Role of Purinergic Receptor in Alpha Fodrin Degradation in Par C5 Cells

Abstract

Autoantibodies specific for alpha-fodrin fragments are found in the tissues of persons afflicted with Sjögren’s syndrome (SS). However, the mechanism for alpha-fodrin degradation remains elusive. The following experiments utilized Par C5 cells to examine the role of P2X7 receptor (P2X7R) in apoptosis, particularly in the cleavage and release of alpha-fodrin, an apparent SS autoantigen. Five mM ATP stimulation induced apoptotic cell death with a sustained Ca²⁺ influx, which was mimicked in HEK cells transfected with P2X7R. ATP also induced cleavage of alpha-fodrin mediated by caspase-3 and calpain, releasing alpha-fodrin fragments through membrane blebs. However, both apoptotic cell death and alpha-fodrin cleavage were inhibited in the presence of 300 μM oxidized-ATP (ox-ATP), an irreversible blocker of P2X7R, or in Ca²⁺-free solution. We concluded that P2X7R plays an important role in apoptosis and alpha-fodrin degradation in salivary epithelial cells, providing an important clue elucidating the presence of alpha-fodrin fragments in SS tissues.

INTRODUCTION

Sjögren’s syndrome (SS) is an autoimmune disease affecting the function of exocrine organs, including the salivary glands. Primary Sjögren’s syndrome (pSS) is characterized by the presence of autoantibodies binding to a number of organ-specific and non-specific autoantigens. Beside anti-Ro/ La RNP complex and anti-muscarinic autoantibodies (Li et al., 2004), auto-antibodies against autoantigens such as alpha- and beta-fodrin and poly (ADP) ribose polymerase (PARP) have been identified in a subpopulation of persons with SS (Routsias and Tzioufas, 2007). The cleaved products of alpha-fodrin in both a mouse model and an SS subpopulation have also been identified (Maruyama et al., 2004; Kahaly et al., 2005; Wang et al., 2006; Willeke et al., 2007). Autoimmune disorders are closely related to aberrant apoptosis, and the by-products of apoptotic cells have been shown to generate both organ-specific and systemic antibodies (Bellone, 2000; Maniati et al., 2008).

Extracellular ATP acts as an excitatory transmitter and regulates many physiological events, such as cytokine release and salivary secretion (Surprenant et al., 1996; Novak, 2003). Purinergic receptors, P2X and P2Y, are expressed in salivary acinar cells. Among the subtypes of purinergic receptors, the persistent activation of P2X7R as a non-selective Ca²⁺-permeable cation channel (North, 2002) induces an increase in intracellular Ca²⁺, considered a key element triggering apoptosis (Tombal et al., 2002; Nobile et al., 2003). However, the role of P2X7R in apoptotic cell death and its signaling pathways in salivary epithelial cells have not been rigorously studied. We hypothesized that apoptotic cell death in the Par C5 cell line, including cleavage of alpha-fodrin and its release through membrane blebs, is P2X7 receptor-mediated. The results of this study may provide an important clue elucidating the presence of the alpha-fodrin fragments in SS tissues.

MATERIALS & METHODS

Reagents

ATP, oxidized ATP (ox-ATP), was obtained from Sigma (St. Louis, MO, USA). Rho-associated kinase (ROCK-I) inhibitor (Y-27632) and calpain inhibitor (PD 151746) were obtained from CALBIOCHEM (San Diego, CA, USA). Fura-2 A/M (fura-2) was obtained from Molecular Probes (Eugene, OR, USA). Caspase-3 antibodies and secondary anti-rabbit antibody conjugated to HRP were purchased from Cell Signaling (Beverly, MA, USA). Mouse anti-calpain and mouse monoclonal antibody were obtained from Chemicon (Temecula, CA, USA). Alpha-fodrin (spectrin alpha-II) goat polyclonal antibody, donkey anti-goat IgG-HRP, and donkey anti-goat IgG-FITC were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Cell Cultures

A previously established and characterized rat parotid acinar cell line, Par C5, was cultured in 60-mm culture dishes in DMEM/F12 (1:1 mixture) supplemented with 2.5% fetal calf serum, 5 μg/mL transferrin, 1.1 μM hydrocortisone, 0.1 μM retinoic acid, 2.0 nM T3, 5 μg/mL insulin, 80 ng/mL epidermal growth factor, 4 mM L-glutamine, 50 μg/mL gentamicin sulfate, and a trace element mixture (Limesand et al., 2003).

