1Department of Pathology and Laboratory Medicine, Children's Hospital of Philadelphia and University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania; and 2Department of Endocrinology, First Affiliated Hospital, Sun Yat-sen University, Guangzhou, China
Submitted 16 March 2005 ; accepted in final form 18 May 2005
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ABSTRACT |
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cytokine; islet; insulin secretion; apoptosis; PANDER
Local production of cytokines may be of key importance in the pathogenesis of type 1 diabetes. The consequences of local intraislet production of a variety of cytokines have been examined using transgenic mouse models. Studies with selective expression of cytokines or mutant cytokine receptors suggest that TNF- and IFN-
play a causative role in the development of insulitis preceding diabetes development. Expression of IFN-
or IFN-
within the pancreatic islets results in increased insulitis and diabetes (13, 26). These results suggest that local production of IFN-
may play a distinctive role in the establishment of insulitis and subsequent
-cell destruction. However, the mechanisms of cytokine-induced pancreatic islet
-cells damage in autoimmune diabetes are still not fully elucidated.
We (32) have recently characterized a novel cytokine-like gene family (FAM3) that includes at least four genes. We have previously demonstrated that a novel cytokine, PANDER (pancreatic derived factor), also named FAM3B (32), is highly expressed in both -cells and
-cells of pancreatic islets (6). PANDER was discovered by searching the genetic databases for novel four-helix bundles by use of structure-based methods. PANDER is a 235-amino acid protein with a secretory signal peptide. Recombinant PANDER fragments have potent cytotoxic effects on the insulin-secreting
-cells, mouse islet cells, rat islet cells, and human islet cells (6), which are similar to those originally described for IL-1. Recombinant PANDER protein secreted from Chinese hamster ovary (CHO) cells exists as two NH2-terminal fragments cleaved at amino acid Glu30 and Ser46, with a mass of 22.9 and 21.1 kDa, respectively (32). In contrast, recombinant PANDER prepared in Pichia pastoris is a mixture of PANDER fragments cleaved at residues Ser46 and Ala55 with a mass of 21.1 and 20.0 kDa, respectively (32). Although the PANDER fragments obtained from Pichia were shown to cause
-cell apoptosis (6), the biological function of full-length PANDER remains unclear.
The aim of the present study was to overexpress a full-length (705 nt) PANDER cDNA by use of adenovirus as a vector in the -cell lines and mouse islets to establish the role of the endogenous PANDER on
-cell viability and function.
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MATERIALS AND METHODS |
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TC3 insulinoma cells were cultured in T175-cm flasks in complete RPMI 1640 (11 mM glucose) supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 100 µg/ml streptomycin, and 2 mM L-glutamine at 37°C under conditions of 95% air-5% CO2. The medium was changed twice a week and on the day before experiment. Cells were trypsinized weekly and were used exclusively between passages 4050.
Pancreatic Islet Isolation and Culture
All animal studies were preapproved by our local Institutional Animal Care and Use Committee. C57/black mice (Charles River Laboratories, Boston, MA) were injected with Nembutal (0.05 mg/g mouse). After the mice were anesthetized, the bile duct was cannulated, and the pancreas was inflated with Hanks' balanced buffer. The inflated pancreas was removed and cleaned of its lymph nodes, fat, blood vessels, and bile duct. Tissue was digested with collagenase P (Roche Molecular Biochemicals) as previously described (22) and purified on a discontinuous Ficoll gradient. Isolated islets were washed and cultured in complete CMRL-1066 medium (11 mM glucose, supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin) at 37°C (95% air, 5% CO2).
Gene Transfer of PANDER to TC3 Cells or Mouse Islets
An adenovirus vector with the full-length mouse PANDER (acc. no. AF494379
[GenBank]
) gene under the control of the CMV promoter (Ad-PANDER) was engineered by Dr. Yuan Zhu and colleagues at GlaxoSmithKline (Collegeville, PA). TC3 cells were cultured in RPMI 1640 (10% FBS) with a fresh medium change 24 h before use. Cells were washed twice with prewarmed serum-free RPMI 1640 supplemented with 1% pencillin-streptomycin before adenoviral infection. Cells were infected with Ad-PANDER, Ad-LacZ, or Ad-GFP in a minimal volume of serum-free RPMI 1640 medium at 80100 plaque-forming units (pfu) per cell for 23 h at 37°C. Cells were then washed twice with serum-free RPMI 1640 and once with RPMI 1640 supplemented with 10% FBS. Cells were further cultured at 37°C for 2 days in RPMI 1640 supplemented with 10% FBS. Mouse islets were treated in a similar fashion using CMRL-1066. The expression efficiency for Ad-GFP is almost 100% in
TC3 cells and islet cells. That of Ad-PANDER is difficult to confirm because the cells have endogenous PANDER. Ad-GFP infection is thus used as an index of infection efficiency, as used routinely for other types of cell.
