Overexpression of SSAT in Kidney Cells Recapitulates Various Phenotypic Aspects of Kidney Ischemia-reperfusion Injury
Zhaohui Wang*,,
Kamyar Zahedi,,
Sharon Barone*,
Kathy Tehrani,
Hamid Rabb,
Karl Matlin¶,
Robert A. Casero# and
Manoocher Soleimani*,||
*Division of Nephrology and Hypertension, Department of Medicine, University of Cincinnati, Division of Nephrology and Hypertension, Department of Pediatrics, Children Hospital Medical Center, and ¶Department of Surgery, University of Cincinnati, Cincinnati, Ohio; Division of Nephrology and #Department of Oncology, Johns Hopkins University School of Medicine, Baltimore, Maryland; and ||Veterans Affairs Medical Center, Cincinnati, Ohio.
Correspondence to Dr. Manoocher Soleimani, Division of Nephrology and Hypertension, Department of Medicine, University of Cincinnati, 231 Albert Sabin Way, MSB 259G, Cincinnati, OH 45267-0585. Phone: 513-558-5463; Fax: 513-558-4309; E-mail: Manoocher.soleimani{at}uc.edu
ABSTRACT. To ascertain the role of spermidine/spermine N-1-acetyl-transferase(SSAT; the rate-limiting enzyme in polyamine catabolism) incell injury, cultured kidney (HEK 293) cells conditionally overexpressingSSAT were generated. The SSAT expression was induced and itsenzymatic activity increased 24 h after addition of tetracyclineand remained elevated over the length of the experiments. Inductionof SSAT upregulated the expression of polyamine oxidase andresulted in the reduction of cellular concentration of spermidineand spermine, increased concentration of putrescine, and inhibitedcell growth. SSAT overexpression increased the expression ofheme oxygenase-1 (HO-1) by 350% 24 h after addition of tetracycline,indicating the induction of oxidative stress. The presence ofcatalase significantly prevented the upregulation of HO-1 inSSAT overexpressing cells, indicating that generation of H2O2is partially responsible for the induction of oxidative stress.Overexpression of SSAT caused rounding and loss of cell anchorageand significantly altered the morphology of actin-containingfilopodia, suggesting an adhesion defect. SSAT upregulationmay mediate majority of the oxidative stress in kidney ischemia-reperfusioninjury (IRI) as manifested by decreased cell growth, generationof toxic metabolites (H2O2 and putrescine), upregulation ofHO-1, disruption of cell anchorage, and defect in cell adhesion.These data point to the inhibition of polyamine catabolism asa therapeutic approach for the prevention of tissue injury inkidney IRI.
Ischemia-reperfusion injury (IRI) is the major cause of morbidityand mortality in diseases such as stroke, myocardial infarction,and acute renal tubular necrosis. Ischemic conditions resultin ATP depletion and accumulation of toxic metabolites. Reperfusionresults in the production of reactive oxygen intermediates (1,2).The resulting alteration in cellular metabolism and generationof toxic molecules contribute to tissue damage in IRI (1,2),which is characterized by the presence of necrotic and apoptoticareas in the affected organs (1,3). Despite important developmentsin our understanding of the pathophysiology of IRI in kidney,heart, and other organs, there is no specific therapy for patientsexcept for supportive care.
Polyamines (spermidine, spermine, and putrescine) are aliphaticcations derived from ornithine (4,5). They play a fundamentalrole in the stabilization of DNA structure; they modulate geneexpression and regulate protein synthesis, signal transduction,and cell growth and differentiation (4,6–8). Spermidine/spermineN-1-acetyl-transferase (SSAT; the rate-limiting enzyme in polyaminecatabolism) acetylates both spermidine and spermine. As a result,the cellular contents of spermidine and spermine are decreasedand the concentrations of N-acetyl spermidine and N-acetyl spermineare increased (9). The subsequent activity of polyamine oxidase(PAO) on acetylated polyamines results in the production ofspermidine or putrescine (depending on the starting substrate)and H2O2, a reactive oxygen intermediate. Figure 1 depicts therole of SSAT in polyamine catabolism.
