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Diabetologia

, Volume 56, Issue 3, pp 520–532 | Cite as

Baculoviral inhibitors of apoptosis repeat containing (BIRC) proteins fine-tune TNF-induced nuclear factor κB and c-Jun N-terminal kinase signalling in mouse pancreatic beta cells

  • B. M. Tan
  • N. W. Zammit
  • A. O. Yam
  • R. Slattery
  • S. N. Walters
  • E. Malle
  • S. T. Grey
Article

Abstract

Aims/hypothesis

For beta cells, contact with TNF-α triggers signalling cascades that converge on pathways important for cell survival and inflammation, specifically nuclear factor κB (NF-κB), c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase pathways. Here, we investigated the function of baculoviral inhibitors of apoptosis repeat containing (BIRC) proteins in regulating TNF signalling cascades.

Methods

TNF regulation of Birc genes was studied by mRNA expression and promoter analysis. Birc gene control of cell signalling was studied in beta cell lines, and in islets from Birc2 −/− and Birc3 −/− mice, and from Birc3 −/− Birc2Δ beta cell mice that selectively lack Birc2 and Birc3 (double knockout [DKO]). Islet function was tested by intraperitoneal glucose tolerance test and transplantation.

Results

TNF-α selectively induced Birc3 in beta cells, which in turn was sufficient to drive and potentiate NF-κB reporter activity. Conversely, Birc3 −/− islets exhibited delayed TNF-α-induced IκBα degradation with reduced expression of Ccl2 and Cxcl10. DKO islets showed a further delay in IκBα degradation kinetics. Surprisingly, DKO islets exhibited stimulus-independent and TNF-dependent hyperexpression of TNF target genes A20 (also known as Tnfaip3), Icam1, Ccl2 and Cxcl10. DKO islets showed hyperphosphorylation of the JNK-substrate, c-Jun, while a JNK-antagonist prevented increases of Icam1, Ccl2 and Cxcl10 expression. Proteosome blockade of MIN6 cells phenocopied DKO islets. DKO islets showed more rapid loss of glucose homeostasis when challenged with the inflammatory insult of transplantation.

Conclusions/interpretation

BIRC3 provides a feed-forward loop, which, with BIRC2, is required to moderate the normal speed of NF-κB activation. Paradoxically, BIRC2 and BIRC3 act as a molecular brake to rein in activation of the JNK signalling pathway. Thus BIRC2 and BIRC3 fine-tune NF-κB and JNK signalling to ensure transcriptional responses are appropriately matched to extracellular inputs. This control is critical for the beta cell’s stress response.

Keywords

Beta cell BIRC Diabetes Gene Inflammation Islet JNK NF-κB TNF 

Abbreviations

ASK1

Apoptosis signal-regulating kinase 1

BIRC

Baculoviral inhibitors of apoptosis repeat containing proteins

DKO

Birc3 −/− Birc2Δ beta cell mice that selectively lack Birc2 and Birc3

eGFP

Enhanced green fluorescent protein

eGFP-Birc3

pIRES2-eGFP-Birc3

IAP

Inhibitors of apoptosis

IκBα

Nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha

JNK

c-Jun N-terminal kinase

NF-κB

Nuclear factor of kappa light polypeptide gene enhancer in B cells

NIK

NF-κB-inducing kinase

p38

p38 mitogen-activated protein kinases

PDTC

Pyrrolidine dithiocarbamate

pIRES2

Plasmid containing an internal ribosome entry site two

RIPK1

Receptor-interacting protein kinase 1

TNFR

TNF receptor

TRAF

TNFR-associated factor

XIAP

X-linked inhibitor of apoptosis protein

Introduction

TNF-α is a pleiotropic cytokine that is involved in the pathogenesis of autoimmune type 1 diabetes and influences cell proliferation, inflammation and cell death. TNF-α mediates its actions through two distinct receptors, TNF receptor (TNFR) 1/p55 and TNFR2/p75, both of which are expressed on virtually all cell types [1]. TNFR1 is predominantly responsible for TNF signalling in most cell types [1], mediated by the sequential formation of two complexes upon TNFR1 ligation [2]. Complex I forms with the recruitment of TNFR1-associated death domain protein, which subsequently recruits TNFR-associated factor (TRAF) 2, TRAF5 and receptor-interacting protein kinase 1 (RIPK1) [2]. Within complex I, RIPK1 is essential for activating the nuclear factor of kappa light polypeptide gene enhancer in B cells (NF-κB) pathway [3], while TRAF2 is required for activation of the c-Jun N-terminal kinase (JNK) pathway [4]. These signalling cascades are imperative for the control of genes involved in immune response, inflammation and cell survival [1]. In addition, a secondary complex, spatially and temporally distinct from complex I, comprises Fas-associated death domain protein and caspase 8, which triggers a pro-apoptotic cascade [2]. Normally, TNF-α-stimulated cells are protected from the pro-apoptotic force of complex II by the expression of NF-κB-induced genes, including A20 (also known as Tnfaip3) and Cflip (also known as Cflar), both of which are regulated via complex I. A20 and Cflip prevent activation of caspase-8, possibly at the level of complex II [5, 6]. Thus, TNF-α triggers a co-ordinated and complex cellular program regulating cell proliferation, inflammation and cell death.

