Involvement of the RNA-binding protein ARE/poly(U)-binding factor 1 (AUF1) in the cytotoxic effects of proinflammatory cytokines on pancreatic beta cells
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Chronic exposure of pancreatic beta cells to proinflammatory cytokines leads to impaired insulin secretion and apoptosis. ARE/poly(U)-binding factor 1 (AUF1) belongs to a protein family that controls mRNA stability and translation by associating with adenosine- and uridine-rich regions of target messengers. We investigated the involvement of AUF1 in cytokine-induced beta cell dysfunction.
Production and subcellular distribution of AUF1 isoforms were analysed by western blotting. To test for their role in the control of beta cell functions, each isoform was overproduced individually in insulin-secreting cells. The contribution to cytokine-mediated beta cell dysfunction was evaluated by preventing the production of AUF1 isoforms by RNA interference. The effect of AUF1 on the production of potential targets was assessed by western blotting.
MIN6 cells and human pancreatic islets were found to produce four AUF1 isoforms (p42>p45>p37>p40). AUF1 isoforms were mainly localised in the nucleus but were partially translocated to the cytoplasm upon exposure of beta cells to cytokines and activation of the ERK pathway. Overproduction of AUF1 did not affect glucose-induced insulin secretion but promoted apoptosis. This effect was associated with a decrease in the production of the anti-apoptotic proteins, B cell leukaemia/lymphoma 2 (BCL2) and myeloid cell leukaemia sequence 1 (MCL1). Silencing of AUF1 isoforms restored the levels of the anti-apoptotic proteins, attenuated the activation of the nuclear factor-κB (NFκB) pathway, and protected the beta cells from cytokine-induced apoptosis.
Our findings point to a contribution of AUF1 to the deleterious effects of cytokines on beta cell functions and suggest a role for this RNA-binding protein in the early phases of type 1 diabetes.
KeywordsAUF1 Apoptosis Cytokine Diabetes Heterogeneous nuclear ribonucleoprotein D Insulin Islet RNA Secretion
AU-rich binding protein
ARE/poly(U)-binding factor 1
B cell leukaemia/lymphoma 2
Chemokine (C-C motif) ligand 2
Chemokine (C-X-C motif) ligand 2
Enhanced green fluorescent protein
Extracellular regulated MAPK
Green fluorescent protein
Human growth hormone
Inhibitor of κB
Inducible nitric oxide synthase
c-Jun N-terminal kinase
Mitogen-activated protein kinase
Myeloid cell leukaemia sequence 1
Monocyte chemotactic protein 1
Macrophage inhibitor protein 2
Small interfering RNA
Type 1 diabetes is an autoimmune disease characterised by a progressive loss of pancreatic beta cells. Insulin, the hormone produced by these cells, plays an essential role in the maintenance of blood glucose homeostasis. During the autoimmune attack, macrophages and lymphocytes infiltrate pancreatic islets and release proinflammatory cytokines with a major impact on the expression of key beta cell genes leading to defective insulin secretion and sensitisation to apoptosis [1, 2].
Most studies investigating the causes of beta cell dysfunction during the early phases of type 1 diabetes have focused on the role of signalling cascades culminating in the activation of transcription factors , but, so far, the potential impact of cytokines on mRNA stability has been poorly investigated. Several RNA-binding proteins are known to bind to specific sequences located on the 3′-untranslated region (3′-UTR) of target mRNAs. A family of RNA-binding proteins called AU-rich (ARE)-binding proteins (AUBPs) interacts specifically with adenosine- and uridine-rich regions located in the 3′-UTR of target mRNAs . Under resting conditions, AUBPs are principally located in the nucleus  but, upon stimulation, translocate to the cytoplasm and bind to their targets, leading to mRNA protection, stabilisation, degradation or inhibition of messenger translation . AUF1 (ARE/poly(U)-binding factor 1) , also known as hnRNP-D (heterogeneous nuclear ribonucleoprotein D), was originally identified as a protein that binds and induces the destabilisation of the mRNAs encoding c-myc and granulocyte–macrophage colony-stimulating factor . AUF1 was later reported to affect the stability of a large variety of mRNAs involved in inflammation, cell cycle control or apoptosis [8, 9]. Although the destabilising function of AUF1 is well documented, some studies suggest that the protein can also exert a positive effect on mRNA stability .
