Oxysterols modulate calcium signalling in the A7r5 aortic smooth muscle cell-line

Biochimie 95 (2013) 568e577

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Research paper

Oxysterols modulate calcium signalling in the A7r5 aortic smooth muscle cell-line Yzzam Hammoud, Tom Rice, John J. Mackrill* Department of Physiology, University College Cork, BioSciences Institute, College Road, Cork, Ireland

a r t i c l e i n f o

a b s t r a c t

Article history: Received 30 April 2012 Accepted 3 August 2012 Available online 1 September 2012

Prolonged exposure to oxidized low density lipoprotein (oxLDL) can alter various aspects of cell biology, including modification of vasomotor responses and downregulation of calcium channel proteins in aortic smooth muscle cells. However, the components of oxLDL responsible for these effects have not been fully elucidated. The study reported here aimed at examining the consequences of extended exposure to oxysterols, cholesterol oxidation products whose levels are elevated in oxLDL as compared to unmodified LDL, on calcium signalling mechanisms in A7r5 cells, a model aortic smooth muscle cell-line. Within 24 h of exposure, all three oxysterol congeners tested caused an elevation in the resting cytoplasmic Ca2þ concentration. These oxysterols also inhibited Ca2þ transients in response to arginine vasopressin and bradykinin, and some but not all congeners ablated Ca2þ signals triggered by platelet activating factor, the ryanodine receptor calcium channel agonist 4-choloro-meta-cresol, or thapsigargin, an inhibitor of endoplasmic reticulum Ca2þ uptake. The effects of long-term exposure to the oxysterol congener 7bhydroxycholesterol on arginine vasopressin stimulated Ca2þ signals were mainly at the level of Ca2þ release from intracellular stores rather than on Ca2þ influx mechanisms. Of the calcium signalling proteins tested, only the type 1 ryanodine receptor and the type 1 inositol 1,4,5-trisphosphate receptor (IP3R1) were significantly downregulated by 24 h exposure to oxysterols. Decreases in IP3R1 protein triggered by 7b-hydroxycholesterol were both time and concentration dependent, occurring over a concentration range encountered within atherosclerotic lesions. IP3R1 downregulation by certain oxysterols is mediated by proteasomal proteolysis, since it can be abolished by co-incubation with epoxomicin. Overall, these data demonstrate that major oxysterol components of oxLDL cause long-term alterations in Ca2þ signalling in a model aortic smooth muscle cell. Such effects could contribute to the pathology of atherosclerotic disease. Ó 2012 Published by Elsevier Masson SAS.

Keywords: Oxysterol Smooth muscle Calcium Inositol 1,4,5-trisphosphate receptor Ryanodine receptor Atherosclerosis

1. Introduction As described within other contributions to this Special Issue, oxysterols are oxidized derivatives of cholesterol that exert a variety of effects on biological systems [1e3]. Examples include initiation of cell-death, alterations in vascular tone [4,5], triggering of inflammatory responses and differentiation of macrophages [6]. It is postulated that such effects underlie a variety of pathologies linked

Abbreviations: 4-CMC, 4-chloro-meta-cresol; 7b-OH, 7b-hydroxycholesterol; 7-keto, 7-ketocholesterol; 25-OH, 25-hydroxcholesterol; AVP, arginine vasopression; b-epoxide, cholesterol-5b,6b-epoxide; BK, bradykinin; DMEMs modified Eagle’s medium, Dulbecco’; DMSO, dimethyl sulfoxide; GPCR, G-protein coupled receptor; IP3R, inositol 1,4,5-trisphosphate receptor; LDL, low-density lipoproteins; oxLDL, oxidized LDL; PAF, platelet activating factor; PMCA, plasmalemmal Ca2þATPase; RyR, ryanodine receptor; SERCA, sarcoplasmic/endoplasmic Ca2þ-ATPase; TG, thapsigargin; VGCC, voltage-gated calcium channel. * Corresponding author. Tel.: þ353 0 21 4902337; fax: þ353 0 21 4205370. E-mail address: [email protected] (J.J. Mackrill). 0300-9084/$ e see front matter Ó 2012 Published by Elsevier Masson SAS. http://dx.doi.org/10.1016/j.biochi.2012.08.003

with elevated oxysterol levels, including atherosclerosis, agerelated macular degeneration and neurodegeneration. Elevated plasma levels of cholesterol and of low-density lipoproteins (LDL) represent major risk factors for the development of atherosclerotic lesions. Oxidized LDL (oxLDL) displays enhanced atherogenic properties relative to unmodified LDL complexes [7], indicating that oxidized components underlie this pathogenicity. Oxysterols are potentially pro-atherogenic components of oxLDL, but their roles in cardiovascular disease have been a matter of debate for nearly two decades [1,3,7e9]. However, it is of note that the concentrations of two major oxysterol congeners, 7b-hydroxycholesterol (7b-OH) and 7-ketocholesterol (7-keto), are substantially elevated in both plasma and arterial plaques from human subjects with atherosclerosis relative to tissues from healthy individuals [10]. The molecular mechanisms by which oxLDL and oxysterols influence the biology of target cells are diverse and have not been fully characterized. Such effects include modification of the biophysical properties of cell membranes, control of gene

