Preventing p38 MAPK-mediated MafA degradation ameliorates β-cell dysfunction under oxidative stress - PubMed (original) (raw)

Preventing p38 MAPK-mediated MafA degradation ameliorates β-cell dysfunction under oxidative stress

Ilham El Khattabi et al. Mol Endocrinol. 2013 Jul.

Abstract

The reduction in the expression of glucose-responsive insulin gene transcription factor MafA accompanies the development of β-cell dysfunction under oxidative stress/diabetic milieu. Humans with type 2 diabetes have reduced MafA expression, and thus preventing this reduction could overcome β-cell dysfunction and diabetes. We previously showed that p38 MAPK, but not glycogen synthase kinase 3 (GSK3), is a major regulator of MafA degradation under oxidative stress. Here, we examined the mechanisms of this degradation and whether preventing MafA degradation under oxidative stress will overcome β-cell dysfunction. We show that under oxidative and nonoxidative conditions p38 MAPK directly binds to MafA and triggers MafA degradation via ubiquitin proteasomal pathway. However, unlike nonoxidative conditions, MafA degradation under oxidative stress depended on p38 MAPK-mediated phosphorylation at threonine (T) 134, and not T57. Furthermore the expression of alanine (A) 134-MafA, but not A57-MafA, reduced the oxidative stress-mediated loss of glucose-stimulated insulin secretion, which was independent of p38 MAPK action on protein kinase D, a regulator of insulin secretion. Interestingly, the expression of proteasomal activator PA28γ that degrades GSK3-phosphorylated (including T57) MafA was reduced under oxidative stress, explaining the dominance of p38 MAPK over the GSK3 pathway in regulating MafA stability under oxidative stress. These results identify two distinct pathways mediating p38 MAPK-dependent MafA degradation under oxidative and nonoxidative conditions and show that inhibiting MafA degradation under oxidative stress ameliorates β-cell dysfunction and could lead to novel therapies for diabetes.

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Figures

Figure 1.

p38 MAPK mediates MafA degradation via threonine 57 independent of GSK3 priming kinase activity. MIN6 cells transfected with WT-MafA, or MafA expression plasmids with mutations in priming kinase (S65) and p38 MAPK (T57, T134) target sites (A65, A57A65, A65A134, or A57A65A134) were cultured in 0.8 mM glucose for the last 16 hours in the presence of indicated combination of 50 μM CHX, DMSO, and 20 μM SB203580 (SB20). Representative gels immunoblotted with α-HSV and α-actin antibodies are shown for each MafA:HSV expression plasmid. Graphs on the right present results from quantification of band intensities from at least 3 independent experiments. Results are presented relative to the expression of respective MafA derivative in the presence of DMSO alone as 1 ± SEM. *, P < .05.

Figure 2.

Under nonoxidative conditions p38 MAPK mediates MafA degradation through the proteasomal pathway. A, MIN6 cells cotransfected with pcDNA or WT-MafA:HSV along with Ub:HA expression plasmids were cultured in 0.8 mM glucose for the last 16 hours in the presence of indicated combination of DMSO, 20 μM SB203580 (SB20), 20 μM SB216763 (SB21), and 10 μM MG132. Whole-cell extracts were immunoprecipitated (IP) with α-HSV antibody followed by immunoblotting (IB) with α-HA antibody. A representative gel from at least 3 independent experiments is shown. B, MIN6 cells cotransfected with pcDNA, WT-MafA:HSV, A57A134-, A65-, or A57A65A134-HSV-tagged MafA and Ub:HA expression plasmids were cultured in 0.8 mM glucose for the last 16 hours in the presence of 10 μM MG132. Whole-cell extracts were immunoprecipitated with α-HSV antibody followed by immunoblotting with α-HA antibody. The reblot with α-HSV shows correct mobility and levels of different MafA derivatives. A representative gel from at least 3 independent experiments is shown.

