TRPC1 is regulated by caveolin‐1 and is involved in oxidized LDL‐induced apoptosis of vascular smooth muscle cells

Reagents

Cell culture reagents were from Invitrogen (Carlsbad, CA, USA). 2‐aminoethoxydiphenyl‐borane (2‐APB), Ethylene glycol tetraacetic acid (EGTA) and cytochalasin D were from Sigma‐Aldrich (St Louis, MO, USA). The following antibodies were used: polyclonal anti‐TRPC1 (Alomone Labs, Jerusalem, Israel), polyclonal anti‐caveolin‐1 and monoclonal anti‐β‐actin (Upstate Biotechnology, Billerica, MA, USA). The cell surface protein biotinylation and purification kit was from Pierce. The enhanced chemoluminescence (ECL) kit was from Amersham Pharmacia (Buckinghamshire, UK). Hiperfect transfection reagent was from Quiagen (Courtaboeuf, France). Fluo‐3/AM, SYTO‐13, propidium iodide were from Molecular Probes (Invitrogen, Carlsbad, CA, USA).

Cell culture

We used different VSMC cells: human primary VSMC (hSMC) and rabbit arterial SMC stably expressing caveolin‐1 because VSMC grown in primary culture are rapidly converted from a contractile to a synthetic phenotype leading to a decrease of cell surface caveolae and caveolin‐1 [14]. In addition, there is a phenotype‐dependent variation in the number of caveolae in VSMC both in vivo and in vitro. To avoid caveolin‐1 and caveolae decrease by passages, we established a stable caveolin‐1 transfected VSMC model, allowing us to obtain stable and constant level of caveolin‐1 protein and caveolae.

Rabbit arterial femoral SMC (obtained from ATCC, Rockville, MD, USA) and stable caveolin‐1 transfectants were established and grown as described previously [7]. To obtain stable transfectants, rabbit arterial SMC were transfected with the pCDNA3/caveolin‐1 plasmid (a generous gift of Dr. P. Fielding, San Francisco, CA, USA) or by the empty plasmid pCDNA3 using LipofectAMINE Plus reagent (Invitrogen). Twenty‐four hours after transfection, the cells were split into 10‐cm dishes in medium containing 500 μg/ml G418, and the medium was changed every 3–4 days until G418‐resistant colonies were clearly evident. Rabbit arterial VSMC expressing the empty plasmid pCDNA or the plasmid pCDNA3/caveolin‐1 were, respectively, denominated SMC/ev and SMC/cav1. The levels of caveolin‐1 expression and cell surface caveolae were similar in primary hSMC and SMC/cav1 (analysed by Western blotting and electron microscopy, [7]).

The hSMC were obtained from human coronary arteries at postmortem examinations. All experiments were conformed to the declaration of Helsinki in compliance with French legislation and written informed consent was obtained from patients for the use of surgery residual tissue for research. Briefly, the arteries were cut longitudinally and small pieces of the media were carefully stripped from the vessel wall and cultured. Within 1–2 weeks, hSMC migrated from the explants; they were capable of being passaged 3 weeks after the first appearance of cells. They were identified as VSMC by their characteristic hill‐and‐valley growth pattern and immunohistochemistry for VSMC‐specific α‐actin. The cultures were maintained in Doulbecco’s Modified Eagle’s Medium (DMEM) containing 10% foetal calf serum at 37°C in a humidified, 5% CO₂/95% air atmosphere. The hSMC were used until the eighth passage. Caveolin‐1 expression is checked every three passages in hSMC and SMC/cav1. For experiments, cells were transferred 24 hrs in G418‐free medium.

LDL isolation and mild oxidation

LDL from human pooled sera were prepared by ultracentrifugation, dialysed against phosphate buffered saline (PBS) containing 100 μM ethylenediaminetetraacetic acid (EDTA). LDL were mildly oxidized by UV‐C + copper/EDTA (5 μM) (oxLDL) as previously reported [15]. OxLDL contained 4.2 to 7.4 nmoles of TBARS (thiobarbituric acid‐reactive substances)/μg apoB. Relative electrophoretic mobility and 2,4,6‐trinitrobenzenesulfonic acid reactive amino groups were 1.2–1.3 times and 85–92% of native LDL, respectively.