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expression by binding to a range of transcription factors and actions exerted via effector proteins called oxysterol-binding protein related proteins [1,2]. Recently, it has been demonstrated that certain oxysterols can also mediate their effects by direct and selective activation of G-protein coupled receptors (GPCRs). The oxysterol 7a,25-dihydroxycholesterol binds to the EpsteineBarr virus induced gene-2 product (EBI2, or GPR183), triggers increases in cytoplasmic Ca2þ levels and promotes immune cell migration [6]. By acting as an agonist for the GPCR smoothened, 20(S)-hydroxycholesterol activates the Hedgehog pathway in target cells [11]. These discoveries suggest that oxysterols act as endogenous ligands in receptor mediated signal transduction cascades. Alterations in cytoplasmic free Ca2þ concentrations ([Ca2þ]c) in response to added oxysterols or oxLDL have been observed in various cell types, over time periods ranging from seconds to days, for a recent review please see [12]. Given that Ca2þ is a ‘universal’ second messenger, regulating processes as diverse as muscle contraction and cell-death [13], it is likely that this could play a part in cellular responses to oxLDL and to oxysterols. For example, minimally oxidized LDL triggers a rapid rise [Ca2þ]c in rabbit aortic smooth muscle cells, that is partially dependent on release of this ion from intracellular stores via inositol 1,4,5-trisphosphate receptor (IP3R) calcium channels [14]. Similarly, in human aortic smooth muscle cells 25-hydroxcholesterol (25-OH) or 7b-OH trigger oscillations in [Ca2þ]c within seconds of addition, followed by apoptotic cell-death over a time period of days [15,16]. In these cells apoptosis triggered by 25-OH could be inhibited by verapamil or nifedipine, antagonists of L-type voltage-gated calcium channels (VGCCs), or by chelation of extracellular Ca2þ [16]. This implies that cell-death triggered by 25-OH in these myocytes is dependent on Ca2þ influx via VGCCs. In contrast, [Ca2þ]c oscillations initiated by 7b-OH were insensitive to verapamil [15], suggesting that distinct oxysterol congeners can exert their biological effects via different mechanisms. In addition to their rapid effects, oxLDL and its oxysterol components have been reported to promote long term alterations in calcium signalling pathways. Such chronic changes in cell biology are likely to be of importance in atherosclerosis, in that this disease progresses slowly [9]. For example, exposure of rabbit aortic smooth muscle cells to oxLDL for 6 days resulted in an inhibition of their [Ca2þ]c responses to noradrenaline and a decrease in the density of IP3R calcium channel proteins [14]. In the same cell type, chronic oxLDL exposure also resulted in reduced [Ca2þ]c elevations in response to the purinoreceptor agonist ATP, the peptide hormone endothelin-1, or ryanodine, a plant alkaloid ligand that binds to the ryanodine receptor (RyR) family of calcium release channels. Prolonged oxLDL exposure also leads to a downregulation of RyR protein abundance [17]. Changes in calcium channel expression and function might explain some alterations in arterial smooth muscle vasomotor responses resulting from chronic oxLDL exposure. It has been proposed that oxysterols might be the components of oxLDL that cause such biochemical and physiological alterations [4,12,18]. However, oxLDL also contains a range of additional oxidized and bioactive substances, including lysophosphatidylcholine, lysophosphatidic acid and 4-hydroxynonenal. The effects of long-term (hours to days) exposure to pure oxysterol congeners on smooth muscle Ca2þ signalling have not been investigated to date. However, in SH-SY5Y neuroblastoma cells, 24S-hydroxycholesterol triggers a rise in [Ca2þ]c that peaks after 30 h of addition [19]. In the U937 monocytic cell line, 7b-OH but not b-epoxide (cholesterol-5b,6b-epoxide) causes a rise in [Ca2þ]c within minutes of exposure, that persists for at least 72 h. In these cells following 24 h of exposure, both oxysterol congeners decrease Ca2þ mobilization in response to the sarcoplasmic/endoplasmic

