the development of new treatment strategies.

Materials and Methods

Primary Astrocyte Culture
Primary cultures of neonatal rat astrocytes were prepared from 1-day-old Sprague-Dawley rats as described in our previous protocol (Dai et al. 2017a). The harvested cells were plated in 60-mm Falcon Primaria dishes or coverslips coated with poly- lysine and cultured in Dulbecco’s modified Eagle medium (DMEM)/F12 supplemented with 10% fetal bovine serum and 5.55 mM glucose. The cells were incubated in a humidi- fied atmosphere of 5% CO2/95% air at 37 °C. At day 9, mi- croglia and oligodendrocyte progenitors were removed by shaking to obtain astrocyte-enriched cultures, which were further passaged two to three times. The preparations contained 95–99% astrocytes (Dai et al. 2017a).

Western Blotting
Total protein content was determined by the Lowry method, and equal amounts of protein were subjected to sodium dode- cyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a polyvinylidene fluoride membrane. The membrane was blocked with 1% BSA for 1 h at room temperature and then incubated for 2 h at room temperature with primary antibodies against ERK (1:1000, Santa Cruz Biotechnology, Inc., Dallas, TX, USA) and phospho-ERK (1:1000, Santa Cruz Biotechnology, Inc.). After three washes (5 min each wash) with Tris-buffered saline supplemented with 0.1% Tween 20 (TBST), the membrane was further in- cubated with horseradish peroxide-conjugated secondary an- tibodies (Santa Cruz Biotechnology, Inc.). The bands were visualized by enhanced chemiluminescence reagents (Thermo Fisher Scientific). The optical density of each band was assessed using Quantity One software (version 4.6.9; Bio- Rad Laboratories, Inc., Hercules, CA, USA).

For the immunoprecipitation of EGFR and Src, whole-cell lysates containing 1 mg of protein were incubated with 8 μg each of anti-EGFR (Upstate Biotechnology, Lake Placid, NY, USA) and anti-Src (Cell Signaling Technology, Danvers, MA, USA) antibodies for 12 h at 4 °C to form an immune complex. Subsequently, 200 μl of washed Protein G agarose bead slurry (Upstate Biotechnology, Lake Placid) was added, and the mix- ture was incubated at 4 °C for another 2 h. The agarose beads were collected by centrifugation at 14,000 ×g for 5 s. After three washes with PBS, the beads were boiled for 5 min, and the supernatant was collected for subsequent analysis by Western blotting. The antibodies used included those specific for EGFR p-Tyr845, EGFR p-Tyr1068, and PKCδ (all pur- chased from Cell Signaling Technology).

PKC Translocation Assay
Astrocyte cultures suspended in a solution containing 10 mM EGTA, 1 mM EDTA, 0.5 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 50 μg/ml leupeptin, 25 μg/ml aprotinin, and 20 mM Tris-HCl (pH 7.5) were ho- mogenized in a Potter-Elvehjem homogenizer. The cell ho- mogenates were subjected to centrifugation at 100,000 ×g for 1 h at 4 °C. The supernatants that constituted the cytosolic fraction were removed. The remaining pellets were resuspend- ed in the abovementioned solution containing 1% Triton X- 100 for 30 min on ice. Thereafter, the mixture was centrifuged at 25,000 ×g, and the supernatant was collected as the particulate fraction. The cytosolic and particulate fractions were subjected to SDS-polyacrylamide gel electrophoresis and immunoblot analysis. An antibody targeting PKCδ was used to analyze the translocation of this PKC isoform.

Cell Volume Determination
Astrocyte volume was determined by measuring the intracel- lular water space using the 3-O-methyl-[3H]-glucose ([3H] OMG) equilibration method (Dai et al. 2017a; Jayakumar et al. 2014; Kletzien et al. 1975), which is a well-established and sensitive method for cell volume determination (Aschner 2011). Briefly, cells were incubated with [3H] OMG (1 mM containing 1 μCi of radioactive OMG) for 12 h. A small aliquot of cell medium was saved for radioactivity determina- tion. After rapid washing with ice-cold buffer containing 290 mM sucrose, 1 mM Tris-nitrate (pH 7.4), 0.5 mM calcium nitrate, and 0.1 mM phloretin, the cells were lysed with 1 N NaOH. The intracellular water space was normalized to the protein level and expressed as μl/mg protein.

