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. 2011 Sep 4;17(10):1251-60.
doi: 10.1038/nm.2449.

p38 MAPK-mediated regulation of Xbp1s is crucial for glucose homeostasis

Jaemin Lee  1 , Cheng Sun, Yingjiang Zhou, Justin Lee, Deniz Gokalp, Hilde Herrema, Sang Won Park, Roger J Davis, Umut Ozcan
Affiliations

p38 MAPK-mediated regulation of Xbp1s is crucial for glucose homeostasis

Jaemin Lee et al. Nat Med. .

Abstract

Here we show that p38 mitogen-activated protein kinase (p38 MAPK) phosphorylates the spliced form of X-box binding protein 1 (Xbp1s) on its Thr48 and Ser61 residues and greatly enhances its nuclear migration in mice, whereas mutation of either residue to alanine substantially reduces its nuclear translocation and activity. We also show that p38 MAPK activity is markedly reduced in the livers of obese mice compared with lean mice. Further, we show that activation of p38 MAPK by expression of constitutively active MAP kinase kinase 6 (MKK6Glu) greatly enhances nuclear translocation of Xbp1s, reduces endoplasmic reticulum stress and establishes euglycemia in severely obese and diabetic mice. Hence, our results define a crucial role for phosphorylation on Thr48 and Ser61 of Xbp1s in the maintenance of glucose homeostasis in obesity, and they suggest that p38 MAPK activation in the livers of obese mice could lead to a new therapeutic approach to the treatment of type 2 diabetes.

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Conflict of interest statement

Authors do not have any conflict of interest.

