Distinct and sequential upregulation of genes regulating cell growth and cell cycle progression during hepatic ischemia-reperfusion injury

Sharon Barone, Tomohisa Okaya, Steve Rudich, Snezana Petrovic, Kathy Tenrani, Zhaohui Wang, Kamyar Zahedi, Robert A. Casero, Alex B. Lentsch, Manoocher Soleimani

Abstract

Ischemia-reperfusion injury (IRI) in liver and other organs is manifested as an injury phase followed by recovery and resolution. Control of cell growth and proliferation is essential for recovery from the injury. We examined the expression of three related regulators of cell cycle progression in liver IRI: spermidine/spermine N-acetyltransferase (SSAT), p21 (a cyclin-dependent kinase inhibitor), and stathmin. Mice were subjected to hepatic IRI, and liver tissues were harvested at timed intervals. The expression of SSAT, the rate-limiting enzyme in the polyamine catabolic pathway, had increased fivefold 6 h after IRI and correlated with increased putrescine levels in the liver, consistent with increased SSAT enzymatic activity in IRI. The expression of p21, which is transactivated by p53, was undetectable in sham-operated animals but was heavily induced at 12 and 24 h of reperfusion and declined to undetectable baseline levels at 72 h of reperfusion. The interaction of the polyamine pathway with the p53-p21 pathway was shown in vitro, where activation of SSAT with polyamine analog or the addition of putrescine to cultured hepatocytes induced the expression of p53 and p21 and decreased cell viability. The expression of stathmin, which is under negative transcriptional regulation by p21 and controls cell proliferation and progression through mitosis, remained undetectable at 6, 12, and 24 h of reperfusion and was progressively and heavily induced at 48 and 72 h of reperfusion. Double-immunofluorescence labeling with antibodies against stathmin and PCNA, a marker of cell proliferation, demonstrated colocalization of stathmin and PCNA at 48 and 72 h of reperfusion in hepatocytes, indicating the initiation of cell proliferation. The distinct and sequential upregulation of SSAT, p21, and stathmin, along with biochemical activation of the polyamine catabolic pathway in IRI in vivo and the demonstration of p53-p21 upregulation by SSAT and putrescine in vitro, points to the important role of regulators of cell growth and cell cycle progression in the pathophysiology and/or recovery in liver IRI. The data further suggest that SSAT may play a role in the initiation of injury, whereas p21 and stathmin may be involved in the resolution and recovery after liver IRI.

  • liver failure
  • heme oxygenase-1
  • polyamines

ischemia-reperfusion injury (IRI) is the major cause of morbidity and mortality in diseases such as shock liver, myocardial infarction, and acute ischemic renal failure. Ischemic conditions result in ATP depletion and the accumulation of toxic metabolites, whereas reperfusion results in the production of reactive oxygen intermediates (3, 6, 19). The resulting alteration in cellular metabolism and the generation of toxic molecules contribute to tissue damage in IRI (3, 6, 19), which is characterized by the presence of necrotic and apoptotic areas in the affected organs (6, 19). Despite significant developments in the understanding of the pathophysiology of IRI in liver, kidney, heart, and other organs, there is no specific therapy for patients except supportive care.

Polyamines (spermidine, spermine, and putrescine) are aliphatic cations derived from ornithine (32, 36) that play a fundamental role in cell growth, differentiation, and protein synthesis regulation (15, 18). Spermidine/spermine N-acetyltransferase (SSAT), the rate-limiting enzyme in polyamine catabolism, acetylates both spermidine and spermine, resulting in the depletion of intracellular spermidine and spermine and increasing the concentrations of N-acetyl spermidine and N-acetyl spermine (9, 39). The subsequent activity of polyamine oxidase (PAO) on acetylated polyamines results in the production of spermidine or putrescine (depending on the starting substrate) and H2O2, a reactive oxygen intermediate. The following schematic depicts the role of SSAT in polyamine catabolism: Embedded Image

As indicated, SSAT upregulation depletes intracellular polyamines and causes the generation of toxic metabolites (putrescine and H2O2) that together result in cell growth arrest and injury. Depletion of polyamines has been shown to upregulate p53 (27), which is a known regulator of p21 (13, 40).p21WAF1/CIP1, which has been well described in many cancer cells (14, 42), is a potent inhibitor of cyclin-dependent kinase (CDK) and regulates the transition from the G1 phase to the S phase of the cell cycle. It is activated by p53, a well-known tumor suppressor and apoptosis-regulating gene (16, 25). As a result, the p21WAF1/CIP1 gene may be linked via p53 to the cell cycle control that can result in growth suppression and terminal cellular differentiation (5). Mutation of the p21WAF1/CIP1 gene might release the cell from these negative growth controls, contributing to transformation of cells to a cancerous or proliferative state (1, 37). p21 has been shown to negatively regulate the transcription of stathmin (4).

