Cyclosporine-A-induced nephrotoxicity in children with minimal-change nephrotic syndrome: long-term treatment up to 10 years

The impact of cyclosporine A (CsA) therapy in patients with steroid-dependent nephrotic-syndrome (SDNS) on long-term renal function is controversial. Data beyond 5 years are rare. Long-term renal function was evaluated in children with SDNS with and without CsA therapy, especially beyond 5 years. Twenty children were treated with CsA (study group) for a mean of 5.4 ± 2.2 years (ten patients for 5–11 years). Glomerular filtration rate (GFR) was calculated before and after 3 and 12 months and at latest follow-up of therapy. Fifteen children with cyclophosphamide-treated SDNS without CsA served as controls. In the study group, GFR decreased within 12 months from 136 ± 19 to 120 ± 31, to 114 ± 14 ml/min per 1.73 m2 at latest follow-up (p < 0.0001). Patients with CsA > 5 years had a GFR of 111 ± 14 ml/min per 1.73 m2 at latest follow-up without a GFR below 90 ml/min per 1.73 m2. No CsA toxicity was found in biopsies. In the control group, GFR dropped within 3 months, from 137 ± 27 to 130 ± 24, to 126 ± 19 ml/min per 1.73 m2 at latest follow-up (p = 0.1). Patients with and without nephrotoxic CsA therapy showed a drop in GFR. In CsA-treated patients, GFR was about 12% lower at latest follow-up compared with patients without nephrotoxic therapy but always remained within normal range. CsA seems to be safe, even in long-term treatment for more than 5 years.
Introduction
Minimal-change nephrotic syndrome (MCNS) in children is characterised by steroid responsiveness, subsequent relapses and a benign prognosis concerning renal function [1]. Patients with steroid-dependent nephrotic syndrome (SDNS) and frequent relapsing nephrotic syndrome (FRNS) with steroid-toxic side effects are recommended for treatment with cyclophosphamide (CP). The overall rate of cumulative sustained remission after therapy with CP, however, was only 24% after 10 years [2]. The efficacy of cyclosporine A (CsA) in the treatment of steroid-sensitive nephrotic syndrome (SSNS) has been well demonstrated [3–8]. Early withdraw of CsA leads to relapses of the SDNS. So patients remain dependent on CsA for years. CsA was identified as being nephrotoxic, inducing tubulointerstitial fibrosis [9], vasoconstriction leading to reduced renal plasma flow and glomerular filtration rate (GFR) [10]. The potential risk of long-term CsA therapy to induce chronic renal failure in patients with MCNS has been discussed controversially. Seikaly et al. [11] found no drop in GFR but a histological progression of tubulointerstitial lesions in patients treated with CsA in comparison with those without CsA. Hulton et al. [5, 12] demonstrated a significant reduction of GFR within the first 3 months after introduction of CsA. Thereafter, the GFR remained stable. In contrast, Inoue et al. [6] could not detect a significant impairment of renal function in patients with CsA therapy. The interpretation of several studies focussing on the impact of CsA therapy on long-term renal function is limited and controversial.
Most authors investigate the influence of CsA on renal function in nephrotic children with different histologic entities, not considering their different risk to progress to chronic renal failure. Thus Niaudet et al., Habib and Niaudet, and Gregory et al. [7, 13, 14] did not differentiate between MCNS and focal segmental glomerulosclerosis (FSGS) in their studies evaluating long-term renal function in nephrotic children with CsA therapy.The physiological drop in GFR after temporary hyperfiltration during a relapse often has been neglected. Relapse-associated hypoproteinemia and hypoalbuminaemia lead to oedema and increased GFR [15]. Hulton et al. [12] considered that concomitant therapy with steroids could contribute to an increase of GFR due to the mineralocorticoid effect. Consequently, steroid cessation could lead to a physiological drop in GFR.Several studies claim to address the long-term effect of CsA therapy on renal function, but actually, many of them do not extend more than 2–3 years [5, 6, 7, 12, 16], depicting the short-term effect of CsA on renal function.No study evaluated an additional control group without nephrotoxic therapy to demonstrate the physiological development of GFR in patients with SDNS.
In this study, we focus on the long-term renal function in 20 children with biopsy-proven MCNS treated with CsA for a mean of 5.36 ± 2.2 (range 2–11) years. The development of GFR in these patients is compared with a control group with SDNS without CsA therapy.
Patients and methods
Between 1993 and 2004, 20 children (eight girls and 12 boys) with biopsy-proven MCNS were treated with CsA for a mean of 5.4 ± 2.2 (median 5.0, range 2.1–11.0) years. Patients only qualified for this retrospective analysis after a minimum CsA therapy duration of 2 years. The definitions and criteria for NS, remission, relapse and steroid dependency were those used by the International Study of Kidney Disease in Children (ISKDC) and the Arbeitsgemeinschaft Pädiatrische Nephrolgie (APN) [17, 18].
