Regulation of dynein-mediated autophagosomes trafficking by ASM in CASMCs

Abstract

Acid sphingomyelinase (ASM; gene symbol Smpd1) has been shown to play a crucial role in autophagy maturation by controlling lysosomal fusion with autophagosomes in coronary arterial smooth muscle cells (CASMCs). However, the underlying molecular mechanism by which ASM controls autophagolysosomal fusion remains unknown. In primary cultured CASMCs, lysosomal Ca²⁺ induced by 7-ketocholesterol (7-Ket, an atherogenic stimulus and autophagy inducer) was markedly attenuated by ASM deficiency or TRPML1 gene silencing suggesting that ASM signaling is required for TRPML1 channel activity and subsequent lysosomal Ca²⁺ release. In these CASMCs, ASM deficiency or TRPML1 gene silencing markedly inhibited 7-Ket-induced dynein activation. In addition, 7-Ket-induced autophagosome trafficking, an event associated with lysosomal Ca²⁺ release and dynein activity, was significantly inhibited in ASM-deficient (Smpd1^−/−) CASMCs compared to that in Smpd1^+/+ CASMCs. Finally, overexpression of TRPML1 proteins restored 7-Ket-induced lysosomal Ca²⁺ release and autophagosome trafficking in Smpd1^−/− CASMCs. Collectively, these results suggest that ASM plays a critical role in regulating lysosomal TRPML1-Ca²⁺ signaling and subsequent dynein-mediated autophagosome trafficking, which leads its role in controlling autophagy maturation in CASMCs under atherogenic stimulation.

2. INTRODUCTION

Autophagy is a tightly-controlled cellular catabolic pathway leading to lysosomal degradation and recycling of proteins and organelles in eukaryotes (1). It has been demonstrated that autophagy processes includes induction and formation of autophagosomes, autophagy maturation and autophagic efflux. During autophagy maturation, autophagosomes traffic and fuse with lysosomes, which leads to the acidification of autophagosomes to matured autophagolysosomes with acidic pH (2, 3). Autophagy maturation is a critical step in late stage of autophagy, which ensures efficient autophagic efflux, the process known as breakdown of autophagic contents in autophagolysosomes and autophagolysosomes themselves by lysosomal proteases (2, 3). Recent studies have indicated a protective role of autophagy in vascular smooth muscle cell (SMC) homeostasis during atherogenesis (4, 5). In the vasculature of progressive atherosclerosis or restenosis after coronary angioplasty, moderately enhanced autophagy play a protective role by preventing an imbalance of vascular SMC homeostasis, which helps vascular smooth muscle in differentiated contractile phenotype, and thereby decreases cell proliferation and prevents fibrosis (6). In contrast, it is relatively understudied about the role of defective or reduced autophagy in the pathogenesis of atherosclerosis. In this regard, we recently demonstrated that impaired autophagy maturation causes defective autophagic efflux in acid sphingomyelinase (ASM; gene symbol Smpd1)-deficient coronary arterial SMCs (CASMCs), which is associated with enhanced protein expression of vimentin (a proliferative phenotype marker) and proliferation rate (7). Our studies for the first time implicated that impaired autophagy maturation may contributes to the pathogenesis of atherosclerosis via promoting SMC dedifferentiation and switching to a more proliferative phenotype. However, it remains unknown the precise mechanism how ASM signaling controls autophagy maturation process, particularly autophagosome trafficking, in CASMCs.