Stathmin, also referred to as Op18, is a ubiquitous cytosolic phosphoprotein that is involved in the control of cell proliferation and differentiation (11, 26, 30). The expression of stathmin is decreased during the upregulation of p53, presumably via p21, to prevent cell proliferation (28). This raises the possibility that reduction in the expression of p53-p21 or mutations in p53-p21 releases the inhibitory effect of p53-p21 on cell growth and results in the upregulation of stathmin, leading to an increased proliferative response (1, 37). Stathmin interacts with several putative downstream target and/or partner proteins. One major action of stathmin that is mediated via changes in its level of phosphorylation is to interfere with microtubular dynamics during cell cycle progression. The phosphorylation of stathmin is regulated in part by CDK (29), which is under the control of p21 and p53 (13, 14, 40, 42). These interactions and modifications allow stathmin to play a critical role in the regulation of the dynamic equilibrium of microtubules during different phases of the cell cycle.

Liver IRI is manifested by an injury phase, which is associated with decreased cell growth, followed by the recovery phase, which is associated with enhanced cell growth and proliferation. Despite the essential role of SSAT, p21, and stathmin in normal cell growth and proliferation, virtually nothing is known about their expression or function in liver IRI. Our results demonstrate distinct and sequential upregulation of SSAT, p21, and stathmin during the reperfusion phase of liver IRI. The data suggest that the early upregulation of SSAT may contribute to cell injury and oxidative stress. Conversely, the data show that later expression of stathmin and p21 is associated with the resolution of injury. These findings suggest that regulation of SSAT, p21, and stathmin may be critical to the injury and/or recovery of the liver after IRI.

METHODS

Model of hepatic IRI.

Male C57BL/6 mice (10–12 wk of age) were obtained from The Jackson Laboratory (Bar Harbor, ME). This project was approved by the University of Cincinnati Institutional Animal Care and Use Committee and conformed to National Institutes of Health guidelines. Partial hepatic ischemia was induced as described previously (21). Briefly, mice were anesthetized with pentobarbital sodium (60 mg/kg ip). A midline laparotomy was performed, and an atraumatic clip was used to interrupt blood supply to the left lateral and median lobes of the liver. After 90 min of partial hepatic ischemia, the clip was removed to initiate hepatic reperfusion. Sham control mice underwent the same protocol without vascular occlusion. Mice were killed after the indicated periods of reperfusion, and blood and liver samples were obtained for analysis.

RNA isolation and Northern hybridization.

Total cellular RNA was extracted from the liver (30 μg/lane), size fractionated on a 1.2% agarose-formaldehyde gel, and transferred to nylon membranes by capillary transfer using 10× sodium chloride sodium phosphate-EDTA buffer. Membranes were cross-linked using UV light or baked. Hybridization was performed according to the method described previously by Gilbert and Church (8). Membranes were washed, blotted dry, and exposed to PhosphorImager screens at room temperature for 24–72 h and scanned using the PhosphorImager. A 32P-labeled cDNA fragment of the mRNA-encoding rat SSAT (corresponding to nt 323–892 of a rat SSAT cDNA; GenBank accession no. NM_009121), p21 (corresponding to nt 23–697; GenBank accession no. BC002043), and stathmin (corresponding to nt 28–393 of mouse stathmin cDNA; GenBank accession no. NM_017166) was used as a specific probe.

Immunoblot analysis of p21 and SSAT.

Total cellular or nuclear protein was prepared from the liver according to established protocols (20). Proteins were resolved using SDS-PAGE (50 μg/lane) and transferred to nitrocellulose membrane. The membrane was blocked with 5% milk proteins and then incubated for 6 h with antibodies against p21 (Research Diagnostics, Flanders, NJ) or SSAT (44). p21-specific antibodies were used for blotting against nuclear proteins, and SSAT antibodies were used against cytoplasmic proteins. The secondary antibody was a donkey anti-rabbit IgG conjugated to horseradish peroxidase (Pierce). The results were visualized using an ECL method (SuperSignal substrate; Pierce) and captured on light-sensitive imaging film (Kodak).

Immunocytochemical labeling.

