Intracellular amyloidogenesis by human islet amyloid polypeptide induces apoptosis in COS-1 cells

Abstract

Human islet amyloid polypeptide (hIAPP) is co-secreted with insulin from pancreatic islet beta cells. This peptide spontaneously aggregates in the form of fibrils, and amyloid deposits are associated with dead or degenerating beta cells, a hallmark of noninsulin-dependent diabetes mellitus. We demonstrated that COS-1 cells transfected with vectors expressing hIAPP exhibited intracellular amyloid deposits that were associated with cell death (O'Brien, Butler, Kreutter, Kane, Eberhardt, Am J Pathol 1995, 147:609-616). To establish the mechanism of cell death, we transfected COS-1 cells with vectors expressing amyloidogenic hIAPP or nonamyloidogenic rat IAPP and mutant hIAPP constructs and assayed them for markers characteristic of apoptosis and necrosis by fluorescence-activated cell sorting analysis. Amyloidogenic hIAPP-transfected COS cells contained up to threefold more apoptotic cells present at 96 hours after transfection compared with the nonamyloidogenic vector controls. The hIAPP-induced apoptosis was negligible at 24 and 48 hours after transfection and was maximal at 96 hours which parallels the time course of amyloidogenesis. Immunohistochemical staining and confocal microscopy showed that hIAPP is localized with distinct clustering in the endoplasmic reticulum and Golgi apparatus with no discernable extracellular staining. These experiments provide direct evidence that intracellular hIAPP amyloid causes cell death by triggering apoptotic pathways.

Figure 1.

Expression of hIAPP induces apoptosis in transfected COS-1 cells. Cells were transfected with 15 μg of pMT2 control (A), pMT2-hIAPP^anti (B), or pMT2-hIAPP (C) expression vectors as described (Materials and Methods) and cultured in complete medium for 96 hours. Apoptosis and necrosis were assessed by annexin-V labeling and 7-AAD staining, respectively, using FACS analysis. Dot plot profiles for annexin-V (x axis) and 7-AAD (y axis) for each treatment group are shown. The percentage of positive cells in the individual quadrants are shown (upper left panel, necrotic; upper right panel, late stage apoptosis; lower right panel, early to mid stage apoptosis).

Figure 2.

Time dependent induction of apoptosis in COS-1 cells expressing hIAPP. COS-1 cells were transfected with 15 μg of either the pMT2-hIAPP (A) or pMT2-hIAPP^anti (B) expression vectors and cultured as described (Materials and Methods). At 24, 48, 72, and 96 hours after transfection, cells were assessed for apoptosis and necrosis by annexin-V and 7-AAD labeling with FACS analysis. Histogram profiles for annexin-V labeling for each group are shown with the apoptotic cells (annexin-V positive) delineated by the bar (M1).

Figure 3.

Expression of the hIAPP polypeptide and its amyloidogenic domain are required for induction of apoptosis in COS-1 cells. A: Expression of the hIAPP antisense mRNA in COS-1 cells blocks hIAPP-induced apoptosis. COS-1 cells were transfected with the pMT2, pMT2-hIAPP^anti, pMT2-hIAPP, or equimolar concentrations of both pMT2-hIAPP and pMT2-hIAPP^anti expression vectors and cultured as described (Materials and Methods). After 96 hours the cells were assessed for apoptosis and necrosis by labeling with annexin-V (cross-hatched bars) and 7-AAD (shaded bars) and analyzed by FACS. B: Expression of hIAPP containing its amyloidogenic domain but not a mutant hIAPP or rIAPP, lacking the amyloidogenic domain, leads to apoptosis of transfected COS-1 cells. Cells were transfected with 15 μg of pMT2, pMT2-hIAPP^anti, pMT2-hIAPP, pMT2-hIAPP^mut, or pMT2-rIAPP expression vectors. Apoptosis was assessed by annexin-V binding and FACS analysis at 72 (open bars) or 96 hours (shaded bars). The data are from three independent experiments; data marked with asterisks were statistically different from control (P < 0.05) by multivariate analysis of variance and post hoc Bonferroni t-test.

Figure 4.