Upon autophagy induction, autophagosomes are formed in multiple sites in the cytoplasm, and these autophagosomes have to be trafficked and transported to meet with lysosomes. The precise molecular mechanisms underlying this transport process are not clear. Dynein ATPase is a multi-subunit microtubule motor protein complex, which is responsible for nearly all minus-end microtubule-based transport of vesicles within eukaryotic cells (8). A variety of intracellular motile processes are regulated by dynein ATPase activity including trafficking of membranous vesicles and other intracellular particles (9). This motor protein has been recently implicated in lysosomes and autophagosomes trafficking to meet and form autophagosomes (10, 11). Indeed, work in our laboratory has demonstrated that inhibition or loss of dynein function causes impaired autophagosome fusion with lysosomes, increased autophagosome accumulation, and reduced autophagolysosome formation in CASMCs (12). Previous studies have indicated that direct binding of Ca²⁺ to a component of the dynein complex regulates dynein motor function and its cytoplasmic distribution (13, 14). CD38 is a multifunctional enzyme responsible for the production and metabolism of a secondary messenger, NAADP (nicotinic acid adenine dinucleotide phosphate), in vascular cells (15). Mechanistically, NAADP can cause lysosomal Ca²⁺ release and thereby regulate lysosome function through its action on the transient receptor potential mucolipin-1 (TRPML1) channels, or through other mechanisms such as two-pore channels (16, 17). We recently demonstrated that CD38/NAADP-mediated lysosomal Ca²⁺ release plays an essential role in regulating dynein ATPase activity and autophagosome trafficking in CASMCs (12, 18). Therefore, based on these previous observations, we hypothesized that in ASM-deficient CASMCs under proatherogenic stimulation, inhibited dynein ATPase activation due to impaired lysosomal Ca²⁺ release via TRPML1 channels contributes to defective autophagosome trafficking leading to impaired autophagy maturation.

To test this hypothesis, we first determined whether the TRPML1-mediated lysosomal Ca²⁺ release response to proatherogenic stimulation is blunted in ASM-deficient CASMCs. Then, we examined whether dynein ATPase activation and autophagosomes trafficking in CASMCs with proatherogenic stimulation are also inhibited due to ASM deficiency. The roles of TRPML1 in lysosomal Ca²⁺ release, dynein activation and autophagosome trafficking in CASMCs are further confirmed by modulating its protein expression level by TRPML1 gene silencing or its cDNA overexpression.

3. MATERIALS AND METHODS

3.1. Mice

ASM-deficient (Smpd1^−/−; Smpd1 is the gene symbol for ASM gene, sphingomyelin phosphodiesterase 1) and wild-type (Smpd1^+/+) mice were used in the present study as we described previously (7, 19). All experimental protocols were reviewed and approved by the Animal Care Committee of Virginia Commonwealth University. All animals were provided standard rodent chow and water ad libitum in a temperature-controlled room.

3.2. Primary cell culture of mouse CASMCs

Mouse CASMCs were isolated as previously described (7, 20). In brief, mice were deeply anesthetized with intraperitoneal injection of pentobarbital sodium (25 mg/kg). The heart was excised with an intact aortic arch and immersed in a petri dish filled with ice-cold Krebs-Henseleit solution. A 25-gauge needle filled with Hanks' buffered saline solution was inserted into the aortic lumen opening while the whole heart remained in the ice-cold buffer solution. The opening of the needle was inserted deep into the heart close to the aortic valve. The needle was tied in place with the needle tip as close to the base of the heart as possible. The infusion pump was started with a 20 ml syringe containing warm HBSS through an intravenous extension set at a rate of 0.1. ml/min for 15 min. HBSS was replaced with warm enzyme solution (1 mg/ml collagenase type I, 0.5. mg/ml soybean trypsin inhibitor, 3% bovine serum albumin (BSA), and 2% antibiotic), which was flushed through the heart at a rate of 0.1. ml/min. Perfusion fluid was collected at 30, 60, and 90-min intervals. At 90 min, the heart was cut with scissors, and the apex was opened to flush out the cells that collected inside the ventricle. The fluid was centrifuged at 1000 rpm for 10 min, the cell-rich pellets were mixed with the media described below, and the cells were plated on 2% gelatin-coated six-well plates and incubated in 5% CO₂ at 37°C. Advanced Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum, 10% mouse serum, and 2% antibiotics were used for isolated CASMCs. The identification of CASMCs was based on positive staining by anti-α-actin antibody and the SMCs morphology. The medium was replaced 3 days after cell isolation and then once or twice each week until the cells grew to confluence. All studies were performed with cells of passage of 3–5. Six-week-old male C57BL/6J ASM-deficient (Smpd1^−/−) mice and their wild-type littermates (Smpd1^+/+) were used in the present study, and mouse genotyping was performed as we described previously (21).

