Introduction
The islet of Langerhans is the core unit of the endocrine pancreas, which regulates blood glucose homeostasis. Regulation is achieved by the release of insulin from beta cells in response to increasing levels of glucose and by the secretion of glucagon from alpha cells under fasting conditions. Imbalance in this circuitry leads to either hyperglycaemia, the hallmark of diabetes mellitus, or hypoglycaemia. Loss of beta cell function coupled to insulin resistance of target tissues, which usually associates with obesity and chronic low-grade inflammation, defines type 2 diabetes [
1]; high-grade T lymphocyte inflammation mediating autoimmune beta cell destruction is characteristic of type 1 diabetes [
2].
Emerging evidence suggests that alterations in endoplasmic reticulum (ER) function contribute to beta cell disarray in both type 1 and 2 diabetes [
3]. In an attempt to restore ER function and prevent apoptosis, cells activate the unfolded protein response (UPR) [
4]. The crucial role of the UPR in balancing beta cell death and survival is illustrated in the human Wolfram and Wolcott–Rallison syndromes, in which mutations in UPR genes result in unresolved ER stress, beta cell death and early-onset diabetes [
5,
6]. Interestingly, MODY genes such as
Pdx1 and
Hnf1a regulate UPR-associated genes [
7,
8]. These clinical conditions suggest that islet-enriched transcription factors involved in insulin biosynthesis and secretion also preserve the BCM by limiting ER stress.
Paired box (
Pax) genes encode transcription factors critical for tissue development and cellular differentiation [
9]. Paired box 4 (PAX4) is necessary for the generation of pancreatic islet cell progenitors and their differentiation towards beta cells [
10,
11].
Pax4 gene mutations have been associated with type 1 and 2 diabetes as well as with ketosis-prone diabetes, suggesting a key role of PAX4 in mature islets [
12,
13]. Accordingly, overexpression of PAX4 in adult beta cells was shown to block streptozotocin (STZ)-induced hyperglycaemia in mice whereas the diabetes-linked variant PAX4R129W was less efficient [
14]. Despite differences in nitric oxide synthase 2 (NOS2) levels, both PAX4- and PAX4R129W-expressing islets exhibited similar levels of cytokine-induced NO production, indicating that the nuclear factor-κB (NF-κB) signalling pathway was fully activated and that additional anti-apoptotic pathways are involved in islet survival. Consistent with this premise, PAX4 islets expressed higher levels of B cell CLL/lymphoma 2 (BCL-2) [
14]. Nonetheless, overexpression of BCL-2 in islets did not prevent autoimmune-mediated beta cell destruction and development of hyperglycaemia [
15]. Thus, although these data highlight the protective function of PAX4 against a chemical acute stress, whether such an effect can also be conveyed in the context of a pathophysiological autoimmune attack and the molecular mechanism involved in this protection remain to be established.
Herein, we investigated whether PAX4 and PAX4R129W could promote beta cell health, preventing the development of hyperglycaemia in the RIP-B7.1 mouse model of experimental autoimmune diabetes (EAD), and sought to characterise the PAX4-regulated pathways implicated in islet survival and expansion.
Discussion
Inflammation is a common denominator in types 1 and 2 diabetes, and leads to beta cell failure and death, predominantly by apoptosis. As yet, there is no ‘unifying hypothesis’ for the mechanisms triggering beta cell deterioration [
31,
32]; deciphering the molecular roadmap regulated by factors such as PAX4—mutations in which are linked to both forms of diabetes—may help identify common pathways. Herein, we provide proof-of-concept that PAX4, but not PAX4R129W, preserves the BCM and delays the development of hyperglycaemia in the RIP-B7.1 mouse model of EAD. This highlights the mechanistic differences between the wild type and mutant variant.
