Interleukin-1{beta} induces posttranslational carboxymethylation and alterations in subnuclear distribution of lamin B in insulin-secreting RINm5F cells

Rajakrishnan Veluthakal, Rajesh Amin, and Anjaneyulu Kowluru

Department of Pharmaceutical Sciences, Wayne State University, and {beta} Cell Biochemistry Research Laboratory, John D. Dingell Veterans Affairs Medical Center, Detroit, Michigan 48201

Submitted 10 February 2004 ; accepted in final form 7 June 2004


    ABSTRACT
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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We examined the effects of interleukin-1{beta} (IL-1{beta}) treatment on the distribution and degradation of lamin B in the nuclear fraction from insulin-secreting RINm5F cells. Western blot analysis indicated that IL-1{beta} treatment caused significant alterations in the redistribution of lamin B, specifically between the Triton X-100-soluble (membrane) and -insoluble (matrix) fractions of the nucleus. IL-1{beta} treatment also increased the lamin carboxymethyltransferase activity and the relative abundance of the carboxymethylated lamin in the nuclear fraction. A significant increase in the relative abundance of lamin B degradation products was also observed in the nuclear fraction from the IL-1{beta}-treated cells. These findings are compatible with a measurable increase in the lamin-degrading caspase-6 activity in IL-1{beta}-treated cells. Confocal microscopic observation of IL-1{beta}-treated cells suggested a significant dissociation of lamin B from the nuclear lamina and its subsequent association with the DNA-rich elements within the nucleus. NG-monomethyl-L-arginine, a known inhibitor of inducible nitric oxide synthetase (iNOS), markedly inhibited IL-1{beta}-induced iNOS gene expression, NO release, caspase-3 and caspase-6 activation, lamin B degradation, and loss of metabolic cell viability, indicating that the observed IL-1{beta}-induced effects on nuclear lamin B involve the intermediacy of NO. Together, our data support the hypothesis that IL-1{beta} treatment results in significant increase in the carboxymethylation of lamin B, which would place lamin B in a strategic location for its degradation mediated by caspases. This could possibly lead to dissolution of the nuclear envelope, culminating in the demise of the effete {beta}-cell.

pancreatic {beta}-cell; lamin carboxymethyltransferase; nitric oxide; nuclear matrix; caspases


THE NUCLEUS IS A COMPLEX ORGANELLE and the center of major activities during the cell cycle progression and development. It undergoes dynamic processing of a number of constituent macromolecules, including lamins and DNA. The nuclear lamina is a fibrillar meshwork of proteins lining the interior of the nuclear envelope. In the mammalian cell, the nuclear lamina consists mainly of three proteins, namely, lamins A, B, and C (38, 53c). These intermediate filament proteins subserve several cellular functions, including DNA replication (39), chromatin organization (21), differentiation, nuclear structural support (37), and nuclear envelope assembly (21). Previous data from several laboratories in multiple cell types, including data from our own investigations of isolated {beta}-cells, indicated that lamin B undergoes posttranslational modifications (e.g., isoprenylation and carboxymethylation) (3, 7, 8, 17, 28, 48, 54, 56). Such modifications appear to play crucial roles in the assembly of the nuclear envelope. The COOH-terminal isoprenylation and carboxymethylation of lamins A and B appear to increase their hydrophobicity, thus promoting their interaction with relevant inner nuclear membrane proteins (23). Several phosphorylation sites on lamins A, B, and C have also been identified. The NH2-terminal domain and the nuclear localization signal (NLS) domain consist of phosphorylation sites for p34cdc2 kinase (38). Phosphorylation by protein kinase C (PKC) of an additional site in the NLS domain appears to "initiate" lamin proteolysis and DNA fragmentation in cells undergoing apoptosis (10, 38). In contrast, phosphorylation of lamins, specifically lamin B, is thought to favor its dissociation from the nuclear lamina (10, 38). Together, these posttranslational modifications appear to contribute to the subnuclear localization of lamin B as well as its vulnerability to proteolytic cleavage by caspase-6.

It is well established that long-term exposure of isolated {beta}-cells to cytokines (e.g., IL-1{beta}) results in their demise via apoptotic mechanisms (2, 16, 34, 45). This has been used as a model for insulin-dependent diabetes mellitus, and the cytotoxicity of interleukin-1{beta} (IL-1{beta}) is attributed primarily to the induction of inducible nitric oxide synthetase (iNOS) and the subsequent generation of nitric oxide (NO) (15). Several recent studies, including our own, have indicated that NO exerts its cytotoxic effects by affecting {beta}-cell metabolism at various levels, including alterations in mitochondrial membrane permeability properties, leading to the release of cytochrome c (Refs. 19, 51, and 57; unpublished observations from our laboratory). It is well accepted that cytochrome c activates caspase-3, which in turn activates a series of caspase isoforms, including caspase-6 (47). Activation of these caspases leads to hydrolysis and thereby functional regulation of several key cellular proteins, including the PKC {delta}-isoform. Extant studies suggest that PKC{delta} and lamin B are hydrolyzed by caspase-3 and caspase-6, respectively (1, 43).

Nuclear envelope disassembly represents one of the hallmark features of IL-1{beta}-induced apoptosis of the islet {beta}-cell. Because lamin B plays a significant role in maintaining the integrity of the nuclear envelope, alterations in the distribution of lamin B are thought to favor the breakdown of the nuclear envelope followed by DNA degradation and chromatin condensation. Despite this existing body of evidence indicating regulatory roles of lamin B in nuclear envelope assembly, very little has been studied thus far with regard to alterations in nuclear lamin B metabolism in a {beta}-cell progressing toward its apoptotic demise after exposure to IL-1{beta}. The present study was undertaken to examine the alterations in the carboxymethyltransferase that regulates the posttranslational carboxymethylation of lamin B in isolated {beta}-cells exposed to cytotoxic levels of IL-1{beta}. We also examined alterations in the subnuclear distribution of lamin B, including colocalization of lamin B with caspase-6 in the nuclear fraction under the duress of IL-1{beta}. Finally, using NG-monomethyl-L-arginine (L-NMMA), a known inhibitor of iNOS, we examined putative regulatory roles for IL-1{beta}-induced iNOS gene expression and NO release in IL-1{beta}-mediated activation of caspase-3 and caspase-6 and lamin B degradation in the {beta}-cell progressing toward apoptotic demise. Our current findings suggest that exposure of isolated {beta}-cells to IL-1{beta} results in a significant increase in the carboxymethylation of lamin B, which would place this key nuclear protein in a strategic location for its degradation mediated by caspases. This could possibly lead to disassembly of the nuclear envelope and chromatin condensation, culminating in the apoptotic demise of the {beta}-cell.


    MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 
Materials. IL-1{beta} was purchased from R&D Systems (Minneapolis, MN), and S-adenosyl-L-[3H-methyl]methionine ([3H]SAM) was obtained from NEN Life Sciences (Boston, MA). N-acetyl-S-trans,trans-farnesyl-cysteine (AFC) was purchased from Biomol Research Laboratories (Plymouth Meeting, PA). Rabbit polyclonal antibody against caspase-3 was obtained from Cell Signaling Technology (Beverly, MA). Affinity-purified polyclonal antisera directed against lamin B, PKC{delta}, iNOS, and caspase-6 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). 4',6-Diamidino-2-phenylindole dihydrochloride (DAPI) nucleic acid stain was purchased from Molecular Probes (Eugene, OR). Rhodamine-conjugated anti-goat IgG, FITC-conjugated anti-goat IgG, and Griess reagent were purchased from Sigma (St. Louis, MO). L-NMMA was purchased from Alexis Biochemicals (San Diego, CA). The colorimetric caspase assay kit was purchased from BioVision (Mountain View, CA). The apoptotic DNA ladder kit was purchased from Roche Applied Science (Indianapolis, IN). All other reagents used in this study were of the highest reagent purity available.

Insulin-secreting cells. RINm5F cells were purchased from American Type Culture Collection (Manassas, VA) and cultured in RPMI 1640 medium containing 2.5 mM glutamine supplemented with 10% fetal calf serum, 100 IU/ml penicillin, and 100 IU/ml streptomycin.

Treatment of cells and isolation of subnuclear fractions. Rat insulinoma (RIN) cells were treated with IL-1{beta} (600 pM) for 48 h. After a brief rinse in phosphate-buffered saline (PBS; pH 7.5), cells were scraped and spun at 100 g for 5 min to remove the medium. The cellular pellet was homogenized in an isotonic medium consisting of 250 mM mannitol, 10 mM Tris base (pH 7.5) at 4°C, and protease inhibitor cocktail, using a Potter-Elvehjem tissue homogenizer. The homogenate was centrifuged for 5 min at 200 g to yield a crude nuclear pellet and the postnuclear supernatant. The crude nuclear pellet was resuspended in the homogenization medium containing 1 mM EDTA, layered on top of 1.5 M sucrose buffered with 10 mM Tris (pH 7.5), and centrifuged for 90 min at 26,000 g. The resulting nuclei-enriched pellet was then resuspended in a hypotonic medium consisting of (in mM) 10 Tris, pH 7.4, 4.5 EDTA, 2.5 EGTA, and 2.3 mercaptoethanol, and 5.0 benzamidine and sonicated at 4°C for 30 s. The nuclear lysate fraction was centrifuged for 30 min at 10,000 g to yield the nuclear cytosol (referred to as the "soluble" fraction) and the nuclear particulate fraction. The nuclear particulate fraction was again sonicated in the same buffer with 0.5% Triton X-100 to solubilize the nuclear membrane. After 15-min incubation at 4°C, this fraction was centrifuged at 16,000 g for 30 min to yield the nuclear membrane (referred to as the "membrane" fraction) and the Triton X-100 insoluble nuclear matrix fractions (referred to as the "matrix" fraction) (27).

Nitrite release. RINm5F cells grown in 24-well plates were treated with IL-1{beta} (600 pM, 48 h) and/or L-NMMA (500 µM) as indicated. The medium was collected after 48 h and centrifuged at 100 g for 5 min. Equal volumes of medium and Griess reagent were mixed, and the absorbance was measured at 540 nm as described previously (49).

Western blot analysis of iNOS. RINm5F cells were treated with IL-1{beta} (600 pM, 48 h) in the absence or presence of L-NMMA (500 µM) as indicated. Extracted proteins (40–60 µg) were separated by SDS-PAGE, and the resolved proteins were transferred to a nitrocellulose membrane by wet transfer. Blots were then probed with anti-iNOS antibody (1:1,000 dilution) and then incubated with secondary antibody conjugated to horseradish peroxidase (HRP). Immune complexes were detected using the enhanced chemiluminescence (ECL) kit.

Metabolic cell viability assay. RINm5F cells, seeded at a density of 1 x 106 cells/ml in round-bottomed, 96-well plates, were treated with or without IL-1{beta} (600 pM, 48 h) in the absence or presence of L-NMMA (500 µM) as indicated. Cell viability was determined by performing a colorimetric assay (at 550–690 nm) using 3-[4,5-dimethylthiazolyl-2]-2,5-diphenyltetrazolium bromide (MTT), which measures the reduction of MTT into the blue formazan product by metabolically active cells.

DNA extraction and gel electrophoresis. Genomic DNA was extracted from the cells treated with IL-1{beta} (600 pM, 48 h) according to the protocol provided with the apoptotic DNA ladder kit (Roche Applied Science, Indianapolis, IN). Briefly, cells treated with IL-1{beta} (600 pM, 48 h) after a brief rinse with PBS were incubated in binding buffer (6 M guanidine-HCl, 10 mM urea, 10 mM Tris·HCl, and 20% Triton X-100, pH 4.4) for 10 min at 15°C. After three washes (20 mM NaCl, 2 mM Tris·HCl, pH 7.5, 80% ethanol), DNA was extracted using elution buffer (10 mM Tris, pH 8.5). The extracted DNA was separated on 1% agarose gel and visualized under UV light.