3.3. Transfection of CASMCs with TRPML1 siRNA or cDNA plasmid

RNA interference of TRPML1 gene was achieved by transfection of double-stranded siRNA of targeting TRPML1 (Accession number: BC118374) consisted of 5′-CAGCUUCCGGCUCCUG-3′ as we previously described (22). The scrambled small RNA (5′-AATTCTCCGAACGTGTCACGT-3′) was used as a negative control. Overexpression of TRPML1 was achieved by transfections of CASMCs with TRPML1 cDNA plasmids as described in our previous study (22, 23). Transfection of siRNA or cDNA was performed using the siLentFect Lipid Reagent or TransFectin Lipid Reagent (Bio-Rad, CA, USA) according to the manufacturer's instructions, as we described previously (24). The efficiency of TRP-ML1 silencing or overexpression in CASMCs was assessed by Western blotting as reported previously (22).

3.4. Fluorescent microscopic measurement of (Ca²⁺)_i in CASMCs

A fluorescence image analysis system was used to determine (Ca²⁺)_i in CASMCs with fura-2 acetoxymethyl ester (fura-2) as an indicator as previously described (17, 20), Having been loaded with 10 μM fura-2 at room temperature for 30 min, the cells were washed three times with Ca²⁺-free Hank's buffer. The ratio of fura-2 emissions, when excited at the wavelengths of 340 and 380 nm, was recorded with a digital camera (Nikon Diaphoto TMD Inverted Microscope). Metafluor imaging and analysis software were used to acquire, digitize, and store the images for off-line processing and statistical analysis (Universal Imaging). The fluorescence ratio of excitation at 340 nm to that at 380 nm (F340/F380) was determined after background subtraction, and (Ca²⁺) was calculated by using the following equation: (Ca²⁺)_i = K_dβ((R − R_min)/(R_max − R)), where K_d for the fura-2-Ca²⁺ complex is 224 nM; R is the fluorescence ratio (F₃₄₀/F₃₈₀); R_max and R_min are the maximal and minimal fluorescence ratios measured by addition of 10 μM of Ca²⁺ ionophore ionomycin to Ca²⁺-replete solution (2.5. mM CaCl₂) and Ca²⁺-free solution (5 mM EGTA), respectively; and β is the fluorescence ratio at 380-nm excitation determined at R_min and R_max, respectively. Lysosomal Ca²⁺ release was monitored indirectly by treating Fura-2-loaded CASMCs with Glycyl-L-phenylalanine 2-naphthylamide (GPN, 200 mM), a tripeptide causing osmotic lysis of cathepsin C-positive lysosomes.

3.5. Fluorescent confocal microscopic measurement of lysosome Ca²⁺ release

To detect lysosome Ca²⁺ release, sub-confluent CASMCs in 35-mm cell culture dishes were incubated with dextran-conjugated tetramethylrhodamine (Rho; 1 mg/ml; Molecular Probes) for 4 h in DMEM medium containing 10% FBS at 37°C, 5% CO₂, followed by a 20-h chase in dye-free medium for lysosomes loaded with Rho as previously described (12, 25). After being washed with Hank's buffered saline solution (HBSS) (in mM: 5.0. KCl, 0.3. KH₂PO₄, 138 NaCl, 4.0. NaHCO₃, 0.3. Na₂HPO₄·7H₂O, 5.6. D-glucose, and 10.0. HEPES, with 2% antibiotics) three times, the Rho-loaded cells were then incubated with the Ca²⁺-sensitive dye fluo-4 at a concentration of 5 μM. Ca²⁺ release and lysosome trace recordings were performed. Lysosome/Rho (Lyso/Rho) fluorescence images were acquired at 568-nm excitation and 590-nm emission. The co-localization coefficiency of Ca²⁺/fluo-4 and Lyso/Rho was analyzed with Image-Pro Plus 6.0. software (26).