RT-PCR

Amplification conditions were 60 sec at 94°C, 30 sec at 55°C, 45 sec at 72°C for 40 cycles, and, finally, 5 min at 72°C. The primers used [forward, reverse, and product size (bp)] were as follows: (P2X1) 5′-TGGGTGGGTGTTTGTCTATG-3′ and 5′-TGAAGTTGAAGCCTGGAGAC-3′ 738; P2X2, 5′-TCCA-TCATCACCAAAGTCAA-3′ and 5′-TTGGGGTAGTGGATGC-TGTT-3′ 391; P2X3, 5′-TTGAGGGTAGGGGATGTGGT-3′ and 5′-GCTGATAATGGTGGGGATGA-3′ 326; P2X4, 5′-AGG-GCTACCAGGAAACGGAC-3′ and 5′-GATTGTGCCAAGA-CGGAATA-3′ 550; P2X5, 5′-TGAAGGGTGGTGTGATAGG- AA-3′ and 5′-GTTGATGACTGTGGGGATGA-3′ 268; P2X6, 5′-CTGTGGGATGTGGCTGACTT-3′ and 5′-TCAAAGTCCC- CTCCAGTCAT-3′ 484; P2X7, 5′-CGACAAGTACACCAATG- AGTCCC-3′ and 5′-GGCTCGTCCACAAAGGACA-3′ 384; P2Y1, 5′-TCCTCTTCATTCCAATGTGCC-3′ and 5′-TCTTC- TTCTTGAGCCTGCCCAG-3′ 391; P2Y2, 5′-GGGACGAACT- GGGTTACAAATGT-3′ and 5′-GGTGTGGCAACTGAGGTC- AAGTG-3′ 785; P2Y4, 5′-CAACCAATGCCAATGGAACT- ACC-3′ and 5′-ACTTGTCCCCCGTGAAGAGATAG-3′ 414; P2Y6, 5′-TGCTTGGGTGGTATGTGGAGTC-3′ and 5′-TGAT-GTGTGAAGTAGAAGAGGATAGGG-3′ 489; P2Y12, 5′-TCA- GCCAACACCACCTCCATTCC-3′ and 5′-CCAGACCAAA- CTCCGACTTCAAG-3′ 544; and GAPDH, 5′-CCATCACC- ATCTTCCAGGAG-3′ and 5′-CCTGCTTCACCACCTTCTTG-3′ 576.

Cytotoxicity Assay and Cell Death Experiments

The cytotoxicity of increasing ATP concentrations (from 0 to 10 mM) was determined by the methyl tetrazolium cytotoxicity (MTT) assay (Young et al., 2005). The numbers of cells in each phase of the cell cycle were determined by means of a FACSort flow cytometer (Becton Dickinson, San Jose, CA, USA) after the DNA was stained with propidium iodide (PI). The formation of oligonucleosome-sized fragments was viewed following DNA (10 μg) fractionation on 1.5% agarose gel and ethidium bromide staining.

Intracellular Ca²⁺ Measurement

Intracellular Ca²⁺ concentration ([Ca²⁺]_i) was measured in Par C5 and HEK293 cells loaded with 2 μM fura-2 for 30 min at 37°C. The cells were incubated in the normal solution for 10 min prior to the experiments. Fura-2-loaded cells were excited at 340/380 nm, and changes in [Ca²⁺]_i were measured under an inverted microscope (Olympus IX70, Tokyo, Japan). The normal solutions contained 140 mM NaCl, 5 mM KCl, 1 mM MgSO₄, 1 mM CaCl₂, 10 mM HEPES, and 10 mM glucose. We prepared the Ca²⁺-free solution by replacing CaCl₂ with EGTA.

