Hyperglycemia Activates p53 and p53-Regulated Genes Leading to Myocyte Cell Death

Hyperglycemia Activates p53 and p53-Regulated GenesLeading to Myocyte Cell DeathFabio Fiordaliso,

1,3Annarosa Leri,

1Daniela Cesselli,

2Federica Limana,

1,3Bijan Safai,

2

Bernardo Nadal-Ginard,1

Piero Anversa,1

and Jan Kajstura1

To determine whether enzymatic p53 glycosylationleads to angiotensin II formation followed by p53 phos-phorylation, prolonged activation of the renin-angioten-sin system, and apoptosis, ventricular myocytes wereexposed to levels of glucose mimicking diabetic hyper-glycemia. At a high glucose concentration, O-glycosyla-tion of p53 occurred between 10 and 20 min, reached itspeak at 1 h, and then decreased with time. AngiotensinII synthesis increased at 45 min and 1 h, resulting in p38mitogen-activated protein (MAP) kinase–driven p53phosphorylation at Ser 390. p53 phosphorylation wasabsent at the early time points, becoming evident at 1 h,and increasing progressively from 3 h to 4 days. Phos-phorylated p53 at Ser 18 and activated c-Jun NH2-terminal kinases were identified with hyperglycemia,whereas extracellular signal-regulated kinase was notphosphorylated. Upregulation of p53 was associatedwith an accumulation of angiotensinogen and AT1 andenhanced production of angiotensin II. Bax quantityalso increased. These multiple adaptations paralleledthe concentrations of glucose in the medium and theduration of the culture. Myocyte death by apoptosisdirectly correlated with glucose and angiotensin II lev-els. Inhibition of O-glycosylation prevented the initialsynthesis of angiotensin II, p53, and p38-MAP kinase(MAPK) phosphorylation and apoptosis. AT1 blockadehad no influence on O-glycosylation of p53, but it inter-fered with p53 phosphorylation; losartan also preventedphosphorylation of p38-MAPK by angiotensin II. Inhibi-tion of p38-MAPK mimicked at a more distal level theconsequences of losartan. In conclusion, these in vitroresults support the notion that hyperglycemia withdiabetes promotes myocyte apoptosis mediated by acti-vation of p53 and effector responses involving the localrenin-angiotensin system. Diabetes 50:2363–2375, 2001

Abiochemical event with diabetes is the forma-

tion of glycosylated products (1). Proteins con-stitute the principal substrate of this reaction,which generates glycoproteins in the extracel-

lular compartment, plasma membrane, cytoplasm, and,ultimately, in the nucleus (2,3). The most frequent type ofintracellular glycosylation is O-linked N-acetylglucos-amine (GlcNAc). This post-translational modification con-sists of single GlcNAc residues that are connected to thehydroxyl group of serine or threonine by a transferasecatalyzing the O-glycosylation (3,4). These sites are clus-tered at the COOH-terminal of intracellular proteins, in theproximity of proline or valine residue (5). They are similarto phosphorylation sites for several protein kinases (3,6).Glycosylation and phosphorylation activate several tran-scription factors (7), including p53 (8). The processes ofglycosylation and phosphorylation are tightly and dynam-ically regulated; they affect the activation and stability ofthe p53 protein (8–10). There is direct competition be-tween glucose and phosphate at a single amino acidresidue, resulting in a decrease in the level of phosphory-lation when glycosylation occurs, and vice versa (3,6).Because p53 binding sites are present in the promoter ofangiotensinogen (Aogen) and AT1 receptor genes (11,12),p53 enhances the myocyte renin-angiotensin system (RAS)and the formation of angiotensin II (Ang II). Moreover, p53reduces the expression of genes opposing cell death, suchas Bcl-2, and upregulates genes promoting apoptosis, suchas Bax (13). In this report, the hypothesis was raised thathigh amounts of glucose lead to enzymatic glycosylation ofp53, which activates the myocyte RAS. Synthesis of Ang IIand binding to AT1 receptors may trigger distal eventsupregulating p38 mitogen-activated protein (MAP) kinase(14), which, in turn, may phosphorylate p53 at the COOH-terminal, sustaining its transcriptional activity (15). Addi-tionally, the possibility of phosphorylation of p53 at theNH2-terminal was examined because this site of activationis implicated in several stress-induced cellular responses(16–18). Phosphorylation of c-Jun NH2-terminal kinases(JNK) and extracellular signal-regulated kinases (ERK)was also evaluated to recognize the role of these enzymesin glucose-mediated apoptosis (19–21). The decreasedBcl-2–to–Bax protein ratio in myocytes, induced by en-hanced p53 function, should depress the resistance of cellsto the death signals transmitted by Ang II (11,12,22).Because type 1 diabetes and type 2 diabetes are charac-terized by elevated plasma glucose concentrations (23),

From the 1Department of Medicine, New York Medical College, Valhalla, NewYork; the 2Department of Dermatology, New York Medical College, Valhalla,New York; and the 2Mario Negri Institute of Pharmacological Research, Milan,Italy.

Address correspondence and reprint requests to Jan Kajstura, Departmentof Medicine, New York Medical College, Vosburgh Pavilion, Room 302A,Valhalla, NY 10595. E-mail: [email protected].

Received for publication 1 December 2000 and accepted in revised form18 July 2001.

Aogen, angiotensinogen; Ang II, angiotensin II; ATF-2, activating transcrip-tion factor-2; BAG, benzyl 2-acetamido-2-deoxy-�-D-galactopyranoside; CM,conditioned medium; ELISA, enzyme-linked immunosorbent assay; ERK,extracellular signal-regulated kinase; GlcNAc, N-acetylglucosamine; HRP,horseradish-peroxidase; JNK, c-Jun NH2-terminal kinase; MAP, mitogen-acti-vated protein; MAPK, MAP kinase; PI, propidium iodide; PMSF, phenylmeth-ylsulfonyl fluoride; RAS, renin-angiotensin system; SFM, serum-free medium;TdT, terminal deoxynucleotidyl transferase; TFA, trifluoroacetic acid; TBST,Tris-buffered saline/Tween 20.

DIABETES, VOL. 50, OCTOBER 2001 2363

ventricular myocytes were exposed in vitro to hyperglyce-mia to identify its role in cell death mechanisms.

