Free Radical Research, March 2015; 49(3): 299–308 © 2015 Informa UK, Ltd. ISSN 1071-5762 print/ISSN 1029-2470 online DOI: 10.3109/10715762.2014.1002495

ORIGINAL ARTICLE

Transglutaminase 2 is involved in homocysteine-induced activation of human THP-1 monocytes M. Currò1§, C. Gangemi1,2§, A. Gugliandolo1,2, R. Risitano1, N. Ferlazzo3, R. Ientile1 & D. Caccamo1 of Biomedical Sciences and Morphofunctional Imaging, University of Messina, Messina, Italy, 2Department of Clinical and Experimental Medicine, University of Messina, Messina, Italy, and 3Department of Drug Sciences and Health Products, University of Messina, Messina, Italy

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Abstract Aberrant transglutaminase 2 (TG2) expression and protein cross-linking activity have been associated with several chronic neurodegenerative disorders in which inflammatory processes triggered by activated microglia and monocytes play a key role, such as Alzheimer’s disease, Parkinson’s disease, amyotrophic lateral sclerosis, and multiple sclerosis. Interestingly, mild-to-moderate hyperhomocysteinemia (HHcy), corresponding to increased plasma homocysteine (Hcy) concentrations in the range 16–60 μM, have recently been associated with the above-cited diseases. Using THP-1 monocytes, here we investigated the role of TG2 in cell response to mildly elevated Hcy concentrations. A five-day incubation with Hcy (∼25 μM) increased reactive oxygen species, peroxide lipids, as well as 8-hydroxyguanosine levels by twofold, and decreased the endogenous cell antioxidant defenses, that is reduced glutathione, by 50% in Hcy-exposed cultures compared with controls (p ⬍ 0.01). Hcy-induced oxidative stress was associated with increases in TG2 expression and activity, as well as nuclear factor kappa B activation. Notably, the latter was reduced in the presence of the TG-specific inhibitor R283. Hcy exposure also significantly increased the mRNA levels of tumor necrosis factor alpha, interleukin (IL)-6, and IL-1β, as well as the level of Hcy-inducible endoplasmic reticulum (ER) stress protein, a marker of ER stress, in Hcy-exposed cultures compared with controls. Notably, these effects were dramatically reduced by R283. These preliminary findings indicate that TG2 plays a key role in Hcy-induced activation of THP-1 monocytes, involving oxidative as well as ER stress and inflammation. This underlines the potential of TG2 inhibition in the therapeutic management of inflammatory processes contributing to neurodegenerative disorders associated with mild HHcy. Keywords: transglutaminase 2, homocysteine, oxidative stress, inflammation, ER stress

Introduction Transglutaminase 2 (TG2) is the most widely distributed member of TGs, a family of calcium-dependent enzymes that catalyze the post-translational modification of proteins by the formation of isopeptide bonds. This occurs through either protein cross-linking via ε-(γ-glutamyl) lysine bonds or primary amine incorporation (polyamination) at selected peptide-bound glutamine residues. The cross-linked products are highly resistant to proteolytic degradation, and play a major role in extracellular matrix stabilization as well as blood clotting and wound healing [1]. However, aberrant TG2 expression and TG crosslinking activity have been associated with a variety of chronic neurodegenerative diseases, such as Alzheimer’s disease (AD), Parkinson’s disease (PD), amyotrophic lateral sclerosis (ALS), and multiple sclerosis (MS), characterized by accumulation of protein aggregates containing isopeptide bonds and/or inflammatory processes in the brain, triggered by activated brain-resident microglial

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cells through the release of pro-inflammatory cytokines and neurotoxic factors [2,3]. Moreover, in some cases bone-marrow-derived cells or circulating monocytes may be repeatedly recruited from the periphery and infiltrate the brain, where they can be subsequently incorporated into the local cellular networks, and become an integral part of the pathology, thereby reinforcing inflammatory processes within the central nervous system [4]. Notably, different studies showed that TG2 upregulation is induced by the exposure to pro-inflammatory molecules in astroglial and microglial cells, accelerates neuroinflammation, and is required for monocyte maturation and pro-inflammatory activation [5–10]. There is good epidemiological evidence that inflammatory stress is associated with vascular impairment that accelerates the progression of cognitive decline and dementia. Interestingly, experimental evidence showed that high concentrations (0.1–1 mM) of homocysteine (Hcy), a sulfur-containing non-protein amino acid, may be a determinant of vascular impairment, since Hcy triggers

authors contributed equally to this work. Correspondence: Riccardo Ientile, Department of Biomedical Sciences and Morphofunctional Imaging, Polyclinic University of Messina, Via C. Valeria 98125 Messina, Italy. Tel: ⫹ 39 090 2213383. Fax: ⫹ 39 090 221 3382. E-mail: [email protected] (Received date: 10 June 2014; Accepted date: 21 December 2014; Published online: 3 February 2015)