Statistical Analysis
The data are expressed as the mean ± standard error of the mean (S.E.M.) of at least three individual experiments. Statistical analysis was performed using one-way analysis of variance followed by Fisher’s least significant difference test. P < 0.05 was considered significant. Results Involvement of PKC in Ammonia-induced EGFR Transactivation Ammonia (NH3) is a nitrogen compound produced by nitro- gen metabolism in the body. Under physiological pH condi- tions, ≥ 98% ammonia is present as ammonium ions (NH +) (Sturgeon and Shawcross 2014), and for this reason, NH4Cl was used in this study to model ammonium toxicity; for sim- plicity, “ammonia” is used to refer to NH3 and NH +, as done by many other researchers in this field (Görg et al. 2019). We designed experiments to identify the involvement of PKC in the ammonia-induced phosphorylation of EGFR at Tyr845 and Tyr1068. Previous studies have shown that bisindolylmaleimide (BIM) inhibits the activation of both novel and classical PKCs, while GF109203X specifically pre- vents the activities of classical PKCs. To test which PKC isoenzymes are involved in ammonia-induced Tyr845 and Tyr1068 phosphorylation, we pretreated the cells with either BIM (1 μM) or GF109203X (3 μM) for 30 min prior to am- monia treatment. It was found that BIM but not GF109203X attenuated ammonia-induced EGFR phosphorylation in cultured rat astrocytes (Fig. 1a and b), suggesting that PKCs other than the classical isoform contribute to ammonia- induced EGFR Tyr845 and Tyr1068 phosphorylation. We fur- ther determined the involvement of the novel PKC isozymes in ammonia-augmented EGFR phosphorylation. Incubating astrocytes with rottlerin (5 μM), a PKCδ-specific inhibitor, blocked the ammonia-induced phosphorylation of EGFR, suggesting that PKCδ might mediate the response to ammonia Mediating Effect of Src in PKC-induced EGFR Transactivation in Astrocytes Following Ammonia Treatment Although PKC seems essential for ammonia-induced EGFR phosphorylation, the tyrosine phosphorylation sites of EGFR are theoretically not directly affected by the serine-threonine kinase PKC. In other words, there should exist an intermediate kinase between PKC and EGFR. The nonreceptor tyrosine kinase Src is a common cotransducer of transmembrane sig- nals, and it has been shown to phosphorylate EGFR at Tyr845 and/or Tyr1068 in multiple types of in various cell types. Additionally, our prior study showed that ammonia-induced EGFR transactivation is a Src activity-dependent process. Furthermore, Src has a poorly conserved unique domain that can be activated by PKC at serine and threonine residues. As such, the question arose as to whether this dependence on Src is exerted downstream of PKC activity. In cultured astrocytes that were incubated with BIM or rottlerin, exposure to ammo- nia did not activate Src, whereas Src activation was observed in cells treated with ammonia only (Fig. 2a). We further explored the association between PKC and Src following ammonia treatment using a coimmunoprecipitation assay. As shown in Fig. 2b, 20 min of treatment with ammonia significantly increased the level of PKCδ pulled down by a Src antibody. These data demonstrate that ammonia promotes the activation of PKCδ and the PKCδ-Src interaction, leading to subsequent phosphorylation and activation of Src, which then functions as a cotransducer of EGFR signaling. EGFR Tyrosine Kinase Activity Is Required for the Ammonia-induced Phosphorylation of EGFR at Tyr1068 but Not at Tyr845 Our previous investigation demonstrated that ammonia- triggered EGFR activity is related to the phosphorylation of EGFR at the Tyr845 and Tyr1068 sites. However, the depen- dence of the phosphorylation of these two tyrosine sites on EGFR kinase activity is still unknown. To evaluate this de- pendence, we