By use of suppression subtractive hybridization, we observeda major increase in SSAT expression levels in kidneys afterIRI (10). The SSAT upregulation correlated with the onset ofinjury and not with a significant increase in uremic toxins(10). Overexpression of SSAT was also observed in cultured cellssubjected to ATP depletion, an in vitro model of IRI. To determinewhether increased expression of SSAT is detrimental to cellsurvival in IRI, the effect of overexpression of SSAT was studiedin cultured kidney cells. The results indicated that SSAT overexpressionper se generates oxidative stress and enhances susceptibilityto injury in cultured cells. We propose that SSAT upregulationcontributes to the tissue damage associated with IRI.
Conditional Overexpression of SSAT in Cultured Kidney Cells
HEK293 cells stably transfected with the regulatory plasmidpcDNA6/TR, which encodes the Tet repressor protein (TetR) underthe control of human cytomegalovirus promoter, were purchasedfrom Invitrogen (Carlsbad, CA). To establish the tetracycline-inducibleSSAT expression, the region spanning SSAT cDNA bases 6 to 800(containing the full coding region) was amplified by reversetranscriptase-PCR from rat kidney cDNA with sense primer 5'-CGGGAAACGAATGAGGAACCACCand antisense primer 5'-ATTCTGCCTCCAAACCACATACATGAC, which weresynthesized on the basis of the mouse SSAT cDNA sequence (accessionno. NM_009121). The PCR product was gel purified, subclonedinto pGEMT-Easy vector (Promega), and transformed into Escherichiacoli. The insert was released by NotI digestion and ligatedinto pcDNA4/TO (a tetracycline-inducible mammalian expressionvector). Sequencing was performed to verify the correct orientationof SSAT insert. The isolated clones were then used to transfectHEK293 cells. Several colonies of HEK 293 cells stably expressingSSAT were isolated and expanded.
Generation and Conditional Overexpression of Inactive Mutant SSAT in Cultured Kidney Cells
To examine the role of SSAT in mediating cell injury in moredetail, we generated a construct that was used to express aninactive SSAT mutant. Briefly, we used the Quick Change PCRbased site directed mutagenesis method (Strategene) to developan expression vector that codes for an inactive SSAT mutantprotein that contains two mutations in residues 101 and 152(R101 A/E152K). This mutated protein does not display any SSATactivity (9). After confirming the sequence of the insert, HEK293cells were transfected with the tetracycline-inducible inactiveSSAT construct. Stable transfectants were isolated and expandedsimilar to the wild type SSAT.
Stable Expression of SSAT-expressing Inducible Construct
HEK293 cells were stably transfected with the inducible constructaccording to established methods with Transfast Transfectionreagent (Progema). The transfectants were selected in the presenceof Zeocin at a final concentration of 200 µg/ml. Singleclones were isolated and expanded for further studies.
RNA Isolation and Northern Hybridization
Total cellular RNA was extracted from cultured cells or kidneysamples with the Tri Reagent method (MRC, Cincinnati, OH) followingthe manufacturer’s protocol. Total cellular RNA (30 µg/lane)was size-fractionated on a 1.2% agarose-formaldehyde gel andtransferred to nylon membranes by capillary transfer with 10xSSPE buffer. Membranes were cross-linked by ultraviolet lightor baked. Hybridization was performed according to Gilbert andChurch (11). Membranes were washed, exposed to PhosphorImagerscreens at room temperature for 24 to 72 h, and scanned by PhosphorImager.A 32P-labeled cDNA fragment of the mRNA-encoding SSAT (correspondingto nucleotides 323 to 892 of a mouse SSAT cDNA; GenBank accessionno. NM_009121) or PAO (corresponding to nucleotides 1 to 861of mouse PAO cDNA (GenBank accession no. XM_113921) was usedas a specific probe. For heme oxygenase-1 (HO-1), a 445-bp PCRfragment encoding nucleotides 21 to 466 of a rat HO-1 cDNA (GenBankaccession no. NM_012580) was used.