Figures

Figure 1
Figure 1
SAPK signaling increases XBP1s mRNA stability and nuclear translocation. (a) XBP1s protein levels in the MEFs infected with adenoviruses expressing LacZ (Ad-LacZ) or XBP1s (Ad-XBP1s) and treated with anisomycin (ANS) at indicated concentrations for 2h. (b) XBP1s protein levels from MEFs infected with Ad-LacZ or Ad-XBP1s and treated with ANS (25 ng ml−1) for 15, 30, 60 and 120 min. (c–d) XBP1s immunoblotting in MEFs infected with Ad-LacZ or Ad-XBP1s and treated with TNFα (c) for 2 h at increasing concentrations or (d) at 10 ng ml−1 concentration for 30, 60 and 120 min. (e) XBP1s protein degradation rate in MEFs infected with Ad-LacZ or Ad-XBP1s, treated with vehicle or ANS (25 ng ml−1) for 2 h then subjected to cycloheximide (CHX) (10 μg ml−1) for indicated time points. XBP1s protein levels were determined by direct immunoblotting of the whole cell lysates. (f) XBP1s protein/actin ratio before and at indicated times after CHX treatment. (g) XBP1s mRNA levels after ANS stimulation. MEFs were infected with Ad-XBP1s and subsequently treated with ANS for 1 h at indicated concentrations. (h) XBP1s mRNA levels at indicated time points after addition of actinomycin D (10 μg ml−1) to the MEFs infected with Ad-XBP1s and stimulated with ANS (25 ng ml−1) for 1 h. (i) Cytoplasmic and nuclear XBP1s protein levels from MEFs infected with Ad-XBP1s and exposed to ANS (25 ng ml−1) for 2 h. Graph adjacent to each blot depicts the ratio of XBP1s in ANS- versus vehicle-treated cells. Each experiment was independently reproduced three times. Error bars are ±S.E.M. Significance was determined by two-way analysis of variance (ANOVA) with Bonferroni multiple-comparison analysis (Figures 1f and 1h) or Student’s t-test (Figure 1g) (**P<0.01, ***P<0.001).
Figure 2
Figure 2
p38 MAPK increases mRNA stability of XBP1s through activation of MK2. (a) XBP1s, phospho-c-Jun (p-cJun) and actin levels from Ad-LacZ- or Ad-XBP1s-infected MEFs that were pre-treated with JNK inhibitor VIII (JNKi, 10 μM) for 30 min and subsequently subjected to ANS (25 ng ml−1) for 2 h. (b) Ad-LacZ- or Ad-XBP1s-infected (left) wild-type (WT) and JNK1,2-deficient (Mapk8,9−/−) or (right) WT and MKK4,7-deficient (Map2k4,7−/−) MEF cells were treated with ANS at indicated concentrations for 2 h. Whole cell lysates were immunoblotted with XBP1s, p-cJun, phospho-p38 MAPK (p-p38), p38 MAPK and actin antibodies. (c) HEK293 cells were transfected with XBP1s or co-transfected with XBP1s and MKK7-JNK1 plasmids (Empty vector was used for normalization of DNA transfection amount). XBP1s, p-cJun and actin levels 24 h after the transfections. (d) Ad-LacZ- or Ad-XBP1s-infected MEFs were pretreated with SB203580 (10 μM), a specific p38 MAPK inhibitor for 30 min and then subjected to ANS (25 ng ml−1) treatment for 2 h. XBP1s, phospho-ATF2 (p-ATF2), p-cJun and actin levels were determined in whole cell lysates. (e) Ad-LacZ- or Ad-XBP1s-infected (left) WT and MKK3,6-deficient (Map2k3,6−/−) or (right) WT and p38α-deficient (Mapk14−/−) cells were treated with ANS at indicated concentrations for 2 h. (f) HEK293 cells were transfected with XBP1s or MKK6Glu, or co-transfected with MKK6Glu and XBP1s together (Empty vector was used for normalization of DNA transfection amount). Transfected cells were either treated with SB203580 (10 μM) or vehicle for 24 h. (g) XBP1s mRNA levels from Ad-XBP1s-infected MEFs that are pretreated with SB203580 (10 μM) for 30 min, stimulated with ANS (25 ng ml−1) for additional 1 h and subjected to actinomycin D (10 μM) for indicated time points. (h) WT and MK2-deficient (Mapkapk2−/−) cells were infected with Ad-LacZ or Ad-XBP1s and subsequently treated with ANS for 2 h at indicated concentrations. (i) mRNA levels of XBP1s from WT and Mapkapk2−/− cells infected with Ad-XBP1s and treated with ANS at different concentrations for 1 h. (j) XBP1s mRNA levels from Ad-XBP1s-infected WT and Mapkapk2−/− MEFs stimulated either with vehicle or ANS (25 ng ml−1) for 1 h and then treated with actinomycin D (10 μg ml−1) for indicated time points. Each experiment was independently performed three times. Error bars are ±S.E.M. Significance was determined by two-way ANOVA (figure 2g), three-way ANOVA (figure 2j) with Bonferroni multiple-comparison analysis, or Student’s t-test (figure 2i) (*P<0.05, ***P<0.001).
Figure 3
Figure 3