examined the effect of tyrphostin AG1478, a immunoprecipitation to analyze the phosphorylation level of EGFR at Tyr845 and Tyr1068. The data are presented as the mean ± S.E.M. of three independent experiments. *p < 0.05 compared with the control group, #p < 0.05 compared with the ammonia group potent and specific inhibitor of EGFR tyrosine kinase activity. As shown in Fig. 3a, AG1478 significantly decreased the phosphorylation of the Tyr845 and Tyr1068 residues follow- ing EGF treatment. Compared to ammonia, this specific EGFR kinase activity inhibitor effectively inhibited phosphor- ylation at Tyr1068 but had no discernible effect on the phos- phorylation of the Tyr845 residue (Fig. 3b). Our data indicate that ammonia first induced the phosphorylation of EGFR at Tyr845, which then led to an increase in EGFR kinase activity and the resultant phosphorylation of the Tyr1068 residue. Based on our previous study, the ammonia-triggered phos- phorylation of Tyr845 might originate from direct activation by Src. Pharmacological Inhibition of PKCδ Abrogates Ammonia-induced ERK Activation The activation of the MEK/ERK pathway functions as an important mechanism that mediates ammonia toxicity in as- trocytes. Our previous study showed that ammonia-induced ERK phosphorylation/activation results from EGFR tyrosine kinase activity, as the administration of AG1478 effectively blocks ERK phosphorylation. Furthermore, the pharmacolog- ical inhibition of Src by PP1 also prevents the ammonia- induced activation of ERK (Dai et al. 2013). In the current study, we investigated the role of PKC activity in ammonia- induced mitogenic signaling. It was found that pretreating astrocytes with BIM or rottlerin abrogated ERK phosphoryla- tion induced by ammonia (Fig. 4). PKC Inhibition Prevents Ammonia-induced Astrocyte Swelling To examine whether PKC activation by ammonia contributes to astrocyte swelling, cells were pretreated with various phar- macological inhibitors of the PKC family and then treated with ammonia for 12 h. As shown in Fig. 5, BIM-, rottlerin-, or GF109203X-treated astrocytes showed a significant reduc- tion in cell swelling compared to that observed after treatment of three independent experiments, *p < 0.05 compared with the control group, #p < 0.05 compared with the ammonia group. b Cultured astro- cytes were incubated with 3 mM NH4Cl for 20 min, and immunoprecip- itation was performed with an antibody against Src. The data are present- ed as the mean± S.E.M. *p < 0.05 compared with the control group with ammonia alone. The inhibitors themselves had no effect on cell size (data not shown). Ammonia-induced Translocation/Activation of PKCα Is Dependent on EGFR Tyrosine Kinase Activity Since GF109203X inhibits astrocyte swelling but not EGFR phosphorylation induced by ammonia, classical PKC iso- forms might mediate the neurotoxic effects of ammonia by acting in parallel with or operating downstream of EGFR sig- naling. As shown in Fig. 6, AG1478 attenuated ammonia- induced PKCα translocation to the particulate fraction, sug- gesting that ammonia-induced PKCα activation resulted from EGFR tyrosine kinase activity. A similar sequence was previ- ously observed for the effect of evodiamine on adipogenesis in 3T3-L1 preadipocytes (T. Wang et al. 2009). The aforemen- tioned results confirm that PKCδ mediates ammonia-induced astrocyte swelling by activating Src and downstream EGF receptor/ERK signaling, which may contribute to the patho- genesis of neuropsychiatric disorders associated with hyperammonemia (Fig. 7). Discussion Brain edema and the subsequent increase in intracranial pres- sure and brain herniation are major complications of acute liver failure (ALF). Normally, blood ammonia concentrations are 10~50 μM. In ALF, however, arterial ammonia levels can rise to as high as 