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Collagen Fragment-Induced Smooth Muscle Cell Death
M Hénaff, J Quignard, N Biendon, J. Morel, N. Macrez
To cite this version:
M Hénaff, J Quignard, N Biendon, J. Morel, N. Macrez. Calcium Signaling T-type Calcium Channels Involved In Collagen Fragment-Induced Smooth Muscle Cell Death. Calcium Signaling, 2014. �hal- 02342273�
T-type Calcium Channels Involved In Collagen Fragment-Induced Smooth Muscle Cell Death
M. Hénaff PhD, J.F. Quignard PhD, N. Biendon, J.L. Morel PhD*, N. Macrez PhD*
Abstract— Apoptosis of smooth muscle cells (SMC) due to changes in extracellular matrix has emerged as an important factor underlying vascular and visceral disease progression. The calcium signal induced by extracellular matrix fragmentation also plays a pivotal role in cell death and disease evolution. Here, we found that KDGEA, a particular motif of type I collagen, induced rat duodenal SMC death in cultures through specific interaction with α2β1 integrin signaling as suggested by (1) the absence of KDGAA effect, an inactive analog of KDGEA, or LDV, a fibronectin fragment that recognizes α4β1 integrin, and (2) the inhibition of SMC death induced by KDGEA when α2β1
expression was knocked down by antisense oligonucleotide.
DNA fragmentation was also detected in KDGEA-treated cells, thus suggesting activation of an apoptotic process.
These KDGEA-activated processes were totally blunted by pharmacological modulation of Ca2+ entry through T-type Ca2+ channels and antisense oligonucleotides directed against these channels. KDGEA also increased caffeine-induced calcium release and this effect was abolished when cultures were treated with either antisense oligonucleotide or T-type channel blockers. These results suggested that type I collagen peptide KDGEA can induce the death of SMC through T-type Ca2+ channel activation and alteration of calcium release from sarcoplasmic reticulum.
Keywords — apoptosis, calcium entry, extracellular matrix, integrin
I. INTRODUCTION
ANY diseases are associated with extracellular matrix (ECM) remodeling characterized in some cases by a
pronounced type I collagen deposition, as observed during tissue fibrosis and remodeling (
The MS was submitted March10th, 2014. This work was supported by the Centre National de la Recherche Scientifique, by the Agence Nationale de la Recherche (05-PCOD-029-03 to NM) and by Association Française contre les Myopathies (AFM-12655 and AFM-11794 to JLM).
Université de Bordeaux, INSERM, U885, 146 rue Léo Saignat, 33076 Bordeaux, France (MH, JFQ); Université de Bordeaux, CNRS, UMR5293, Institut des maladies neurodégénératives, 146 rue Léo Saignat, 33076 Bordeaux, France (NB, JLM, NM).
*Correspondence to Nathalie Macrez (e-mail:
nathalie.macrez@u-bordeaux.fr) and Jean-Luc Morel (e-mail:
jean-luc.morel@u-bordeaux.fr).
1, 2). For example, in scleroderma, collagen degradation coincides with SMC rarefaction surrounded by areas of focal fibrosis (3) resulting in hypomotility, lumen dilatation, tensile rigidity and loss of organ functions (4) in pulmonary, peripheral vascular, renal, cardiac and gastrointestinal systems. In vessels, atherosclerosis lesions are rich in type I and III collagens constituting up to 60% of the total plaque proteins (5) that contribute to plaque stability. As atherosclerosis progresses, degradation of fibrous cap collagens and SMC apoptosis coincide with sites of plaque rupture and have both been implied in pathogenesis (6, 7). In other vascular diseases such as aneurysms and dissections of ascending aorta, the inflammatory process leads to protease-mediated degradation of ECM and collagen disruption. These processes act in concert with SMC death to progressively weaken the aortic wall, resulting in dilatation and aneurysm formation or aortic dissections (8, 9).
Although contribution of collagen remodeling and SMC death to various diseases has been well documented, precise mechanisms involved in this phenomenon remain unclear.