RESEARCH DESIGN AND METHODS

Cell culture. Left ventricular myocytes were isolated from male Sprague-Dawley rats at 3 months. Under chloral hydrate anesthesia (300 mg/kg bodywt), hearts were excised, and myocytes were dissociated by collagenase(11,24). Trypan-blue–excluding cells constituted 85%. Nonmyocytes ac-counted for 1–2%. Myocytes were cultured in serum-free medium (SFM) andin the absence of insulin. After 30 min, the medium was changed with SFMcontaining 5.5, 12.5, or 25 mmol/l glucose, corresponding to plasma levels of100, 225, and 450 mg/dl, respectively. Hyperosmolarity was corrected bydecreasing salts, and acidosis was corrected by adding 20 mmol/l HEPES.Conversely, hyperosmolarity was mimicked by including 20 mmol/l mannitol.The effect of the AT1 antagonist losartan (10�7 mol/l), O-glycosylation blocker,benzyl 2-acetamido-2-deoxy-�-D-galactopyranoside (BAG, 4 mmol/l; Sigma, St.Louis, MO), and p38-MAP kinase (MAPK) inhibitor (SB 202190, 1 �mol/l;Calbiochem, San Diego, CA) were determined; these compounds were addedto the medium 1 h before glucose. Cultures were studied at 10, 20, and 45 minand at 1, 3, 12, 24, 48, and 96 h. Myocytes were fixed in 1% formaldehyde forcytochemistry, and they were frozen at �80°C for biochemistry (11,12,22,24).p53 glycosylation. Myocytes were suspended in lysis buffer consisting of 50mmol/l Tris-HCl, pH 7.5, 5 mmol/l EDTA, 250 mmol/l NaCl, 0.1% Triton X-100,2 mmol/l phenylmethylsulfonyl fluoride (PMSF), 2 �g/ml aprotinin, 5 mmol/ldithiothreitol, and 1 mmol/l Na3VO4. A volume of 50 �g proteins wereseparated on 12% SDS-PAGE and transferred onto nitrocellulose. Carbohy-drate moieties were oxidized with 10 mmol/l sodium periodate, followed byincorporation of biotin hydrazide. Biotinylated complexes were detected bystreptavidine-alkaline phosphatase and BCIP/NBT color development re-agents (Bio-Rad, Hercules, CA). Protein glycosylation was confirmed byperforming enzymatic deglycosylation with O-glycosidase D and hexosamini-dase, before periodate oxidation (25). Glycosylated p53 was detected byshort-term labeling of adherent myocytes with [35S]methionine followed byimmunoprecipitation of cell lysates with a p53 monoclonal antibody, 3 �g pAb240 (Santa Cruz Biotechnology, Santa Cruz, CA). Glycosylated residues boundto p53 were recognized by immunoprecipitation of proteins with 3 �g pAb 240;Western blot was done with the monoclonal antibody RL2 anti–O-linkedGlcNAc (Alexis, San Diego, CA) (26).p53 phosphorylation. A volume of 50 �g proteins were separated on 12%SDS-PAGE, transferred to nitrocellulose, and exposed to three distinctphosphorylated p53 antibodies with 1 �g/ml in Tris-buffered saline/Tween 20(TBST). Rabbit phosphorylated p53 antibody (New England BioLabs, Beverly,MA) recognizes phosphorylation of p53 at Ser 390 only (15). This site, Ser 390,in rodents corresponds to Ser 392 in humans. This polyclonal antibody isproduced by immunizing rabbits with a synthetic phosphorylated Ser 392peptide corresponding to residues around Ser 392 of human p53. Mouse p53antibody 421 (Calbiochem) is a monoclonal antibody generated by immuniz-ing mice with p53 protein. The epitope against which the antibody is raised islocated at the COOH terminus of p53, from amino acids 372 to 381 (27,28).This region overlaps with the phosphorylation site of protein kinase C (PKC)in the regulatory domain of p53. Thus, antibody 421 does not react with p53after phosphorylation by PKC (9). Rabbit phosphorylated p53 Ser 15 antibody(Ab-3; Oncogene, Boston, MA) is generated by immunizing rabbits with asynthetic phosphorylated Ser 15 peptide corresponding to residues surround-ing Ser 15 of human p53. Human Ser 15 corresponds to Ser 18 in rodents.Phosphorylated p53 Ser 15 antibody detects p53 only when phosphorylated atSer 15 and does not react with nonphosphorylated p53 (17). Bound antibodieswere detected by horseradish peroxidase (HRP)-conjugated anti-mouse oranti-rabbit IgG. Phosphorylated p53 and nonactivated p53 were detected as53-kDa bands.Phosphorylation of p38-MAPK, JNK, and ERK. Myocyte lysates (80 �g),were exposed to rabbit phosphorylated p38-MAPK antibody (New EnglandBioLabs) or anti-phosphorylated MAPK and anti-phosphorylated JNK poly-clonal antibodies (Promega, Madison, WI) to identify the active forms ofp38-MAPK, ERK1 and -2, and JNK1 and -2, respectively. Phosphorylatedp38-MAPK was seen as a band at 38 kDa. Active ERK1 and -2 were notdetected, whereas active JNK1 and -2 were recognized as two close bands atnearly 50 kDa. To evaluate the enzymatic activity of p38-MAPK, proteinlysates, 60 �g, were exposed to immobilized phosphorylated p38-MAPKantibody. Pellets were resuspended in kinase buffer containing 200 �mol/lATP and 2 �g activating transcription factor-2 (ATF-2) and were kept at 30°Cfor 30 min. Samples were separated on 12% SDS-PAGE, transferred ontonitrocellulose, and exposed to phosphorylated ATF-2 antibody (15).p53 DNA binding activity. To prepare a double-stranded probe for Bax,oligonucleotides 5�-AGCTTGCTCACAAGTTAGAGACAAGCCTGGGCGTGGC