100~450 μM. Furthermore, the brain:blood ammonia concentration also increases from ~ 2 in healthy in- dividuals to up to 8 in ALF patients (Butterworth 2002); i.e., brain ammonia levels can vary from 0.8~3.6 mM. In addition, in previous reports, ammonia levels as high as 5 mM were observed in the brains of experimental animals with ALF (Jayakumar et al. 2016; Jayakumar et al. 2011). In this work, as in our previous study (Dai et al. 2013), 3 mM ammonia was used to model in vitro ALF. Elevated brain ammonia-triggered astrocyte swelling represents an important mechanism of these complications. However, the mechanism by which ammonia causes astrocyte swelling in ALF is largely unknown. Among other mechanisms, EGFR transactivation and associated sig- naling events have been strongly implicated in ammonia- induced astrocyte swelling (Dai et al. 2017a, 2013; Song and Du 2014). Our previous study (Dai et al. 2013) and current study showed that ammonia stimulation induces a prominent increase in EGFR phosphorylation of Tyr845 and Tyr1068. Intriguingly, our present study showed that the phosphoryla- tion of Tyr1068 but not of Tyr845 is dependent on EGFR kinase activity. This suggests that ammonia-induced Tyr845 phosphorylation precedes EGFR kinase activity and the acti- vation of Tyr1068. In other words, ammonia first phosphory- lates EGFR at Tyr845, which then leads to the augmentation of EGFR kinase activity and ultimately causes the phosphor- ylation of Tyr 1068. Although both Tyr845 and Tyr1068 are likely Src phosphorylation sites, we herein provide evidence that only Tyr845 is a direct target of Src following ammonia stimulation. This is consistent with our previous finding of the ammonia-induced association of EGFR with Src at the Tyr845 residue of EGFR (Dai et al. 2013), the regulation of EGFR Samples were collected for immunoprecipitation to analyze the phosphor- ylation level of EGFR at Tyr845 and Tyr1068. The data are presented as the mean ± S.E.M, of three independent experiments, *p < 0.05 compared with the control group. #p < 0.05 compared with the EGF/ammonia group Tyr845 phosphorylation by Src by the COOH terminus of TM4SF5 in hepatoma cell lines (Jung et al. 2013), and the role of the c-Src–mediated phosphorylation of EGFR Tyr845 in the regulation of receptor function and tumor progression (Biscardi et al. 1999). Mounting evidence shows that Na,K-ATPase, in addition to having ion pump activity, functions as a receptor. Na,K- ATPase and Src can combine to form a complex receptor, and cardiotonic steroids, such as ouabain, telocinobufagin, and marinobufagin, are the most common ligands for Na,K- ATPase. Upon binding to and activating Na,K-ATPase, car- diotonic steroids can regulate cell functions via Src activation in a variety of physiological and pathological conditions (Amaral et al. 2018; Chen et al. 2018). Of relevance, it has been shown that chronic ammonia stimulation increases the content of ouabain-like compounds in astrocyte cultures (Kala et al. 2000), that canrenone, an inhibitor of ouabain, signifi- cantly inhibits ammonia-triggered signaling and pathophysio- logical effects, and that ammonia-induced signaling transduc- tion and astrocyte swelling are effectively prevented by Src inhibition (Dai et al. 2013; Liang et al. 2014; Wang et al. 2015). These findings strongly support the notion that ammo- nia exerts its neurotoxic effect on astrocytes via the initiation of Na,K-ATPase/ouabain/Src signaling. Although Na,K- ATPase–mediated Src activation has been well established in ammonia toxicity in astrocytes, other mechanisms by which ammonia induces Src activation should also be addressed. The PKC family is a phospholipid-dependent serine/ threonine kinase family regulated by Ca2+, diacylglycerol (DAG), and phosphatidylserine (Amos et al. 2005). PKC activation