Polyamines play an important role in cell growth and differentiation, while their overproduction has potentially oncogenic consequences. Polyamine homoeostasis, a critical determinant of cell fate, is precisely tuned at the level of biosynthesis, degradation and transport. The enzymes ODC (ornithine decarboxylase), AdoMetDC (S-adenosylmethionine decarboxylase) and SSAT (spermidine/spermine N1-acetyltransferase) are critical for polyamine pool maintenance. Our experiments were designed to examine the expression of these enzymes in testosterone-induced hypertrophic and antifolate-induced hyperplastic mouse kidney, characterized by activation of AR (androgen receptor) and HGF (hepatocyte growth factor) membrane receptor c-Met respectively. The expression of these key enzymes was up-regulated by antifolate CB 3717 injury-evoked activation of HGF/c-Met signalling. In contrast, activation of the testosterone/AR pathway remarkably induced a selective increase in ODC expression without affecting other enzymes. Studies in catecholamine-depleted kidneys point to a synergistic interaction between the signalling pathways activated via cell membrane catecholamine receptors and AR, as well as c-Met. We found that this cross-talk modulated the expression of ODC and AdoMetDC, enzymes limiting polyamine biosynthesis, but not SSAT. This is in contrast with the antagonistic cross-talk between AR- and c-Met-mediated signalling which negatively regulated the expression of ODC, but affected neither AdoMetDC nor SSAT.
- membrane receptor
- nuclear receptor
- polyamine biosynthesis
- receptor-mediated signalling
Polyamines have been widely implicated in the growth and development of mammalian tissues. They play important roles in the remodelling processes associated with tissue repair or apoptosis, depending on environmental signals. Polyamine homoeostasis is regulated by ODC (ornithine decarboxylase) and AdoMetDC (S-adenosylmethionine decarboxylase), enzymes responsible for biosynthesis, and SSAT (spermidine/spermine N1-acetyltransferase) crucial for polyamine catabolism. These enzymes respond to a variety of stimuli affecting cell growth and differentiation. Over recent years, the link between polyamines and cancer has been well established. The synthesis and catabolism of polyamines is modified in response to tumour promoters and carcinogens, leading to increased polyamine levels in tumour cells. Attempts are in progress to use various inhibitors of polyamine metabolism for cancer treatment and prevention [1,2].
The development of transgenic mice with altered polyamine metabolism has contributed to our understanding of the precise physiological functions of individual polyamines [3,4]. Overexpression of the enzymes involved in polyamine metabolism, as well as antizyme, has demonstrated specific roles for polyamines in organ hypertrophy, spermatogenesis, cell cycle regulation, promotion of tumorigenesis and skin physiology [1,4]. Recent studies have focused on how distinct signalling pathways, activated by various extracellular signals, lead to modified expression of the genes coding for enzymes involved in polyamine metabolism. Signal transduction pathways known to modify polyamine homoeostasis can be activated at the cell membrane by a variety of stimuli including growth factors and also by hormones acting through their intracellular receptors. To date, the majority of studies have been conducted in vitro using cultured cells, with relatively few reports from in vivo studies. To address this issue, we have compared the effect of extracellular [HGF (hepatocyte growth factor)-activated] and intracellular (androgen-activated) signalling on the regulation of enzymes which modulate polyamine homoeostasis in the mouse kidney.
Renal polyamine metabolism – experimental models and activation of signalling pathways
The effect of activation of the two major signalling pathways known to modulate the expression of three key regulatory enzymes of polyamine homoeostasis (ODC, AdoMetDC and SSAT) was examined in the female mouse kidney. Kidney hypertrophy was induced by testosterone (125 mg/kg subcutaneously for 5 days). Regenerative kidney hyperplasia was induced by antifolate or folate (300 mg/kg or 250 mg/kg intraperitoneally respectively for 1 day), which is nephrotoxic due to precipitation of the drug in renal tubules at physiological pH [5,6]. Although multiple growth factors participate in the complex process of organ regeneration, HGF and its c-Met tyrosine kinase receptor seem to have a pivotal role in renal regeneration after acute injury . HGF, identical with scatter factor, is a pleiotropic mesenchymal-derived growth factor implicated in the control of several biological processes such as embryogenesis, cell growth, wound healing and organ regeneration. In the injured kidney, HGF, via its cell membrane c-Met receptor, acts as morphogen and mitogen to reconstruct the renal tubule epithelium and to accelerate recovery from acute renal failure [8,9]. In contrast with HGF, c-Met is induced exclusively in the damaged organ and has an important role in the activation of signalling pathways leading to specific physiological cellular responses [8,9].
HGF and its receptor mRNA were significantly increased in antifolate CB 3717/folate injured mouse kidney (Table 1). Notably, the induction of HGF mRNA preceded that of c-Met . Moreover, we observed that increased HGF and c-Met receptor expression preceded the rise in DNA synthesis observed in kidneys damaged by antifolate CB 3717 [6,10].
