Inhibition of GAPDH activity by poly(ADP-ribose) polymerase activates three major pathways of hyperglycemic damage in endothelial cells (original) (raw)
Specificity of GAPDH antisense ODN. To test our hypothesis that hyperglycemia-induced mitochondrial superoxide overproduction activates pathways of hyperglycemic damage by partially inhibiting GAPDH and thereby diverting upstream glycolytic metabolites into these signaling pathways, we first needed to determine the specificity and efficacy of the GAPDH antisense ODNs. As previously reported (6), incubation of cells in 30 mM glucose reduced GADPH activity by 73%, from 157.9 ± 17.6 nmol/s/mg protein (5 mM glucose) to 42.2 ± 10.5 nmol/s/mg protein (Figure 1). After transfection with GAPDH antisense, cells incubated in 5 mM glucose showed a similar reduction in GAPDH activity, from 157.9 ±17.6 nmol/s/mg protein (5 mM glucose) to 34.3 ± 8.8 nmol/s/mg protein (5 mM glucose + antisense). In contrast, after transfection with GAPDH scrambled ODNs, GAPDH activity in cells incubated in 5 mM glucose was unchanged from that of cells incubated in 5 mM glucose alone (184.3 ± 23.6 nmol/s/mg protein (5 mM glucose + scrambled olgios) versus 157.9 ± 17.6 nmol/s/mg protein (5 mM glucose alone). That this reduction in activity by GAPDH antisense reflected a decrease in GAPDH protein was confirmed by immunoblotting (data not shown).
Effect of GAPDH antisense ODNs on GAPDH activity in BAECs. Each bar represents the mean ± SEM of four separate experiments. *P < 0.01 compared with cells incubated in 5 mM glucose alone.
Effect of GAPDH antisense ODN on PKC activation, hexosamine pathway activation, intracellular AGE formation, and NF-κB activation in cells cultured in 5 mM glucose. Incubation of bovine aortic endothelial cells with 30 mM glucose increased the membrane fraction of intracellular PKC activity from 104.45 ± 10.06 pmol/min/mg protein in cells incubated in 5 mM glucose (Figure 2a, bar 1) to 224.54 ± 23.94 pmol/min/mg protein (Figure 2a, bar 2), as previously described (5). After transfection with GAPDH antisense (Figure 2a, bar 3), cells incubated in 5 mM glucose showed a similar increase in PKC activity, from 104.45 ± 10.06 pmol/min/mg protein (5 mM glucose) to 196.63.35 ± 11.09 pmol/min/mg protein (5 mM glucose + antisense). In contrast, after transfection with GAPDH scrambled ODNs (Figure 2a, bar 4), PKC activity in cells incubated in 5 mM glucose was unchanged from that of cells incubated in 5 mM glucose alone 81.13 ± 13.18 pmol/min/mg protein (5 mM glucose + scrambled ODN) versus 104.45 ± 10.06 pmol/min/mg protein (5 mM glucose alone).
Effect of GAPDH antisense ODNs on pathways of hyperglycemic damage in BAECs. (a) PKC activation; (b) hexosamine pathway activation; (c) intracellular AGE formation; (d) NF-κB activation. *P < 0.01 compared with cells incubated in 5 mM glucose. AU, arbitrary units. For a through c, each bar represents the mean ± SEM of four separate experiments. For d, each bar represents the mean ± SEM of fluorescence from 40 cells measured in an in situ DNA–protein binding assay.
Similarly, incubation of BAECs with 30 mM glucose increased the UDP-N-acetylglucosamine concentration, an indicator of hexosamine pathway flux, from 1.35 ± 0.08 nmol/mg protein in cells incubated with 5 mM glucose (Figure 2b, bar 1) to 2.82 ± 0.12 nmol/mg protein (Figure 2b, bar 2), as previously described (6). After transfection with GAPDH antisense (Figure 2b, bar 3), cells incubated in 5 mM glucose showed a similar increase in UDP-N-acetylglucosamine, from 1.35 ± 0.08 nmol/mg protein (5 mM glucose) to 2.38 ± 0.30 nmol/mg protein (5 mM glucose + antisense). In contrast, after transfection with GAPDH scrambled ODNs (Figure 2a, bar 4), UDP-N-acetylglucosamine concentration in cells incubated in 5 mM glucose was unchanged from that of cells incubated in 5 mM glucose alone, 1.06 ± 0.03 nmol/mg protein (5 mM glucose + scrambled ODN) versus 1.35 ± 0.08 nmol/mg protein (5 mM glucose alone).