Immunofluorescent labeling of hIAPP, hIAPP variants, and rIAPP in COS-1 cells. Cells were transfected with the pMT2-hIAPP (A and B), pMT2-hIAPP^anti (C), and co-transfected pMT2-hIAPP and pMT2-hIAPP^anti expression vectors as described in Materials and Methods and cultured in 8-well chamber slides for 48 hours. Immunofluorescent labeling on permeabilized cells was performed with rabbit anti-amylin antibodies and FITC-labeled GAR secondary antibodies. Phase contrast (B) and immunoflouresence microscopy (A, C, and D) were performed concurrently on a Zeiss laser scanning confocal microscope. Intense immunofluoresence (green) is seen localized in clusters in the perinuclear regions of hIAPP-expressing cells only (A). Cells were also transfected with the nonamyloidogenic pMT2-hIAPP^mut (E) or pMT2-rIAPP (F) expression vectors. After 48 hours, the permeabilized cells were labeled with rabbit anti-IAPP antibodies and GAR-FITC to detect intracellular IAPP (Materials and Methods) by LSCM. Apoptotic bodies (G) and membrane blebbing (H) extruding from cells undergoing apoptosis in pMT2-hIAPP-transfected COS-1 cells at 48 hours after transfection. Translocated extracellular phosphatidylserine residues were labeled by annexin-V and Rhodamine Red fluorescence and analyzed by LSCM. Annexin positive labeling on apoptotic cells (red staining) and formation of apoptotic bodies (arrows) are shown in G. Intracellular hIAPP is also evident in apoptotic blebs (green staining and arrows) as labeled with anti-amylin antibodies and FITC fluorescence in H.

Figure 5.

Immunofluorescent labeling of amylin in permeabilized and nonpermeabilized hIAPP-expressing COS-1 cells. COS-1 cells were transfected with the pMT2-hIAPP (A and B) or pMT2-hIAPP^anti (C and D) expression vectors and cultured in multiwell chamber slides for 72 hours. Fixed cells were either permeabilized with 0.05% saponin (B and D) or left untreated (A and C) prior to immunofluorescent staining with anti-amylin antibodies. Immunofluorescent labeling (green) is seen only in permeabilized hIAPP-expressing cells (B). Nonpermeabilized cells show no evidence of amylin immunolabeling (A) above the background levels (C and D). E depicts a conditioned media experiment in which the medium from pMT2 control (CTRL) or pMT2-hIAPP (hIAPP) transfected cells was added to COS-1 cells that were freshly transfected with pMT2 (CTRL) or pMT2-hIAPP (hIAPP) plasmids and then incubated for 96 hours to determine whether extracellular amylin/amyloid was cytotoxic. The data represent the average results from two independent experiments and the error bars reflect the average deviation from the mean in the two experiments.

Figure 6.

Double labeling of intracellular hIAPP and specific markers of apoptosis is found only in hIAPP-expressing COS-1 cells. A: Intracellular accumulation of hIAPP is associated with extracellular annexin-V labeling indicative of apoptosis in pMT2-hIAPP-transfected COS-1 cells. Extracellular annexin was fluorescently labeled with streptavidin-rhodamine (red) and intracellular IAPP with FITC (green). The majority (>90%) of control pMT2-hIAPP^anti-transfected cells (B) have only background levels of annexin labeling; the single brightly labeled cell in B is undergoing apoptosis. Intracellular accumulation of hIAPP (green) is also associated with TUNEL labeling of fragmented DNA (red) in the nucleus of pMT2-hIAPP- (C) but not control pMT2-transfected cells (D). Double labeling of adherent COS-1 cells was performed as described in Materials and Methods.

Figure 7.

Intracellular accumulation of hIAPP in COS-1 cells is found co-localized with both Golgi- and ER-specific markers. Dual immunolabeling with antibodies against the Golgi p58K marker protein (A; red), Mann II ER-Golgi marker (D; red), or the ER marker, PDI (G; red), and anti-amylin (green in C, F, and I), demonstrates a co-localization with both ER and Golgi markers (yellow in B, E, and H). Immunolabeling was performed on COS-1 cells 48 hours after transfection with pMT2-hIAPP and pMT2-hIAPP^anti control plasmids. Permeabilized cells were concurrently labeled with rabbit anti-IAPP and mouse anti-marker antibodies, then sandwich labeled with GAR-FITC and GAM-RHOD, and analyzed by LSCM (Materials and Methods).

Figure 8.

Amino acid sequences of hIAPP, hIAPP^mut, and rIAPP. The amyloidogenic domain of hIAPP, GAILSS , is indicated in bold.