All patients had SDNS and were treated with cyclophosphamide (CP) prior to CsA without achieving sustained remission. CsA treatment was started with a dosage of 100–150 mg/m2 per body surface area (BSA) in two divided doses. CsA dose was adjusted to a target blood level of 80–120 ng/ml. Side effects such as hypertension, hypertrichosis and gingival hyperplasia were not consistently documented and could not be considered in this study. Renal function (GFR) was calculated by the Schwartz formula ([19], κ-factor 0.55) before and after 3 and 12 months and at the latest follow-up of CsA therapy. Renal biopsy under CsA therapy was performed in five patients after 4.9–7.0 years. All patients starting CsA therapy were still on a standard relapse treatment with prednisone (40 mg/m2 every 48 h) that was discontinued after 4 weeks. At the reviews after 3 and 12 months and latest follow-up, all patients were in remission and had no steroid therapy.
In the control group, 15 children (seven girls, eight boys) with SDNS treated with CP were evaluated for long-term renal function. GFR was calculated before and after therapy with CP and at the latest follow-up (mean 4.9 ± 3.4 years). According to APN recommendations, these patients were treated with CP with 2 mg/kg body weight for 12 weeks [18, 20]. As concomitant medication, all patients in the control group received prednisone 60 mg/m2 every 48 h tapered to 10 mg/m2 every 48 h and discontinued when CP was stopped. At latest follow-up, all patients were in remission without further steroid therapy.
Statistical analysis
Statistical analysis was performed employing SPSS 12.0 for windows. Data were expressed as mean ± standard deviation. Student’s t test was performed, and a p value <0.05 was regarded as significant.
Results
Twenty paediatric patients with SDNS were treated with CsA due to failing CP therapy for a mean of 5.4 ± 2.2  (median 5.0, range 2.1–11.0) years. The mean starting dosage was 130 ± 32 mg/m2 BSA per day. The mean CsA dosage after 12 months was 126 ± 26 mg/m2 BSA per day, with a trough level of 99 ± 27 ng/ml. The mean age at the beginning of CsA therapy was 8.4 ± 3.0  (median 8.6, range 3.1–14.2) years. GFR calculated at the beginning, at months 3 and 12 and at the latest follow-up of CsA treatment was 136 ± 19 (median 136), 130 ± 20 (median 135), 120 ± 31 (median 113) and 114 ± 14 (median 114) ml/min per 1.73 m2, respectively (Table 1 and Fig. 1). Unfortunately, data of only 12 patients were available for the calculation of GFR at month 3. The decline in GFR from the beginning to month 12 and to the latest follow-up was significant, with p = 0.025 and p < 0.0001, respectively (Fig. 2). The drop in GFR within the first 12 months was 11.8% and during the whole follow-up 14.9%. Three patients reestablished their GFR after cessation of CsA. No patient had a GFR below 90 ml/min per 1.73 m2, and 17 patients had a GFR above 105 ml/min per 1.73 m2 at latest follow-up. CsA trough level at latest follow-up was 85 ± 62 ng/ml;  mean CsA dosage was 105 ± 34 mg/m2 BSA per day.
Table 1Patient characteristics [mean ± standard deviation (SD)] and renal function calculated by glomerular filtration rate (GFR in ml/min per 1.73 m2; mean ± SD) in patients with cyclosporine A (CsA, study group) and with cyclophosphamide (CP, control group) Study group n = 20Control group n = 15Age at start of MCNS (years)4.4 ± 2.2 4.0 ± 2.9Age at start of CP (years)5.7 ± 2.26.0 ± 3.2Age at start of CsA (years)8.4 ± 3.0–CsA trough level at  month 12 (ng/ml)98.8 ± 26.9–Follow-up time (years)5.4 ± 2.24.9 ± 3.4GFR at start of CP138.7 ± 24.6p = 0.4137.7 ± 27.6p = 0.3Median 132Median 131.6Range 119–181Range 95–196GFR at stop of CP130 ± 30.9130.3 ± 24.7Median 135.9Median 131.1Range 82–164Range 93–178GFR at start of CsA136.3 ± 19.0p<0.0001Median 136.5Range 97–186GFR at latest follow-up114.5 ± 14.5126.4 ± 19.8p = 0.1Median 114.2Median 124.6Range 89–135Range 92–156MCNS minimal-change nephrotic syndrome, GFR glomerular filtration rateFig. 1Development of the glomerular filtration rate (GFR) of all patients on cyclosporin A (CsA) therapy (n = 20)Fig. 2Boxplot demonstrating the development of glomerular filtration rate (GFR) in patients on cyclosporin A (CsA) therapy (n = 20)
CsA was effective in 19 of 20 patients, leading to long-term remission or reduction of relapses to an infrequent relapsing NS (IRNS). Chlorambucil was introduced in one patient due to frequent relapses after CsA cessation. In three patients, arterial hypertension was treated; in one patient, antihypertensive therapy could be stopped after CsA cessation. Ten patients were treated with CsA for more than 5 (median 6.5, mean 7.0 ± 1.8,  range 5.0–11.0) years. GFR at latest follow-up was 111 ± 14   (median 109, range 90–132) ml/min per 1.73 m2. Five patients underwent renal biopsy after 5.1–7.3 years after CsA therapy had been started before they were transferred to adult nephrologists. CsA-associated nephrotoxicity was not reported by an experienced renal pathologist (Prof. U. Helmchen, Hamburg, Germany). CsA was stopped in nine patients (mean age 13.1 ± 2.5 years) after a mean of 4.9 ± 1.9 (median 4.4, range 2.3–9.0) years due to long-term sustained remission in all but one patient. GFR remained normal after CsA cessation, with 120 ± 25 (median 116) and 123 ± 10 (median 125, range 105.18–134.98) ml/min per 1.73 m2 at time of therapy cessation and at latest follow-up, respectively.
