This Article Abstract Full Text (PDF) All Versions of this Article: 145/5/2319    most recent Author Manuscript (PDF) Purchase Article View Shopping Cart Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Tejedo, J. R. Articles by Bedoya, F. J. Search for Related Content PubMed PubMed Citation Articles by Tejedo, J. R. Articles by Bedoya, F. J. Pubmed/NCBI databases Compound via MeSH Substance via MeSH Hazardous Substances DB NITRIC OXIDE

Nitric Oxide Triggers the Phosphatidylinositol 3-Kinase/Akt Survival Pathway in Insulin-Producing RINm5F Cells by Arousing Src to Activate Insulin Receptor Substrate-1

Laboratory of Biochemistry of the Immune System, Department of Medical Biochemistry and Molecular Biology, University of Sevilla, 41009 Sevilla, Spain

Address all correspondence and requests for reprints to: Francisco J. Bedoya, Laboratory of Biochemistry of the Immune System, Department of Medical Biochemistry and Molecular Biology, University of Sevilla, Avenida Sanchez Pizjuan 4, 41009 Sevilla, Spain. E-mail: bedoya{at}us.es.

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Mechanisms involved in the protective action of nitric oxide (NO) in insulin-producing cells are a matter of debate. We have previously shown that pharmacological inhibition of c-Src cancels the antiapoptotic action of low and sustained concentrations of exogenous NO. In this study, using insulin-producing RINm5F cells that overexpress Src either permanently active (v-Src) or dominant negative (dn-Src) forms, we determine that this tyrosine kinase is the principal mediator of the protective action of NO. We also show that Src-directed activation of insulin receptor substrate-1, phosphatidylinositol 3-kinase (PI3K), Akt, and Bad phosphorylation conform a substantial component of the survival route because pharmacological inhibition of PI3K and Akt canceled the antiapoptotic effects of NO. Studies performed with the protein kinase G (PKG) inhibitor KT-5823 revealed that NO-dependent activation of c-Src/ insulin receptor substrate-1 is not affected by PKG activation. By contrast, Akt and Bad activation are partially dependent on PKG activation. Endogenous production of NO after overexpression of endothelial nitric oxide synthase in RINm5F cells mimics the effects produced by generation of low amounts of NO from exogenous diethylenetriamine/NO. In addition, we found that NO produces c-Src/PI3K- and PKG-dependent activation of ERK 1/2. The MAPK kinase inhibitor PD 98059 suppresses NO-dependent protection from DNA fragmentation induced by serum deprivation. The protective action of low and sustained concentration of NO is also observed in staurosporine- and Taxol-induced apoptosis. Finally, NO also protects isolated rat islets from DNA fragmentation induced by serum deprivation. These data strengthen the notion that NO production at physiological levels plays a role in protection from apoptosis in pancreatic ß-cells.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
APOPTOTIC DEATH OF pancreatic ß-cells plays an important role in the pathogenesis of diabetes (1, 2). The apoptotic cascade can be triggered by intracellular events such as metabolic dysfunction, perturbations in the cell cycle, DNA damage, and by external factors such as activation of death receptors and inflammatory cytokines or the lack of trophic factors (3). Activation of pancreatic ß-cell survival pathways is crucial for facing noxious challenges that eventually lead to destruction of the endocrine pancreas in both types 1 and 2 diabetes (4). Recent studies have shown the involvement of insulin/IGF-1 signaling in the control of ß-cell survival and growth (5, 6, 7, 8). It is also established that low concentrations of nitric oxide (NO) protects from apoptosis in several cell systems including ß-cells (9, 10, 11, 12, 13). NO-induced survival in neurons and in endothelial cells is dependent on phosphorylation of the apoptotic protein Bad through activation of cGMP/protein kinase G (PKG)/phosphatidylinositol 3-kinase (PI3K)/Akt system (10, 14, 15). The protective action of NO in serum-deprived insulin producing RINm5F cells also involves the activation of c-Src (9). This soluble tyrosine kinase is directly activated by NO and regulates apoptosis and cell survival in a number of cell systems (16, 17, 18, 19, 20, 21). It is entirely possible that NO generation mediates the insulin/IGF-1 survival pathway in ß-cells. In the present report, we document that the protective action of NO involves the activation of PI3K/Akt/Bad system in the pancreatic ß-cell line RINm5F. The survival pathway triggered by NO is dependent on activation of c-Src and phosphorylation of insulin receptor substrate-1 (IRS-1) and activation of ERK 1/2. Activation of the PI3K/Akt system is also dependent on the soluble guanylate cyclase (GC)/PKG system.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Materials
KT-5823, PD 98059, monoclonal antiphosphotyrosine (PY20), and monoclonal anti-c-Src (clone 327) were from Calbiochem (La Jolla, CA); PP1 and SH6 were from Alexis Biochemicals (San Diego, CA), monoclonal anti-cytochrome c (7H8.2C12) was from PharMingen (San Diego, CA); monoclonal anti-endothelial nitric oxide synthase (eNOS) was from Transduction Laboratories (Lexington, KY); protein G Sepharose, [-³²P] ATP, and polyvinyl difluoride (PVDF) transfer membrane were from Amersham Pharmacia Biotech (Uppsala, Sweden); diethylenetriamine (DETA)/NO was from Research Biochemicals International (Natick, MA); RPMI 1640 was from BioWhittaker (Verviers, Belgium); streptomycin, penicillin, glutamine, amphotericin B, cell death detection ELISA^plus, 4-(2-aminoethyl)-benzenesulfonyl fluoride (pefablock), pepstatin A, aprotinin, and leupeptin were from Roche (Stockholm, Sweden); polyclonal anti-p-ERK 1/2 (Thr202/Tyr204) was from Cell Signaling (Beverly, MA); polyclonal anti IRS-1 (C-20), monoclonal anti-Bcl-2 (C-2), monoclonal anti-Akt-1, polyclonal anti-p-Akt (Ser473), monoclonal anti-Bad, polyclonal anti-p-Bad (Ser136), and polyclonal anti-ERK-1 were from Santa Cruz Biotechnology (Santa Cruz, CA); c-Src (K296R/Y528F) dominant-negative in pUSEamp(+) were from Upstate Biotechnology (Lake Placid, NY); Taxol, staurosporine, enolase, antimouse and antirabbit IgG peroxidase conjugate, trypsin and other chemicals were from Sigma (St. Louis, MO).