3.6. Assay of cytoplasmic dynein ATPase activity

Dynein in mouse CASMCs was purified using a published protocol with slight modification (18, 27). Cytoplasmic protein of mouse CASMCs was extracted with ice-cold extraction buffer (250 ml of 0.0.5 M PIPES-NaOH, 0.0.5 M HEPES, pH 7.0., containing 2 mM MgC1₂, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride (PMSF), 10 μg/ml leupeptin, 10μg/ml tosyl arginine methyl ester (TAME), 1 μg/ml pepstatin A, and 1 mM dithiothreitol (DTT). Exogenous taxol (20 μM) was added to 20 mL of cell extract containing 4 mg/mL cytoplasmic protein, which was incubated in a 37°C water bath (with occasional swirling) for 12 min. The cell extract was underlayered with a prewarmed 7.5.% sucrose solution, and then centrifuged at 60,000 g for 30 min at 35°C. The supernatant was removed and the pellet was resuspended in 10 mL of extraction buffer containing 3 mM MgGTP and 5 μM taxol to release kinesin and dynamin. The resuspended pellet was incubated for 15 min prior to centrifugation at 60,000 g for 30 min. The supernatant was removed, and the pellet was resuspended in 1.2.5 mL of extraction buffer containing 10 mM Mg-ATP for 10 min at 37°C. The resuspended pellet was centrifuged at 200,000 g for 30 min at 25°C. The supernatant containing ATP-released cytoplasmic dynein was used for sucrose density gradient fractionation. Cytoplasmic dynein may constitute up to 50% of total protein in the ATP extract, the remainder consisting of tubulin and a low level of fibrous microtubule-associated proteins (MAPs). 1 mL ATP extract was further centrifuged on 10 mL of a 5–20% sucrose gradient in fractionation buffer (20 mM Tris-HCl, pH 7.6., 50 mM KCl, 5 mM MgSO₄, 0.5. mM EDTA and 1 mM DTT) at 125,000 g for 16 h at 4°C. Eleven 1 mL fractions were collected from the bottom of the tube. The dynein fraction peak at about fraction 5, well resolved from the other tubulin and MAPs.

The assays of dynein ATPase activity were performed in 50 μL reaction mixtures containing 20 mM Tris-HCl (pH 7.6.), 50 mM KCl, 5 mM MgSO₄, 0.5. mM EDTA and 1 mM DTT (28). In a standard assay condition, 10 μL of enzyme fractions and 4 mM of ATP were incubated with assay buffer at 37 °C for 40 min. The reaction was then stopped using highly acidic malachite green reagent and the absorbance was read at 660 nm in spectrophotometer (Elx800, Bio-Tek). The amount of inorganic phosphate release in the enzymatic reaction was calculated using the standard calibration curve generated with inorganic phosphate. The control in this assay contained all ingredients of the reaction mixture but the reaction was stopped at 0 time.

3.7. Dynamic analysis of autophagosome movement in CASMCs

CASMCs (2×10⁴/ml) cultured in 35 mm dish were incubated with 12 μl BacMam GFP-LC3B virus particles at 37°C for 16 h to express the LC3B-GFP gene (18). The confocal fluorescent microscopic recording was conducted with an Olympus Fluoview System. The fluorescent images for autophagosomes (LC3B-GFP) of the CASMCs were continuously recorded at an excitation/emission (nm) of 485/520 by using XYT recording mode with a speed of 1 frame/10 second for 10 min. Vesicle tracking was performed in MAGEJ using the LSM reader and Manual tracking plugins according to the published protocol (11). Ten vesicles with GFP-LC3B were chosen at random for each cell. These vesicles were then tracked manually for as long as they were visible, while the program calculated velocities for each frame. All the results were further calculated and analyzed in Excel. The number of cells with different velocity of autophagosomes was calculated.

3.8. Statistics

Data are presented as means ± SE. Significant differences between and within multiple groups were examined using ANOVA for repeated measures, followed by Duncan's multiple-range test. Student's t-test was used to evaluate the significance of differences between two groups of observations. P < 0.0.5. was considered statistically significant.

4. RESULTS

4.1. Attenuation of 7-Ket-induced lysosomal Ca²⁺ release in Smpd1^−/− CASMCs

To test whether impaired autophagy maturation in Smpd1^−/− CASMCs was associated reduced lysosomal Ca²⁺ signaling, we conducted fluorescent imaging analysis to monitor lysosomal Ca²⁺ release by treating Fura-2 loaded CASMCs with 200 μM GPN. GPN was used to release Ca²⁺ from lysosomes by inducing their selective osmotic swelling. 7-Ketocholesterol (7-Ket) is a major oxidized cholesterol product and more atherogenic than cholesterol (29, 30). Since 7-Ket also induces autophagy process in SMCs (31), this oxidized cholesterol product (10 μM, 24 h treatment) was chosen as atherogenic stimulus in the present study as we reported recently (7). As shown in Figure 1A and 1B, GPN-induced Ca²⁺ release was significantly enhanced in Smpd1^+/+ CASMCs with 7-Ket stimulation, which was markedly attenuated in Smpd1^−/− CASMCs. We recently reported that lysosomal Ca²⁺ release in CASMCs is primarily through the transient receptor potential mucolipin-1 (TRPML1) channels in lysosomes (16, 17). As shown in Figure 1C, we demonstrated that downregulation of TRPML1 by gene silencing blocked 7-Ket-enhanced lysosomal Ca²⁺ release in Smpd1^+/+ CASMCs, which indicates that TRPML1 mediates 7-Ket-enhanced lysosomal Ca²⁺ release in Smpd1^+/+ CASMCs. In contrast, overexpression of TRPML1 by its cDNA transfection markedly restored 7-Ket-enhanced lysosomal Ca²⁺ release in Smpd1^−/− CASMCs (Figure 1D), which suggests that TRPML1-meidated lysosomal Ca²⁺ release is impaired in Smpd1^−/− CASMCs.

