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Involvement of c-Jun N-terminal Kinase in Oxidative Stress-mediated Suppression of Insulin Gene Expression*

Open AccessPublished:May 14, 2002DOI:https://doi.org/10.1074/jbc.M202066200
      Oxidative stress, which is found in pancreatic β-cells in the diabetic state, suppresses insulin gene transcription and secretion, but the signaling pathways involved in the β-cell dysfunction induced by oxidative stress remain unknown. In this study, subjecting rat islets to oxidative stress activates JNK, p38 MAPK, and protein kinase C, preceding the decrease of insulin gene expression. Adenovirus-mediated overexpression of dominant-negative type (DN) JNK, but not the p38 MAPK inhibitor SB203580 nor the protein kinase C inhibitor GF109203X, protected insulin gene expression and secretion from oxidative stress. Moreover, wild type JNK overexpression suppressed both insulin gene expression and secretion. These results were correlated with changes in the binding of the important transcription factor PDX-1 to the insulin promoter; adenoviral overexpression of DN-JNK preserved PDX-1 DNA binding activity in the face of oxidative stress, whereas wild type JNK overexpression decreased PDX-1 DNA binding activity. Furthermore, to examine whether suppression of the JNK pathway can protect β-cells from the toxic effects of hyperglycemia, rat islets were infected with DN-JNK expressing adenovirus or control adenovirus and transplanted under renal capsules of streptozotocin-induced diabetic nude mice. In mice receiving DN-JNK overexpressing islets, insulin gene expression in islet grafts was preserved, and hyperglycemia was ameliorated compared with control mice. In conclusion, activation of JNK is involved in the reduction of insulin gene expression by oxidative stress, and suppression of the JNK pathway protects β-cells from oxidative stress.
      PDX-1
      pancreatic and duodenal homeobox factor-1
      ROS
      reactive oxygen species
      JNK
      c-Jun N-terminal kinase
      MAPK
      mitogen-activated protein kinase
      PKC
      protein kinase C
      WT
      wild type
      DN
      dominant-negative type
      GFP
      green fluorescent protein
      pfu
      plaque-forming unit
      STZ
      streptozotocin
      NAC
      N-acetyl-l-cysteine
      Ad
      adenovirus
      Some of the β-cell failure that is fundamental to diabetes appears to be because of the adverse effects of chronic hyperglycemia on β-cells, a process called glucose toxicity, which leads to suppression of insulin gene transcription and glucose-stimulated insulin secretion (
      • Weir G.C.
      • Laybutt D.R.
      • Kaneto H.
      • Bonner-Weir S.
      • Sharma A.
      ,
      • Bonner-Weir S.
      • Trent D.F.
      • Weir G.C.
      ,
      • Sharma A.
      • Olson L.K.
      • Robertson R.P.
      • Stein R.
      ,
      • Olson L.K.
      • Sharma A.
      • Peshavaria M.
      • Wright C.V.E.
      • Towle H.C.
      • Robertson R.P.
      • Stein R.
      ,
      • Tokuyama Y.
      • Sturis J.
      • DePaoli A.M.
      • Takeda J.
      • Stoffel M.
      • Tang J.
      • Sun X.
      • Polonsky K.S.
      • Bell G.I.
      ,
      • Zangen D.H.
      • Bonner-Weir S.
      • Lee C.H.
      • Latimer J.B.
      • Miller C.P.
      • Habener J.F.
      • Weir G.C.
      ,
      • Jonas J.-C.
      • Sharma A.
      • Hasenkamp W.
      • Iikova H.
      • Patane G.
      • Laybutt R.
      • Bonner-Weir S.
      • Weir G.C.
      ). The reduction of expression or DNA binding activity of the pancreatic and duodenal homeobox factor-1 (PDX-1)1 (also known as IDX-1/STF-1/IPF1) (
      • Ohlsson H.
      • Karlsson K.
      • Edlund T.
      ,
      • Leonard J.
      • Peers B.
      • Johnson T.
      • Ferreri K.
      • Lee S.
      • Montminy M.R.
      ,
      • Miller C.P.
      • McGehee R.E.
      • Habener J.F.
      ,
      • Stoffers D.A.
      • Thomas M.K.
      • Habener J.F.
      ,
      • Weir G.C.
      • Sharma A.
      • Zangen D.H.
      • Bonner-Weir S.
      ) is often observed simultaneously with suppression of insulin gene transcription (
      • Sharma A.
      • Olson L.K.
      • Robertson R.P.
      • Stein R.
      ,
      • Olson L.K.
      • Sharma A.
      • Peshavaria M.
      • Wright C.V.E.
      • Towle H.C.
      • Robertson R.P.
      • Stein R.
      ,
      • Jonas J.-C.
      • Sharma A.
      • Hasenkamp W.
      • Iikova H.
      • Patane G.
      • Laybutt R.
      • Bonner-Weir S.
      • Weir G.C.
      ). PDX-1, a member of the homeodomain family of transcription factors, plays an important role in pancreas development (
      • Jonsson J.
