Calcium elevations in MIN6 cells evoked by extracellular human islet amyloid polypeptide involve activation of the mechanosensitive ion channel Trpv4

Introduction

Islet amyloid polypeptide (IAPP), also known as amylin, is the main component of islet amyloid (1,2), which is found within the pancreatic islets of Langerhans in subjects with type 2 diabetes mellitus (T2DM) (3,4). Because deposits of islet amyloid colocalize with areas of cell degeneration, the process of amyloidosis has been strongly associated with the progressive loss of pancreatic β-cell mass that is one of the hallmarks of type 2 diabetes (5–8). A target region of human IAPP (hIAPP) between positions 20 and 29 is thought to be responsible for the formation of amyloid fibrils (9). Rodent IAPP (rIAPP), which differs from the human sequence at several residues within this critical region, is not amyloidogenic (9).

The process of ordered aggregation into amyloid fibrils is a common event in many cell-degenerative diseases, including Alzheimer’s disease, Pakinson’s disease, prion-protein related encephalopathies and several other local and systemic amyloidoses (10). Although the peptides that form amyloid deposits show no apparent primary sequence homology, they all adopt a β-strand secondary structure after conversion to fibrils (11), suggesting that partially folded states serve as the precursors for nucleation (12–14). However, the toxicity of amyloidogenic peptides appears to lie in the oligomeric intermediates rather than the mature fibrils (15,16). Thus, the conversion of a normally soluble protein into amyloid can give rise to a gain in toxic function, providing a common mechanism by which amyloid contributes to disease pathogenesis through cell toxicity and cell death (17).

Amyloid peptides have been reported to induce death cell by apoptosis (6,18–20), which may result from the disruption of calcium homeostasis (21). Indeed, in MIN6 model pancreatic β-cells, we recently showed that hIAPP triggered calcium changes that were associated with the induction of apoptosis via a pathway involving activation of the endoplasmic reticulum (ER) stress response (22). The elevation of intracellular calcium in neurons and astrocytes has been reported to follow the formation and extracellular deposition of several amyloid peptides, including hIAPP (23–27), and a range of potential underlying mechanisms for the increased calcium permeability have been proposed. In cell-free systems such as phospholipid bilayers, hIAPP amyloid has been reported to form cation-permeable channels as a result of its insertion into membranes (28), but also to destabilize the membrane structure in parallel with the measured kinetics of hIAPP fibril growth (29) and to cause a non-specific increase in conductance (16,30–33). In addition, however, activation of cell surface receptors coupled to calcium influx has been reported in a neural cell line exposed to amyloid β peptide aggregation (34). Although diverse, these mechanisms are compatible with the idea that the cytotoxicity of hIAPP is initiated on the cell surface and requires close contact between the aggregation process and the β-cell plasma membrane (6). In regions where this occurs, the plasma membrane of pancreatic β-cells in amyloidogenic islets in vivo is usually characterized by ultrastructural morphological abnormalities (2,35–37).

The aim of the present study was to investigate the mechanism by which hIAPP fibril formation results in calcium influx across the plasma membrane of MIN6 cells and its association with apoptosis.

Methods

Results

As reported previously, hIAPP was observed to aggregate spontaneously in solution over the course of 2 hours (39). Electron microscopy of freshly prepared solutions containing 10 μM hIAPP showed that prefibrillar hIAPP structures were first observed after 10 min, with maximum formation after 30-45 min (Fig. 1A, left panel). At 2 hours we also detected fibrils of hIAPP (Fig. 1A, right panel). The development of hIAPP fibrils was confirmed by monitoring the fluorescence of ThT at 482 nm, the intensity of which reflects the binding to, and hence the formation of, amyloid fibrils. In solutions containing 10 μM hIAPP, but not 10 μM rIAPP, ThT fluorescence increased throughout the 2 hour incubations, indicating the progressive formation of fibrils (Fig. 1B).

