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Aldose reductase mediates cytotoxic signals of hyperglycemia and TNF- in human lens epithelial cells¹

Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston, Texas, USA; and
^* Divison of Cardiology, Department of Medicine, University of Louisville, Louisville, Kentucky, USA

²Correspondence: Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch, 6.644 Basic Science Bldg., Galveston, TX 77555-0647, USA. E-mail: ssrivast{at}utmb.edu

SPECIFIC AIM

Although the development of secondary diabetic complications is associated with increased apoptosis in several target tissues, the mechanisms by which diabetes induces cell death remain unclear. The aim of this study was to examine the role of the aldose reductase (AR), an enzyme that metabolizes excess glucose to sorbitol and contributes to the osmotic and oxidative effects of diabetes in cell death induced by two main instigators of diabetic injury: high glucose and TNF-.

PRINCIPAL FINDINGS

1. Inhibition of AR attenuates high glucose and TNF--induced apoptosis in HLEC
Incubation of the serum-starved transformed human lens epithelial cells -B3 (HLEC) with high glucose (50 mM) or TNF- (2 nm) for 24 h decreased cell growth, viability, and DNA synthesis ([³H]-thymidine incorporation) and increased caspase-3 activity, nuclear fragmentation and degradation of nucleosomal histones measured using Roche’s Cell Death ELISA kit), consistent with increased apoptosis. Preincubation of these cells with two structurally unrelated AR inhibitors—sorbinil and tolrestat (10 µM each)—attenuated high glucose or TNF--induced apoptosis, suggesting that AR may be an essential mediator of cell death-induced by high glucose or TNF-.

2. Inhibition of AR abrogates high glucose and TNF--induced activation of NF-B in HLEC
The transcription factor NF-B regulates the expression of genes involved in cell growth, differentiation, inflammation, and apoptosis and is activated by oxidants, cytokines, and growth factors. Therefore, we examined whether the proapoptotic role of AR relates to NF-B activation. Incubation of serum-starved HLEC with high glucose (50 mM) for 4 h or TNF- (0.1 nm) for 1 h resulted in significant activation of NF-B as measured by electrophoretic mobility gel shift assay (EMSA). Preincubation with sorbinil caused a dose-dependent inhibition of NF-B activated by TNF- or high glucose. However, 10 µM sorbinil caused > 60% inhibition of NF-B activity stimulated by high glucose whereas 20 µM sorbinil was required to cause the same extent of inhibition of NF-B activated by TNF-, suggesting a greater AR dependence of high glucose signaling (Fig. 1 A). Preincubation with AR inhibitor for at least 12 h was required to inhibit NF-B induction by TNF- or high glucose (Fig. 1B ), indicating that sorbinil by itself does not directly react with components of NF-B signaling but that inhibition of AR prevents metabolic changes permissive of NF-B activation.

View larger version (102K): [in this window] [in a new window]   Figure 1. Concentration and time dependence of sorbinil-induced inhibition of high glucose- and TNF--stimulated NF-B activity. A) Quiescent HLEC cells were preincubated without or with the indicated concentrations of sorbinil for 24 h, then left unstimulated (control) or stimulated with 0.1 nM TNF- for 1 h or 50 mM glucose for 4 h. B) HLEC were preincubated with sorbinil 10 µM and left untreated (control) or were stimulated with 0.1 nM TNF- for 1 h or 50 mM glucose for 4 h. After the indicated times, nuclear extracts were prepared and NF-B activity was measured by EMSA as shown.

2. Inhibition of AR attenuates high glucose and TNF--induced NF-B translocation, IB- phosphorylation, and degradation
To further elucidate the involvement of AR, we examined events upstream of NF-B activation. In unstimulated cells, NF-B is present as a heteromeric form of p65, p50, and inhibitory partner IB, which is phosphorylated, ubiquitinated, and degraded, leaving active NF-B dimer of p65 and p50 to translocate into the nucleus. Incubation of serum-starved HLEC with high glucose or TNF- caused translocation and accumulation of active NF-B in the nuclear region. However, preincubation of serum-starved HLEC with AR inhibitors prevented nuclear migration of NF-B. Both high glucose and TNF- induced phosphorylation of IB- within 120 and 45 min of exposure, respectively. This was followed by degradation and rapid resynthesis of IB-. Preincubation of the cells with sorbinil (10 or 20 µM) attenuated glucose and TNF--induced IB- phosphorylation and degradation, indicating that inhibition of AR prevents events upstream to the activation sequelae of IB-.

3. Involvement of protein kinase C (PKC) in the activation of NF-B in HLEC induced by high glucose and TNF-
Serum kinases, including PKC, can phosphorylate IB- and initiate NF-B activation. Since IB- phosphorylation is mediated by upstream kinases such as PKC, MAPK, and IKK, we measured the effect of inhibiting AR on high glucose and TNF--induced activation of PKC using Promega’s SignaTECT PKC assay system. Incubation of the cells with high glucose (50 mM) or TNF- (2 nM) for 4 h led to nearly a twofold increase in membrane-bound PKC activity (Fig. 2 A), whereas preincubation with AR inhibitors attenuated the increase in the membrane-bound PKC induced by high glucose or TNF-. Inhibition of AR did not prevent the activation of PKC or NF-B caused by stimulating the cells with 10 nM phorbol ester (PMA) for 4 h, indicating that AR probably mediates high glucose and TNF- signals upstream of PKC.