Western blot analysis

Cells were lysed in solubilizing buffer (10 mM Tris pH 7.4, 150 mM NaCl, 1% Triton X‐100, 1% sodium deoxycholate, 0.1% sodium dodecyl sulphate, 1 mM sodium orthovanadate, 1 mM sodium pyrophosphate, 5 mM sodium fluoride, 1 mM phenylmethylsulfonyl fluoride, 1 μg/ml leupeptin, 1 μg/ml aprotinin) for 30 min. on ice. Forty micrograms of protein cell extracts were resolved by SDS‐polyacrylamide gel electrophoresis, transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). Then membranes were probed with the indicated primary antibodies and revealed with the secondary antibodies coupled to horseradish peroxidase using the ECL chemoluminescence kit. Membranes were then stripped and reprobed with anti‐β‐actin antibody to control equal loading of proteins.

Detergent‐free purification of caveolae/raft membrane fractions

Lipid raft‐enriched domains were purified from cultured cells using a modified carbonate method [16]. Cells were lysed in 2 ml 0.5 M Na₂CO₃ (pH 11), containing protease inhibitors and homogenized sequentially with a tightly fitting Dounce homogenizer (10 strokes) and three 10‐sec. bursts using a sonicator Soniprep 150 (MSE, London, UK). Equal amounts of cell lysates were then adjusted to 45% sucrose by mixing with 2 ml 90% sucrose prepared in 25 mM 2‐(N‐Morpholino)ethanesulfonic acid (MES) (pH 6.5) and 150 mM NaCl. This suspension was placed at the bottom of an ultracentrifuge tube and overlaid with a 5–35% discontinuous sucrose gradient. After centrifugation at 39,000 rpm for 18 hrs at 4°C in a swinging bucket rotor SW41 (Beckman, Roissy, France), a total of 12 fractions (1 ml each) were collected from the top of each gradient and analysed by Western blotting.

Evaluation of cytotoxicity, necrosis and apoptosis

Cytotoxicity was evaluated using the MTT [3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide] test, as previously used [4]. This method is based on the transformation and colorimetric quantification of MTT. The respiratory chain and other electron transport systems reduce MTT and other tetrazolium salts and thereby form non‐water‐soluble violet formazan crystals within the cell. The amount of these crystals can be determined spectrophotometrically (OD measured at 570 nm) and serves as an estimate for the number of mitochondria and hence the number of living cells in the sample. For cytotoxicity experiments cells were serum starved for 24 hrs and stimulated for the indicated times at 37°C. Cell lysis (necrosis) was evaluated by lactate dehydrogenase release. Apoptotic and necrotic cells were counted after fluorescent staining by two vital fluorescent dyes, the permeant DNA intercalating green fluorescent probe SYTO‐13 (0.6 μM) and the non permeant DNA intercalating red fluorescent probe propidium iodide (15 μM) using an inverted fluorescence microscope (Fluovert FU, Leitz, Stuttgart, Deutschland). Normal nuclei exhibit a loose green coloured chromatin. Nuclei of primary necrotic cells exhibit a loose red coloured chromatin. Apoptotic nuclei exhibited condensed yellow/green‐coloured chromatin associated with nucleus fragmentation, whereas post‐apoptotic necrotic cells exhibited the same morphological features, but were red coloured.

Measurement of intracellular Ca²⁺

Cytosolic Ca²⁺ was determined using fluo3/AM according to Jagnadan et al.[17]. Briefly, cells were incubated with a loading solution consisting of HEPES‐buffered saline (HBS; 135 mM NaCl, 5.9 mM KCl, 1.2 mM MgCl₂, 1.5 mM CaCl₂, 11.6 mM HEPES, and 11.5 mM glucose, pH 7.3) supplemented with 5 μM fluo‐3/AM, 0.02% pluronic F‐127 and 1 mg/ml bovine serum albumin for 30 min., and then incubated in the loading solution without Fluo‐3 for 30 min. to allow de‐esterification of the probe. Loading solution was then replaced with HBS, and cells were placed in a fluorometer and fluorescence was recorded (495 and 525 nm excitation and emission wavelength, respectively). Results were normalized on protein levels and expressed in ratio of control.

Biotinylation of cell surface proteins

Following oxLDL treatment, cells were washed twice with ice‐cold PBS and then incubated in 1 mg/ml sulfo‐NHS‐SS‐Biotin (Pierce) in PBS for 30 min. at 4°C. The biotinylation reaction was terminated with washing the cells three times with ice‐cold PBS containing 10 mM glycine. The cells were then lysed in solubilizing buffer, isolation of labelled proteins were preceded using Cell Surface Protein Biotinylation and Purification Kit (Pierce) according to the manufacturer’s instructions. The recovered proteins were analysed by Western blot as described previously, blots were probed with anti‐TRPC1 antibody.

siRNA transfection

The selected siRNA specific to TRPC1 is 5′‐AAG CUU UUC UUG CUG GCG UGC‐3′ and to caveolin‐1 are 5′‐CAG GGC AAC AUC UAC AAG C‐3′ and 5′‐CCA GAA GGG ACA CAC AGU U‐3′ (Dharmacon, Waltham, MA, USA). SiRNAs were transfected using the Hiperfect reagent (Quiagen) according to the manufacturer’s recommendations.