Perifusion of Islets for Insulin Secretion
Islets (100200) were collected under a stereomicroscope, washed twice with Krebs-Ringer bicarbonate (KRB; 115 mM NaCl, 24 mM NaHCO3, 5 mM KCl, 1 mM MgCl2, 2.5 mM CaCl2, 25 mM HEPES, and 1% BSA, pH 7.4), and loaded into a 13-mm chamber containing a nylon membrane filter. Islets were first perifused with KRB containing 3 mM glucose (G3) for 1 h and challenged with various conditions, as indicated in the figure legends. Effluent fractions were then collected at 1-min intervals and stored at 20°C before determination of their insulin content. Insulin was assayed by the Radioimmunoassay Core of the Penn Diabetes Center.
Cell Viability Determination
MTT assay.
The assay is based on the ability of viable cells to reduce MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) to insoluble colored formazan crystals (15). It is used as an indirect measure of -cell viability.
TC3 cells plated in 24-well plates (1 x 106) were infected with Ad-PANDER or Ad-GFP. After culture for 25 days, cells were washed twice with Krebs-HEPES buffer and then incubated in 1 ml of Krebs-HEPES buffer (0 mM glucose) with 0.5 mg/ml MTT for 60 min at 37°C. The supernatant was discarded, and cells in each well were lysed with 500 µl of 2-propanol and incubated for 1 h at room temperature. The optical density of the resultant colored 2-propanol was measured at 560 nm on a MicroKinetics plate reader (PerkinElmer).
Propidium iodide nuclear staining.
Propidium iodide (PI) is a highly polar dye, which penetrates cells with damaged membranes and stains the nuclei red. TC3 cells plated in 12-well plates (
2 x 105 cells/well) were infected with Ad-PANDER or Ad-GFP and cultured in RPMI 1640. After 2 days, PI was added to each well (final 10 µg/ml) and incubated with the cells for 15 min at room temperature in the dark. Cell images were acquired using a Nikon fluorescence microscope (Diaphot) connected with a Hamamatsu digital camera, C4742-95, at x40 magnification and were analyzed with SimplePCI software (Compix Imaging Systems, Cranberry, PA) with excitation at 488 nm and emission at >510 nm. Pictures were acquired under both bright field and fluorescence settings. For quantitative analysis of the cell viability, the PI fluorescence intensity of the total cells in each condition was measured with a Victor 2000 fluorimeter (PerkinElmer).
Annexin V Staining.
Annexin V is a Ca2+-dependent phospholipid-binding protein with a high affinity for phosphatidylserine (PS) (1). In normal cells, PS is located on the cytoplasmic surface of the cell membrane. In apoptotic cells, PS is translocated from the inner to the outer surface of the cell membrane. Annexin V labeled with a fluorophore can identify apoptotic cells by binding to the PS exposed on the outer leaflet. TC3 cells or mouse islets infected with Ad-PANDER or Ad-LacZ were washed once with ice-cold PBS and then once with annexin V binding buffer (10 mM HEPES, 140 mM NaCl, and 2.5 mM CaCl2, pH 7.4). Cells or islets were then rinsed with annexin V binding buffer and incubated in 100 µl of annexin V binding buffer supplemented with 10 µl of PI and 25 µl of annexin V for 15 min at room temperature in the dark. Cells or islets were washed again with annexin V binding buffer and dried out for 20 min in the dark. The fluorescent images were captured using the Leica TCS SP2 confocal microscope with a HeNe-argon laser as a light source. Each image was collected with excitation at 488 nm and emission at 520 nm for annexin V and with excitation at 543 nm and emission at 610 nm for PI concurrently. The images were analyzed using Leica Confocal Software.
TUNEL assay.