The contribution of TNF-α to type 1 diabetes pathogenesis is multifaceted. A characteristic feature of diabetes is the infiltration of the pancreatic islets with T cells, with TNFRI-null islets being protected from destruction by T lymphocytes [7]. TNF-α is an early cytokine detected within the immune infiltrate surrounding islets [8]. In the context of type 1 diabetes, TNF-α exerts detrimental effects, including heightened expression of inflammatory genes in pancreatic beta cells [9], the impairment of insulin secretion [10] and the triggering of beta cell apoptosis [5]. TNF-α’s ability to induce and perpetuate diabetes has been clearly demonstrated in 3-week-old NOD mice, which upon administration of exogenous TNF-α had accelerated onset of diabetes and increased disease frequency [11]. In contrast, diabetes progression can be ameliorated by anti-TNF-α monoclonal antibody treatment [11]. Despite the importance of TNF-α in the pathogenesis of type 1 diabetes, the exact molecular machinery governing the TNF signalling cascade in islets and pancreatic beta cells remains poorly understood. An improved understanding of TNF signalling could lead to novel diagnostics or therapies for patients with type 1 diabetes.

Inhibitors of apoptosis (IAP) or baculoviral IAP repeat containing proteins (BIRC) are implicated in the regulation of downstream TNF signalling networks [12]. A total of eight BIRC proteins has been identified, namely: neuronal AIP (BIRC1), cellular IAP1 (BIRC2), cellular IAP2 (BIRC3), X-linked inhibitor of apoptosis protein (XIAP/BIRC4), survivin (TIAP/BIRC5), baculoviral IAP repeat-containing ubiquitin-conjugating enzyme (BRUCE/Apollon/BIRC6), Melanoma-IAP (ML-IAP/Livin/kidney-IAP/BIRC7) and ILP-2 (Testis Specific-IAP)/BIRC8). A key cellular function of BIRC proteins is the regulation of apoptosis [12]. Indeed, XIAP can prevent apoptosis in a number of cell types, including islets [13, 14, 15, 16] by preventing direct IAP binding protein with low pI (DIABLO)-mediated cleavage of caspase 9 [17]. While BIRC proteins were thought to primarily act as regulators of apoptosis, this function has not been established for all its members; it is now recognised that BIRC proteins are also involved in the regulation of diverse cellular functions, including the regulation of signalling and inflammation [12]. Further to this, BIRC2 and BIRC3, together with TRAF2, have been identified as key components of the TNF signalling pathway that are necessary for TNF-mediated NF-κB activation [18]. With reference to beta cells, some studies have shown that beta cells express Birc3, and that Birc3 expression is regulated by the TNF-α-mediated NF-κB pathway [5, 19]; however, its binding partner, Birc2, has not been examined. This suggested to us that in beta cells, BIRC3 may participate in a TNF signalling feedback loop, an idea that has not been tested to date. Here we examined the requirements for BIRC3 and BIRC2 in TNF signalling in beta cells.

Methods

Mouse strains

BALB/c and C57BL/6 mice were from Australian BioResource (Mossvale, NSW, Australia). Birc2 −/− and Birc3 −/− mice were a kind gift of D. Vaux (Cell Signalling and Cell Death division, Walter and Eliza Hall Institute, Parkville, VIC, Australia). Birc3 −/− mice containing Birc2 loxP/lox-P were crossed with RIP-Cre mice (Cre driven by the rat insulin promoter) (Jax Mice, Bar Harbour, ME, USA) to generate beta cell-specific double knockout Birc3 −/− Birc2Δ beta cell mice that selectively lack Birc2 and Birc3 (double knockout [DKO]). Procedures complied with the Australian Code of Practice for Care and Use of Animals for Scientific Purposes.

Cytokines

Mouse islets were isolated as described [20]. Either 70–100 islets or 1 × 106 MIN6 beta cells [21] were stimulated with 200 U/ml of TNF-α for 4 h (RNA analysis) and 8 h (promoter analysis) (R&D Systems, Minneapolis, MN, USA). β-TC3 cells were used for some transfection studies [22]. In some cases, cells were pretreated for 1 h with 1 μmol/l actinomycin D, 50 μmol/l SP600125, 1 μmol/l MG-132 and 50 μmol/l pyrrolidine dithiocarbamate (PDTC) (all from Sigma-Aldrich, St Louis, MO, USA), and with 20 μmol/l SB203580 (Cell Signalling Technology, Beverly, MA, USA), prior to cytokine stimulation. Western blotting was performed using standard protocols with IκBα antibody (9242L), phospho-c-Jun (Ser73, 9164S) (Cell Signalling Technology), β-actin (Clone AC-15; A5441; Sigma-Aldrich) and horseradish peroxidase-conjugated antibodies (Pierce, Rockford, IL, USA), and chemiluminescence (GE Healthcare, Uppsala, Sweden).

Quantitative RT-PCR

Total RNA and cDNA were generated from beta cell lines and mouse islets using standard techniques previously described by this research group [5]. Relative gene expression is the ratio to the housekeeping gene, Cyclophilin A (Ppia); fold-changes were analysed using the \( {{2}^{{ - \Delta \Delta {{{\rm{C}}}_{{\rm{t}}}}}}} \) method. Primers used for PCR are provided in electronic supplementary material (ESM) Table 1.

Cloning of Birc3 and promoter

The Birc3 coding region was cloned from cytokine-stimulated mouse islets, using the primers shown in ESM Table 2, with a PCR system (Expand High Fidelity PCR; Roche, Indianapolis, IN, USA), and subcloned into a pIRES2-eGFP vector (Clontech Laboratories, Mountain View, CA, USA). Putative transcription factor binding sites in the Birc3 proximal promoter were identified using PROMO3.0 [23]. The mouse Birc3 5′ untranslated region was amplified from genomic DNA using the primers shown in ESM Table 3 with KOD Hot Start DNA Polymerase (Merck, Darmstadt, Germany) and subcloned into a pGL3 basic vector (Promega, Sydney, NSW, Australia).

Transient transfections

Transfection of MIN6 and β-TC3 cells was performed using techniques and expression plasmids encoding NF-κB.Luc (Promega), RelA/p65, A20.Luc, and plasmid containing beta galactosidase under the control of the Rous Sarcoma virus (pRSV-β-galactosidase) as previously described by us [5]. Luciferase values (Luciferase Assay System; Promega) were normalised to β-galactosidase activity (Galacto-Star; Applied Biosystems, Bedford, MA, USA) for relative luciferase activity.