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Ca2þ-ATPase (SERCA) pump inhibitor thapsigargin (TG) [20], implying that they cause a chronic depletion of intracellular Ca2þ stores [21]. Finally, an oxysterol mixture promoted 45Ca2þ uptake into human endothelial cells over a 72 h period [22]. The central objective of the current study was to investigate effects of long-term exposure to pure oxysterol congeners on Ca2þ signalling in a smooth muscle cell-line. This study focussed on mechanisms involved in Ca2þ release from intracellular stores, given that oxysterols potentially underlie the reported downregulation of IP3R and RyR caused by prolonged exposure to oxLDL [14,17]. To achieve this, A7r5 cells were treated with 7b-OH, 7-keto or b-epoxide (30 mM for 24 h), key cholesterol oxidation products that are enriched in oxLDL and within atherosclerotic plaques [9,10]. The A7r5 cell-line is derived from embryonic rat aorta and has been widely used as a model of vascular smooth muscle. Furthermore, this cell-line expresses multiple Ca2þ signalling proteins that are characteristic of vascular myocytes, including IP3Rs, RyRs and SERCA pumps [23e25]. The effect of prolonged oxysterol exposure on Ca2þ signals in A7r5 cells triggered by hormones, channel agonists or TG was investigated using fura-2 Ca2þ-videomicroscopy. The influence of oxysterol treatment on the expression of key Ca2þ signalling proteins in smooth muscle cells was examined by western blotting. Following 24 h of exposure, all three oxysterols significantly elevated resting [Ca2þ]c. They also reduced Ca2þ increases in elicited by arginine vasopressin (AVP) or bradykinin (BK), whereas increases in [Ca2þ]c in response to platelet activating factor (PAF), TG or the RyR agonist 4-chlorometa-cresol (4-CMC) were selectively inhibited by some, but not all, oxysterol congeners. The effects of 7b-OH on AVP stimulated Ca2þ signals were predominantly at the level of Ca2þ release from the SR/ ER, rather than on Ca2þ influx across the plasma membrane. None of the oxysterols tested significantly downregulated protein expression of SERCA pumps, the plasmalemmal Ca2þ-ATPase (PMCA), the VGCC Cav1.2, or of the ER-plasmalemmal linker protein junctophilin-1. However, both 7b-OH and b-epoxide significantly downregulated levels of the RyR1 Ca2þ release channel. All three oxysterols decreased the expression of the IP3R1 Ca2þ channel protein; on further investigation of the effects of 7b-OH, this downregulation proved to be concentration and time-dependent. Co-incubation with epoxomicin abolished the effects of 7b-OH and b-epoxide, but not those of 7-keto, on IP3R1 protein density, indicating a role for proteasomal degradation in this process. Overall, these findings demonstrate that prolonged oxysterol exposure exerts multiple effects on Ca2þ signalling in smooth muscle cells, that might account for some of the pathological changes observed in vascular myocytes within atherosclerotic lesions. 2. Materials and methods 2.1. Materials The rat embryonic aorta smooth muscle A7r5 cell-line was obtained from the European Collection of Animal Cell Cultures (ECACC, Porton Down Salisbury, UK). Dulbecco’s modified Eagle’s medium (DMEM), arginine vasopressin (AVP), bradykinin, epoxomicin, 4-chloro-meta-cresol (4-CMC, 4-chloro-3-methylphenol), triton X-100, protease inhibitors, cholesterol and oxysterols (>95% pure) were purchased from SigmaeAldrich (Dublin, Ireland). An acetoxymethyl ester of the ratiometric calcium sensing fluorophore fura-2 (fura-2-AM) was obtained from Invitrogen (Dublin, Ireland). Thapsigargin (TG) and platelet activating factor-16 (PAF) were from Merck Biosciences (Darmstadt, Germany). Uncoated 35 mm diameter glass-bottom (10 mm grade ‘0’ cover-slip) dishes were purchased from MatTek Corporation (Ashland, MA, USA). All other

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reagents were of the highest grade available and were obtained from SigmaeAldrich (Dublin, Ireland). 2.2. Maintenance and treatment of A7r5 cells A7r5 cells were maintained in a growth medium consisting of DMEM, 10% fetal bovine serum, 50 U/mL penicillin and 50 mg/mL streptomycin at 37  C in a humidified atmosphere of 95% air/5% CO2. For subculture, cells were detached from 75 cm2 flasks using 0.05% trypsin/0.02% EDTA solution, were centrifuged at 200 gmax for 5 min, resuspended in growth medium then transferred to either 35 mm glass-bottom dishes for videomicroscopy, or 100 mm plastic dishes for preparation of cell lysates. For treatment with oxysterols, cells were generally incubated with 30 mM of each oxysterol for 24 h, except for concentrationeresponse experiments (see Figure legends for details). All experiments incorporated vehicle negative controls (0.3% ethanol for oxysterols, 0.5% dimethyl sulfoxide (DMSO) for epoxomicin). 2.3. Fura-2 videomicroscopy Videomicroscopy with the ratiometric fluorophore fura-2 was used to assess the effects of prolonged oxysterol treatment on Ca2þ signalling in A7r5 smooth muscle. Cells were grown on glassbottom dishes to about 50% confluency and prior to experimentation were washed twice with 1 mL of a modified Krebs-Henseleit Buffer (KHB) (120 mM NaCl, 4.8 mM KCl, 2 mM CaCl2, 1.2 mM MgSO4, 1.2 mM KH2PO4, 25 mM NaHCO3, 5 mM HEPES, 1 mM probenecid). Cells were then incubated with 2 mM fura-2acetoxymethyl ester (250 mL/dish) for 30 min in a 37  C incubator. Cells were then washed twice with 1 mL of KHB, immersed in 1 mL of this buffer and dishes were placed on the stage of an Olympus IX51 inverted fluorescence microscope within an encapsulating incubator (Solent Scientific, Segensworth, UK) maintained at 37  C. To detect alterations in resting Ca2þ levels and responses to agonists, fura-2 loaded cells were alternately excited peak wavelengths of 340 nm and 380 nm (Cairn Monochromator and 75-W Xenon lamp, Cairn, Faversham, Kent, UK). Emitted light was collected via an Olympus UplanF1 1.3 NA 100x oil-immersion objective, filtered through a dichroic mirror (400 nm cut-off) and recorded using a Hamamatsu ORCA-ER CCD videocamera (Hamamatsu Photonics Ltd., Hertfordshire, UK), set at exposure time of 500 ms per channel. Hardware was controlled and images were acquired using Andor IQ v1.9 software (Andor, Belfast, Northern Ireland). Following recording of resting values for approximately 50 s (40 frames), agonists such as hormones, KCl, 4-CMC or TG were introduced via a micropipette as a 20.4 mL bolus of a stock 50 times more concentrated than the desired value. Images were collected for another 200 frames (about 250 s), then were processed within the Andor IQ v1.9 software. Firstly, background fluorescence was subtracted by masking, then ratio images were generated by dividing each image collected at 340 nm excitation by the subsequent image illuminated at 380 nm. The perimeter of each cell was defined as a region of interest and the mean fura-2 ratio from within this region against time was exported to Microsoft Excel 2003 for further analyses. These data were plotted and used to calculate the average resting ratio, peak value attained and increase in fura-2 ratio following agonist addition. In order to assess any distinct effects of oxysterol pretreatment on AVP stimulated Ca2þ release compared with those on Ca2þ entry, fura-2 loaded cells were pre-incubated for 10 min in Ca2þ-free KHB containing 0.5 mM of the chelator EGTA (extracellular free [Ca2þ] of approximately 100e400 nM, estimated