An in situ Cell Death Detection Kit (Boehringer Mannhein, Indianapolis, IN) was used to detect apoptotic cells. In this method, terminal deoxynucleotidyltransferase (TdT) was used to catalyze the polymerization of fluorescein-labeled nucleotides to free 3-OH termini of DNA strand breaks. After infection of cells with Ad-PANDER or Ad-LacZ for 2 days, TC3 cells were trypsinized and washed twice with cold PBS-1% BSA. Cells were then fixed with 200 µl of 4% paraformaldehyde and incubated for 30 min at room temperature. After being rinsed with PBS, cells were resuspended with 250 µl of permeabilization solution (0.1% Triton X-100 in 0.1% sodium citrate) and incubated on ice for 10 min. Then, 50 µl of TUNEL reaction mixture were added to the samples and the positive controls, (50 µl of label solution only were added to the negative controls), and cells were incubated at 37°C for 1 h. Apoptotic cells were identified by FITC staining and analyzed by flow cytometry. A Coulter EPICS Elite Flow cytometer (Beckman-Coulter, Hialeah, FL) equipped with a 5-W argon laser operated at 488 nm and 260-mW output was used for all studies. Monomeric forms of the
TC3 cells were electronically gated on the basis of forward and side scatter measurements to exclude cell aggregates from evaluation. Fluorescence signals were collected with a photomultiplier tube configured with 550-nm dichroic and 525-nm band pass filters. Ten thousand events were collected into a four-decalog single-parameter histogram for each sample. The percentage of positive cells was determined on the basis of the evaluation of cells treated with TUNEL reagents lacking TdT by using a cursor setting that yielded fewer than 2% positive cells.
Immunoblotting
-TC3 cells were cultured in 10-cm dishes in RPMI 1640 supplemented with 10% FBS, 100 U/ml penicillin, 100 µg/ml streptomycin, and 2 mM L-glutamine at 37°C under condition of 95% air-5% CO2. Cells were used at 70% confluence, and lysates from the cells were prepared and mixed with SDS sample buffer. Samples were then subjected to SDS-PAGE and electrotransferred to Hybond nitrocellulose membranes, which were then probed with antibodies to Akt, phospho-Akt (Ser473), phospho-Bcl-2 (Ser70), NF-
B p65, Fas, STAT1, phospho-STAT1 (Tyr701), caspase-3, cleaved caspase-3 (all from Cell Signaling), and PANDER (GlaxoSmithKline). Immunoblot analysis was performed using the ECL Western Blotting Detection Reagents (Amersham Phamacia Biotech UK).
Measurement of Caspase-3/7 Activity in Mouse Islets
Islets were placed in a 35-mm nontissue culture-treated dish containing CMRL-1066 medium. Approximately 100 murine islets were placed into each dish and exposed to the treatment conditions. The islets were then cultured for 24 h at 37°C, 5% CO2, to allow for islet recovery. Thereafter, 4 nM exogenous murine PANDER was added. Untreated islets were supplemented with medium only. After 48 h, islets were hand counted again to ensure equal numbers of islets among samples and transferred to a 1.5-ml Eppendorf tube. Tubes were centrifuged for 2 min at 2,000 rpm, and islets were resuspended in 100 µl of CMRL-1066. The activity of caspase-3 was measured using the Caspase-Glo 3/7 Assay (Promega). All reagents were supplied by the manufacturer. Specifically, islets were lysed using the Caspase-Glo 3/7 reagent and incubated at room temperature for 1 h. Luciferase activity was measured using a Monolight 3010 luminometer (Analytical Luminescence Laboratory). Caspase-3/7 activity was normalized to total protein content, which was measured using a standard bBicinchoninic acid (Sigma) assay.
Statistical Analysis
Data are presented as means ± SE. Statistical significance of differences between groups was analyzed by unpaired Student's t-test or by one-way analysis of variance when more than two groups were compared.
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RESULTS |
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Mouse pancreatic islets were treated with recombinant PANDER (rPANDER, 4 nM) for 3 days (Fig. 1A) and subsequently perifused. Under basal conditions of 3 mM glucose, PANDER-treated islets secreted insulin at the same rate as control. However, 20 mM glucose plus 0.5 mM carbachol (G20+CCH)-stimulated insulin secretion was significantly decreased by 37.3% compared with control. The area under curve (total insulin secretion during 10 min of stimulation) in the PANDER-treated islets was 63.2 ± 12.0 ng/100 islets (vs. control 100.9 ± 9.2 ng/100 islets, P < 0.05). Furthermore, 30 mM K+-induced insulin secretion was also significantly decreased in PANDER-treated islets (area under curve is 31.4 ± 4.5 ng/100 islets vs. control 46.6 ± 4.8 ng/100 islets, P < 0.05). Overexpression of PANDER in mouse islets (Fig. 1B) for 3 days caused a significant decrease of insulin secretion in response to insulin secretagogues. The area under curve of insulin secretion stimulated by 20 mM glucose plus 0.5 mM carbachol in the Ad-PANDER-treated islets was 42.6 ± 15.3 ng/100 islets (vs. control 66.6 ± 18.6 ng/100 islets, P < 0.05). The area under curve of insulin secretion stimulated by 3 mM glucose plus 30 mM KCl in the Ad-PANDER-treated islets was 9.3 ± 2.5 ng/100 islets (vs. control 13.9 ± 2.9 ng/100 islets, P < 0.05). Overexpression of PANDER did not affect the basal insulin secretion at 3 mM glucose (Fig. 1, B and C). When islets were stimulated with 25 mM glucose alone, insulin secretion was increased only slightly, and there was no difference between Ad-PANDER and Ad-LacZ islets (Fig. 1C).