Adenovirus transduction

MIN6 cells (1 × 106) were infected for 1.5 h with recombinant adenovirus expressing GFP (rAd.GFP) or rAd.IκBα [24] at a multiplicity of infection of 100:1 in serum-free medium before adding FCS-enriched medium to achieve a 10% (vol./vol.) FCS concentration. Cells were incubated overnight and the medium was replaced prior to stimulation with TNF-α for 4 h.

In vivo studies

Intraperitoneal glucose tolerance tests were performed on 12-week-old male mice administered 2 g/kg glucose following a 16 h fast. For islet transplantation, islets from donor (H-2b) mice were transplanted into recipient mice (H-2k) that had been rendered diabetic with 200 mg/kg streptozotocin (Sigma-Aldrich) as previously described [25].

Statistics

Statistical analyses were performed using GraphPad Prism 5 software (GraphPad Software, San Diego, CA, USA).

Results

Birc3 is an early immediate-response gene regulated by de novo transcription

In the steady state, Birc3 was highly expressed (Fig. 1a) and was selectively increased by TNF-α in primary islets (p ≤ 0.0001) (Fig. 1b) and MIN6 cells (Fig. 1c, d). Once induced, high levels of Birc3 mRNA were maintained in primary islets (p ≤ 0.01 and p ≤ 0.0001) (Fig. 1e) and MIN6 cells (p ≤ 0.01 and p ≤ 0.001) (Fig. 1f).
Fig. 1

Birc3 is an early immediate-response gene. (a) Expression of Birc genes, as indicated, in primary mouse islets that were left untreated or (b) were stimulated with TNF-α for 4 h, and in (c) MIN6 cells left untreated or (d) stimulated with TNF-α for 4 h. (e) Expression of Birc3 in primary islets and (f) in MIN6 cells stimulated with TNF-α for 1 to 8 h. Data represent mean ± SEM of gene expression relative to Ppia from at least three independent experiments. Statistical comparisons were by ANOVA with pair-wise multiple comparisons made using an unpaired t test or single unpaired t test between two groups. **p ≤ 0.01, ***p ≤ 0.001 and ****p ≤ 0.0001. Birc1a, also known as Naip1; Birc1b, also known as Naip2; Birc1e, also known as Naip5; Birc1f, also known as Naip6; Birc4, also known as Xiap

To determine the mechanism of Birc3 induction, primary islets and MIN6 cells were pretreated with the transcription inhibitor, actinomycin D, prior to TNF-α stimulation. Pre-treatment with an optimal dose of actinomycin D (1 μmol/l) resulted in >70% suppression of TNF-α-induced Birc3 expression in islets (p ≤ 0.0001) (Fig. 2a) and MIN6 cells (p ≤ 0.001) (Fig. 2b). Thus Birc3 is an inflammation-regulated, early immediate-response gene regulated via de novo gene transcription.
Fig. 2

Birc3 is regulated by de novo transcription. Birc3 expression in primary islets (a) or MIN6 cells (b) that were left untreated or were pretreated for 1 h with actinomycin D (AcD), with or without TNF-α stimulation for 4 h. (c) Diagram of putative transcription elements in the Birc3 proximal promoter ∼500 bp upstream of the transcription start site (START), identified using PROMO 3.0 software. AP-1, activator protein 1; STAT, signal transducer and activator of transcription. (d) Induction of the Birc3 reporter in MIN6 cells by TNF-α exposure for 8 h. RLA, relative luciferase activity. (e) Expression of Birc3 in MIN6 cells that were treated with or without TNF-α for 4 h, and were either left untreated or had been pretreated for 1 h with SB203580, (f) SP600125 or (g) PDTC. (h) Expression of Birc3 in primary islets that were left untreated or were pretreated for 1 h with PDTC, with or without TNF-α stimulation for 4 h. (i) Induction of the Birc3 reporter in MIN6 cells by RelA/p65. EV, control vector. (j) Induction by RelA/p65 of endogenous Birc genes mRNA, as indicated, in β-TC3 cells. Birc1a, also known as Naip1; Birc1b, also known as Naip2; Birc1e, also known as Naip5; Birc1f, also known as Naip6; Birc4, also known as Xiap. (k) Western blot analysis of IκBα levels in MIN6 cells transduced with rAD.GFP or rAD.IκBα. β-Actin was used as a loading control. Representative blots are shown. IκBα (39 kDa) was quantified by densitometry and expressed relative to β-actin. (l) Induction of Birc3 by MIN6 cells left non-infected (NI), or transduced with rAD.GFP or rAD.IκBα prior to TNF-α stimulation for 4 h. (a, b, eh, j, l) Data represent mean ± SEM of gene expression relative to Ppia from three independent experiments or (d, k) mean ± SEM from three independent experiments. Statistical comparisons were made by single unpaired t test between two groups. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 and ****p ≤ 0.0001

NF-κB controls TNF-induced Birc3 expression

The 5′ untranslated region of the Birc3 proximal promoter contained three putative consensus NF-κB binding sites and a TATA-like box with the sequence ‛TTTAAA’ (Fig. 2c). Potential putative activator protein 1, signal transducers and activators of transcription 1 and protein 53 (p53) binding sites were also identified. This region was cloned to generate the Birc3 reporter, which showed an approximately twofold increase upon TNF-α stimulation (p ≤ 0.01) (Fig. 2d). These results indicate that the Birc3 proximal promoter contains cytokine responsive elements that direct Birc3 gene expression.