Integrins represent a large family of 24 ECM receptors with different ligand specificity, in which α1β1, α2β1, α10β1 and α11β1 form a subset of collagen-binding integrins (10). ECM binding to integrin can change intracellular Ca2+ concentration ([Ca2+]i) in many cell types including platelets, neutrophils, monocytes, lymphocytes, fibroblasts, endothelial, epithelial and SMC. As a result, a myriad of cellular functions such as differentiation, contraction, relaxation or apoptosis are affected (11-13). In vascular smooth muscle for example, damage to ECM after tissue injury or remodeling could be involved in vascular tone modulation. Indeed, the use of peptides and specific antibodies showed that ligands of α5β1 or α4β1 (fibronectin receptors) and αvβ3 (vitronectin receptor) integrins reciprocally regulated Ca2+ flow through L-type Ca2+ current in myocytes of rat skeletal muscle arterioles (12-14).
Disturbances of Ca2+ regulation by sarcoplasmic reticulum (SR) can play an important role in cell death (15). SR Ca2+ overload or depletion can lead to calpain activation, which induces apoptosis through BAD, Bid and caspase-12 (16).
These studies suggest a strong link between abnormal myocyte Ca2+ handling, SR and cell death.
Here, we investigated the role of KDGEA (Lys-Asp-Gly-Glu-Ala), a short soluble collagen type I-derived motif specifically recognized by the collagen-binding integrin
M
15
α2β1 (17) on SMC survival and Ca2+ signaling. We found that KDGEA induced SMC death via Ca2+ entry through T-type Ca2+ channels causing SR Ca2+ signal alteration.
II. METHODS
Cell culture - Investigations conform to the European Community and French guiding principles for care and use of animals. Authorization to perform animal experiments (A-33-00031 to NM) was obtained from the Préfecture de la Gironde (France). Rat duodenum SMC were obtained as previously described (18). Then, SMC were seeded on glass slides (density: 103 cells/mm2) in 24-well plates and maintained in short term primary culture (37°C, 95% air, 5% CO2) for 6 days in M199 medium containing 10% fetal calf serum, 20U/ml penicillin and 20µg/ml streptomycin. Before treatment, SMC cultures were serum-deprived to 0.5% serum overnight.
Cell Treatment - KDGEA (Lys-Asp-Gly-Glu-Ala), KDGAA (Lys-Asp-Gly-Ala-Ala) and LDV-containing peptide (Glu-Ile-Leu-Asp-Val-Ser-Pro-Thr) were purchased from Eurogentec (Serain, Belgium). For survival assessment, peptides were added to cultures (day 6) at different concentrations (10 to 200 µM) for 24 hours before analysis by WST-1 test or DNA gel electrophoresis. In some experiments, 1,2-bis(2-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid tetra acetoxymethyl ester (BAPTA-AM, 10 μM, Fluoprobes-Interchim, France) was added for 30 minutes and removed before KDGEA addition. In other experiments, pharmacological drugs such as Nickel (Ni2+, 50 µM), Mibefradil (1 µM), Pimozide (1 µM), Isradipine (1 µM), Verapamil (10 µM), purchased from Sigma-Aldrich, were applied 30 minutes before KDGEA-treatment. In calcium and electrophysiological experiments, peptides were added 3 hours before analyzes.
Survival assessment - SMC cultures grown to confluence in 24-well plates were treated in quadruplicate for 24 hours as indicated above. At the end of experiment, cells were washed with PBS. Remaining viable adherent cells were assessed using WST-1 test following manufacturer’s instructions (Roche diagnostic, France). Briefly, WST-1 diluted to 1/10 in culture medium was added and plates were incubated (37°C, 30 minutes). The absorbance was measured at 440 nm. Each experiment was performed in quadruplicate in 3 independent cultures.
DNA ladders - In order to determine DNA ladder fragmentation, a hallmark of apoptosis, cell cultures used for WST-1 test were further submitted to total genomic DNA extraction using DNA preparation kit from Epicentre (Madison, WI, USA) and according to manufacturer’s instructions. DNA concentration was analyzed by biophotometer (Eppendorf, Le Pecq, France) and 10 µg was separated by 2% agarose gel electrophoresis containing 0.5 µg/mL ethidium bromide. Gels were photographed under UV illumination by EDAS120 and analyzed with KDS1D 2.0 software (Kodak Digital Science, Paris, France). Experiments were performed in duplicate in 3 independent cultures.