using fluo-3 acid fluorimetry). Following recording of the effects of AVP on [Ca2þ]c as described, CaCl2 was added to a final concentration of 2 mM and fura-2 ratios were recorded for an additional 160 s. 2.4. Western blotting for detection of calcium signalling proteins Following incubation with various substances for the time periods indicated in the figure legends, A7r5 cells were washed twice with 5 mL of ice-cold phosphate buffered saline (PBS), then were detached from each dish into another 5 mL of ice-cold PBS using a rubber policeman. Cells were collected by centrifugation at 18,000 gmax for 5 min at 4  C, then were lysed in approximately five times the pelleted cell volume of RIPA buffer (150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1 mM Na3VO4, 1 mM NaF, 50 mM TriseHCl pH7.4, with 2 mg/mL of peptstatin A, leupeptin, aprotinin and 0.5 mM phenylmethylsulfonyl fluoride) by incubation on ice for 30 min. Cell debris was removed by centrifugation at 22,000 gmax for 30 min at 4  C and the protein concentration of each lysate was determined using the Bradford method with bovine serum as a standard. Lysates were then either analysed immediately or snap frozen in liquid nitrogen and stored at 80  C. Equal quantities of protein (45 mg/lane) from each lysate were resolved using 7.5% reducing SDS-PAGE minigels, then were transferred onto polyvinyldifluoride membranes (Millipore, Carrigtwohill, County Cork, Ireland) as described previously [26]. Proteins greater than 200 kDa in molecular weight were transferred for 2 h at 70 V; those of less than 200 kDa were transferred for 1 h. In general, blots were blocked with 5% non-fat milk in PBS (mPBS) for 1 h at room temperature, then were incubated with primary antibodies overnight at 4  C, shaking at 60 rpm. The following primary antibodies were utilized: for detection of the type 1 IP3R (IP3R1), a 1:1000 dilution of polyclonal antibody (pAb) H-80; for RyR1, 1:500 mouse monoclonal antibody (mAb) clone XA7; for SERCA2, 1:500 mouse mAb clone IID8 (all from Santa Cruz Biotechnology, California, USA); for calnexin, 1:1000 mouse mAb clone TO-5; for PMCA, 1:500 anti-erythrocyte Ca2þ-ATPase mouse mAb clone 5F10 (from SigmaeAldrich); for VGCC Cav1.2, 1:500 rabbit pAb Cav1.2 (Alomone Labs, Israel); and for the C-terminus of the PM-ER/SR linker protein junctophilin-1, 1:500 rabbit pAb 405100 (Invitrogen, Dublin, Ireland). Following overnight incubation with these antibodies, blots were washed for three times 5 min with PBS, then were incubated with a 1:1000 dilution of either antirabbit IgG or anti-mouse Ig horse-radish peroxidase conjugate (SigmaeAldrich). Following another three 5 min washes with PBS, immunoreactivity was detected using enhanced chemiluminescence (Pierce Western Blot Substrates, Medical Supply Co. Ltd., Dublin, Ireland) and exposure to X-ray film. Uniformity of protein loading was verified by probing stripped blots with a 1:20,000 dilution of mouse mAb clone AC-15 (SigmaeAldrich) to detect b-actin. This employed a protocol identical to that described above, except that washes, dilutions and blocking were performed using tris buffered saline containing 0.05% Tween 20, instead of PBS. To quantify levels of protein expression, X-ray films developed from blots were photographed and band densities determined using NIH ImageJ software (http://rsbweb.nih.gov/ij/). For each blot, band density values were divided by the corresponding densities of b-actin bands and the resulting ratios normalized to that of the vehicle-treated control sample. Concentrationeresponse and timedependency of 7b-OH induced IP3R1 downregulation were analysed using GraphPad Prism software version 4.03. Concentrationdependency data were fitted to a four parameter logistic equation (sigmoidal relationship with variable slope) by non-linear regression:

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IP3 R proteinð% controlÞ ¼

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minimum þ ðmaximum  minimum % IP3 RÞ
1 þ 10ðlogEC50log½7bOHÞ  slope factor

Where the EC50 is the half-maximally effective concentration of 7b-OH for IP3R downregulation and the slope factor indicates the steepness of this sigmoidal relationship. Time-dependency data were fitted to a monoexponential decay relationship. 2.5. Statistical analyses All experiments were repeated at least three times. Where numerical values were obtained, mean values are expressed plus or minus the standard error of this mean. Values were compared using either the unpaired Student’s t-test (for pairs of values) or one way analysis of variance followed by post hoc multiple comparisons using Dunnett’s test. Statistical analyses were performed using GraphPad Prism software version 4.03. Mean values were considered to be significantly different at a p value of less than 0.05.