To investigate whether hIAPP increases cytosolic calcium concentrations in the insulinoma cell line MIN6, we monitored the 340/380 nm fluorescence ratio in cells loaded with the calcium indicator dye, fura-2. When MIN6 cells were incubated in 10 μM hIAPP for 2 hours, we observed a >1.4-fold elevation of the fluorescence ratio in 21.3% of cells (70/328), indicative of a rise in the intracellular calcium concentration (Fig. 2A-C). By contrast, no calcium changes of this magnitude were observed in 128 cells treated with rIAPP or in 87 cells incubated without additions (Fig. 2D,E) (p<0.001 vs hIAPP). In the cells incubated with hIAPP, the latency of the calcium rise had a bimodal distribution, with a first peak occurring at 25-30 min and a second at 75-90 min (Fig. 2B). The timing of the first peak coincides with the first appearance of IAPP deposits by light microscopy. In those cells that exhibited a rise in [Ca²⁺]_i, the 340/380 nm ratio increased progressively until, in many cases, the cell was observed to detach from the glass dish, thereby preventing calibration of the calcium signal in individual responsive cells. The mean peak ratio before detachment in the cells that exhibited a calcium rise was 2.0 ± 0.39 (n=41), similar to the maximum ratio of 1.9 ± 0.54 (n=72) in the remaining cells treated at the end of the experiment with ionomycin and 5 mM Ca²⁺. This suggests that the calcium concentration in affected cells rose to at least 1 μM.

We next investigated whether the rise in [Ca²⁺]_i was dependent on calcium entry through voltage gated Ca²⁺ channels. Inclusion of 5 μM nifedipine in the incubation medium significantly abolished the hIAPP-triggered rise in the fura-2 ratio (0/198 cells responded, p<0.001 compared with hIAPP alone) (Fig. 3A), suggesting that L-type Ca²⁺ channels contribute to the calcium rise in MIN6 cells. As L-type Ca²⁺ channels normally only open at a threshold membrane potential of approx -40 mV, the most likely explanation for their recruitment by hIAPP is as a consequence of membrane depolarisation. To investigate the role of Na⁺ ions in any initial depolarisation, we repeated the experiments in buffer containing choline⁺ in place of Na⁺. In the absence of hIAPP, the calcium concentration in MIN6 cells was less stable in this Na⁺-free buffer, and tended to rise slightly during the 2 hour incubation, with 4/187 cells (2.1%) showing a fluorescence ratio change of >1.4-fold (Fig. 3B). This may be explained by Na⁺/Ca²⁺ exchangers operating in reverse when extracellular Na⁺ is removed, resulting in persistent Ca²⁺ influx, as described previously in β-cells (40). Removal of extracellular Na⁺ did not, however, prevent the larger calcium responses to hIAPP, which were observed in 8.2% of cells (7/85, p<0.01 compared with Na⁺-free buffer alone) (Fig. 3C). Interestingly, however, in Na⁺-free buffer nifedipine was no longer able to abolish hIAPP-triggered calcium responses, which were still observed in 8.4% of cells (13/154: not significantly different from the response rate with hIAPP in Na⁺-free buffer alone) (Fig. 3D). This suggests that under Na⁺-free conditions, hIAPP activates a Ca²⁺ influx pathway distinct from the opening of L-type Ca²⁺ channels.

The results raised the possibility that a non voltage-dependent Ca²⁺ channel, such as a TRP channel, might be implicated in responses to hIAPP, and we speculated that a mechanosensitive member of the TRP channel family might play a role. To test this hypothesis, we first investigated the effect of the relatively non-specific TRP channel inhibitor, gadolinium (Gd³⁺, 100 μM). In support of our hypothesis, addition of Gd³⁺, in the presence of Na⁺ free buffer and nifedipine, abolished the calcium responses to hIAPP (0.3% responders, 1/311 cells, p<0.001 compared with hIAPP in Na⁺ free buffer without Gd³⁺) (Fig. 3E).

We next examined our microarray database (41) for mechanosensitive TRP channels expressed in MIN6 cells, and the search identified the Ca²⁺-permeable channel Trpv4 as a possible candidate. To investigate the potential role of Trpv4, we tested the effect of Ruthenium Red (RR), a general inhibitor of TRPV channels. Both in standard buffer and Na⁺ free buffer, RR (10 μM) abolished the effect of hIAPP on [Ca²⁺]_i (only 0.6% of cells responded in standard bath and 1% responded in Na⁺ free buffer, p<0.001 compared with hIAPP in standard or Na⁺-free buffer without RR, respectively) (Fig. 3F,G).

By electron microscopy, we observed that hIAPP aggregates interacted with the cell membrane of MIN6 cells early after treatment (Fig. 4A). In these regions, the plasma membrane became irregular and exhibited membrane protrusions, which were clearly evident after 2 hours. hIAPP-induced membrane changes were also observed in the presence of RR, indicating that the effect of RR was not to prevent membrane changes or fibril formation (Fig. 4A).