Cholesterol and 7‐ketocholesterol analysis

The cholesterol and 7‐ketocholesterol content of the sucrose gradient fractions were assayed by GC‐MS [18].

Determination of LDL cellular uptake

The uptake of native LDL and oxLDL was studied by using LDL labelled with the fluorescent lipid dye 3,3′‐dioctadecyl‐indocarbocyanine (DiI) (Molecular Probes), according to Negre‐Salvayre et al.[19]. The DiI content was determined by fluorometry (spectrofluorometer Jobin Yvon JY3C; excitation 520 nm, emission 568 nm) using a standard of DiI similarly treated.

Statistical analysis

Data are given as means ± S.E.M. Estimates of statistical significance was performed by ANOVA analysis (Tukey test, SigmaStat software). Values of P < 0.05 were considered statistically significant.

TRPC1 is involved in oxLDL‐induced Ca²⁺ influx and apoptosis

We first investigated the molecular mechanisms involved in oxLDL‐induced Ca²⁺ rise using VSMC stably expressing caveolin‐1 (SMC/cav1), recently developed and characterized in our laboratory because VSMC grown in primary culture are rapidly converted from a contractile to a synthetic phenotype leading to a decrease of cell surface caveolae and caveolin‐1 (see ‘Materials and methods’ section for cell line details). As previously reported, oxLDL triggered a higher Ca²⁺ rise in SMC/cav1 compared to control VSMC expressing an empty vector (SMC/ev) [7]. The cytosolic Ca²⁺ rise was precluded by the extracellular Ca²⁺ chelator EGTA thus suggesting that the sustained Ca²⁺ rise requires an influx of extracellular Ca²⁺ (Fig. 1A, upper panel). Interestingly, the sustained Ca²⁺ rise was also inhibited by aminoethoxydiphenylborane (2‐APB), an inhibitor of TRPC channels (Fig. 1A, upper panel). Time course analysis of cell death following oxLDL treatment showed that the number of apoptotic cells increases after 12 hrs oxLDL stimulation in accordance with the kinetic of the Ca²⁺ rise (Fig. 1A, lower panel). As expected, pre‐treatment with EGTA or 2‐APB before oxLDL exposure, reduces the apoptotic effect of oxLDL (Fig. 1B). Moreover, under the same conditions, the number of apoptotic cells, counted by Syto13/PI fluorescent staining, was significantly reduced (Fig. 1C and D). In addition, silencing TRPC1, the major TRPC channel expressed in VSMC, by small interfering RNA (siRNA) specific to TRPC1 (Fig. 2A) inhibited the oxLDL‐induced Ca²⁺ rise (Fig. 2B) and reduced the toxic effect of oxLDL (Fig. 2C and D). To evaluate whether TRPC1 is implicated in oxLDL‐induced apoptosis under more physiological experimental conditions, we performed the same experiment on hSMC. As shown in Fig. 2E, we observed that the cellular toxicity induced by oxLDL in hSMC was significantly decreased by TRPC1 silencing, thus supporting the major role of TRPC1 channel in oxLDL‐induced Ca²⁺ influx and VSMC apoptosis.

Altogether, these data support the critical role of Ca²⁺ influx through TRPC1 channel in oxLDL‐induced VSMC apoptosis. However, it may be noted that the Ca²⁺‐dependent apoptotic pathway induced by oxLDL is not the sole apoptotic mechanism, because the extrinsic apoptotic pathway mediated by death receptors has been shown to be activated by oxLDL [20]. But in our experimental model, the intrinsic Ca²⁺‐dependent apoptotic pathway is prevailing (≈60% of apoptotic cells), on the basis of the protective effect of Ca²⁺ chelators.

Caveolin‐1 is involved in TRPC1 expression and oxLDL‐induced apoptosis

We have previously reported that caveolin‐1 sensitizes VSMC to oxLDL‐induced apoptosis by potentiating the Ca²⁺ influx and the mitochondrial Ca²⁺‐dependent apoptotic pathway [7]. It has also been shown that caveolin‐1 plays a critical role in the functional expression of TRPC1 in the plasma membrane and may serve as a scaffold to integrate TRPC1 regulation of Ca²⁺ influx pathways [10].