RT-PCR reactions - Total RNA was extracted using RNA preparation kit (Epicentre, Madison,WI), following supplier instructions. Three conditions were tested: RNA from longitudinal layers of duodenal smooth muscle (DR), from SMC freshly isolated (J0) or from our model of cell cultured for 6 days in control (J6) or after antisense oligonucleotide treatment. RNA concentration was determined by biophotometer (Eppendorf, Le Pecq, France). Reverse transcription and polymerase chain reactions were performed as described previously using Qiagen kits (Qiagen, Hilden, Germany) (18). PCR annealing temperatures were: 59.2°C, 57.5°C or 60°Cfor α2, α4 integrins or T-type channel primer pairs, respectively. Sense (s) and antisense (as) oligonucleotides were designed using lasergene software (DNASTAR, Madison, WI, USA) according to sequences deposited in GenBank (accession numbers BC099151, NM001107737 for α2 and α4 integrins, respectively).
Nucleotide sequences for each primer pair were: α2: s:
5'-TGACCGGATACAAACAGACAAG-3', as: 5'-CATAGC CAACAGCAAAAGGATTC-3', α4: s:5'-CAATGCCTCGGTG GTCAATCCT-3', as: 5'-GGGCCCCCATCACAATCAAA T-3', β2-microglobuline: s: 5'-ATTCAGAAAACTCCCCAA ATTCAAGT-3', as: 5'-GATTACATGTCTCGGTCCCAGG T-3'. In some experiments, amplicon relative amounts were determined and normalized to that of β2-microglobuline. Each experiment was repeated twice in 3 independent experiments.
Antisense oligonucleotide treatment - Phosphorothioate antisense oligonucleotides labelled by 5′-Cy5 indocarbocyanin to visualize cellular uptake were designed based on rat α2 integrin sequence in regions lacking known splice variants (asα2: 5'-CCCGGTCAGATACAGGAGTG-3') and synthesized by Eurogentec. T-type calcium channel phosphorothioate antisense oligonucleotide has been previously published (asCaV3: 5'-TCCACCACCACGCCCACAAACATGTT-3') (19). To set free results from Jet-Pei™ transfection, we used as control the sequence 5'-TCACCCAGCACCCCCAACAC ATAGTT-3' named scrambled (SCR) which was not complementary to any rat registered nucleotide sequences.
Antisense oligonucleotides (50 nM) were reconstituted in NaCl 150 mM before transfection in myocytes cultured for 2-4 days with Jet-Pei™ solution according to manufacturer’s instructions. Experiments were performed in 3-5 independent cultures.
Membrane current recordings - Voltage-clamp and current-clamp recordings were made with a standard patch-clamp technique using List EPC-7 patch-clamp amplifier (Darmstadt-Eberstadt, Germany). Whole-cell recordings were performed at room temperature with patch pipettes (resistances:
2-5 MΩ). Membrane potential and current records were analyzed using Pclamp system software (Molecular Devices, Foster City, CA). Junction potential was electronically compensated before acquisition with EPC-7 patch-clamp amplifier (7.1 ± 1 mV, n=5 for K+ solution and 4.5 ± 0.8 mV for Cs+ solution). T-type currents were measured by test pulse from -80 to -30 mV, a potential where T-type channels were mostly activated. L-type currents were measured at 0 mV from a holding potential of -60 mV where T-type channels are inactivated (20). Cells were superfused with extracellular 16
solution (Ca2+ solution) containing (mM): 130 NaCl, 5.6 KCl, 1 MgCl2, 2 CaCl2, 11 glucose, 10 HEPES, pH 7.4 with NaOH.
The basic pipette solution (K+ solution) contained (mM): 5 NaCl, 50 KCl, 65 K2SO4, 2 MgCl2, 2 ATP, 10 HEPES, pH 7.3 with KOH. To record the T-type current more accurately, without contamination by K+ currents, an intracellular Cs+ solution was used (mM): 130 CsCl, 10 EGTA, 5 ATPNa2, 2 MgCl2, 10 HEPES, pH 7.3 with CsOH. Drugs were applied on cells via pressure ejection from a glass pipette.
[Ca2+]i measurement - Measurements of free [Ca2+]i were assessed as described previously (21, 22). Briefly, myocytes were loaded (30 minutes, 37°C) with membrane permeant Ca2+
dye indo-1-AM (10 μM, Molecular probes-Interchim, France).