Incubation of BAECs with 30 mM glucose increased intracellular AGE formation from 33,315 ± 1,750 arbitrary units (AU) in cells incubated in 5 mM glucose (Figure 2c, bar 1) to 85,954 ± 7,431 AU (Figure 2c, bar 2), as previously described (5). This hyperglycemia-induced 2.6-fold increase was reproduced in cells transfected with GAPDH antisense (Figure 2c, bar 3) (74,837 ± 3,828 AU), whereas in cells transfected with GAPDH scrambled ODN (Figure 2c, bar 4), AGE formation was not different (27,238 ± 3,819 AU) from that of cells incubated in 5 mM glucose alone.
Lastly, incubation of BAECs with 30 mM glucose increased NF-κB activation by twofold, from 188,636 ± 13,333 AU in cells incubated in 5 mM glucose (Figure 2d, bar 1) to 379,053 ± 9,734 AU in cells incubated in 30 mM glucose (Figure 2d, bar 2), as previously described (5). This hyperglycemia-induced increase was reproduced in cells transfected with GAPDH antisense (Figure 2d, bar 3) (465,044 ± 31,474 AU), whereas in cells transfected with GAPDH scrambled ODN (Figure 2d, bar 4), NF-κB activation was not increased (160,009 ± 13,388 AU) above that of cells incubated in 5 mM glucose alone.
Effect of PARP inhibition on GAPDH activity. We next evaluated the extent of poly(ADP-ribosyl)ation on GAPDH in BAECs. Immunoprecipitation of GAPDH, followed by Western blotting with anti-poly(ADP-ribose) IgG showed that incubation of cells in 30 mM glucose increased covalent modification of GAPDH by 2.2-fold, from 57,155 ± 5,288 AU (Figure 3a, bar 1) to 125,532 ± 9,577 AU (Figure 3a, bar 2). This increased modification of GAPDH by poly(ADP-ribosyl)ation was completely prevented in cells exposed to 30 mM glucose by overexpression of either UCP-1 (Figure 3a, bar 4), a specific protein uncoupler of oxidative phosphorylation capable of collapsing the proton electrochemical gradient that drives superoxide production (5), or McSOD (21), the mitochondrial isoform of this enzyme (Figure 3a, bar 5). The poly(ADP-ribosyl)ation of GADPH induced by 30 mM glucose was also completely prevented by addition of the potent PARP inhibitor, PJ34 (Figure 3a, bar 6).We then assessed the effect of this covalent modification on GAPDH activity (Figure 3b). As described previously (6), the inhibitory effect of incubation in 30 mM glucose was completely prevented by overexpression of either UCP-1 (Figure 3b, bar 4) or MnSOD (Figure 3 b, bar 5), in parallel with the changes in poly(ADP-ribosyl)ation of GAPDH. The prevention of hyperglycemia-induced inhibition of GAPDH activity by PJ34 (Figure 3b, bar 6) directly demonstrated that hyperglycemia-induced overproduction of superoxide-inhibited GAPDH activity by causing poly(ADP-ribosyl)ation of the enzyme via PARP. Because PARP is exclusively in the nucleus, we examined the subcellular distribution of both poly(ADP-ribosyl)ated and unmodified GAPDH. In response to 30 mM glucose, there was a 1.7-fold increase of GAPDH modification in both the cytoplasmic and nuclear compartments, whereas the unmodified GAPDH decreased 64% in cytoplasm and 27% in the nucleus (data not shown). Approximately 60% of the poly(ADP-ribosyl)ated GAPDH was in the nucleus, and 40% in the cytoplasm, both in cells exposed to 30 mM glucose, and in cells exposed to 5 mM glucose (data not shown).