      • Carlsson L.
      • Edlund T.
      • Edlund H.
      ,
      • Ahlgren U.
      • Jonsson J.
      • Edlund H.
      ,
      • Offield M.F.
      • Jetton T.L.
      • Labosky P.
      • Ray M.
      • Stein R.
      • Magnuson M.
      • Hogan B.L.M.
      • Wright C.V.E.
      ,
      • Kaneto H.
      • Miyagawa J.
      • Kajimoto Y.
      • Yamamoto K.
      • Watada H.
      • Umayahara Y.
      • Hanafusa T.
      • Matsuzawa Y.
      • Yamasaki Y.
      • Higashiyama S.
      • Taniguchi N.
      ) and differentiation (
      • Sharma A.
      • Zangen D.H.
      • Reitz P.
      • Taneja M.
      • Lissauer M.E.
      • Miller C.P.
      • Weir G.C.
      • Habener J.F.
      • Bonner-Weir S.
      ,
      • Bonner-Weir S.
      • Taneja M.
      • Weir G.C.
      • Tatarkiewicz K.
      • Song K.-H.
      • Sharma A.
      • O'Neil J.J.
      ,
      • Watada H.
      • Kajimoto Y.
      • Miyagawa J.
      • Hanafusa T.
      • Hamaguchi K.
      • Matsuoka T.
      • Yamamoto K.
      • Matsuzawa Y.
      • Kawamori R.
      • Yamasaki Y.
      ,
      • Serup P.
      • Jensen J.
      • Andersen F.G.
      • Jorgensen M.C.
      • Blume N.
      • Holst J.J.
      • Madsen O.D.
      ) and in maintaining normal β-cell function by regulating the expression of multiple genes, including insulin, GLUT2, and glucokinase (
      • Ahlgren U.
      • Jonsson J.
      • Jonsson L.
      • Simu K.
      • Edlund H.
      ,
      • Wang H.
      • Maechler P.
      • Ritz-Laser B.
      • Hagenfeldt K.A.
      • Ishihara H.
      • Philippe J.
      • Wollheim C.B.
      ,
      • Waeber G.
      • Thompson N.
      • Nicod P.
      • Bonny C.
      ,
      • Watada H.
      • Kajimoto Y.
      • Umayahara Y.
      • Matsuoka T.
      • Kaneto H.
      • Fujitani Y.
      • Kamada T.
      • Kawamori R.
      • Yamasaki Y.
      ).
      It has been postulated that oxidative stress (
      • Finkel T.
      • Holbrook N.J.
      ) which is found in the diabetic state (
      • Ihara Y.
      • Toyokuni S.
      • Uchida K.
      • Odaka H.
      • Tanaka T.
      • Ikeda H.
      • Hiai H.
      • Seino Y.
      • Yamada Y.
      ,
      • Nishikawa T.
      • Edelstein D., Du, X.L.
      • Yamagishi S.
      • Matsumura T.
      • Kaneda Y.
      • Yorek M.A.
      • Beebe D.
      • Oates P.J.
      • Hammes H.-P.
      • Giardino I.
      • Brownlee M.
      ,
      • Laybutt D.R.
      • Kaneto H.
      • Hasenkamp W.
      • Grey S.
      • Jonas J.-C.
      • Sgroi D.C.
      • Groff A.
      • Ferran C.
      • Bonner-Weir S.
      • Sharma A.
      • Weir G.C.
      ) is involved in the progression of β-cell deterioration (
      • Kaneto H.
      • Fujii J.
      • Myint T.
      • Miyazawa N.
      • Islam K.N.
      • Kawasaki Y.
      • Suzuki K.
      • Nakamura M.
      • Tatsumi H.
      • Yamasaki Y.
      • Taniguchi N.
      ,
      • Matsuoka T.
      • Kajimoto Y.
      • Watada H.
      • Kaneto H.
      • Kishimoto M.
      • Umayahara Y.
      • Fujitani Y.
      • Kamada T.
      • Kawamori R.
      • Yamasaki Y.
      ,
      • Maechler P.
      • Jornot L.
      • Wollheim C.B.
      ,
      • Kaneto H.
      • Kajimoto Y.
      • Miyagawa J.
      • Matsuoka T.
      • Fujitani Y.
      • Umayahara Y.
      • Hanafusa T.
      • Matsuzawa Y.
      • Yamasaki Y.
      • Hori M.
      ,
      • Tanaka Y.
      • Gleason C.E.
      • Tran P.O.T.
      • Harmon J.S.
      • Robertson R.P.
      ,
      • Ihara Y.
      • Yamada Y.
      • Toyokuni S.
      • Miyawaki K.
      • Ban N.
      • Adachi T.
      • Kuroe A.
      • Iwakura T.
      • Kubota A.
      • Hiai H.
      • Seino Y.
      ,
      • Kaneto H., Xu, G.
      • Song K.-H.
      • Suzuma K.
      • Bonner-Weir S.
      • Sharma A.
      • Weir G.C.