Islet isolation, ß-cell line culture, and transfection
Rat pancreatic islet were isolated under aseptic conditions from collagenase-digested pancreas of Wistar adult male rats. Batches of 10 islets were cultured in RPMI 1640 medium containing 2 mg/ml glucose, 100 µg/ml streptomycin, 100 U/ml penicillin G, 2.5 µg/ml amphotericin B, 2 mM glutamine, and 10% fetal bovine serum for 24 h before experimental treatment. RINm5F cells were maintained in RPMI 1640 supplemented with 100 µg/ml streptomycin, 100 U/ml penicillin G, 2.5 µg/ml amphotericin B, 2 mM glutamine, and 10% fetal bovine serum in a humidified atmosphere of 5% CO₂ at 37 C. For transfection, cells (6 x 10⁶) in 800 µl serum-free RPMI 1640 were electroporated with a single pulse of 300 V and 1200 µF capacitance with 5 µg of the expression plasmids: c-Src (K296R/Y528F) dominant negative, pCDNA3-v-Src (provided by Dr. P. Reddy, Temple University School of Medicine, Fels Institute for Cancer Research and Molecular Biology, Philadelphia, PA), pCDNA3.1-eNOS (provided by Dr. D. J. Stewart, Department of Medicine, University of Toronto, Toronto, Ontario, Canada). After the pulse, cells were seeded in RPMI 1640 medium supplemented with 10% fetal bovine serum for 48 h. For selection, transfected cells were cultured in RPMI 1640 medium containing geneticin (400 mg/liter).

Detection of histone-associated DNA
After treatment, islets were trypsinized for 10 min at 37 C. Dispersed islet cells were washed with PBS and suspended in 100 µl of lysis buffer. RINm5F cells were scraped of the plates and centrifuged at 700 x g for 10 min, washed with PBS, and suspended in lysis buffer. DNA fragmentation was determined with the cell death detection ELISA^plus according to the manufacturer’s instructions

Western blot
Cells were collected by centrifugation at 700 x g for 3 min at 4 C, washed twice with ice-cold PBS and centrifuged at 700 x g for 3 min. The cytochrome c and Bcl-2 were determined by Western blotting, as previously described (22, 23).

For analysis of Src, cells were placed in 50 µl lysis buffer [20 mM HEPES (pH 7.5), 10 mM EGTA, 40 mM glycerophosphate, 140 mM NaCl, 25 mM MgCl₂, 1 mM dithiothreitol, 2% Nonidet P-40, 2 mM sodium orthovanadate, 50 µM phenarsine oxide, 1 mM pefablock, 10 µg/ml pepstatin A, 10 µg/ml leupeptin, and 100 U/ml aprotinin]. Lysis was carried out at 4 C for 1 h. Cell lysates were sonicated three times for 10 sec on ice (Branson sonifier, duty cycle 100%, output control 1) and centrifuged at 15,000 x g for 15 min. Twenty-five micrograms of supernatant protein were resolved on 10% SDS-PAGE electrophoresis and blotted to PVDF membranes that were incubated for 1 h at room temperature with anti c-Src clone 327 and anti c-Src clone 28 (24). After removal of the excess of primary antibody by three washes with TBST buffer [containing 50 mM Tris (pH 7.5), 150 mM NaCl, and 0.1% Tween 20] membranes were incubated with antimouse IgG conjugated with peroxidase (1:20,000) for 1 h. Bound antibodies were detected by enhanced chemiluminescence.