responses in these cells. Although 7b-OH pre-treatment caused a slight decrease in the amplitude of the Ca2þ transient in response to calcium re-addition (Fig. 2), this effect was not statistically significant. Overall, these observations indicate that 24 h exposure to 7b-OH modulates A7r5 cell Ca2þ responses to AVP, predominantly by actions on the intracellular Ca2þ stores. Prolonged incubation with these oxysterols also modified [Ca2þ]c responses to other hormones and to pharmacological modulators of Ca2þ signalling, often in a congener selective fashion. For example, all three congeners significantly ablated the increase in [Ca2þ]c stimulated by 1 mM AVP (Figs. 1a and 3a), or 10 mM BK (Fig. 3b), whereas the Ca2þ response to 1 mM PAF was inhibited by 7b-OH and b-epoxide, though not by 7-keto (Fig. 3c). The predominant effect of these oxysterols on hormone triggered Ca2þ responses appears to be a reduction in the initial rapid transient

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Incubation of A7r5 aortic smooth muscle cells with 30 mM of 7bOH for a period of 24 h elevated their resting [Ca2þ]c (as indicated by a rise in fura-2 ratio), relative to cells treated with vehicle (0.3% ethanol) alone (Fig. 1a and b.). This treatment also decreased Ca2þ transients in response to hormones such as AVP (Fig. 1a; also see Fig. 3aec.). This effect was not restricted to 7b-OH: 24 h preincubation with 30 mM of either b-epoxide or 7-keto also resulted in significant increases in fura-2 ratio, to levels of between 15 and 30% greater than vehicle treated cells (Fig. 1b.). The effects of oxysterols on A7r5 cell Ca2þ signalling were only apparent on a time-scale of hours, rather than minutes. Preliminary experiments indicated that 30 mM 7b-OH only exerted significant effects on resting [Ca2þ]c following exposures of greater than 2 h (Supplemental Fig. 1a); and that it only had an effect on the amplitude of the Ca2þ transient in response to AVP following exposure times of greater than 1 h (Supplemental Fig. 1b). In common with many hormones coupled to increases in [Ca2þ]c, AVP stimulates both Ca2þ release from the SR/ER and influx of this ion across the plasma membrane [27]. Such hormones generate biphasic Ca2þ responses, with an initial transient depending on Ca2þ release from the SR/ER (dependent on IP3Rs sometimes working in concert with other intracellular channels, such as the RyRs) and on influx of this ion; followed by a sustained plateau phase dependent on Ca2þ entry via a number of pathways, including store-operated calcium entry involving the STIM/Orai protein families [28]. In order to assess the relative effects of prolonged exposure to 7b-OH on these two components of AVP stimulated Ca2þ signals, fura-2 ratios were monitored in A7r5 cells under low extracellular Ca2þ conditions (buffered with 0.5 mM EGTA, 100e400 nM free Ca2þ). Pre-treatment with 7b-OH (24 h, 30 mM) had no significant effect on the resting fura-2 ratio in these cells (0.292  0.006 versus 0.282  0.020, V versus 7b-OH treated, n ¼ 6). However, 7b-OH significantly reduced the initial Ca2þ transient in response to AVP under these low extracellular Ca2þ conditions (Fig. 2), suggesting that extended exposure to this oxysterol impairs Ca2þ storage and/or release by the SR/ER. Re-addition of extracellular Ca2þ unveils the Ca2þ entry component of AVP

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3.1. Prolonged exposure to oxysterols alters Ca2þ signalling in A7r5 cells

Mean fura-2 ratio (340/380nm)

3. Results

Fig. 1. Exposure of A7r5 cells to oxysterols for 24 h alters their resting Ca2þ levels and Ca2þ responses to AVP. A7r5 cells were exposed to 30 mM of 7b-OH, 7-keto, b-epoxide, or vehicle (‘V’, 0.3% ethanol) for a period of 24 h. Alterations in Ca2þ responses to hormones such as AVP (panel a and Fig. 2) or resting [Ca2þ]c were monitored using fura-2 videomicroscopy. Panel a shows representative fura-2 ratio traces in cells treated with either V (black trace) or 7b-OH (grey trace), then stimulated with 1 mM AVP (grey bar). Panel b indicates the effect of oxysterols on mean resting fura-2 ratio (SEM, n ¼ 5e8 experiments; *p < 0.05, **p < 0.01 compared to ‘V’ by ANOVA).