To study the downstream effect of hIAPP cytotoxicity, we measured apoptosis by FACS analysis. Cells incubated for 24 hours in the presence of hIAPP exhibited only 68% viability compared with control cells (cf 93% viability using rIAPP) (Fig. 4B). Addition of RR in the presence of hIAPP improved cell viability to 79% (p=0.006), representing a 45% reduction in hIAPP-induced cell death (Fig. 4B). We next investigated whether we could confirm the key observed effects of RR on hIAPP cytotoxicity in primary cultures of mouse pancreatic islets (Fig.4C). After 72 hours treatment with 10 μM hIAPP, viability of mouse islet cells decreased significantly to 22% compared to 73% in the control (p<0.001) (Fig. 4D). Treatment with RR in the presence of hIAPP improved islet cell viability to 45% (p<0.05), representing a 52% reduction in hIAPP-induced apoptosis (Fig. 4D).

We further evaluated the potential involvement of Trpv4 in hIAPP- triggered calcium changes and apoptosis using siRNA technology. By RT-PCR, we detected Trpv4 expression in MIN6 cells, as well as liver, testis and pancreas (Fig. 5A). Transient transfection of MIN6 cells with siRNA against Trpv4 (siTrpv4) decreased expression of Trpv4, as assessed by RT-PCR, whereas the control siRNA against amylase (siAmy2) had no effect on Trpv4 expression (Fig. 5B). When fura-2-loaded, siAmy2-treated MIN6 cells were incubated in hIAPP for 2 hours, elevation of the 340/380 nm fluorescence ratio was detected in 10% of cells (14/156), whereas siTrpv4-treated cells showed a significant protection against hIAPP induced [Ca²⁺]_i elevation (0.87% responders, x/y cells,: p<0.001 compared with siAmy2) (Fig. 5C).

We also investigated whether silencing the Trpv4 gene had a protective effect on hIAPP-induced apoptosis, by measuring cell viability after 24 hours of hIAPP treatment. In the apoptosis assay using FACS, cell viability was ~78% in control cells transfected with either siAmy2 or siTrpv4. Whilst hIAPP reduced viability in the control siAmy2 cells to 26%, it only decreased viability to 52% in the siTrpv4 cells (p=0.002 for comparison between siAmy2 and siTrpv4 cells each treated with hIAPP). This represents a 50% reduction in hIAPP-triggered cell death by apoptosis in the cells with reduced expression of Trpv4 (Fig. 5D).

Recent findings revealed that the hIAPP-triggered calcium elevation and apoptosis in MIN6 cells were associated with activation of the ER stress response (22). To attempt to distinguish between the possibilities that hIAPP-induced ER stress might be either a trigger of, or a consequence of, the observed cytoplasmic [Ca²⁺] changes, we assessed the degree of ER stress in siTrpv4-treated cells, as these exhibited significantly reduced hIAPP-triggered [Ca²⁺] changes. ER stress was quantified by measuring the activation of Xbp1. The spliced active form of Xbp1 is a key transcription factor for upregulation of ER chaperones during ER stress responses (42), and has been shown to be important for maintenance of pancreatic β-cell ER function (43). Splicing of Xbp1 mRNA in MIN6 cells under control conditions was 23.24±1.9% of the total Xbp1 (Fig. 5E). Exposure of siAmy2-treated cells to 10 μM hIAPP for 2 hour significantly increased the levels of sXbp1 to 41.26±3.23% (p<0.05 vs basal condition), however, no increment was observed in cultures treated with siTrpv4 (21.35±2.14% of total Xbp1) (Fig. 5E). Twenty-four hours after treatment, the increase of sXbp1 product had returned near to basal levels in siAmy2 and remained normal in siTrpv4-treated cells (30.25±4.4% and 20.64±0.42%, respectively) (Fig. 5E). Moreover, we studied the expression level of Hsp90b1, which encodes a resident ER luminal molecular chaperone, known to be elevated by the upstream activity of sXbp1 as a consequence of ER stress (42). siAmy2-treated cells incubated in the presence of hIAPP showed significantly increased expression of Hsp90b1 after both 2 and 24 hours (p=0.02 and p=0.01 at 2 and 24 hours, compared with untreated siAmy2 cells). In cells treated with siTrpv4 and incubated with hIAPP, expression of Hsp90b1 was normal at 2 and 24 hours (Fig. 5F). These data represent a significant reduction in hIAPP-triggered ER stress in the cells with reduced expression of Trpv4 (Fig. 5E-F), which is in agreement with the observed protection against hIAPP-triggered cell death (Fig. 5D).

Discussion

Our results show that hIAPP triggers calcium changes in a model of the pancreatic β-cell, the MIN6 cell line. Calcium elevation was progressive and severe, and frequently resulted in cell detachment. The calcium changes corresponded in time with the appearance of hIAPP aggregates in the solution, alterations in the surface membrane morphology, and a reduction in cell viability. Pharmacological and siRNA experiments suggested that activation of both Trpv4 and L-type calcium channels contributed to the hIAPP-triggered calcium rise.

