Department of Pharmaceutical Sciences, Wayne State University, and 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
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ABSTRACT |
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pancreatic -cell; lamin carboxymethyltransferase; nitric oxide; nuclear matrix; caspases
It is well established that long-term exposure of isolated -cells to cytokines (e.g., IL-1
) 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
(IL-1
) 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
-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in mitochondrial membrane permeability properties, leading to the release of cytochrome c (Refs. 19, 51, and 57; unpublished observations from our laboratory).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
-isoform. Extant studies suggest that PKC
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-induced apoptosis of the islet
-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
-cell progressing toward its apoptotic demise after exposure to IL-1
. The present study was undertaken to examine the alterations in the carboxymethyltransferase that regulates the posttranslational carboxymethylation of lamin B in isolated
-cells exposed to cytotoxic levels of IL-1
. 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
. Finally, using NG-monomethyl-L-arginine (L-NMMA), a known inhibitor of iNOS, we examined putative regulatory roles for IL-1
-induced iNOS gene expression and NO release in IL-1
-mediated activation of caspase-3 and caspase-6 and lamin B degradation in the
-cell progressing toward apoptotic demise. Our current findings suggest that exposure of isolated
-cells to IL-1
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
-cell.
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MATERIALS AND METHODS |
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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 (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 (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 (600 pM, 48 h) in the absence or presence of L-NMMA (500 µM) as indicated. Extracted proteins (4060 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 (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 550690 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 (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
(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 (2830), using [3H]SAM as the methyl donor. The release of base-labile methanol from the AFC methyl ester was measured using scintillation spectrometry (2830). 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. 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 (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 (4060 µg protein as indicated) from cells treated with either diluent alone or IL-1 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-treated cells.
RINm5F cells were grown on glass coverslips for 2 days and then incubated with or without IL-1
(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, 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 was visualized in both control and IL-1
-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).
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RESULTS |
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To further examine whether IL-1 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
-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
. A modest but insignificant increase in this activity was also demonstrable in the nuclear membrane fraction isolated from the IL-1
-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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We next verified whether IL-1-induced increase in carboxymethylation of lamin B results in its degradation. To accomplish this, protein lysates from the control and IL-1
-treated cells were subjected to SDS-PAGE, and the relative abundance of lamin B in control and IL-1
-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
-treated cell lysates. Furthermore, a protein in the molecular weight region of 45 kDa was prominent in IL-1
-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
-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 plus L-NMMA were comparable to those of control cells (Fig. 4D). These data imply that IL-1
-induced degradation of lamin B involves the intermediacy of NO, presumably via the activation of
-cell caspases, and this was examined in experiments described below.
IL-1-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
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
, a substrate for caspase-3). It has also been suggested that caspase-3-mediated hydrolysis of PKC
results in its functional activation (1, 43). The data shown in Fig. 5A demonstrate that exposure of RIN cells to IL-1
(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
-mediated activation of each of these caspases, implicating NO in this signaling step. IL-1
-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
-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
-induced caspase activation and subsequent lamin B degradation in isolated
-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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Using confocal microscopy to gain further understanding of the role of IL-1-induced caspase-6 activation (Fig. 5), we examined the effects of IL-1
on colocalization of caspase-6 with lamin B. Figure 8A demonstrates the localization of lamin B (green) within the nucleus of IL-1
-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
localization within the nucleus (open arrow) in an IL-1
-treated cell. Together, these data provide the first evidence for significant alterations in lamin B metabolism in IL-1
-treated, insulin-secreting cells.
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DISCUSSION |
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It is well established that IL-1-induced dysfunction and demise of the pancreatic
-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
, 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
. 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
. Our findings also suggest that IL-1
-induced caspase activation is a NO-dependent step because L-NMMA markedly attenuated IL-1
-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 -cell lines (28), in the present study we examined whether the carboxymethylation status is altered in these cells after exposure to IL-1
. 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
-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
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 treatment. In this context, earlier studies demonstrated that the target of caspase-3 is procaspase-6 as well as PKC
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
. 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
-treated cells to further determine the status of these proteins after IL-1
treatment. We observed that IL-1
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 -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
, 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
. 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
, and these data correlate well with the MTT assay, in which we observed a significant decrease in viable cells from IL-1
-treated cells.
It is germane to point out recent studies of Eitel et al. (14), who recently reported changes in PKC and lamin B signaling cascade in insulin-secreting RIN1046-38 cells after exposure to palmitate. These investigators reported translocation of this PKC
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
to the nuclear fraction in
-cells after exposure to IL-1
(600 pM, 48 h). These findings are all the more important in the context of
-cell apoptosis because PKC
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
-cell demise are comparable and might involve similar signal transduction mechanisms. Furthermore, IL-1
(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 induces posttranslational carboxymethylation and alterations in subnuclear distribution of lamin B in insulin-secreting RINm5F cells. We have also demonstrated that IL-1
-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
-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
, if any, in lamin B-demethylating enzymes (i.e., prenylcysteine carboxymethyl esterase) in the
-cell. These roles are being investigated in our laboratory.
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GRANTS |
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ACKNOWLEDGMENTS |
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Portions of this work were previously published in abstract form (53a, 53b).
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FOOTNOTES |
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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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