Carboxymethylation of AFC and lamin B. The activity of methyltransferase was measured using AFC as the substrate as reported previously (28–30), using [3H]SAM as the methyl donor. The release of base-labile methanol from the AFC methyl ester was measured using scintillation spectrometry (28–30). In brief, the assay buffer (total volume 25 µl; 100 mM Tris·HCl, pH 7.5, 1 mM EDTA, 1 mM dithiothreitol, 10 µM unlabeled SAM, and 1 µCi [3H]SAM) and 200 µM AFC were mixed with an equal volume of RIN cell fractions (60 µg of protein) and incubated at 37°C for 30 min. The AFC methyl ester formed was extracted by adding 500 µl of chloroform-methanol (1:1; vol/vol) and 250 µl of water and vortexed vigorously. The organic phase was transferred to an Eppendorf tube and allowed to dry at 40°C. NaOH (1 M) containing 1% SDS was added to the dried tubes, and the tubes were placed carefully in a vial containing scintillation fluid and left at 37°C for an additional 24 h to allow equilibration of the released [3H]methanol vapor into the scintillation fluid. The amount of [3H]methanol released was quantitated using scintillation spectrometry.

The carboxymethylation of lamin was studied in subnuclear fractions isolated from cells treated with either diluent alone or IL-1{beta}. The assay mixture in a total volume of 100 µl containing 50 mM sodium phosphate buffer, pH 6.8, 1 mM EGTA, 75 µg of protein, and 100 µCi/ml [3H]SAM were incubated at 37°C for 1 h. The reaction was terminated by adding the sample buffer. Labeled proteins were separated by SDS-PAGE and visualized by autoradiography after drying the gels previously soaked in EN3HANCE autoradiography enhancer (NEN Life Sciences) and exposing them to X-ray film for 6 wk at –70°C.

For immunoprecipitation experiments, the methylated protein samples were extracted with cholate buffer (in mM: 20 HEPES, pH 7.4, 1% sodium cholate, 2 MgCl2, 1 DTT, 1 EGTA, 1 benzamidine, 1 PMSF, and 1 protease inhibitor cocktail), sonicated three times, precleared with goat serum, and incubated overnight with anti-lamin B antibody (1:200 dilution). Immunoprecipitated proteins were captured using protein G agarose, and the degree of carboxymethylation of lamin B in the immunoprecipitates was quantitated by vapor phase equilibration assay as described above.

Assay of caspase-3 and caspase-6 activities. RINm5F cells were treated with IL-1{beta} (600 pM) in either the absence or presence of L-NMMA (500 µM) for 48 h as indicated. Quantitation of caspase-3 and caspase-6 in protein lysates was accomplished by measuring the proteolytic cleavage of the chromogenic substrate acetyl-Asp-Glu-Val-Asp-p-nitroanilide (Ac-DEVD-pNA) and acetyl-Val-Glu-Ile-Asp-p-nitroanilide (Ac-VEID-pNA) for caspase-6 at 400 nm (9).

Western blot analysis for lamin B and caspase-3. RIN cell fractions (40–60 µg protein as indicated) from cells treated with either diluent alone or IL-1{beta} in the presence or absence of L-NMMA (500 µM) were separated by SDS-PAGE and transferred to a nitrocellulose membrane. The membrane was blocked (with 5% nonfat dry milk) in 20 mM Tris·HCl, 150 mM NaCl, and 0.1% Tween (TBST), washed twice for 15 min each time with TBST, and then incubated with primary polyclonal goat anti-lamin B antibody or primary polyclonal rabbit anti-caspase-3 antibody (1:1,000 dilution) for 1 h at room temperature. The blot was then washed twice with TBST for 15 min each time and incubated with secondary antibodies (anti-goat IgG HRP or anti-rabbit IgG HRP) at room temperature for 1 h. Immune complexes were visualized using an ECL detection kit.

Confocal microscopic determination of nuclear changes in IL-1{beta}-treated cells. RINm5F cells were grown on glass coverslips for 2 days and then incubated with or without IL-1{beta} (600 pM) for an additional 2 days. After fixation in ice-cold methanol for 20 min, the cells were blocked for 30 min in a solution consisting of 5% nonimmune rabbit serum and 1% BSA in TBS (pH 7.5) and incubated further for 2 h with primary polyclonal goat anti-lamin B (1:200 dilution). The cells were washed again three times for 5 min each time in TBS with 0.1% Tween and then exposed for 1 h to rabbit anti-goat rhodamine-conjugated secondary antibody (1:400 dilution) for 1 h, washed, and exposed to a DAPI solution as indicated. The cells were then mounted in an aqueous mounting medium and photographed using a Zeiss LSM 510 laser scanning confocal microscope.

Colocalization of lamin B with caspase-6. RINm5F cells were treated with diluent alone or IL-1{beta}, fixed, and blocked with 3% BSA in TBS. The cells were then incubated with primary goat anti-lamin B polyclonal antibody (1:200 dilution) for 2 h, washed, and incubated with mouse anti-caspase-6 monoclonal antibody (1:300 dilution) for an additional 2 h. After several washes with TBS, the cells were incubated with anti-goat conjugated to FITC to detect the lamin B. For the detection of caspase-6, the cells were incubated for 1 h with an anti-mouse serum conjugated to rhodamine. The cells were then mounted in an aqueous mounting medium. Caspase-6 colocalization with lamin B was photographed using a Zeiss LSM 510 laser scanning confocal microscope. Verification of lamin B and caspase-6 colocalization was achieved using a z-stacking technique. Briefly, in this technique, 1-µm optical sections through the cell are used, which allows better visualization of both proteins and their specific colocalization within the cell.

To verify the specificity of the anti-lamin B serum that we used in these studies, we incubated control cells with the antibody against lamin B, which was immunoneutralized using the control peptide (purchased from Santa Cruz Biotechnology), and detected no staining for lamin B microscopically. PKC{delta} was visualized in both control and IL-1{beta}-treated cells using a rabbit polyclonal antibody (1:200 dilution) and a secondary anti-rabbit IgG conjugated to rhodamine (1:300 dilution).

Protein assay. Protein concentration in the lysates and subnuclear fractions was determined using the dye-binding method of Bradford with BSA used as the standard (5).