Immunoprecipitation and in vitro kinase assay
Batches of cells (3 x 10⁶) were placed in 50 µl of lysis buffer for 1 h. at 4 C. After centrifugation, clarified supernatants were immunoprecipitated with primary antibody (2 µg/mg protein) for 4 h at 4 C. Twenty microliters of 50% (vol/vol) protein G Sepharose were then added, and the mixture was incubated for 4 h at 4 C, followed by centrifugation to recover the protein G Sepharose pellets. The immunoprecipitates were washed three times with PBS and once with kinase reaction buffer [10 mM 3[N-morholino]propanesulfonic acid (pH 7.5), 12.5 µM ß-glycerophosphate, 7.5 mM MgCl₂, 0.5 mM EGTA, 0.5 mM NaF, 0.5 mM Na₃VO₄] and then suspended in 20 µl of kinase reaction buffer. The immunoprecipitates were used for detection of total protein and phosphorylated protein by Western blot (IRS-1, ERK 1/2, Akt, Bad) and kinase assay (c-Src).

Src kinase assay was performed in 30 µl kinase buffer containing 20 µM ATP, 1 µCi[-³²P] ATP, and 10 µg acid-denatured enolase for 20 min at 30 C (9). Samples were analyzed using SDS-PAGE. After electrophoresis, the proteins were transferred to PVDF membranes and subjected to autoradiography.

Other analyses
Protein concentration was determined by Bradford’s technique (Bio-Rad Laboratories, Hercules, CA). Data are mean ± SD of at least three independent experiments, except for results of blots and autoradiographies, in which case a representative experiment is depicted in the figures. Comparisons between group values were analyzed using one-way ANOVA. Differences were considered significant when P 0.05.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Src mediates the antiapoptotic action of NO in serum-deprived RINm5F cells
Figure 1A shows that a significant release of histone-bound DNA to cytosol is apparent 9 h after removal of serum in control, nontransfected RINm5F cells. Transfection of cells with a plasmid containing Src permanently active (RINm5F/v-Src cells) protects significantly from DNA fragmentation induced by serum deprivation. On the other hand, when cells are transfected with the dominant-negative (K296R/Y528F) Src plasmid (RINm5F/dn-Src cells), DNA fragmentation was significantly apparent 4 h after serum removal, and the overall amount of fragmented DNA after 24 h of serum starvation doubled the values in control, nontransfected RINm5F cells. The upper blot in Fig. 1A shows that expression levels of Src protein are higher in transfected cells than in nontransfected cells. Lower blot in Fig. 1A shows that a fraction of endogenous Src is active in control cells; transfection with permanently active (v-Src) increases the relative amount of activated Src, whereas the amount of active Src is lower in cells transfected with dominant-negative Src (dn-Src), which contains a kinase domain incapable of binding ATP (mutation K296R) and the mutation Y528F that renders Src constitutively active. dn-Src interacts with activators of Src, titrating them competitively from endogenous Src, which consequently does not become activated. Clone 28 antibody recognizes a region adjacent to Y528 in the C terminal of activated protein. Figure 1B shows that addition of DETA/NO (10 µM) to serum-deprived cells leads to significant decrease in DNA fragmentation both in control and RINm5F/v-Src cells. DETA/NO fails to protect from serum deprivation-induced DNA fragmentation in RINm5F/dn-Src cells, thus suggesting that Src mediates NO protective action. DNA fragmentation in cells transfected with pUSEamp(+) is not significantly different from control, nontransfected cells, either in the presence or absence of serum (data not shown). Figure 1C shows that the PI3K inhibitor LY-294002 (20 µM) and the MAPK kinase (MEK) inhibitor PD 98059 (20 µM) cancel NO-protective action on serum withdrawal-induced DNA fragmentation. These findings raise the possibility that PI3K and MEK survival pathways are involved in NO protective action.

View larger version (34K): [in this window] [in a new window]   FIG. 1. Effect of Src gene manipulation on apoptosis induced by serum removal: effect on DNA fragmentation. A, Nontransformed RINm5F cells (control cells), RINm5F cells transformed with Src gene permanently active (RINm5F/v-Src), and cells transformed with dominant-negative (K296R/Y528F) Src gene (RINm5F/dn-Src) were cultured in serum-free RPMI 1640 medium for the indicated times. The images inserted show the level of expression of total Src (detected with clone 327 antibody) and the Src active form (detected with clone 28 antibody). B, Control RINm5F cells and cells transfected with either permanently active Src gene (RINm5F/v-Src) or dn-Src gene (RINm5F/dn-Src) were deprived of serum for 15 h and exposed to DETA/NO, as indicated in the figure. C, RINm5F cells were serum starved for 15 h either in the absence or presence of 10 µM DETA/NO, 20 µM LY 294002, and 20 µM PD 98059. DNA fragmentation was determined as in Materials and Methods. D, Effect on cytochrome c release and Bcl-2 down-regulation. Cells were cultured in serum-free medium for 15 h and cytochrome c levels in cytosol and Bcl-2 protein in whole-cell homogenates were detected by immunoblot as in Materials and Methods. A–C, Data are mean ± SD of three to five independent experiments. Images shown in A and D are representative from three independent experiments. A, *, P 0.005 vs. control cells with serum; **, P 0.005 vs. RINm5F cell-deprived serum; ***, P 0.005 vs. RINm5F and RINm5F/v-Src cells treated with DETA/NO. C, *, P 0.005 vs. control cells with serum; **, P 0.005 vs. deprived serum condition; ***, P 0.005 vs. DETA/NO condition.

