Activation of endoplasmic reticulum stress response by enhanced polyamine catabolism is important in the mediation of cisplatin-induced acute kidney injury.
ABSTRACT: Cisplatin-induced nephrotoxicity limits its use in many cancer patients. The expression of enzymes involved in polyamine catabolism, spermidine/spermine N1-acetyltransferase (SSAT) and spermine oxidase (SMOX) increase in the kidneys of mice treated with cisplatin. We hypothesized that enhanced polyamine catabolism contributes to tissue damage in cisplatin acute kidney injury (AKI). Using gene knockout and chemical inhibitors, the role of polyamine catabolism in cisplatin AKI was examined. Deficiency of SSAT, SMOX or neutralization of the toxic products of polyamine degradation, H2O2 and aminopropanal, significantly diminished the severity of cisplatin AKI. In vitro studies demonstrated that the induction of SSAT and elevated polyamine catabolism in cells increases the phosphorylation of eukaryotic translation initiation factor 2? (eIF2?) and enhances the expression of binding immunoglobulin protein BiP/GRP78) and CCAAT-enhancer-binding protein homologous protein (CHOP/GADD153). The increased expression of these endoplasmic reticulum stress response (ERSR) markers was accompanied by the activation of caspase-3. These results suggest that enhanced polyamine degradation in cisplatin AKI may lead to tubular damage through the induction of ERSR and the consequent onset of apoptosis. In support of the above, we show that the ablation of the SSAT or SMOX gene, as well as the neutralization of polyamine catabolism products modulate the onset of ERSR (e.g. lower BiP and CHOP) and apoptosis (e.g. reduced activated caspase-3). These studies indicate that enhanced polyamine catabolism and its toxic products are important mediators of ERSR and critical to the pathogenesis of cisplatin AKI.
Project description:The expression of catabolic enzymes spermidine/spermine N(1)-acetyltransferase (SSAT) and spermine oxidase (SMO) increases after ischemic reperfusion injury. We hypothesized that polyamine catabolism is upregulated and that this increase in catabolic response contributes to tissue damage in endotoxin-induced acute kidney injury (AKI). SSAT mRNA expression peaked at threefold 24 h following LPS injection and returned to background levels by 48 h. The activity of SSAT correlated with its mRNA levels. The expression of SMO also increased in the kidney after LPS administration. Serum creatinine levels increased significantly at approximately 15 h, peaking by 24 h, and returned to background levels by 72 h. To test the role of SSAT in endotoxin-induced AKI, we injected wild-type (SSAT-wt) and SSAT-deficient (SSAT-ko) mice with LPS. Compared with SSAT-wt mice, the SSAT-ko mice subjected to endotoxic-AKI had less severe kidney damage as indicated by better preservation of kidney function. The role of polyamine oxidation in the mediation of kidney injury was examined by comparing the severity of renal damage in SSAT-wt mice treated with MDL72527, an inhibitor of both polyamine oxidase and SMO. Animals treated with MDL72527 showed significant protection against endotoxin-induced AKI. We conclude that increased polyamine catabolism through generation of by-products of polyamine oxidation contributes to kidney damage and that modulation of polyamine catabolism may be a viable approach for the treatment of endotoxin-induced AKI.
Project description:We have previously showed that platinum drugs up-regulate SSAT and SMO and down-regulate ODC and SAMDC in the polyamine pathway. Several studies including our own established that platinum drugs combined with polyamine analog DENSPM produces synergistic increase in SSAT activity with polyamine depletion. Since polyamine pathway is an important therapeutic target, we investigated whether agents containing both platinum and polyamines have similar effects on the polyamine pathway. Two complexes i) Pt-spermine with two cisplatin molecules linked to a spermine in the center and ii) Pd-spermine with similar structure i, but Pd (II) substituted for Pt (II) were analyzed with respect to their effect on the expression of genes in polyamine pathway, SSAT and SMO protein expression, SSAT activity and polyamine pools. Pt-, Pd-spermine complexes induced significant down-regulation of SMO, arginase 2 and NRF-2, with no change in SSAT, while cisplatin as a single agent or in combination with DENSPM induced significant up-regulation of SSAT and SMO. The SSAT activity was not induced by either Pt- or Pd-spermine in A2780 cells; SMO protein levels were significantly elevated compared to the no-drug control and to a similar extent as cisplatin/DENSPM. The Pd-spm treatment induced a fall in putrescine levels to 33%, spermidine to 62% and spermine to 72% while Pt-spm did not induce such a decline. Comparative cytotoxicity studies in A2780 cells indicated the potency to be cisplatin> Pd-Spm>Pt-Spm. Although both complexes exhibit a lower potency, the degree of resistance itself is much lower for Pt-spermine and Pd-spermine in that order (2.5 and 7.5, respectively) compared to cisplatin ( approximately 12) as tested in cisplatin resistant A2780/CP cells. These studies suggest that Pd (II)-polyamine complexes may constitute a promising group of inorganic compounds for further studies in the development of novel chemotherapy/adjuvant chemotherapy strategies.