TATATTGA-3� and 5�-AGCTTCA-ATATAGCCCACGCCCAGGCTTGTCTCTAACTTGTGAGCA-3�, were annealed and labeled with [�-32P]ATP and T4 poly-nucleotide kinase. Oligonucleotides 5�-GCTGAGCTTGGATCTGG-AAGGCGACACTGGG-3� and 5�-CCCAGTGTCGCCTTCCAGATCCAAGCTCAGC-3� wereused to prepare a AT1 probe. Oligonucleotides 5�-AGCCTCTGTACAGAGTAGCCTGGGAATA-GATCCATCTTC-3� and 5�-GAAGATGGATCTATTCCCAGGCTACTCTGTACAGAGGCT-3� were used for Aogen probe (11,22). Nuclearextracts were incubated with [32P]-labeled probes and were separated on 4%PAGE. For specificity, nuclear extracts were exposed to a p53 antibody, 0.5 �gpAb 240 (Santa Cruz Biotechnology), or to an irrelevant antibody, mousemonoclonal anti-human Mdm2 (SMP14; Santa Cruz Biotechnology), and sub-ject to competition with an excess of unlabeled self-oligonucleotide (11,22).Western blot. Proteins (50 �g) were separated on SDS-PAGE, transferredonto nitrocellulose, and exposed to mouse monoclonal p53 (pAb 240; SantaCruz Biotechnology), rabbit polyclonal Bcl-2 (�C21; Santa Cruz Biotechnolo-gy), rabbit polyclonal Bax (P19; Santa Cruz Biotechnology), mouse monoclo-nal Aogen (Swant, Bellinzona, Switzerland), rabbit polyclonal AT1 (306; SantaCruz Biotechnology), goat polyclonal AT2 (C-18; Santa Cruz Biotechnology)antibodies, and 1 �g/ml TBST. Bound antibodies were detected by HRP-conjugated anti-mouse, anti-rabbit, or anti-goat IgG. p53 was detected as a53-kDa band, Bax as a 19-kDa band, Bcl-2 as a 25-kDa band, Aogen as a 56- to58-kDa band, AT1 as a 41-kDa band, and AT2 as a 42- to 44-kDa band (11,12,22).Ang II. Ang II concentration in conditioned medium (CM) was measured byenzyme-linked immunosorbent assay (ELISA) (Peninsula, Belmont, CA). CMwas treated with 10% trifluoroacetic acid (TFA) and centrifuged. The super-natant was dried, and the residue was dissolved in 0.1% TFA and purified inC-18 Sep-Pak column. Ang II fraction was eluted with 30% acetonitrile in 5 mlof 0.1% TFA, dried, and dissolved in TBST. Samples (50 �l) were analyzed ina microtiter plate using Ang II antibody (1:32,000 dilution) and a tracer,biotinylated Ang II. Color reaction was developed with tetrametyl-benzidine.Absorbance was recorded at 450 nm, and the concentration was calculatedfrom standard curves (12,22).Terminal deoxynucleotidyl transferase assay. Cells were incubated with50 �l cacodylate buffer containing 5 units terminal deoxynucleotidyl trans-ferase (TdT) and biotinylated dUTP. dUTP was detected by fluroesceinisothiocyanate–avidin. Cytoplasm was stained with �-sarcomeric actin andnuclei with propidium iodide (PI). In each condition, 2000–3000 cells wereexamined by confocal microscopy (11,29).In situ ligation with hairpin probe. An oligonucleotide probe with single-base 3� overhang (11,22,29) was used to detect double-strand DNA breakswith single-base 3� overhangs. Cells were incubated in 10 mmol/l sodiumcitrate and were treated with a solution containing 1 unit/�l T4 ligase and 35ng/�l hairpin probe (Synthetic Genetics, San Diego, CA).Data analysis. Results are the means � SD. The significance between twomeasurements was determined by Student’s t test and by multiple compari-sons by ANOVA and Bonferroni method. P values of �0.05 were consideredsignificant. Unless indicated in the text or legends, n equaled five separate cellisolations in each case.

RESULTS

p53 glycosylation. To evaluate whether p53 becameglycosylated, myocytes were cultured in SFM at 25 mmol/lglucose; 5.5 mmol/l glucose was used as a control. Withhigh glucose, osmolarity was adjusted to 290 mOsm.Glycoprotein detection by periodate oxidation showedglycosylation of a 55.6-kDa protein (Fig. 1A and B). Theincrease in protein glycosylation seen at 10 min with highglucose was not statistically significant. However, a three-fold increase in optical density at 20 min (0.50 � 0.20 vs.1.7 � 0.5, P � 0.001) and a sixfold increase at 45 min(0.42 � 0.20 vs. 2.6 � 0.8, P � 0.001) was demonstrated;glycosylation peaked sevenfold (0.44 � 0.12 vs. 3.1 � 0.7,P � 0.001) at 1 h and remained elevated fourfold (0.56 �0.29 vs. 2.2 � 0.5, P � 0.001) at 3 h. A progressive decreasein glycosylation of the 55.6-kDa protein was noted at 12,24, 48, and 96 h. Enzymatic deglycosylation before perio-date oxidation prevented the appearance of the 55.6-kDaglycoprotein (Fig. 1A and B).

In the presence of high glucose, losartan did not inter-fere with protein glycosylation at 45 min and 1 h (Fig. 1C).Conversely, inhibition of O-glycosylation by BAG mark-

HYPERGLYCEMIA, p53, AND MYOCYTE APOPTOSIS

2364 DIABETES, VOL. 50, OCTOBER 2001

edly attenuated the 55.6-kDa protein (Fig. 1D). The 55.6kDa glycoprotein was identified as p53 by radiolabelingmyocytes exposed to 5.5 and 25 mmol/l glucose with[35S]methionine for 1, 2, and 3 h. Myocyte lysates werethen immunoprecipitated with pAb 240 anti-p53 (Fig. 1E).At high glucose, two proteins at 55.6 and 53 kDa weredetected. The lower–molecular weight band correspondedto p53, and the higher molecular weight band reflected theaddition of carbohydrates to the p53 protein.

To identify the sugar moiety bound to p53, myocytelysates obtained from cells cultured in the presence of 5.5and 25 mmol/l glucose were immunoprecipitated with pAb240 anti-p53. Immunoprecipitated proteins were subjectedto Western blot with the monoclonal antibody RL2, whichrecognized O-GlcNAc linked to p53 (Fig. 1F). The O-glycosylated form of p53 was apparent at 55.6 kDa. O-GlcNAc-p53 was detected in myocytes exposed to highglucose for 20 min (0.8 � 0.2, n 5). O-GlcNAc-p53increased at 45 min (2.2 � 0.3, n 5; P � 0.001), peakingat 1 h (4 � 0.57, n 5; P � 0.001). The level ofO-GlcNAc-p53 remained stable at 3 h (3.5 � 0.7, n 5; P �0.001) and decreased progressively at 12 h (1.6 � 0.4, n 5; P � 0.001) and 24 h (1.2 � 0.3, n 5; P � 0.001).p53 glycosylation and Ang II formation. To establishwhether O-glycosylation of p53 upregulated the myocyteRAS, Ang II concentration, expressed as picograms perhour per 106 cells, was measured by ELISA in CM collected

from myocytes cultured at 5.5 and 25 mmol/l glucoseunder isosmotic conditions. In comparison with 5.5 mmol/lglucose, Ang II formation at 25 mmol/l glucose did notincrease at 10 and 20 min (values were combined), butincreased 1.4-fold at 45 min, 1.6-fold at 1 and 3 h (valueswere combined), and 1.8-fold at 12 h (Fig. 2). Inhibition ofO-glycosylation by BAG prevented the increase in Ang II ateach time point.p53 phosphorylation at the COOH-terminus. The nextquestion concerned whether p53 glycosylation–mediatedAng II formation resulted in p53 phosphorylation. Phos-phorylation of p53 at the COOH-terminal is implicated inthe upregulation of the myocyte RAS and the proapoptoticprotein Bax as well as downregulation of the antiapopto-tic protein Bcl-2 (10 –13,22). This may initiate cell death(11,22). For this purpose, cell lysates, obtained from myo-cytes cultured at 5.5 and 25 mmol/l glucose, were exposedto p53 antibodies recognizing, respectively, the site wherephosphorylation by PKC at Ser 376 occurred and the sitewhere phosphorylation by p38-MAPK at Ser 390 occurred.p53 phosphorylation at Ser 390 was undetectable at 5.5mmol/l glucose, nor was it detectable at 25 mmol/l glucoseafter 10, 20, and 45 min (Fig. 3A and B). With high glucose,p53 phosphorylation was clearly visible at 1 h (2.4 � 0.7,n 5). A progressive increase in phosphorylated p53 atSer 390 was noted at 3 h (3.6 � 0.9, n 5; P � 0.05), 12 h(4.5 � 1.1, n 5; P � 0.01), 24 h (6.4 � 1.8, n 5; P �

FIG. 1. Glycosylation and metabolic labeling of p53. A and B: At high glucose (Glu), glycoprotein detection by periodate oxidation showsglycosylation of a 55.6-kDa protein that peaks at 1 h and decreases thereafter. Enzymatic deglycosylation (Degly) prevents the appearance of the55.6-kDa band. C and D: Losartan (Los) does not interfere with glycosylation of the 55.6-kDa protein, whereas BAG markedly attenuates thisenzymatic reaction. E: Metabolic labeling at high glucose leads to the identification of nonglycosylated p53 (53 kDa) and glycosylated p53 (55.6kDa). At low glucose, only one band corresponding to nonglycosylated p53 (53 kDa) is detected. F: The sugar moiety bound to p53 is shown byimmunoprecipitation of myocyte proteins with anti-p53, followed by Western blot with RL2 antibody, which recognizes O-GlcNAc.

F. FIORDALISO AND ASSOCIATES


0.005), 48 h (11 � 3, n 5; P � 0.001), and 96 h (15 � 4,n 5; P � 0.001). Losartan and BAG prevented phosphor-ylation (Fig. 3C and D).

At 25 mmol/l glucose, there was an increase with time,from 1 h to 4 days, in the level of nonactivated p53 at Ser376 that corresponds to the site of p53 activation by PKC(Fig. 3E). The increase in the latent form of p53 with highglucose followed the decrease in p53 glycosylation, be-cause pAb 421 anti-p53 does not recognize glycosylatedp53. Glycosylated p53 peaked at 1 h, when nonactivatedp53 at Ser 376 was not detectable. As expected, theamount of nonactivated p53 at 5.5 mmol/l glucose wassimilar to that at 25 mmol/l, reflecting the lack of p53phosphorylation at Ser 376 with low and high glucoseconcentrations. Losartan did not modify the level of non-activated p53 at Ser 376 (Fig. 3F), indicating that translo-cation of PKC by Ang II was not involved in p53phosphorylation. Optical density values for nonactivatedp53 are not shown.p53 phosphorylation at the NH2-terminus. Phosphor-ylated p53 at Ser 18 represents an additional site ofactivation of the tumor suppressor in response to DNAdamage (16–18). Thus, phosphorylated p53 at Ser 18 wasevaluated. Phosphorylated p53 was not detectable at 5.5mmol/l glucose. At 25 mmol/l glucose, phosphorylated p53at Ser 18 was not apparent at 45 min, but its quantityincreased progressively at 1 h (0.11 � 0.02, n 5), 3 h(0.37 � 0.09, n 5; P � 0.05), 12 h (0.52 � 0.11, n 5; P �0.001), 24 h (0.69 � 0.12, n 5; P � 0.001), 48 h (0.91 �0.13, n 5; P � 0.001), and 96 h (1.36 � 0.25, n 5; P �0.001) (Fig. 3G).Phosphorylated p53 at Ser 390 and Ang II formation.

Phosphorylation of p53 at the COOH-terminal and at Ser390 is mediated by the activation of p38-MAPK (15). Tostrengthen the notion that p53 phosphorylation by thisMAPK followed p53 glycosylation–induced Ang II forma-tion, myocytes were cultured at 5.5 and 25 mmol/l glucosein the presence of an inhibitor of p38-MAPK (SB 202190)or an AT1 antagonist (losartan). AT1 blockade preventsactivation of p38-MAPK by Ang II. Ang II concentration

was then measured under two conditions of suppressedp38-MAPK. Values at 10 and 20 min and at 1 and 3 h werecomparable and, thus, combined. When phosphorylationwas blocked, the production of Ang II was not decreasedfor up to 3 h but was inhibited at 12 h (Fig. 3H). At 12 h,p53 glycosylation was significantly decreased (Fig. 1A–C),whereas p53 phosphorylation at Ser 390 was upregulated(Fig. 3A–C). Losartan produced effects similar to SB 202190.The attenuated synthesis of Ang II at 12 h by SB 202190and losartan documented that p53 phosphorylation was asecondary event triggered by p53 glycosylation.p38-MAPK activity. To confirm that p38-MAPK wasstimulated by Ang II and was responsible for p53 phos-phorylation at Ser 390, phosphorylated p38 was measuredby Western blot in myocytes cultured at 5.5 and 25 mmol/lglucose in isosmotic media (Fig. 4A and B). Phosphory-lated p38 was undetectable at all time points with 5.5mmol/l glucose; this was also the case at 25 mmol/l glucoseafter 10, 20, and 45 min. At 25 mmol/l glucose, phosphor-ylated p38 became apparent at 1 h (0.5 � 0.2, n 5) and3 h (0.7 � 0.3, n 5). Phosphorylated p38 progressivelyincreased at 12 h (1.0 � 0.3, n 5; P � 0.005), 24 h (1.5 �0.5, n 5; P � 0.003), 48 h (2.1 � 0.7, n 5; P � 0.002),and 96 h (2.8 � 0.9, n 5; P � 0.001). p38-MAPKenzymatic activity was also assessed by measuring ATF-2phosphorylation. ATF-2 is a physiological substrate ofp38-MAPK. Phosphorylated ATF-2 was not visible in myo-cytes at 5.5 mmol/l glucose. However, in cells treated with25 mmol/l glucose, activated ATF-2 increased from 1 to96 h (Fig. 4C; optical density values not shown). Earlierintervals were not examined because phosphorylated p38was not present. We thus established that activation ofp38-MAPK was caused by Ang II synthesis induced by p53glycosylation. Western blots were performed with myo-cytes treated with the inhibitor of O-glycosylation, BAG,1 h before the exposure of the cells to low and highglucose concentrations. The active form of the enzymewas undetectable in these samples (Fig. 4D).ERK and JNK kinases. To complete the analysis of theeffects of glucose on the MAPK family of proteins, theactive forms of ERK and JNK were examined. Phosphor-ylated ERK1 and -2 levels were not detected in myocytescultured in the presence of low and high glucose (data notshown). In contrast, the active forms of JNK1 and JNK2were recognized at high glucose (Fig. 4E). This wasapparent at 20 min (0.61 � 0.08, n 5) and consistentlyincreased at 1 h (1.1 � 0.11, n 5; P � 0.05), 3 h (1.27 �0.22, n 5; P � 0.01), 12 h (2.38 � 0.31, n 5; P � 0.001),24 h (2.67 � 0.34, n 5; P � 0.001), and 48 h (2.9 � 0.4,n 5; P � 0.001). Phosphorylated JNK was not detectedat 5.5 mmol/l glucose.p53 DNA binding. To document that p53 phosphorylationenhanced p53 function, gel mobility assays were per-formed. In comparison with myocytes at 5.5 mmol/l glu-cose, the optical density of the p53-shifted band for theAogen promoter increased 5-fold (P � 0.001) and 12-fold(P � 0.001) in cells exposed to 25 mmol/l glucose for 1 day(0.42 � 0.13 vs. 2.2 � 0.6) and 2 days (0.45 � 0.21 vs. 5.3 �1.9), respectively (Fig. 5A). Osmolarity did not affect p53binding. Controls included preincubation of nuclear ex-tracts with a p53 antibody or with an excess of unlabeledself-oligonucleotide; both conditions prevented the forma-