NO activates ERK 1/2 in a PI3K-dependent manner as a part of the antiapoptotic action
When RINm5F cells are deprived of serum for 4 h, the degree of ERK 1/2 activation decreases notably. The addition DETA/NO (10 µM) leads to ERK 1/2 activation in serum-deprived cells, and the PI3K inhibitors wortmannin and LY 294002 suppress this action (Fig 2A). When cells are transfected with the dn-Src, DETA/NO-induced activation of ERK 1/2 is abolished. Transfection with v-Src gene protects from serum withdrawal-induced ERK 1/2 inactivation, and PI3K inhibitors block this action (Fig. 2B).

View larger version (19K): [in this window] [in a new window]   FIG. 2. Role of PI3K in the action of DETA/NO on the activation ERK 1/2 in serum-deprived cells. Cells were deprived of serum for 4 h. DETA/NO (10 µM) was added to cells for 15 min. The PI3K inhibitors were added 2 h before DETA/NO. A, NO induces the activation of ERK 1/2 in a PI3K-dependent manner. Nontransformed RINm5F cells were exposed to the additions as indicated above. Cells were then homogenized and immunoprecipitated with anti-ERK 1/2 antibody. B, Src mediates the action of PI3K in NO-triggered activation of ERK 1/2. RINm5F/v-Src cells and RINm5F/dn-Src cells were treated as indicated before. Immunoprecipitated ERK1/2 from cell homogenates was then used for Western blot. Immunoblot was performed as in Materials and Methods. Images are representative from three independent experiments.

View larger version (33K): [in this window] [in a new window]   FIG. 3. Role of PKG in the actions of NO in serum-deprived cells. Nontransfected RINm5F cells were deprived of serum for 4 h. DETA/NO (10 µM) was added to cells for 15 min. The PKG inhibitor was added 2 h before DETA/NO. A, The activation of Src is independent of PKG. Homogenates from RINm5F cells were immunoprecipitated with anti-c-Src antibody (clone 327). Immunoprecipitates were blotted with either anti-c-Src (clone 28) (top panel) or anti-c-Src (clone 327) monoclonal antibodies (middle panel). In vitro c-Src kinase activity was tested in immunoprecipitates with exogenous enolase as substrate (bottom panel) as in Materials and Methods. B, Activation of Akt, Bad, and ERK 1/2 is partially dependent on PKG. Homogenates from RINm5F cells were immunoprecipitated with anti-Akt antibody, anti-Bad antibody, and anti-ERK 1/2 antibody and blotted with antibodies against p-ERK 1/2, ERK 1/2, p-Akt, Akt, p-Bad, and Bad. Images are representative from three independent experiments. Densitometry values were normalized for the control values (defined as 1).

View larger version (39K): [in this window] [in a new window]   FIG. 4. NO-induced phosphorylation of IRS-1 is dependent on c-Src and independent of PKG and PI3K. A, RINm5F cells were deprived of serum for 4 h and exposed to DETA/NO for 15 min. PKG and PI3K inhibitors were added 2 h before DETA/NO. B, v-Src and dn-Src RINm5F cells were deprived of serum for 4 h and exposed to DETA/NO for 15 min. PI3K inhibitors were added 2 h before DETA/NO. A and B, Cell homogenates were immunoprecipitated with anti-IRS-1 antibody and blotted with either antiphosphotyrosine antibody or IRS-1 antibodies. Images are representative from three independent experiments.

Akt and Bad phosphorylation are involved in the protective action of NO
When RINm5F cells are deprived of serum, activation levels of Akt are low (Fig. 5A). Exposure to DETA/NO (10 µM) restores Akt activation in a PI3K-dependent manner because wortmannin and LY-294002 were able to block DETA/NO-induced Akt activation. Bad phosphorylation is decreased in serum-deprived cells, and addition of DETA/NO (10 µM) counteracts partially this effect. In addition, experiments with PI3K and Akt inhibitors (SH6) show that DETA/NO-dependent phosphorylation is dependent on PI3K/Akt activation (Fig. 5B).

View larger version (30K): [in this window] [in a new window]   FIG. 5. NO-induced phosphorylation of Bad is dependent on activation of PI3K/Akt system. Cells were deprived of serum for 4 h and exposed to DETA/NO for 15 min. Inhibitors were added 2 h before DETA/NO. A, RINm5F cell homogenates were immunoprecipitated with anti-Akt antibody and blotted with anti-p-Akt and anti-Akt. B, RINm5F cells homogenates were immunoprecipitated with anti-Bad antibody and blotted with anti-p-Bad and anti-Bad. Images are representative from three independent experiments.