    RESULTS
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 MATERIALS AND METHODS
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Exposure of RIN cells to IL-1{beta} results in an increase in NO production and subsequent loss in metabolic cell viability. The main objective of this study was to examine IL-1{beta}-induced, NO-dependent alterations in the nuclear fraction, specifically at the level of degradation of lamin B, in the isolated {beta}-cell. Therefore, in the initial experiments, we determined the sensitivity to IL-1{beta} of the RINm5F cells that we used in the current investigation. As a functional measure, we quantitated NO release from the control and IL-1{beta}-treated cells. The data in Fig. 1A show that exposure of RIN cells to IL-1{beta} (600 pM, 48 h) significantly increased NO release, which is compatible with earlier observations at our laboratory (49). IL-1{beta}-induced NO release was markedly decreased by L-NMMA, a known inhibitor of iNOS. Furthermore, we observed a significant inhibition in IL-1{beta}-induced iNOS gene expression in RIN cells treated with L-NMMA (Fig. 1B). In subsequent experiments, we observed a significant (nearly 40%) loss in the metabolic viability of these cells after exposure to IL-1{beta}, indicating significant metabolic dysfunction (Fig. 1C). The observed decrease in metabolic cell viability in IL-1{beta}-treated cells was largely prevented by coprovision of these cells with L-NMMA, implicating a direct involvement of NO in IL-1{beta}-induced metabolic dysfunction of the {beta}-cell. We used these experimental conditions (600 pM, 48 h) in subsequent studies aimed at examining the effects of IL-1{beta} on nuclear lamin B metabolism in these cells.



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Fig. 1. Inhibition by NG-monomethyl-L-arginine (L-NMMA) of interleukin (IL)-1{beta}-induced nitric oxide (NO) release, inducible nitric oxide synthetase (iNOS) gene expression, and decrease in metabolic viability in rat insulinoma (RIN) cells. A: RIN cells were incubated in the presence of diluent alone or IL-1{beta} (600 pM) in the simultaneous absence or presence of L-NMMA (500 µM) for 48 h as indicated. NO released in the medium was quantitated using Griess reagent. Data are means ± SE from 3 independent experiments *P < 0.05 vs. control. **P < 0.05 vs. IL-1{beta}-treated group. B: RIN cells were incubated in the presence of diluent alone or IL-1{beta} (600 pM) in the simultaneous absence or presence of L-NMMA (500 µM) for 48 h as indicated. Proteins were separated by SDS-PAGE, and the resolved proteins were transferred to a nitrocellulose membrane. Blots were probed with anti-iNOS antibody, followed by incubation with secondary antibody conjugated to horseradish peroxidase (HRP). Immune complexes were visualized using the enhanced chemiluminescence (ECL) kit. A representative blot from 2 experiments yielding similar results is shown. C: RIN cells were incubated in the presence of diluent alone or IL-1{beta} (600 pM) in the simultaneous absence or presence of L-NMMA (500 µM) for 48 h as indicated. The metabolic cell viability was determined using 3-[4,5-dimethylthiazolyl-2]-2,5-diphenyltetrazolium bromide (MTT) assay (see METHODS). Data are means ± SE from 3 independent experiments. *P < 0.05 vs. control. **P< 0.05 vs. IL-1{beta}-treated group.

 
IL-1{beta} induces DNA fragmentation in RIN cells. To further confirm data from our cell viability assays, we studied the effects of IL-1{beta} on DNA degradation. The data in Fig. 2 depict the electrophoretic fractionation of DNA extracted from cells treated with IL-1{beta} (600 pM, 48 h). These data reveal a marked degradation of DNA as evidenced by the DNA laddering profile in cells treated with IL-1{beta}. However, we detected no fragmentation of DNA extracted from RIN cells treated with the diluent alone (Fig. 2).



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Fig. 2. IL-1{beta} treatment causes DNA fragmentation in RIN cells. DNA was isolated from cells treated with either diluent alone (–) or IL-1{beta} (600 pM, 48 h; +) according to the procedure provided with the kit (Apoptotic DNA ladder kit; Roche Diagnostic). Purified DNA at a concentration of 1–3 µg was loaded onto 1% agarose gel and run at 75 V for 90 min. The bands were visualized and photographed under UV light.

 
IL-1{beta} significantly increases prenylcysteine methyltransferase activity and carboxymethylation of lamin B in RIN cells. Several earlier studies (3, 7, 8, 17, 48, 54, 56), including our own (28), in isolated rat islets and clonal {beta}-cells demonstrated that lamin B undergoes isoprenylation and carboxymethylation at its COOH-terminal cysteine residue. Such modification steps are known to promote the translocation of lamin B to the membranous sites (e.g., nuclear lamina or membrane) for the assembly of the nuclear envelope (7, 10, 17, 56). The carboxymethylation of nuclear lamin B and low-molecular-weight G proteins, as well as the {gamma}-subunits of trimeric G proteins, is mediated by a prenylcysteine methyltransferase enzyme that we previously characterized in isolated {beta}-cells (33).

To further examine whether IL-1{beta} treatment has any effect on the methylation status and hence the membrane association of lamin B, we first quantitated prenylcysteine carboxymethyltransferase activity in the soluble and membrane fractions derived from the nuclei isolated from the control and IL-1{beta}-treated cells. This activity was measured using AFC as the methyl group acceptor. The data in Fig. 3 indicate a substantial degree of stimulation of AFC methyltransferase activity in the nuclear soluble fraction isolated from cells treated with IL-1{beta}. A modest but insignificant increase in this activity was also demonstrable in the nuclear membrane fraction isolated from the IL-1{beta}-treated cells (Fig. 3). These data imply that the methylation site for lamin may be the nuclear soluble compartment, after which it moves to the nuclear membrane fraction in a manner akin to small G proteins (35). Moreover, the data in Fig. 3 indicate much lower specific activity of the methylating enzyme in the nuclear membrane fraction than that of its counterpart in the nuclear soluble fraction.



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Fig. 3. IL-1{beta} treatment stimulates the N-acetyl-S-trans,trans-farnesyl-cysteine (AFC)-carboxymethyltransferase activity in the nuclear soluble and membrane compartments. RIN cells were incubated in the presence of diluent alone or IL-1{beta} (600 pM, 48 h). Membrane and soluble fractions were isolated, and the activity of methyltransferase was measured using AFC as substrate and S-adenosyl-L-[3H-methyl]methionine ([3H]SAM) as the methyl donor (see METHODS). Data are means ± SE from 3 independent experiments and are expressed as nmol [3H]methanol liberated/mg protein. *P < 0.05.