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300 M. Currò et al. endothelial dysfunction through oxidative, inflammatory, as well as ER stress [11]. Moreover, epidemiological studies show that a state of mild-to-moderate hyperhomocysteinemia (HHcy), that is, Hcy plasma levels ranging between 16 and 60 μM, has been associated with vascular dementia as well as AD, PD, ALS, and MS [12,13], diseases in which TG2 plays a key role as discussed above. Interestingly, we demonstrated that the exposure to Hcy is able to induce TG2 upregulation associated with oxidative stress in Neuro2A cells [14,15]. It has recently been shown, in murine models, that mildly increased Hcy levels (20–30 μM) induce blood– brain barrier disruption, cerebral microvascular integrity alterations, monocyte transmigration into the brain as well as inflammatory transcriptional signaling and activation, and neuroinflammation leading to vascular dementia [16–21]. In the light of these observations, we first aimed to investigate whether TG2 is involved in monocyte response to mildly elevated Hcy concentrations (∼25 μM). Methods Materials The human pre-monocytic cell line, THP-1, was obtained from DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen-Braunschweig, Germany). Roswell Park Memorial Institute (RPMI)-1640, L-glutamine, 4-(2hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), sodium pyruvate, glucose, D,L-Hcy, protease inhibitor cocktail, dimethylsulfoxide (DMSO), phosphate-buffered saline (PBS), 2′,7′-dichlorofluoresceindiacetate (DCF-DA), mouse monoclonal antibodies against TG2 and β-actin, and other chemicals of analytical grade were from Sigma (Milan, Italy). EZ-Link 5-(biotinamido) pentylamine (BAPA) was from Thermo Scientific Pierce (EuroClone, Milan Italy). The synthetic fluorescent probes diphenyl-1pyrenylphosphine (DPPP) as well as t-butoxycarbonylLeu-Met-7-amino-4-chloromethylcoumarin (CMAC-Invitrogen Molecular Probes), and fluorescein isothiocyanate (FITC)-labeled avidin-fluorochrome were a generous gift from Prof. Angela Di Pietro of University of Messina (Italy). Rabbit polyclonal antibody against the Hcy-inducible endoplasmic reticulum (ER) stress protein (Herp) was from SantaCruz Biotechnology (DBA-Milan, Italy). Horseradish peroxidase (HRP)-conjugated secondary antibodies against mouse and rabbit immunoglobulin G, and ECL Chemiluminescence Detection Kit were from GE Healthcare Life Sciences (Milan, Italy). Developer, fixer, and Kodak X-ray film were from Kodak (Milan, Italy). Fetal bovine serum (FBS); TRIzol for RNA extraction; high-capacity cDNA archive kit; TaqMan Gene Expression Master Mix; TaqMan Gene Expression Assays (Assays-on-Demand) for human β-actin (ID: Hs99999903_ m1), TG2 (ID: Hs00190278_m1), interleukin (IL)-6 (ID: Hs00985639_m1), IL-1β (ID: Hs01555410_m1), and

tumor necrosis factor alpha (TNF-α) (ID: Hs00174128_ m1); and SYBR Green polymerase chain reaction (PCR) Master Mix were from Life Technologies (Milan, Italy). Oligonucleotides used as primers for real-time PCR with SYBR Green were from Eurofins (Carlo Erba, Milan, Italy). R283 (1,3-dimethyl-2-[(2-oxopropyl)thio]imidazolium chloride) was kindly provided by Prof. Martin Griffin of the School of Life & Health Sciences at Aston University in Birmingham (UK). Cell culturing and treatment THP-1 monocytes were maintained in RPMI-1640 supplemented with L-glutamine (2 mM), HEPES (10 mM), sodium pyruvate (1 mM), glucose (2.5 g/L), and 10% FBS, at 37°C in a 5% CO2/95% air humidified atmosphere. Medium was renewed every 2 days, and split was performed when cells reached maximum density (1 ⫻ 106 cells/ml). After a suitable number of passages, THP-1 monocytes were exposed to 50 μM D,L-Hcy for 2–5 days. Given that only the L-enantiomer was reported to be active, the effective Hcy concentration may have been half of that used, that is, ∼25 μM, a concentration similar to mean plasma Hcy levels observed in subjects with neurodegenerative diseases. To avoid cell stress by serum deprivation, RPMI medium was supplemented with 5% FBS. All cell experiments were independently replicated three times. Cell viability To assess the effects of incubation with Hcy on cell viability, a 3-(4,5-methylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) reduction assay was performed. At the end of incubation with/without Hcy, THP-1 cells, grown in 96-well culture plates, were incubated with fresh medium containing MTT (0.5 mg/ml) at 37°C for 4 h. Then, insoluble formazan crystals were dissolved in 100 μl of a 10% (w/v) sodium dodecyl sulfate (SDS) solution in HCl (0.01 M). The optical density in each well was evaluated by spectrophotometrical measurement at 540 nm with a Sunrise microplate reader (Tecan Italia, Cologno Monzese, Italy). The percent cell viability was assessed by the absorbance ratio of treated versus untreated cells. All experiments were made in triplicate. Evaluation of reactive oxygen species levels The intracellular levels of reactive oxygen species (ROS) were evaluated using the membrane-permeable lipophilic fluorochrome DCF-DA (1 μM) as described by Di Pietro et al. [22]. At the end of each treatment, the cells were incubated with 5 μM H2DCF-DA (dissolved in DMSO) for 30 min at 37°C. Then, the cells were washed twice with PBS, and fluorescence data were acquired using a Dako Galaxy Flow Cytometer (DakoCytomation, Glostrup, Denmark) in the green fluorescence channel (530 ⫾ 20 nm).