To determine the molecular mechanisms driving Birc3 transcription, MIN6 cells were left untreated, or were pretreated with SP600125, SB203580 or PDTC to selectively target the JNK, p38 mitogen-activated protein kinases (p38) or NF-κB pathways respectively, prior to TNF-α stimulation. Birc3 mRNA levels were measured by quantitative RT-PCR. Neither SB203580 (Fig. 2e) nor SP600125 (Fig. 2f), but only PDTC pre-treatment inhibited TNF-α-induced Birc3 expression, e.g. by ∼90% for MIN6 cells (p ≤ 0.05) (Fig. 2g) and ∼75% for islets (p ≤ 0.05) (Fig. 2h). These data show that NF-κB is necessary for TNF-α-induced Birc3 expression.

NF-κB is sufficient to drive de novo Birc3 expression

When MIN6 cells were co-transfected with Birc3 reporter and a RelA/p65 expression vector, Birc3 reporter activity was increased by twofold (p ≤ 0.001) (Fig. 2i). To determine whether RelA/p65 was sufficient to drive endogenous Birc3 gene expression, β-TC3 cells were transfected with the RelA/p65 expression vector and Birc family gene expression determined by quantitative RT-PCR. In this case, Birc3 was selectively induced (∼sixfold, p ≤ 0.05) (Fig. 2j). Moreover, overabundance of IκBα in MIN6 cells (Fig. 2k) resulted in a 77% suppression of TNF-α-induced Birc3 expression (p ≤ 0.01) (Fig. 2l). Thus, NF-κB is necessary and sufficient to drive de novo transcription of Birc3 mRNA.

Increased Birc3 regulates NF-κB signalling

Our results imply a model whereby TNF-α activates NF-κB, which subsequently induces increased Birc3 expression. To elucidate the function of increased Birc3 in pancreatic beta cells, MIN6 cells were transfected with the pIRES2-eGFP control vector or 0.3 to 0.6 μg pIRES2-eGFP-Birc3 (eGFP-Birc3). eGFP-Birc3-transfected MIN6 cells expressed Birc3 in a range similar to TNF-α-stimulated MIN6 cells (i.e. 12-fold, p ≤ 0.001) (Fig. 3a). Subsequently, MIN6 cells were transfected with a luciferase reporter containing two tandem NF-κB responsive elements (NF-κB.Luc), plus either 0.1 to 0.6 μg eGFP-Birc3, or 0.6 μg control vector. In MIN6 cells, eGFP-Birc3-expressing cells showed a dose-dependent increase in NF-κB reporter activity (Fig. 3b), while a combination of TNF-α stimulation and 0.6 μg eGFP-Birc3 showed a further increase of NF-κB reporter activity, from 3.4-fold (p ≤ 0.05) with TNF-α alone to sevenfold with eGFP-Birc3 (p ≤ 0.01).
Fig. 3

Increased Birc3 regulates NF-κB signalling. (a) Endogenous expression of Birc3 in MIN6 cells transfected either with 0.6 μg control vector (EV) for 24 h, in the presence or absence of TNF-α for 4 h, or with 0.3 μg – 0.6 μg eGFP-Birc3. Data represent mean ± SEM of gene expression relative to Ppia from six independent experiments. (b) Induction of the NF-κB reporter in MIN6 cells transfected with either 0.6 μg EV or 0.1 to 0.6 μg eGFP-Birc3, in the absence or presence of TNF-α for 8 h. Data represent mean ± SEM from three independent experiments. (c) Induction of A20 reporter in β-TC3 cells transfected with either 0.3 μg EV or 0.3 μg eGFP-Birc3, in the absence or presence of TNF-α for 8 h. Data represent mean ± SEM from four independent experiments. Statistical comparisons were made by single unpaired t test between two groups or by ANOVA with pair-wise multiple comparisons made using an unpaired t test. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 and ****p ≤ 0.0001

To elucidate how Birc3 modulates a native NF-κB promoter, the effect of Birc3 activity on the NF-κB-driven A20 reporter [5] was examined. β-TC3 cells were transfected with 2 μg A20.Luc and either 0.25 μg pIRES2-eGFP control vector or eGFP-Birc3. A20 reporter activity increased from sevenfold (p ≤ 0.001) for TNF-α alone to 9.5-fold for TNF-α and eGFP-Birc3 (Fig. 3c). These data demonstrate that increased Birc3 expression is sufficient to drive NF-κB activity and enhance TNF-α-induced NF-κB signalling.

A redundant role for Birc3 in NF-κB signalling

The effect of Birc3 loss-of-function on beta cell TNF-α-mediated NF-κB signalling was examined in Birc3 −/− mice (Fig. 4a). Birc3 is not necessary for beta cell function as indicated by the normal glucose tolerance of Birc3 −/− mice (Fig. 4b). We analysed TNF-α-induced NF-κB signalling in wild-type or Birc3 −/− islets via western blot analysis of IκBα levels [26]. Compared with wild-type islets, TNF-α-stimulated Birc3 −/− islets showed delayed IκBα degradation, but a normal return to baseline levels at 60 min (Fig. 4c). Analysis of NF-κB-regulated and TNF-α-induced genes in Birc3 −/− islets revealed an abnormal expression pattern. While normal A20 induction (Fig. 4d) was observed, expression of Ccl2 (p ≤ 0.01) (Fig. 4e) and Cxcl10 (p ≤ 0.01) (Fig. 4g) was reduced, with Icam1 levels being modestly increased (p ≤ 0.01) (Fig. 4f). These data indicate that BIRC3 is necessary to fine-tune cellular responses to TNF-α.
Fig. 4