p38 MAPK phosphorylates XBP1s at Thr48 and Ser61. (a) MS/MS analysis of XBP1s after 2-h anisomycin (25 ng ml−1) stimulation (see online methods for experimental details). (b–c) HEK293 cells were transfected with WT or mutant XBP1s; (b) XBP1s-T48A or (c) XBP1s-S61A. Subsequently, cells were stimulated with ANS (25 ng ml−1) for 2 h. p-XBP1sThr48, p-XBP1sSer61, XBP1s and actin levels were determined via direct immunoblotting. (d) Coomassie blue staining (left panel) and western blotting (right panel) for recombinant His-TF-XBP1s protein. (e) In vitro kinase assay of p38 MAPK for XBP1s and ATF2 at the presence or absence of SB203580 (10 μM). Total XBP1s, p38 and ATF2 protein levels were detected by western blotting, and phospho-XBP1s, phospho-p38 and phospho-ATF2 were detected by autoradiography. (f) p-XBP1sThr48, p-XBP1sSer61 and XBP1s protein levels on His-TF-XBP1s subjected to p38 MAPK in vitro kinase assay. (g) XBP1s protein levels in whole cell, cytoplasmic and nuclear extracts of HEK293 cells that were transfected with indicated XBP1s plasmids and further stimulated with vehicle or ANS (25 ng ml−1) for 2 h. Each experiment was independently reproduced three times. #: non-specific band.
Figure 4
Figure 4
Inhibition of p38 MAPK blocks XBP1s nuclear translocation. (a–b) (a) Eight-week-old WT lean male mice or (b) age-matched ob/ob males were fasted for 24 h and refed for 1 h. p-XBP1sThr48, p-XBP1sSer61, XBP1s, p-p38, p38 and tubulin levels in total liver extracts. Liver nuclear extracts were used to investigate XBP1s and lamin A/C protein levels. (c–e) Eight-week-old WT lean male mice were intraperitoneally injected with SB203580 (2 mg kg−1 per day) for 3 d. Subsequently, mice were fasted for 24 h and refed for 1 h. (c) Total lysates, nuclear and cytoplasmic protein fractions were prepared from the liver and indicated parameters were analyzed via western blotting. (d–e) (d) XBP1s and (e) Hspa5 mRNA levels at fasting and refed conditions in the livers of the mice either treated with vehicle or SB203580. Each experiment was independently reproduced three times. Error bars are ±S.E.M. Significance was determined by Student’s t-test (**P<0.01, ***P<0.001). NS: Non-significant.
Figure 5
Figure 5
Reactivation of p38 MAPK in the liver of ob/ob mice greatly enhances XBP1s nuclear translocation. (a) p-p38, p38 and actin levels in the liver, muscle and adipose tissues of eight-week-old WT and ob/ob mice. (b) MKK6 and tubulin protein levels in MEFs infected with Ad-LacZ or Ad-MKK6Glu. (c–j) Eight-week-old male ob/ob mice were infected with 8 x 106 plaque-forming units (PFU) g−1 of Ad-LacZ (n=5) or Ad-MKK6Glu (n=5) via tail vein injection. (c) (left) Six-hour fasted blood glucose (mg dl−1) levels on day 3 and (right) circulating insulin levels at six-hour fasted state on day 7 after virus injections. (d–e) (d) Glucose tolerance test (GTT) on day 5 and (e) insulin tolerance test (ITT) on day 3 after virus injections. (f–j) On post-injection day 7, mice were sacrificed after 6-h fasting and liver tissues were collected. (f) MKK6, p-p38, p38, p-ATF2 and actin levels, (g) total XBP1s and tubulin as well as nuclear XBP1s, p-XBP1sThr48, p-XBP1sSer61 and lamin A/C levels. (h) (top) mRNA levels of XBP1s, Hspa5 and Dnajb9, (bottom) p-IRE1Ser724, total IRE1 and actin protein levels. (i) IR and IRS1 tyrosine and Akt Thr308 phosphorylations together with their total protein levels after insulin infusion (0.75 IU kg−1) through the portal vein on post-injection day 7. Graphs next to the blots depict the ratio between phospho- and total-protein. (j) mRNA levels of Gck, G6pc, Pck1 and Ppargc1a in the livers. Three independent groups of mice (n=15 total in each group) were used in the experiments. Error bars are ±S.E.M. Significance was determined by two-way ANOVA with Bonferroni multiple-comparison analysis (figure 5e) or Student’s t-test (*P<0.05, **P<0.01, ***P<0.001). AUC: Area under a curve.
Figure 6
Figure 6
XBP1s-T48A/S61A cannot migrate to the nucleus in the liver and regulate glucose homeostasis. (a–f) Eight-week-old male ob/ob mice were injected with Ad-LacZ (n=5) or Ad-XBP1s (n=5) or Ad-XBP1s-T48A/S61A (n=5) (3.6 x 107 PFU g−1 for each) via the tail vein. (a–c) (a) Six-hour fasting blood glucose levels on day 3, (b) GTT on day 5 and (c) ITT on day 7 after adenovirus injections. (d) On post-injection day 9, mice were sacrificed after 6-h fasting. XBP1s protein levels were determined both in the whole liver lysates and in nuclear extracts. (e) XBP1s and (f) Hspa5 and Dnajb9 mRNA levels in the liver. Three independent groups of mice (n=15 total in each group) were used in the experiments. Error bars are ±S.E.M. Significance was determined by one-way ANOVA except figure 6c (two-way ANOVA) with Bonferroni multiple-comparison analysis (*P<0.05, **P<0.01, ***P<0.001).
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