Transfection of HEK293 Cells and Selection of Stable Clones

HEK293 cells and HEK-P2X7R cells were maintained in DMEM supplemented with 5% fetal calf serum and antibiotics (50 U/mL penicillin and 50 μg/mL streptomycin). Hygromycin (25 μg/mL) was added to the media containing HEK-P2X7R cells. Cells were grown in 24-well culture plates in a humidified atmosphere of 5% CO₂ at 37^oC.

Immunoblotting

Par C5 cells were homogenized in lysis buffer [50 mM Tris, pH 7.5, 1% Triton X-100, 100 mM NaCl, 10 mM tetrasodium pyro-phosphate, 10 mM NaF, 1 mM EDTA, 1 mM NaV, 1 mM EGTA, 1 mM phenylmethylsulphonyl fluoride (PMSF), and 1 μg/mL aprotinin, leupeptin, and pepstatin], followed by gentle sonification in ice. Following protein concentration determination, the proteins were separated on SDS-polyacrylamide gel, transferred electrophoretically to polyvinylidene fluoride (PVDF) membranes, and incubated with primary antibody.

Immunocytochemistry

Par C5 cells were grown in DMEM/F12 medium and allowed to adhere to coverslips for 2 hrs at 37^oC in 5% CO₂. Treated (5 mM ATP) and untreated cells were fixed in ice-cold 96% ethanol for 5 min at 4^oC or by 4% paraformaldehyde for 10 min at room temperature. Fixed cells were washed in PBS 3X for 5 min and then placed in blocking solution (2% BSA, 5% FBS, and 0.1% Triton X 100). The cells were then incubated overnight at 4^oC with primary Ab, alpha-fodrin goat polyclonal Ab. After being washed, the cells were incubated at room temperature for 1 hr with secondary Ab, anti-goat IgG-FITC. For negative control, cells were stained only with secondary Ab.

TCA Precipitation

Proteins were precipitated by the addition of trichloroacetic acid (TCA) and cholic acid to each medium sample. The precipitated proteins were washed twice with 100% acetone to extract residual TCA and then dissolved in SDS-PAGE sample buffer. Triton X-100 detergent-extracted cell lysates and TCA-precipitated proteins from the extracellular medium were separated via SDS-PAGE electrophoresis with 8% gels, and Western blot analyses were performed.

RESULTS

Cell Death in Par C5 Cells Mediated by P2X7 Receptor (P2X7R)

We investigated the subtypes of P2X and P2Y receptors expressed in Par C5 cells. Par C5 cells expressed both transcripts of P2X4 and P2X7 receptors, and various P2Y receptors, including P2Y1, P2Y2, P2Y4, P2Y6, and P2Y12 (Fig. 1A). We then stimulated Par C5 cells with increasing concentrations of ATP (2, 4, 6, 8, and 10 mM) at various incubation periods (4, 8, 12, and 16 hrs) to find the critical concentration of ATP-induced cell death, as assessed by the methyl tetrazolium (MTT) assay (Fig. 1B ). The total cell death population was increased in proportion to both ATP concentration and incubation periods. The EC₅₀ value was estimated as 5.0 ± 0.1 mM (n = 20, mean ± SEM); thus, we used 5 mM ATP to induce cell death throughout the experiments. Stimulation of Par C5 cells with 5 mM ATP for 2 hrs increased the proportion of sub-G1 population cells assessed by FAC analysis (shifting the axis to the left), indicating increased cell death (Fig. 1C ). However, ATP stimulation in the presence of 300 μM oxidized-ATP or in Ca²⁺-free bath solutions did not change the proportion of sub-G1 cells. ATP stimulation for 6 hrs induced DNA fragmentation in Par C5 cells (Fig. 1D ). However, DNA fragmentation was also inhibited either in the presence of 300 μM ox-ATP or in Ca²⁺-free bath solutions. We found that 500 μM Bz-ATP, a specific P2X7R agonist, and 5 mM ATP induced membrane blebbing in 43% and 61% of cells, respectively. However, 1 mM UTP (P2Y2, P2Y4, and P2Y6 agonists) and 1 mM ADP (P2Y1 and P2Y12 agonists) did not induce membrane blebs (data not shown).