View larger version (23K): [in this window] [in a new window]   FIG. 6. Protection from serum-deprived apoptosis by eNOS overexpression involves activation of Src, PI3K/Akt, and PKG. Cells were cultured for 15 h in serum-free medium in the absence or in the presence of the inhibitors. A, DNA fragmentation was determined in RINm5F cells transfected with pcDNA 3,1 vector alone and cells transfected with pcDNA 3,1-eNOS vector as in Materials and Methods. When appropriated, cells were also exposed to the inhibitors for 15 h. Data are mean ± SD from three to five independent experiments. B, Cytosolic fraction and homogenates from vector cells and eNOS cells were blotted with anti-cytochrome c and anti-Bcl-2 antibodies, respectively. Images are representative from three independent experiments. A, *, P 0.005 vs. RINm5F/pcDNA 3.1 control cells with serum; **, P 0.005 vs. RINm5F/pcDNA3.1 cell-deprived serum; ***, P 0.005 vs. RINm5F/eNOS cell-deprived serum condition.

Involvement of c-Src, IRS-1, PI3K/Akt, and PKG in the protection from apoptosis provided by eNOS overexpression
The upper and middle blots in Fig. 7A show that IRS-1 phosphorylation is dependent on NO and involves c-Src activation because L-NMMA and PP1 inhibited significantly (60 and 50%, respectively) IRS-1 phosphorylation. The blots also show that IRS-1 phosphorylation is not dependent on PKG activation. PI3K inhibition significantly increases IRS-1 phosphorylation.

View larger version (27K): [in this window] [in a new window]   FIG. 7. Involvement Src, PKG, and PI3K in the activation of IRS-1, Akt, ERK , and Bad in eNOS-transfected cells. RINm5F cells transfected with pcDNA 3,1 vector alone and cells transfected with pcDNA 3,1-eNOS vector were deprived of serum for 4 h. The inhibitors were added 2 h before extraction. A, Homogenates were precipitated with anti-IRS-1, anti-Akt, and anti-ERK 1/2 and blotted with anti-phosphotyrosine antibody (pIRS-1) or antibodies that recognize phosphorylated forms of proteins (pAkt, pERK 1/2). Densitometry values were normalized for the control values (defined as 1). B, Homogenates are precipitated with anti-Bad and blotted with anti-p-Bad. Images are representative from three independent experiments.

Lower blots in Fig. 7A show that ERK 1/2 activation is dependent on eNOS, c-Src, and PI3K and partially dependent on PKG.

Bad phosphorylation induced by eNOS overexpression is significantly suppressed by eNOS, c-Src, PI3K, and Akt inhibitors (Fig. 7B).

The protective action of NO is also apparent in Taxol and staurosporin-induced apoptosis and serum-deprived rat islets
Apoptosis inducers such as Taxol and staurosporin lead to a concentration-dependent increase in the amount of DNA fragmentation in RINm5F cells, and DETA/NO (10 µM) significantly diminishes this effect (Fig. 8A). On the other hand, isolated rat islets undergo fragmentation of DNA when deprived of serum for 24 h, and the presence of DETA/NO (10 µM) reduces partially and significantly this effect (Fig. 8B).

View larger version (33K): [in this window] [in a new window]   FIG. 8. Effect of DETA/NO in Taxol and staurosporin-induced apoptosis and serum-deprived rat islets. A, RINm5F cells were exposed to the apoptosis inducers for 24 h in the presence of serum at the indicated concentrations either in the absence or presence of 10 µM DETA/NO. B, Batches of 10 islets were serum starved for 24 h either in the absence or presence of 10 µM DETA/NO. DNA fragmentation was measured as in Materials and Methods. A and B, *, P 0.005 vs. control condition; **, P 0.005 vs. treated condition

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Transfection with the permanently active form of Src confers to RINm5F cells substantial protection from apoptotic fragmentation of DNA, cytochrome c release to cytosol, and Bcl-2 degradation, whereas transfection with dn-Src enhances apoptosis in serum-deprived cells. The finding that low concentrations (10 µM) of DETA/NO failed to confer protection from DNA fragmentation in cells transfected with the inactive form of Src whereas conferring protection in control cells and in v-Src-transfected cells suggest that this tyrosine kinase is a relevant target for NO in the antiapoptotic response. Additional actions of NO are suggested by the fact that a significant protective action is still present in cells transfected with the permanently active form of Src. The participation of Src in the antiapoptotic response has been documented in the literature (19, 20, 21). Thus, vascular endothelial growth factor-triggered protection from apoptosis in endothelial cells depends on Raf-1 activation by Src and is linked to PI3K activation in fibroblasts (20, 21).

Our results support the notion that PI3K is also implied in the antiapoptotic action of NO because the inhibitor LY 294002 blocks the protective action of NO on DNA fragmentation in serum-deprived cells. Our data show that NO also signals through MAPK pathway. Protection from DNA fragmentation conferred by NO exposure in serum-depleted cells was canceled by the MAPK kinase inhibitor PD 58059. In addition, serum-exposed cells show activated ERK 1/2 as previously reported (25, 26) and removal of serum led to substantial decrease of ERK 1/2 phosphorylation. Experiments performed with cells expressing v-Src and dn-Src show that Src activates ERK. This kinase is involved in survival responses in several cell types, including islets (25, 27, 28). It is thus entirely possible that the activation of ERK by NO is a component of the survival response in RINm5F cells as previously shown in other cell types. Controversy exists, however, on this issue. We reported the activation of ERK in RINm5F cells exposed to apoptotic concentrations of NO (23). It is conceivable that under such circumstances the protective role of activated ERK is overruled by direct apoptotic actions of NO at the mitochondria. The involvement of the sGC/PKG in the antiapoptotic action of NO has been substantiated in a variety of cell systems (10, 11, 14, 15). We previously reported that both soluble GC and c-Src mediate NO-dependent activation of PKG (9). In this work, we show that PKG activation by NO participates in the activation of ERK 1/2 and Akt and phosphorylation of Bad. On the other hand, the PKG inhibitor KT-5823 does not affect Src activation by NO. Taken together, these results suggest that concerted activation of Src and GC by NO triggers an antiapo-ptotic pathway in RINmF cells that involves Akt and Bad phosphorylation and the ERK 1/2 activation.