      ) as well as in the development of diabetic complications (
      • Nishikawa T.
      • Edelstein D., Du, X.L.
      • Yamagishi S.
      • Matsumura T.
      • Kaneda Y.
      • Yorek M.A.
      • Beebe D.
      • Oates P.J.
      • Hammes H.-P.
      • Giardino I.
      • Brownlee M.
      ,
      • Baynes J.W.
      ,
      • Baynes J.W.
      • Thorpe S.R.
      ,
      • Du X.-L.
      • Edelstein D.
      • Rossetti L.
      • Fantus I.G.
      • Goldberg H.
      • Ziyadeh F., Wu, J.
      • Brownlee M.
      ). In fact, in the presence of diabetes, reactive oxygen species (ROS) are produced in islets (
      • Ihara Y.
      • Toyokuni S.
      • Uchida K.
      • Odaka H.
      • Tanaka T.
      • Ikeda H.
      • Hiai H.
      • Seino Y.
      • Yamada Y.
      ) as well as in various tissues (
      • Nishikawa T.
      • Edelstein D., Du, X.L.
      • Yamagishi S.
      • Matsumura T.
      • Kaneda Y.
      • Yorek M.A.
      • Beebe D.
      • Oates P.J.
      • Hammes H.-P.
      • Giardino I.
      • Brownlee M.
      ,
      • Laybutt D.R.
      • Kaneto H.
      • Hasenkamp W.
      • Grey S.
      • Jonas J.-C.
      • Sgroi D.C.
      • Groff A.
      • Ferran C.
      • Bonner-Weir S.
      • Sharma A.
      • Weir G.C.
      ) through several processes such as the non-enzymatic glycosylation reaction (
      • Baynes J.W.
      • Thorpe S.R.
      ,
      • Sakurai T.
      • Tsuchiya S.
      ,
      • Ookawara T.
      • Kawamura N.
      • Kitagawa Y.
      • Taniguchi N
      ), the electron transport chain in mitochondria (
      • Nishikawa T.
      • Edelstein D., Du, X.L.
      • Yamagishi S.
      • Matsumura T.
      • Kaneda Y.
      • Yorek M.A.
      • Beebe D.
      • Oates P.J.
      • Hammes H.-P.
      • Giardino I.
      • Brownlee M.
      ), and the hexosamine pathway (
      • Kaneto H., Xu, G.
      • Song K.-H.
      • Suzuma K.
      • Bonner-Weir S.
      • Sharma A.
      • Weir G.C.
      ). Furthermore, it has been reported that levels of 8-hydroxy-2′-deoxyguanosine, a marker for oxidative stress, are increased in the blood of type 2 diabetic patients (
      • Dandona P.
      • Thusu K.
      • Cook S.
      • Snyder B.
      • Makowski J.
      • Armstrong D.
      • Nicotera T.
      ,
      • Leinonen J.
      • Lehtimaki T.
      • Toyokuni S.
      • Okada K.
      • Tanaka T.
      • Hiai H.
      • Ochi H.
      • Laippala P.
      • Rantalaiho V.
      • Wirta O.
      • Pasternack A.
      • Alho H.
      ). Because expression levels of the antioxidant enzymes, superoxide dismutase, catalase, and glutathione peroxidase, are relatively low in islets as compared with other tissues (
      • Tiedge M.
      • Lortz S.
      • Drinkgern J.
      • Lenzen S.
      ), β-cells can be expected to be vulnerable to oxidative stress. Indeed, studies have shown that oxidative stress exerts deleterious effects upon β-cells in the diabetic state, suppressing insulin gene transcription and glucose-stimulated insulin secretion, and even producing apoptosis (
      • Kaneto H.
      • Fujii J.
      • Myint T.
      • Miyazawa N.
      • Islam K.N.
      • Kawasaki Y.
      • Suzuki K.
      • Nakamura M.
      • Tatsumi H.
      • Yamasaki Y.
      • Taniguchi N.
      ,
      • Matsuoka T.
      • Kajimoto Y.
      • Watada H.
      • Kaneto H.
      • Kishimoto M.
      • Umayahara Y.
      • Fujitani Y.
      • Kamada T.
      • Kawamori R.
      • Yamasaki Y.
      ,
      • Maechler P.
      • Jornot L.
      • Wollheim C.B.
      ,
      • Kaneto H.
      • Kajimoto Y.
      • Miyagawa J.
      • Matsuoka T.
      • Fujitani Y.
      • Umayahara Y.
      • Hanafusa T.
      • Matsuzawa Y.
      • Yamasaki Y.
      • Hori M.
      ,
      • Tanaka Y.
      • Gleason C.E.
      • Tran P.O.T.
      • Harmon J.S.
      • Robertson R.P.
      ,
      • Ihara Y.
      • Yamada Y.
      • Toyokuni S.
      • Miyawaki K.
      • Ban N.
      • Adachi T.
      • Kuroe A.
      • Iwakura T.
      • Kubota A.
      • Hiai H.
      • Seino Y.
      ,
      • Kaneto H., Xu, G.