Four different isoforms of AUF1 resulting from differential splicing of exons 2 and 7 have been described: p37, p40, p42 and p45 . The production of these isoforms varies between cell types and developmental stages and can be modified in response to different stimuli . Moreover, AUF1 isoforms are subjected to post-translational modifications that affect the activation state of the RNA-binding protein in a cell-type and treatment-dependent manner [13, 14].
Since many proteins involved in stress and immune responses or in cellular growth are encoded by mRNAs containing ARE sequences, AUF1 is a good candidate for mediating the alterations in gene expression underlying the impairment in beta cell activities observed in the presence of proinflammatory cytokines.
The aim of this study was to investigate the possible involvement of AUF1 in cytokine-mediated beta cell dysfunction and in the development of type 1 diabetes. We found that AUF1 is indeed activated upon exposure to cytokines and contributes to beta cell apoptosis elicited by these proinflammatory mediators.
The extracellular regulated MAP kinase (ERK) inhibitor, PD98059, was obtained from Calbiochem–Novabiochem (San Diego, CA, USA). The c-Jun N-terminal kinase (JNK) inhibitor, SP600125, was from Enzo Life Sciences (Plymouth Meeting, PA, USA). IL-1β and the p38 mitogen-activated protein kinase (MAPK) inhibitor, SB 239063, were purchased from Sigma (Buchs, Switzerland). Recombinant mouse IFNγ was obtained from R&D Systems (Minneapolis, MN, USA), and TNFα from Alexis Corporation (Lausen, Switzerland). Hoechst 33342 was purchased from Invitrogen (Carlsbad, CA, USA).
Culture and transfection of insulin-secreting cell lines and human pancreatic islets
The insulin-secreting cell line, MIN6 clone B1 , was cultured at a density of 1.5 × 105 cells/cm2 in DMEM/Glutamax medium (Invitrogen) supplemented with 15% FCS, 50 IU/ml penicillin, 50 μg/ml streptomycin and 70 μmol/l β-mercaptoethanol. The insulin-producing cell line, INS-1E , was cultured at the same density as MIN6 cells in RPMI 1640 (Invitrogen) supplemented with 10% FCS, 50 IU/ml penicillin, 50 μg/ml streptomycin, 1 mmol/l sodium pyruvate and 50 μmol/l β-mercaptoethanol.
Transient transfections of MIN6 and INS-1E cells were performed with Lipofectamine 2000 (Invitrogen) following the instructions provided by the manufacturer. For a 24-well plate, 60 pmol small interfering RNA (siRNA) duplexes and 0.8 μg plasmids were used.
Human pancreatic islets were provided by the Cell Isolation and Transplantation Center at the University of Geneva, School of Medicine, thanks to the Islets for Research distribution programme of the European Consortium for Islet Transplantation (ECIT) sponsored by the Juvenile Diabetes Research Foundation. The purity of islet preparations used for this study ranged from 80% to 95%, and the samples contained 51 ± 7% (mean±SD) insulin-positive cells, as revealed by immunofluorescence analysis using an antibody against insulin (Linco Research, St Charles, MO, USA). The islets were cultured in CMRL medium (Invitrogen) supplemented with 10% FCS, 100 IU/ml penicillin, 100 μg/ml streptomycin, 2 mmol/l l-glutamine and 250 μmol/l HEPES. Islet cell monolayers were prepared by treating the islets for 7–9 min with trypsin (0.25 mg/ml)/EDTA at 37°C. The trypsinisation was terminated by adding serum-containing culture medium. The cells were seeded at a density of 5.5 × 104 cells/cm2. Human islet cells were transfected using the same conditions as used for MIN6 cells.
AUF1 overproduction and downregulation
To experimentally increase AUF1 levels, MIN6 cells were transiently transfected with plasmids expressing enhanced green fluorescent protein (EGFP)-labelled constructs of each individual AUF1 isoform . Reduction of the level of the selected isoforms was achieved using siRNA duplexes directed against: exon 2, targeting p45 and p40 (siAUF1p45p40); exon 7, targeting p45 and p42 (siAUF1p45p42); and a sequence astride exon 3 and 4, targeting all isoforms (siAUF1all). The sequences were the following: siAUF1p45p40, 5′-ACU CCU CCC CAC GAC ACA CTT-3′; siAUF1p45p42, 5′-UCA AGG CUA UGG CAA CUA UTT-3′; and siAUF1all, 5′-AGA AAG AUC UGA AGG ACU ATT-3′. An siRNA duplex directed against green fluorescent protein (GFP; 5′-GAC GUA AAC GGC CAC AAG UUC-3′), which has no effect on pancreatic beta cell functions, was used as control.