Figure 1.

Lysosome Ca²⁺ release in response to proatherogenic stimuli in Smpd1^+/+ and Smpd1^−/− CASMCs. Glycyl-l-phenylalanine-β-naphthylamide (GPN, 200 μM) was used to release Ca2+ from lysosomes. Representative lysosomal Ca2+ traces (A) and summarized data (B) showing the effects of Smpd1 gene deficiency on GPN-induced lysosomal Ca²⁺ release in CASMCs (n=7). *P<0.0.5 vs. Ctrl; ^#P<0.0.5 vs. Smpd1^+/+ CASMCs with 7-Ket (10 μM). (C) Effects of TRPML1 gene silencing using TRPML1 siRNA on GPN-induced lysosomal Ca²⁺ release in Smpd1^+/+ CASMCs (n=7). *P<0.0.5 vs. Ctrl; ^#P<0.0.5 vs. Smpd1^+/+ CASMCs Scr with 7-Ket. (D) Effects of TRPML1 gene overexpression using TRPML1cDNA on GPN-induced lysosomal Ca2+ release in Smpd1^−/− CASMCs (n=7). *P<0.0.5 vs. Ctrl; ^#P<0.0.5 vs. Smpd1^−/− CASMCs Scr with 7-Ket.

4.2. Decreased colocalization of Ca²⁺ regions with lysosomes in Smpd1^−/− CASMCs

We further confirmed that lysosomal Ca²⁺ release is impaired in Smpd1^−/− CASMCs by visualizing the lysosomal Ca²⁺ release locally in lysosomes, i.e. we measured the localization of Ca²⁺ around lysosomes by confocal microscopy after labeling cells with fluo-4. As shown in Figure 2A, the colocalization of Ca²⁺ release regions with lysosomes was indicated by yellow spots, which was formed by green fluo-4 signals with rhodamine-red lysosomal marker (Lyso/Rho). Quantification of the colocalization data revealed a significant increase in the colocalization coefficiency of fluo-4-Ca²⁺ and Lyso/Rho in in Smpd1^+/+ CASMCs when they were treated with 7-Ket. However, such colocalization of Ca²⁺ release regions with lysosomes by 7-Ket was markedly reduced in Smpd1^−/− CASMCs. Similarly, we examined the effect of TRPML1 gene silencing or overexpression on the colocalization of Ca²⁺ regions with lysosomes in Smpd1^+/+ or Smpd1^−/− CASMCs, respectively. As shown in Figure 2B, silencing of TRPML1 gene blocked 7-Ket-enhanced colocalization of Ca²⁺ release regions with lysosomes in Smpd1^+/+ CASMCs. As shown in Figure 2C, TRPML1 overexpression significantly restored 7-Ket-enhanced increases in the colocalization of Ca²⁺ release regions with lysosomes in Smpd1^−/− CASMCs.

Figure 2.