The activation of IRS-1 by c-Src as a part of the signaling system triggered by IGF and insulin receptor activation has been reported previously (29, 30, 31). We have found that the presence of serum in culture media regulates IRS-1 phosphorylation in RINm5F cells. Exogenously generated NO was able to induce IRS-1 phosphorylation in serum-deprived cells in a PI3K- and PKG-independent manner. Most interestingly, v-Src cells display IRS-1 phosphorylation in the absence of external serum, whereas dn-Src cells did not. Neither DETA/NO nor PI3K and PKG inhibitors modify IRS-1 phosphorylation in transfected cells cultured in the presence of serum. Apoptotic susceptibility to serum deprivation has been described in human islets carrying Arg⁹⁷² IRS-1, thus suggesting an important role for this system in ß-cell survival (6). Our finding that Src governs IRS-1 activation widens the scope of extracellular survival signals that might operate through the IRS-1 system.

NO antiapoptotic action implies Bad phosphorylation through PI3K/Akt-dependent activation. The relevance of the role of NO in protecting cells from serum deprivation was substantiated by the finding that eNOS transfected cells display resistance to DNA fragmentation, cytochrome c release, and Bcl-2 degradation when exposed to serum-free media. In fact, eNOS transfection was able to restore IRS-1 phosphorylation in serum-starved cells in a Src-dependent manner. PI3K/Akt and PKG transduce the antiapoptotic signaling that involves Bad phosphorylation. The participation of PI3K/Akt in survival induced by IGF-1 and other factors is well substantiated (19, 32, 33, 34). eNOS overexpression in cells has been used as a tool to study the effect of near physiological concentrations of NO on apoptosis (13, 14). The results we report here with eNOS transfected cell strengthen the notion that NO production at physiological levels plays a role in protection from apoptosis in insulin-producing RINm5F cells. The protective action of NO was also observed in Taxol and staurosporin-apoptosis in RINm5F cells and serum-deprived isolated rat islets, thus indicating that the mechanism reported here operates in a variety of apoptotic situations and also operates in differentiated ß-cells.

The scheme presented in Fig. 9 summarizes the tentative NO-induced signaling pathways in pancreatic ß-cells, as derived from the results reported in the present work. The contribution of ERK 1/2 to ß-cell survival deserves further exploration. With regard to the possible physiological relevance of the present data, production of NO at low levels could be involved in the activation of ß-cell survival pathways. A combination of acquired and genetic alterations in these pathways may be a significant factor in the loss of ß-cell mass in types 1 and 2 diabetes.

View larger version (20K): [in this window] [in a new window]   FIG. 9. Hypothetical scheme of antiapoptotic NO signaling in insulin producing RINmF cells. Generation of sustained low levels of NO after activation of eNOS leads to activation of c-Src and PKG. c-Src-dependent activation of IRS-1 triggers PI3K/Akt activation. Akt is partially activated by PKG. Antiapoptotic phosphorylation of Bad is dependent on Akt. PI3K- and PKG-dependent phosphorylation of ERK-1/2 also occurs. The contribution of this pathway to the overall control of apoptosis in this cell line remains to be characterized. Bold lines denote evidence provided for this cell line in preceding paper (9 ).

	Acknowledgments

 
Monoclonal anti-c-Src (clone 28) was kindly donated by Dr. H. Kawakatsu (University of California, San Francisco Lung Biology Center, San Francisco, CA).

	Footnotes

 
This work was supported by grants from Dirección General de Investigación Científica y Técnica (SAF 2000/117 and 2003/367, Junta de Andalucía (CV286), and Instituto de Salud Carlos III, RGDM(G03/212). J.R.T. and G.M.C. are postdoctoral fellows from Fundación Carolina.

Abbreviations: DETA, Diethylenetriamine; dn-Src, dominant negative Src; eNOS, endothelial nitric oxide synthase; GC, guanylate cyclase; IRS-1, insulin receptor substrate-1; L-NMMA, N^G-monomethyl L-arginine; MEK, MAPK kinase; NO, nitric oxide; PI3K, phosphatidylinositol 3-kinase; PKG, protein kinase G; PVDF, polyvinyl difluoride; v-Src, permanently active Src.

Received November 3, 2003.