 
These data prompted us to determine the degree of carboxymethylation of lamin B in control and IL-1{beta}-treated cells. This was accomplished using [3H]SAM as the methyl donor, and the labeled proteins in total lysates were resolved using SDS-PAGE. Figure 4A represents the labeling of a protein with an apparent molecular weight of 75 kDa (similar to the molecular mass of lamin B) as detected by autofluorography. Furthermore, the labeling of this protein appeared to increase significantly in IL-1{beta}-treated cells. Extant data from our laboratory (29, 44, 50) demonstrate that in addition to nuclear lamin B, at least three types of proteins underwent carboxymethylation after incubation of cellular lysates with [3H]SAM. These proteins included the catalytic subunit of protein phosphatase 2A (36 kDa), low-molecular-weight G proteins belonging to the Ras superfamily (22–25 kDa), and the {gamma}-subunits of trimeric GTPases (5–7 kDa). However, under our current experimental conditions, IL-1{beta} treatment affected the carboxymethylation of only lamin B (data not shown).



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Fig. 4. IL-1{beta} treatment induces methylation of lamin B. A: total lysate proteins (75 µg) from control and IL-1{beta}-treated cells were incubated with [3H]SAM as the methyl donor and the degree of incorporation of label into a protein with an apparent molecular weight of 75 kDa was visualized using autofluorography. B: nuclear lysate protein (60 µg) from the control and IL-1{beta}-treated RIN cells were incubated in sodium phosphate buffer (pH 6.8) with [3H]SAM as the methyl donor (see METHODS). The methylated lamin B was immunoprecipitated using anti-lamin serum, and the degree of carboxymethylation of lamin was determined using vapor-phase equilibration assay. Data are means ± standard deviations from 2 independent immunoprecipitation experiments and are expressed as %control. *P < 0.05. C: total protein lysates (50 µg) prepared from control and IL-1{beta}-treated cells were resolved by SDS-PAGE and transferred to nitrocellulose membrane. The membrane was probed for lamin B using goat anti-lamin B antibody (1:1,000 dilution), and immune complexes were identified using an ECL detection kit. Data represent 3 blots yielding comparable results. D: total protein lysates (50 µg) prepared from control, IL-1{beta}-treated, and IL-1{beta} plus L-NMMA-treated cells were resolved using SDS-PAGE and transferred to nitrocellulose membrane. The membrane was probed for lamin B using goat anti-lamin B antibody (1:1,000 dilution), and immune complexes were identified using an ECL detection kit. Data represent 2 blots yielding comparable results.

 
To further confirm and quantitate the degree of carboxymethylation of lamin B, the methylated proteins derived from the nuclear fractions isolated from control and IL-1{beta}-treated cells were immunoprecipitated using an antiserum directed against lamin B. The degree of carboxymethylation of lamin B in the immunoprecipitates was quantitated using a vapor-phase equilibration assay (see METHODS for additional details). The data in Fig. 4B indicate a significant increase in the carboxymethylation of lamin B in RIN cells after exposure to IL-1{beta}, which is compatible with data shown in Fig. 4A.

We next verified whether IL-1{beta}-induced increase in carboxymethylation of lamin B results in its degradation. To accomplish this, protein lysates from the control and IL-1{beta}-treated cells were subjected to SDS-PAGE, and the relative abundance of lamin B in control and IL-1{beta}-treated cell lysates was verified by Western blotting. The data in Fig. 4C show that a protein in the molecular weight region of 75 kDa, corresponding to the size of lamin B, was observed in control cell lysates. The intensity of this 75-kDa band was markedly reduced, however, in IL-1{beta}-treated cell lysates. Furthermore, a protein in the molecular weight region of 45 kDa was prominent in IL-1{beta}-treated cell lysates, presumably corresponding to a degradation product of lamin B. These findings are compatible with the observations of Buendia et al. (6), who reported the emergence of a 45-kDa lamin B degradation product in cells undergoing apoptosis, presumably after proteolytic cleavage of the full-length lamin B by caspases (see IL-1{beta}-induced caspase-3 and caspase-6 activation in a NO-dependent manner).

Furthermore, the protein profiles in lysates derived from cells treated with IL-1{beta} plus L-NMMA were comparable to those of control cells (Fig. 4D). These data imply that IL-1{beta}-induced degradation of lamin B involves the intermediacy of NO, presumably via the activation of {beta}-cell caspases, and this was examined in experiments described below.

IL-1{beta}-induced caspase-3 and caspase-6 activation in a NO-dependent manner. In the next series of studies, we examined the effects of IL-1{beta} treatment on caspase -3 and -6 activities because these proteases were shown to hydrolyze several cellular proteins (e.g., lamin B, a substrate for caspase-6; and PKC{delta}, a substrate for caspase-3). It has also been suggested that caspase-3-mediated hydrolysis of PKC{delta} results in its functional activation (1, 43). The data shown in Fig. 5A demonstrate that exposure of RIN cells to IL-1{beta} (600 pM, 48 h) results in significant increases in the activities of caspase-3 (85%) and caspase-6 (40%). Furthermore, coprovision of L-NMMA significantly reduced IL-1{beta}-mediated activation of each of these caspases, implicating NO in this signaling step. IL-1{beta}-induced NO-mediated activation of caspase-3 was also confirmed by Western blot analysis (Fig. 5B), which demonstrated the emergence of a band in the molecular weight region of 17 kDa in IL-1{beta}-treated cells, representing the active fragment of full-length caspase-3 (35 kDa). The formation of a cleaved active fragment of caspase-3 was inhibited by L-NMMA, again implicating NO in caspase-3 activation. Together, these findings clearly implicate NO in IL-1{beta}-induced caspase activation and subsequent lamin B degradation in isolated {beta}-cells. Furthermore, they are compatible with recent studies that demonstrated NO-mediated alterations in the mitochondrial membrane potential leading to the release of mitochondrial cytochrome c into the cytosolic fraction, culminating in the activation of caspase-3 and caspase-6 (Ref. 47 and unpublished observations from this laboratory).