Increase in Cytoplasmic Free Calcium Concentration ([Ca²⁺]_i) Mediated by P2X7 Receptors in Par C5 and HEK-P2X7 Cells

Our results from FACS analysis and DNA fragmentation suggest that cell death depends on the increased [Ca²⁺]_i. Thus, we characterized P2X7 receptor-mediated Ca²⁺ mobilization in Par C5 cells. In a normal bath solution containing 1 mM Ca²⁺, 100 μM and 5 mM ATP increased [Ca²⁺]_i in a biphasic manner (Fig. 2A ). The initial increase in [Ca²⁺]_i was followed by a sustained elevation, which was higher than the basal [Ca²⁺]_i level. Compared with 100 μM ATP, the sustained increase in [Ca²⁺]_i was more prominent following stimulation with the 5 mM ATP. Since we could not rule out that the ATP-stimulated sustained [Ca²⁺]_i increase was mediated by other types of P2X receptors, we cloned the P2X7R and transfected it into the HEK cells which do not endogenously express P2X subtype receptors. In the wild-type (W/T) HEK cells (Fig. 2B ), both 100 μM and 5 mM ATP induced a transient [Ca²⁺]_i increase; however, a sustained [Ca²⁺]_i increase was not measured. In the transduced HEK (HEK-P2X7R) cells, the sustained [Ca²⁺]_i increase was measured following stimulation with 5 mM ATP, but was not induced by the lower dose of 100 μM ATP (Fig. 2C ). In Ca²⁺-free bath solution, the sustained [Ca²⁺]_i increase following 5 mM ATP stimulation in HEK-P2X7R cells was absent (Fig. 2D ).

ATP-induced Cleavage of Alpha-fodrin and Its Signaling Pathway

We next examined whether 5 mM ATP induced the cleavage of alpha-fodrin in Par C5 cells. Following ATP stimulation for 2 hrs, the 240-kDa alpha-fodrin protein was cleaved into smaller fragments, including 180 kDa, 150 kDa, and 120 kDa (Fig. 3A ). However, the ATP-induced cleavage of alpha-fodrin was inhibited by pre-treatment with 300 μM ox-ATP or in Ca²⁺-free solution. Further investigating the signaling pathways associated with alpha-fodrin cleavage, we found that 5 mM ATP induced caspase-3 activation after 100 min (Fig. 3B ), which was markedly attenuated in the presence of Ox-ATP and Ca²⁺-free solution (Fig. 3C ). Five mM ATP also induced calpain activation in amounts proportional to the incubation periods (Fig. 3D ). Similarly, ATP-stimulated calpain activation was also inhibited by 300 μM ox-ATP, or in Ca²⁺-free solution (Fig. 3E ). These results demonstrate that P2X7R stimulation by ATP induces activation of caspase-3 and calpain in the presence of extracellular Ca²⁺. Utilizing specific inhibitors, we investigated further the effect of caspase-3 or calpain on the cleavage of alpha-fodrin. Caspase-3 inhibitor, 300 μM DEVE-fmk, or calpain inhibitor, 20 μM PD 151746, partially blocked cleavage of alpha-fodrin (Fig. 3F ). However, simultaneous application of both inhibitors completely blocked cleavage of alpha-fodrin (Fig. 3F , lane 5).