      • Song K.-H.
      • Suzuma K.
      • Bonner-Weir S.
      • Sharma A.
      • Weir G.C.
      ). Expression and DNA binding activity of PDX-1 are also suppressed by oxidative stress (
      • Matsuoka T.
      • Kajimoto Y.
      • Watada H.
      • Kaneto H.
      • Kishimoto M.
      • Umayahara Y.
      • Fujitani Y.
      • Kamada T.
      • Kawamori R.
      • Yamasaki Y.
      ,
      • Kaneto H.
      • Kajimoto Y.
      • Miyagawa J.
      • Matsuoka T.
      • Fujitani Y.
      • Umayahara Y.
      • Hanafusa T.
      • Matsuzawa Y.
      • Yamasaki Y.
      • Hori M.
      ,
      • Tanaka Y.
      • Gleason C.E.
      • Tran P.O.T.
      • Harmon J.S.
      • Robertson R.P.
      ,
      • Kaneto H., Xu, G.
      • Song K.-H.
      • Suzuma K.
      • Bonner-Weir S.
      • Sharma A.
      • Weir G.C.
      ). Furthermore, some toxic effects of hyperglycemia on β-cells in rodent models are reduced by antioxidant treatment (
      • Kaneto H.
      • Kajimoto Y.
      • Miyagawa J.
      • Matsuoka T.
      • Fujitani Y.
      • Umayahara Y.
      • Hanafusa T.
      • Matsuzawa Y.
      • Yamasaki Y.
      • Hori M.
      ,
      • Tanaka Y.
      • Gleason C.E.
      • Tran P.O.T.
      • Harmon J.S.
      • Robertson R.P.
      ,
      • Ihara Y.
      • Yamada Y.
      • Toyokuni S.
      • Miyawaki K.
      • Ban N.
      • Adachi T.
      • Kuroe A.
      • Iwakura T.
      • Kubota A.
      • Hiai H.
      • Seino Y.
      ). Thus, it is likely that oxidative stress mediates some of the toxic effects of hyperglycemia.
      Several signal transduction pathways including c-Jun N-terminal kinase (JNK) (also known as stress-activated protein kinase) (
      • Hibi M.
      • Lin A.
      • Smeal T.
      • Minden A.
      • Karin M.
      ,
      • Derijard B.
      • Hibi M., Wu, I.-H.
      • Barrett T., Su, B.
      • Deng T.
      • Karin M.
      • Davis R.J.
      ), p38 mitogen-activated protein kinase (MAPK), and protein kinase C (PKC) are known to be activated by oxidative stress (
      • Finkel T.
      • Holbrook N.J.
      ,
      • Koya D.
      • King G.L.
      ) or high glucose (
      • Koya D.
      • King G.L.
      ,
      • Liu W.
      • Schoenkerman A.
      • Lowe W.L., Jr.
      ,
      • Purves T.
      • Middlemas A.
      • Agthong S.
      • Jude E.B.
      • Boulton A.J.
      • Fernyhough P.
      • Tomlinson D.R.
      ) in several cell types. However, it is not known which of these kinases is activated in pancreatic islets and involved in oxidative stress-mediated suppression of insulin gene transcription. In this study, we show that activation of JNK is involved in reduction of insulin gene expression by oxidative stress and that suppression of the JNK pathway can protect β-cells from oxidative stress.

      DISCUSSION

      It has been postulated that oxidative stress provoked under diabetic conditions is involved in β-cell dysfunction characterized by decreases in insulin gene transcription and glucose-stimulated insulin secretion (
      • Kaneto H.
      • Fujii J.
      • Myint T.
      • Miyazawa N.
      • Islam K.N.
      • Kawasaki Y.
      • Suzuki K.
      • Nakamura M.
      • Tatsumi H.
      • Yamasaki Y.
      • Taniguchi N.
      ,
      • Matsuoka T.
      • Kajimoto Y.
      • Watada H.
      • Kaneto H.
      • Kishimoto M.
      • Umayahara Y.
      • Fujitani Y.
      • Kamada T.
      • Kawamori R.
      • Yamasaki Y.
      ,
      • Maechler P.
      • Jornot L.
      • Wollheim C.B.
      ,
      • Kaneto H.
      • Kajimoto Y.
      • Miyagawa J.
      • Matsuoka T.
      • Fujitani Y.
      • Umayahara Y.
      • Hanafusa T.
      • Matsuzawa Y.
      • Yamasaki Y.
      • Hori M.
      ,
      • Tanaka Y.
      • Gleason C.E.
      • Tran P.O.T.
      • Harmon J.S.
      • Robertson R.P.
      ,
      • Ihara Y.
      • Yamada Y.
      • Toyokuni S.
      • Miyawaki K.
      • Ban N.
      • Adachi T.
      • Kuroe A.
      • Iwakura T.
      • Kubota A.
      • Hiai H.
      • Seino Y.
      ,
      • Kaneto H., Xu, G.
      • Song K.-H.
      • Suzuma K.