Paraformaldehyde-fixed, paraffin-embedded sections were washed twice in PBS (pH 7.4) and blocked with 10% rabbit serum and 0.3% Triton X-100 in PBS for 1 h. Single- and double-immunofluorescence labeling with antistathmin and anti-PCNA antibodies was performed as described previously by investigators at our laboratories (45). For double labeling, tissue sections were incubated with rabbit anti-stathmin polyclonal antibodies (Calbiochem, San Diego, CA) and mouse anti PCNA monoclonal antibodies (Santa Cruz Biotechnology, Santa Cruz, CA). Sections were washed and then incubated with appropriate secondary antibodies (rabbit anti-goat IgG conjugated with Oregon Green 488 for anti-stathmin and goat anti-mouse IgG conjugated with Alexa Fluor 568 dye for anti-PCNA) for 2 h at room temperature. Sections were examined and images were acquired on a Nikon PCM 2000 laser confocal scanning microscope as 0.5-μm optical sections of the stained cells. The 488-nm line of the argon laser, isolated with the standard argon laser excitation filter supplied with the PCM 2000, was used for green dye excitation. The PCM 2000 standard 515/30-nm emission filter was used for the green-emitting dye. Red dye was excited with the 543.5-nm single-line output of the HeNe laser. The PCM 2000 standard red-channel, long-pass, 565-nm filter was used as the emission filter for the red dye. Digital images of the green and red dyes were acquired simultaneously through a single illumination and detection pinhole. The images were resolved discretely into two channels and analyzed separately.

Measurement of polyamines in liver samples.

The concentrations of spermine, spermidine, and putrescine in liver samples from normal and IRI groups were measured by performing HPLC according to established methods and as described previously (22, 35, 44).

Effect of SSAT induction by polyamine analog DENSPM or putrescine addition on hepatocyte morphology, p53-p21 pathway, and viability.

Mouse hepatocytes (AML-12; American Type Culture Collection, Manassas, VA) were cultured in a 1:1 mixture of Dulbecco's modified Eagle's medium (DMEM) and Ham's F-12 medium, supplemented with 10% fetal bovine serum, 1% insulin-transferrin-selenium G, 1% penicillin-streptomycin, and 40 ng/ml dexamethasone. Cells (n = 2 × 106) were plated in a 100-mm tissue culture dish. Twenty-four hours later, cells were treated with medium or 40 μM polyamine analog N1,N11-diethylnorspermine (DENSPM; Tocris Cookson, Ellisville, MO), a potent and specific inducer of SSAT (7, 12, 24). Twenty-four or 48 h later, cells were harvested and examined for the expression of SSAT and p21 as well as alteration in morphology and viability assayed using Trypan blue exclusion. The AML-12 cell line was generated by Dr. Nelson Fausto from mice transgenic for transforming growth factor-α (43). These cells have typical hepatocyte features such as peroxisomes and biliary canaliculi-like structures, do not grow in soft agar, and are nontumorigenic in nude mice. Similarly to normal hepatocytes, AML-12 cells express high levels of mRNA for serum (albumin, α1-antitrypsin, and transferrin) and gap junction proteins (connexins 26 and 32), secrete albumin, and contain solely isozyme 5 of lactate dehydrogenase. After extensive passaging, AML-12 cells continue to strongly coexpress hepatocyte connexin mRNA but do not display nonparenchymal cell markers.

To examine the effect of putrescine on cell morphology, cell viability, and the p53-p21 pathway, putrescine at 1 or 2 mM was added to cultured hepatocytes. Cells were examined 24 h later for the expression of p53, p21, and cellular morphology and viability.

Statistical analyses.

Values are expressed as means ± SE. The significance of differences between mean values was examined using ANOVA. P < 0.05 was considered statistically significant. For quantitation of the expression levels of SSAT, PAO, the p53-p21 pathway, and stathmin in Northern hybridization experiments, the intensity of each transcript on the membrane was measured using the PhosphorImager and normalized by dividing by the intensity of labeling of its corresponding 28S rRNA. For Western blot analysis, the intensity of bands was assessed by densitometric scanning and normalized by dividing by the intensity of the corresponding β-actin.

RESULTS

Induction of liver IRI.