Project description:BACKGROUND:Polyamine catabolism plays a key role in maintaining intracellular polyamine pools, yet its physiological significance is largely unexplored. Here, we report that the disruption of polyamine catabolism leads to severe cerebellar damage and ataxia, demonstrating the fundamental role of polyamine catabolism in the maintenance of cerebellar function and integrity. METHODS:Mice with simultaneous deletion of the two principal polyamine catabolic enzymes, spermine oxidase and spermidine/spermine N1-acetyltransferase (Smox/Sat1-dKO), were generated by the crossbreeding of Smox-KO (Smox-/-) and Sat1-KO (Sat1-/-) animals. Development and progression of tissue injury was monitored using imaging, behavioral, and molecular analyses. RESULTS:Smox/Sat1-dKO mice are normal at birth, but develop progressive cerebellar damage and ataxia. The cerebellar injury in Smox/Sat1-dKO mice is associated with Purkinje cell loss and gliosis, leading to neuroinflammation and white matter demyelination during the latter stages of the injury. The onset of tissue damage in Smox/Sat1-dKO mice is not solely dependent on changes in polyamine levels as cerebellar injury was highly selective. RNA-seq analysis and confirmatory studies revealed clear decreases in the expression of Purkinje cell-associated proteins and significant increases in the expression of transglutaminases and markers of neurodegenerative microgliosis and astrocytosis. Further, the ?-Synuclein expression, aggregation, and polyamination levels were significantly increased in the cerebellum of Smox/Sat1-dKO mice. Finally, there were clear roles of transglutaminase-2 (TGM2) in the cerebellar pathologies manifest in Smox/Sat1-dKO mice, as pharmacological inhibition of transglutaminases reduced the severity of ataxia and cerebellar injury in Smox/Sat1-dKO mice. CONCLUSIONS:These results indicate that the disruption of polyamine catabolism, via coordinated alterations in tissue polyamine levels, elevated transglutaminase activity and increased expression, polyamination, and aggregation of ?-Synuclein, leads to severe cerebellar damage and ataxia. These studies indicate that polyamine catabolism is necessary to Purkinje cell survival, and for sustaining the functional integrity of the cerebellum.
Project description:Polyamines spermine and spermidine are highly regulated, ubiquitous aliphatic cations that maintain DNA structure and function as immunomodulators and as antioxidants. Polyamine homeostasis is disrupted after brain injuries, with concomitant generation of toxic metabolites that may contribute to secondary injuries. To test the hypothesis of increased brain polyamine catabolism after traumatic brain injury (TBI), we determined changes in catabolic enzymes and polyamine levels in the rat brain after lateral controlled cortical impact TBI. Spermine oxidase (SMO) catalyzes the degradation of spermine to spermidine, generating H2O2 and aminoaldehydes. Spermidine/spermine-N(1)-acetyltransferase (SSAT) catalyzes acetylation of these polyamines, and both are further oxidized in a reaction that generates putrescine, H2O2, and aminoaldehydes. In a rat cortical impact model of TBI, SSAT mRNA increased subacutely (6-24 h) after TBI in ipsilateral cortex and hippocampus. SMO mRNA levels were elevated late, from 3 to 7 days post-injury. Polyamine catabolism increased as well. Spermine levels were normal at 6 h and decreased slightly at 24 h, but were normal again by 72 h post-injury. Spermidine levels also decreased slightly (6-24 h), then increased by approximately 50% at 72 h post-injury. By contrast, normally low putrescine levels increased up to sixfold (6-72 h) after TBI. Moreover, N-acetylspermidine (but not N-acetylspermine) was detectable (24-72 h) near the site of injury, consistent with increased SSAT activity. None of these changes were seen in the contralateral hemisphere. Immunohistochemical confirmation indicated that SSAT and SMO were expressed throughout the brain. SSAT-immunoreactivity (SSAT-ir) increased in both neuronal and nonneuronal (likely glial) populations ipsilateral to injury. Interestingly, bilateral increases in cortical SSAT-ir neurons occurred at 72 h post-injury, whereas hippocampal changes occurred only ipsilaterally. Prolonged increases in brain polyamine catabolism are the likely cause of loss of homeostasis in this pathway. The potential for simple therapeutic interventions (e.g., polyamine supplementation or inhibition of polyamine oxidation) is an exciting implication of these studies.