      • Bonner-Weir S.
      • Sharma A.
      • Weir G.C.
      ). For example, ROS are produced in β-cells in the diabetic state (
      • Ihara Y.
      • Toyokuni S.
      • Uchida K.
      • Odaka H.
      • Tanaka T.
      • Ikeda H.
      • Hiai H.
      • Seino Y.
      • Yamada Y.
      ), and antioxidant treatment can provide some protection against this dysfunction and improve glucose tolerance (
      • Kaneto H.
      • Kajimoto Y.
      • Miyagawa J.
      • Matsuoka T.
      • Fujitani Y.
      • Umayahara Y.
      • Hanafusa T.
      • Matsuzawa Y.
      • Yamasaki Y.
      • Hori M.
      ,
      • Tanaka Y.
      • Gleason C.E.
      • Tran P.O.T.
      • Harmon J.S.
      • Robertson R.P.
      ,
      • Ihara Y.
      • Yamada Y.
      • Toyokuni S.
      • Miyawaki K.
      • Ban N.
      • Adachi T.
      • Kuroe A.
      • Iwakura T.
      • Kubota A.
      • Hiai H.
      • Seino Y.
      ). Thus, it is likely that oxidative stress mediates some of the toxic effects of hyperglycemia upon β-cells. In this study, we examined the mechanism for oxidative stress-mediated suppression of insulin gene expression. We have shown that in isolated rat islets several pathways (JNK, p38 MAPK, and PKC) are activated by oxidative stress, preceding suppression of insulin gene expression (Figs. 1 and2). Furthermore, oxidative stress-mediated suppression of insulin gene expression and secretion was prevented by overexpression of DN-JNK (FIG. 3, FIG. 4, FIG. 5), suggesting that JNK is involved in oxidative stress-mediated suppression of insulin gene expression and secretion found in diabetes.
      Suppression of insulin gene expression by JNK overexpression was accompanied by a decrease of PDX-1 DNA binding activity (Fig. 6), which may explain at least part of the suppression of insulin gene expression. PDX-1 (
      • Ohlsson H.
      • Karlsson K.
      • Edlund T.
      ,
      • Leonard J.
      • Peers B.
      • Johnson T.
      • Ferreri K.
      • Lee S.
      • Montminy M.R.
      ,
      • Miller C.P.
      • McGehee R.E.
      • Habener J.F.
      ,
      • Stoffers D.A.
      • Thomas M.K.
      • Habener J.F.
      ,
      • Weir G.C.
      • Sharma A.
      • Zangen D.H.
      • Bonner-Weir S.
      ) plays an important role in pancreas development (
      • Jonsson J.
      • Carlsson L.
      • Edlund T.
      • Edlund H.
      ,
      • Ahlgren U.
      • Jonsson J.
      • Edlund H.
      ,
      • Offield M.F.
      • Jetton T.L.
      • Labosky P.
      • Ray M.
      • Stein R.
      • Magnuson M.
      • Hogan B.L.M.
      • Wright C.V.E.
      ,
      • Kaneto H.
      • Miyagawa J.
      • Kajimoto Y.
      • Yamamoto K.
      • Watada H.
      • Umayahara Y.
      • Hanafusa T.
      • Matsuzawa Y.
      • Yamasaki Y.
      • Higashiyama S.
      • Taniguchi N.
      ) and differentiation (
      • Sharma A.
      • Zangen D.H.
      • Reitz P.
      • Taneja M.
      • Lissauer M.E.
      • Miller C.P.
      • Weir G.C.
      • Habener J.F.
      • Bonner-Weir S.
      ,
      • Bonner-Weir S.
      • Taneja M.
      • Weir G.C.
      • Tatarkiewicz K.
      • Song K.-H.
      • Sharma A.
      • O'Neil J.J.
      ,
      • Watada H.
      • Kajimoto Y.
      • Miyagawa J.
      • Hanafusa T.
      • Hamaguchi K.
      • Matsuoka T.
      • Yamamoto K.
      • Matsuzawa Y.
      • Kawamori R.
      • Yamasaki Y.
      ,
      • Serup P.
      • Jensen J.
      • Andersen F.G.
      • Jorgensen M.C.
      • Blume N.
      • Holst J.J.
      • Madsen O.D.
      ) and in the regulation of cell-specific expression of insulin and various other genes essential for β-cell function (
      • Ahlgren U.
      • Jonsson J.
      • Jonsson L.
      • Simu K.
      • Edlund H.
      ,
      • Wang H.
      • Maechler P.
      • Ritz-Laser B.
      • Hagenfeldt K.A.
      • Ishihara H.
      • Philippe J.
      • Wollheim C.B.
      ,
      • Waeber G.
      • Thompson N.
      • Nicod P.
      • Bonny C.
      ,
      • Watada H.
      • Kajimoto Y.
      • Umayahara Y.
      • Matsuoka T.
      • Kaneto H.
      • Fujitani Y.
      • Kamada T.
      • Kawamori R.
      • Yamasaki Y.