Analysis of the expression of protein-coding genes
Total RNA extraction was performed with the RNeasy mini kit (Qiagen, Hilden, Germany). Conventional quantitative RT-PCR (qRT-PCR) was carried out as described previously . Real-time PCRs were performed on a Bio-Rad MyiQ Single-Color Real-Time PCR Detection System (Bio-Rad Laboratories, Hercules CA, USA). The list of primers can be found in electronic supplementary material (ESM) Table 1. Samples were tested in triplicate, and the results were normalised using cDNA amplified with 18S primers in the same samples.
For assessment of the secretory capacity, MIN6 cells (2 × 105) plated in 24-well dishes were transiently co-transfected with AUF1-overexpressing plasmids and with a construct encoding the human growth hormone (hGH) (pXGH5; Nichols Institute Diagnostics, San Juan Capistrano, CA, USA). After 72 h, the cells were washed and preincubated for 30 min in Krebs buffer (127 mmol/l NaCl, 4.7 mmol/l KCl, 1 mmol/l CaCl2, 1.2 mmol/l KH2PO4, 1.2 mmol/l MgSO4, 5 mmol/l NaHCO3, 0.1% BSA and 25 mmol/l HEPES, pH 7.4) containing 2 mmol/l glucose. The medium was then discarded, and the cells were incubated for 45 min in either the same buffer (basal condition) or Krebs buffer containing 20 mmol/l glucose (stimulatory condition). After collection of the supernatant fractions, the cells were lysed in PBS containing 0.5% Triton X-100 to evaluate total cellular hGH content. The amount of hGH in the samples was assessed using an hGH ELISA kit (Roche Diagnostics, Rotkreuz, Switzerland). The same was performed for measurement of insulin secretion except that the cells were not co-transfected with hGH and the cells were lysed in ethanol/acid (75% ethanol, 1.5% HCl and 23.5% water) to evaluate total insulin content. The amount of insulin in the samples was assessed using Insulin ELISA (EIA) (SPI-bio, Montigny-le-Bretonneux, France).
Subcellular fractionation and protein extraction
Subcellular fractionation was performed as described by Li et al. . Briefly, cells were lysed for 15 min on ice using a Triton X-100 lysis buffer (50 mmol/l Tris/HCl, pH 7.5, 0.5% Triton X-100, 137.5 mmol/l NaCl, 10% glycerol, 1 mmol/l sodium vanadate, 50 mmol/l NaF, 10 mmol/l sodium pyrophosphate, 5 mmol/l EDTA and the Protease Inhibitors Cocktail [Sigma, St Louis, MO, USA]). After a 15 min centrifugation at 12,000 g, the supernatant fraction was collected and stored as ‘cytoplasmic fraction’. The pellet was washed and resuspended in Triton X-100 lysis buffer containing 0.5% SDS. After sonication, the tube was centrifuged at 12,000 g for 15 min. The supernatant fraction yielded the ‘nuclear fraction’. To obtain whole cell extracts, the cells were directly scraped into SDS-containing lysis buffer.
Protein extracts (25–50 μg) were separated on polyacrylamide gels and transferred to poly(vinylidine fluoride) membranes. The membranes were incubated overnight at 4°C with primary antibodies. Immunoreactive bands were visualised by chemiluminescence (Amersham Biosciences, Piscataway, NJ, USA) after incubation with horseradish peroxidase-coupled secondary antibodies for 1 h at room temperature. The antibody against AUF1 (07-260) was purchased from Upstate (Temecula, CA, USA). The antibodies against myeloid cell leukaemia sequence 1 (MCL1 [sc-819]) and lamin B (sc-6216) were purchased from Santa-Cruz Biotechnology (Santa-Cruz, CA, USA). The antibodies against B cell leukaemia/lymphoma 2 (BCL2 ), inhibitor of kappa B (IκBα; 4814) and phospho-IκBα (2859) were purchased from Cell Signaling Technologies (Danvers, MA, USA). The antibody against actin (MAB1501) was from Chemicon International (Temecula, CA, USA). Finally, the antibody against α-tubulin (T5168) was obtained from Sigma (Buchs, Switzerland).