FIG. 2. Ang II generation. Ang II levels in CM of myocytes exposed tolow and high glucose are shown at 10–20 min, 45 min, 1–3 h, and 12 h.BAG prevents the increase in Ang II formation at 25 mmol/l glucose atall time points. Results are the means � SD. *P < 0.05 vs. 5.5 mmol/lglucose and vs. 5.5 and 25 mmol/l glucose at 10–20 min; †P < 0.05 vs. 25mmol/l glucose at the same time point. Values of n vary from 7 to 17.



tion of a p53-shifted complex. Conversely, the addition ofan irrelevant antibody did not interfere with the p53 band.

With respect to cells at 5.5 mmol/l glucose, myocytescultured at 25 mmol/l glucose showed a fourfold (P �0.001) and an eightfold (P � 0.001) increase in the p53-shifted complex for the AT1 promoter at one (0.13 � 0.05vs. 0.53 � 0.18) and two (0.18 � 0.10 vs. 1.4 � 0.33) d,respectively (Fig. 5B). Similarly, high glucose increasedp53 DNA binding to the Bax promoter fourfold (P � 0.001)and ninefold (P � 0.001) at one (1.1 � 0.4 vs. 4.2 � 1.2) andtwo (1.3 � 0.9 vs. 12 � 4) d (Fig. 5C). Consistency inprotein loading, lack of protein degradation, and unifor-mity in the relative purity of nuclear extracts were evaluat-ed by SDS-PAGE and Coomassie blue staining (not shown).Western blot. The expression of p53, Bax, Bcl-2, Aogen,AT1, and AT2 receptors was evaluated in myocytes ex-posed to 5.5, 12.5, and 25 mmol/l glucose for 4 days. Wehave previously shown that conditions characterizedby hyperglycemia-induced Ang II formation (30 –32), orenhanced Ang II synthesis alone, independent from hy-

perglycemia (11,12,22), led to upregulation of p53 andp53-associated genes. Currently, p53 protein increased inproportion to glucose concentration (0.23 � 0.07, 1.0 �0.22, and 2.2 � 0.6 for 5.5, 12.5, and 25 mmol/l, respec-tively, n 6 in all cases; P � 0.05–0.001 vs. 5.5 mmol/l)and losartan, at 10�7 mol/l, completely abolished the effectof hyperglycemia on p53 quantity (data not shown). p53activation was paralleled by comparable increases in Bax(2.3 � 0.5, 7.9 � 1.7, and 13 � 2.3 for 5.5, 12.5, and 25mmol/l, respectively, n 6 in all cases; P � 0.001 vs. 5.5mmol/l), whereas an inverse correlation was observedbetween glucose and Bcl-2 expression (9.3 � 1.6, 6.6 � 1.0,and 3.7 � 0.7 for 5.5, 12.5, and 25 mmol/l, respectively, n 6 in all cases; P � 0.005–0.001 vs. 5.5 mmol/l); losartanprevented the changes in Bax and Bcl-2. Aogen (0.73 �0.18, 2.4 � 0.4, and 3.8 � 0.7 for 5.5, 12.5, and 25 mmol/l,respectively, n 6 in all cases; P � 0.001 vs. 5.5 mmol/l)and AT1 receptors (1.7 � 0.3, 4.0 � 0.8, and 6.0 � 0.7 for5.5, 12.5, and 25 mmol/l, respectively, n 6 in all cases;P � 0.001 vs. 5.5 mmol/l) mimicked the adaptations of p53,

FIG. 3. p53 phosphorylation and Ang II generation. A and B: At high glucose (Glu), phosphorylation of p53 at Ser 390 is apparent at 1 h andincreases progressively thereafter. C and D: Losartan (Los) and BAG prevent p53 phosphorylation at Ser 390. E and F: p53 Detected withantibody 421 corresponds to nonactivated p53 at Ser 376. G: At high glucose, phosphorylation of p53 at Ser 18 is apparent at 1 h and increasesprogressively with time in culture. H: Ang II levels in CM of myocytes exposed to low and high glucose are shown at 10–20 min, 45 min, 1–3 h,and 12 h. SB 202190 and Los prevent the increase in Ang II formation only at 12 h. Results are the means � SD. *P < 0.05 vs. 5.5 mmol/l glucoseand vs. 5.5 and 25 mmol/l glucose at 10–20 min; †P < 0.05 vs. 25 mmol/l glucose at the same time point. Values of n vary from 7 to 16.



showing higher levels with higher glucose concentrations.As expected, AT2 receptors were not modified by glucose(3.7 � 0.5, 4.0 � 0.7, and 3.6 � 0.5 for 5.5, 12.5, and 25mmol/l, respectively, n 6 in all cases; NS) or losartan.Hyperosmolarity did not significantly change any of thelisted values.Ang II. To demonstrate that enhanced p53 function andupregulation of Aogen with hyperglycemia resulted in along-term increase in Ang II synthesis, Ang II was mea-sured in CM after exposure of myocytes to 5.5, 12.5, and 25

FIG. 4. p38-MAPK and JNK activation. A and B: At 25 mmol/l glucose(Glu), p38-MAPK is phosphorylated at threonine 180 and tyrosine 182(phospho-p38 MAPK) at 1 h, and its expression increases progressivelywith time. Anisomycin-treated NIH-3T3 cells are used as positivecontrol (�), whereas untreated NIH-3T3 cells are used as negativecontrol (�). C: The enzymatic activity of phosphorylated p38-MAPK isassessed by the phosphorylation of ATF-2 at threonine 71 (phosphor-ylated ATF-2). D: BAG prevents phosphorylation of p38-MAPK at 25mmol/l glucose. E: The active forms of JNK1 and -2 are detectable withhigh glucose at 20 min and consistently increase with time. Phosphor-ylated JNK is not seen at 5.5 mmol/l glucose.