Effect of genes that alter mitochondrial superoxide production and of PARP inhibition on poly(ADP-ribosyl)ation of GAPDH (a), and on GAPDH activity (b), in BAECs. Cells were incubated in 5 mM glucose or 30 mM glucose alone, in 30 mM glucose plus either control, UCP-1– or MnSOD-expressing adenoviral vectors, and in 30 mM glucose plus 3 μM PJ34. Each bar represents the mean ± SEM of four separate experiments. *P < 0.01 compared with cells incubated in 5 mM glucose alone.
Treatment of the cells with a second, structurally unrelated PARP inhibitor, INO-1001 (300 nM), yielded results identical to those seen with PJ34 (data not shown). These data are consistent with an unaltered translocation of modified GAPDH from the nuclear compartment to the cytosolic compartment. In vivo, aortas from PARP-1 KO mice (10) showed no increase in GAPDH poly(ADP-ribosyl)ation after incubation in high glucose, whereas aortas from WT mice showed a nearly fourfold increase (Figure 4).
Effect of hyperglycemia on poly(ADP-ribosyl)ation of GAPDH in aortas from WT and PARP-1 KO mice. Each bar represents the mean ± SEM of four separate experiments. *P < 0.01 compared with aortas incubated in 5 mM glucose.
Effect of hyperglycemia-induced mitochondrial superoxide overproduction on PARP activity. To confirm directly that hyperglycemia-induced mitochondrial superoxide overproduction activated PARP, activity of this enzyme was determined (Figure 5). Incubation of BAECs in 30 mM glucose increased PARP activity 1.7-fold, from 147.5 ± 4.7 pmol/min/mg protein in cells incubated with 5 mM glucose (Figure 5, bar 1) to 254.9 ± 15.6 pmol/min/mg protein (Figure 5, bar 2). The activation of PARP by 30 mM glucose was completely inhibited by overexpression of either UCP-1 (Figure 5, bar 4) or MnSOD (Figure 5, bar 5) to 152.1 ± 15.1 pmol/min/mg protein and 165.5 ± 9.5 pmol/min/mg protein, respectively, whereas vector alone had no effect.
Effect of hyperglycemia and genes that alter mitochondrial superoxide production on PARP activity in BAECs. Cells were incubated in 5 mM glucose or 30 mM glucose alone, in 30 mM glucose plus either control, UCP-1– or MnSOD-expressing adenoviral vectors, and in 30 mM glucose plus 3 μM PJ34. Each bar represents the mean ± SEM of four separate experiments. *P < 0.01 compared with cells incubated in 5 mM glucose alone.
Effect of hyperglycemia-induced mitochondrial superoxide overproduction on DNA strand breaks. Because PARP is activated by single- or double-strand breaks in DNA (22), the effect of hyperglycemia-induced mitochondrial superoxide overproduction on DNA strand breaks was determined using the COMET single-cell electrophoresis assay (Figure 6). Incubation in 30 mM glucose increased the length of the DNA tail twofold, compared to 5 mM glucose, from 13.65 ± 0.76 μm (Figure 6, a and f, bar 1) to 30.99 ± 0.7 μm (Figure 6, b and f, bar 2). The increase in DNA strand breaks induced by 30 mM glucose was completely inhibited by overexpression of either UCP-1 (Figure 6, d and f, bar 4) or MnSOD (Figure 6, e and f, bar 5) to 15.67 ± 0.77 μm and 18.27 ± 0.57 μm, respectively, whereas vector alone had no effect (Figure 6, c and f, bar 3).
Effect of hyperglycemia and genes that alter mitochondrial superoxide production on DNA strand breaks in BAECs. Cells were incubated in 5 mM glucose (a) or 30 mM glucose alone (b), or in 30 mM glucose plus either control (c), UCP-1–expressing (d) or MnSOD-expressing (e) adenoviral vectors. Fluorescent micrographs from single-cell electrophoresis assay. (f) Quantitation of DNA strand breaks from single-cell electrophoresis assay. Each bar represents the mean ± SEM of 40 cells for each incubation condition. *P < 0.01 compared with cells incubated in 5 mM glucose alone.