Accepted for publication January 28, 2004.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Mandrup-Poulsen T 2003 Apoptotic signal transduction pathways in diabetes. Biochem Pharmacol 66:1433–1440[CrossRef][Medline]
2. Chandra J, Zhivotovsky B, Zaitsev S, Juntti-Berggren L, Berggren PO, Orrenius S 2001 Role of apoptosis in pancreatic ß-cell death in diabetes. Diabetes 50(Suppl 1):S44–S47
3. Mandrup-Poulsen T 2001 ß-Cell apoptosis. Stimuli and signaling. Diabetes 50(Suppl 1):S58–S63
4. Sesti G 2002 Apoptosis in the ß-cells: cause or consequence of insulin secretion defect in diabetes? Ann Med 34:444–450[CrossRef][Medline]
5. Rakatzi I, Seipke G, Eckel J 2003 [LysB3, GluB29] insulin: a novel insulin analog with enhanced ß-cell protective action. Biochem Biophys Res Commun 310:852–859[CrossRef][Medline]
6. Federici M, Hribal ML, Ranalli M, Marselli L, Porzio O, Lauor D, Borboni P, Lauro R, Marchetti P, Melino G, Sesti G The common Arg 972 polymorphism in insulin receptor substrate-1 causes apoptosis of human pancreatic islets. FASEB J 10.1096/fj. 2000-00–04fje
7. Tuttle RL, Gill NS, Pugh W, Lee JP, Koeberlein B, Furth EE, Polonsky KS, Naji A, Birnbaum MJ 2001 Regulation of pancreatic ß-cell growth and survival by the serine/threonine protein kinase Akt1/PKB. Nat Med 10:1133–1137
8. Welsh N, Makeeva N, Welsh M 2002 Overexpression of the Shb SH2 domain-protein in insulin-producing cells leads to altered signaling through the IRS-1 and IRS-2 proteins. Mol Med 8:695–704[Medline]
9. Tejedo J, Ramírez R, Cahuana G, Rincón P, Sobrino F, Bedoya FJ 2001 Evidence for involvement of c-Src in the anti-apoptotic action of nitric oxide in serum-deprived RINm5F cells. Cell Signal 13:809–817[CrossRef][Medline]
10. Sata M, Kakpki M, Nagata D, Nishimatsu H, Suzuki E, Aoyagi T, Sugiura S, Kojima H, Nagano K, Kabgawa K, Matsuo H, Omata M, Nagai R, Hirata Y 2000 Adrenomedullin and nitric oxide inhibit human endothelial cell apoptosis via cyclic GMP-independent mechanism. Hypertension 36:83–88[Abstract/Free Full Text]
11. Dash PR, Cartwright JE, Baker PN, Johnstone AP, Whitley GSJ 2003 Nitric oxide protects human extravillous trophoblast cells from apoptosis by cyclic GMP-dependent mechanism and independently of caspase 3 nitrosylation. Exp Cell Res 287:314–324[CrossRef][Medline]
12. Ozaki M, Kawashima S, Hirase T, Yamashita T, Namiki M, Inoue N, Hirata K, Yokoyama M 2002 Overexpression of endothelial nitric oxide synthase in endothelial cells is protective against ischemia-reperfusion injury in mouse skeletal muscle. Am J Pathol 160:1335–1344[Abstract/Free Full Text]
13. Kwon YG, Min JK, Kim KM, Lee DJ, Billiar TR, Kim YM 2001 Sphingosine 1-phosphate protects human umbilical vein endothelial cells from serum-deprived apoptosis by nitric oxide production. J Biol Chem 276:10627–10633[Abstract/Free Full Text]
14. Ha KS, Kim KM, Kwon YG, Bai SK, Nam WD, Yoo YM, Kim PKM, Chung HT, Billiar TR, Kim YM 2003 Nitric oxide prevents 6-hyidroxydopamine-induced apoptosis in PC12 cells through cGMP-dependent PI3 kinase/Akt activation. FASEB J 17:1036–1047[Abstract/Free Full Text]
15. Ciani E, Virgili M, Contestabile A 2002 Akt pathway mediates a cGMP-dependent survival role of nitric oxide cerebellar granule neurones. J Neurochem 81:218–228[CrossRef][Medline]
16. Akhand AA, Pu M, Senga T, Kato M, Suzuki H, Miyata T, Hamaguchi M, Nakashima I 1999 Nitric oxide controls Src kinase activity through a sulfhydryl group modification-mediated Tyr-527-independent and Tyr-416-linked mechanism. J Biol Chem 274:25821–25826[Abstract/Free Full Text]
17. Minetti M, Mallozi C, Di Stasi AMM 2002 Peroxynitrite activates kinases of the Src family and up-regulates tyrosine phosphorylation signaling. Free Radic Biol Med 33:744–754[CrossRef][Medline]
18. MacMillan-Crow LA, Greendorfer JS, Vickers SM, Thompson JA 2000 Tyrosine nitration of c-Src tyrosine kinase in human pancreatic ductal adenocarcinoma. Arch Biochem Biophys 377:350–356[CrossRef][Medline]
19. Johnson D, Agochiya M, Samejima K, Earnshaw W, Frame M, Wyke J 2000 Regulation of both apoptosis and cell survival by the v-Src oncoprotein. Cell Death Differ 7:685–696[CrossRef][Medline]