      ). Moreover, mutations in PDX-1 are known to cause some cases of maturity-onset diabetes of the young (
      • Stoffers D.A.
      • Ferrer J.
      • Clarke W.L.
      • Habener J.F.
      ). Thus, it is likely that JNK-mediated suppression of PDX-1 DNA binding activity accounts for some of the suppression of insulin gene transcription and of β-cell function, which fits with reports that PDX-1 expression of DNA binding activity is decreased in association with reduction of insulin gene transcription after chronic exposure to a high glucose concentration (
      • Sharma A.
      • Olson L.K.
      • Robertson R.P.
      • Stein R.
      ,
      • Olson L.K.
      • Sharma A.
      • Peshavaria M.
      • Wright C.V.E.
      • Towle H.C.
      • Robertson R.P.
      • Stein R.
      ,
      • Zangen D.H.
      • Bonner-Weir S.
      • Lee C.H.
      • Latimer J.B.
      • Miller C.P.
      • Habener J.F.
      • Weir G.C.
      ,
      • Jonas J.-C.
      • Sharma A.
      • Hasenkamp W.
      • Iikova H.
      • Patane G.
      • Laybutt R.
      • Bonner-Weir S.
      • Weir G.C.
      ,
      • Kaneto H.
      • Kajimoto Y.
      • Miyagawa J.
      • Matsuoka T.
      • Fujitani Y.
      • Umayahara Y.
      • Hanafusa T.
      • Matsuzawa Y.
      • Yamasaki Y.
      • Hori M.
      ,
      • Tanaka Y.
      • Gleason C.E.
      • Tran P.O.T.
      • Harmon J.S.
      • Robertson R.P.
      ). Thus, we postulate that activation of JNK pathway leads to decreased PDX-1 activity and subsequent suppression of insulin gene transcription in the diabetic state. Although not examined in this study, there are several possible mechanisms for suppression of PDX-1 DNA binding activity by the JNK pathway. One possibility is that activation of the JNK pathway influences nuclear accumulation of PDX-1, as has been shown for NFAT4, a member of the REL domain family transcription factors that are important mediators of immune response (
      • Chow C.-W.
      • Rincon M.
      • Cavanagh J.
      • Dickens M.
      • Davis R.J.
      ). Another is that JNK activation changes the phosphorylation state of PDX-1 (
      • Elrick L.J.
      • Docherty K.
      ), which could influence the PDX-1 DNA binding activity. As discussed later, we do not propose that suppression of insulin gene expression by oxidative stress is exclusively mediated through lowering of PDX-1; c-Jun, c-Myc, and other factors are likely to make contributions.
      To examine the potential therapeutic application for islet transplantation, we transplanted DN-JNK overexpressing islets under kidney capsules of STZ diabetic nude mice and found that DN-JNK provides some protection for β-cells, thus ameliorating hyperglycemia (Fig. 7). Under hyperglycemic conditions oxidative stress is provoked in β-cells (
      • Ihara Y.
      • Toyokuni S.
      • Uchida K.
      • Odaka H.
      • Tanaka T.
      • Ikeda H.
      • Hiai H.
      • Seino Y.
      • Yamada Y.
      ) and some other cell types (
      • Nishikawa T.
      • Edelstein D., Du, X.L.
      • Yamagishi S.
      • Matsumura T.
      • Kaneda Y.
      • Yorek M.A.
      • Beebe D.
      • Oates P.J.
      • Hammes H.-P.
      • Giardino I.
      • Brownlee M.
      ,
      • Laybutt D.R.
      • Kaneto H.
      • Hasenkamp W.
      • Grey S.
      • Jonas J.-C.
      • Sgroi D.C.
      • Groff A.
      • Ferran C.
      • Bonner-Weir S.
      • Sharma A.
      • Weir G.C.
      ) through processes such as the non-enzymatic glycosylation reaction (
      • Baynes J.W.
      ,
      • Baynes J.W.
      • Thorpe S.R.
      ,
      • Sakurai T.
      • Tsuchiya S.
      ,
      • Ookawara T.
      • Kawamura N.
      • Kitagawa Y.
      • Taniguchi N
      ), the electron transport chain in mitochondria (
      • Nishikawa T.
      • Edelstein D., Du, X.L.
      • Yamagishi S.
      • Matsumura T.
      • Kaneda Y.
      • Yorek M.A.
      • Beebe D.
      • Oates P.J.
      • Hammes H.-P.
      • Giardino I.
      • Brownlee M.
      ), and the hexosamine pathway (
      • Kaneto H., Xu, G.
      • Song K.-H.
      • Suzuma K.
      • Bonner-Weir S.
      • Sharma A.
      • Weir G.C.
      ). Moreover, the JNK pathway has been found to be activated by oxidative stress (
      • Finkel T.
      • Holbrook N.J.
      ) or high glucose (
      • Liu W.
      • Schoenkerman A.
      • Lowe W.L., Jr.
      ,
      • Purves T.
      • Middlemas A.
      • Agthong S.
      • Jude E.B.