FIG. 5. p53 DNA binding activity. A, B, and C: At 25 mmol/l glucose(Glu), p53 phosphorylation enhances p53 binding to the promoter forAogen, AT1, and Bax. Osmolarity has no effect on p53 binding. I,isosmotic medium; H, hyperosmotic medium. Preincubation of nuclearextracts with a p53 antibody (Ab) or with an excess of unlabeled selfoligonucleotide (Co) prevents the formation of a p53-shifted complex.Conversely, the addition of an irrelevant antibody (Irr) does notinterfere with the p53 band. Ao, AT1, and Bax: angiotensinogen, AT1,and Bax probes in the absence of nuclear extracts. Arrows point to thep53-shifted complex.



mmol/l glucose for 1, 2, and 4 days (Fig. 6). Osmolarity didnot affect Ang II levels, and values were combined. Incomparison with 5.5 mmol/l glucose, 25 mmol/l glucoseincreased Ang II formation 1.9-fold at 1 day. Additionally,Ang II generation increased 1.9- and 2.5-fold at 2 days and2.6- and 3.9-fold at 4 days with 12.5 and 25 mmol/l glucose,respectively.Myocyte apoptosis. This form of cell death is character-ized first by nuclear damage, which is frequently not de-tectable morphologically. However, double DNA strandbreaks can be identified histochemically. This initial phaseof the apoptotic process is followed by nuclear fragmenta-tion and later by cytoplasmic alterations (33). To determinewhether Ang II activated cell death, myocyte apoptosis wasmeasured. Figure 7A–I illustrates by confocal microscopythree myocytes undergoing apoptosis. Nuclear morphol-ogy was preserved in Fig. 7A–C, whereas nuclear fragmen-tation was apparent in Fig. 7D–I. Cell striation was evidentin the first example. Loss of cytoplasmic structure and cellshrinkage were seen in the other two cases.

Apoptosis was measured in myocytes cultured at 5.5,12.5, and 25 mmol/l glucose for periods of 1, 2, and 4 days.Cell death increased with time and glucose concentration(Fig. 7J). Hyperosmolarity of the medium did not alter thedegree of apoptosis at any time point or glucose concen-tration, including 4 days and 25 mmol/l glucose. Valuescollected under isosmotic and hyperosmotic conditionswere combined. The lack of impact of increased osmolar-ity on apoptosis was confirmed by measuring cell death at5.5 mmol/l glucose in the presence of 20 mmol/l mannitolfor 4 days. Osmolarity increased to a value comparablewith 25 mmol/l glucose, but apoptosis was essentiallyidentical to that found at 5.5 mmol/l glucose, with 2.8 � 1.2and 3.6 � 1.7% for control and mannitol, respectively (n 6; P 0.6). At 4 days of hyperglycemia, apoptosis com-prised nearly 3, 8, and 15% of myocytes at 5.5, 12.5, and 25mmol/l glucose, respectively. Moreover, to evaluate the im-pact of BAG on myocyte apoptosis, 4 mmol/l of this inhib-itor of O-glycosylation was added to myocytes cultured at25 mmol/l glucose in isosmotic medium. Myocyte apopto-sis was measured 4 days later, with 2.5 � 1.1% for 5.5mmol/l glucose (n 6), 13 � 4% for 25 mmol/l glucose and

no BAG (n 6; P � 0.001), and 3.5 � 1.7% for 25 mmol/lglucose plus BAG (n 6; P 0.7). Thus, hyperosmolarityhad no effect on the magnitude of myocyte apoptosis, andBAG blocked high glucose–mediated cell death.Apoptosis, losartan, and pH. To examine the effects ofAT1 blockade on cell death, losartan was added 1 h beforeglucose and maintained for up to 4 days. Losartan attenu-ated apoptosis by nearly 60% in all conditions but failedto decrease cell death to baseline (Fig. 7K). These valuesof apoptosis were obtained by TdT. Enhanced uptake ofglucose by myocytes leads to the generation of lactic acid,thereby decreasing extracellular pH (34). The effects ofglucose concentration and time on the pH of the mediumare shown in Fig. 7L. Essentially, 5.5 mmol/l glucose didnot decrease pH during the 4-day period. However, 25mmol/l glucose reduced pH throughout the duration of theculture. To prevent the development of acidosis, 20 mmol/lHEPES were added to the medium (Fig. 7L). The influenceof pH on hyperglycemia-induced apoptosis was deter-mined. The impact of losartan on cell death was alsore-examined under low and physiological pH. TdT detectsdouble-strand DNA cleavage induced by Ca2-dependentDNase I and pH-dependent DNase II (11,29), but it cannotseparate the effects of these two DNases. Conversely, thehairpin probe identifies double-strand DNA cleavage witha single-base 3� overhang, which is the form of apoptosismediated by Ang II exclusively via the stimulation ofCa2-dependent DNase I (11,29).

Figure 7M illustrates the extent of myocyte apoptosis incell cultures at 5.5 and 25 mmol/l glucose in the presenceand absence of losartan for 4 days. These experimentswere performed in isosmotic media. At 25 mmol/l glucoseand pH 6.9, myocyte apoptosis measured by the hairpinprobe was significantly lower than that assessed by TdT.As expected, losartan did not abolish cell death in thepresence of acidosis, resulting in a significant TdT labelingof myocyte nuclei. In contrast, at 25 mmol/l glucose andpH 7.4, apoptosis evaluated by hairpin probe was almostidentical to that determined by TdT. As expected, losartanreduced cell death to control values when measured byeither TdT or hairpin probe. Thus, losartan completelyinhibited myocyte apoptosis triggered by Ang II formationwith hyperglycemia.Acidosis, phosphorylated p53 at Ser 390, and phos-

phorylated p38 MAPK. In view of the presence of acido-sis with hyperglycemia, we evaluated the potential effectsof low pH on the main pathways implicated in the activa-tion of p53 and p53-mediated apoptosis in this modelsystem. The levels of phosphorylated p53 at Ser 390 andphosphorylated p38 MAPK were not affected by pH inmyocytes cultured for 2 and 4 days at 25 mmol/l glucose.The presence or absence of HEPES did not change the ac-tivity of these two proteins (Fig. 8A and B). These obser-vations confirmed that the biochemical results obtainedwith high glucose concentration were not influenced by pH.