Fluorescence intensities (F405and F485) were measured for one excitation (λex 340 nm) at two emission wavelengths (λem
405 and 485 nm). Changes in [Ca2+]i were estimated from the changes in the F405/F480 fluorescence ratio (R) as described previously (23). Rbasal is the R value obtained in basal conditions (during 10 s before stimulation). Response amplitudes are expressed as ΔR /Rbasal (deltaR = maximal R value obtained after stimulation - Rbasal). Caffeine was applied by pressure ejection from a glass pipette. All experiments were carried out at 26±1°C.
Statistical analysis - Student's t-tests or for multiple comparisons, one way ANOVA combined with Dunnett and Student-Newman-Keuls post-hoc test were used and considered significant at p<0.05. Results are presented as mean±s.e.m. and n is the number of independent experiments.
III. RESULTS
KDGEA peptide induced smooth muscle cell death.
KDGEA is a particular soluble motif of type I collagen that specifically recognizes α2β1 integrin [17]. To assess peptide effects on cell survival, duodenal SMC primary cultures were treated by KDGEA increasing concentrations for 24 hours.
Fig. 1. KDGEA-induced apoptosis of duodenal SMC in culture. A. Statistical representation of myocyte survival assessed by WST-1 method after 24h treatment with KDGEA, KDGAA (100 µM) or LDV-containing peptide (100 µM), (n=3 in quadruplicate; ***p<0.001). B. KDGEA-induced internucleosomal DNA cleavage. C. RT-PCR expression of α2 and α4 integrins in duodenal smooth muscle tissue (DR), and duodenal smooth muscle cells freshly isolated (J0) or cultured for 6 days (J6).
For low concentrations (10, 50 µM), KDGEA did not modify cell survival (96.8±1.1% and 94.7±2.4%, respectively, n=3) assessed using WST-1 test (Fig. 1A). However, for higher concentrations (100, 200 µM), KDGEA dramatically reduced myocyte survival (62.7±2.5%, n=10 and 54.3±1.2%, n=3, respectively, p<0.001 vs control). Oligonucleosomal-length DNA fragmentation, a hallmark of apoptosis (24), was detected by agarose gel electrophoresis (Fig. 1B) suggesting that KDGEA (100 µM) induces cell death through an apoptotic mechanism.
Other peptides were tested on myocyte survival to check whether KDGEA cell death was mediated specifically by α2β1
integrin. These experiments showed that neither KDGAA (100 µM), the inactive analog of KDGEA, nor LDV-containing peptide (100 µM), a fibronectin sequence that specifically binds α4β1 integrin, induced cell death (104.6±4.9%, n=4 and 92.0±5.8%, n=3, respectively, NS vs control) (Fig. 1A). This 17
result was corroborated by the absence of oligonucleosomal DNA fragmentation (Fig. 1B). α2 and α4 integrin expressions in longitudinal layers of duodenal smooth muscle as well as in SMC freshly isolated or cultured for 6 days were confirmed by RT-PCR (Fig. 1C).
To confirm that KDGEA-induced cell death was mediated through α2β1 integrin activation, we used two antisense oligonucleotides, one to inhibit α2β1 expression, and a scrambled one used as control to set results free from Jet-Pei™
transfection. To demonstrate antisense oligonucleotide efficiency, we analyzed endogenous α2 mRNA expression by RT-PCR in tested conditions. Values are expressed as relative α2 mRNA amounts compared to β2-microglobuline levels (Fig. 2A). The α2 expression was comparable in control and SCR conditions, whereas it was dramatically decreased in asα2-treated cultures.
F ig. 2. A. Evaluation of antisense oligonucleotides directed against α2 integrin by RT-PCR expressed as the ratio of α2 integrin to β2-microglobuline (Falpha2/Fbeta2M) expression (n=3). B. Statistical representation of SMC survival assessed by WST-1 method, in control- or KDGEA-treated cells for 24h, following 3 days of asα2-treatment (n=3 in quadruplicate; ***p<0.001).
In these conditions, in the absence of KDGEA, cell survival showed no difference between SCR and as-α2-treated conditions (Fig. 2B). In contrast, after KDGEA application myocyte survival was highly reduced in SCR conditions (55.4±7.1% n=3, p<0.001) but not in as-α2-knockdown cultures (99.9±4.4%, n=3; Fig. 2B).
Taken together, these results indicated that KDGEA induced duodenal SMC death through specific interaction with α2β1
integrin signaling.
KDGEA-induced cell death was sensitive to calcium entry through T-type calcium channels.