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signalling proteins, as reported for oxLDL [14,17]. To test this hypothesis, lysates prepared from A7r5 cells treated for 24 h with these oxysterols, vehicle or 30 mM cholesterol, were analysed by western blotting. Since AVP, BK and PAF all trigger Ca2þ increases via the IP3 signalling pathway and oxysterols generally suppressed Ca2þ transients elicited by these hormones (Fig. 3aec.), the effects of prolonged exposure to these cholesterol oxidation products on IP3R1 protein density were examined. IP3R1 is a major Ca2þ release channel subtype expressed in the SR/ER of the A7r5 cell-line [25]. All three oxysterols caused a significant downregulation of this protein within 24 h of exposure (Fig. 4a.). Another Ca2þ release channel present in SR/ER of A7r5 cells is RyR1 [24], which can be gated by physical coupling to plasmalemmal VGCCs, or which can act amplify Ca2þ signals from other sources, in a process termed Ca2þ-induced Ca2þ-release [13]. RyR1 protein expression was suppressed by exposure to 7b-OH and b-epoxide, but not 7-keto (Fig. 4b). Despite the inhibition by 7b-OH of Ca2þ signals triggered by TG, none of the oxysterols tested significantly altered the expression of SERCA2 (Fig. 4c.), an SR/ER Ca2þ ATPase pump present in these cells [23]. Similarly, there was no detectable effect of any of the oxysterols tested on the protein density of PMCA, the plasmalemmal Ca2þ pump (Fig. 4d); calnexin, an ER integral membrane Ca2þ-binding chaperone (Fig. 4e); or the ER-plasmalemmal linker protein junctophilin-1 (Fig. 4f). As anticipated from their lack of detectable effects on depolarization-induced Ca2þ transients, these oxysterols did not significantly alter the protein expression of Cav1.2, a VGCC present in this cell-line (Fig. 4g). Cholesterol (30 mM for 24 h) had no significant effect on the expression levels of any of the proteins examined (Fig. 4).

Fig. 2. The oxysterol 7b-OH decreases Ca2þ release from intracellular stores, but not Ca2þ influx, in A7r5 cells in response to AVP. A7r5 cells were exposed to 30 mM of 7bOH or vehicle (‘V’, 0.3% ethanol) for a period of 24 h. Cells were then loaded with fura2, then were transferred to KHB containing 0.5 mM EGTA to chelate extracellular Ca2þ. Changes in fura-2 ratio in response to 1 mM AVP (mid-grey bar) followed by readdition of extracellular Ca2þ (dark grey bar) were monitored using fluorescent videomicroscopy. Panel a shows representative traces from individual cells pretreated with V (black trace) or 7b-OH (grey trace). Panel b shows the average increase in fura-2 ratio triggered by AVP or Ca2þ readdition (n ¼ 6; *p < 0.01 by Student’s t-test; n.s, not significant).

that is considered to be dependent on Ca2þ release from intracellular stores via IP3Rs [13,27], rather than subsequent plateau phases (Supplemental Fig. 2). None of the oxysterols tested had an effect on the amplitude of Ca2þ transients elicited by application of 60 mM KCl (Fig. 3d), which triggers Ca2þ influx due to depolarization of cells and activation of VGCCs [29]. Exposure of A7r5 myocytes to 500 nM TG, a reagent that inhibits SERCA pumps thereby depleting ER Ca2þ stores [20], caused a gradual rise in [Ca2þ]c that reached a plateau within several minutes of addition. Of the oxysterols tested, only 7b-OH significantly inhibited the TG induced increase in [Ca2þ]c (Fig. 3e, Supplemental Fig. 2e). Both 7b-OH and 7-keto, but not b-epoxide, significantly reduced transients triggered by 500 mM 4-CMC (Fig. 3f), a pharmacological agonist of the RyR family of Ca2þ release channels [30]. 3.2. Effects of 24 h exposure to oxysterols on expression of Ca2þ signalling proteins in A7r5 cells One mechanism by which 7b-OH, 7-keto or b-epoxide could exert long-term changes in Ca2þ signal transduction in A7r5 myocytes would be by altering the levels of key calcium

3.3. Concentration- and time-dependence of 7b-OH downregulation of IP3R1 Given that both the Ca2þ responses to phosphatidylinositollinked GPCRs (AVP, BK and PAF, Fig. 3aec.) and IP3R1 protein density (Fig. 4a.) are diminished by prolonged incubation with oxysterols, these effects were investigated in greater detail. The concentration-dependence of IP3R1 protein downregulation resulting from incubation with 7b-OH was examined. This effect was concentration dependent, with 24 h exposure to different concentrations of 7b-OH causing a maximal downregulation of IP3R1 to 47  4% of control values, with a half-maximal effective concentration (EC50) of 2.5  0.9 mM and a slope factor of 1.0  0.5 (Fig. 5a). Downregulation of IP3R1 protein due to exposure to 30 mM 7b-OH was also time-dependent, fitting a one phase exponential decay with a half-life of 9.8  0.8 h, reaching a plateau of 29  4% within 72 h (Fig. 5b).

3.4. Involvement of proteasomal degradation in 7b-OH induced downregulation of IP3R1 Prolonged stimulation of cells, including A7r5 smooth muscle, with agonists of phosphatidylinositol-linked GPCRs leads to proteolysis of their IP3Rs via the ubiquitin-/proteasome-dependent ER-associated degradation (ERAD) pathway [25,31]. Given that the time-scale of the effect of 7b-OH on IP3R1 density is consistent with a ubiquitin-/proteasome-dependent mechanism, a selective blocker was used to explore this possibility. The a0 , b0 -expoxyketone, epoxomicin is an irreversible blocker of the proteasome [32]. Epoxomicin (100 nM) inhibited the downregulation of IP3R1 due to 24 h incubation with 30 mM of 7b-OH or b-epoxide, but not that caused by 7-keto (Fig. 6).