TG2 involvement in Hcy-induced monocyte activation

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Analysis of TG2 expression After RNA isolation with TRIzol reagent from suspended cells, RNA (1 μg) was reverse transcribed with HighCapacity cDNA Archive Kit according to the manufacturer’s instructions. Then, mRNA levels of TG2, as marker of monocyte functional activation [7,9], were quantified by real-time PCR, using TaqMan-based Gene Expression Assay according to the manufacturer’s instructions. β-Actin was used as a reference gene. Quantitative real-time PCR reactions were set up in a 96-well plate and were carried out in 10 μl reactions containing 1X TaqMan Gene Expression Master Mix, 1X TaqMan-specific assay, and 25 ng of RNA converted into cDNA. Real-time PCR was performed in a 7900HT Fast Real-Time PCR System with the following profile: one cycle at 95°C for 10 min, followed by 40 cycles at 95°C for 15 s and 60°C for 1 min. Data were collected and analyzed using SDS 2.3 and RQ manager 1.2 software (Applied Biosystems, Foster City, CA) using the 2⫺ΔΔCT relative quantification method. Values were expressed as n-fold change relative to control cells. For TG2 Western blot analysis, after incubation with/ without Hcy, THP-1 monocytes were homogenized with hypotonic and hypertonic buffer on ice to obtain cytosolic and nuclear proteins. Protein concentration was determined using the Bradford method and then cytosolic proteins (25–30 μg) were separated by SDS-polyacrylamide gel electrophoresis (PAGE) onto 10% gel, and transferred to nitrocellulose membranes. Blots were blocked for 1 h at room temperature, on a rotating device, with 5% non-fat dry milk in Tris-buffered saline, supplemented with Tween-20 (TBS-T). Then, membranes were incubated overnight at 4°C, on a rotating device, with mouse monoclonal antibodies against either TG2 or β-actin (diluted 1:1000–1:15000 in 5% non-fat dry milk in TBS-T, respectively). After extensive washing by TBS-T (4 times), blotted membranes were incubated for 2 h with HRP-conjugated anti-mouse (1:2000 and 1:25000 in 5% non-fat dry milk in TBS-T) secondary antibodies. Blots were developed by ECL chemiluminescence detection system kit using Kodak film. Bands were scanned and quantified by densitometric analysis with an AlphaImager 1200 System (Alpha Innotech, San Leandro, CA, USA), after normalization against β-actin. Flow cytometric analysis of oxidative stress markers The assessment of oxidative stress markers in Hcyexposed as well as control THP-1 monocytes was performed by flow cytometric analysis. Reduced glutathione (GSH) levels were measured by means of the fluorescent dye CMAC. This probe is highly specific since the reaction with intracellular thiols other than GSH, particularly protein sulfhydryls, is negligible and the background labeling of proteins is nearly absent. Specificity is warranted by the presence of a chloromethyl group that makes the catalytic glutathione S-transferase

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activity essential to form a fluorescent adduct with GSH. After incubation, cell suspensions of controls and Hcy-exposed cultures, prepared as reported above, were incubated with 5 μM CMAC for 30 min at 37°C. Then, cell fluorescence was analyzed using a Dako Galaxy Flow Cytometer at 351 nm Ex and 380 nm Em. Lipid peroxidation was assessed using DPPP, having a high reactivity with lipid hydroperoxides in the membranes of live cells, as previously described by Di Pietro et al. [22]. The DPPP is a non-fluorescent probe, reacting stoichiometrically with hydroperoxide to yield a fluorescent phosphine oxide and its corresponding hydroxide. After incubation of DPPP-loaded cells at 37°C for 3 h, the fluorescent phosphine oxide signals were collected in the green channel at 495 nm Ex and 530 nm Em. DNA oxidation was assessed by measurement of 8-hydroxyguanosine levels. Briefly, 8-hydroxyguanosine was detected by binding of the FITC-labeled avidin fluorochrome. The probe binds to 8-hydroxyguanosine with a high specificity due to the structural analogies between the keto form of the oxidized base and biotin. The method, previously adapted to flow cytometric analysis, was performed in THP-1 monocytes permeabilized in methanol (15 min at ⫺ 20°C) and loaded with the avidin–FITC conjugate (1 h at 37°C). The emission signals were collected in the green fluorescence channel. TG2 inhibition experimental setting In order to elucidate TG2 role in cell response to Hcy, cell cultures were incubated for 2–5 days with Hcy in the presence or absence of R283 (250 μM), a synthetic small peptide capable of permeating the cell membrane and acting as a competitive inhibitor of TG2 active site at intracellular level; additionally, R283 causes a TG2 conformational change thus altering the enzyme’s ability to interact with other cell proteins [23]. The chosen R283 concentration was previously shown as the minimal concentration useful for inhibiting TG activity without cell viability alterations [9]. All experiments were independently replicated three times. Assessment of NF-κB activation and cytokine upregulation The presence of DNA-binding activity of nuclear factor kappa B (NF-κB) in nuclear extracts of THP-1 monocytes incubated with Hcy was evaluated by electrophoretic mobility shift assay (EMSA) as described by Caccamo et al. [24]. After RNA isolation with TRIzol reagent from suspended cells, RNA (1 μg) was reverse transcribed with High-Capacity cDNA Archive Kit according to the manufacturer’s instructions. Then, mRNA levels of IL-6, TNF-α, and IL-1β were quantified by real-time PCR using TaqMan Gene Expression Assays according to the manufacturer’s instructions. β-Actin was used as an endogenous control. Quantitative real-time PCR reactions were carried out as described above for TG2.

302 M. Currò et al. Assessment of ER stress ER stress was assessed by evaluation of protein amount of Herp. Briefly, protein isolation and quantification, and Western blot analysis were carried out as described above, except for the use of rabbit polyclonal primary antibody against Herp, diluted 1:5000 in 5% non-fat dry milk in TBS-T, and anti-rabbit secondary antibody, diluted 1:10000 in 5% non-fat dry milk in TBS-T.