DISCUSSION

The findings of the present study indicate that high glucoseconcentrations led to O-glycosylation of p53 and synthesisof Ang II in adult cardiac myocytes. Binding to AT1 recep-tors promoted the activation of p38-MAPK, which in turninduced p53 phosphorylation at Ser 390 in the COOH ter-

FIG. 6. Ang II generation. Ang II levels in CM of myocytes exposed tolow and high glucose are shown at 1, 2, and 4 days. Ang II formationincreased at 25 mmol/l glucose at all time points. Results are means �SD. *P < 0.05 vs. 5.5 mmol/l; †P < 0.05 vs. 12.5 mmol/l; ‡P < 0.05 vs. 1day; §P < 0.05 vs. 2 days. Values of n vary from 6 to 15.



minus. This post-translational modification sustained p53function and the upregulation of p53-dependent and -reg-ulated genes. p53 stability and transcriptional activitywere also influenced by its phosphorylation at Ser 18 in the

NH2 terminus. Conversely, hyperglycemia induced a dualphosphorylation of JNK at Thr 183 and Tyr 185 (19,21),most likely facilitating the degradation of p53 (35,36).However, these multiple responses enhanced p53 activity,

FIG. 7.



increasing the susceptibility of myocytes to undergo AngII–mediated apoptosis. The extent of myocyte death di-rectly correlated with glucose and Ang II levels. Withhyperglycemia, intracellular acidosis became apparent,and this metabolic defect accounted for nearly 40% of the

myocyte death. The AT1 blocker losartan prevented theeffects of Ang II on cell death but had no influence onapoptosis triggered by acidosis. Importantly, inhibition ofO-glycosylation counteracted this cascade of events byinterfering with the initiation of p53 function and the

FIG. 7. Glucose, losartan, pH, and myocyte apoptosis. A– I: Confocal images of different aspects of apoptosis in myocytes cultured at 25 mmol/lglucose under isosmotic conditions for 1 day (A–C: �1,200) and 4 days (D–I: �1,200). In the upper panels (A, D, and G), blue fluorescencecorresponds to PI labeling of nuclei. In the central panels (B, E, and H), green fluorescence reflects apoptotic nuclei detected by hairpin one (B

and E) and TdT (H). In the lower panels (C, F, and I), bright fluorescence corresponds to the combination of PI and hairpin 1 (C and F) and PIand TdT (I) staining of apoptotic nuclei. In the lower panels (C, F, and I), red fluorescence shows �-sarcomeric actin–labeling of myocytecytoplasm. J: Hyperglycemia and apoptosis. M: Hyperglycemia, losartan, and apoptosis. Bar graphs show the means � SD. Values of n vary from13 to 16. The level of apoptosis increases (J) with glucose concentrations and time in culture. *P < 0.05 vs. 5.5 mmol/l; †P < 0.05 vs. 12.5 mmol/l;‡P < 0.05 vs. 1 day; §P < 0.05 vs. 2 days. K: Losartan attenuates apoptosis in all conditions. *P < 0.05 vs. 5.5, 12.5, and 25 mmol/l in the absenceof Los. L: pH decreases with high glucose and time in culture. The addition of HEPES corrects acidosis restoring physiological pH. *P < 0.05 vs.12 h; †P < 0.05 vs. 1 day; ‡P < 0.05 vs. 3 days; §P < 0.05 vs. 4 days. M: Hyperglycemia, pH, losartan, and apoptosis. Apoptosis induced by bothacidosis and hyperglycemia is attenuated only in part by losartan. At physiological pH (pH 7.4), losartan completely inhibits cell death promotedby high glucose. *P < 0.05 vs. TdT alone (�).



FIG. 7—Continued.



generation of Ang II. Thus, hyperglycemia upregulated themyocyte RAS, which is implicated in the development of adiabetic cardiomyopathy in vivo (30–32).Hyperglycemia and p53 activation. Glycosylation ofproteins with hyperglycemia in vivo and in vitro occursthrough the addition of carbohydrates, without involvingan enzymatic reaction (1,2). This process is commonlydefined as glycation or nonenzymatic glycosylation. How-ever, the formation of glycation products requires timeand is not evident before cells are kept in high glucose–containing media for at least 1 week (37). The rate ofglycation is so slow that rapidly turning-over intracellularproteins would not be present long enough to undergononenzymatic glycosylation (38). This is relevant to p53,which has a half-life of 20 min (10). The enzymaticO-glycosylation of p53 occurred between 10 and 45 minand progressively decreased after 3 h. The addition ofO-GlcNAc to the COOH-terminal of p53 was replaced byphosphorylation, which became apparent only after 1 h ofhyperglycemia. Phosphorylated p53 increased consistentlyfrom 3 h to 4 days. Inhibition of O-glycosylation preventedp53 phosphorylation at Ser 390, p38 MAPK activation, AngII synthesis, and apoptosis. Thus, O-glycosylation andphosphorylation were sequentially implicated in the mod-ulation of p53 function and transactivation of p53-depen-dent and -regulated genes in myocytes. Hyperglycemia-mediated O-glycosylation of p53 has never been shownbefore. Similarly, enzymatic glycosylation of p53 has neverbeen documented in normal cells. The only example wasobtained in a carcinoma-derived cell line in which wild-type glycosylated p53 is constitutively expressed and isbiologically active (8). Glycosylated p53 promotes tran-scription of target genes and induces apoptosis.

Glycosylation and phosphorylation activate several tran-scription factors (7,8), �20 of which, including SP1, AP-1,SRF, and p53, are known to be O-glycosylated (8). Thebasic region at the COOH-terminus of p53 represses DNAbinding, maintaining p53 in its latent form (9). This inhi-bition can be relieved by various mechanisms: introduc-tion of bulky groups, including sugars like GlcNAc (9), orneutralization of charge through phosphorylation by PKC,casein kinase II, and p38-MAPK (9,10). The sites of glyco-sylation do not possess a common consensus sequence,

but some shared characteristics exist (5). These sites areclustered at the COOH-terminus in the proximity of pro-line and valine residues. They are located in sequencesrich in serine and threonine (5) that are similar to phos-phorylation sites for protein kinases (4–6). These sitesconstitute the substrate for the catalytic activity of glyco-syl transferase and protein kinases (6). The role of thehexosamine pathway in p53 glycosylation has been dem-onstrated here by the identification of O-GlcNAc residuesbound to the transcription factor.