The synthetic IAPP peptide concentration used in the present study is within the range of concentrations used in the literature to spontaneously form IAPP oligomers and fibrils in culture (6,9,16,22). In T2DM, IAPP fibrils are often found accumulated between β-cells and islet capillaries (2,35–37), indicating that there may be insufficient clearance of secreted IAPP, making it difficult to estimate the IAPP concentrations that accumulate locally throughout the years of disease progression. Nevertheless, we showed here that the ultrastructural morphology of MIN6 cells in the amyloidogenic conditions was characterized by plasma membrane alterations compared with the control cells. These alterations included cell membrane irregularities and invaginations filled with fibrils, similar to those described in human T2DM islets in vivo (2,35–37).

Amyloid oligomers (including hIAPP) have been reported to increase [Ca²⁺] in neurons and astrocytes (23–27), although the effect of hIAPP on pancreatic β-cells is not well documented. We showed recently that increases in intracellular [Ca²⁺] could be detected in hIAPP-treated MIN6 cells, with associated activation of the ER stress response and induction of apoptosis (22). An earlier study, by contrast, suggested that hIAPP does not trigger changes in [Ca²⁺] in insulinoma cells (44), but the conclusions were derived from measurements of [Ca²⁺] in cell populations which might have excluded any non-attached cells. Our finding that individual MIN6 cells detached soon after experiencing a hIAPP-triggered Ca²⁺ rise might explain the previous failure to detect Ca²⁺ changes in insulinoma cells.

The mechanisms previously implicated in calcium responses to amyloid oligomers in other cell types and cell-free systems include direct formation of Ca²⁺-permeable pores by the amyloid oligomers themselves and the activation of voltage gated calcium channels (28,34). Amyloid-pores are unlikely to underlie our findings in MIN6 cells, as the Ca²⁺ changes were inhibited by nifedipine, Gd³⁺ and ruthenium red, none of which has been found to block amyloid channels. The inhibition of hIAPP-triggered Ca²⁺ responses by nifedipine in MIN6 cells indicated, however, that L-type Ca²⁺ channels were activated by hIAPP, either as a result of changes in the properties of the L-type Ca²⁺ channels themselves, or as a secondary consequence of membrane depolarisation. The latter idea is supported by the findings in Na⁺-free buffer, as an additional nifedipine-insensitive Ca²⁺-elevating pathway was identified under these conditions. This was found to be blocked by Gd³⁺ or ruthenium red, indicating the likely involvement of specific members of the TRP channel family. siRNA mediated knock down of the mechanosensitive TRP channel, Trpv4, protected against the toxic effects of hIAPP on [Ca²⁺] as well as on induction of ER stress and apoptosis. Our subsequent Trpv4 knock down experiments, using loss of cell viability as a marker of hIAPP-induced apoptosis, supported the notion that activation of this channel may be an early response to hIAPP.

Trpv4 is a mechano- and osmo-sensitive channel located on the cell surface, with a Ca²⁺/Na⁺ permeability ratio of ~6 (45), which is inhibited by Gd³⁺ and ruthenium red, and is not blocked by 2-APB. The observed morphological deformations in the plasma membrane following hIAPP treatment suggest that the deposition of hIAPP insoluble aggregates, which can reach high molecular weights (46), might weaken the mechanical strength of the plasma membrane making it susceptible to Trpv4 activation. We propose that it may provide a link between hIAPP-induced mechanical perturbations of the β-cell plasma membrane by hIAPP and the depolarising Ca²⁺ influx. The finding that siTrpv4 protected against both hIAPP-induced ER stress and [Ca²⁺] changes suggests that this channel is involved at a step that precedes the ER stress response, perhaps at the level of cytoplasmic [Ca²⁺] modulation. However, as RR did not protect completely against amyloid cytotoxicity, our data cannot exclude the involvement of other early mechanisms in the induction of islet amyloid cytotoxicity. Nevertheless, activation of Trpv4 may play a more widespread role in mediating some of the effects of amyloid on intracellular [Ca²⁺] that have been previously described in the literature. Amyloid-induced Ca²⁺ entry has, for example, been attributed to activation of channels with a relatively high selectivity for Ca²⁺ over Na⁺ (47), which are insensitive to L-type Ca²⁺ channel blockers (48). The potential involvement of Trpv4 and alternative mechanosensitive channels in mediating amyloid-induced Ca²⁺ changes, ER stress and apoptosis in other cell types will require further evaluation.