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In situ TG activity assay Given that TG2 enzyme activation is calcium dependent, we aimed to evaluate changes of in situ TG activity as a marker of Hcy-induced ER stress. For this purpose, we measured the levels of BAPA incorporation into cell proteins by spectrophotometrical methods reported by Zhang et al. [25], with some modifications. Briefly, 4 h prior to the end of incubation period, 1 mM BAPA was added to the culture medium. Then, cells were homogenized, sonicated on ice, and protein concentration was determined using the Bradford method. Ten micrograms of protein was diluted with coating buffer, loaded into each well of a 96-well microtiter plate and incubated overnight at 4°C. After blocking non-specific binding sites, 100 μl of HRPconjugated streptavidin (1:1000) in 1% bovine serum albumin (BSA) and 0.01% Tween-20 in borate saline buffer was added to each well and incubated at room temperature for 1 h. After washing, 200 μl of substrate solution (0.4 mg of o-phenylenediamine dihydrochloride/ml of 0.05 M sodium citrate phosphate buffer, pH: 5.0) was added to each well. The reactions were stopped using 3 N HCl, and the presence of biotinylated proteins was quantified by measuring the absorbance at 492 nm on a microplate spectrophotometer (Tecan). All measurements were done in triplicate and repeated at least three times. Analysis of LC3 expression The mRNA levels of LC3 were quantified by real-time PCR using SYBR Green-based gene expression analysis according to the manufacturer’s instructions. β-Actin was used as an endogenous control. The following primer sequences were used: LC3 sense primer 5′-cggtgataataga acgatacaag-3′ and anti-sense primer 5′-ctgagattggtgtgg agac-3′; β-actin sense primer 5′-ttgttacaggaagtcccttgcc-3′ and anti-sense primer 5′-atgctatcacctcccctgtgtg-3′. Quantitative real-time PCR reactions were set up in duplicate in a 96-well plate and were carried out in 20 μl reactions containing 1x SYBR Green PCR Master Mix, 0.1 μM specific primers, and 25 ng of RNA converted into cDNA. Reverse transcription PCR was performed in a 7900HT Fast Real-Time PCR System with the following profile: one cycle at 95°C for 10 min, followed by 40 cycles at 95°C for 15 s and 60°C for 1 min. A standard dissociation stage was added to assess primer specificity. For LC3 Western blot analysis, THP-1 cells were lysed in 65 mM Tris, pH: 6.8, 4% SDS, and 1.5% β-mercaptoethanol, completed with protease inhibitor cocktails and

held at 100°C for 5 min. Proteins were separated by SDSPAGE onto 15% gel, and transferred to a polyvinylidene difluoride membrane. Blots were blocked for 45 min at room temperature, on a rotating device, with 5% non-fat dry milk in PBS, supplemented with Tween-20 (PBS-T). Then, membranes were incubated overnight at 4°C, on a rotating device, with mouse monoclonal antibody against β-actin (1:1000 in 5% BSA in PBS-T) and rabbit monoclonal antibody against LC3 (1:1000 in 5% BSA in PBS-T). After three washes with PBS-T, blotted membranes were incubated for 1 h with HRP-conjugated anti-mouse (1:2000 in 5% non-fat dry milk in PBS-T) or anti-rabbit (1:2000 in 5% non-fat dry milk in PBS-T) secondary antibodies. Blots were developed by ECL chemiluminescence detection system kit using Kodak film. Bands were scanned and quantified by densitometric analysis with an AlphaImager 1200 System (Alpha Innotech, San Leandro, CA, USA), after normalization against β-actin. Statistical analysis All measurements were done in triplicate and repeated at least three times. Data are presented as means ⫾ standard error of the mean (SEM). Data were tested for normal distribution by Kolmogorov–Smirnov test and then appropriate statistical analysis was performed. Results were analyzed by one-way analysis of variance (ANOVA), followed by Newman–Keuls post hoc test, or by Student’s t-test (for comparisons between two groups). P values ⬍ 0.05 were considered significant. Results Our experimental approach involved the use, in all experimental settings, of 50 μM D,L-Hcy, corresponding to an active concentration of ∼25 μM that matches the mean plasma Hcy concentrations found in patients with chronic neuroinflammatory disorders. To assess the time-dependent effects of Hcy, THP-1 cells were incubated for 2 and 5 days. Then, Hcy’s ability to produce alterations at cellular and/or molecular level was evaluated by analysis of cell viability, oxidative stress, and TG2 upregulation, the latter is used as a marker of monocyte functional activation [7,9]. Under the chosen incubation conditions, THP-1 viable cell number was not significantly reduced in Hcy-exposed cultures compared with control (Figure 1A). On the contrary, after five, but not two, days of incubation, Hcy was able to induce a twofold increase in ROS concentration and TG2 mRNA levels, respectively, compared with control (p ⬍ 0.01) (Figure 1B, C). On the basis of these observations, a period of five days of incubation was chosen for subsequent experiments as standard incubation time of THP-1 monocytes with Hcy. Flow cytometric analyses showed that after five days of incubation, Hcy triggered significant redox status alterations, involving, other than ROS increases (Figure 1B), a twofold increase in lipid peroxide and 8-hydroxyguanosine

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Figure 1. Dose-dependent effects of Hcy on THP-1 cells, evaluated as changes in cell viability (% MTT reduction) (A), increase in oxidative stress (B), and upregulation of TG2 mRNA levels (C). **p ⬍ 0.01, ***p ⬍ 0.001, significant differences in comparison with control cells; §§p ⬍ 0.01, §§§p ⬍ 0.001, significant differences in comparison with Hcy-exposed cells, evaluated by one-way ANOVA, followed by Newman– Keuls post hoc test.