Release of Cleaved Alpha-fodrin through Membrane Blebs

Following treatment with 5 mM ATP, the cleaved alpha-fodrin was localized by immunocytochemistry in the Par C5 membrane blebs (Fig. 4A , arrow). No staining was evident in negative control cells (data not shown). Pre-treatment with 300 μM ox-ATP, 10 μM Y-27632 (Rho-associated kinase ROCK-I inhibitor), or in Ca²⁺-free solution prevented the localization of the cleaved alpha-fodrin fragments in the membrane blebs following ATP stimulation (Fig. 4A ). Utilizing TCA precipitation techniques, we isolated intact (240 kDa) alpha-fodrin protein from the untreated Par C5 pellets (Fig. 4B , lane 1). The partially cleaved (150 kDa) alpha-fodrin was found in both the cell pellets and extra-cellular medium following ATP stimulation (Fig. 4B , lanes 2 and 3). However, in the presence of 300 μM ox-ATP or 10 μM Y-27632, ATP-stimulated release of 150 kDa alpha-fodrin into the extracellular medium was completely inhibited (Fig. 4C , lanes 1 and 3). ATP-induced alpha-fodrin release was also not observed in Ca²⁺-free solution (Fig. 4C , lane 2).

DISCUSSION

Extracellular ATP is not a specific P2X7 agonist; however, ATP does activate P2X7R in various tissues (Surprenant et al., 1996; North, 2002; Novak, 2003). Our results strongly suggest that ATP induced cell death in Par C5 cells through P2X7R. First, we found that Par C5 cells expressed P2X7R transcript. Second, the P2X7R antagonist, ox-ATP, completely inhibited the cell-death-associated changes stimulated by ATP. Third, with the exception of Bz-ATP, a specific P2X7R agonist, other types of P2 agonist did not induce membrane blebs. Finally, ATP induced a sustained [Ca²⁺]_i increase in Par C5 cells, which was measured in the HEK cells only following transfection with P2X7R.

The ATP-induced increase of DNA fragmentation and the proportion of cells in the sub-G1 phase in our experiment were Ca²⁺-dependent, since this apoptotic phenomenon was not observed in Ca²⁺-free solution. These results are consistent with previous reports demonstrating that both endonucleases responsible for DNA fragmentation and an increase in sub-G1 population were dependent on calcium (Orrenius et al., 2003; Jung et al., 2008). In the Par C5 cells, we also observed that P2X7 receptor-mediated Ca²⁺ mobilization by 5 mM ATP induced a sustained increased in [Ca²⁺]_i, which was not observed in the presence of ox-ATP, the irreversible blocker of P2X7R, or in Ca²⁺-free bath solution. The sustained Ca²⁺ influx mediated by P2X7R was further confirmed in transfected HEK cells. Thus, our results strongly suggest that sustained Ca²⁺ influx is mediated by P2X7R, and that Ca²⁺ plays an important role in apoptotic cell death. Changes in intracellular Ca²⁺ also played a putative role in inducing cytotoxicity and apoptotic cell death in various cell types (Tombal et al., 2002; Mattson and Chan, 2003; Wang et al., 2004; Zhang et al., 2005).

Analysis of our data demonstrates that P2X7R mediates the cleavage of alpha-fodrin through caspase-3 and calpain in Par C5 cells. Although we cannot rule out that alpha- fodrin is cleaved due to other mechanisms associated with apoptosis, proteolysis, specifically of alpha-fodrin, has been characterized by a variety of stimuli, including calcium-dependent activation of calpain and caspases-3 (Wang, 2000; Nagaraju et al., 2001; Sergeev et al., 2006). We also found that 150-kDa alpha-fodrin fragments are released into the extracellular medium through Par C5 membrane blebs. The presence of 150- or 120-kDa alpha-fodrin fragments in the surrounding interstitium has also been detected in apoptotic acinar/ductal epithelial cells in the SS mouse model (Maruyama et al., 2004; Miyazaki et al., 2005; Sisto et al., 2006; Wang et al., 2006; Willeke et al., 2007).

Thus, all our results strongly suggest that alpha-fodrin in Par C5 cells is cleaved and released into the extracellular medium by P2X7R stimulation. Release of alpha-fodrin fragments may act as an autoantigen, stimulating the production of autoantibodies and mediating various autoimmune responses found in SS. To our knowledge, this is the first report characterizing P2X7R-mediated alpha-fodrin degradation and release in salivary epithelial cells. Our understanding of the role of P2X7R-mediated triggering of apoptotic cell death and degradation of alpha-fodrin may provide a rational basis for effective therapies directed toward SS-associated xerostomia (dry mouth) and oral health deterioration.