Confocal microscopic detection of Ca²⁺ release from lysosomes locally in Smpd1^+/+ and Smpd1^−/− CASMCs. (A) Representative confocal microscopy images showing Ca²⁺ release regions that colocalized with lysosomes as shown by yellow spots formed by green fluo-4 signals with rhodamine-red lysosomal marker (Lyso/Rho). (B) Summarized data showing the effects of Smpd1 gene deficiency on the colocalization coefficiency of Ca²⁺/fluo-4 with Lyso/Rho signals (n=6). *P<0.0.5 vs. Ctrl; ^#P<0.0.5 vs. Smpd1^+/+ CASMCs with 7-Ket (10 μM). (C) Effects of TRPML1 gene silencing using TRPML1 siRNA on 7-Ket-induced colocalization of Ca²⁺/fluo-4 with Lyso/Rho signals in Smpd1^+/+ CASMCs (n=6). *P<0.0.5 vs. Ctrl; ^#P<0.0.5 vs. Smpd1^+/+ CASMCs Scr with 7-Ket. (D) Effects of TRPML1 gene overexpression using TRPML1cDNA on 7-Ket-induced colocalization of Ca²⁺/fluo-4 with Lyso/Rho signals in Smpd1^−/− CASMCs (n=6). *P<0.0.5 vs. Ctrl; ^#P<0.0.5 vs. Smpd1^−/− CASMCs Scr with 7-Ket.

4.3. Inhibited dynein ATPase activation in Smpd1^−/− CASMCs upon 7-Ket stimulation

Dynein is a motor protein responsible for nearly all minus-end microtubule-based transport of vesicles in eukaryotic cells. Recent studies have implicated this protein in autophagosomes trafficking to meet with the lysosomes to form autophagolysosomes, which is a critical step during autophagy maturation (2, 10, 11, 32, 33). We have also recently showed that dynein is involved in autophagosome trafficking in CASMCs (12). Thus, we examined whether ASM deficiency results in deregulated dynein activation during atherogenic stimulation. As shown in Figure 3A, treatment of CASMCs with 7-Ket resulted in a markedly increase in dynein ATPase activity in Smpd1^+/+ CASMCs, which was significantly attenuated in Smpd1^−/− CASMCs or in Smpd1^+/+ CASMCs with ASM inhibitor amitriptyline. As shown in Figure 3B, silencing of TRPML1 gene blocked 7-Ket-induced dynein activation in Smpd1^+/+ CASMCs suggesting that TRPML1-mediated Ca²⁺ is the primary source of lysosomal Ca²⁺ release leading to dynein enhanced activation. As shown in Figure 3C, TRPML1 overexpression significantly restored 7-Ket-enhanced increases in the dynein activation in Smpd1^−/− CASMCs, which further confirms that TRPML1-mediated lysosomal Ca²⁺ signaling controls dynein activity in CASMCs.

Figure 3.

7-Ket-induced dynein activation in Smpd1^+/+ and Smpd1^−/− CASMCs. (A) Summarized data showing the dynein ATPase activity in Smpd1^+/+ and Smpd1^−/− CASMCs or Smpd1^+/+ treated with amitriptyline (Ami, 1 μM) under control or with 7-Ket stimulation (n=6). *P<0.0.5 vs. Ctrl; ^#P<0.0.5 vs. Smpd1^+/+ CASMCs with 7-Ket. (C) Effects of TRPML1 gene silencing using TRPML1 siRNA on 7-Ket-induced dynein activity in Smpd1^+/+ CASMCs (n=6). *P<0.0.5 vs. Ctrl; ^#P<0.0.5 vs. Smpd1^+/+ CASMCs Scr with 7-Ket. (D) Effects of TRPML1 gene overexpression using TRPML1cDNA on 7-Ket-induced dynein activity in Smpd1^−/− CASMCs (n=6). *P<0.0.5 vs. Ctrl; ^#P<0.0.5 vs. Smpd1^−/− CASMCs Scr with 7-Ket.

4.4. Inhibited autophagosome movement in Smpd1^−/− CASMCs upon 7-Ket stimulation

We next examine whether reduced dynein activation contributes to impaired autophagosomes trafficking Smpd1^−/− CASMCs. Autophagosomes are formed randomly throughout the cytoplasm and then transported to lysosomes. However, the majority of lysosomes are localized in the perinuclear region. Therefore, autophagosomes need to be transported in the cytoplasm to meet and fuse with lysosomes. To monitor the autophagosomes movement to lysosomes, we labeled autophagosomes with LC3B-GFP by BacMam technique and determined the velocity of autophagosomes movement in Smpd1^+/+ and Smpd1^−/− CASMCs with 7-Ket. As shown in Figure 4, autophagosomes movement was markedly reduced in the velocity-range between 0.0.1 mm/second and 0.0.9 mm/second in Smpd1^−/− CASMCs compared to that in Smpd1^+/+CASMCs. Moreover, TRPML1 gene silencing has similar inhibitory effects to Smpd1 gene deletion on autophagosome movement in Smpd1^+/+CASMCs, whereas overexpression of TRPML1 recovers the enhanced autophagosome movement in response to 7-Ket stimulation in Smpd1^−/− CASMCs.