In the first series of experiments, we established a long-term time course of IRI and recovery. Mice were subjected to partial hepatic ischemia for 90 min and reperfusion for 6–72 h. Liver histology was assessed using hematoxylin and eosin staining of liver sections. As shown in Fig. 1A, the livers obtained from sham-operated mice had normal liver histology. After ischemia and 6, 12, or 24 h of reperfusion, there was marked hepatocellular necrosis that was associated with intense neutrophil accumulation (Fig. 1, BD). After 48 or 72 h of reperfusion, there was evidence of a return to normal hepatocellular structure, but necrotic areas remained present (Fig. 1, E and F). To assess the extent of hepatocellular necrosis during this time course, serum levels of alanine aminotransferase (ALT) were determined. Serum levels of ALT peaked at 6–12 h of reperfusion (Fig. 1G), indicating that the majority of the injury to hepatocytes occurred during this time. After 24 h, serum ALT levels were diminished, and by 48 h, serum ALT levels were near baseline (Fig. 1G), indicating that there was no recurrent liver injury at these times. Coupled with the histological evidence, it appears that after 48 h of reperfusion, the liver is in a recovery and/or proliferative phase.

Fig. 1.

Histological and biochemical assessment of liver ischemia-reperfusion injury (IRI). Hematoxylin and eosin-stained liver sections were obtained from sham-operated controls (A) and from mice that underwent 90 min of hepatic ischemia and 6 h (B), 12 h (C), 24 h (D), 48 h (E), or 72 h (F) of reperfusion. Arrows indicate edges of necrotic regions, and arrowheads indicate areas of neutrophil accumulation. Serum samples were obtained at the same time points and analyzed for alanine aminotransferase (ALT), a specific marker of hepatocellular injury (G).

Expression of SSAT in liver IRI.

In the next series of experiments, total RNA from the livers of control animals and those from animals subjected to IRI (90 min of ischemia followed by 6, 12, 24, 48, or 72 h of reperfusion) were size fractionated and subjected to Northern blot analysis using radiolabeled SSAT cDNA probe. A representative Northern hybridization is shown in Fig. 2A, which indicates significant upregulation of SSAT in early reperfusion intervals. When adjusted for loading, the SSAT mRNA levels had increased ∼9.6 ± 1.6-fold at 6 h of reperfusion (n = 3; P < 0.001). As shown in Fig. 2A, the mRNA levels of SSAT had declined rapidly at 48 h of reperfusion and had returned to levels twofold above baseline at 72 h of reperfusion. To determine whether the increase in SSAT mRNA levels were associated with increased protein abundance, we performed Western blot analysis of SSAT on cytoplasmic extracts of liver samples. A representative blot shown in Fig. 2B, top, demonstrates a significant increase in SSAT protein expression. The equity in protein loading was shown using blotting against β-actin antibody (Fig. 2B, bottom). When normalized on the basis of protein loading, the results indicated that SSAT abundance increased 4.2 ± 0.6-fold at 6 h (P < 0.001, n = 3) and 3.2 ± 0.4-fold at 12 h of reperfusion (n = 3 for each group; P < 0.001).

Fig. 2.

Examination of the time course of spermidine/spermine N-acetyltransferase (SSAT) expression in liver IRI. A: Northern blot analyses were performed (30 μg/well total RNA) to examine the time course of expression of SSAT in IRI. The expression of SSAT increased ∼15-fold and fivefold at 6 h of reperfusion (n = 3; P < 0.05 for each zone) and threefold at 12 h of reperfusion (n = 3; P < 0.05 vs. time-matched control). Equal loading was confirmed by examination of the 28S rRNA bands (bottom). B: immunoblot analysis. A representative Western blot analysis of SSAT on cytoplasmic extract of IRI samples (top). As shown, the expression of SSAT is increased at 6, 12, and 24 h of reperfusion. The expression of β-actin is shown as control for loading (bottom). C: measurement of putrescine concentration in IRI liver tissues. As shown, the concentration of putrescine increased approximately ninefold at 6 h of reperfusion in liver IRI.

Measurement of polyamines in liver tissue in IRI.

To determine whether the increase in SSAT abundance reflects an increase in its activity, the concentration of polyamines (spermine, spermidine, and putrescine) was measured in liver samples using HPLC. The concentration of putrescine was 0.34 in sham-operated animals and increased to 3.21 nmol/mg protein at 6 h of reperfusion (n = 3 in each group; P < 0.03). The concentrations of spermine and spermidine were 7.42 and 10.62 nmol/mg of protein, respectively, in the sham-operated group and decreased to 4.92 and 3.89 nmol/mg protein, respectively, at 6 h of reperfusion (n = 3 for each group; P < 0.05 for each polyamine in IRI vs. sham). Figure 2C shows the results of putrescine measurement in liver IRI. Taken together, these results indicate that SSAT is activated in liver IRI and results in the depletion of polyamines (spermine and spermidine) and increased concentrations of putrescine.