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Islets were cultured with or without (control) rPANDER for 3 days. They were then incubated for 30 min without glucose. While islets were perifused with various concentrations of glucose, NAD(P)H autofluorescence was measured following excitation at 360 nm with emission recorded at 410 nm. All fluorescent values were normalized to that without glucose as 100%. In control islets, glucose at 2, 5, 10, and 30 mM increased NAD(P)H to 115.0 ± 5.0, 126.8 ± 5.6, 140.6 ± 9.6, and 167.5 ± 14.4% compared with that without glucose. Glucose effects (Fig. 1D) in rPANDER-treated islets were not significantly different (P > 0.05).
Overexpression of PANDER Inhibits -TC3 Cell Viability
In previous studies (6), we showed that rPANDER causes -cell apoptosis. However, rPANDER is a mix of two NH2-terminal fragments, cleaved at amino acid Ala55 and Ser46, and the biological function of endogenously produced longer isoforms of PANDER remains unclear. PANDER protein was overexpressed in insulin-secreting cells with Ad-PANDER. Infection with Ad-PANDER in
TC3 cells resulted in the production of two PANDER proteins, presumably the full-length PANDER (
26 kDa) and an NH2-terminal Glu30-truncated PANDER (
23 kDa) (Fig. 2). The molecular mass of full-length PANDER was slightly heavier than expected, probably due to posttranslational modifications in the
-cells. The molecular mass of endogenous full-length PANDER in control cells (Fig. 2, lanes 4 and 5) or Ad-LacZ (data not shown) -infected cells was the same as that of Ad-PANDER cells (
26 kDa). Overexpression of PANDER using adenovirus for 2, 4, or 5 days had a significant (P < 0.05) inhibitory effect on
TC3 cell viability compared with that of the Ad-GFP-treated or the noninfected cells (Fig. 3). The optical densities of the Ad-PANDER group (presented as percentages of untreated control) in the MTT assay were 51.3 ± 3.3% (vs. Ad-GFP 78.3 ± 3.0%, P < 0.0001) at day 2, 63.7 ± 3.4% (vs. Ad-GFP 92.1 ± 3.9%, P < 0.0001) at day 4, and 57.7 ± 8.6% (vs. Ad-GFP 119.9 ± 8.8%, P < 0.001) at day 5, respectively.
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PI fluorescence staining was also used to analyze cell death (both apoptosis and necrosis) of TC3 cells. Cells infected with Ad-PANDER (Fig. 4, C and D) had a much higher percentage of dead cells than those infected with Ad-GFP (Fig. 4, A and B). To obtain a quantitative assessment, the PI fluorescence intensity of
TC3 cells was measured in a fluorometer. Compared with the Ad-GFP-treated cells, there was a significant increase of PI fluorescence intensity in the Ad-PANDER-treated cells (160 ± 13% vs. Ad-GFP infected cells 100 ± 7%, P = 0.001), indicating a significantly increased number of dead cells.
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Immunoblotting of TC3 cell lysates with anti-Akt, anti-phospho-Akt (Ser473), anti-phospho-Bcl-2 (Ser70), anti-NF-
B, anti-Fas, anti-STAT1, and anti-phospho-STAT1 (Tyr701) antibodies showed no significant difference in the amount of these proteins between the cells treated with or without 4 nM rPANDER (data not shown). Similar results were obtained when cells were infected with Ad-PANDER (data not shown). However, immunoblotting of
TC3 cell lysates with anti-cleaved caspase-3 antibodies showed that the two cleaved caspase-3 fragments (19 and 17 kDa) were increased in the Ad-PANDER-infected cells compared with Ad-LacZ-treated cells after 48 h of incubation (Fig. 8A). No caspase-3 activation was observed after 20 h of incubation in both groups of cells. Ad-PANDER in mouse islets mainly increased the level of 19-kDa cleaved caspase-3 (Fig. 8, A and B). Caspase-3/7 activity was increased (P < 0.05) 1.7 ± 0.2-fold after rPANDER treatment for 48 h in mouse islets (Fig. 8C). The effect of Ad-PANDER on the caspase-3 activation in
TC3 cell and mouse islets is consistent with that of our previous study using rPANDER (6).