leads to the phosphorylation and activation of downstream signaling proteins that control a variety of bio- logical responses, including cell cycle regulation, autophagy, apoptosis, and bipolar disorder (Saxena et al. 2017; T. Wang et al. 2018; Wang et al. 2017; Yi et al. 2019). PKC-regulated signaling proteins include MAPK, AKT, Src, and EGFR (Amos et al. 2005; Cai et al. 2017; Davis and Czech 1985; Wang et al. 2019). c-Src is phosphorylated by PKC at a poorly conserved unique domain rich in serine and threonine residues (Amos et al. 2005; Brown and Cooper 1996). PKC-dependent Src activation is well established in many biological processes (Hsieh et al. 2008; Huang et al. 2013; Yang et al. 2009). In addition, it was previously documented that a pathological concentration of ammonia can trigger a transient increase in PKC activity, contributing to delayed apoptosis of C6 glioma cells (Buzanska et al. 2000). Therefore, we were prompted to explore the possible involvement of PKC in Src-mediated EGFR phosphorylation following ammonia toxicity in astro- cytes, as reported in our previous study (Dai et al. 2013). Our current study suggests that PKCδ is an important isoform for the sequential activation of Src/EGFR/ERK. Notably, PKCα also seems to mediate ammonia-induced astrocyte signaling and functional alterations, as reflected by the profound activation of PKCα by ammonia treatment as well as the effect of GF109203X on cell swelling. The finding that ammonia-induced PKCα is sensitive to AG1478 pretreat- ment, as observed in the present study, indicates that PKCα activation occurs following EGFR activation. Similarly, evodiamine inhibits adipogenesis via the activation of the EGFR-PKCα-ERK signaling cascade (T. Wang et al. 2009). In conclusion, our results indicate that PKCδ mediates ammonia-induced astrocyte swelling by activating Src and downstream EGF receptor/ERK signaling, which may con- tribute to the pathogenesis of neuropsychiatric disorders asso- ciated with hyperammonemia, such as hepatic encephalopa- thy, urea cycle deficiencies, and Reye’s syndrome. References Amaral LS, Martins Ferreira J, Predes D, Abreu JG, Noel F, Quintas LEM (2018) Telocinobufagin and marinobufagin produce different effects in LLC-PK1 cells: a case of functional selectivity of bufadienolides. Int J Mol Sci 19(9). https://doi.org/10.3390/ijms19092769 Amos S, Martin PM, Polar GA, Parsons SJ, Hussaini IM (2005) Phorbol 12-myristate 13-acetate induces epidermal growth factor receptor transactivation via protein kinase Cdelta/c-Src pathways in glioblas- toma cells. J Biol Chem 280(9):7729–7738. https://doi.org/10.1074/ jbc.M409056200 Aschner M (2011) Volume measurements in cultured primary astrocytes. Methods Mol Biol 758:391–402. https://doi.org/10.1007/978-1- 61779-170-3_26 Atef ME, Anand-Srivastava MB (2016) Oxidative stress contributes to the enhanced expression of Gqalpha/PLCbeta1 proteins and hyper- trophy of VSMC from SHR: role of growth factor receptor transactivation. Am J Physiol Heart Circ Physiol 310(5):H608– H618. https://doi.org/10.1152/ajpheart.00659.2015 Biscardi JS, Maa MC, Tice DA, Cox ME, Leu TH, Parsons SJ (1999) c- Src-mediated phosphorylation of the epidermal growth factor recep- tor on Tyr845 and Tyr1101 is associated with modulation of receptor function. J Biol Chem 274(12):8335–8343 Brown MT, Cooper JA (1996) Regulation, substrates and functions of src. Biochim Biophys Acta 1287(2–3):121–149 Butterworth RF (2002) Pathophysiology of hepatic encephalopathy: a new look at ammonia. Metab Brain Dis 17(4):221–227. https:// doi.org/10.1023/a:1021989230535 Buzanska L, Zablocka B, Dybel A, Domanska-Janik K, Albrecht J (2000) Delayed induction of apoptosis by ammonia in C6 glioma cells. Neurochem Int 37(2–3):287–297 Cai L, Stevenson J, Geng X, Peng C, Ji X, Xin R, Rastogi