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A20 inhibits cytokine-induced apoptosis and nuclear factor kappaB-dependent gene activation in islets.

Grey ST, Arvelo MB, Hasenkamp W, Bach FH, Ferran C - J. Exp. Med. (1999)

Bottom Line: The cytoprotective effect of A20 against apoptosis correlates with and is dependent on the abrogation of cytokine-induced NO production.The inhibitory effect of A20 on cytokine-stimulated NO production is due to transcriptional blockade of inducible NO synthase (iNOS) induction; A20 inhibits the activation of the transcription factor nuclear factor kappaB at a level upstream of IkappaBalpha degradation.This qualifies A20 as part of the physiological cytoprotective response of islets.

View Article: PubMed Central - PubMed

Affiliation: Immunobiology Research Center, Harvard Medical School, Beth Israel Deaconess Medical Center, Boston, Massachusetts 02215, USA. sgrey@caregroup.harvard.edu

ABSTRACT
Insulin-dependent diabetes mellitus (IDDM) is an autoimmune disease resulting from apoptotic destruction of beta cells in the islets of Langerhans. Low expression of antioxidants and a predilection to produce nitric oxide (NO) have been shown to underscore beta cell apoptosis. With this perspective in mind, we questioned whether beta cells could mount an induced protective response to inflammation. Here we show that human and rat islets can be induced to rapidly express the antiapoptotic gene A20 after interleukin (IL)-1beta activation. Overexpression of A20 by means of adenovirus-mediated gene transfer protects islets from IL-1beta and interferon gamma-induced apoptosis. The cytoprotective effect of A20 against apoptosis correlates with and is dependent on the abrogation of cytokine-induced NO production. The inhibitory effect of A20 on cytokine-stimulated NO production is due to transcriptional blockade of inducible NO synthase (iNOS) induction; A20 inhibits the activation of the transcription factor nuclear factor kappaB at a level upstream of IkappaBalpha degradation. These data demonstrate a dual antiapoptotic and antiinflammatory function for A20 in beta cells. This qualifies A20 as part of the physiological cytoprotective response of islets. We propose that A20 may have therapeutic potential as a gene therapy candidate to achieve successful islet transplantation and the cure of IDDM.

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NO mediates islet apoptosis induced by IL-1β and IFN-γ. (a) NO donors induce apoptosis in rat islets. Islets were left untreated or were stimulated with GSNO (1.0 mM) or NONOate (0.1 mM) for 16 h, and the percentage of apoptotic cells was determined by flow cytometry. The percentage of apoptotic events was calculated as described and is given in the upper right corner. Data are from a representative experiment of three independent experiments conducted. (b) The l-arginine analogue L-NIO inhibits both apoptosis and NO generation in rat islets. Islets were cultured in the presence or absence of IL-1β (10 U/ml) and IFN-γ (300 U/ml) for 40 h with or without L-NIO (2.2 μM), and the percentage of apoptosis for each condition was measured by flow cytometry. Data from three independent experiments were pooled and are given as the percentage of apoptosis (mean ± SD). NO production (mean ± SD, [nitrite] μM) was measured in the culture medium from each condition and is given in the chart. Suppression of NO production correlated with protection from apoptosis.
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Figure 5: NO mediates islet apoptosis induced by IL-1β and IFN-γ. (a) NO donors induce apoptosis in rat islets. Islets were left untreated or were stimulated with GSNO (1.0 mM) or NONOate (0.1 mM) for 16 h, and the percentage of apoptotic cells was determined by flow cytometry. The percentage of apoptotic events was calculated as described and is given in the upper right corner. Data are from a representative experiment of three independent experiments conducted. (b) The l-arginine analogue L-NIO inhibits both apoptosis and NO generation in rat islets. Islets were cultured in the presence or absence of IL-1β (10 U/ml) and IFN-γ (300 U/ml) for 40 h with or without L-NIO (2.2 μM), and the percentage of apoptosis for each condition was measured by flow cytometry. Data from three independent experiments were pooled and are given as the percentage of apoptosis (mean ± SD). NO production (mean ± SD, [nitrite] μM) was measured in the culture medium from each condition and is given in the chart. Suppression of NO production correlated with protection from apoptosis.