Figure 4.

Analysis of autophagosome dynamic movements in 7-Ket stimulated Smpd1^+/+ and Smpd1^−/− CASMCs. The summarized data show the velocity of autophagsomoes in 7-Ket stimulated Smpd1^+/+ and Smpd1^−/− CASMCs, or in Smpd1^+/+ CASMCs with TRPML1 siRNA transfection as well as in Smpd1^−/− CASMCs with TRPML1 cDNA transfection (n=6). *P<0.0.5 vs. Ctrl; ^#P<0.0.5 vs. Smpd1^+/+ CASMCs; ^#P<0.0.5 vs. Smpd1^−/− CASMCs Scr with 7-Ket.

5. DISCUSSION

The purpose of the present study is to explore the roles of TRPML1-mediated lysosomal Ca²⁺ signaling and motor protein dynein in impaired autophagy maturation in ASM-deficient (Smpd1^−/−) CASMCs. In this study, we demonstrated that in CASMCs, ASM deficiency results in deregulated TRPML1-mediated lysosomal Ca²⁺ release signaling. Such impaired lysosomal Ca²⁺ signaling was associated with inhibited dynein activation and defective autophagosome trafficking in ASM-deficient CASMCs.

Accumulating evidence suggest that lysosome serves as another important intracellular Ca²⁺ store similar to the sarcoplasmic reticulum (34). Lysosomal Ca²⁺ can be released to mediate molecular trafficking or recycling and to control vesicular fusion events associated with lysosomes (35, 36). NAADP, a CD38-ADP-ribosylcyclase product, is one of the most potent intracellular Ca²⁺ mobilizing molecules, which mobilizes intracellular Ca²⁺ release in a two-pool mechanism depending upon a lysosome-dependent Ca²⁺ store in arterial SMCs (37, 38). Our previous studies have demonstrated that NAADP antagonist treatment markedly blocked 7-Ket enhanced lysosomal Ca²⁺ release response implicating a role of NAADP in mobilizing lysosomal Ca²⁺ in CASMCs upon proatherogenic stimulation (12). In the present study, we first observed that ASM deficiency abolished in 7-Ket enhanced lysosomal Ca²⁺ release in CASMCs by analyzing lysosomal Ca²⁺ release capacity using fura-2 fluorescence imaging. Further, we confirmed that ASM deficiency reduced colocalization of Ca²⁺ release regions with lysosomes by 7-Ket in CASMCs by directly visualizing the lysosomal Ca²⁺ release locally in lysosomes. Thus, our data for the first time reveal that in ASM-deficient CASMCs, lysosomal Ca²⁺ release response is impaired upon proathergenic stimulation. Together, the data from previous and present studies suggest that NAADP-mediated lysosomal Ca²⁺ release response is impaired in ASM-deficient CASMCs. Such impaired lysosome Ca²⁺ release response may be due to reduced numbers of lysosome Ca²⁺ stores via downregulation of lysosome biogenesis or increased lysosomal alkalizations (34). However, ASM deficiency did not alter either the number of lysosomes or expression of lysosome marker protein Lamp1 in CASMCs indicating that lysosome biogenesis remains unchanged in these ASM-deficient cells (7). Further, ASM deficiency had no effects on the lysosome acidic pH-dependent protease activity indicating that lysosomal pH is independent of ASM expression in CASMCs (7). Therefore, it seems that NAADP-mediated lysosomal release machinery is defective in ASM-deficient CASMCs. In vascular cells, NAADP-mediated regulation of lysosomal Ca²⁺ release and lysosome function is primarily through the activity of the TRPML1 channels (16, 22). ASM is the key enzyme to hydrolyze sphingomyelin to ceramide in lysosomes and its deficiency commonly causes altered `sphingolipid rheostat' with abnormal levels of sphingolipid species including sphingomyelin, ceramide, sphingosine and their phosphorylated metabolites (39), which can be major regulators for Ca²⁺-dependent lysosomal trafficking function (40–42). A recent study has demonstrated that TRPML1-mediated lysosomal Ca²⁺ release is dramatically reduced in cells from Niemann-Pick disease patients (genetically deficient in Smpd1 gene) (42), which is attributed to the inhibitory effects of sphingomyelin accumulation in lysosomal membranes by ASM deficiency on the TRPML1 channel activity. In the present study, we found that downregulation of TRPML1 expression by TRPML1 gene silencing mimics the inhibitory effect of ASM deficiency on 7-Ket enhanced lysosomal Ca²⁺ release, whereas overexpression of TRPML1 could restore the lysosomal Ca²⁺release response in ASM-deficient cells. Therefore, similar inhibitory mechanism for TRPML1 activity may be present in ASM-deficient CASMCs.