View larger version (42K): [in this window] [in a new window]   Figure 2. Inhibition of AR abrogates PKC activation. A) Quiescent HLEC were incubated with 10 µM sorbinil or tolrestat for 24 h. B) HLEC were transiently transfected with AR antisense or scrambled control oligonucleotides. Subsequently, the cells were stimulated with high glucose (50 mM), TNF- (0.1 nM), or PMA (10 nM) for 4 h and the membrane-bound PKC activity was determined as described in the text. Bars represent mean ± SE (n=4). **P < 0.001 vs. activity without the inhibitor (A) or with the scrambled control oligonucleotides transfected cells (B). The inset in panel B shows AR expression as determined by Western blot analysis after HLEC transfections. C, control; L, treated with lipofectamine alone; S, treated with scrambled; A, antisense oligonucleotides. Corresponding levels of the housekeeping enzyme, glyceraldehydes-3-phosphate dehydrogenase, determined by Western analysis of the same gel are also shown in the inset.

To rule out the nonspecific effects of AR inhibitors, we transfected the HLEC with AR antisense oligonucleotides. This treatment led to a significant decrease in AR activity and AR protein (as quantified by Western blot analysis using recombinant AR antibodies), whereas treatment with scrambled oligonucleotides had no effect. Compared with untransfected cells or cells transfected with scrambled oligonucleotides, the AR antisense-transfected cells displayed less PKC activation on stimulation by high glucose or TNF-. Transfection with AR antisense did not affect PKC activation by PMA (Fig. 2B ). Antisense ablation of AR also attenuated apoptosis induced by high glucose and TNF-. These observations confirm that AR plays a critical role in PKC-NF-B signaling leading to apoptosis and that the changes observed with AR inhibitors are not due to nonspecific effects of these drugs.

4. Inhibition of AR specifically attenuates redox-sensitive signals
We examined the effect of AR inhibition on other apoptotic signaling events such as phosphorylation of JNK, p38, and the activation of AP1, SP1, and OCT1. Incubation of HLEC with high glucose or TNF- induced phosphorylation of JNK and p38 but did not affect the total cellular abundance of these proteins. Preincubation of the cells with AR inhibitors attenuated high glucose and TNF--induced phosphorylation of JNK and p38 but did not affect total JNK and p38. The high glucose and TNF--induced activation of transcription factor AP1, downstream of JNK/p38, was also attenuated by AR inhibitors. However, the AR inhibitors had no effect on the high glucose or TNF--stimulated redox-insensitive transcription factors SP1 or OCT1, further indicating that inhibition of AR specifically affects redox-sensitive signaling events initiated by high glucose and TNF-.

CONCLUSIONS AND SIGNIFICANCE

To the best of our knowledge this is the first report demonstrating that AR activity is essential for the apoptotic signaling events associated with high glucose or TNF- stimulation and that inhibition of this enzyme prevents apoptosis as well as the activation of the PKC/NF-B pathway. Aldose reductase represents the first and the rate-limiting step in the polyol pathway, which is a subsidiary route for glucose metabolism. Although under normal physiological conditions the AR catalyzed transformation represents only a minor fate of glucose, under hyperglycemia, where the glucose concentration is increased, or under stress when AR is activated, reduction to sorbitol may be an important route of glucose metabolism. However, because the AR consumes NADPH and generates osmotically active polyols, increased flux of glucose via AR has been linked with oxidative and osmotic stress. In agreement, inhibition of AR has been shown to prevent tissue injury and dysfunction associated with chronic exposure to high glucose or galactose or due to long-term diabetes.

The mechanisms by which diabetes leads to progressive tissue injury are unclear, but are relevant to the clinical development of a host of secondary complications leading to an increased incidence of cataracts, blindness, kidney failure, and heart diseases. Recently, apoptosis has been suggested to be a key cellular mechanism by which long-term diabetes induces injury to tissues that do not require insulin for glucose uptake and consequently face recurrent and severe intracellular hyperglycemia. In agreement with these observations, we found that exposure to high glucose or TNF- induces cell death in HLEC with features characteristic of apoptosis. Inhibition of AR by using specific inhibitors or antisense oligonucleotides prevented apoptosis in these cells, suggesting that AR is essential for the metabolic and signaling events that precede programmed cell death. Inhibition of AR prevented the activation of cellular kinases JNK, p38, and PKC and the activation of redox-sensitive transcription factors like NF-B and AP1. Inhibition of AR did not prevent activation of redox-insensitive transcription factors SP1 and OCT1 nor did it prevent the direct activation of PKC by phorbol ester. Based on these observations, we conclude that AR-dependent metabolism is essential for cytokine and high glucose-mediated cell death and that inhibition of this enzyme prevents redox-sensitive events preceding the activation of PKC and NF-B (Fig. 3 ). Because oxidative stress has been suggested to be a causative factor in the development of diabetic and hyperglycemic injury, the results of our study may be important to the understanding and treatment of diabetic complications.

View larger version (31K): [in this window] [in a new window]   Figure 3. Proposed role of AR in the mediation of cytotoxic signals of high glucose and TNF- in HLEC. Reactive oxygen species (ROS) generated by binding of TNF- to its cognate receptor or by high glucose by either auto-oxidation or decreasing the ratio of NADPH/NADP can directly or through phopholipase/DAG/Ca2+ signals activate PKC and caspase-3. Activation of PKC could in turn activate redox-sensitive transcription factors NF-B and AP1 by initiating phosphorylation cascades involving IKK and MAPK. Transcription factors could translocate to the nucleus and transcribe proinflammatory genes leading to cytotoxicity. Possible sites of AR mediation in the cytotoxic signals of TNF- and high glucose are denoted by dotted line arrows. Solid line arrows and broken line arrows denote direct and a cascade of signals, respectively.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0568fje; to cite this article, use FASEB J. (December 18, 2002) 10.1096/fj.02-0568fje

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