Pannexin-2-deficiency sensitizes pancreatic β-cells to cytokine-induced apoptosis in vitro and impairs glucose tolerance in vivo

Highlights

• Pannexin 2 is expressed in pancreatic islets and β-cells.

• Pannexin 2-deficiency is associated with aggravated islet apoptosis.

• Pro-inflammatory cytokines suppress pannexin 2 expression.

• Pannexin 2-deficiency increases the severity of chemically-induced diabetes in mice.

Abstract

Pannexins (Panx's) are membrane proteins involved in a variety of biological processes, including cell death signaling and immune functions. The role and functions of Panx's in pancreatic β-cells remain to be clarified. Here, we show Panx1 and Panx2 expression in isolated islets, primary β-cells, and β-cell lines. The expression of Panx2, but not Panx1, was downregulated by interleukin-1β (IL-1β) plus interferon-γ (IFNγ), two pro-inflammatory cytokines suggested to contribute to β-cell demise in type 1 diabetes (T1D). siRNA-mediated knockdown (KD) of Panx2 aggravated cytokine-induced apoptosis in rat INS-1E cells and primary rat β-cells, suggesting anti-apoptotic properties of Panx2. An anti-apoptotic function of Panx2 was confirmed in isolated islets from Panx2^-/- mice and in human EndoC-βH1 cells. Panx2 KD was associated with increased cytokine-induced activation of STAT3 and higher expression of inducible nitric oxide synthase (iNOS). Glucose-stimulated insulin release was impaired in Panx2^-/- islets, and Panx2^-/- mice subjected to multiple low-dose Streptozotocin (MLDS) treatment, a model of T1D, developed more severe diabetes compared to wild type mice. These data suggest that Panx2 is an important regulator of the insulin secretory capacity and apoptosis in pancreatic β-cells.

Introduction

Pannexins (Panx1-3) are a family of integral membrane proteins sharing topological similarities with the connexin gap junction proteins (Penuela et al., 2013, Sosinsky et al., 2011). Whereas connexins form intercellular junctions between adjacent cells, pannexins assemble into single membrane channels, allowing for the passage of small molecules such as ATP and glutamate, thereby eliciting biological effects different from gap junctions (Sosinsky et al., 2011, Locovei et al., 2006a). Panx1 and Panx3 are ubiquitously expressed at the mRNA and protein levels, and have been detected in the central nervous system (CNS), skeletal muscle, pancreas, spleen, skin, osteoblasts, and chondrocytes (Baranova et al., 2004, Penuela et al., 2007, Ishikawa et al., 2011, Bond et al., 2011). The evaluation of Panx2 tissue expression at the protein level has been hampered by the lack of adequate antibodies (Cigliola et al., 2015). Initial gene expression studies suggested that Panx2 transcriptional activity is largely restricted to the CNS (Baranova et al., 2004, Bruzzone et al., 2003), while a more recent report indicated ubiquitous expression of Panx2 (Le et al., 2014).

Numerous physiological roles have been ascribed to pannexins which may be implicated in several human diseases (Penuela et al., 2014). Panx1 is the most widely studied Pannexin member and is involved in variety of biological functions including Ca²⁺ wave initiation (Locovei et al., 2006b, Scemes et al., 2009), apoptotic signalling via the release of ATP (Chekeni et al., 2010), regulation of vasoconstriction (Billaud et al., 2011), and in facilitating HIV-1 viral infections (Seror et al., 2011). Panx1 may also be involved in the activation of the inflammasome and IL-1β release from macrophages in vitro (Pelegrin and Surprenant, 2006), although this has not been proven in vivo (Bargiotas et al., 2012, Wang et al., 2013). Recently, whole exome sequencing identified a patient with a homozygous missense and loss-of-function PANX1 variant, who suffers from multiple organ pathologies thereby providing the first direct link between pannexin function and human disease (Shao et al., 2016).

Panx2 is involved in neuronal differentiation (Swayne et al., 2010) and growth regulation in glioma cells (Lai et al., 2009), whereas Panx3 is important for osteoblast, keratinocyte, and chondrocyte differentiation (Ishikawa et al., 2011, Iwamoto et al., 2010, Celetti et al., 2010). Functional studies utilizing Panx1 knockout (Panx1^-/-), Panx2 knockout (Panx2^-/-) and double knockout (Panx1^−/−Panx2^-/-) mice in a model of ischemia-induced neurodegeneration, showed that while Panx2^-/- mice are partially protected from neurodegeneration, double knockout mice (Panx1^−/−Panx2^-/-) exhibit the most pronounced protection against ischemic neurological deficits (Bargiotas et al., 2012).

The role and functions of pannexins in pancreatic islets and β-cells remain unexplored. Recently, however, we identified Panx2 as an interaction partner in a type 1 diabetes (T1D) network generated from genetic data and protein-protein interactions (Bergholdt et al., 2012). Interestingly, we also found that Panx2 transcripts were up-regulated in human islets in response to treatment with pro-inflammatory cytokines (IL-1β + IFNγ + TNFα) (Bergholdt et al., 2012), suggesting that Panx2 might be involved in mediating the detrimental effects of the cytokines which are early mediators of β-cell apoptosis in T1D (Eizirik et al., 2009). Based on these findings, we set out to examine the expression and potential functional roles of pannexins in β-cells and islets in relation to immune-mediated β-cell apoptosis. We document the presence of Panx1 and Panx2 transcripts in islets and β-cells, and show that Panx2-deficiency leads to impaired insulin secretion, increased apoptosis and more severe diabetes in a mouse model of T1D.