Effect of SSAT induction in cultured mouse hepatocytes.

To ascertain the effect of SSAT upregulation on cell viability and morphology, cultured mouse hepatocytes were grown to confluence and exposed to the polyamine analog DENSPM at 40 μM for 48 h. DENSPM is a specific and potent inducer of SSAT and causes polyamine depletion (7, 12, 24). Cells were harvested after 48 h of exposure to DENSPM and examined for SSAT induction and cell morphology and viability. As shown in Fig. 3A, top, treatment with DENSPM caused significant induction of SSAT mRNA. To determine whether SSAT induction results in the upregulation of p21, the above membrane was stripped and reprobed. As shown in Fig. 3A, middle, SSAT induction by the polyamine analog DENSPM enhanced the expression of p21.

Fig. 3.

Effect of polyamine analog N1,N11-diethylnorspermine (DENSPM) on SSAT and p21 expression and morphology and viability in cultured hepatocytes. A: SSAT and p21 upregulation by polyamine analog DENSPM in cultured mouse hepatocytes. As shown, addition of DENSPM caused upregulation of SSAT (A, top) and p21 (A, middle). The equity in RNA loading is shown by comparable 18S rRNA abundance in both groups (A, bottom). B: SSAT overexpression decreases cell viability. As shown, cells treated with DENSPM showed >24% cell death vs. 8% for control as examined using Trypan blue exclusion (n = 4; P < 0.05).

Light microscopic analysis of live hepatocytes with or without DENSPM showed significant alteration in cell morphology in polyamine analog-treated cells. To determine the effect of SSAT induction on cell viability, vehicle-treated and DENSPM-treated cells were examined for Trypan blue exclusion. The results (Fig. 3B) demonstrate that the induction of SSAT was associated with increased cell death (24% vs. 8.5% cell death in DENSPM-treated cells vs. control hepatocytes, respectively, n = 4 experiments; P < 0.05). Together, these results demonstrate that the upregulation of SSAT decreases cell viability in hepatocytes.

The results of the experiments shown in Fig. 3 demonstrate that the upregulation of SSAT enhances the expression of p21 and increases cell death. Because SSAT upregulation caused polyamine depletion and increased putrescine generation, we sought to ascertain the effect of putrescine on cell function and morphology in liver cells in vitro. Toward this end, normal mouse liver hepatocytes were grown to confluence and then were exposed to 1 or 2 mM putrescine for 24 h. We first tested whether putrescine can increase the expression of p53 and p21. Figure 4A, top, indicates that the expression of p53 increased two- and fourfold in the presence of 1 and 2 mM putrescine (lanes 2 and 3), respectively, for 24 h. The expression of p21 (Fig. 4A, middle) was also significantly increased by putrescine in the same membrane. Figure 4A, bottom, shows 28S rRNA for loading equity. Figure 4B indicates that putrescine, at 2 mM, decreased cell viability in hepatocytes as assayed using Trypan blue exclusion (P < 0.001, n = 4). Together, these results demonstrate that putrescine causes the activation of the p53-p21 pathway and decreases cell viability in cultured hepatocytes.

Fig. 4.

Effect of putrescine on p53-p21 pathway expression and cell viability in cultured hepatocytes. A: p53 and p21 mRNA upregulation by putrescine. Lane 1: control; lane 2, 1 mM putrescine; and lane 3, 2 mM putrescine. As shown, addition of 2 mM putrescine for 24 h resulted in upregulation of p53 (A, top) and p21 (A, middle). The equity in RNA loading is shown by comparable 18S rRNA abundance in both groups (A, bottom). B: effect of putrescine on cell viability. Lane 1, control; lane 2, 1 mM putrescine; and lane 3, 2 mM putrescine. As shown, addition of 2 mM putrescine for 24 h significantly decreased cell viability as examined using Trypan blue exclusion (n = 4; P < 0.01).

We propose that SSAT increases the generation of putrescine, which in turn enhances the expression of p21 via activation of p53. On the basis of these studies, we suggest that putrescine is a possible link between SSAT activation and cell injury in liver cells in IRI.

Expression of p21 in liver IRI.