levels, and a decrease in GSH levels by around 50% in Hcy-exposed cultures compared with controls (p ⬍ 0.01) (Figure 2). However, as reported above, these toxic effects were not associated with a loss of cell viability. We previously observed that 250 μM Hcy induced TG2 upregulation in Neuro2A neuroblastoma cells [15]. Thus, we evaluated whether a mildly elevated Hcy concentration (∼25 μM) could affect TG2 expression in THP-1 monocytes. Real-time PCR and Western blot analysis showed that Hcy exposure for five days induced a slight increase in TG2 expression (Figures 1C and 3A). The assessment of NF-κB DNA binding activity by EMSA showed a significant increase in NF-κB activation in THP-1 monocytes exposed to Hcy for five days; notably, this effect was strongly reduced in the presence of the TG2 inhibitor R283 (Figure 4A), in agreement with the recently accepted role for TG2 in NF-κB activation [26]. Given the well-known involvement of activated NF-κB in the upregulation of pro-inflammatory cytokines, we also evaluated whether the incubation with Hcy could induce changes in the expression of TNF-α, IL-6, and IL-1β. The exposure of THP-1 monocytes to Hcy for five days increased the mRNA levels of TNF-α by around twentyfold (p ⬍ 0.001), those of IL-6 by sixfold (p ⬍ 0.001), and those of IL-1β by twofold (p ⬍ 0.01) in comparison with control cells. Notably, Hcy-mediated cytokine upregulation was significantly reduced in the presence of the TG2 inhibitor R283 (Figure 4B). This suggests the involvement

of TG2 in cytokine upregulation, most likely through NF-κB activation. Given the reported toxic effects of a short exposure to high Hcy concentrations (100–500 μM) on ER in different cell types [11], we wondered whether even a prolonged cell exposure to ∼25 μM Hcy could trigger ER stress in THP-1 monocytes. Thus, we assessed the protein amount of Herp, a marker of ER-stress and homeostatic regulator of ER-resident calcium release channel proteins. We observed a fourfold increase in Herp levels in THP-1 monocytes incubated with Hcy for five days compared with that in controls (p ⬍ 0.001); notably, Hcy-induced Herp increase was suppressed in the presence of R283 (Figure 5). It is well known that ER stress is associated with calcium release from intracellular stores. Given that TG2 enzyme activation is calcium dependent, we aimed to evaluate changes of in situ TG activity as an indirect proof of intracellular calcium level elevation. Indeed, TGmediated BAPA incorporation into cell proteins was increased by twofold in THP-1 monocytes incubated for five days with ∼25 μM Hcy compared with controls (Figure 3B), suggesting that mildly elevated Hcy concentrations are also able to induce calcium release from ER stores. Given the reported linkage between autophagy and ER stress [27], we evaluated the modifications of LC3, a key marker of autophagosome assembly, in Hcy-exposed cells. After a five-day incubation with Hcy, LC3 mRNA levels

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Figure 2. Flow cytometric analysis of oxidative stress-related markers in THP-1 cells incubated with/without Hcy for five days. A) Measurement of GSH by means of the fluorescent dye CMAC. B) Assessment of lipid peroxidation by the fluorophore DPPP. C) Evaluation of DNA oxidation by measurement of 8-hydroxyguanosine levels. All measurements were done in triplicate and repeated at least three times. **p ⬍ 0.01, ***p ⬍ 0.001, significant differences in comparison with control cells (Ctr) evaluated by Student’s t-test.

were increased by 50% (p ⬍ 0.05); moreover, in the presence of R283 LC3 mRNA levels were more significantly increased in comparison with cells exposed to Hcy alone (p ⬍ 0.01); interestingly, R283 alone also significantly increased LC3 mRNA levels in comparison with control cells (p ⬍ 0.001) (Figure 6A). Densitometric analysis of immunoblots of LC3-II form, being directly linked to

autophagosome maturation, also showed that Hcy induced a protein increase of around 50% (Figure 6B). Discussion Epidemiological studies correlate mild-to-moderate HHcy (16–60 μM) with a range of chronic neurodegenerative

Figure 3. Analysis of TG2 expression and enzyme activity in THP-1 cells incubated with/without Hcy for five days. A) Immunoblots and densitometric analysis of TG2 protein amounts. B) In situ TG activity evaluated by spectrophotometric measurement of BAPA incorporated into cell proteins. Data are means ⫾ SEM from three independent experiments performed in triplicate. *p ⬍ 0.05, ***p ⬍ 0.001, significant differences in comparison with control cells (Ctr); §§§p ⬍ 0.001, significant differences in comparison with Hcy-exposed cells (Student’s t-test or one-way ANOVA, followed by Newman–Keuls post hoc test).

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Figure 4. Evaluation of NF-κB activation and cytokine induction in THP-1 cells incubated with/without Hcy for five days, in the presence or absence of R283. A) Assessment of NF-κB DNA binding activity by EMSA. B) Relative quantification by real-time PCR of TNF-α, IL-6, and IL-1β levels. Data are the mean ⫾ SEM of three independent experiments performed in triplicate. **p ⬍ 0.01, ***p ⬍ 0.001, significant differences in comparison with control cells (Ctr); §§p ⬍ 0.01, §§§p ⬍ 0.001, significant differences in comparison with cells exposed to Hcy alone (one-way ANOVA, followed by Newman–Keuls post hoc test).