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Fig. 5. IL-1{beta} induces caspase-3 and caspase-6 activation in a NO-dependent manner. A: cells were incubated in the absence or presence of IL-1{beta} (600 pM, 48 h) with and without L-NMMA (500 µM). Lysate proteins (300 µg) were incubated in a buffer containing appropriate substrates, and the cleaved chromophore was quantitated spectrophotometrically at 400 nm. Data are means ± SE from 3 independent experiments and are expressed as %control. *P < 0.05 vs. control. **P < 0.05 vs. IL-1{beta}-treated group. B: RIN cells were incubated in the presence of diluent alone or IL-1{beta} (600 pM) in the absence or presence of L-NMMA (500 µM) for 48 h as indicated. Proteins were separated by SDS-PAGE, and the resolved proteins were transferred to a nitrocellulose membrane. Blots were probed with anti-caspase-3 antibody, followed by incubation with secondary antibody conjugated to HRP. Immune complexes were visualized using an ECL kit. A representative blot from 2 experiments yielding similar results is shown.

 
Exposure of RIN cells to IL-1{beta} results in altered subnuclear distribution of lamin B. The data shown in Figs. 3 and 4 indicate significant alterations in the carboxymethylation of lamin B in the nuclear fraction derived from IL-1{beta}-treated cells. Because these modification steps are known to alter the subnuclear distribution of lamin B (48), we assessed the association of lamin B within the subnuclear fractions isolated from the control and IL-1{beta}-treated cells. For this purpose, we determined the relative abundance of lamin B by performing Western blot analyses in soluble, membrane, matrix, and postnuclear supernatant fractions isolated from control and IL-1{beta}-treated cells. The data shown in Fig. 6 indicate that lamin B is localized in all three fractions of the nucleus. The rank order of distribution was the membrane fraction (~52%) was greater than the matrix fraction (~38%), which was greater than the soluble fraction (~10%). As expected, lamin B was least detectable in the postnuclear supernatant. Interestingly, membrane-associated lamin B appears to be smaller in size than its counterparts in the other two fractions. Also, the relative abundance of this protein in the membrane fraction appears to be lower in the IL-1{beta}-treated cells (~38%) than in control membrane preparations (~52%). We also observed a significant increase in the matrix fraction after IL-1{beta} treatment (~51%) compared with the control matrix fraction (~38%). We consistently observed (as shown in Fig. 6) that in the nuclear matrix fraction, the relative abundance of low-molecular-weight proteins or peptides is much higher in IL-1{beta}-treated cells. At present, it is difficult to speculate whether these represent the "degradation products" of lamin B, but they appear to yield positive bands with an antiserum raised against lamin B. We also think that these bands are not nonspecific, because the antiserum used in above studies (Fig. 4C) yielded one major band corresponding to the size of lamin B and another band of less intensity corresponding to its degradative product of caspase-6. For these reasons, we speculate that these bands represent the degradation products of lamin B. Together, these data suggest specific alterations in the abundance of lamin in various compartments of the nucleus (specifically the matrix and membrane fractions) in cells exposed to IL-1{beta}. These data were further confirmed by confocal microscopy (see next subsection).



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Fig. 6. IL-1{beta}-induced alterations in the distribution of lamin B in different nuclear compartments. RIN cells were treated with diluent alone or IL-1{beta} (600 pM, 48 h) and protein form (50 µg) soluble, membrane, matrix, and postnuclear supernatant (PNS) fractions were resolved by SDS-PAGE (10% acrylamide) and transferred to nitrocellulose membrane. The membrane was probed for lamin B using goat anti-lamin B antibody (1:1,000 dilution). A representative blot from 3 experiments yielding similar results is shown.

 
Exposure of RIN cells to IL-1{beta} results in distinct changes in subnuclear localization of lamin B: evidence for colocalization of lamin B and caspase-6. Our immunological findings regarding the subnuclear distribution of lamin B and its methylating enzyme indicate that IL-1{beta} treatment facilitates degradation of lamin B, followed by its dissociation from the nuclear lamina. To further confirm this, we investigated the nuclear distribution of lamin B in control and IL-1{beta}-treated cells by confocal microscopy. The data in Fig. 7A show that in control cells, lamin B is associated with the nuclear envelope, which is demonstrated by the confluent red halo. This observation was further verified using DAPI (Fig. 7B), a stain that binds specifically to the chromatin material within the nucleus, for the same cell shown in Fig. 7A. After incubation with IL-1{beta} (600 pM, 24 h), lamin B becomes dissociated from the nuclear envelope and starts internalizing (Fig. 7, C and D).



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Fig. 7. Confocal microscopic evidence suggesting changes in the distribution of lamin B in control and IL-1{beta}-treated cells. A: control RINm5F cells show a halo pattern of lamin indirect immunolocalization associated with the nuclear envelope. A1: enlarged version of image shown in A. B: 4',6-diamidino-2-phenylindole dihydrochloride (DAPI) labeling of chromatin material in the nucleus from cells shown in A verifies that lamin B is localized to the nuclear region. C and D: IL-1{beta} treatment (600 pM, 24 h) created gaps of lamin B localization with the nuclear envelope as indicated by arrows. Note that more lamin B is associated with the internal region of the nucleus in IL-1{beta}-treated cells than is associated with the same region in control cells. Arrows indicate gaps in the association of lamin B with the nuclear envelope. E: after exposure to IL-1{beta} (600 pM, 48 h), lamin is mostly within the nucleus and is no longer visible within the nuclear envelope (lack of halo pattern). Also, the cells appear to form small fragments with lamin B association, as shown by arrows. E1: enlarged version of the image shown in E. F: DAPI stain for cells shown in E. Note that chromatin material is condensed internally, indicating significant cellular changes.

 
Together, these data provide the first evidence to suggest altered subnuclear distribution of lamin B in cells incubated with IL-1{beta}. Figure 7E shows a relative lack of the confluent halo pattern, suggesting that lamin B became internalized and that small bodies with lamin association had formed. Figure 7F represents the same group of cells shown in Fig. 7E and shows that the small lamin-associated bodies also contain chromatin material, as evidenced by the DAPI stain. Together, these data indicate significant changes in the lamin distribution pattern in IL-1{beta}-treated cells, which is compatible with immunological data described above (Fig. 6).

