Suppressor of cytokine signaling 3 (SOCS-3) protects beta -cells against interleukin-1beta - and interferon-gamma -mediated toxicity

Abstract

Suppressor of cytokine signaling 3 (SOCS-3) is a negative feedback regulator of IFN-gamma signaling, shown up-regulated in mouse bone marrow cells by the proinflammatory cytokines interleukin-1beta (IL-1beta), tumor necrosis factor-alpha (TNF-alpha), and IFN-gamma. IL-1beta and IFN-gamma alone, or potentiated by TNF-alpha, are cytotoxic to the insulin producing pancreatic beta-cells and beta-cell lines in vitro and suggested to contribute to the specific beta-cell destruction in Type-1 diabetes mellitus (T1DM). Using a doxycycline-inducible SOCS-3 expression system in the rat beta-cell line INS-1, we demonstrate that the toxic effect of both IL-1beta or IFN-gamma at concentrations that reduced the viability by 50% over 3 days, was fully preventable when SOCS-3 expression was turned on in the cells. At cytokine concentrations or combinations more toxic to the cells, SOCS-3 overexpression yielded a partial protection. Whereas SOCS-3-mediated inhibition of IFN-gamma signaling is described in other cell systems, SOCS-3 mediated inhibition of IL-1beta signaling has not previously been described. In addition we show that SOCS-3 prevention of IL-1beta-induced toxicity is accompanied by inhibited transcription of the inducible nitric oxide synthase (iNOS) by 80%, resulting in 60% decreased formation of the toxic nitric oxide (NO). Analysis of isolated native rat islets exposed to IL-1beta revealed a naturally occurring but delayed up-regulated SOCS-3 transcription. Influencing SOCS-3 expression thus represents an approach for affecting cytokine-induced signal transduction at a proximal step in the signal cascade, potentially useful in future therapies aimed at reducing the destructive potential of beta-cell cytotoxic cytokines in T1DM, as well as other cytokine-dependent diseases.

Figure 1

Inducible SOCS-3 expression in the INS-r3#2 clone. A clear induction of SOCS-3 mRNA measured by RNase protection assay was induced by increasing concentrations of doxycycline (A), with a maximal induction, relative to the internal standard cyclophilin (Cyclo), obtained at 0.5–1 μg/ml doxycycline. The induced mRNA correlated well with the induced SOCS-3 protein measured by Western blot (B).

Figure 2

Effect of recombinant SOCS-3 expression on cytokine-mediated β-cell impairment. Viability was determined by the MTT assay in INS-r3#2 cells cultured in the absence or presence of cytokines for 3 days, with or without 0.5 μg/ml doxycycline-induced SOCS-3 expression, starting 1 day before exposure to the cytokines. (A) The cells were exposed to TNF-α (200 units/ml), IFN-γ (200 units/ml), IL-1β (150 pg/ml), 10× IL-1β (1500 pg/ml), or the mix of all three cytokines (200 units/ml, 200 units/ml, and 150 pg/ml, respectively). The INS-r3#2 cells were exposed to IFN-γ (B; 200 units/ml, n = 3) and to IFN-γ + IL-1β (C; 200 units/ml and 150 pg/ml, respectively, n = 5), in the presence of increasing concentrations of doxycycline. Data are mean values ± SD. Significant P values are indicated in A and in Results.

Figure 3

Effect of recombinant SOCS-3 expression on cytokine-induced NO production in INS-r3#2 cells. NO production (measured as accumulated nitrite; A) was determined from the same experimental conditions detailed in Fig. 2A. (B) A dose-dependent reduction of NO production in response to increasing doxycycline concentration is shown for the INS-r3#2 cells exposed to IL-1β (150 pg/ml) for 3 days. Data are mean values ± SD of three separate experiments. Significant P values are indicated in A.

Figure 4

Effect of recombinant SOCS-3 expression on IL-1β-induced iNOS promoter activity. Following transfection with an iNOS promoter construct, promoter activity was measured in INS-r3#2 cells (A) or the parental INS-1 cells (B) exposed to increasing concentrations of IL-1β in the absence (white bars) or presence (black bars) of doxycyclin. INOS promoter activity induced by 150 pg/ml IL-1β in the absence of doxycycline is set to 100% for both cell lines. Data are mean ± SD for n = 3.

Figure 5

SOCS-3 inhibited IFN-γ phosphorylation-dependent activation of STAT-1 signaling. EMSA of nuclear extracts demonstrated activation of STAT-1 following IFN-γ exposure of both the parental INS-1 cell line and the INS-r3#2 clone (A), which was clearly inhibited when SOCS-3 was induced by 1 μg/ml doxycycline in the INS-r3#2 clone. STAT-1 specificity of the activated STAT is evident from supershifting only by the STAT-1 specific antibody (B). In addition, only a competition with a specific oligo (sc) and not with a nonspecific oligo (nsc) was detected.

Figure 6

SOCS-3 mRNA expression in rat islets exposed to IL-1β. Rat islets were exposed to different concentrations of IL-1β for 1 or 24 h and semiquantitative analysis of SOCS-3 mRNA expression relative to the internal standard TBP was performed. Data are mean ± SD of n = 5–10 separate experiments. *, P ≤ 0.01 and **, P ≤ 0.001 compared with control at 24 h, and +, P ≤ 0.001 between 1 and 24 h exposure to 1500 pg/ml IL-1β.