Changes in [Ca2+]i are involved in numerous intracellular events, including triggering of cell death [24,25]. By buffering intracellular Ca2+ to assess its putative role in our apoptotic model, we found that BAPTA-AM (10 µM), a permeant Ca2+
chelator, totally blunted KDGEA-induced cell death (102.9±15.3%, n=3 independent cultures, NS vs control) (Fig.. 3A) and suppressed oligonucleosomal DNA fragmentation (data not shown).
Fig. 3. Pharmacological assessment of calcium sensitivity and entry in KDGEA-induced apoptosis. A. Statistical representation of myocyte survival assessed by WST-1 method in conditions that modulated [Ca2+]i and calcium entry without (open bars) or with KDGEA treatments (black bars; n=4 experiments in quadruplicate. ***p<0.001 as compared to CTL). B. RT-PCR expression of T-type Ca2+ channel pore-forming α1 subunits (α1G, α1H, α1I) of duodenal smooth muscle tissue (DR), and duodenal smooth muscle cells freshly isolated (J0) or cultured for 6 days (J6).
The putative extracellular origin of Ca2+ was first assessed pharmacologically. In control conditions, Ca2+ channel blockers did not modify cell survival (Fig. 3A). In the presence of KDGEA, inhibitors of L-type Ca2+ channels, namely Verapamil (10 µM) and Isradipine (1 µM) did not modify KDGEA-induced apoptosis while T-type Ca2+ channel blockers Pimozide (1 µM), Mibefradil (1 µM) or Nickel (50 µM) totally abrogated it (Fig. 3A). Moreover, RT-PCR analysis revealed that in duodenal smooth muscle as well as in SMC freshly isolated or cultured for 6 days, expression of α1G and α1H subunits but not of α1I were detected (Fig. 3B).
Voltage-gated Ca2+ currents were recorded by a standard patch-clamp technique to functionally confirm their presence in duodenal smooth muscle cells. Figure 4A represents a typical whole-cell Ca2+ current with a two-component inward current evoked by a long depolarization pulse from -80 to -10 mV.
Each component could be isolated on the basis of their voltage dependence of activation and inactivation. The first component was a transient peak inward current (open square) that presented typical features of T-type Ca2+ currents (22). This 18
transient component was followed by a sustained inward component (filled square) that was activated for higher voltage step and inactivated more slowly (Fig. 4A-B), as described for L-type calcium currents (22).
Fig. 4. Functional and pharmacological assessment of T- and L-type Ca2+
channels in rat duodenal SMC. A. Typical current elicited by a step depolarization from -80 to -10 mV, showing a transient peak current (□, due to T-type channels) followed by a sustained current (■, due to L-type channels) and current-voltage relationship measured at the peak (□) and the end of the pulse (■): transient current was activated for lower voltage step (characteristics of T-type channels) than for sustained high voltage-activated L-type channels (holding potential -80 mV). For a step depolarization to -30 mV, the current was mainly due to T-type channels activities. B. 50 µM Ni2+ almost totally inhibited T-type currents (evoked by a step depolarization from -80 to -30 mV, open bars in C) while it weakly inhibited L-type currents (evoked by a step depolarization to 0 mV just after the first step, black bars in C). C.
Concentration-dependent effects of Ni2+, Mibefradil, Isradipine and Verapamil on T-type (open bars) and L-type (black bars) Ca2+ currents using the protocol illustrated in B. Experiments were performed in extracellular Ca2+ solution, and intracellular Cs+ solution.
Having demonstrated that T- and L-type currents coexist in duodenal SMC, we tested if their pharmacological profile was consistent with survival data obtained previously. T-type currents were elicited by test pulse from -80 to -30 mV, a potential where they were mostly activated. L-type calcium currents were measured at 0 mV from a holding potential of -30 mV, a holding potential where T-type channels were inactivated (22). In these conditions, Nickel application (50 µM) inhibited T-type currents but partially reduced L-type currents (Fig. 4B). The ability of Calcium channel blockers to discriminate between T-type and L-type currents is summarized in Figure 4C. These results showed that Mibefradil (1 µM) inhibited preferentially (~70%) T-type currents, whereas Isradipidine (1 µM) and Verapamil (10 µM) produced a significant inhibition of L-type currents with a lower alteration of T-type currents.