Light Microscopy
Cells were exposed to tetracycline (1 µg/ml) for 24 hand light microscopic images were obtained at x100 magnificationwith a Zeiss inverted microscope.
Measurement of SSAT Activity and the Intracellular Concentration of Polyamines
The enzymatic activity of SSAT and the intracellular concentrationof spermine, spermidine, and putrescine were measured on celllysate by HPLC, as described previously (12).
Cell Growth
To determine cell growth, cultured cells were plated and assayedby CellTiter 96 Non-Radioactive Cell Proliferation Assay fromPromega (Madison, WI).
IRI in Rats
IRI was induced as described previously (10,13). Briefly, bilateralIRI was induced in male Sprague-Dawley rats (200 to 250 g) byoccluding the renal pedicles with microvascular clamps for 15min (mild ischemia) or 30 min (severe ischemia) under ketamine-xylazineanesthesia (150 µg/g as ketamine, 3 µg/g as xylazine).Completeness of ischemia was verified by blanching of the kidneys,signifying the stoppage of blood flow. The blood flow to thekidneys was reestablished by removal of the clamps (reperfusion)with visual verification of blood return. Animals subjectedto sham operation (identical treatment except the renal pedicleswere not clamped) were used as controls. During the procedure,animals were well hydrated, and their body temperature was controlledto about 94°F with an adjustable heating pad. After ischemia,animals were kept under veterinary observation. At 12 h afterischemia, animals were killed and their kidneys harvested.
Cell Attachment Assay
HEK cells, stably transfected with tetracycline inducible wild-typeSSAT expression vector, were seeded at 1.0 x 105 cells per wellin six-well tissue culture plates (Falcon, Franklin Lakes, NJ).Forty-eight hours after seeding, the growth medium was removedand replaced with fresh growth medium with or without tetracycline.Twenty-four hours after changing the medium, light microscopicimages of the cells were obtained. To examine the effect ofSSAT induction on cell attachment, the unattached cells wereharvested by aspirating the culture medium, and the attachedcells were harvested by treating the adherent cells with trypsin-EDTAsolution (Life Technologies BRL, Grand Island, NY). The numberof nonadherent and adherent cells was determined by performingcell counts with a hemacytometer.
Filamentous Actin Staining
Flasks of cells expressing SSAT under the control of the tetracyclinetransactivator were induced with tetracycline for approximately24 h, then trypsinized and replated on coverslips coated withfibronectin (10 µg/ml for 2 h at 37°C). After 3 hor overnight incubation, the cells were fixed with 3% formaldehydein PBS, permeabilized with 0.1% Triton-X100 in PBS, and stainedwith a 1:100 dilution of Alexafluor 488-phalloidin (MolecularProbes) for 30 min to visualize filamentous actin accordingto established methods (14,15). Coverslips were mounted in VectashieldHard Set mounting medium containing DAPI (Vector Laboratories)as a nuclear counterstain and viewed in a Zeiss confocal fluorescencemicroscope with a x63 PlanApo lens. Both confocal and Nomarskidifferential interference contrast images were digitally photographed.
Materials 32P-dCTP was purchased from New England Nuclear (Boston, MA).Nitrocellulose filters and chemicals were purchased from SigmaChemical (St. Louis, MO). High Prime labeling kits were purchasedfrom Roche Diagnostics Gmbh (Mannheim, Germany).
Statistical Analyses
Values are expressed as mean ± SEM. The significanceof difference between mean values was examined by ANOVA. P <0.05 was considered statistically significant.
Conditional Overexpression of SSAT Leads to PAO Upregulation Figure 2 depicts a Northern blot analysis for the expressionof SSAT mRNA in several clones stably transfected with an SSATinducible construct. Our results indicate that addition of tetracyclineinduces the expression of SSAT mRNA. To determine whether SSATinduction upregulates the expression of PAO, the same blot wasstripped and reprobed with radiolabeled PAO cDNA. PAO mRNA levelsincrease in tetracycline-treated cells. Taken together, thesedata indicate that increased SSAT expression causes the inductionof PAO. For the purpose of the following studies, one of theclones expressing high levels of SSAT (clone 1) was chosen andstudied in detail.