conditions, including vascular dementia, AD, PD, ALS, and MS [12,13]. Under these conditions, inflammatory processes, that is, activation of microglial cells and the recruitment/acti-

vation of circulating monocytes into the brain occur. Moreover, inflammatory conditions are reflected by cellular changes in peripheral blood monocytes/macrophages, enabling their use for diagnosis, finding therapeutic targets, and prognosis of the disease [3]. To our knowledge, there are no previous investigations on the effects of a prolonged exposure to mildly elevated Hcy concentrations (∼25 μM) on monocytes, similar to those found in patients with chronic neurodegenerative diseases. In the present study, we show that a five-day incubation of human THP-1 monocytes with ∼25 μM Hcy produced several alterations at cellular and molecular level, without affecting cell viability. Mildly elevated Hcy concentrations induce oxidative stress in THP-1 monocytes

Figure 5. Immunoblot and densitometric analysis of Herp protein amounts in THP-1 cells incubated with/without Hcy for 5 days in the presence or absence of R283. Data are expressed as mean ⫾ SEM of three independent experiments performed in triplicate. ***p ⬍ 0.001, significant differences in comparison with control cells (Ctr); §§§p ⬍ 0.001, significant differences in comparison with Hcy-exposed cells (one-way ANOVA, followed by Newman–Keuls post hoc test).

The exposure of THP-1 cells to Hcy also resulted in a significant increase in ROS levels, lipid peroxidation, as well as DNA oxidation; and a strong decrease of GSH, participating directly in the neutralization of free radicals. Moreover, Hcy increased TG2 expression and in situ TG activity. This first suggests a possible causal link between the mild HHcy associated with chronic neuroinflammatory disorders and the pathognomic sign of these diseases, that is, oxidative stress and the accumulation of protein aggregates, within which TG-catalyzed isopeptide bonds have been found. Mildly elevated Hcy concentrations induce a TG-mediated pro-inflammatory activation in THP-1 monocytes Given the known involvement of activated NF-κB in the induction of pro-inflammatory cytokines, we evaluated

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Figure 6. Expression analysis of autophagy-related LC3 protein marker in THP-1 monocytes incubated with/without Hcy for 5 days in the presence or absence of R283. A) Relative quantification by real-time PCR of LC3 mRNA levels. B) Immunoblots and densitometric analysis of LC3-II cleaved isoform protein amounts. Data are expressed as mean ⫾ SEM of three independent experiments performed in triplicate. *p ⬍ 0.05, ***p ⬍ 0.001 significant differences in comparison with control cells (Ctr); §§p ⬍ 0.01, significant differences in comparison with Hcy-exposed cells; #p ⬍ 0.05 in comparison with Ctr ⫹ R283 (one-way ANOVA, followed by Newman–Keuls post hoc test).

whether a prolonged incubation with ∼25 μM Hcy could also induce NF-κB activation and cytokine upregulation in THP-1 monocytes. Concomitant with the increase in TG2 expression and activity, we observed that Hcy triggered NF-κB activation and upregulation of pro-inflammatory genes, such as TNF-α, IL-6, and IL-1β, and that these effects were significantly reduced in the presence of the specific TG2 inhibitor, R283. Our findings suggest that Hcy-induced NF-κB activation takes place through an alternative pathway involving TG2 as mediator, as demonstrated previously [6,26]. Moreover, the pro-inflammatory action of Hcy involved TG2, since inhibition of TG activity by R283 reduced cytokine mRNA levels, most likely through the reduction of activated NF-κB nuclear levels. These results are in line with those of Su and coworkers [28], who reported that the incubation with Hcy enhanced monocyte activation and dose-dependently induced cytokine upregulation. ER stress induced by mildly elevated Hcy concentrations in THP-1 monocytes involves TG activity Recent evidence indicate that the production of inflammatory cytokines and ROS can trigger ER stress; moreover, other than through ROS generation, inflammatory pathways and ER stress are integrated also via the intracellular calcium and NF-κB activation [29–31]. These observations suggest that ER stress is a key factor in the inflammatory response, and a potential mediator of inflammation. Here we show that the exposure for five days to ∼25 μM Hcy was able to trigger the ER stress response in THP-1 monocytes, as demonstrated by the upregulation of Herp protein, a Hcy inducible marker of ER stress and homeostatic regulator of ER-resident calcium release channel proteins [11,32], and the increase in calcium-dependent TG-mediated BAPA incorporation compared with control cells. Interestingly, the most recent observations showed that TG2 specifically co-localizes with typical ER-resident chaperones (protein disulfide isomerase, ERp57, and calreticulin) suggesting a direct link to the ER [33]; moreover, ER stress activated TG2 in various cell types, dependent on ER stress-induced increase in intracellular

calcium concentrations, but not on TG2 protein levels [34]. Together, our findings indicate that even cell exposure to mildly elevated Hcy concentrations is able to trigger ER stress leading to calcium release from intracellular stores. Notably, TG activity inhibition by R283 also reduced Herp, suggesting a previously not reported cross talk between these two proteins. Mildly elevated Hcy concentrations induce autophagy activation in THP-1 monocytes ER stress, associated with various pathological conditions, including oxidative stress and inflammation, is known to stimulate the assembly of the pre-autophagosomal structure as a reaction promoting cellular survival [27,35]. Indeed, in our experimental conditions Hcyinduced ER stress was concomitant with the increase in LC3-II level, the active form of LC3 protein that is commonly used as a marker of autophagosome, because it is the essential part of the vesicle and stays associated until the last moment before its fusion with the membrane. This suggests that autophagy induction is likely a pro-survival response of THP-1 monocytes upon toxic stimulation with Hcy, as suggested by the lack of Hcy effects on cell viability despite Hcy-evoked oxidative, inflammatory, and ER stress. Notably, LC3 mRNA levels, but not protein levels, were significantly increased, rather than diminished, in the presence of the TG inhibitor R283 both in Hcy-exposed and control THP-1 cells. This suggests that LC3 upregulation mediated by R283 is a not specific effect, and that TG activity is not involved in Hcy-induced autophagy. The increase in LC3 mRNA levels could be explained taking into account that TG2 conformational changes induced by R283, specifically targeting the TG2 active site [23], likely disrupt the interaction between TG2 and other cytosolic proteins, that is, transcription factors, involved in LC3 upregulation. The results obtained here by targeting TG2 active site with R283 confirm previous experimental observations, suggesting that TG2-mediated regulation of autophagy is most likely related to the physical presence of TG2, acting as protein scaffold within the cells [36,37]. Interestingly, it has been reported that Herp acts as a