To confirm T-type channels’ involvement in KDGEA-induced cell death, we used two antisense oligonucleotides, one to inhibit the expression of T-type channels (asCav3), and a scrambled one used as control to set results free from Jet-Pei™ transfection. RT-PCR expression of T-type channels detected previously was used to assess antisense oligonucleotide efficiency (Fig. 5A). Values are expressed as relative α1G and α1H mRNA amounts compared to β2-microglobuline levels. α1G and α1H mRNA expressions were comparable in control and SCR conditions, whereas they were dramatically decreased in asCav3-treated cultures.
Fig. 5. Inhibition of T-type channel expression by antisense oligonucleotide (asCav3) suppressed KDGEA-induced apoptosis. A. RT-PCR assessment of asCav3 treatment against α1G and α1H isoforms previously detected (maximal effect on mRNA expression measured 3 days after transfection). Fluorescence ratios of α1G (Fα1G/Fbeta2M, open bars) and α1H (Fα1H/Fbeta2M, black bars) to β2-microglobuline indicated the relative mRNA expression in control, scrambled- or asCav3-treated cells (***P<0.001; n= 3 in duplicate). B.
Statistical representation of SMC survival assessed by WST-1 method, in control- or KDGEA-treated cells, following a 3-day asCaV3 treatment (***p<0.001; n= 3 in quadruplicate).
In these conditions, myocyte survival assessment showed no significant differences between SCR and asCav3 cultures (Fig. 5B). However, in KDGEA-treated cells myocyte survival was highly reduced in SCR condition, whereas in asCav3 condition KDGEA did not modify myocyte survival.
All together, these results suggested that the KDGEA impairment of SMC survival was highly dependent upon calcium entry through T-type calcium channels.
19
KDGEA treatment enhanced calcium entry and modified sarcoplamic reticulum calcium signaling.
We further examined the regulation of calcium entry through T-type calcium channel by measuring the effect of KDGEA peptide on T-type current activated by a step depolarisation from -80 to -30 mV. The transient increase in [Ca2+]i in 3h-KDGEA-treated cells was higher than in control cells, as illustrated in Figure 6A and 6B.
Fig. 6. KDGEA-treated cells exhibited a higher [Ca2+]i increase induced by T-type channel activities. A. Typical Ca2+ response elicited by a step depolarization from -80 to -30 mV in control condition or after a 3h-KDGEA treatment. B. Statistical representation of basal [Ca2+]i after a 3h-KDGEA treatment (left) and KDGEA calcium response elicited by a step depolarization (right). Calcium responses were measured as difference between resting basal level and peak of Ca2+ transient. Cells were loaded with indo-1-AM (*p<0.05;
n=7 experiments).
Since calcium entry could induce apoptosis through alteration of [Ca2+]i, mitochondrial and/or SR Ca2+ contents (25), [Ca2+]i changes were recorded using indo-1-AM, in resting conditions or following short applications (10 seconds) of either (1) CCCP, a mitochondrial uncoupler that triggers Ca2+ release from mitochondria (25), or (2) caffeine, an activator of ryanodine receptor that triggers Ca2+ release from SR. In 3h-KDGEA-treated cultures, basal [Ca2+]i did not differ from control conditions (1.37±0.03, n=16 cells in KDGEA-treated conditions vs 1.34±0.04, n=20 cells in control) (Fig. 6B), suggesting that cytosolic calcium homeostasis was conserved during the first hours of KDGEA application.
Application of CCCP caused a slow and transient rise in [Ca2+]i with no difference between control and KDGEA-treated cells (data not shown). In contrast, caffeine application induced a transient increase in [Ca2+]i with a magnitude greater in KDGEA-treated cells than in controls (Fig. 7A). This signal depended upon α2 integrin activation since asα2-treated cultures presented a magnitude of caffeine-induced calcium increase similar to control levels (Fig. 7B). Moreover, in the presence of calcium channel blockers, SR function assessment by caffeine application showed that Isradipine did not prevent calcium signal increase. In contrast, Mibefradil reduced this phenomenon to a control level (Fig. 7B). Furthermore, asCav3 prevented SR calcium increase evoked by caffeine (Fig. 7B).
Fig. 7. SR-calcium alteration evoked by caffeine (10 mM) in KDGEA-treated SMC. A. Intracellular calcium signal recorded following a 10-sec caffeine application in control or KDGEA-treated cells loaded with indo-1-AM. B.
