Connexin36 contributes to INS-1E cells survival through modulation of cytokine-induced oxidative stress, ER stress and AMPK activity

Abstract

Cell-to-cell communication mediated by gap junctions made of Connexin36 (Cx36) contributes to pancreatic b-cell function. We have recently demonstrated that Cx36 also supports b-cell survival by a still unclear mechanism. Using specific Cx36 siRNAs or adenoviral vectors, we now show that Cx36 downregulation promotes apoptosis in INS-1E cells exposed to the pro-inflammatory cytokines (IL-1b, TNF-a and IFN-c) involved at the onset of type 1 diabetes, whereas Cx36 overexpression protects against this effect. Cx36 overexpression also protects INS-1E cells against endoplasmic reticulum (ER) stress-mediated apoptosis, and alleviates the cytokine-induced production of reactive oxygen species, the depletion of the ER Ca 2 þ stores, the CHOP overexpression and the degradation of the anti-apoptotic protein Bcl-2 and Mcl-1. We further show that cytokines activate the AMP-dependent protein kinase (AMPK) in a NO-dependent and ER-stress-dependent manner and that AMPK inhibits Cx36 expression. Altogether, the data suggest that Cx36 is involved in Ca 2 þ homeostasis within the ER and that Cx36 expression is downregulated following ER stress and subsequent AMPK activation. As a result, cytokine-induced Cx36 downregulation elicits a positive feedback loop that amplifies ER stress and AMPK activation, leading to further Cx36 downregulation. The data reveal that Cx36 plays a central role in the oxidative stress and ER stress induced by cytokines and the subsequent regulation of AMPK activity, which in turn controls Cx36 expression and mitochondria-dependent apoptosis of insulin-producing cells.

Low Level Pro-inflammatory Cytokines Decrease Connexin36 Gap Junction Coupling in Mouse and Human Islets through Nitric Oxide-mediated Protein Kinase Cδ

“…Recent studies have shown that Cx36 gap junctions can protect islets from cytokine or streptozotocin-induced cell death and oxidative stress (30,31). Similarly, deletion of PKC␦ in mice protects islets from cytokine-induced cell death (38), which is consistent with the mechanism we describe by which gap junctions are disrupted.…”

“…Future studies measuring the dynamics of Cx36 turnover will be needed to further characterize this disruption. Previous measurements in INS-1E cells have also shown decreases in Cx36 gene expression, total protein, and Cx36 staining with similar levels of pro-inflammatory cytokines (30). Decreased Cx36 gene expression was also observed in islets with hyperglycemia and hyperlipidemia, conditions that produce high levels of pro-inflammatory cytokines (25,26), and may represent an additional mechanism of Cx36 regulation.…”

“…Human islets were received from the Integrated Islet Distribution Program (donor IDs: AAEZ340, AAJF122, ABAF490, ABCQ166, ABD1375, ABEI419, ABFV041, ABGK387A, ABHC283, ABJR111, and ABJ5204) with a median viability of 90% and an average stimulation index (fold-increase insulin secretion from 2 to 20 mM glucose) of 2.5 for all donors, and were cultured overnight in CMRL medium 1066 (Fisher Scientific) before commencing experiments. Islets were treated for 1 h, 24 h, or 30 min in RPMI medium with 0, 0.001, 0.01, 0.1, or 1 times dilutions of a cytokine mixture (termed "relative cytokine concentration" or RCC) consisting of 10 ng/ml of recombinant mouse tumor necrosis factor-␣ (TNF-␣, R&D Systems, Minneapolis, MN), 5 ng/ml of recombinant mouse interleukin-1␤ (IL-1␤, R&D Systems), and 100 ng/ml of recombinant mouse interferon-␥ (IFN-␥, R&D Systems) (10,30). Islets were also treated with individual or combinations of two cytokines at 0.1 RCC for 24 h.…”

“…Changes in Cx36 gap junction coupling have been shown to be protective against cytokine-induced damage in INS-1E cells (30) and against apoptosis in mouse islets (31). Despite this protective mechanism and the importance of Cx36 in regulating insulin release, the regulation of Cx36 gap junction coupling in the islet is poorly understood (32).…”

Endoplasmic Reticulum Stress in Hearing Loss

“…Treatment of cells with ER stress inducers resulted in decreased expression of connexins such as Cx36 and Cx43, as well as reduced gap junctional intercellular communication [93,94]. Selective down-regulation of gap junction proteins might help to alleviate the burden of ER and prevent the transmission of 'stress' signals to adjacent cells, hence providing an important protective mechanism under ER stress [93].…”