TG2 involvement in Hcy-induced monocyte activation

modulator of autophagic process. Indeed, under basal conditions and increasingly after stress, higher LC3-II levels were observed when knocking down Herp cells compared with control cells [38]. In agreement with these observations, we found that the reduction of Herp protein levels was concomitant with increase in LC3 mRNA level.

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Conclusions The present preliminary findings indicate that TG2 plays a key role in THP-1 cell response to Hcy toxic stimulus, involving oxidative as well as ER stress, and inflammation. These observations underline the potential of TG2 inhibition in the therapeutic management of HHcy-related neurodegenerative disorders involving microglia and monocyte activation. Acknowledgements We thank Prof. Martin Griffin of the School of Life & Health Sciences at Aston University in Birmingham (UK) for generously providing us R283. We thank Prof A. Di Pietro and Dr G. Visalli from University of Messina for generously providing us fluorescent probes and helping with flow cytometric analysis of oxidative stress markers. We also thank Prof M.T. Sciortino and Dr G. Siracusano from University of Messina for generously providing us mouse monoclonal antibody against LC3 and helping with Western blot analysis. Declaration of interest The authors declare that there is no conflict of interest. The authors alone are responsible for the content and writing of the paper. References [1] Griffin M, Casadio R, Bergamini CM. Transglutaminases: nature’s biological glues. Biochem J 2002;368:377–396. [2] Hoffner G, André W, Vanhoutteghem A, Souès S, Djian P. Transglutaminase-catalyzed crosslinking in neurological diseases: from experimental evidence to therapeutic inhibition. CNS Neurol Disord Drug Targets 2010;9:217–231. [3] Wu W, Shao J, Lu H, Xu J, Zhu A, Fang W, Hui G. Guard of delinquency? A role of microglia in inflammatory neurodegenerative diseases of the CNS. Cell Biochem Biophys 2014;70:1–8. [4] Prinz M, Tay TL, Wolf Y, Jung S. Microglia: unique and common features with other tissue macrophages. Acta Neuropathol 2014;128:319–331. [5] Monsonego A, Shani Y, Friedmann I, Paas Y, Eizenberg O, Schwartz M. Expression of GTP-dependent and GTPindependent tissue-type transglutaminase in cytokine-treated rat brain astrocytes. J Biol Chem 1997;272:3724–3732. [6] Lee J, Kim YS, Choi DH, Bang MS, Han TR, Joh TH, Kim SY. Transglutaminase 2 induces nuclear factor-kappaB activation via a novel pathway in BV-2 microglia. J Biol Chem 2004;279:53725–53735.

307

[7] Currò M, Ferlazzo N, Condello S, Caccamo D, Ientile R. Transglutaminase 2 silencing reduced the beta-amyloid-effects on the activation of human THP-1 cells. Amino Acids 2010; 39:1427–1433. [8] van Strien ME, Brevé JJ, Fratantoni S, Schreurs MW, Bol JG, Jongenelen CA, et al. Astrocyte-derived tissue transglutaminase interacts with fibronectin: a role in astrocyte adhesion and migration? PLoS One 2011;6:e25037. [9] Currò M, Ferlazzo N, Risitano R, Condello S, Vecchio M, Caccamo D, Ientile R. Transglutaminase 2 and phospholipase A interactions in the inflammatory response in human THP-1 monocytes. Amino Acids 2014 46:759–766. [10] Oono M, Okado-Matsumoto A, Shodai A, Ido A, Ohta Y, Abe K, et al. Transglutaminase 2 accelerates neuroinflammation in amyotrophic lateral sclerosis through interaction with misfolded superoxide dismutase 1. J Neurochem 2014;128: 403–418. [11] Boldyrev AA. Molecular mechanisms of homocysteine toxicity. Biochemistry (Mosc) 2009;74:589–598. [12] Selhub J. Public health significance of elevated homocysteine. Food Nutr Bull 2008;29:S116–S125. [13] Wald DS, Kasturiratne A, Simmonds M. Serum homocysteine and dementia:meta-analysis of eight cohort studies including 8669 participants. Alzheimers Dement 2011;7:412–417. [14] Ferlazzo N, Condello S, Currò M, Parisi G, Ientile R, Caccamo D. NF-kappaB activation is associated with homocysteine-induced injury in Neuro2a cells. BMC Neurosci 2008;9:62. [15] Currò M, Condello S, Caccamo D, Ferlazzo N, Parisi G, Ientile R. Homocysteine-induced toxicity increases TG2 expression in Neuro2a cells. Amino Acids 2009;36: 725–730. [16] Zhang D, Jiang X, Fang P, Yan Y, Song J, Gupta S, et al. Hyperhomocysteinemia promotes inflammatory monocyte generation and accelerates atherosclerosis in transgenic cystathionine beta-synthase-deficient mice. Circulation 2009; 120:1893–1902. [17] Hohsfield LA, Humpel C. Homocysteine enhances transmigration of rat monocytes through a brain capillary endothelial cell monolayer via ICAM-1. Curr Neurovasc Res 2010;7: 192–200. [18] Tyagi N, Givvimani S, Qipshidze N, Kundu S, Kapoor S, Vacek JC, Tyagi SC. Hydrogen sulfide mitigates matrix metalloproteinase-9 activity and neurovascular permeability in hyperhomocysteinemic mice. Neurochem Int 2010; 56: 301–307. [19] Beard RS Jr, Reynolds JJ, Bearden SE. Hyperhomocysteinemia increases permeability of the blood-brain barrier by NMDA receptor-dependent regulation of adherens and tight junctions. Blood 2011;118:2007–2014. [20] Meng S, Ciment S, Jan M, Tran T, Pham H, Cueto R, et al. Homocysteine induces inflammatory transcriptional signaling in monocytes. Front Biosci 2013;18:685–695. [21] Sudduth TL, Powell DK, Smith CD, Greenstein A, Wilcock DM. Induction of hyperhomocysteinemia models vascular dementia by induction of cerebral microhemorrhages and neuroinflammation. J Cereb Blood Flow Metab 2013;33:708–715. [22] Di Pietro A, Visalli G, Munaò F, Baluce B, La Maestra S, Primerano P, et al. Oxidative damage in human epithelial alveolar cells exposed in vitro to oil fly ash transition metals. Int J Hyg Environ Health 2009;212:196–208. [23] Bergamini CM, Collighan RJ, Wang Z, Griffin M. Structure and regulation of type 2 transglutaminase in relation to its physiological functions and pathological roles. Adv Enzymol Relat Areas Mol Biol 2011;78:1–46. [24] Caccamo D, Campisi A, Currò M, Aguennouz M, Li Volti G, Avola R, Ientile R. Nuclear factor-kappab activation is associated with glutamate-evoked tissue transglutaminase up-regulation in primary astrocyte cultures. J Neurosci Res 2005; 82:858–865.