Figure 1.

ATP-induced apoptosis in Par C5 cells. (A) RT-PCR analyses with total RNA. Par C5 cells expressed both transcripts of P2X4 and P2X7 receptors, and various P2Y receptors, including P2Y1, P2Y2, P2Y4, P2Y6, and P2Y12. Lane ‘M’ indicates kb markers, and GAPDH was used as the positive control. (B) Viability test for ATP-treated cells with the MTT assay. Cells were treated with 2 to 10 mM ATP for 4 hrs (▪), 8 hrs (♦), 12 hrs (▴), and 16 hrs (•), EC₅₀ value was estimated as 5.0 ± 0.1 mM (n = 20, mean ± SEM). (C) ATP-induced shift of sub-G1 population in Par C5 cells. Flow cytometric analysis for untreated cells (Control), cells stimulated with 5 mM ATP alone (ATP), or in the presence of 300 μM ox-ATP (ATP + ox-ATP), or in Ca²⁺-free solution (ATP + Ca²⁺-free). ATP stimulation for 2 hrs shifted the axis of the sub-G1 cell population to the left, from 3.99% to 91.42%. (D) DNA fragmentation in Par C5 cells after ATP stimulation for 6 hrs. DNA fragmentation was visualized on 1.5% agarose gel stained with ethidium bromide (left M, Lambda/EcoRI+HindIII; right M, pWON600/EcoRI+HinfI). Control cells were incubated with media alone.

Figure 2.

Measurement of cytoplasmic free Ca²⁺ concentration ([Ca²⁺]_i) in Par C5 and P2X7R transfected HEK (HEK-P2X7R) cells loaded with 2 μM fura-2. (A) 5 mM ATP induced a sustained increase in [Ca²⁺]_i in Par C5 cells. (B) An ATP-induced increase in [Ca²⁺]_i in wild-type HEK cells (HEK-W/T) and (C) transfected HEK-P2X7R cells in the presence of Ca²⁺, and (D) the absence of [Ca²⁺]_i. Each tracing is a representative of at least 10 separate experiments.

Figure 3.

ATP-induced cleavage of alpha-fodrin in Par C5 cells. (A) Cleavage of 240-kDa alpha-fodrin by 5 mM ATP in the presence or absence of 300 μM ox-ATP or Ca²⁺-free solution. (B) ATP induced caspase-3 activation in a time-dependent manner. (C) Partial inhibition of ATP-induced caspase-3 activation by pre-treatment with 300 μM ox-ATP or in Ca²⁺-free solution. (D) ATP induced calpain activation in a time-dependent manner. (E) Inhibition of calpain activation by pre-treatment with ox-ATP or in Ca²⁺-free solution. (F) Inhibition of alpha-fodrin cleavage by DEVE-fmk (lane 3), PD 151746 (lane 4), or simultaneous application of both inhibitors (lane 5).

Figure 4.

Subcellular distribution and release of cleaved alpha-fodrin. (A) Subcellular location of cleaved alpha-fodrin in Par C5 cells following 5 mM ATP stimulation for 2 hrs. Cleaved alpha-fodrin is present only in the cell pre-treated with ATP alone, and fragments are localized in the membrane blebs (indicated by the arrow). Membrane blebs filled with alpha-fodrin are not present in cells pre-treated with ox-ATP, Y-27632, or in Ca²⁺-free solution. Scale bar, 20 μm. (B) Cleaved alpha-fodrin (150 kDa) in apoptotic cells (lane 2) and in extracellular medium (lane 3) detected by the TCA precipitation technique. (C) 5 mM ATP-stimulated release of 150-kDa alpha-fodrin into the extracellular medium (Ext.med) in Ca²⁺-containing solution (lane 4) or after pre-treatment with ox-ATP (lane 1), in Ca²⁺-free solution (lane 2), or by Y-27632 (lane 3).

Footnotes

Notes

Acknowledgements

This work was supported by the Korea Science & Engineering Foundation through the Oromaxillofacial Dysfunction Research Center for the Elderly (R13-2008-008-01001-0) at Seoul National University.

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