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Y. Hammoud et al. / Biochimie 95 (2013) 568e577

Fig. 3. Prolonged exposure of A7r5 cells to oxysterols alters their Ca2þ responses to hormones, thapsigargin and the RyR agonist 4-CMC, but not to depolarisation by KCl. A7r5 cells were exposed to V, or 30 mM of 7b-OH, 7-keto, or b-epoxide for 24 h. Responses to known Ca2þ mobilising reagents were then monitored using fura-2 videomicroscopy: a. 1 mM AVP; b. 10 mM BK; c. 1 mM PAF; d. 60 mM KCl; e. 500 nM TG; and f. 4-CMC (note the larger responses to this reagent). The data shown are mean increases (SEM) in fura-2 ratio above resting values and were derived from between 3 and 9 independent experiments. Statistical differences from the values in vehicle pretreated cells are represented by *p < 0.05 and **p < 0.01).

4. Discussion The involvement of oxLDL in the pathology of atherosclerosis has long been appreciated. Pathological changes that occur in this disease include accumulation of lipids, inflammatory responses, cell differentiation and death, and alterations in vasomotor responses of vascular smooth myocytes. All of these processes can be regulated by Ca2þ in its role as a second messenger. Alterations that occur in response to prolonged exposure to oxLDL include changes in the density and function of calcium signalling proteins, such as downregulation of IP3R and RyR Ca2þ release channels in aortic smooth muscle cells [14,17]. The current study sought to

examine the effects of 7b-OH, 7-keto and b-epoxide, three major oxysterol components of oxLDL and atherosclerotic plaques, on Ca2þ signalling in the A7r5 aortic myocyte model cell-line. Prolonged exposure to each of the three oxysterols tested promoted a sustained elevation in cytoplasmic Ca2þ concentration. In the case of 7b-OH, these increases in resting Ca2þ level did not occur acutely, since they were not apparent within 2 h of exposure. This indicates that oxysterols can modulate Ca2þ signalling in A7r5 cells via mechanisms acting over a time-scale of hours or more. Similar increases in ‘resting’ Ca2þ levels have been observed in differentiated U937 macrophage-like cells following 24 h exposure to 7b-OH. In this macrophage model, 72 h exposure to 7b-OH

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a

b

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Fig. 4. Prolonged exposure of A7r5 cells to oxysterols downregulates IP3R1 and RyR1 calcium release channels, but not other calcium signalling proteins. A7r5 cells were exposed to V, or 30 mM of cholesterol (‘C’), 7b-OH, 7-keto, or b-epoxide for 24 h, then were lysed and subjected to western blotting in order to assess changes in expression levels of various calcium signalling proteins: a. IP3R1; b. RyR1; c. SERCA2; d. PMCA; e. Cav1.2; f. calnexin and g. JP1. The band density of each of these proteins was expressed as a ratio of the density of b-actin in the same sample; then expressed as a percentage of this ratio in a parallel experiment using untreated cells. Data shown represent mean expression levels (SEM) derived from between 5 and 12 independent experiments. Statistical differences from the values in vehicle pretreated cells are represented by *p < 0.05 and **p < 0.01).

caused a decrease in [Ca2þ]c. In the same system, b-epoxide failed to detectably influence cytoplasmic Ca2þ levels for up to 72 h [21]. The time-dependent, cell type-dependent and congener-selective effects of oxysterols on [Ca2þ]c suggest that these changes occur through specific mechanisms, rather than through general alterations in the biophysical properties of cell membranes [12].

However, the nature of the mechanisms that underlie increases in [Ca2þ]c triggered by oxysterols have not been unambiguously discerned. All three oxysterols modified Ca2þ transients in response to hormones that activate phosphatidylinositol hydrolysis in A7r5 cells, namely AVP, BK and PAF. The only exception to this was

Y. Hammoud et al. / Biochimie 95 (2013) 568e577

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time (h) Fig. 5. Downregulation of IP3R1 protein by 7b-OH is concentration and timedependent. Panel a: A7r5 cells were exposed to various concentrations of 7b-OH for a period of 24 h, then were subjected to western blotting for IP3R1 and b-actin. Each data point represents the mean (SEM, n ¼ 3) IP3R1/b-actin ratio normalised to that in untreated cells. Data are fitted to a sigmoidal concentrationeresponse relationship with a variable slope. Panel b: cells were exposed to 30 mM of 7b-OH for different periods of time. Each data point represents the mean (SEM, n ¼ 3) IP3R1/b-actin ratio normalised to that in cells at time zero. Data are fitted to a monoexponential decay relationship.

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Fig. 6. Effect of proteasomal inhibition on oxysterol-induced downregulation of IP3R1 protein. A7r5 cells were exposed to V, or 30 mM of C, 7b-OH, 7-keto, or b-epoxide for 24 h, in the presence or absence of 500 nM epoxomicin (‘E’). Data represent mean (SEM) IP3R1/b-actin ratios normalised to that in untreated cells (n ¼ 7). Statistical differences from the values in vehicle pretreated cells are represent by **p < 0.01.