Figure 3.

p38 MAPK directly interacts with MafA. MIN6 cells cotransfected with recombinant p38 MAPKα and each of the following HSV-tagged WT-, A57-, A134-, or A57A134-MafA expression plasmids were cultured in the absence (A) or presence of 100 μM tBHP (B) and 10 μM of MG132 during the last 6 hours. Whole-cell extracts were immunoprecipitated with α-HSV or normal rabbit IgG antibodies followed by immunoblotting with αp38 MAPK antibody (third row). The bottom row shows the reblots using α-HSV antibody. The first and second rows represent 10% of input samples used for immunoprecipitation (IP) experiments, immunoblotted (IB) with α-HSV and αp38 MAPK antibody, respectively, to detect the amount of MafA and p38 MAPK in input. Representative gels from 2–3 independent experiments are shown.

Figure 4.

p38 MAPK mediates degradation of endogenous MafA under oxidative stress. A, Inhibitor of p38 MAPK, SB203580, rescues oxidative stress-mediated degradation of endogenous MafA in isolated pancreatic islets. Adult rat islets were cultured for the last 5 hours in the presence of DMSO or 20 μM SB203580 (SB20) in the presence or absence of CHX and 100 μM tBHP. Whole-cell extracts were subjected to immunoblots with α-MafA and α-actin antibodies. An image of a representative gel from 3 independent experiments is shown. Immunoblot of extracts from a second independent islet preparation for the effects of tBHP and SB20 is also shown. Graph on the right presents quantification of MafA band intensity normalized to the loading control (actin) from 3 independent experiments. Results are presented relative to the expression of MafA in CHX+DMSO as 1 ± SEM both in the presence and absence of tBHP. *, P < .01. B, p38 MAPK mediates degradation of MafA in the presence of oxidative stress. MIN6 cells infected with either GFP or dominant-negative (DN)p38 MAPK adenoviruses cultured for 24 hours were split into 6-well plates and cultured for an additional 24 hours with the last 5 hours in the presence or absence of 100 μM tBHP. Whole-cell extracts were subjected to immunoblots with α-MafA and α-actin antibodies. An image of a representative gel from 3 independent experiments is shown. Immunoblot from a second independent set of extracts from MIN6 cells infected with AdDNp38 MAPK is also shown. Graph on the right presents quantification of band intensity of endogenous MafA from 3 independent experiments. The results are presented relative to the expression of MafA (normalized to the loading control, actin) in MIN6 cells infected with GFP adenovirus (AdGFP) and treated with DMSO in the absence of tBHP as 1 ± SEM. *, P < .01. MIN6 cells infected with DNp38 MAPK adenovirus (AdDNp38 MAPK) had no significant difference in MafA protein levels in the presence or absence of tBHP.

Figure 5.

Under oxidative stress p38 MAPK mediates MafA degradation through the proteasomal pathway. A, MIN6 cells were cultured in 0.8 mM glucose for the last 6 hours in the presence of indicated combination of DMSO, 20 μM SB203580 (SB20), and 10 μM MG132 in the presence or absence of 100 μM tBHP. Whole-cell extracts were subjected to immunoblots (IB) with α-MafA and α-actin antibodies. A representative gel from 3 independent experiments is shown. Graph on the right presents quantification of band intensity of endogenous MafA corresponding to lanes 4–7 from 3 independent experiments. The results are presented relative to the expression of MafA (normalized to the loading control, actin) as 1 ± SEM in MIN6 cells treated with DMSO in the presence of tBHP. *, P < .01. B, MIN6 cells cotransfected with pcDNA or MafA:HSV and Ub:HA expression plasmids were treated with a combination of DMSO, 10 μM MG132, and 100 μM tBHP for the last 6 hours in the presence of 0.8 mM glucose. Whole-cell extracts were immunoprecipitated (IP) with α-HSV antibody followed by immunoblotting with α-HA antibody. Whole-cell extracts from 2 independent experiments of MIN6 cells cotransfected with MafA:HSV and Ub:HA and treated with tBHP is shown. Reblot using α-HSV antibody is shown. A representative gel from at least 3 independent experiments is shown.