One of important findings from the present study is that dynein activation in response to proatherogenic stimulation is inhibited in ASM-deficient CASMCs. To our knowledge, this is the first report showing that ASM controls dynein activity in mammalian cells. Dynein is a multi-subunit microtubule motor protein complex containing two identical heavy chains and the ATPase activity, which are responsible for generating movement of cargo along the microtubules. Previous studies have demonstrated that in mammalian cells, activation of this motor protein promotes nearly all minus-end microtubule-based transport of vesicles and more recently, lysosome and autophagosome trafficking (10, 11). Dynein activity inhibition or its function disruption abolishes lysosome fusion with autophagosomes and causes autophagosome accumulation in a variety of mammalian cells including glioma cells, neuronal cells, or SMCs (10, 12). Previous studies have reported that dynein motor function can be modulated by direct binding of Ca²⁺ to a component of the dynein complex, which causes redistribution of cytoplasmic dynein (13, 14). Therefore, dynein would be a potential target protein modulated by TRPML1-mediated lysosomal Ca²⁺ signaling. In the present study, 7-Ket enhanced dynein activity was blocked in ASM-deficient CASMCs. Similarly, transfection of wild-type CASMCs with TRPML1 siRNA also blocked dynein activation by 7-Ket, whereas TRPML1 overexpression corrected impaired dynein activation in ASM-deficient CASMCs. Thus, our data support the view that ASM controls TRPML1-mediated lysosomal Ca²⁺ signaling and subsequently modulates dynein activity in CASMCs upon proatherogenic stimulation.

Finally, we tested whether ASM deficiency impairs autophagosome trafficking and whether this process is regulated by TRPML1 activity. By quantifying the movement of LC3-GFP-labeled autophagosomes, we demonstrated that autophagosome movement within a cell was enhanced in 7-Ket treated Smpd1^+/+ CASMCs, which was markedly prevented in Smpd1^−/− CASMCs (Figure 4). These results implicate that ASM function is needed for controlling autophagosome trafficking under proatherogenic stimulation. Deficiency of TRPML1 results in impaired lysosome trafficking and autophagolysosomal degradation in human fibroblasts (43). In the present study, we also demonstrated that silencing of the lysosomal Ca²⁺ channel TRPML1 mimics the inhibitory effects of ASM deficiency on 7-Ket-enhanced autophagosome trafficking in CASMCs. Moreover, TRMPL1 overexpression is sufficient to correct the defective autophagosome trafficking in Smpd1^−/− CASMCs. Thus, these results indicate that defective autophagosome trafficking in Smpd1^−/− CASMCs is a consequence of impaired TRPML1-mediated lysosomal Ca²⁺ signaling. Our data also support the view that upon atherogenic stimulation, ASM activity controls the TRPML1-mediated lysosomal Ca²⁺ release response and subsequent dynein-regulated autophagosome trafficking in CASMCs.

In summary, we for the first time link the impaired TRPML1-mediated lysosomal Ca²⁺ signaling due to ASM deficiency to the inhibited dynein activity and consequent defective autophagosome trafficking in CASMCs. Our study provides novel mechanistic insight about how ASM controls the autophagy maturation via regulating TRMPL1-mediated Ca²⁺ and dynein activity.

Abbreviations

7-Ket

7-ketocholesterol

ASM

acid sphingomyelinase

CASMC

coronary arterial smooth muscle cell

GPN

Glycyl-L-phenylalanine 2-naphthylamide

NAADP

Nicotinic acid adenine dinucleotide phosphate

Smpd1

sphingomyelin phosphodiesterase 1

TRPML1

transient receptor potential mucolipin-1