Statistical representation of caffeine-induced [Ca2+]i amplitude in various tested conditions (***p<0.001 as compared with CTL, p<0.001 as compared with KDGEA; n=7 experiments).
Finally, the acute application of KDGEA could increase the T-type Ca2+ current by 5% (not shown).
All together, these results suggested that, in duodenal SMC, SR calcium signalling was altered in KDGEA-induced cell death through α2 integrin interaction without alterations of cytosolic or mitochondrial calcium contents. This phenomenon involved Ca2+ entry through T-type calcium channels.
IV. DISCUSSION
In this study, we provide several lines of evidence that the collagen fragment KDGEA peptide impaired cell survival of cultured duodenal SMC through T-type Ca2+ channel activation and alteration of SR Ca2+ release.
We observed a dramatic reduction of cell survival after treatment of duodenal SMC with KDGEA (100 µM), a fragment of type I collagen. This is in line with previous studies which showed that degraded type I collagen can promote initiated calpain-mediated disassembly of focal adhesions and loss of vascular SMC attachment to ECM (1). This phenomenon is at least partly dependent on α2- and αv-integrins and could promote SMC proliferation, migration and invasion into arterial intima (26, 27). Recently, two studies showed that the same type I collagen fragments could either activate apoptosis of vascular SMC by calpain-mediated inactivation of anti-apoptotic proteins such as xIAP (X-chromosome-linked inhibitor of apoptosis), or promote survival by a mechanism involving αvβ3-dependent NF-κB activation with consequent activation of IAPs (28, 29). Here we showed that, as vascular SMC, duodenal SMC also activated a cellular death process in the presence of a collagen fragment. This SMC death presented the DNA fragmentation feature of apoptosis. Moreover, by our peptidic approach, we have identified that the KDGEA sequence was sufficient to induce this process. KDGEA 20
contains the minimal active recognition sequence for α2β1
integrin that corresponds to the 435-438 residues of alpha 1(I)-CB3 fragment of type I collagen (17). This collagen often predominates in neomatrix of fibrotic smooth muscle tissues such as peripheral vascular and gastrointestinal systems of systemic sclerosis patients or during progression of atherosclerosis (3-9).
KDGEA-induced cell death seemed independent from cellular adhesion since α2β1 integrin knockdown treatment did not modify cell survival, as compared to the scrambled condition. This is consistent with the fact that α2β1-deficient mice are viable, fertile and develop normally (30). This suggests that in our experimental model KDGEA played a pivotal role in cell death induction through its interaction with α2β1 integrin signaling. However fibrosis, and especially collagen degradation and turnover, could produce several peptides which can present different biological activities. The cellular decision between life and death would then be governed by integration of signals from all engaged integrins and could have an impact on disease progression.
Whatever was the cell death activated by KDGEA, the important feature of this study is that this process could be totally blunted by calcium signal modulation. In SMC, as in many other cell types, calcium signaling is a universal signal transduction mediating extracellular effects of environmental signals into a specific cellular response such as contraction, proliferation or apoptosis (31). In the osteoblast-like cell line SaOS-2 (32) as in human dermal fibroblasts (33), the DGEA peptide, which is more acidic than KDGEA, induced Ca2+
transient by mobilizing calcium from intracellular stores when applied at millimolar ranges. In our study, KDGEA at lower concentration (100 µM) impaired cell survival by a calcium-dependent mechanism as shown by the suppression of cell death in cultures pre-treated with the intracellular calcium chelator BAPTA-AM. In SMC, calcium homeostasis is a complex process highly controlled by various sarcolemmal calcium channels and intracellular calcium stores which may be altered during apoptosis. In our model, pharmacological modulation of membrane calcium channels or reduction of T-type calcium channel expression by antisense oligonucleotides blunted KDGEA-induced cell death. This suggested that KDGEA interaction with α2β1 integrin reduced cell survival via calcium entry through T-type calcium channels. Few studies have reported that pharmacological inhibition of T-type calcium channels could protect against apoptosis. For example, T-type calcium channel blockers Mibefradil, Kurtoxin, Nickel, Zinc or Pimozide protected neurons from delayed ischemia-induced damage (34). In a spermatogenic cell model, Mishra et al. showed that 2,5-hexanedione, a metabolite of the industrial solvent n-hexane, caused a significant increase in reactive oxygen species followed by [Ca2+]i enhancement inhibited by Pimozide. This calcium entry through T-type channels altered the Bcl-xS to Bcl-xL ratio, leading to mitochondrial changes resulting in apoptotic death (35). Because of the weak specificity of T-type channel blockers, our results using antisense oligonucleotide were the first direct demonstration
that, in SMC, T-type channel activation was involved in cell death induction. Moreover, interaction between α2β1 integrin and its ligand KDGEA was needed for their activation.