Using confocal microscopy to gain further understanding of the role of IL-1{beta}-induced caspase-6 activation (Fig. 5), we examined the effects of IL-1{beta} on colocalization of caspase-6 with lamin B. Figure 8A demonstrates the localization of lamin B (green) within the nucleus of IL-1{beta}-treated cells, which was overlaid with a phase-contrast image to better determine the nuclear region. The open arrow in Fig. 8A indicates a damaged cell, which is evidenced by lamin B being internalized into the nucleus and consequently lacking the normal halo pattern of lamin B seen in Fig. 8A (solid arrow). Figure 8B represents caspase-6 localization in the same cells shown in Fig. 8A. Here again, the open arrow in the phase-contrast image indicates a damaged cell in which caspase-6 is associated with the internal region of the nucleus, which differs from the halo pattern of the normal cell. The images shown in Fig. 8, A and B, were overlaid to produce Fig. 8C, in which lamin B (green) and caspase-6 (red) in the damaged cell (open arrow) are shown colocalizing within the nucleus as evidenced by the yellow color. The solid arrow in Fig. 8C represents a cell that does not colocalize lamin B (green) with caspase-6 (red), presumably because they are not in the same plane, and consequently have not migrated to a destination where lamin B is cleaved and/or internalized within the nucleus. Figure 8D represents a phase-contrast image of PKC{delta} localization within the nucleus (open arrow) in an IL-1{beta}-treated cell. Together, these data provide the first evidence for significant alterations in lamin B metabolism in IL-1{beta}-treated, insulin-secreting cells.



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Fig. 8. Colocalization of lamin B and caspase-6 after IL-1{beta} treatment. A: lamin (green) is detected by FITC-labeled secondary antibody after IL-1{beta} treatment (600 pM, 48 h). Solid arrow indicates a cell with normal lamin B staining pattern, as in Fig. 7A. Open arrow indicates damaged cell identified by lack of a halo lamin staining pattern and internalized staining pattern within the nucleus. B: caspase-6 (red) is detected by rhodamine labeling in same cells shown in A. Note that solid arrow indicates normal circular staining pattern associated with the nuclear envelope. Open arrow points to a damaged cell in which caspase-6 is more internalized within the nucleus. C: overlay image of lamin B (green) and caspase-6 (red). Open arrow indicates a damaged cell wherein the lamin B and caspase-6 colocalized to form a yellow coloration. Solid arrow points to a normal cell in which little, if any, colocalization of lamin and caspase-6 occurs. Insets 1 and 2: enlarged versions of same images shown in C. D: phase-contrast image in which protein kinase C {delta}-isoform (PKC{delta}) is localized in cells after IL-1{beta} (600 pM, 48 h) treatment. Open arrows indicate a damaged cell, and PKC{delta} is in the nuclear region.

 

    DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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One of the main objectives of this study was to determine the regulatory effects and the signaling steps involved in IL-1{beta}-induced metabolic dysfunction of the insulin-secreting {beta}-cell, specifically at the level of nuclear lamin B metabolism. The salient features of this study are that IL-1{beta} treatment of RIN cells results in 1) a significant increase in the prenylcysteine carboxymethyltransferase activity and subsequent methylation of lamin B in the nuclear fraction; 2) significant alterations in the subnuclear distribution of lamin B; 3) increased activation of caspase-3 and caspase-6, which are known to mediate proteolysis of PKC{delta} and lamin B, respectively; and 4) a significant association of caspase-6 and lamin B, leading to degradation of lamin B. Our data also provide the first evidence to suggest that IL-1{beta}-induced activation of caspases and subsequent degradation of lamin B were markedly reduced by coprovision of cells with L-NMMA, an inhibitor of iNOS gene expression and NO release. These data thus establish a link between signaling steps involving IL-1{beta}-induced iNOS gene expression, NO release, caspase activation, and nuclear lamin B degradation leading to apoptotic demise of the islet {beta}-cell.

It is well established that IL-1{beta}-induced dysfunction and demise of the pancreatic {beta}-cell is attributable primarily to increased expression of iNOS and subsequent generation of NO (2, 15, 16, 34, 45). NO has been shown to play a dual role in cellular function because moderate levels of NO have been shown to be cytoprotective in some cases (12, 18), while higher levels of NO have been shown to be cytotoxic, leading to cell death (4, 42). One of the known functions of NO is to elicit changes in the mitochondrial electron transport system culminating in the release of cytochrome c, which, in turn, activates a family of cysteine proteases known as caspases (20). These proteases are fairly specific and have been shown to cleave several signaling proteins, resulting in modulation of their function. For example, caspase-3 has been shown to hydrolyze PKC{delta}, resulting in the generation of a constitutively active form of the kinase (11). Our current data demonstrate that under conditions of increased NO generation, significant stimulation of caspase-3 and caspase-6 activities is demonstrable in RIN cells after exposure to IL-1{beta}. In this context, it is germane to point out recent studies by Kwon et al. (32), who reported a significant increase in caspase-3 activity after exposure of RINm5F cells with increasing concentrations of the saturated fatty acid, palmitate. Previous studies implicated NO for alteration in the mitochondrial membrane potential properties and further release in cytochrome c and subsequent activation of caspases (20). In support of this, our current data demonstrate a significant activation of caspase-3 and caspase-6 in cells exposed to IL-1{beta}. Our findings also suggest that IL-1{beta}-induced caspase activation is a NO-dependent step because L-NMMA markedly attenuated IL-1{beta}-induced caspase activation.

There are several important targets within the cell that are also affected by NO, such as enzymes, structural and integral membrane proteins, and DNA (13, 22, 36). As a logical extension of our earlier observations demonstrating that lamin B undergoes methylation in the normal rat islets, human islets, and clonal {beta}-cell lines (28), in the present study we examined whether the carboxymethylation status is altered in these cells after exposure to IL-1{beta}. Our data indicate a significant increase in carboxymethyltransferase activity (using AFC as the substrate) as well as methylation of lamin B in the nuclear fraction derived from IL-1{beta}-treated cells. To our knowledge, this study is the first to demonstrate a significant increase in the protein-methylating enzymes in the nuclear fraction. In this context, Hmadcha et al. (24) showed that NO selectively increases the activity of DNA methyltransferase, which is fairly distinct from carboxymethyltransferase and which we quantitated in the present study. Our observations are important because methylesterification of lamin B, an intermediate filament protein, has been shown to be prerequisite for the reassembly of the nuclear envelope after mitosis in mammalian cells (7, 26). This modification of lamin B enhances the hydrophobicity of the protein (presumably via neutralization of the carboxylate anion) and facilitates its movement toward the membrane, which is evident from our Western blot analysis, in which its distribution was found to be in the order of membrane > matrix > cytosol. Interestingly, we noticed that the basal activity of this enzyme is much higher in the soluble fraction than in the membrane fraction and that IL-1{beta} treatment significantly increased the activity of the soluble enzyme compared with the membrane-associated enzyme.