SSAT activation causes polyamine depletion and leads to the upregulation of p21, a CDK inhibitor, which causes cell cycle arrest (46). Furthermore, the addition of putrescine in hepatocytes in vitro increases the expression of p21 (Fig. 4A, top). In the next series of experiments, we sought to determine the onset of expression of p21 in liver IRI. The results are shown in Fig. 5 and indicate a pattern of expression that is distinct from SSAT in that the peak expression of SSAT precedes the induction of p21. As shown in Fig. 5A, mRNA levels of p21 were undetectable in control animals but became detectable at 6 h of reperfusion, were markedly induced at 12 h of reperfusion, and remained elevated at 24 h of reperfusion. Interestingly, the expression of p21 mRNA declined rapidly to levels barely above baseline at 48 h of reperfusion. These experiments were repeated a total of three times with samples from three different animals. The induction of p21 mRNA was highly significant at each of the 6-, 12-, and 24-h reperfusion time points (n = 3 separate samples; P < 0.001 vs. sham). To determine whether the increase in p21 mRNA levels also reflected an increase in p21 protein abundance, we performed Western blot analysis of nuclear extracts of liver samples. A representative blot is shown in Fig. 5B and demonstrates that p21 protein abundance was undetectable in control samples but was induced at 12 and 24 h of reperfusion. p21 protein expression remained elevated at 48 h but declined significantly at 72 h of reperfusion. The equity in protein loading is shown in Fig. 5B, bottom, where the membrane was reprobed with β-actin-specific antibody. The analysis of the results demonstrated that the induction of p21 was highly significant at each of the 6, 12, and 24 h time points of reperfusion vs. sham control (n = 3 separate samples; P < 0.001 for each time point vs. sham control).

Fig. 5.

Examination of the time course of p21 expression in liver IRI. A: Northern blot analyses were performed (30 μg/well total RNA) to examine the time course of p21 expression in IRI. The expression of p21 was undetectable in sham-operated animals and mildly detectable at 6 h of reperfusion. It was induced at 12 and 24 h of reperfusion and declined significantly at 48 and 72 h of reperfusion. Equal loading was confirmed by examination of 28S rRNA bands (bottom). B: immunoblot analysis. Representative Western blot analysis of p21 in nuclear extract of IRI samples. As shown, the expression of p21 was undetectable in sham-treated animals but was induced at 12 and 24 h of reperfusion. The expression of p21 became undetectable at 72 h of reperfusion. The equity in protein loading is shown by β-actin labeling with specific antibodies.

Expression of stathmin in liver IRI.

p21 negatively regulates the transcription of stathmin (4). The profound induction of p21 expression at 12 and 24 h of reperfusion and its sharp reduction at 48 and 72 h of reperfusion raised the possibility that its expression level might inversely regulate the expression of stathmin, a phospho-oncoprotein that controls cell proliferation. We therefore examined the expression of stathmin in liver IRI. A representative Northern blot analysis is shown in Fig. 6 and indicates a pattern of expression that is distinct from p21 and SSAT expression. As noted in Fig. 6, the mRNA levels of stathmin remained undetectable in control animals and at 6, 12, and 24 h of reperfusion. However, stathmin expression was heavily induced at 48 and 72 h of reperfusion. The experiments were repeated and verified in three separate samples (data not shown). Analysis of the results demonstrated that the induction of stathmin at 48 and 72 h was highly significant compared with sham controls (n = 3 separate Northern blot analyses; P < 0.001).

Fig. 6.

Expression of stathmin in the livers of mice subjected to IRI. Northern blot analyses were performed (30 μg/well total RNA) to examine the time course of expression of SSAT mRNA in IRI. The mRNA levels of SSAT (left and right). Equal loading was confirmed by examining 28S rRNA bands (bottom).

To examine the cellular distribution and regulation of stathmin, immunocytochemical staining was performed in livers with IRI. Accordingly, paraffin-embedded sections from control livers and livers from mice subjected to IRI were harvested at timed intervals (0, 6, 12, 24, 48, and 72 h of reperfusion) and examined for the expression of stathmin using immunofluorescent microscopy. Figure 7 demonstrates the expression of stathmin in sham-operated controls and at 72 h of reperfusion. The results indicate that in the livers of sham-operated controls (time 0) stathmin was expressed by a very limited population of hepatocytes (Fig. 7, left). However, the expression of stathmin had increased significantly at 72 h of reperfusion (Fig. 7, right). A survey of various fields demonstrated that at least 35–40% of hepatocytes were expressing stathmin at 72 h of reperfusion, whereas in control animals, only <1% of cells were positive for stathmin.