For the assessment of the apoptotic activity, MIN6 cells (1 × 105) and human dissociated islet cells plated in 24-well dishes were transiently transfected with plasmids overexpressing AUF1 isoforms or with siRNAs reducing their endogenous level. Apoptosis was assessed 2 days later by staining the cells with Hoechst 33342 (Invitrogen) and scoring the cells displaying pyknotic nuclei. The experiments were carried out blindly, and at least 800 cells per condition were analysed.
Transfected MIN6 cells were incubated overnight at 4°C with primary antibody directed against cleaved caspase 3 (9661; Cell Signaling). Immunolabelled proteins were visualised by incubating the cells for 1 h at room temperature with fluorescent secondary antibodies (Invitrogen). Images were obtained by fluorescence microscopy.
Statistical differences were tested by ANOVA. The experiments including more than two groups were first analysed by ANOVA, and multiple comparisons of the means were then carried out using the post hoc Dunnett’s test, with a discriminating p value of 0.05.
We first examined whether changes in the level of AUF1 alter the capacity of pancreatic beta cells to synthesise and secrete insulin. Overproduction of each isoform independently did not significantly affect proinsulin mRNA levels (ESM Fig. 4a). Moreover, blocking the production of AUF1 isoforms using siRNAs was not sufficient to restore proinsulin production (ESM Fig. 4b) and insulin content (ESM Fig. 4c) in MIN6 cells treated with IL-1β (10 ng/ml) for 24 h.
We then assessed whether AUF1 overproduction affects the secretory properties of beta cells. Overproduction of AUF1 isoforms did not influence insulin secretion under basal (2 mmol/l glucose) or stimulated (20 mmol/l glucose) conditions (ESM Fig. 5a). To verify that failure to detect changes in insulin secretion is not caused by the relatively low transfection efficiency, the cells were transiently co-transfected with AUF1-overproducing constructs and with an hGH-encoding plasmid. hGH is specifically targeted to beta cell secretory granules and is co-released with insulin , allowing selective monitoring of exocytosis from the transfected cells. AUF1 overproduction affected neither basal nor glucose-induced hGH release, confirming that the RNA-binding protein does not regulate the production of essential components of the beta cell secretory machinery (ESM Fig. 5b).
The initial phases of type 1 diabetes are characterised by a cytokine-mediated inflammatory reaction directed against pancreatic islets, which culminates in a progressive loss of beta cells. Proinflammatory cytokines released by T lymphocytes and macrophages invading the islets and by the endocrine cells themselves alter beta cell gene expression by affecting the activity of important transcription factors leading to impairment of key signalling pathways. The involvement of transcription factors such as NFκB, signal transducers and activators of transcription 1 (STAT1) and activator protein 1 (AP-1) in cytokine-mediated beta cell damage has been extensively investigated . However, it is becoming increasingly clear that other regulatory molecules, acting upstream or downstream of transcription factors, make an important contribution to the control of gene expression. This is the case for RNA-binding proteins, such as AUF1, that bind to specific regions of their target mRNAs, thereby influencing their stability and translation. There is mounting evidence that AUF1 plays a central role in inflammation. Indeed, AUF1-binding motifs are present in the messengers of important inflammatory mediators such as the cytokines, TNFα and granulocyte–macrophage colony-stimulating factor [27, 28], the chemokines, CXCL2/MIP2 and CCL2/MCP1 , and those of some interleukins [29, 30]. Moreover, AUF1 affects the production of important components of the signalling pathways initiated by cytokines [31, 32].
Here, we demonstrate that beta cells produce four AUF1 isoforms that are differentially distributed between the cytosolic and nuclear compartments. The localisation of AUF1 can be influenced by a variety of factors including: the presence or absence of exons containing nuclear localisation signals [33, 34]; interaction with transporters  or chaperones [36, 37]; protein ubiquitination ; and phosphorylation of specific residues [7, 38]. At steady-state, most AUF1 isoforms accumulate in the nucleus but are partially relocalised upon exposure of insulin-secreting cells to proinflammatory cytokines. The precise mechanisms underlying the nucleocytoplasmic shuttling of the protein have not yet been defined. However, since the effect of the cytokines can be prevented by inhibiting the MAPK, ERK, this process is likely to involve the phosphorylation of one or more residues of AUF1. In agreement with this hypothesis, nuclear ERK has recently been reported to stimulate nucleocytoplasmic translocation and activation of AUF1p42 . In contrast, in our hands, neither JNK nor p38 MAPK appeared to affect the localisation of AUF1 isoforms.