R, Sy C, Rafols JA, Ding Y (2017) Combining normobaric oxygen with ethanol or hypothermia prevents brain damage from thromboembolic stroke via PKC-Akt-NOX modulation. Mol Neurobiol 54(2):1263–1277. https://doi.org/10.1007/s12035-016-9695-7 Cattaneo F, Iaccio A, Guerra G, Montagnani S, Ammendola R (2011) NADPH-oxidase-dependent reactive oxygen species mediate EGFR transactivation by FPRL1 in WKYMVm-stimulated human lung cancer cells. Free Radic Biol Med 51(6):1126–1136. https://doi. org/10.1016/j.freeradbiomed.2011.05.040 Chen Y, Peng FF, Jin J, Chen HM, Yu H, Zhang BF (2017) Src-mediated ligand release-independent EGFR transactivation involves TGF- beta-induced Smad3 activation in mesangial cells. Biochem Biophys Res Commun 493(2):914–920. https://doi.org/10.1016/j. bbrc.2017.09.121 Chen L, Jiang P, Li J, Xie Z, Xu Y, Qu W, Feng F, Liu W (2018) Periplocin promotes wound healing through the activation of Src/ ERK and PI3K/Akt pathways mediated by Na/K-ATPase. Phytomedicine 57:72–83. https://doi.org/10.1016/j.phymed.2018.12.015 Dai H, Song D, Xu J, Li B, Hertz L, Peng L (2013) Ammonia-induced Na,K-ATPase/ouabain-mediated EGF receptor transactivation, MAPK/ERK and PI3K/AKT signaling and ROS formation cause astrocyte swelling. Neurochem Int 63(6):610–625. https://doi.org/ 10.1016/j.neuint.2013.09.005 Dai H, Jia G, Wang W, Liang C, Han S, Chu M, Mei X (2017a) Genistein inhibited ammonia induced astrocyte swelling by inhibiting NF- kappaB activation-mediated nitric oxide formation. Metab Brain Dis 32(3):841–848. https://doi.org/10.1007/s11011-017-9975-6 Dai H, Jia G, Wang H, Yang J, Jiang H, Chu M (2017b) Epidermal growth factor receptor transactivation is involved in the induction of human hepatoma SMMC7721 cell proliferation by insufficient radiofrequency ablation. Oncol Lett 14(2):2463–2467. https://doi. org/10.3892/ol.2017.6463 Davis RJ, Czech MP (1985) Tumor-promoting phorbol diesters cause the phosphorylation of epidermal growth factor receptors in normal hu- man fibroblasts at threonine-654. Proc Natl Acad Sci U S A 82(7): 1974–1978 Görg B, Karababa A, Schütz E, Paluschinski M, Schrimpf A, Shafigullina A, Castoldi M, Bidmon HJ, Häussinger D (2019) O-GlcNAcylation- dependent upregulation of HO1 triggers ammonia-induced oxida- tive stress and senescence in hepatic encephalopathy. J Hepatol 71(5):930–941. https://doi.org/10.1016/j.jhep.2019.06.020 Heidari R (2018) Brain mitochondria as potential therapeutic targets for managing hepatic encephalopathy. Life Sci. https://doi.org/10.1016/ j.lfs.2018.12.030 Hsieh HL, Sun CC, Wang TS, Yang CM (2008) PKC-delta/c-Src-medi- ated EGF receptor transactivation regulates thrombin-induced COX-2 expression and PGE(2) production in rat vascular smooth muscle cells. Biochim Biophys Acta 1783(9):1563–1575. https:// doi.org/10.1016/j.bbamcr.2008.03.016 Huang CY, Lin HJ, Chen HS, Cheng SY, Hsu HC, Tang CH (2013) Thrombin promotes matrix metalloproteinase-13 expression through the PKCdelta c-Src/EGFR/PI3K/Akt/AP-1 signaling path- way in human chondrocytes. Mediat Inflamm 2013:326041. https:// doi.org/10.1155/2013/326041 Jayakumar AR, Panickar KS, Murthy Ch R, Norenberg MD (2006) Oxidative stress and mitogen-activated protein kinase phosphoryla- tion mediate ammonia-induced cell swelling and glutamate uptake inhibition in cultured astrocytes. J Neurosci 26(18):4774–4784. https://doi.org/10.1523/jneurosci.0120-06.2006 Jayakumar AR, Valdes V, Norenberg MD (2011) The Na-K-cl cotransporter in the brain edema of acute liver failure. J Hepatol 54(2):272–278. https://doi.org/10.1016/j.jhep.2010.06.041 Jayakumar AR, Tong XY, Curtis KM, Ruiz-Cordero R, Abreu MT, Norenberg MD (2014) Increased toll-like receptor 4 in cerebral en- dothelial cells contributes to the astrocyte swelling and brain edema in acute hepatic encephalopathy. J Neurochem 