Effect of PARP inhibition on PKC activation, hexosamine pathway activation, intracellular AGE formation, and NF-κB activation in cells cultured in 30 mM glucose. Having demonstrated that hyperglycemia-induced mitochondrial superoxide overproduction inhibits GAPDH activity by PARP-mediated poly(ADP-ribosyl)ation of the enzyme as a result of reactive oxygen species–induced (ROS-induced) DNA strand breaks, and having shown that inhibiting GAPDH with antisense ODN activates multiple pathways of hyperglycemic damage in cells cultured in 5 mM glucose to the same extent as does culturing these cells in 30 mM glucose, we sought to determine whether this sequence of events explained the activation of these pathways by hyperglycemia in vivo (Figure 7). We therefore evaluated the effect of PARP inhibition by PJ34 on each of the pathways of hyperglycemic damage in cells incubated with 30 mM glucose.
Effect of PARP inhibition on hyperglycemia-induced pathways of vascular damage in BAECs. (a) PKC activation; (b) hexosamine pathway activation; (c) intracellular AGE formation; (d) NF-κB activation. *P < 0.01 compared with cells incubated in 5 mM glucose. For a through c, each bar represents the mean ± SEM of four separate experiments. For d, each bar represents the mean ± SEM of fluorescence from 40 cells measured in an in situ DNA–protein binding assay.
Incubation of BAECs in 30 mM glucose increased the membrane fraction of intracellular PKC activity from 123.79 ± 17.3 pmol/min/mg protein in cells incubated in 5 mM glucose (Figure 7a, bar 1) to 280.14 ± 6.52 pmol/min/mg protein (Figure 7a, bar 2). Overexpression of either UCP-1 or MnSOD completely inhibited the effect of 30 mM glucose to 75.6 ± 28.1 pmol/min/mg protein and 107.15 ± 19.9 pmol/min/mg, respectively (Figure 7a, bars 4 and 5), as shown previously (5). Inhibition of PARP by PJ34 also completely prevented the activation of PKC by 30 mM glucose (Figure 7a, bar 6). Similarly, incubation of BAECs with 30 mM glucose increased the UDP-N-acetylglucosamine concentration, an indicator of hexosamine pathway flux from 1.18 ± 0.13 nmol/mg protein in cells incubated with 5 mM glucose (Figure 7b, bar 1) to 2.4 ± 0.3 nmol/mg protein (Figure 7b, bar 2). Overexpression of either UCP-1 or MnSOD completely inhibited the effect of 30 mM glucose to 1.2 ± 0.1 nmol/mg protein and 1.2 ± 0.2 nmol/mg protein, respectively (Figure 7b, bars 4 and 5), as shown previously (6). Inhibition of PARP by PJ34 also completely prevented the effect of 30 mM glucose (Figure 7b, bar 6). Incubation of BAECs with 30 mM glucose also increased intracellular AGE formation from 58,107 ± 3,765 AU in cells incubated in 5 mM glucose (Figure 7c, bar 1) to 92,707 ± 12,906 AU (Figure 7c, bar 2). Overexpression of either UCP-1 or MnSOD completely inhibited the effect of 30 mM glucose to 56,527 ± 6,319 AU and 48,094 ± 8,739 AU, respectively (Figure 7c, bars 4 and 5), as shown previously (5). Inhibition of PARP by PJ34 also completely prevented the effect of 30 mM glucose (Figure 7c, bar 6).
Lastly, incubation of BAECs with 30 mM glucose increased NF-κB activation by 2.6-fold, from 1014 ± 12 AU in cells incubated in 5 mM glucose (Figure 7d, bar 1) to 2653 ± 40 AU in cells incubated in 30 mM glucose (Figure 6d, bar 2). Overexpression of either UCP-1 or MnSOD completely inhibited the effect of 30 mM glucose to 997 ± 4.9 AU and 798 ± 3.8 AU, respectively (Figure 7d, bars 4 and 5), as shown previously (5). Inhibition of PARP by PJ34 also completely prevented this effect of 30 mM glucose (Figure 7d, bar 6). PJ34, unlike earlier generation PARP inhibitors, has no antioxidant properties and acts by competitive blockade of the NAD+ binding site in the enzyme. Nevertheless, to further verify that the observed effects were due to PARP inhibition, a structurally unrelated PARP inhibitor, INO-1001, was used. The effects of INO-1001 on hyperglycemia-induced PKC activation and NF-κB activation were identical to those with PJ34 (data not shown).