A redundant role for Birc3 in NF-κB signalling. (a) Expression of Birc3 in wild type (WT) primary islets in the absence (black circles) or presence (white circles) of TNF-α for 4 h, and in Birc3 −/− islets under the same conditions (absence, black triangles; presence, white triangles). Values from individual experiments are shown (n ≥ 5 per group). Bars represent mean ± SEM of gene expression relative to Ppia. (b) Blood glucose concentrations during intraperitoneal glucose tolerance tests on 12-week-old male wild-type (black circles, solid line) and Birc3 −/− (white triangles, dotted line) mice. Data represent mean ± SEM from four mice per group. (c) Western blot analysis of NF-κB activation measured by IκBα degradation in wild-type (WT) and Birc3 −/− islets that were either left untreated or were stimulated with TNF-α at the indicated times. β-Actin was used as a loading control. Representative blots are shown. IκBα (39 kDa) was quantified by densitometry and expressed relative to β-actin. Data represent mean ± SEM from three independent experiments. (d) Expression of A20, (e) Ccl2, (f) Icam1 and (g) Cxcl10 in wild-type islets in the absence (black circles) or presence (white circles) of TNF-α for 4 h, and in Birc3 −/− islets under the same conditions (absence, black triangles; presence, white triangles). Values from individual experiments are shown (n ≥ 11 per group). Bars represent mean ± SEM of gene expression relative to Ppia. Statistical comparisons were made by single unpaired t test between two groups. *p ≤ 0.05, **p ≤ 0.01 and ****p ≤ 0.0001

Birc2 provides negative control of TNF pathways in beta cells

BIRC2 can compensate for BIRC3 [3, 27], therefore we examined NF-κB signalling in single Birc2 −/− islets (Fig. 5a). Birc2 −/− mice had normal glucose homeostasis (Fig. 5b). Regarding NF-κB signalling, Birc2 −/− islets had delayed kinetics of TNF-α-stimulated IκBα degradation (Fig. 5c). Noticeably, in the absence of a pro-inflammatory stimulus, Birc2 −/− islets expressed high levels of Ccl2 (ninefold greater, p ≤ 0.01) (Fig. 5e) and Cxcl10 (sevenfold greater, p ≤ 0.05) (Fig. 5g) compared with wild-type islets. Furthermore, while TNF-α-stimulated Birc2 −/− islets expressed normal induced levels of A20 (Fig. 5d), induction of Ccl2 (threefold, p ≤ 0.001) and Icam1 (threefold, p ≤ 0.0001) mRNA was significantly higher than for wild-type islets (Fig. 5e, f). These findings suggest that Birc2 functions as a negative control factor, dampening expression of TNF target genes in islet cells.
Fig. 5

Birc2 provides negative control of TNF-pathways in beta cells. (a) Expression of Birc2 in wild-type islets in the absence (black circles) or presence (white circles) of TNF-α for 4 h, and in Birc2 −/− islets under the same conditions (absence, black squares; presence, white squares). Values from each experiment are shown (n ≥ 6 per group). Bars represent mean ± SEM of gene expression relative to Ppia. (b) Blood glucose concentrations during intraperitoneal glucose tolerance tests on 12-week-old male wild-type (black circles, solid line) and Birc2 −/− (white squares, dotted line) mice. Data represent mean ± SEM from four mice per group. (c) Western blot analysis of NF-κB activation measured by IκBα degradation in wild-type (WT) and Birc2 −/− islets that were left untreated or were stimulated with TNF-α at the indicated times. β-Actin was used as a loading control. Representative blots are shown. IκBα (39 kDa) was quantified by densitometry and expressed relative to β-actin. Data represent mean ± SEM from three independent experiments. (d) Expression of A20, (e) Ccl2, (f) Icam1 and (g) Cxcl10 in wild-type islets in the absence (black circles) or presence (white circles) of TNF-α for 4 h, and in Birc2 −/− islets under the same conditions (absence, black squares; presence, white squares). Values from individual experiments are shown (n ≥ 5 per group). Bars represent mean ± SEM of gene expression relative to Ppia. Statistical comparisons were made by single unpaired t test between two groups. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 and ****p ≤ 0.0001

Dysregulated gene expression in the absence of Birc2 and Birc3

As BIRC2 and BIRC3 can compensate for each other to allow NF-κB signalling [3, 27], we generated DKO mice. DKO islets exhibited loss of BIRC2 and BIRC3 (Fig. 6a, b), but normal glucose homeostasis, indicating that these two proteins are dispensable for physiological beta cell function (Fig. 6c). Analysis of TNF-α-induced IκBα degradation revealed that NF-κB signalling kinetics were significantly delayed in the absence of BIRC2 and BIRC3 (Fig. 6d). Moreover, DKO islets showed significant disturbances in the regulation of TNF target genes, expressing significantly higher stimulus-independent levels of A20 (fourfold, p ≤ 0.0001), Ccl2 (eightfold, p ≤ 0.001), Icam1 (22-fold, p ≤ 0.0001) and Cxcl10 (17-fold, p ≤ 0.05) compared with resting wild-type islets (Fig. 6e–h). Analysis of TNF-α-induced responses showed hyperinduction of A20 (threefold, p ≤ 0.0001), Ccl2 (fourfold, p ≤ 0.0001) and Icam1 (18-fold, p ≤ 0.001), but not of Cxcl10 mRNA compared with wild-type islets (Fig. 6e–h). Therefore, Birc2 −/−- and Birc3 −/−-deficient beta cells show aberrant NF-kB signalling and dysregulated control of TNF target genes.
Fig. 6