Free Radic Res Downloaded from informahealthcare.com by University of Connecticut on 03/31/15 For personal use only.

308 M. Currò et al. [25] Zhang J, Lesort M, Guttmann RP, Johnson GV. Modulation of the in situ activity of tissue transglutaminase by calcium and GTP. J Biol Chem 1998;273:2288–2295. [26] Park KS, Kim DS, Ko C, Lee SJ, Oh SH, Kim SY. TNF-alpha mediated NF-kappaB activation is constantly extended by transglutaminase 2. Front Biosci (Elite Ed) 2011;3:341–354. [27] Yorimitsu T, Nair U, Yang Z, Klionsky DJ. Endoplasmic reticulum stress triggers autophagy. J Biol Chem 2006; 281:30299–30304. [28] Su SJ, Huang LW, Pai LS, Liu HW, Chang KL. Homocysteine at pathophysiologic concentrations activates human monocyte and induces cytokine expression and inhibits macrophage migration inhibitory factor expression. Nutrition 2005;21: 994–1002. [29] Zhang K, Kaufman RJ. From endoplasmic-reticulum stress to the inflammatory response. Nature 2008;454:455–462. [30] Garg AD, Kaczmarek A, Krysko O, Vandenabeele P, Krysko DV, Agostinis P. ER stress-induced inflammation: does it aid or impede disease progression? Trends Mol Med 2012;18:589–598. [31] Nakajima S, Kitamura M. Bidirectional regulation of NF-κB by reactive oxygen species: a role for unfolded protein response. Free Rad Biol Med 2013;65:162–174

[32] Kokame K, Agarwala KL, Kato H, Miyata T. Herp, a new ubiquitin-like membrane protein induced by endoplasmic reticulum stress. J Biol Chem 2000;275:32846–32853. [33] Wilhelmus MM, Verhaar R, Andringa G, Bol JG, Cras P, Shan L, et al. Presence of tissue transglutaminase in granular endoplasmic reticulum is characteristic of melanized neurons in Parkinson’s disease brain. Brain Pathol 2011;21:130–139. [34] Lee JH, Jeong J, Jeong EM, Cho SY, Kang JW, Lim J, et al. Endoplasmic reticulum stress activates transglutaminase 2 leading to protein aggregation. Int J Mol Med 2014;33:849–855. [35] Arroyo DS, Gaviglio EA, Peralta Ramos JM, Bussi C, Rodriguez-Galan MC, Iribarren P. Autophagy in inflammation, infection, neurodegeneration and cancer. Int Immunopharmacol 2014;18:55–65. [36] D’Eletto M, Farrace MG, Falasca L, Reali V, Oliverio S, Melino G, et al. Transglutaminase 2 is involved in autophagosome maturation. Autophagy 2009;5:1145–1154. [37] Gundemir S, Colak G, Feola J, Blouin R, Johnson GV. Transglutaminase 2 facilitates or ameliorates HIF signaling and ischemic cell death depending on its conformation and localization. Biochim Biophys Acta 2013;1833:1–10. [38] Quiroga C, Gatica D, Paredes F, Bravo R, Troncoso R, Pedrozo Z, et al. Herp depletion protects from protein aggregation by up-regulating autophagy. Biochim Biophys Acta 2013; 1833:3295–3305.

Transglutaminase 2 is involved in homocysteine-induced activation of human THP-1 monocytes.

Aberrant transglutaminase 2 (TG2) expression and protein cross-linking activity have been associated with several chronic neurodegenerative disorders ...
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