Figure 2. Northern hybridization of SSAT in clones stably expressing the SSAT-containing inducible construct. (top) Addition of tetracycline for 24 h induces the expression of SSAT. (middle) SSAT induction causes the induction of polyamine oxidase (PAO). (bottom) Equity in RNA loading.
To determine the optimal time course of SSAT induction by tetracycline,cells from clone 1 were exposed to tetracycline for varioustime intervals (1, 2, 3, and 4 d) and processed for RNA isolation.As indicated in Figure 3, the expression of SSAT peaks at 24h and remains elevated at 96 h after the addition of tetracyclineversus time-matched controls.
Figure 3. Time course of conditional overexpression of SSAT by tetracycline in cultured kidney cells. SSAT expression peaked at 24 h and remained elevated at 96 h after addition of tetracycline. Equal loading of RNA in various lanes was confirmed by the examination of 28s rRNA bands (bottom).
Measurement of Enzymatic Activity of SSAT and Concentration of Polyamines
The overexpression of SSAT is associated with a dramatic inductionof SSAT enzymatic activity (Figure 4a), induction of PAO (Figure 2),and a significant reduction in the intracellular concentrationof spermidine and spermine throughout the experiment (Figure 4b).The decrease in the concentration of polyamines was alsoassociated with a significant reduction in cell growth (Figure 4c).
Figure 4. Measurement of enzymatic activity of SSAT and concentration of polyamines in SSAT overexpressing cells. (a) SSAT enzymatic activity increased significantly and remained elevated at 96 h after addition of tetracycline. (b) Concentration of spermidine and spermine was significantly reduced as early as 24 h after overexpression of SSAT. (c) Cell growth was significantly inhibited in response to overexpression of SSAT.
Upregulation of SSAT and HO-1 In Vivo Is an Early Event and Correlates with the Severity of Kidney Ischemic Injury In vivo kidney IRI results in the upregulation of SSAT and causesthe generation of H2O2 and putrescine, which are both toxicmetabolites (10). HO-1 is upregulated in nephrotoxicity, andits enhanced expression is regarded as a marker of oxidativestress (16–22). It is increased in response to a numberof injury-causing chemicals, including H2O2, and may provideprotection against cell injury (23,24). The purpose of the nextseries of experiments was to examine the expression of SSATand HO-1 with respect to each other in two groups of rats withmild and severe renal ischemic injury. Animals were subjectedto 15 min (mild injury) or 30 min (severe injury) of ischemiaand killed after 12 h of reperfusion. As demonstrated in Figure 5,the onset and the magnitude of expression of HO-1 and SSATare similar and correlate with the severity of injury.
Figure 5. SSAT upregulation parallels enhanced expression of heme oxygenase-1 (HO-1) in in vivo kidney IRI. The onset and the magnitude of enhanced expression of SSAT in in vivo kidney IRI (top) correlates with the onset and the severity of injury (15 and 30 min of ischemia), respectively, and parallels the expression of HO-1 (bottom). Three rats were killed for each time point (12 or 24 h of reperfusion) and for sham-operated controls. Each lane represents RNA from an individual animal.
To determine the SSAT enzymatic activity in kidneys with IRI,tissue samples (cortex or medulla) from sham-operated animalsand rats with 30 min ischemia and 12 h of reperfusion were harvested,homogenized in ice-cold isolation solution, and centrifugedat low speed as described. The extracts were processed for SSATactivity as described (above) (10). SSAT enzymatic activitieswere 1.96 ± 0.21 and 2.36 ± 0.23 pmol/mg protein/minin cortex and medulla, respectively, in sham-operated animalsand increased to 37.5 ± 2.4and 47.6 ± 2.7 pmol/mgprotein/min, respectively, at 12 h of reperfusion (P < 0.01in both cortex and medulla versus sham). These results demonstratethat IRI is associated with an approximately 20-fold increasein SSAT activity in kidney tissue in in vivo IRI.