Dysregulated gene expression in the absence of Birc2 and Birc3. (a) Expression of Birc2 and (b) Birc3 in wild-type islets (black circles) and DKO islets (black diamonds). Values from individual experiment are shown (n ≥ 7 per group). Bars represent mean ± SEM of gene expression relative to Ppia. (c) Blood glucose concentrations during intraperitoneal glucose tolerance tests on 12-week-old male wild-type (black circles, solid line) and DKO (white diamonds, dotted line) mice. Data represent mean ± SEM from at least seven mice per group. (d) Western blot analysis of NF-κB activation measured by IκBα degradation in wild-type (WT) and DKO islets that were left untreated or were stimulated with TNF-α at the indicated times. β-Actin was used as a loading control. Representative blots are shown. IκBα (39 kDa) was quantified by densitometry and expressed relative to β-actin. Data represent mean ± SEM from three independent experiments. (e) Expression of A20, (f) Ccl2, (g) Icam1 and (h) Cxcl10 in wild-type islets in the absence (black circles) or presence (white circles) of TNF-α for 4 h, and in DKO islets under the same conditions (absence, black diamonds; presence, white diamonds). Values from individual experiments are shown (n ≥ 6 per group). Bars represent mean ± SEM of gene expression relative to Ppia. Statistical comparisons were made by single unpaired t test between two groups. *p ≤ 0.05, ***p ≤ 0.001 and ****p ≤ 0.0001

Birc2 and Birc3 fine-tune inflammatory gene expression in islet cells

We examined the relative contribution of BIRC2 and BIRC3 to the phenotypes observed for DKO islets. We found that in the absence of a TNF-stimulus, A20 (p ≤ 0.0001) (Fig. 7a) and Icam1 (p ≤ 0.0001) (Fig. 7c) expression was increased in DKO islets, but not in Birc2 −/− or Birc3 −/− islets, indicating that neither BIRC2 nor BIRC3 alone can exert molecular control over A20 and Icam1 expression. In contrast, molecular control of Ccl2 expression was strictly controlled by BIRC2, as the phenotype of Birc2 −/− islets was identical to DKO islets (p ≤ 0.001 and p ≤ 0.05) (Fig. 7b). Like Ccl2, control of Cxcl10 expression appears to be more strongly dependent on the presence of BIRC2 (p ≤ 0.05 and p ≤ 0.05) (Fig. 7d).
Fig. 7

Birc2 and Birc3 fine-tune inflammatory gene expression in islet cells. (a) Basal levels of A20, (b) Ccl2, (c) Icam1 and (d) Cxcl10 mRNA from wild-type (WT) (white circles), Birc2 −/− (white squares), Birc3 −/− (white triangles) or DKO (white diamonds) islets. Data sourced from Figs. 4, 5 and 6. Values from individual experiments are shown (n ≥ 5 per group). (e) Basal levels of A20, (f) Ccl2, (g) Icam1 and (h) Cxcl10 from DKO islets either left untreated (NT) (white diamonds) or treated for 8 h with actinomycin D (AcD) (black diamonds). Values from individual experiments are shown (n = 4 per group). Statistical comparisons were made by single unpaired t test between two groups. Bars represent mean ± SEM of gene expression relative to Ppia. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 and ****p ≤ 0.0001

BIRC proteins regulate gene transcription through JNK

When DKO islets were treated with actinomycin D, the stimulus-independent expression of A20 (13-fold, p ≤ 0.0001) and Cxcl10 (tenfold, p ≤ 0.01), but not of Ccl2 or Icam1, was suppressed (Fig. 7e–h), indicating that basal A20 and Cxcl10 mRNA levels are regulated by active transcription in DKO islets.

We hypothesised that the increased stimulus-independent transcription rates of A20 and Cxcl10 in DKO islets might be due to loss of control of the normal regulatory paths that control TNF-α-induced gene expression. We first sought to identify the possible pathways involved in normal TNF-α-induced gene expression. To do this, MIN6 cells were left untreated, or were pretreated with PDTC, SP600125 or SB203580 to inhibit NF-κB, JNK or p38 respectively, prior to TNF-α stimulation for 4 h. Our findings show that, in MIN6 cells, TNF-α-stimulated A20 was regulated by NF-κB, but not by the JNK or p38 pathways (Fig. 8a), whereas TNF-α-induced Ccl2, Icam1 and Cxcl10 expression was regulated by the NF-κB and the JNK pathways (Fig. 8b–d).
Fig. 8

Birc2 and Birc3 fine-tune TNF signalling in islet cells. (a) Expression of A20, (b) Ccl2, (c) Icam1 and (d) Cxcl10 in MIN6 cells left untreated (black circles) or pretreated for 1 h with PDTC (white inverted triangles), SP600125 (white squares) or SB203580 (white diamonds) prior to TNF-α stimulation for 4 h. Values from individual experiments are shown (n ≥ 3 per group). (e) Expression of A20, (f) Ccl2, (g) Icam1 and (h) Cxcl10 in DKO islets left untreated (black circles) or treated for 8 h with PDTC (white inverted triangles), SP600125 (white squares) or SB203580 (white diamonds). Values from individual experiments are shown (n = 4 per group). ah Bars represent mean ± SEM of gene expression relative to Ppia. (i) Western blot analysis of JNK activation by c-Jun phosphorylation in resting wild-type (WT) and DKO islets. β-Actin was used as a loading control. Representative blots are shown. Phosphorylated c-Jun (48 kDa) was quantified by densitometry and expressed relative to β-actin. Data represent mean ± SEM from three independent experiments. Statistical comparisons were made by single unpaired t test between two groups. *p ≤ 0.05 and **p ≤ 0.01

We next tested the idea that the increased stimulus-independent gene expression in DKO islets might be due to hyperactivation of those same pathways involved in the regulation of normal TNF-α-induced gene expression. To test this, DKO islets were treated with inhibitors and gene expression was analysed by quantitative RT-PCR. Notably, the stimulus-independent expression of Ccl2, Icam1 and Cxcl10 was sensitive to SP600125, but not to SB203580 or PDTC treatment (Fig. 8e–h). Moreover, examination of JNK activation by analysis of c-Jun phosphorylation showed hyperphosphorylation of c-Jun in DKO islets compared with wild-type islets (Fig. 8i). These findings indicate that loss of Birc2 and Birc3 dysregulates fine control over the JNK pathway, such that beta cells are hypersensitive to the triggering of JNK signalling, resulting in the loss of fine control over gene transcription.