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DISCUSSION |
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PANDER is a newly identified cytokine that is specifically expressed in both -cells and
-cells of the pancreatic islets of Langerhans (6). We (6) previously demonstrated that rPANDER inhibits pancreatic
-cell and
-cell viability. The rPANDER that we used in the previous study was a mixture of two isoforms of 21.1 and 20.0 kDa produced from P. pastoris, but it remained unclear whether overexpressed PANDER had any biological function. In this study, we expressed the full-length cDNA of PANDER in a
TC3 cell line as well as mouse islets by use of an adenovirus vector. Our data showed that an endogenous increase in two PANDER isoforms in
-cells caused apoptosis in a manner similar to that of the recombinant PANDER isoforms.
In general, an apoptotic cascade may be elicited by a number of extracellular stimuli, including withdrawal of growth factors, certain hormones, and inflammatory mediators such as cytokines, activation of death receptors, or metabolic factors. Fas, Akt, STATs, NF-B, and the Bcl-2 family are all thought to be important intracellular mediators in the cytokine-induced
-cell apoptosis in type 1 diabetes. Fas is expressed on activated lymphocytes and some cells in the target tissues. Studies have documented that cytokine-induced Fas upregulation in cultured murine and human islets can transmit apoptosis signals (28). NOD mice homozygous for Fas gene mutation do not develop diabetes (8), indicating that Fas-mediated apoptosis is an important pathway leading to
-cell destruction in type 1 diabetes.
The serine/threonine kinase Akt is a major mediator of cell survival by directly inhibiting different proapoptotic signals such as Bad (10), preventing cytochrome c release from the mitochondria (18) or indirectly activating NF-B (17). The activation of NF-
B protects the
-cell from TNF-
-induced apoptosis (7). Phosphorylation of Akt prevents apoptosis and promotes cell survival (16). The STAT family has numerous isoforms, and they are necessary components of cytokine receptor signaling (9). IFN-
binds to its receptor and causes STATs to be phosphorylated by JAK, dissociated from the receptor, dimerized, and translocated to the nucleus. Once in the nucleus, STAT dimers bind specific enhancers to regulate the transcription of target genes (14).
In the current study, we find that both rPANDER and Ad-PANDER activate caspase-3, which is consistent with our previous study (6). Unlike other cytokines, such as TNF-, IL-1
, and IFN-
, secreted from inflammatory cells, PANDER is expressed in the target
-cells. Apoptosis can be triggered at different points of the apoptotic cascade depending on the initiating stimulus. The signals inducing apoptosis under some conditions can induce differentiation and proliferation under different conditions and/or in different cell types (13). But the final pathway leading to cell death seems to be common with activation of cysteine proteases (caspases) and endonucleases that cleave nuclear DNA into oligosomal fragments (20). Our study indicated that caspase activation of caspases correlates with PANDER-induced
-cell death. On the one hand, the proximal steps of PANDER-induced
-cell apoptosis are unique without changes in the protein levels of STAT, Akt, Bcl-2, NF-
B, or Fas (changes in activity and subcellular location/translocation are unknown), but its distal steps are similar to that of others with the possible involvement of caspase (21).
Our preliminary data have shown that IFN- treatment of islets results in upregulation of PANDER gene expression, presumably in the
-cells (31), suggesting that PANDER may mediate the effects of inflammatory cytokine-induced
-cell dysfunction and death. Furthermore, we now show, using a mouse perifusion model, that full-length as well as recombinant PANDER inhibit insulin secretion. Treatment of mouse islets with recombinant or overexpressed PANDER induced a significantly decreased insulin secretion in response to carbachol plus glucose stimulation or potassium stimulation but not the stimulation of glucose alone. Thus PANDER probably affects mainly the amplifying signals of insulin secretion rather than the triggering signals. It is conceivable that PANDER modulates insulin secretion by affecting intracellular Ca2+ homeostasis in
-cells.
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GRANTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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