Mentions: Our data demonstrate that A20 can protect islets from cytokine-induced apoptosis. Furthermore, they show that the antiapoptotic effect of A20 correlates with suppression of cytokine-induced NO production, suggesting that A20 is protecting islets through effects on NO generation. This hypothesis is in accordance with data from the literature showing that NO is a key mediator of cytokine-induced islet cytotoxicity 91433. To determine whether the antiapoptotic effect of A20 was a direct result of its ability to suppress NO production, we examined the role of NO in cytokine-induced apoptosis of islets. We first examined if NO could directly induce apoptosis in rat islets. Rat islets were cocultured with one of two NO donors, NONOate or GSNO, at various concentrations ranging from 0.01 μM to 10 mM. 16 h later, islets were examined for induction of apoptosis (Fig. 5 a). Both NONOate and GSNO, in a dose-dependent manner, induced significant levels of apoptosis in rat islets. However, NONOate was 10-fold more potent than GSNO due to its higher release of NO in the medium (Fig. 5 a, and data not shown). Given that NO is able to directly induce apoptosis in rat islets, we next examined whether NO was the agent responsible for islet apoptosis after cytokine stimulation. The NOS inhibitor L-NIO (500 μM) was added to cytokine-stimulated islets. Islets stimulated with IL-1β and IFN-γ underwent apoptosis and generated high levels of NO (Fig. 5 b). In contrast, islets stimulated with IL-1β and IFN-γ in the presence of L-NIO were completely protected from apoptosis (P < 0.001, n = 3), and NO generation was suppressed to below background levels (P < 0.01, n = 3; Fig. 5 b). Taken together, these data demonstrate that NO is the central mediator of cytokine-induced islet apoptosis.


A20 inhibits cytokine-induced apoptosis and nuclear factor kappaB-dependent gene activation in islets.

Grey ST, Arvelo MB, Hasenkamp W, Bach FH, Ferran C - J. Exp. Med. (1999)

NO mediates islet apoptosis induced by IL-1β and IFN-γ. (a) NO donors induce apoptosis in rat islets. Islets were left untreated or were stimulated with GSNO (1.0 mM) or NONOate (0.1 mM) for 16 h, and the percentage of apoptotic cells was determined by flow cytometry. The percentage of apoptotic events was calculated as described and is given in the upper right corner. Data are from a representative experiment of three independent experiments conducted. (b) The l-arginine analogue L-NIO inhibits both apoptosis and NO generation in rat islets. Islets were cultured in the presence or absence of IL-1β (10 U/ml) and IFN-γ (300 U/ml) for 40 h with or without L-NIO (2.2 μM), and the percentage of apoptosis for each condition was measured by flow cytometry. Data from three independent experiments were pooled and are given as the percentage of apoptosis (mean ± SD). NO production (mean ± SD, [nitrite] μM) was measured in the culture medium from each condition and is given in the chart. Suppression of NO production correlated with protection from apoptosis.
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Related In: Results  -  Collection