Several lines of evidence suggest that L-type channels are regulated by integrin-dependent signaling. In vascular SMC for example, at least three different integrins modulate their activity and alter vascular tone in arterioles. Whole-cell recordings show that insoluble and soluble αvβ3 integrin ligands (RGD peptides, vitronectin and fibronectin fragments) inhibit L-type currents and elicit vasodilatation (13). In contrast, soluble α4β1 (LDV peptide) as well as insoluble α5β1 ligands enhance L-type calcium currents and vasoconstriction (13, 14), indicating that ECM interactions with integrins modulate vascular tone.
However, T-type channels are quantitatively a weak source of calcium entry compared with L-type channels or with SR calcium release during smooth muscle contraction. Thus, our observations raise the question as to how T-type channels can have a specific effect on SMC death. In cardiac myocytes for example, persistent increases in calcium influx through L-type channels induced SR calcium overload, correlated with apoptosis induction (36). In another study, SR calcium overload or depletion activated calpains, which induced apoptosis through BAD, Bid and caspase-12 (16). Interestingly, T-type channels can sustain a continuous calcium influx by a window current mechanism, corresponding to a voltage range where a channel population fraction is always opened and does not inactivate (20). This property could be responsible for the increase in caffeine-induced calcium release observed in KDGEA-treated SMC. Indeed, in cells treated with T-type inhibitors or antisense oligonucleotides, KDGEA-induced cell death was blunted and caffeine-induced SR calcium signal returned to a control level. Taken together, these results suggest a possible increase in SR calcium loading. This is in agreement with studies showing that SR is a component of inducible apoptosis (37). Indeed, SERCA overexpression in non-myocytes was shown to be proapoptotic (38), whereas cells devoid of SR Ca2+ release channels (IP3 receptor) are resistant to apoptosis (39). In addition, proapoptotic Bcl-2 proteins (BAX and BAK) induced apoptosis in part by increasing SR content, whereas antiapoptotic Bcl-2 proteins protected by decreasing SR calcium content (15, 37, 40). More recently, a study performed on spermatogenic cells showed that Ca2+ entry through T-type channels could regulate the ratio of proapoptotic Bcl-xS and antiapoptotic Bcl-xL expression (35).
However, our study could not exclude an enhanced ryanodine receptor activity. Indeed, in SMC, RGD-containing ligands can also mobilize ryanodine-sensitive Ca2+ stores through increased cyclic ADP ribose, the endogenous activator of ryanodine receptors (41).
T-type Ca2+ channels have been suggested to be implicated in cell death via integrin pathway because Mibefradil could activate apoptosis via the modulation of membrane potential and the modification of integrin expression in HLE-B3 cell line (42). Our results showed thatintegrin activation by KDGEA increases the window current of T-type Ca2+ channels the SR 21
Ca2+ loading and/or Ca2+-induced Ca2+ release known to activate apoptosis. The pathway between integrin and T-type Ca2+ channel activation remains unknown.However, integrin-activated increases of L-type Ca2+ channel activity involved phosphorylation of the channel (43) by src and PKA (44, 45). T-type Ca2+ channels has also been shown to be activated via PKA in SMC (46) and src in neuroblastoma cell line (47). Therefore, PKA and src are good candidates to link T-type Ca2+ channels activation to integrin stimulation by KDGEA in SMC.
V. CONCLUSIONS
Collectively, these data suggest a crucial role for T-type channel activation and SR calcium signal in smooth muscle cell death induced by KDGEA. This study could give new perspectives of treatment in pathogenesis processes in which SMC rarefaction coincide with type I collagen deposition and alteration of Ca2+ signaling.
VI. ACKNOWLEDGMENTS
Acknowledgments
We thank Pr. Chantal Mironneau and Dr. Jean Mironneau for scientific support and helpful discussion, and Dr. Anne Prévot for proof reading. All authors declare that they have no conflict of interest.
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