Phosphorylation of p53 was examined at three distinctsites: two at the COOH-terminus and one at the NH2terminus. In the first two cases, phosphorylation of the tu-mor suppressor was restricted to Ser 390 and was medi-ated by p38-MAPK. In contrast, there was no apparent p53phosphorylation at Ser 376, indicating the lack of involve-ment of PKC in the activation of p53 with hyperglycemia.This conclusion was based on the reactivity detected withantibody 421, which recognizes p53 when phosphate groups(9,27,28,39) do not occupy the epitope 372–381. At theNH2-terminus, p53 was phosphorylated at Ser 18, a sitethat is critical for p53 function in response to DNA damage(17). After DNA injury, phosphorylation at the NH2 termi-nus of p53 is relevant to both the stabilization of thisprotein and its role in apoptosis and growth arrest (18).Cell death and block of the cell cycle in G1 depend on p53conformational changes associated with phosphorylationof Ser residues distributed at the NH2 terminus (18). Ser 6,9, 15, 20, and 46 are selectively phosphorylated by differentkinases activated by DNA-damaging agents (16–18,40).DNA injury promotes a consistent phosphorylation of p53at Ser 15 in humans and at its homolog in rodents, Ser 18(17,18,41). We elected to evaluate phosphorylated p53 atSer 18 for its critical role in the initiation of cell death asa result of DNA damage. Our findings on myocyte apopto-sis and double DNA strand breaks with hyperglycemia areconsistent with this possibility.Hyperglycemia and MAPK. p38-MAPK, JNK, and ERKwere investigated to identify their effects on hypergly-cemia-mediated responses in myocytes. Downstreamevents triggered by ligand binding to AT1 receptor wereinvolved in phosphorylation of p38-MAPK (14). This signal-ing pathway was confirmed by the use of the AT1 antago-nist losartan. Treatment of myocytes with the AT1 blockerhad no influence on p53 O-glycosylation, but it abolishedp53 phosphorylation. Losartan also prevented p38-MAPKactivation. Inhibition of p38-MAPK by SB 202190 mim-icked, at a more distal level, the consequences of losartanby preventing phosphorylated p53 and thus the synthesisand secretion of Ang II. The three MAPK families reactdifferently to hormonal and metabolic stimuli, and thevariability in their functional characteristics reflects thecell type involved (19–21). As shown here, hyperglycemiastimulates p38-MAPK and JNK in myocytes in a mannersimilar to vascular endothelial cells (19,21,42,43) andsmooth muscle cells (44,45). Conversely, ERK is notactivated by high glucose concentrations in these cellpopulations, but it is upregulated in adipocytes and fibro-blasts (20). Ang II activates JNK through the AT1 receptorin vascular smooth muscle cells and it inhibits ERKthrough the AT2 receptor (45). Whether AT2 receptorshave a similar effect on myocytes is currently unknown.

FIG. 8. pH and phosphorylation of p53 and p38-MAPK. A and B: At highglucose (Glu), pH does not influence the levels of phosphorylated(phospho) p53 at Ser 390 and phosphorylated p38-MAPK at 2 and 4days. Acidosis was corrected by 4 mmol/l HEPES (HE).



Because of the primary role played by p38-MAPK inmyocytes exposed to hyperglycemia, we chose to selec-tively block this enzyme instead of using a more generalinhibitor of MAPK, such as a MEK inhibitor. The latterwould have affected the proapoptotic action of p38-MAPKand the antiapoptotic function of JNK (14,15,35,36), com-plicating the interpretation of the accumulated results.Hyperglycemia and p53-mediated RAS activation. Di-abetes is characterized by upregulation of RAS (30,32),and interventions decreasing Ang II attenuate morbidityand mortality of diabetic patients (46,47). Inhibition ofACE positively modifies cardiovascular events and ne-phropathy in subjects with severe diabetes (47). However,the mechanism by which diabetes upregulates the sys-temic and/or local RAS remains to be identified. Ventricu-lar myocytes possess a cellular RAS and synthesize andsecrete Ang II (11). Additionally, Ang II stimulates celldeath and cellular hypertrophy (11,24). The possibility thatdiabetes enhances the local RAS, resulting in the develop-ment of cardiac myopathy, has been shown in vivo (30,32);myocyte apoptosis and myocyte hypertrophy have beenidentified in the diabetic heart. AT1 blockade preventsthese phenomena as well as the induction of p53-regulatedgenes interfering with cell death and cell growth.

p53 has been linked to the transactivation of Aogen andAT1 receptor, leading to the formation and release of AngII from myocytes (11,12,22). p53 binding to the promoterof Aogen and AT1 increased with high glucose concentra-tions, and this adaptation resulted in the accumulation ofAogen and AT1 receptor proteins in myocytes. Aogen is thelimiting factor in the generation of Ang II in these cells(22). Inhibition of p53 downregulates the expression ofAogen, attenuating the myocyte RAS and the synthesis ofAng II (12,22). In this study, glucose levels, p53 expression,and Ang II quantity have been found to be directly corre-lated. The increased Ang II in the medium 45 min afterhyperglycemia most likely reflects de novo synthesis andrelease of Ang II from myocytes. This notion is consistentwith the short half-life of the octapeptide, which is 6–12min in the cells and 15 s in the blood (48,49). In the cur-rent experiments, the formation of Ang II followed theO-glycosylation of p53. Therefore, it is reasonable to as-sume that p53-mediated activation of the local RAS stim-ulated the generation and secretion of the peptide from thestressed myocytes.Hyperglycemia and Ang II-mediated apoptosis. Li-gands binding to surface AT1 receptors trigger myocytedeath by a mechanism involving an increase in cytosolicCa2 and the activation of Ca2-dependent DNase I (11,22). pH-dependent DNase II is not implicated in Ang II-mediated apoptosis (11,50). The present results are consis-tent with these observations, emphasizing the importanceof hyperglycemia in enhancing the synthesis of Ang II andAT1 receptor, which constitute the death signal and theexecutor of myocyte apoptosis, respectively. AT1 and AT2receptors have distinct roles in the initiation of apoptosisin various cell types. In PC12W and R3T3 cells, which lackAT1 receptors, Ang II stimulation of AT2 results in inter-nucleosomal DNA cleavage through dephosphorylation ofthe survival factor MAPK (51,52). In vascular smooth mus-cle cells, AT2 receptor activation opposes ERK function,promoting apoptosis (53). AT1 receptors induce cell survi-

val by interfering with the effects of AT2 on ERK activity(54). The claim has been made that neonatal and adultcardiac myocytes behave in a comparable manner in thepresence of Ang II: AT2 triggers apoptosis, and AT1 inhibitscell death (55,56). The discrepancy between these resultsand our observations is difficult to explain. However, culturesystems were not comparable, and much higher doses ofAng II, losartan, and PD123319 were used in these otherexperiments. These factors may account for the difference.

In conclusion, these in vitro results, in combination within vivo observations (30–32), support the notion thathyperglycemia is responsible for apoptotic myocyte deathwith diabetes via activation of effector responses involvingthe local RAS. Importantly, a strong association betweenthe upregulation of cellular RAS and the apoptosis of myo-cytes, endothelial cells, and fibroblasts has been identifiedin the diabetic human heart (31). Our work in diabetic pa-tients (31) and experimental animals (30,32) is the onlyone that has emphasized the role of cell dropout in theinitiation and progression of the diabetic myopathy. Be-cause of the critical role of hyperglycemia in cell death, thecurrent findings in vitro may apply to both juvenile type 1diabetes and adult type 2 diabetes.

ACKNOWLEDGMENTS

This work was supported by National Institutes of Healthgrants HL-38132, HL-39902, HL-43023, AG-15756, AG-17042,HL-66923, HL-65573, and HL-65577 and Juvenile DiabetesFoundation International grant 2000-62.

The initial helpful suggestions of Dr. Ashwani Malhotraare acknowledged.

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Hyperglycemia Activates p53 and p53-Regulated Genes Leading to Myocyte Cell Death