Prolonged exposure of beta cells to cytokines sensitises them to apoptosis. Overproduction of p40, p42 and p45 AUF1 isoforms mimicked the effect of the cytokines and led to a similar impact on beta cell survival. Moreover, blockade of AUF1 production protected the beta cells from apoptosis elicited by the cytokines, indicating that this RNA-binding protein contributes to the deleterious events that lead to death of insulin-secreting cells upon chronic exposure to these inflammatory mediators. We were able to partially elucidate the mechanisms through which AUF1 affects beta cell survival. Indeed, the reduction of the production of two anti-apoptotic proteins, BCL2 and MCL1, elicited by cytokines was efficiently prevented by silencing AUF1. Both of these anti-apoptotic proteins have been shown to play a role in beta cell survival [22, 40]. The effect of AUF1 on BCL2 may be mediated, at least in part, by direct association of the RNA-binding protein to the AU-rich elements present in the 3′-UTR of Bcl2 mRNA, resulting in messenger destabilisation [21, 41]. Indeed, we observed that silencing of AUF1 results in a small but significant increase in Bcl2 mRNA stability. So far, consensus sequences for AUF1 binding have not been identified in the Mcl1 mRNA. Moreover, blockade of AUF1 production by RNA interference appears not to influence Mcl1 mRNA stability. Thus the level of this anti-apoptotic protein is likely to be modulated indirectly through the modification of other regulatory factors.
Our results suggest that one of the key signalling pathways involved in beta cell apoptosis is also regulated by AUF1. The transcription factor NFκB contributes to cytokine-mediated beta cell dysfunction and to the development of type 1 diabetes. Indeed, Eldor and collaborators showed that mice producing an NFκB repressor are protected from diabetes induced by streptozotocin injections . Recently, inhibition of AUF1 was reported to prevent lipopolysaccharide-induced NFκB signalling in monocytes and macrophages . In agreement with these data, we found that phosphorylation of IκBα elicited by a mix of cytokines, an essential step in the activation of the NFκB pathway, is strongly impaired in cells in which AUF1 isoforms are downregulated. The attenuation of the NFκB signalling resulted in reduced induction of iNOS and diminished production of the chemokines, CXCL2/MIP2 and CCL2/MCP1. In other cell types, AUF1 destabilises iNos mRNA . Although our data did not reach statistical significance, in MIN6 cells treated with siRNAs directed against AUF1, iNos mRNA tended to be more stable (ESM Fig. 6 bottom panel). Therefore we cannot rule out the possibility that a similar mechanism also takes place in beta cells. However, even if present, in MIN6 cells the effect on mRNA stability appears to have less impact on iNOS levels than the attenuation of the NFκB signalling. NO production plays a pivotal role in the deleterious action exerted by proinflammatory cytokines on beta cells . Therefore, at least part of the protective effect of AUF1 silencing can probably be attributed to the smaller induction of iNOS. Chemokines such as CXCL2/MIP2 and CCL2/MCP1 released by beta cells chronically exposed to cytokines are thought to constitute an immunogenic signal that favours the recruitment of immune cells and contributes to the amplification of the inflammatory reaction . Our findings indicate that activation of AUF1 can modulate this inflammatory signal that promotes the development of type 1 diabetes.
In this study, we highlighted an important contribution of AUF1 to the deleterious effects of proinflammatory cytokines on beta cell survival. We were able to partially dissect the events leading to AUF1 redistribution and to characterise the signalling cascades downstream of the activation of the RNA-binding protein. Future studies need to precisely identify the molecular mechanisms regulating the nucleocytoplasmic shuttling of AUF1 in cytokine-treated beta cells and to definitively assess its contribution to the development of type 1 diabetes. Our findings suggest that a better knowledge of the role played by AUF1 and other RNA-binding proteins in the islet inflammatory reaction could potentially open the way to new strategies to prevent beta cell death and the development of type 1 diabetes.
This work was supported by the Swiss National Science Foundation grant 31003A-127254 (to R. Regazzi).
ER generated the researched and analysed the data, wrote the manuscript and approved its final version. SG generated the researched and analysed the data, critically reviewed the manuscript and approved its final version. AP contributed to interpretation of the data, critically reviewed the manuscript and approved its final version. RR conceived and designed the experiments, analysed the research data, wrote the manuscript and approved its final version.
Duality of interest
The authors declare that there is no duality of interest associated with this manuscript.