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Figure 5: NO mediates islet apoptosis induced by IL-1β and IFN-γ. (a) NO donors induce apoptosis in rat islets. Islets were left untreated or were stimulated with GSNO (1.0 mM) or NONOate (0.1 mM) for 16 h, and the percentage of apoptotic cells was determined by flow cytometry. The percentage of apoptotic events was calculated as described and is given in the upper right corner. Data are from a representative experiment of three independent experiments conducted. (b) The l-arginine analogue L-NIO inhibits both apoptosis and NO generation in rat islets. Islets were cultured in the presence or absence of IL-1β (10 U/ml) and IFN-γ (300 U/ml) for 40 h with or without L-NIO (2.2 μM), and the percentage of apoptosis for each condition was measured by flow cytometry. Data from three independent experiments were pooled and are given as the percentage of apoptosis (mean ± SD). NO production (mean ± SD, [nitrite] μM) was measured in the culture medium from each condition and is given in the chart. Suppression of NO production correlated with protection from apoptosis.
Mentions: Our data demonstrate that A20 can protect islets from cytokine-induced apoptosis. Furthermore, they show that the antiapoptotic effect of A20 correlates with suppression of cytokine-induced NO production, suggesting that A20 is protecting islets through effects on NO generation. This hypothesis is in accordance with data from the literature showing that NO is a key mediator of cytokine-induced islet cytotoxicity 91433. To determine whether the antiapoptotic effect of A20 was a direct result of its ability to suppress NO production, we examined the role of NO in cytokine-induced apoptosis of islets. We first examined if NO could directly induce apoptosis in rat islets. Rat islets were cocultured with one of two NO donors, NONOate or GSNO, at various concentrations ranging from 0.01 μM to 10 mM. 16 h later, islets were examined for induction of apoptosis (Fig. 5 a). Both NONOate and GSNO, in a dose-dependent manner, induced significant levels of apoptosis in rat islets. However, NONOate was 10-fold more potent than GSNO due to its higher release of NO in the medium (Fig. 5 a, and data not shown). Given that NO is able to directly induce apoptosis in rat islets, we next examined whether NO was the agent responsible for islet apoptosis after cytokine stimulation. The NOS inhibitor L-NIO (500 μM) was added to cytokine-stimulated islets. Islets stimulated with IL-1β and IFN-γ underwent apoptosis and generated high levels of NO (Fig. 5 b). In contrast, islets stimulated with IL-1β and IFN-γ in the presence of L-NIO were completely protected from apoptosis (P < 0.001, n = 3), and NO generation was suppressed to below background levels (P < 0.01, n = 3; Fig. 5 b). Taken together, these data demonstrate that NO is the central mediator of cytokine-induced islet apoptosis.

Bottom Line: The cytoprotective effect of A20 against apoptosis correlates with and is dependent on the abrogation of cytokine-induced NO production.The inhibitory effect of A20 on cytokine-stimulated NO production is due to transcriptional blockade of inducible NO synthase (iNOS) induction; A20 inhibits the activation of the transcription factor nuclear factor kappaB at a level upstream of IkappaBalpha degradation.This qualifies A20 as part of the physiological cytoprotective response of islets.

View Article: PubMed Central - PubMed

Affiliation: Immunobiology Research Center, Harvard Medical School, Beth Israel Deaconess Medical Center, Boston, Massachusetts 02215, USA. sgrey@caregroup.harvard.edu

ABSTRACT
Insulin-dependent diabetes mellitus (IDDM) is an autoimmune disease resulting from apoptotic destruction of beta cells in the islets of Langerhans. Low expression of antioxidants and a predilection to produce nitric oxide (NO) have been shown to underscore beta cell apoptosis. With this perspective in mind, we questioned whether beta cells could mount an induced protective response to inflammation. Here we show that human and rat islets can be induced to rapidly express the antiapoptotic gene A20 after interleukin (IL)-1beta activation. Overexpression of A20 by means of adenovirus-mediated gene transfer protects islets from IL-1beta and interferon gamma-induced apoptosis. The cytoprotective effect of A20 against apoptosis correlates with and is dependent on the abrogation of cytokine-induced NO production. The inhibitory effect of A20 on cytokine-stimulated NO production is due to transcriptional blockade of inducible NO synthase (iNOS) induction; A20 inhibits the activation of the transcription factor nuclear factor kappaB at a level upstream of IkappaBalpha degradation. These data demonstrate a dual antiapoptotic and antiinflammatory function for A20 in beta cells. This qualifies A20 as part of the physiological cytoprotective response of islets. We propose that A20 may have therapeutic potential as a gene therapy candidate to achieve successful islet transplantation and the cure of IDDM.

Show MeSH
Related in: MedlinePlus