In the present study, we have also demonstrated that caspase-3 and caspase-6 activities are increased significantly after IL-1{beta} treatment. In this context, earlier studies demonstrated that the target of caspase-3 is procaspase-6 as well as PKC{delta} and that that of caspase-6 is lamin B (1, 43). Thomas et al. (52) also demonstrated significant activation of caspase-6 followed by cleavage of lamin during apoptosis of the isolated islet. Our current data also demonstrate a significant increase in lamin B degradation in cells treated with IL-1{beta}. Furthermore, lamin B is associated with the lamina by folding the COOH-terminal domain into globular heads, which is essential for this protein to dimerize and associate within the nuclear lamina (55). Therefore, we examined the subnuclear distribution of lamin B derived from IL-1{beta}-treated cells to further determine the status of these proteins after IL-1{beta} treatment. We observed that IL-1{beta} alters the intranuclear distribution of lamin B because the nuclear membrane-associated protein is reduced significantly, and subsequently the matrix-associated lamin B is elevated considerably. These findings are compatible with the studies of Buendia et al. (6), who demonstrated significant association of lamin B with the nuclear matrix fraction in the human T lymphoblastic cells after exposure to actinomycin D.

Our biochemical and Western blot data correlate well with the confocal microscopic data, which show that control {beta}-cells have lamin B associated with the nuclear envelope (Fig. 7), as evidenced by DAPI staining for nuclear material (Fig. 7). However, after exposure to IL-1{beta}, lamin B becomes dissociated from the nuclear envelope and is internalized into the nucleus. These results are compatible with the observations of Kaufmann et al. (25), who demonstrated degradation and loss of lamins after proteolysis in HL-60 cells treated with anticancer agents. This work was substantiated further by Neamati et al. (40) and Oberhammer et al. (41), who demonstrated that chromatin condensation and DNA fragmentation lead to apoptosis of the cell. Rao et al. (46) showed no measurable DNA degradation in cells expressing genetically modified or "uncleavable" lamin, suggesting that lamin degradation is critical for DNA degradation and chromatin condensation to take place. Our confocal experiments using DAPI staining clearly indicate marked nuclear damage associated with chromatin granularity and condensation in cells after exposure to IL-1{beta}. Recent studies by Krystosek (31) also suggested that the appearance of small vesicles (which are also demonstrable in our confocal microscopic experiments) is indicative of a "pinching off" mechanism by which the separation of chromatin into discrete areas associated with lamin may be influenced by damaged DNA as well as the degraded lamina. Our DNA fragmentation data demonstrate significant damage in DNA from cells treated with IL-1{beta}, and these data correlate well with the MTT assay, in which we observed a significant decrease in viable cells from IL-1{beta}-treated cells.

It is germane to point out recent studies of Eitel et al. (14), who recently reported changes in PKC{delta} and lamin B signaling cascade in insulin-secreting RIN1046-38 cells after exposure to palmitate. These investigators reported translocation of this PKC{delta} from the cytosolic compartment to the nuclear fraction in palmitate-treated cells, followed by disintegration of lamin B from the nuclear lamina. They also observed significant activation of caspase activity under conditions in which PKC translocation and lamin B disintegration were demonstrable. Our current findings are in complete agreement with these data. Moreover, unpublished observations from our laboratory suggest a significant translocation of PKC{delta} to the nuclear fraction in {beta}-cells after exposure to IL-1{beta} (600 pM, 48 h). These findings are all the more important in the context of {beta}-cell apoptosis because PKC{delta} has been shown to phosphorylate lamin B, and phosphorylated lamin B is more vulnerable to degradation by caspases (11, 43). Together, it seems likely that the distal events, specifically at the level of the nucleus, involved in cytokine-mediated and fatty acid-induced {beta}-cell demise are comparable and might involve similar signal transduction mechanisms. Furthermore, IL-1{beta} (and palmitate) treatment places lamin B in a strategic location for caspase-mediated hydrolysis by promoting posttranslational carboxymethylation and phosphorylation of this protein. Additional studies are needed to verify this formulation.

In conclusion, our studies provide the first direct evidence to suggest that IL-1{beta} induces posttranslational carboxymethylation and alterations in subnuclear distribution of lamin B in insulin-secreting RINm5F cells. We have also demonstrated that IL-1{beta}-treatment facilitates the dissociation of lamin B from the nuclear lamina and promotes its association with the DNA-containing material leading to DNA degradation and chromatin condensation culminating in {beta}-cell demise. Our findings also establish a regulatory role for NO in these signaling events. It will be interesting to examine the regulatory roles of IL-1{beta}, if any, in lamin B-demethylating enzymes (i.e., prenylcysteine carboxymethyl esterase) in the {beta}-cell. These roles are being investigated in our laboratory.


    GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
These studies were supported by Department of Veterans Affairs Merit Review and Research Enhancement Awards (to A. Kowluru), National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-56005, and by grants from the American Diabetes Association and the Grodman Foundation. A. Kowluru is the recipient of a Research Career Scientist Award from the Department of Veterans Affairs.


    ACKNOWLEDGMENTS
 
We thank Hai-Qing Chen for assistance with cell cultures and Dr. Marie Tannous for help in caspase activity measurements.

Portions of this work were previously published in abstract form (53a, 53b).


    FOOTNOTES
 

Address for reprint requests and other correspondence: A. Kowluru, Dept. of Pharmaceutical Sciences, Eugene Applebaum College of Pharmacy and Health Sciences, Wayne State Univ., 259 Mack Ave., Detroit, MI 48201 (E-mail: akowluru{at}med.wayne.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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