Project description:Fourteen polyamine analogues, asymmetric or symmetric substituted spermine (1-9) or methoctramine (10-14) analogues, were evaluated as potential inhibitors or substrates of two enzymes of the polyamine catabolic pathway, spermine oxidase (SMOX) and acetylpolyamine oxidase (PAOX). Compound 2 turned out to be the best substrate for PAOX, having the highest affinity and catalytic efficiency with respect to its physiological substrates. Methoctramine (10), a well-known muscarinic M2 receptor antagonist, emerged as the most potent competitive PAOX inhibitor known so far (Ki?=?10?nM), endowed with very good selectivity compared with SMOX (Ki=1.2??M vs SMOX). The efficacy of methoctramine in inhibiting PAOX activity was confirmed in the HT22 cell line. Methoctramine is a very promising tool in the design of drugs targeting the polyamine catabolism pathway, both to understand the physio-pathological role of PAOX vs SMOX and for pharmacological applications, being the polyamine pathway involved in various pathologies.
Project description:BACKGROUND:Airway epithelial injury is a crucial component of acute and severe asthma pathogenesis and a promising target for treatment of refractory asthma. However, the underlying mechanism of epithelial injury remains poorly explored. Although high levels of polyamines, mainly spermine, have been found in asthma and comorbidity, their role in airway epithelial injury and the cause of their altered levels in asthma have not been explored. METHODS:We measured key polyamine metabolic enzymes in lung samples from normal and asthmatic subjects and in mice with OVA-induced allergic airway inflammation (AAI). Polyamine metabolism was modulated using pharmacologic/genetic modulators. Epithelial stress and apoptosis were measured by TSLP levels and TUNEL assay, respectively. RESULTS:We found loss of the polyamine catabolic enzymes spermidine/spermine-N (1)-acetyltransferase-1 (SAT1) and spermine oxidase (SMOX) predominantly in bronchial epithelial cells (BECs) of human asthmatic lung samples and mice with AAI. In naïve mice, SAT1 or SMOX knockdown led to airway hyper-responsiveness, remodeling, and BEC apoptosis. Conversely, in mice with AAI, overexpression of either SAT1 or SMOX alleviated asthmatic features and reduced TSLP levels and BEC apoptosis. Similarly, while pharmacological induction of SAT1 and SMOX using the polyamine analogue bis(ethyl)norspermine (BENSPM) alleviated asthmatic features with reduced TSLP levels and BEC apoptosis, pharmacological inhibition of these enzymes using BERENIL or MDL72527, respectively, worsened them. Spermine accumulation in lungs correlated with BEC apoptosis, and spermine treatment caused apoptosis of human BEAS-2B cells in vitro. CONCLUSIONS:Spermine induces BEC injury. Induction of polyamine catabolism may represent a novel therapeutic approach for asthma via reversing BEC stress.
Project description:We recently generated a transgenic mouse line overexpressing spermidine/spermine N1-acetyltransferase (SSAT) gene under its own promoter. The tissue polyamine pools of these animals were profoundly affected and the mice were hairless from early age. We have now generated another transgenic-mouse line overexpressing the SSAT gene under the control of a heavy-metal-inducible mouse metallothionein I (MT) promoter. Even in the absence of heavy metals, changes in the tissue polyamine pools indicated that a marked activation of polyamine catabolism had occurred in the transgenic animals. As with the SSAT transgenic mice generated previously, the mice of the new line (MT-SSAT) suffered permanent hair loss, but this occurred considerably later than in the previous SSAT transgenic animals. Liver was the most affected tissue in the MT-SSAT transgenic animals, revealed by putrescine overaccumulation, significant decrease in spermidine concentration and >90% reduction in the spermine pool. Even though hepatic SSAT mRNA accumulated to massive levels in non-induced transgenic animals, SSAT activity was only moderately elevated. Administration of ZnSO4 further elevated the level of hepatic SSAT message and induced enzyme activity, but not more than 2- to 3-fold. Treatment of the transgenic animals with the polyamine analogue N1,N11-diethylnorspermine (DENSPM) resulted in an immense induction, more than 40000-fold, of enzyme activity in the liver of transgenic animals, and minor changes in the SSAT mRNA level. Liver spermidine and spermine pools were virtually depleted within 1-2 days in response to the treatment with the analogue. The treatment also resulted in a marked mortality (up to 60%) among the transgenic animals which showed ultrastructural changes in the liver, most notably mitochondrial swelling, one of the earliest signs of cell injury. These results indicated that, even without its own promoter, SSAT is powerfully induced by the polyamine analogue through a mechanism that appears to involve a direct translational and/or heterogenous nuclear RNA processing control. It is likewise significant that overexpression of SSAT renders the animals extremely sensitive to polyamine analogues.