20. Alavi A, Hood JD, Frausto R, Stupack DG, Cheresh DA2003 Role of Raf in vascular protection from distinct apoptotic stimuli. Science 301:94–96
21. Shih WL, Kuo ML, Chuang SE, Cheng AL, Doong SL 2003 Hepatitis B virus X protein activates a survival signaling by linking Src to phosphatidylinositol-3-kinase. J Biol Chem 278:31807–31813[Abstract/Free Full Text]
22. Tejedo J, Bernabé JC, Ramírez R, Sobrino F, Bedoya FJ 1999 NO induces a cGMP-independent release of cytochrome c from mitochondria which precedes caspase 3 activation in insulin producing RINm5F cells. FEBS Lett 459:238–243[CrossRef][Medline]
23. Bernabé JC, Tejedo J, Rincón P, Cahuana G, Ramírez R, Sobrino F, Bedoya FJ 2001 Sodium nitroprusside-induced mitochondrial apoptotic events in insulin-secreting RINm5F cells are associated with MAP kinases activation. Exp Cell Res 269:222–229[CrossRef][Medline]
24. Kawakatsu H, Sakai T, Takagaki Y, Shinoda Y, Saito M, Owada MK, Yano J 1996 A new monoclonal antibody which selectively recognizes the active form of Src tyrosine kinase. J Biol Chem 271:5680–5685[Abstract/Free Full Text]
25. Saldeen J, Lee JC, Welsh N 2000 Role of p38 mitogen-activated protein kinase (p38 MAPK) in cytokine-induced rat islet cell apoptosis. Biochem Pharmacol 61:1561–1569
26. Cousin SP, Hugl SR, Myers MG, Whitte MF, Reifel-Miller A, Rodees CJ 1999 Stimulation of pancreatic ß-cell proliferation by growth hormone is glucose-dependent: signal transduction via Janus kinase 2 (JAK2)/signal transducer and activator of transcription 5 (STAT 5) with no cross-talk to insulin receptor substrate-mediated mitogenic signalling. Biochem J 344:649–658
27. Xia Z, Dickens M, Raingeaud J, Davis RJ, Greenberg ME 1995 Opposing effects of Erk and JNK-p38 MAP kinases on apoptosis. Science 270:1326–1331[Abstract/Free Full Text]
28. Paraskevas S, Aikin R, Maysinger D, Lakey JRT, Cavanagh TJ, Hering B, Wang R, Rosenberg L 1999 Activation and expression of Erk, JNK, and p38 MAP-kinases in isolated islets of Langerhans: implications for cultured islet survival. FEBS Lett 455:203–208[CrossRef][Medline]
29. Prisco M, Romano G, Peruzzi F, Valentinis B, Baserga R 1999 Insulin and IGF-I receptors signaling in protection from apoptosis. Horm Metab Res 31:80–89[Medline]
30. Boney CM, Sekimoto H, Gruppuso PA, Frackelton Jr R 2001 Src family tyrosine kinases participate in insulin-like growth factor I mitogenic signaling in 3T3–L1 cells. Cell Growth Differ 12:379–386[Abstract/Free Full Text]
31. Valentinis B, Morrione A, Taylor SJ, Baserga R 1997 Insulin-like growth factor signaling in transformation by src oncogenes. Moll Cell Biol 17:3744–3754[Abstract]
32. Tack I, Elliot SJ, Potier M, Rivera A, Striker GE, Stricker LJ 2002 Autocrine activation of the IGF-I signaling pathway in mesangial cell isolated diabetic NOD mice. Diabetes 51:182–188[Abstract/Free Full Text]
33. Suzuki K, Takahashi K 2000 Anchorage-independent activation of mitogen-activated protein kinase through PI3K by insulin-like growth factor. Biochem Biophys Res Commun 272:111–115[CrossRef][Medline]
34. Vincent AM, Feldman EL2002 Control of cell survival by IGF signaling pathways. Growth Hor IGF Res 12:193–197

This article has been cited by other articles:

				F. Zhang, Q. Zhang, A. Tengholm, and A. Sjoholm Involvement of JAK2 and Src kinase tyrosine phosphorylation in human growth hormone-stimulated increases in cytosolic free Ca2+ and insulin secretion Am J Physiol Cell Physiol, September 1, 2006; 291(3): C466 - C475. [Abstract] [Full Text] [PDF]

				S. Itoh, S. Lemay, M. Osawa, W. Che, Y. Duan, A. Tompkins, P. S. Brookes, S.-S. Sheu, and J.-i. Abe Mitochondrial Dok-4 Recruits Src Kinase and Regulates NF-{kappa}B Activation in Endothelial Cells J. Biol. Chem., July 15, 2005; 280(28): 26383 - 26396. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: 145/5/2319    most recent Author Manuscript (PDF) Purchase Article View Shopping Cart Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Tejedo, J. R. Articles by Bedoya, F. J. Search for Related Content PubMed PubMed Citation Articles by Tejedo, J. R. Articles by Bedoya, F. J. Pubmed/NCBI databases Compound via MeSH Substance via MeSH Hazardous Substances DB NITRIC OXIDE