It is well established that testosterone, an inducer of kidney hypertrophy, acts through the AR (androgen receptor) a member of a superfamily of nuclear receptors . The precise regulation of these ligand-activated transcription factors depends not only on their hormonal ligands but also on a variety of other extracellular signals acting via cell membrane receptors . We found that AR protein but not AR mRNA content was elevated in testosterone-induced hypertrophic kidney. In contrast, in the CB 3717-induced hyperplastic kidney a decrease in both AR protein and mRNA content occurred (M. Dudkowska, T. Jaworski, B. Grzelakowska-Sztabert and M. Manteuffel-Cymborowska, unpublished work). It should be mentioned that testosterone also up-regulated renal c-Met mRNA, an effect that was abolished by the anti-androgen casodex . Together, these results demonstrate that both HGF/c-Met and testosterone/AR signalling pathways are activated in the examined hypertrophic and hyperplastic mouse kidney.
Regulation of the expression of enzymes involved in polyamine metabolism
The activity and mRNA level of the three enzymes crucial for polyamine metabolism (ODC, AdoMetDC and SSAT) and corresponding polyamine levels following stimulation of the testosterone/AR- and HGF/c-Met-mediated signalling pathways are shown in Table 1.
ODC is responsive to diverse stimuli including growth factors and hormones. The expression of ODC is regulated at the level of translation and degradation, and transcriptionally via transcription factors including AP (activator protein) -1, AP-2, Sp1 (specificity protein 1), nuclear receptors and c-Myc . The significant increase in expression of the gene coding for ODC is a characteristic feature of androgeninduced hypertrophic mouse kidney (Table 1), as shown previously by us [6,10,14,15] and others [16–18]. Similarly, oestradiol, another steroid hormone, has been reported to induce ODC expression in the mouse kidney , and also in breast cancer cells, acting in this case through non-genomic cAMP-dependent pathways .
The stimulation factor of testosterone-induced ODC activity in the mouse female hypertrophic kidney reached 870 while in the male mice kidney this was much lower, not exceeding 20 . The weaker responsiveness to testosterone of renal ODC in males is due to their much larger endogenous testosterone pool, which affects basal ODC expression, almost undetectable in the kidneys of female mice but at a considerable level in males.
In contrast with the well-established androgen receptor-mediated induction of ODC expression in the mouse kidney, polyamine metabolism and the signalling pathway activated by antifolate CB 3717 have been investigated exclusively by our group [5,6,10]. We found in both hyperplastic and hypertrophic kidneys that the increase in ODC was much greater at the level of activity rather than at the mRNA level, although these changes were on a smaller scale in the hyperplastic kidney (Table 1). A high dose of folate induced a comparable increase in renal expression of ODC and HGF/c-Met as observed with CB 3717 [5,8,10].
The HGF/c-Met signalling pathway activated at the cell membrane is not the only one which affects renal ODC expression. Indeed, in vitro studies have shown that the application of EGF (epidermal growth factor) can either lead to a reduction in the activity of ODC and AdoMetDC  or stimulation . Moreover, activation of dopamine and adrenergic receptors by selective agonists can induce ODC expression in normal female mouse kidney . ODC induction as a result of stimulation of adrenergic signalling has also been documented in β-adrenoreceptor-mediated cardiac hypertrophy in vitro .
AdoMetDC, the second rate-limiting enzyme of polyamine biosynthesis, was significantly up-regulated at both mRNA and activity levels in hyperplastic but not hypertrophic kidney (Table 1). These results confirm the previous report showing the lack of testosterone responsiveness of AdoMetDC in the mouse and rat kidney in contrast with AdoMetDC induction by testosterone in accessory sex organs .
The regulation of AdoMetDC activity is complex and strongly depends on polyamine content. Translational regulation of AdoMetDC by polyamines, in particular the activating effects of putrescine, has been the subject of intensive investigation [27–29]. Our results have demonstrated that polyamine regulation of AdoMetDC activity also occurs in vivo, a finding in line with those reported by other groups [30,31]. Consistent with this notion, in our experimental model of kidney hyperplasia, we found that the activity of AdoMetDC correlated positively with renal putrescine content and negatively with spermine content.
At present, it is not known whether the HGF/c-Met pathway regulates expression of AdoMetDC. However, it is possible that Akt, activated in hyperplastic kidney , may affect some of the transcription factors [e.g. AP-1, AP-2, CREB (cAMP-response-element-binding protein), Sp1] known to regulate AdoMetDC. AdoMetDC expression is also known to be modulated by insulin in rat hepatoma cells  and by several growth factors, partly through post-transcriptional mechanisms, documented in transformed cells capable of malignant progression .
SSAT is one of the enzymes crucial for polyamine homoeostasis. It prevents the accumulation of polyamines by facilitating their interconversion, excretion and degradation. Basal SSAT activity is usually very low in the cell, but this enzyme may be induced by many physiological and nonphysiological stimuli, including natural polyamines, and superinduced by a plethora of polyamine analogues [35,36]. We have documented that both mRNA content and the activity of SSAT were significantly increased by antifolate CB 3717 (Table 1) and folate injury . In contrast, despite an increase in SSAT mRNA synthesis, a finding consistent with reports from other groups [30,31], SSAT activity was down-regulated in the testosterone-induced hypertrophic kidney. Testosterone-induced reduction in SSAT activity (over 40%) was completely abolished by the anti-androgen casodex, implying a role for androgen receptors in the regulation of SSAT . In line with this finding is a report documenting increase in SSAT activity after the administration of gossypol, an inhibitor of testosterone biosynthesis, and its decrease following testosterone application in canine prostate epithelial cells .