Overexpression of SSAT Upregulates the Expression of HO-1 in Cultured Cells
On the basis of the time course of HO-1 upregulation with respectto SSAT in kidney IRI (Figure 5), and on the basis of polyaminecatabolic pathway (schematic diagram) indicating that SSAT upregulationincreases the generation of H2O2, we hypothesized that SSAToverexpression increases the expression of HO-1 via the releaseof H2O2. Accordingly, we first determined whether SSAT overexpressionin vitro results in the upregulation of HO-1. Toward this end,we examined the expression of HO-1 in HEK cells in responseto SSAT overexpression after 24 h of exposure to tetracycline.Figure 6 is a representative Northern hybridization and demonstratesthat the overexpression of SSAT for 24 h upregulates the expressionof HO-1, with mRNA levels increasing by approximately 3.5-fold(P < 0.02, n = 4). To examine the time course of upregulationof HO-1, membrane from Figure 3 was stripped and reprobed withradiolabeled HO-1 cDNA. As demonstrated in Figure 7a, HO-1 expressionpeaked at 24 h after SSAT induction and gradually returned towardbaseline after 96 h. These results indicate that SSAT inductionper se (and in the absence of IRI) increases the expressionof HO-1. The induction of SSAT directly correlated with putrescineconcentration, which peaked at 24 h and returned toward baselinelevels at 96 h after the addition of tetracycline (Figure 7b).
Figure 6. Conditional overexpression of SSAT upregulates the expression of heme oxygenase-1 (HO-1). HO-1 expression was increased after 24 h of SSAT overexpression induced by tetracycline.
Figure 7. Time course of heme oxygenase-1 (HO-1) upregulation in response to SSAT overexpression. (a) The HO-1 expression peaked at 24 h and returned to baseline 96 h after the addition of tetracycline. (b) Putrescine concentration in SSAT overexpressing cells. The peak of HO-1 upregulation at 24 h correlated with the highest concentration of putrescine.
Effect of Catalase on HO-1 Upregulation in Response to SSAT Overexpression
To determine whether the upregulation of HO-1 in response toSSAT overexpression (Figures 6 and 7) is due to the generationof H2O2, the effect of catalase on the induction of HO-1 wasexamined. Cells were exposed to tetracycline in the presenceor absence of catalase (500 units/ml, added to the media 1 hrbefore the addition of tetracycline) and examined 24 h later.As shown in Figure 8, presence of catalase significantly inhibitedthe upregulation of HO-1, with HO-1 mRNA levels decreasing by63% in the catalase treated cells (P < 0.05, n = 4). Theabove studies demonstrate that the upregulation of HO-1 in culturedcells with conditional overexpression of SSAT is partly mediatedvia H2O2 release (Figure 8) and directly correlates with theconcentration of putrescine (Figure 7), the toxic metabolicproduct of polyamine catabolism generated by SSAT overexpression.
Figure 8. Effect of catalase on the upregulation of heme oxygenase-1 (HO-1) in response to overexpression of SSAT. Presence of catalase, 500 IU/ml, at the time of addition of tetracycline significantly prevented the upregulation of HO-1 in response to SSAT overexpression. Left and right panels are two different experiments.
Effect of SSAT Overexpression on Cell Attachment, Morphology, and Cytoskeletal Structure
In the next series of experiments, we examined and comparedthe effect of overexpression of wild-type and inactive SSATmutant on cell morphology. The inactive SSAT mutant was generatedas described in Materials and Methods. The expression of SSATmRNA increased in both the wild-type and inactive SSAT transfectantsin response to tetracycline treatment. After 24 h of stimulationby tetracycline, putrescine concentration increased by sevenfoldin cells expressing the wild-type SSAT and by only 0.8-foldin cells expressing the inactive mutant SSAT, a more than 80%inhibition in polyamine catabolic pathway activity.