Proteosome inhibition mimics BIRC2 and BIRC3 deficiency

BIRC proteins function as ubiquitin modifying enzymes that target proteins for proteosomal degradation [12]. We tested the idea that, in the absence of BIRC proteins, the accumulation of signalling components normally targeted for degradation, perhaps regulatory kinases, could trigger activation of signalling pathways without appropriate external inputs. To do this, we treated MIN6 cells with the proteosome inhibitor, MG-132, to examine whether loss of proteosome activity could replicate the DKO islet phenotype. Compared with control MIN6 cells, MG-132-treated cells exhibited increased expression of Ccl2 (50%, p ≤ 0.05), Icam1 (50%, p ≤ 0.001) and Cxcl10 (80%, p ≤ 0.05), but not of A20 (Fig. 9a–d). Moreover, based on analysis of c-Jun phosphorylation, MG-132-treated MIN6 cells also showed increased JNK pathway activation (Fig. 9e). Thus blockade of the proteosome resulted in dysregulated JNK pathway activation in a stimulus-independent manner, with hyperexpression of Ccl2, Icam1 and Cxcl10 inflammatory genes.
Fig. 9

Proteosome inhibition mimics BIRC2 and BIRC3 deficiency. Expression of A20 (a), Ccl2 (b), Icam1 (c) and Cxcl10 (d) in MIN6 cells left untreated (white diamonds) or treated for 8 h with MG-132 (black diamonds). Values from individual experiments are shown (n ≥ 5 per group). Bars represent mean ± SEM of gene expression relative to Ppia. (e) Western blot analysis of JNK activation by c-Jun phosphorylation in MIN6 cells with or without MG-132 treatment for 8 h. β-Actin was used as a loading control. Representative blots are shown. Phosphorylated c-Jun (48 kDa) was quantified by densitometry and expressed relative to β-actin. Data represent mean ± SEM from three independent experiments. Statistical comparisons were made by single unpaired t test between two groups. *p ≤ 0.05, **p ≤ 0.01 and ***p ≤ 0.001

DKO islets have impaired function after transplantation

JNK is acutely activated in islet cells during the process of islet isolation [28] and antagonisation of JNK signalling is beneficial for islet survival after isolation [29]. Therefore, we predicted that DKO islets would fair worse in vivo following transplantation. To test this idea, wild-type or DKO islets were transplanted into diabetic allogeneic recipients. Analysis of blood glucose levels showed that wild-type and DKO islets provided good metabolic control immediately after transplantation (Fig. 10). As early graft function can be affected by loss of islet mass due to cell death [30, 31], this suggests that DKO islets were not hypersensitive to death stimuli. However, by post-operative day 6, the function of DKO islets began to deteriorate, a process that worsened with time. In contrast, wild-type islets showed good metabolic control until at least post-operative day 18. The earlier loss of DKO islet function could reflect loss of control of inflammatory signalling.
Fig. 10

DKO islets have impaired function after transplantation. Blood glucose values over time of diabetic CBA mice receiving allogeneic wild-type (solid line) or DKO islets (dotted line); tx, transplant day. Values are mean ± SEM (n ≥ 7 per group). Statistical comparisons were made by single unpaired t test between two groups. *p ≤ 0.05 and **p ≤ 0.01

Discussion

Our studies present novel insights into the molecular control of TNF signalling in primary pancreatic beta cells and the role of BIRC proteins in this process. Surprisingly, both single Birc2 −/− and DKO islets, as well as MIN6 cells treated with proteosome inhibitors, exhibited dysregulated expression of select TNF target genes. The augmented stimulus-independent expression of Ccl2, Cxcl10 and Icam1 mRNA observed for DKO islets was substantially reduced by antagonising JNK signalling. Also, DKO islets showed stimulus-independent hyperphosphorylation of c-Jun. We were also able to demonstrate a role for the JNK pathway in the normal de novo regulation of these same genes in islets and beta cells. These data could indicate that BIRC2 and BIRC3 negatively regulate the JNK pathway and subsequent JNK/activator protein-1-regulated gene expression in islet cells.

Interestingly, for B lymphocytes, loss of BIRC2 also leads to prolonged TNF-mediated JNK signalling [32]. BIRC2 and BIRC3 are recruited to the TNFR1 complex via interaction with TRAF2 [18, 33]. TRAF2 is necessary for TNF-α-mediated JNK signalling, but is redundant in the NF-κB or p38 pathways [4, 34, 35]. BIRC2 modulates the duration of TNF-α-induced JNK signalling by targeting TRAF2 and the mitogen-activated protein kinase kinase kinase protein, apoptosis signal-regulating kinase 1 (ASK1), for proteosomal degradation [32, 36]. Our data indicate that this pathway may operate in beta cells. Indeed, treatment of MIN6 cells with the proteosome inhibitor, MG-132, phenocopied DKO islets, lending support to this concept. Hence, one possibility is that, in the absence of BIRC2 and/or BIRC3, TRAF2 and ASK1 accumulate, subsequently triggering hyperactivation of c-Jun, with dysregulated expression of downstream target genes. Hyperactivation of c-Jun might also affect the duration of signalling, contributing to the enhanced gene expression observed for DKO islets following TNF-ligation.