      • Boulton A.J.
      • Fernyhough P.
      • Tomlinson D.R.
      ) in several cell types. Because we used immune-deficient nude mice as recipients, we assume DN-JNK protected islet grafts from oxidative stress induced by chronic hyperglycemia.
      Because it has been reported that expression of c-Jun, a basic Zip transcription factor that is known to be phosphorylated by JNK, is up-regulated by oxidative stress in several cell types (
      • Maki A.
      • Berezesky I.K.
      • Fargnoli J.
      • Holbrook N.J.
      • Trump B.F.
      ,
      • Li D.W.
      • Spector A.
      ) and suppresses insulin gene transcription (
      • Inagaki N.
      • Maekawa T.
      • Sudo T.
      • Ishii S.
      • Seino Y.
      • Imura H.
      ,
      • Henderson E.
      • Stein R.
      ) by affecting the transactivation potential of the E2A gene products (
      • Robinson G.L.W.G.
      • Henderson E.
      • Massari M.E.
      • Murre C.
      • Stein R.
      ), it is possible that c-Jun expression itself is involved in suppression of insulin gene expression by oxidative stress. Actually, as shown in Fig.3B, total c-Jun expression levels were moderately increased by H2O2, which should, at least in part, account for suppression of insulin gene expression by oxidative stress. As shown in Fig. 3, C and D, however, JNK overexpression suppressed insulin gene expression without affecting c-Jun expression levels, and DN-JNK protected insulin gene expression from oxidative stress without apparently affecting c-Jun expression levels. Thus, we propose that activation of the JNK pathway is also, at least in part, involved in suppression of insulin gene expression by oxidative stress in addition to c-Jun expression. However, we also suspect that c-Jun, even at low levels, is contributing to this effect. Additionally, c-Myc, a basic helix-loop-helix-leucine zipper transcription factor, is also up-regulated by oxidative stress (
      • Maki A.
      • Berezesky I.K.
      • Fargnoli J.
      • Holbrook N.J.
      • Trump B.F.
      ,
      • Li D.W.
      • Spector A.
      ) or high glucose per se (
      • Jonas J.-C.
      • Laybutt D.R.
      • Steil G.M.
      • Trivedi N.
      • Pertusa J.G.
      • Casteele M.V.D.
      • Weir G.C.
      • Henquin J.-C.
      ,
      • Kaneto H.
      • Suzuma K.
      • Sharma A.
      • Bonner-Weir S.
      • King G.L.
      • Weir G.C.
      ) and suppresses insulin gene expression by inhibiting NeuroD-mediated transcriptional activation (
      • Kaneto H.
      • Sharma A.
      • Suzuma K.
      • Laybutt D.R., Xu, G.
      • Bonner-Weir S.
      • Weir G.C.
      ). We assume, however, that some of the suppressive effects of JNK on the insulin gene transcription are independent of c-Jun or c-Myc, because JNK activation reduces insulin gene transcription in association with suppression of PDX-1 DNA binding activity (Fig. 6), whereas both c-Jun and c-Myc suppress the insulin gene transcription through mechanisms other than PDX-1 (
      • Robinson G.L.W.G.
      • Henderson E.
      • Massari M.E.
      • Murre C.
      • Stein R.
      ,
      • Kaneto H.
      • Sharma A.
      • Suzuma K.
      • Laybutt D.R., Xu, G.
      • Bonner-Weir S.
      • Weir G.C.
      ).
      Although JNK activation by oxidative stress preceded suppression of insulin gene expression (Fig. 2, A and D), there was some difference in timing between activation of JNK and suppression of insulin gene expression. We think this is due to stability of insulin mRNA (about 24–48 h) (
      • Welsh M.
      • Nielsen D.A.
      • MacKrell A.J.
      • Steiner D.F.
      ); even if insulin gene transcription is completely suppressed, insulin mRNA levels should not fall very quickly. Also, although WT-JNK suppressed insulin gene expression and DN-JNK protected some of the reduction of insulin gene expression from oxidative stress (Fig. 3D), the extent of insulin gene expression was not completely correlated with phosphorylated c-Jun levels, which is regulated by JNK pathway. Whereas insulin gene expression was clearly decreased by H2O2 treatment alone or Ad-WT-JNK overexpression alone, expression levels of total and phosphorylated c-Jun were only slightly induced by these treatments (Fig. 3, C and D). We think this may be due to very low expression levels of c-Jun in islets; c-Jun expression in islets is quite low, and thus H2O2 treatment alone or Ad-WT-JNK overexpression alone was not enough to induce clear bands of phosphorylated c-Jun. Also, although phosphorylated c-Jun expression was markedly induced by H2O2 in the presence of WT-JNK, the extent of decrease in insulin gene expression was similar to that after H2O2 treatment alone or Ad-WT-JNK overexpression alone. This may also be due to stability of insulin mRNA (half-life is 24–48 h) (
      • Welsh M.
      • Nielsen D.A.
      • MacKrell A.J.
      • Steiner D.F.