To examine the effect of SSAT overexpression on cell morphology,light microscopic analysis of HEK cells was performed in theabsence (control) or presence of tetracycline (SSAT induction).Figure 9a is a light microscopy image of live HEK cells before(control) and after the induction of wild-type SSAT with tetracyclinefor 24 h. Low and high magnification images clearly indicatethat conditional overexpression of SSAT causes the roundingand loss of cell attachment. Morphologic examination of thecells revealed that the induction of SSAT leads to loss of celladhesion processes even in the cells that remain attached tothe substratum. Comparison of induced and uninduced cultures(cell attachment assay studies in Materials and Methods) revealedthat overexpression of SSAT disrupts cell attachment and leadsto a significant increase in the number of nonadherent cells(Figure 9b). As demonstrated, the number of adherent cells significantlydecreased (from 98% to 32%, P < 0.001, n = 3 separate experimentsinvolving a total of nine dishes) and the number of nonadherentcells significantly increased (from 2% to 68%, P < 0.001,n = 3 separate experiments involving a total of nine dishes)in response to SSAT induction. These effects were specific tothe wild-type SSAT because the overexpression of the inactiveSSAT mutant had no significant effect on cell attachment ormorphology (data not shown).
Figure 9. Light microscopic analysis of cell morphology and attachment in response to the induction of SSAT. (a) Cell morphology. As shown, overexpression of SSAT with tetracycline (right) causes the rounding up of cells and detachment of cell anchorage when compared with control (left). Lower magnification (upper) and higher magnification (lower) images were obtained before (left) and after (right) the induction of SSAT. (b) Cell attachment assay. HEK cells were seeded at 1.0 x 105 cells per well in six-well tissue culture plates and treated with tetracycline 48 h later. Twenty-four hours after the addition of tetracycline, the unattached cells were harvested by aspirating the growth medium and the attached cells were harvested by treating the adherent cells with trypsin-EDTA solution. The number of nonadherent and adherent cells was counted with a hemacytometer. As demonstrated, the number of adherent cells were significantly decreased (from 98% to 32%, P < 0.001, n = 3 separate experiments involving a total of nine dishes) and the number of non adherent cells were significantly increased (from 2% to 68%, P < 0.001, n = 3 separate experiments involving a total of nine dishes) in response to SSAT induction. The approximate number of adherent and nonadherent cells in the absence of tetracycline was 9.6 x 105 and 0.195 x 105,/ml, respectively. In the presence of tetracycline, the approximate number of adherent and nonadherent cells was 1.98 x 105 and 4.28 x 105, respectively.
Defects in cell anchorage (Figure 9) point to the cytoskeletonas a major cell structure affected by SSAT overexpression. Inthe last series of experiments, we examined the arrangementof filamentous actin, a major cell cytoskeletal structure, inresponse to conditional overexpression of SSAT in HEK 293 cells.Control and tetracycline-treated cells (Materials and Methods)were plated on fibronectin-coated coverslips for 3 h, then fixedand stained with fluorescence phalloidin (green) to visualizefilamentous actin and DAPI (blue) to visualize nuclei. Nomarskiimages of cells with or without tetracycline induction are depictedin Figure 10 (taken with Zeiss Axioskop, x63 PlanApo lens).As demonstrated, cells are well spread in the control groupbut are significantly less spread in SSAT overexpressing cells.The confocal fluorescence images of cells with or without tetracyclineinduction were stained with Alexafluor 488-phalloidin to visualizefilamentous actin. The results are depicted in Figure 10 anddemonstrate that the thick peripheral band of actin and thefilopodia in the noninduced cells are absent in cells with SSATinduction. These pictures were taken with a Zeiss Confocal 510,x63 PlanApo lens. Figure 10, C and D, are projections of a completethrough-focus series of images.