Consistent with previous reports exploiting the RIP-B7.1 model, 90–100% of immunised non-DOX-treated RIP-B7.1 mice bearing either the
Pax4 or
Pax4R129W transgene developed hyperglycaemia within 3 weeks, correlating with insulitis and beta cell destruction [
33]. DOX-mediated induction of
Pax4 prevented hyperglycaemia development in BPTL animals up to 4 weeks after immunisation, whereas
Pax4R129W induction partially reduced the hyperglycaemic incidence in mutBPTL mice. Thus, PAX4 blunts hyperglycaemia in an autoimmune context, in contrast to several other factors such as caspase-3-generated RAS p21 protein activator 1 (RasGAP) N-terminal fragment (fragment N), cytokine response modifier A (CRMA) or BCL-2 which, despite increasing BCM, could not prevent hyperglycaemia in animal models of type 1 diabetes [
15,
22,
34]. Our data imply that in addition to inhibiting apoptosis, PAX4 is involved in additional regulatory pathways, possibly including immune modulation. Correspondingly, insulitis was reduced after PAX4 overexpression, an effect not attributable to a non-specific repression of the
Cd80 transgene that facilitates the immune response. Our observations extend the analogous findings that inhibition of vascular endothelial growth factor receptor 2 (VEGFR-2) in NOD mice reversed hyperglycaemia by abrogating insulitis and restoring islet cell function [
35]. Although the mechanism by which PAX4 acts at the interface of beta cells and the immune system to blunt insulitis and improve islet recovery remains undefined, our genetic analysis revealed that
Lgals9 was specifically upregulated in PAX4 islets. This gene induces apoptosis of differentiated T helper 1 (Th1) cells [
36], and its overexpression in NOD mice reduced insulitis and hyperglycaemia [
37], prolonging the survival of grafts [
38]. It is therefore plausible that, by enhancing
Lgals9 expression, PAX4 may downregulate Th1 function, partially impeding insulitis and improving islet survival.
Transcriptome profiling revealed that cell-cycle-associated genes upregulated by PAX4 were downregulated by PAX4R129W, despite comparable expression levels of both transgenes. Specifically, the type 2 diabetes-associated cyclin-dependent kinase inhibitor 2A, which strongly inhibits the proliferative cyclin-dependent kinase 4 (CDK4) [
39], was enriched in PAX4R129W islets whereas it was decreased in PAX4 islets. Reciprocally, cyclin D3, which promotes beta cell survival, was increased in PAX4- but not in PAX4R129W-overexpressing islets [
40]. These findings provide some molecular insights into the pathogenic effect of the R129W mutation on beta cell plasticity.
Our data also reveal that PAX4, but not PAX4R129W, is a key regulator of ER function by a combined targeting of genes involved in UPR, Ca
2+ homeostasis, ER–Golgi translocation, and ERAD. The functional importance of the transcriptional changes in these genes was validated by the capacity of PAX4 to prevent thapsigargin-induced ER ultrastructural abnormalities and apoptosis of beta cells, consistent with the finding that PAX4-binding sites are enriched within the promoter region of palmitate-modified ER stress response genes [
41]. In contrast, repression of PAX4-sensitised MIN6 cells to thapsigargin cell death correlates with reduced CALR levels. Calreticulin is a Ca
2+ chaperone of the ER, which contributes to the quality control of protein folding [
42]. Overexpression of CALR in MIN6 cells enhances ER Ca
2+ stores and prevents NO-induced apoptosis [
43]. In this context, the production of NO and ROS induced by inflammatory cytokines in the diabetic environment promotes beta cell death by a variety of mechanisms, including the induction of irreversible double-strand DNA breaks [
27,
44]. We found that PAX4 blunts DNA damage in two models of experimental diabetes, pointing to a general protective mechanism, possibly through preserved ER homeostasis, implicating CALR [
43]. The finding that PAX4-overexpressing islets exhibited improved glucose-induced Ca
2+ oscillations points to this premise. Loss of oscillatory capacity is associated with diminished islet glucose sensitivity and increased ER dysfunction [
45]. Thus, amplified Ca
2+ content in the CALR-enriched ER of PAX4-overexpressing beta cells may impact cytosolic calcium dynamics [
43,
46], thereby preventing activation of downstream apoptotic pathways under stress conditions.
We conclude that PAX4 favours beta cell survival and regeneration in various deleterious inflammatory and high-grade inflammatory environments, such as autoimmunity, through the coordinated regulation of immune modulation, cell cycle, cell survival, ER homeostasis and DNA repair. While both PAX4 and PAX4R129W modulate these pathways, it is the wild-type transcription factor that conveys pro-survival properties by increasing the expression of selected adaptive genes.
Acknowledgements
We thank F. Cortes (Department of Stem Cells, CABIMER, Seville, Spain) for kindly providing the 53BP1 antibody as well as his expert advice on DNA damage. We also thank J. Vallejo Ortega (Department of Stem Cells, CABIMER, Seville, Spain), E. Nadujar and M. Pérez (Genomic Core Facility, CABIMER, Seville, Spain) for excellent technical assistance.