Project description:We have generated a hybrid transgenic mouse line overexpressing both ornithine decarboxylase (ODC) and spermidine/spermine N(1)-acetyltransferase (SSAT) under the control of the mouse metallothionein (MT) I promoter. In comparison with singly transgenic animals overexpressing SSAT, the doubly transgenic mice unexpectedly displayed much more striking signs of activated polyamine catabolism, as exemplified by a massive putrescine accumulation and an extreme reduction of hepatic spermidine and spermine pools. Interestingly, the profound depletion of the higher polyamines in the hybrid animals occurred in the presence of strikingly high ODC activity and tremendous putrescine accumulation. Polyamine catabolism in the doubly transgenic mice could be enhanced further by administration of zinc or the polyamine analogue N(1),N(11)-diethylnorspermine. In tracer experiments with [(14)C]spermidine we found that, in comparison with syngenic animals, both MT-ODC and MT-SSAT mice possessed an enhanced efflux mechanism for hepatic spermidine. In the MT-ODC animals this mechanism apparently operated in the absence of measurable SSAT activity. In the hybrid animals, spermidine efflux was stimulated further in comparison with the singly transgenic animals. In spite of a dramatic accumulation of putrescine and a profound reduction of the spermidine and spermine pools, only marginal changes were seen in the level of ODC antizyme. Even though the hybrid animals showed no liver or other organ-specific overt toxicity, except an early and permanent loss of hair, their life span was greatly reduced. These results can be understood from the perspective that catabolism is the overriding regulatory mechanism in the metabolism of the polyamines and that, even under conditions of severe depletion of spermidine and spermine, extremely high tissue pools of putrescine are not driven further to replenish the pools of the higher polyamines.
Project description:Spermidine/spermine N(1)-acetyltransferase (SSAT), a key enzyme in mammalian polyamine catabolism, undergoes rapid turnover (half-life approx. 30 min) and is highly inducible in response to polyamine analogues such as bis(ethyl)spermine (BE-3-4-3), which greatly stabilize the enzyme. Rapid degradation of SSAT in reticulocyte lysates was preceded by formation of a ladder of ubiquitinated forms, and required the production of high-molecular-mass complexes with ubiquitin (HMM-SSAT-Ubs). Mutation of all 11 lysines in SSAT separately to arginine demonstrated that no single lysine residue is critical for its degradation in vitro, but mutant K87R had a significantly longer half-life, suggesting that lysine-87 may be the preferred site for ubiquitination. Mutations at the C-terminus of SSAT, such as E171Q, resulted in marked stabilization of the protein, due to the lack of formation of the HMM-SSAT-Ubs. Addition of BE-3-4-3 prevented the accumulation of ubiquitin conjugates and the proteasomal degradation of wild-type SSAT. These results indicate that conformational changes brought about by the binding of polyamine analogues prevent the efficient polyubiquitination of SSAT, leading to a major increase in the amount of SSAT protein, and that alteration of the C-terminal end of the protein has a similar effect in preventing the productive interaction with an E2 or E3 component of the ubiquitin pathway.
Project description:It has been shown that in a mouse model overexpressing spermidine/spermine N1-acetyltransferase (SSAT) in whole body the mice have improved glucose homeostasis, reduced white adipose tissue (WAT) mass and high basal metabolic rate. In this study we investigated the glucose and energy metabolism in another SSAT overexpressing mouse model (MT-SSAT) and in addition created a novel mouse model that has adipose tissue specific SSAT overexpression (aP2-SSAT) in order to elaborate the role of enhanced polyamine catabolism in WAT. In this experiment, gene expression profiles of WAT from wild type, SSAT, MT-SSAT and aP2-SSAT mice were studied.