Figure 10. Phalloidin staining of actin cytoskeleton in response to SSAT overexpression. Cells were treated with tetracycline (tet) or vehicle (control) for 24 h before processing for plating on coverslips. Control (A and C) and tetracycline-treated (B and D) cells were plated on fibronectin-coated coverslips for 3 h, then fixed and stained with fluorescence phalloidin to visualize filamentous actin and DAPI (blue) to visualize nuclei. (A, B) Nomarski images of cells without (A) or with (B) tet induction, illustrating the reduced spreading with SSAT expression. Bar = 20 mm. Taken with Zeiss Axioskop, x63 PlanApo lens. (C, D) Confocal fluorescence images of cells without (C) or with (D) tet induction stained with Alexafluor 488-phalloidin to visualize filamentous actin. Note the thick peripheral band of actin in the noninduced cells and the filopodia (arrows) in (C) that are absent in (D). Bar = 20 mm. Taken with a Zeiss Confocal 510, x63 PlanApo lens. (C) and (D) are projections of a complete through-focus series of images.
Polyamines play a fundamental role in cell growth and proliferation.Under the effect of SSAT, polyamines are catabolized to putrescine.The reactive oxygen intermediate H2O2, is also generated asa toxic metabolic byproduct. The reduction in polyamines decreasescell proliferation, whereas generation of putrescine and H2O2causes cell injury and ultimately death. As a result, variouslaboratories have focused on SSAT as a novel target for thetreatment of solid tumors, including breast cancer, where thedesired reduction in cell growth can be achieved by SSAT upregulation.Enhanced expression of SSAT by spermine analogue in the breastcancer cell line L56Br-C1 resulted in the depletion of the cellularpools of polyamine within 24 to 48 h (25). Cell proliferationappeared to be totally inhibited, and within 48 h of treatment,there was an extensive apoptotic response (25). In Ehrlich ascitestumor cells, treatment with the antitumor drug 1'-acetoxychavicolacetate resulted in increased activity of SSAT with subsequentlowering of intracellular polyamines (26). Apoptosis immediatelyfollowed. Administration of exogenous polyamines prevented acetoxychavicolacetate-induced apoptosis (26). A similar observation has beendocumented in human leukemic cells, where depletion of intracellularpolyamines secondary to the overexpression of SSAT resultedin decreased cell growth and caused the induction of apoptosis(27). These studies indicate that enhanced expression of SSATdepletes the cellular polyamine pools, decreases cell growth,and leads to cell injury.
Enhanced expression of SSAT in kidney IRI was recently reported(10). It was postulated that the upregulation of SSAT in IRIis likely detrimental to cell survival by decreasing growthand causing the release of toxic metabolic products such asH2O2. This was tested in the current studies by developing celllines that conditionally overexpress SSAT in response to tetracycline.The most salient feature of the study presented here was theupregulation of HO-1 and alteration in cell morphology in responseto overexpression of SSAT. It is well known that the expressionof HO-1 is enhanced in conditions associated with oxidativestress (23,24). HO-1 is involved in the degradation of hememolecules to iron, carbon monoxide, and biliverdin, productsthat provide protection against the injury through their antioxidant,antiinflammatory, and cytoprotective activities (17,28). Theupregulation of HO-1 in response to overexpression of SSAT directlycorrelated with putrescine concentration, which is increasedas a result of the sudden breakdown of polyamines (Figure 7).The reduction in HO-1 levels, and presumably oxidative stress,after its peak at 24 h may be the result of a decline in thecatabolic reaction of PAO that yields putrescine and H2O2, mostlikely as a result of the depletion of polyamines, which arethe substrates for SSAT and PAO activities. Alternatively, itis plausible that the return of HO-1 to its baseline after itspeak at 24 h is the result of the generation of antioxidantproducts of HO-1 activity such as bilirubin or carbon monoxide(17,28).
In addition to putrescine, SSAT overexpression results in thegeneration of H2O2, a toxic byproduct of polyamine catabolism.Several studies have demonstrated that the addition of H2O2in cultured cells upregulates the expression of HO-1 (23). Wetested the possibility that the generation of H2O2 by SSAT overexpression(Figures 6 and 7) was responsible for the upregulation of HO-1by incubating the HEK cells with catalase at the time of additionof tetracycline. The results demonstrated that the presenceof catalase decreased the expression of HO-1 by 63% in cellsoverexpressing SSAT (Figure 8). The above experiments demonstratethat the generation of H2O2 and putrescine by SSAT overexpressioncauses oxidative stress in cultured kidney cells.