Fig. 7.

Immunofluorescence labeling of stathmin in liver IRI. Paraffin-embedded liver sections were examined for the expression of stathmin using immunofluorescence microscopy (magnification, ×400) in control animals (i.e., sham operated) and at 72 h of reperfusion.

Because 48–72 h of reperfusion corresponded to the onset of a proliferative response, we decided to examine the proliferation status of hepatocytes that express stathmin. Accordingly, immunofluorescent double staining of liver sections with antibodies against stathmin and PCNA, a marker of cell proliferation, were performed at time 0 and at 48 and 72 h of reperfusion. Figure 8 shows that stathmin (green) and PCNA (red) were expressed, albeit at very low levels, in the hepatocytes of sham-operated controls (Fig. 8A). The expression of stathmin and PCNA increased at 48 and 72 h of reperfusion (Fig. 8, B and C). The merged images (Fig. 8, B and C, middle) clearly demonstrate coexpression of stathmin and PCNA at both 48 and 72 h of reperfusion in a majority of the hepatocytes recovering from IRI. The number of stathmin- and PCNA-expressing cells was significantly lower at 48 h vs. 72 h of reperfusion.

Fig. 8.

Double-immunofluorescence labeling of stathmin and PCNA in liver IRI. Paraffin-embedded liver sections were examined for the expression of stathmin (green) and PCNA (red) using immunofluorescence microscopy (magnification, ×400) of reperfusion at time 0 (A) and at 48 h (B) and 72 h (C). The merged images (B and C, middle) indicate that increased stathmin expression preceded the induction of PCNA expression, and all PCNA-positive cells in D expressed stathmin.

DISCUSSION

The present study results demonstrate distinct patterns of expression for genes that regulate cell growth and proliferation in rat liver IRI. SSAT, which causes polyamine depletion and results in cell growth arrest, showed remarkable upregulation at 6 h of reperfusion (Fig. 2), along with increased putrescine and decreased spermidine and spermine in liver samples (see Fig. 2C and results). Furthermore, SSAT induction with the use of the polyamine analog DENSPM was associated with alteration in cell morphology, increased expression of p21, and decreased viability in liver cells in vitro (Fig. 3). The addition of putrescine at 2 mM to hepatocytes increased the expression of the p53-p21 pathway, caused alteration in cell morphology, and decreased cell viability in vitro (Fig. 4). The expression of p21, a cell cycle regulator that controls the G1-to-S transition phase, was undetectable in sham-treated animals but heavily induced at 12 and 24 h of reperfusion. It declined to undetectable baseline levels at 72 h of reperfusion (Fig. 5). The expression of stathmin, which is involved in the control of cell proliferation and progression through mitosis and is under negative transcriptional control by p21, remained undetectable at 6, 12, and 24 h of reperfusion and was heavily induced at 48 and 72 h of reperfusion (Fig. 7). Single- and double-immunofluorescence labeling demonstrated enhanced upregulation and colocalization of stathmin and PCNA, a marker of cell proliferation, at 48 and 72 h of reperfusion in hepatocytes, indicating the initiation of cell proliferation (Figs. 8 and 9).

Polyamines (spermidine and spermine) are essential for cell growth and differentiation (15, 18, 32, 36). Activation of SSAT depletes the intracellular polyamines and increases the production of toxic metabolites, therefore causing cell growth arrest and injury in tumor cells and in kidney IRI (22, 35, 41, 44). Recent studies performed at our laboratory have indicated that the conditional overexpression of SSAT in cultured cells causes growth arrest, increases the expression of heme oxygenase (HO)-1, and alters the arrangement of the cell cytoskeleton (41). The enhanced expression of HO-1 clearly points to the generation of oxidative stress in response to SSAT overexpression (41). Interestingly, the upregulation of HO-1 was significantly prevented in the presence of catalase, strongly indicating that SSAT overexpression leads to the generation of toxic metabolites such as H2O2 (41). The current data demonstrate that upregulation of SSAT is an early event (Fig. 2) and corresponds to the peak period of injury in liver cells. The association of SSAT upregulation and liver injury is further supported in cultured hepatocytes treated with the polyamine analog DENSPM (Fig. 3). This synthetic polyamine, which is a specific and potent inducer of SSAT (7, 12, 24) (Fig. 3), caused significant upregulation of SSAT, increased the expression of p21, altered cell morphology, and increased cell death in liver cells (Fig. 3). On the basis of these results and our recent studies in cultured cells demonstrating that the induction of SSAT is detrimental to cell growth and cytoskeleton arrangement and causes oxidative stress (41), we propose that the upregulation of SSAT in early stages of reperfusion may play an important role in mediating many phenotypic aspects of oxidative stress and injury in liver.