Interestingly, when we compared the levels of TRPC1 proteins in VSMC expressing different levels of caveolin‐1 proteins, we observed that the expression of TRPC1 and caveolin‐1 was correlated (Fig. 3A). In order to investigate whether TRPC1 expression was dependent on caveolin‐1 expression, we induced a decrease of caveolin‐1 expression by specific siRNA and examined the effect on TRPC1 expression. As shown in Fig. 3B, silencing of caveolin‐1 induced a dramatic decrease in TRPC1 level, thus supporting the hypothesis that TRPC1 level may depend on caveolin‐1 expression.

Because TRPC1 is involved in oxLDL‐induced apoptosis, caveolin‐1 expression should regulate the toxic effect of oxLDL through the regulation of TRPC1 level. As expected, caveolin‐1 silencing reduced the toxicity induced by oxLDL treatment in SMC/cav1 (Fig. 4A) and in primary hSMC (Fig. 4B).

TRPC1 channels translocate to caveolar compartment upon oxLDL stimulation

The activity of TRPC1 channel is dependent on its expression and on its insertion in the plasma membrane [21]; this led us to investigate whether oxLDL may regulate either the expression of TRPC1 or its subcellular location. As oxLDL treatment did not modify the whole cellular level of TRPC1 (Fig. 5A), we investigated whether oxLDL may regulate the level of TRPC1 located at the plasma membrane. This was evaluated by using the membrane‐impermeant biotinylation reagent sulfo‐NHS‐SS‐Biotin (that reacts with cell surface proteins) followed by the recovery of biotin‐labelled proteins with streptavidin beads and Western blot analysis. Under resting conditions, biotinylated TRPC1 was barely detectable at the cell surface in SMC/cav1 in spite of the high whole cellular level of TRPC1 (Fig. 5B). This suggests that, under basal conditions, the major part of TRPC1 is not located at the plasma membrane, but is rather located inside the cell (not accessible to the impermeant biotinylation reagent). In contrast, in cells treated for 8 to 14 hrs with oxLDL, the level of biotinylated TRPC1 (i.e. located at the plasma membrane) increased dramatically (Fig. 5B). This strongly suggests that oxLDL induce the translocation of TRPC1 to the plasma membrane. As functional TRPC1 has been shown to be located in caveolar lipid raft domains [21], we investigated whether oxLDL induced the translocation of TRPC1 into caveolar compartment. Using caveolae fractionation methods, we showed that, in untreated cells, TRPC1 was found in non‐caveolar fractions (H₁ and H₂, high‐density fractions) (Fig. 5C), in agreement with the intracellular location suggested above. Interestingly, oxLDL treatment of SMC/cav1 induced the translocation of TRPC1 into caveolar domains (light‐density fractions L) (Fig. 5C). It may be noted that, as expected, caveolin‐1 was localized in the caveolae (light‐density fractions L), both in resting and in oxLDL‐stimulated SMC/cav1 (Fig. 5C).

Given that translocation of TRPC1 to the plasma membrane appears to be induced by oxLDL, we next sought to determine whether TRPC1 translocation is required for oxLDL‐induced apoptosis. It has been shown that local changes in the cytoskeleton or microtubules can contribute to channel trafficking and facilitate plasma membrane insertion [22]. The pre‐treatment of SMC/cav1 with cytochalasin D, a widely utilized membrane‐permeant inhibitor of actin polymerization, prevents TRPC1 translocation observed after 8 hrs oxLDL stimulation (Fig. 6A, upper panel). Accordingly to our hypothesis, we also measured a reduction in oxLDL‐induced apoptosis after cytochalasin D treatment (Fig. 6B). In control experiments, we showed the integrity of cell membranes upon 8 hrs oxLDL and cytochalasin D treatment (Fig. 6A, lower panel), and we confirmed that cytochalasin D did not interfere with oxLDL uptake as measured by oxLDL labelled with the fluorescent lipid dye 3,3′‐dioctadecyl‐indocarbocyanine (Fig. 6C).

Because recent reports have shown that 7‐ketocholesterol [18] or cholesterol [23] induce TRPC1 redistribution to raft fractions, and because oxLDL contain oxysterols (including 7‐ketocholesterol) and can induce cholesterol changes [24], we investigated whether, in our conditions, mildly oxLDL may alter the level of these compounds in subcellular fractions, and more specifically in caveolar fractions. Mildly oxLDL used here induced no appreciable change in the cholesterol content of the three density gradient fractions (Fig. 7A), but increased by 300% the level of 7‐ketocholesterol in the light‐density L caveolar fractions (Fig. 7B).