The light microscopic analysis of live HEK 293 cells that aretransfected with SSAT demonstrate that conditional overexpressionof SSAT significantly altered the morphology of HEK cells (Figure 9a,right panels) when compared with control (Figure 9a, rightpanels). The addition of tetracycline for 24 h caused roundingup and detachment of cell anchorage in cells overexpressingthe SSAT (Figure 9, a and b). This time point (24 h) correlateswith the highest concentration of putrescine and H2O2 (Figure 7).No significant alteration in cell morphology was observedin HEK cells overexpressing the inactive SSAT mutant (data notshown). Cell attachment assays demonstrated increased numberof non adherent and floating cells in response to SSAT induction(Figure 9b). The rounding of the HEK cells and their loss ofanchorage after induction of SSAT expression resembles the detachmentof kidney tubular epithelial cells from the basement membraneobserved in IRI (29–31).
Interestingly, cell cytoskeleton staining revealed significantalterations in assembly of actin (Figure 10). Cells are wellspread in the control group but are significantly less spreadin SSAT-overexpressing cells (Figure 10, A and B). The confocalfluorescence images of cells stained with Alexafluor 488-phalloidinto visualize filamentous actin demonstrated that the thick peripheralband of actin and the filopodia in the noninduced cells areabsent in cells with SSAT induction (Figure 10, C and D). Inadditional studies not shown, we observed the rearrangementof focal adhesion molecule paxicillin in response to SSAT overexpression.In summary, tetracycline induction of SSAT expression renderedcells less able to spread over a fibronectin substratum andaltered the morphology of actin-containing filopodia, suggestingan adhesion defect.
In addition to the kidney, IRI in heart is a major cause ofmorbidity and mortality. We have examined the expression ofSSAT in mice subjected to cardiac IRI. Our results indicatethat the occlusion of left anterior descending artery for 30min causes significant upregulation of SSAT expression in theinjury zone at 6 h after reperfusion (data not shown), indicatingthat the activation of SSAT and polyamine catabolism may bea universal pathway in IRI. As such, SSAT upregulation may playan important role in mediating tissue injury in IRI in a numberof organs in mammals. It is therefore intriguing to speculatethat prevention or inhibition of polyamine catabolic pathwayis a potential target for the treatment of IRI in kidney andheart, which are major causes of morbidity and mortality inthe United States.
In conclusion, conditional overexpression of SSAT in culturedkidney cells resulted in decreased cell growth, altered cellmorphology, and upregulated HO-1 expression, indicating thatSSAT overexpression per se generates cellular phenotypic changesthat mimic those that are observed in IRI. We propose that enhancedSSAT expression in kidney mediates majority of cell damage inIRI. Future studies on the role of SSAT and polyamine catabolicpathway may provide novel treatments for kidney failure in IRI.
Acknowledgments
These studies were supported by NIH grants DK 66589 and 54220(M.S.), CA88843 (R.A.C.), DK54770 (H.R.), a Greater CincinnatiKidney Foundation (K.Z.), a Merit Review Award (M.S.), and grantsfrom Dialysis Clinic Incorporated (M.S.).
Footnotes
Drs. Zhaohui Wang and Kamyar Zahedi contributed equally to thismanuscript.
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Received for publication January 7, 2004.
Accepted for publication April 24, 2004.
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Abstract
Introduction
) is due to the generation of H2O2, the effect of catalase on the induction of HO-1 was examined. Cells were exposed to tetracycline in the presence or absence of catalase (500 units/ml, added to the media 1 hr before the addition of tetracycline) and examined 24 h later. As shown in Figure 8, presence of catalase significantly inhibited the upregulation of HO-1, with HO-1 mRNA levels decreasing by 63% in the catalase treated cells (P < 0.05, n = 4). The above studies demonstrate that the upregulation of HO-1 in cultured cells with conditional overexpression of SSAT is partly mediated via H2O2 release (Figure 8) and directly correlates with the concentration of putrescine (Figure 7), the toxic metabolic product of polyamine catabolism generated by SSAT overexpression. 