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that 7-keto failed to significantly inhibit the Ca2þ increase triggered by PAF. Ablated Ca2þ responses to these hormones were probably not due to decreased expression or function of VGCCs, since neither Ca2þ responses to depolarization nor expression levels of Cav1.2 protein were significantly influenced by oxysterol treatment. Furthermore, under conditions of low extracellular Ca2þ, the predominant effect of 7b-OH on AVP-induced Ca2þ signals in A7r5 cells was a reduction in the amplitude of the initial transient, corresponding to Ca2þ release from intracellular stores. This suggests that the function of Ca2þ influx mechanisms were not drastically altered by long-term exposure to this oxysterol. No oxysterol detectably altered expression levels of SERCA2 pump protein and only 7b-OH significantly reduced the size of the TG-sensitive Ca2þ pool. It is unclear how extended exposure to 7bOH could decrease the magnitude of the TG-sensitive Ca2þ pool without altering the abundance of the SERCA2 proteins. Possibilities include: 1) alteration in SERCA pump function, without changing its levels. For example, SERCA Ca2þ uptake can be modulated by membrane composition (including by changes in cholesterol levels), oxidation, glycosylation, phosphorylation and glutathionylation [33]; 2) alterations in the leakage of Ca2þ from the SR/ER, due to changes in the permeability of this bilayer; and 3) alterations in the expression or function of proteins that modulate SERCA activity, such as phospholamban. Which of these possibilities plays a major role in the effect of 7b-OH on the TG-sensitive Ca2þ pool will be discerned in future studies. However, given the slow time-scale of these effects, it is unlikely that oxysterols act by directly modifying ER/SR membrane composition or by increasing Ca2þ permeability. Both 7b-OH and 7-keto significantly reduced Ca2þ increases in response to the RyR agonist 4-CMC and only 7-keto failed to significantly decrease the level of RyR1 protein over a 24 h period. The reasons for this discrepancy between changes RyR1 protein level (decreased by 7b-OH and b-epoxide) and function (inhibited by 7b-OH and 7-keto) are unclear, but suggest that modifications of calcium signalling proteins more subtle than changes in their density might be occurring. As in the case of SERCA, the activity of RyRs can be influenced by post-translational modifications, alterations in bilayer composition and changes in the function of accessory proteins [34]. Overall, these findings indicate that oxysterol components of oxLDL might be sufficient for the downregulation of RyRs in vascular smooth muscle [17] and for the consequences of this on Ca2þ signalling. All three oxysterols caused a downregulation of the IP3R1 protein, a major Ca2þ release channel in these cells that is coupled to phosphatidylinositol-linked GPCRs via the second messenger IP3. Downregulation of IP3R1 protein by 7b-OH is concentrationdependent, with a half-maximal effect (2.5 mM) within the range encountered by cells within atherosclerotic plaques [10]. The profile of this concentration-dependency is consistent within a receptor-mediated mechanism, rather than a general biophysical effect of this oxysterol, since it displays a good fit to a sigmoidal concentrationeresponse relationship with a biologically relevant degree of negative co-operativity. The time-dependence of downregulation of IP3R proteins by prolonged exposure to 7b-OH is slightly slower than that caused by hormones linked to sustained phosphatidylinositol hydrolysis and production of the second messenger IP3. For example, stimulation of SH-SY5Y neuroblastoma cells with an agonist of muscarinic acetylcholine receptors, or A7r5 myocytes with AVP, initiates degradation of IP3R1 protein that is detectable within 1 h and is maximal by 6 h [25,35]. This downregulation depends on prolonged IP3 production and activation of IP3R channels, resulting in their ubiquitination and degradation by via the ERAD

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proteasomal pathway [31]. In the current study, blockade of IP3R1 proteolysis triggered by 7b-OH and b-epoxide with the proteasomal inhibitor epoxomicin supports such a mechanism. However, epoxomicin failed to abolish the downregulation of IP3R1 elicited by 7-keto. The reasons for this discrepancy are not clear and again suggest that the effects of oxysterols on Ca2þ signalling proteins are mediated through specific molecular mechanisms, in a congener-selective manner. In aortic smooth muscle cells, oxLDL treatment also promotes phosphatidylinositol turnover and degradation of IP3R proteins [14], suggesting a role for oxysterol components of these lipoprotein complexes in this process. Potentially, oxysterols could elevate cytosolic levels of IP3 via GPCRs, such as smoothened or EBI-2, provided that these receptors are coupled to phospholipase C. Prolonged activation of IP3Rs would then lead to their ubiquitination and degradation via the ERAD pathway. In P388D1 monocyte/macrophages, 7-keto or 25OH promote the ubiquitination and proteasomal degradation of the kinase Akt [36], in keeping with this mechanism as a potential pathway for oxysterol-stimulated IP3R downregulation. Future studies will investigate the effects of oxysterols on IP3 production and IP3R ubiquitination in A7r5 cells. Overall, this study has revealed that the three major oxysterols present within oxLDL and atherosclerotic plaques initiate longterm changes in the expression and function of Ca2þ signalling proteins present in a model aortic smooth muscle cell-line. The data presented indicate that these effects are mediated through specific, often congener-selective mechanisms that act predominantly on Ca2þ signalling components residing in the SR/ER. Given the fundamental role of Ca2þ as a second messenger, future characterization of these mechanisms could result in the identification of drug targets for treatment of the pathological changes occurring in atherosclerosis. Acknowledgements This work was partially supported by funding from the Health Research Board of Ireland (HRB PD27/2001 to JJM). TR was funded by an Undergraduate Summer Bursary from the College of Science, Engineering and Food Science, University College Cork. The authors would like to thank members of the European Network on Oxysterols Research (ENOR) for advice and suggestions. YH gave an oral presentation including many of these findings at the 1st ENOR Synposium in Rome, Italy in September 2011.

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[22]

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