Overexpression of SSAT causes the upregulation of p21 (46), likely via p53 (Fig. 4), which has been shown to be activated in response to polyamine depletion (27), a well-documented outcome of SSAT upregulation (22, 35, 41). We have further demonstrated that putrescine, a toxic metabolic product of SSAT activation can upregulate the expression of p53 and p21 directly and decrease cell viability, all known phenotypic parameters in liver IRI. p21WAF1/CIP1, which is a potent inhibitor of CDK, regulates the transition from the G1 phase to the S phase of the cell cycle (14). As a result, p21 and pharmacological inhibitors of CDK have been studied as potential therapeutic targets in the prevention and/or treatment of human neoplasms. The upregulation of p21 at 12 and 24 h of reperfusion (Fig. 4) is consistent with cell cycle arrest, leading to the inhibition of hepatocyte growth in the early phase of IRI in the liver.

Microtubules play an indispensable role in vesicular transport, regulation of cell polarity, and mitosis (33). Previous studies have demonstrated that stathmin is expressed in mitotically active cells such as cancer cells (17, 31, 38) and in response to partial hepatectomy (23). Increased expression of stathmin was shown to be associated with the reentry of the cells into the cell cycle and with the onset of cell proliferation (38). Once expressed, stathmin function (i.e., its ability to bind to tubulin) is regulated via its phosphorylation on four serine residues (at positions 16, 25, 38, and 63) in its COOH-terminal regulatory domain in a cell cycle-dependent manner (2, 10). The role of stathmin in the regulation of cell division is supported by recent studies indicating that the downregulation of stathmin leads to the disruption of spindle structure and difficulties in completing mitosis (17, 34). Together, these results indicate that stathmin is involved in cell division and that its phosphorylation by kinases such as mitogen-activated protein kinase, ERK, and Cdc2 is necessary for the modulation of its activity and completion of mitosis (2, 10, 17, 31, 38).

Immunofluorescence studies have shown that only a minor fraction of cells in the normal liver (∼0.5–1%) express stathmin (Figs. 6 and 7). Stathmin expression increased dramatically in the livers subjected to IRI in the present study (Figs. 6 and 7). The lack of expression of stathmin in differentiated hepatocytes that are in G0 arrest and coexpression of stathmin with PCNA in hepatocytes indicate that stathmin is expressed by mitotically active cells during the repair phase of IRI. On the basis of the colocalization of stathmin and PCNA (Figs. 6 and 7) and its role of in cell mitosis, we suggest that stathmin may play an important role in the regulation of cell proliferation and recovery from liver IRI.

The most salient feature of these studies is the distinct expression pattern of SSAT, p21, and stathmin in liver IRI, along with biochemical activation of polyamine catabolic pathway in the early phase of IRI in vivo. Furthermore, the demonstration of p53-p21 pathway upregulation by putrescine in vitro points to the important role of a polyamine catabolic pathway in the regulation of the p53-p21 pathway and cell growth and cell cycle progression in the pathophysiology and/or recovery from injury in liver IRI. The onset of the upregulation of each gene is sharp and distinct and does not overlap with the expression of the other two genes. Coupled with studies indicating that SSAT activation by the polyamine analog DENSPM or that addition of putrescine was associated with the upregulation of p53-p21 pathway, these results support the possibility that the expression of SSAT may be a prerequisite for the upregulation of p21, which in turn negatively controls the regulation of stathmin. In further support of the stimulatory role of SSAT expression on p21 activation, our studies in kidney cells have demonstrated that conditional overexpression of SSAT in cultured cells upregulates the expression of p21 (46).

In conclusion, liver IRI is associated with distinct, time-dependent upregulation of genes regulating cell growth and cell cycle progression. We propose that SSAT, p21, and stathmin are involved in the pathophysiology and/or recovery from liver IRI, with SSAT causing injury and likely leading to the activation of p21 and stathmin playing an important role in the resolution and/or recovery from injury.

GRANTS

These studies were supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-66589 (to M. Soleimani), National Institutes of Health Grants DK-56029 and HL-72552 (to A. B. Lentsch), a Merit Review Award, and grants from Dialysis Clinic Inc. (to M. Soleimani).

Footnotes

  • 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.

REFERENCES

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