The stimulus-independent expression of Cxcl10 and A20 observed in DKO islets showed different sensitivities to JNK inhibition; hyperexpression of A20 was not sensitive to JNK inhibition. In islets, A20 is an NF-κB-regulated gene [5] that forms a feedback loop to modulate NF-κB activation [20, 37] and control inflammation [38]. Some data show that BIRC antagonists promote degradation of BIRC proteins, which can subsequently trigger stimulus-independent NF-κB signalling. This may be mediated by increased availability of RIPK1, allowing RIPK1–TNFR1 interactions to trigger NF-κB activation [39], subsequently driving expression of NF-κB target genes [40]. Another possibility is regulation of the non-canonical NF-κB component, v-rel reticuloendotheliosis viral oncogene homolog B (RelB). Indeed, Relb −/− fibroblasts exhibit increased NF-κB activity with stimulus-independent expression of NF-κB target genes [41]. Moreover, the non-canonical pathway is regulated at the level of NF-κB-inducing kinase (NIK) [42]. Birc2 −/− mouse embryonic fibroblasts cells (MEFs) and Birc2 −/− Birc3 −/− cancer cells accumulate high NIK levels, and NIK in turn phosphorylates IκB kinase (IKK)α, inducing activation of the non-canonical NF-κB pathway [39, 40]. Hence BIRC2 and BIRC3 provide negative regulatory control over non-canonical NF-κB signalling.

With regard to NF-κB, DKO islets, interestingly, exhibit altered kinetics of IκBα degradation, which presumably reflects a necessary role for BIRC proteins in controlling the speed of NF-κB signalling in beta cells. NF-κB activation is sensitive to proteosome inhibition [43, 44], and BIRC proteins, via their Really Interesting New Gene (RING) domains, can target substrates for proteosomal degradation through ubiquitin editing [3]. One BIRC substrate is RIPK1, which recruits the transforming growth factor (TGF)-beta activated kinase 1 (TAK1)–TGF-beta activated kinase 1/mitogen-activated protein kinase kinase kinase 7 (MAP3K7) binding protein 2 (TAB2)–TAB3 and IKKα–IKKβ–NF-κB essential modulator (NEMO) complexes, triggering TAK1-dependent phosphorylation of IKKβ [45]. The activated NF-κB signalosome [46] subsequently phosphorylates IκBα, initiating the necessary steps for NF-κB translocation [47]. Thus, by controlling the availability of RIPK1 through ubiquitin editing [39], BIRC proteins have the capacity to fine-tune the speed of NF-κB signalling.

Our data provide new insights into the mechanisms by which BIRC proteins control TNF signalling. TNF-α stimulation induces NF-κB activation, which drives early immediate expression of Birc3 in beta cells. The increased levels of BIRC3 may be required to fine-tune TNF-induced inflammatory signalling for full expression of TNF-α-induced genes like Ccl2 and Cxcl10, while suppressing others, including Icam1. Thus, increased BIRC3, in combination with BIRC2, acts simultaneously as a positive factor for NF-κB signalling, but also as a molecular brake that provides modulatory control over the JNK signalling axis. BIRC proteins prevent activation of this pathway in the absence of an overt extracellular signal normally provided by TNF. Thus under inflammatory conditions, pancreatic islets rapidly induce Birc3 to fine-tune NF-κB and JNK signalling pathways to ensure beta cell transcriptional responses are appropriately matched to extracellular inputs. Maintaining a balanced response may be critical for beta cell function under conditions of cellular stress. Thus, for example, beta cell failure in the context of type 2 diabetes is associated with exacerbated CXCL10 expression [48], while activation of the JNK pathway impairs islet survival and function [49, 50]. Moreover, as shown here in the context of islet transplantation, the absence of BIRC proteins impairs islet grafts, resulting in a more rapid loss of glucose homeostasis.

Notes

Acknowledgements

The authors thank J. Cantley and R. Laybutt, (Diabetes and Obesity Research Program, Garvan Institute, Darlinghurst, NSW, Australia) for their insightful critique and helpful discussion. We also acknowledge the technical expertise of staff from the Australian Bioresources and Biological Testing Facility, Garvan Institute, Australia. We thank D. Vaux and H. Carter (Cell Signalling and Cell Death Division, Walter and Eliza Hall, Parkville, VIC, Australia) for providing the Birc2 −/− and Birc3 −/− mice.

Funding

This project was supported by National Health and Medical Research (NHMRC) grants 427695 to S.T. Grey. B.M. Tan was supported by an Australian Postgraduate Award. S.T. Grey is an Australian Research Council Future Fellow and Honorary NHMRC Senior Research Fellow.

Duality of interest

The authors declare that there is no duality of interest associated with this manuscript.

Contribution statement

BMT, NWZ, AOY, RS, SNW, EM and STG made substantial contributions to the study conception and design, the acquisition of data, and to the analysis and interpretation of data. They were all involved in drafting the article and or revising it critically for important intellectual content. All authors made final approval of the version to be published. STG directed the research and is the guarantor of the study.

Supplementary material

125_2012_2784_MOESM1_ESM.pdf (46 kb)
ESM Table 1 (PDF 45 kb)
125_2012_2784_MOESM2_ESM.pdf (36 kb)
ESM Table 2 (PDF 35 kb)
125_2012_2784_MOESM3_ESM.pdf (43 kb)
ESM Table 3 (PDF 43 kb)

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Copyright information

© Springer-Verlag Berlin Heidelberg 2012

Authors and Affiliations

  • B. M. Tan
    • 1
  • N. W. Zammit
    • 1
  • A. O. Yam
    • 1
  • R. Slattery
    • 2
  • S. N. Walters
    • 1
  • E. Malle
    • 1
  • S. T. Grey
    • 1
  1. 1.Gene Therapy and Autoimmunity Group, Immunology ProgramGarvan Institute of Medical ResearchDarlinghurstAustralia
  2. 2.Department of Immunology, Faculty of Medicine, Nursing and Health SciencesMonash University, The Alfred HospitalMelbourneAustralia

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