      ); even if insulin gene transcription is strongly suppressed, insulin mRNA levels should not be markedly decreased in 48 h. Thus, we assume that because H2O2 treatment alone or Ad-WT-JNK alone exerts maximal suppressive effects on insulin gene transcription, the combination of H2O2 and WT-JNK overexpression would not be expected to show additional effects (Fig.3C).
      It has been reported that β-cell destruction by cytokines such as interleukin-1β (
      • Corbett J.A.
      • Wang J.L.
      • Sweetland M.A.
      • Lancaster J.R., Jr.
      • McDaniel M.L.
      ,
      • Eizirik D.L.
      • Sandler S.
      • Welsh N.
      • Cetkovic-Cvrlje M.
      • Nieman A.
      • Geller D.A.
      • Pipeleers D.G.
      • Bendten K.
      • Nellerstrom
      ,
      • Ankarcrona M.
      • Dypbukt J.M.
      • Brune B.
      • Nicotera P.
      ,
      • Kaneto H.
      • Fujii J.
      • Seo H.G.
      • Suzuki K.
      • Matsuoka T.
      • Nakamura M.
      • Tatsumi H.
      • Yamasaki Y.
      • Kamada T.
      • Taniguchi N.
      ,
      • Delaney C.A.
      • Pavlovic D.
      • Hoorens A.
      • Pipeleers D.G.
      • Eizirik D.L.
      ) can be prevented by inhibition of the JNK pathway (
      • Ammendrup A.
      • Maillard A.
      • Nielsen K.
      • Anderson A.N.
      • Serup P.
      • Madsen O.D.
      • Mandrup-Poulsen T.
      • Bonny C.
      ,
      • Bonny C.
      • Oberson A.
      • Steinmann M.
      • Schorderet D.F.
      • Nicod P.
      • Waeber G.
      ,
      • Mandrup-Poulsen T.
      ,
      • Bonny C.
      • Oberson A.
      • Negri S.
      • Sause C.
      • Schorderet D.F.
      ), implying that JNK plays a role in autoimmune β-cell destruction. It should be noted, however, because in this study we used immune-deficient nude mice as recipients, it is not likely that DN-JNK protected islet grafts from oxidative stress induced by immune reaction, although we cannot totally exclude the possibility that DN-JNK exerted some beneficial effects on less obvious oxidative injury induced during the transplantation operation. It has been reported that under hyperglycemic conditions production of ROS is increased in β-cells (
      • Ihara Y.
      • Toyokuni S.
      • Uchida K.
      • Odaka H.
      • Tanaka T.
      • Ikeda H.
      • Hiai H.
      • Seino Y.
      • Yamada Y.
      ) and some other cell types (
      • Nishikawa T.
      • Edelstein D., Du, X.L.
      • Yamagishi S.
      • Matsumura T.
      • Kaneda Y.
      • Yorek M.A.
      • Beebe D.
      • Oates P.J.
      • Hammes H.-P.
      • Giardino I.
      • Brownlee M.
      ,
      • Laybutt D.R.
      • Kaneto H.
      • Hasenkamp W.
      • Grey S.
      • Jonas J.-C.
      • Sgroi D.C.
      • Groff A.
      • Ferran C.
      • Bonner-Weir S.
      • Sharma A.
      • Weir G.C.
      ) and that levels of 8-hydroxy-2′-deoxyguanosine, a marker for oxidative stress, are increased in the blood of type 2 diabetic patients (
      • Dandona P.
      • Thusu K.
      • Cook S.
      • Snyder B.
      • Makowski J.
      • Armstrong D.
      • Nicotera T.
      ,
      • Leinonen J.
      • Lehtimaki T.
      • Toyokuni S.
      • Okada K.
      • Tanaka T.
      • Hiai H.
      • Ochi H.
      • Laippala P.
      • Rantalaiho V.
      • Wirta O.
      • Pasternack A.
      • Alho H.
      ). Also, as shown in this study, JNK is activated by oxidative stress in islets, and the JNK activation reduces the PDX-1 DNA binding activity and insulin gene transcription. Thus, we assume that JNK is involved in deterioration of β-cell function in both type 2 diabetes and the early stages of type 1 diabetes.
      In conclusion, the present results provide new insights into the mechanism through which oxidative stress suppresses insulin gene transcription in pancreatic β-cells, and the finding that this adverse outcome can be prevented by DN-JNK overexpression suggests that the JNK pathway in β-cells could become a new therapeutic target for diabetes.

      ACKNOWLEDGEMENTS

      We thank Dr. Bert Vogelstein (The Johns Hopkins Oncology Center) for kindly providing the AdEasy system, Dr. Yoshitaka Kajimoto (Osaka University School of Medicine) for the PDX-1 antibody, and Dr. Hitoshi Miyazaki (Gene Experiment Center, University of Tsukuba, Japan) for helpful suggestions. We also thank the RIA Core Laboratory of the Diabetes and Endocrinology Research Center of the Joslin Diabetes Center for the insulin radioimmunoassays.

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