SSAT expression is regulated by several transcription factors including PMF1-1 (polyamine modulated factor-1), Nrf-2 (nuclear factor-E2-related factor-2) , Sp1  and NF-κB (nuclear factor κB) . A parallel increase in SSAT mRNA content and SSAT protein occurs during kidney ischemia-reperfusion injury in rats, and therefore may be considered a new marker of tubular cell injury . Moreover, the results of in vitro studies examining SSAT expression in cultured kidney cells point to inhibition of polyamine catabolism as a therapeutic approach for the prevention of tissue injury in the above model of kidney ischemia-reperfusion injury .
Studies examining the regulation of SSAT expression by distinct signalling pathways have identified stimulatory effects of β-adrenoreceptor in the rat parotid gland  and the possible involvement of PPARγ (peroxisome-proliferator-activated receptor γ) in colorectal cancer .
The cross-talk between signalling pathways activated at the cell membrane affects polyamine biosynthetic enzymes intracellularly
In the mouse kidney, reserpine-evoked catecholamine depletion dramatically impaired both testosterone- and antifolate-induced increases in ODC expression. In both cases, the activity of ODC was almost completely abolished (Figure 1) and mRNA content decreased by 50% and 60% respectively [24,45]. The down-regulation of ODC expression did not affect the extent of kidney hypertrophy, however, it substantially reduced antifolate-induced kidney enlargement. These findings confirm our earlier reports that ODC induction is essential for renal hyperplasia .
Catecholamine depletion also reduced folate-induced renal AdoMetDC activity, exerting however no effect on SSAT activity (Figure 1) . Together, these results suggest a synergistic effect of catecholamine receptor activation on HGF and androgen signalling pathways modulating the expression of both polyamine biosynthetic enzymes but not SSAT. In contrast with these synergistic interactions, the cross-talk between HGF/c-Met and testosterone/AR pathways is antagonistic, and results in the negative regulation of c-Met and ODC expression . Indeed, when both pathways were activated by sequential administration of testosterone and antifolate/folate, we observed a substantial severalfold decrease in CB 3717/folate-induced c-Met mRNA expression (Table 1). Similarly, testosterone-induced ODC mRNA content and activity were decreased by 79% and 73% respectively when CB 3717 treatment followed testosterone application. Antagonism between these pathways was also observed under physiological conditions in male kidneys in which, owing to elevated endogenous testosterone levels, neither ODC activity nor mRNA levels was induced by antifolate/folate, whereas the c-Met mRNA response to these drugs was significantly decreased .
Figure 2 shows a simplified network of testosterone- and HGF-activated pathways and their interconnections. Two different transcription factors, AR and c-Myc, are postulated to be involved in the induction of the odc gene in the kidneys under conditions of testosterone/AR- and HGF/c-Met-activated signalling respectively . Although in the studies on HGF-induced transcription of the mouse odc gene c-Myc regulation has recently been questioned in favour of AP-1 transcription factor , it appears that murine ODC transactivation can be mediated by both transcription factors, c-Myc  and AP-1 .
The negative regulation of ODC expression could be connected with, among others, a decreased level of AR and/or c-Myc. The expression of these transcription factors can be regulated by Akt, a serine–threonine kinase that regulates multiple cellular processes including cell proliferation and survival . Recent reports document that AR is one of the numerous substrates of Akt . The resulting AR phosphorylation leads to the inhibition of AR transactivation and suppression of AR target genes . Moreover, Akt can affect AR degradation  and is indirectly responsible for c-Myc transcription and stability . Our preliminary results show that CB 3717 evokes a 2-fold increase in phosphorylated Akt and therefore it is indeed a downstream effector of the HGF/c-Met pathway . Furthermore, we document that activation of both examined pathways up-regulates, nearly 2-fold, the level of c-Myc protein when compared with that evoked solely by testosterone. This significant increase in c-Myc protein might have functional consequences mirrored by down-regulated expression of ODC. Other effectors involved in the cross-talk between studied signalling pathways are at present under investigation.
Our own studies summarized in this paper were supported by grant no. 2 PO4C 09627 from the Ministry of Education and Science, Poland, with meeting support provided by COST 922.
Health Implications of Dietary Amines: A joint COST Action 922 and Biochemical Society Focused Meeting held at Medico-Chirurgical Hall, University of Aberdeen, U.K., 19–21 October 2006. Organized and Edited by H.M. Wallace (Aberdeen, U.K.).
Abbreviations: AdoMetDC, S-adenosylmethionine decarboxylase; AP, activator protein; AR, androgen receptor; HGF, hepatocyte growth factor; ODC, ornithine decarboxylase; Sp1, specificity protein 1; SSAT, spermidine/spermine N1-acetyltransferase
- © 2007 The Biochemical Society