128(6):890–903. https://doi.org/10.1111/jnc.12516 Jayakumar AR, Curtis KM, Panickar KS, Shamaladevi N, Norenberg MD (2016) Decreased STAT3 phosphorylation mediates cell swell- ing in ammonia-treated astrocyte cultures. Biology 5(4):48. https:// doi.org/10.3390/biology5040048 Jung O, Choi YJ, Kwak TK, Kang M, Lee MS, Ryu J, Kim HJ, Lee JW (2013) The COOH-terminus of TM4SF5 in hepatoma cell lines regulates c-Src to form invasive protrusions via EGFR Tyr845 phos- phorylation. Biochim Biophys Acta 1833(3):629–642. https://doi. org/10.1016/j.bbamcr.2012.11.026 Kala G, Kumarathasan R, Peng L, Leenen FH, Hertz L (2000) Stimulation of Na+,K+-ATPase activity, increase in potassium up- take, and enhanced production of ouabain-like compounds in ammonia-treated mouse Tyrphostin AG-1478 astrocytes. Neurochem Int 36(3):203–211
Kletzien RF, Pariza MW, Becker JE, Potter VR (1975) A method using 3- O-methyl-D-glucose and phloretin for the determination of intracel- lular water space of cells in monolayer culture. Anal Biochem 68(2): 537–544
Liang C, Du T, Zhou J, Verkhratsky A, Peng L (2014) Ammonium in- creases Ca(2+) signalling and up-regulates expression of TRPC1 gene in astrocytes in primary cultures and in the in vivo brain. Neurochem Res 39(11):2127–2135. https://doi.org/10.1007/ s11064-014-1406-z
Marti-Carvajal AJ, Gluud C, Arevalo-Rodriguez I, Marti-Amarista CE (2019) Acetyl-L-carnitine for patients with hepatic encephalopathy. Cochrane Database Syst Rev 1:CD011451. https://doi.org/10.1002/ 14651858.CD011451.pub2
Saxena A, Scaini G, Bavaresco DV, Leite C, Valvassoria SS, Carvalho AF, Quevedo J (2017) Role of protein kinase C in bipolar disorder: a review of the current literature. Mol Neuropsychiatry 3(2):108–124. https://doi.org/10.1159/000480349
Song D, Du T (2014) Ammonium activates ouabain-activated signalling pathway in astrocytes: therapeutic potential of ouabain antagonist. Curr Neuropharmacol 12(4):334–341. https://doi.org/10.2174/ 1570159×12666140828222115
Sturgeon JP, Shawcross DL (2014) Recent insights into the pathogenesis of hepatic encephalopathy and treatments. Expert Rev Gastroenterol Hepatol 8(1):83–100. https://doi.org/10.1586/17474124.2014.
Wang T, Wang Y, Yamashita H (2009) Evodiamine inhibits adipogenesis via the EGFR-PKCalpha-ERK signaling pathway. FEBS Lett 583(22):3655–3659. https://doi.org/10.1016/j.febslet.2009.10.046
Wang F, Du T, Liang C, Verkhratsky A, Peng L (2015) Ammonium increases Ca(2+) signalling and upregulates expression of Cav1.2 gene in astrocytes in primary cultures and in the in vivo brain. Acta Physiol (Oxf) 214(2):261–274. https://doi. org/10.1111/apha.12500
Wang X, Tan C, Wang G, Cai JJ, Wang LP, Imperato-McGinley J, Zhu YS (2017) Dual action of NSC606985 on cell growth and apoptosis mediated through PKCdelta in prostatic cancer cells. Int J Oncol 51(5):1601–1610. https://doi.org/10.3892/ijo.2017. 4138
Wang T, Liu C, Jia L (2018) The roles of PKCs in regulating autophagy. J Cancer Res Clin Oncol 144(12):2303–2311. https://doi.org/10. 1007/s00432-018-2731-4
Wang R, Zheng C, Jiang W, Xie X, Liao R, Zhou G (2019) Neuropeptide W regulates proliferation and differentiation of ATDC5: possible involvement of GPR7 activation, PKA and PKC-dependent signalling cascades. J Cell Mol Med. https:// doi.org/10.1111/jcmm.14118
Yang CM, Lee IT, Lin CC, Yang YL, Luo SF, Kou YR, Hsiao LD (2009) Cigarette smoke extract induces COX-2 expression via a PKCalpha/ c-Src/EGFR, PDGFR/PI3K/Akt/NF-kappaB pathway and p300 in tracheal smooth muscle cells. Am J Physiol Lung Cell Mol Physiol 297(5):L892–L902. https://doi.org/10.1152/ajplung.00151.2009
Yi ZY, Liang QX, Meng TG, Li J, Dong MZ, Hou Y et al (2019) PKCbeta1 regulates meiotic cell cycle in mouse oocyte. Cell Cycle:1–18. https://doi.org/10.1080/15384101.2018.1564492

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