Figure 6.

Oxidative stress reduces PA28γ expression and triggers MafA degradation only via T134 and not T57. A, p38 MAPK mediates MafA degradation via T134 and not T57 under oxidative stress_:_ MIN6 cells cotransfected with WT, A57-, A134-, or A57A134-MafA expression plasmids and pEGPN1 were cultured in 0.8 mM glucose for the last 6 hours in the presence of indicated combination of 50 μM CHX, DMSO, and 20 μM SB203580 (SB20) in the presence or absence of 100 μM tBHP. Representative gels immunoblotted with α-HSV and α-GFP antibodies are shown for each MafA:HSV expression plasmid. Graphs on the right present results from quantification of band intensities from at least 3 independent experiments. Results are presented relative to the expression of respective MafA derivative in the presence of CHX and DMSO as 1 ± SEM. *, P < .05; **, P < .01. B, Oxidative stress reduces PA28γ expression: MIN6 cells were cultured in 0.8 mM glucose for the last 6 hours in the presence of the indicated combination of DMSO and 10 μM MG132 in the presence or absence of 100 μM tBHP. Whole-cell extracts were subjected to immunoblots with αPA28γ, α-MafA, and α-actin antibodies. A representative gel from 3 independent experiments is shown. Graph on the right presents quantification of band intensity of PA28γ in MIN6 cells in the presence or absence of tBHP from 3 independent experiments. Results are presented relative to the expression of PA28γ (normalized to the loading control, actin) in cells treated with DMSO and in the absence of tBHP as 1 ± SEM. *, P < .01.

Figure 7.

Under oxidative stress, substitution of A134 for T134 in MafA improves insulin secretion independent of PKD activation. A, Expression of MafA under oxidative stress does not affect PKD activity. MIN6 cells cotransfected with recombinant p38 MAPKα and each of the indicated MafA expression plasmids including oxidative stress-resistant A134-MafA were cultured in the presence of 100 μM tBHP and 10 μM MG132 during the last 6 hours. Whole-cell extracts were subjected to immunoblotting with the following antibodies αP-PKD to detect active PKD, αtotal-PKD or α-actin. Representative gels from 3 independent experiments are shown. B, Oxidative stress-resistant A134-MafA, but not A57-MafA, retains greater GSIS in the presence of oxidative stress. MIN6 cells were transfected with the indicated MafA expression plasmids followed by treatment with or without 75 μM tBHP during the last 6 hours, and GSIS was determined by ratio of insulin secreted in response to 16.7 and 0.8 mM glucose during 60 minutes. Data represent mean ± SEM of insulin secretion expressed as fold induction from 3–6 independent experiments. *, P < .05.

Figure 8.

A schematic model for p38 MAPK-mediated MafA degradation under oxidative and nonoxidative conditions. Red circles represent GSK3 phosphorylation sites, yellow circles represent common T57 phosphorylation site for p38 MAPK and GSK3, green circles represent T134 phosphorylation site, and P denotes phosphorylation. The absence of phosphorylation at the A134 site is shown by an empty circle. Potential pathways for degradation of WT-MafA and A134-MafA under oxidative and nonoxidative conditions are shown. Circles with U indicate ubiquitination; intact and degraded MafA are shown by single ovals and 2 structures, respectively. Reduced expression of PA28γ under oxidative stress is shown by downward arrow and red font; higher expression levels are indicted by bold green font for PA28γ, and dark green arrow shows increased p38 MAPK expression under oxidative stress. Darker green arrow indicates dominant MafA degradation pathway under oxidative stress.

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Preventing p38 MAPK-mediated MafA degradation ameliorates β-cell dysfunction under oxidative stress - PubMed (original) (raw)