The control group consisted of 15 patients with SDNS who were treated with CP at a mean age of 6.0 ± 3.2 (median 4.6) years. At the start of CP, GFR was 138 ± 28 (median 132) ml/min per 1.73 m2. After 3 months, GFR dropped to 130 ± 25 ml (median 131) ml/min per 1.73 m2 (p = 0.3).   GFR at latest follow-up (mean follow-up 4.9 ± 3.4 years) was 126 ± 20 (median 125) ml/min per 1.73 m2  (p = 0.1, Figs. 3 and 4). This drop was in the order of 8% but not statistically significant.
Fig. 3Development of glomerular filtration rate (GFR) of patients without cyclosporin A (CsA) therapy (n = 15)Fig. 4Boxplot demonstrating the development of glomerular filtration rate (GFR) in patients without cyclosporin A (CsA) therapy (n = 15)
Table 1 summarises the development of GFR in both patient groups, starting with the GFR before CP therapy. Patients of both groups had an initial decline in GFR during CP therapy.
Discussion
Histologically, the nephrotoxic effect of CsA has been defined by Mihatch et al. [9]: most frequent lesions attributable to CsA are tubular atrophy, interstitial fibrosis and arteriolar hyalinosis. However, a review of the literature demonstrates that it is difficult to predict the influence of CsA on long-term renal function in patients with MCNS. Some authors did not find a significant reduction in renal function under CsA therapy in nephrotic children [4, 8] and only minor changes in renal histology without progression of CsA tubulointerstitial lesions [14, 21]. Others emphasised reduced GFR [5, 12], reduced renal plasma flow [22] or progression of tubulointerstitial lesions histologically [11, 23] in patients with NS and CsA therapy. Histology progression could not be associated with decrease in renal function [11, 23] or duration of CsA therapy [7]. Myers et al. [24] postulated that in case of striped interstitial fibrosis, some nephrons undergo atrophy while their function could be compensated for by unaffected nephrons. This may mask nephrotoxicity by measurements of normal GFR. In summary, a clear proven association between impaired renal function and long-term CsA therapy in patients with MCNS could not be found. But hints permit the question regarding the safety of long-term CsA therapy in children with a benign renal disease.
Comparison of results of this study with established opinions (e.g. [8, 12, 23]) is difficult:
Most studies mix different aetiological entities with different potential to progress to chronic renal failure (MCNS vs. SRNS and FSGS; [4, 7, 13, 16, 21, 22]). The impairment of renal function in patients with FSGS and other types of SRNS cannot reliably be distinguished from CsA nephrotoxicity, as those entities have a high risk for chronic renal failure on their own.In most studies, the follow-up period is restricted to 2–3 years; in single studies up to 5 years. In this study, at least ten patients underwent long-term CsA therapy > 5 years. No other study investigated patients with such a long treatment period [5–8, 11–14, 23].
Neuhaus et al. and Ganesan et al.  [8, 23] evaluated patients with MCNS only and an extended follow-up time of 5 years. They evaluated nine patients, with one being identified with FSGS as the underlying disease in the follow-up biopsy. All patients received CsA with higher trough levels and higher CsA dosage (level: mean of 220 ng/ml, range 141–270 ng/ml, CsA dosage 126 ± 29 mg/m2 per day) than accepted for our study group. The method for CsA level measurements was a polyclonal assay resulting in higher levels compared with monoclonal assays. Nevertheless, the CsA levels and dosage were still higher than in our study group. CsA toxicity was proven in 3/9 patients histologically. Ganesan et al. [23] found histological signs for CsA toxicity in 79% and renal insufficiency with a GFR < 80 in 4/19 patients without correlation to histological changes. Again, that study mixed patients with steroid-resistant and steroid-responsive NS, not distinguishing between the different clinical entities.
In our study, only patients with histologically proven MCNS were evaluated for long-term renal function under CsA therapy. Patients qualified for the study only after a minimum treatment duration of 2 years. The follow-up period extended over a mean of 5.4 years, with ten patients who had been treated between 5 and 11 years. At least in half of the patients with long-term CsA therapy > 5 years, CsA toxicity was excluded by biopsy. Also in our study group, the GFR of patients with CsA therapy remained within the normal range, with a drop within the first 12 months (mean drop 11.8%) and remaining stable thereafter, with a mean GFR of 115 ± 16 ml/min per 1.73 m2. No patient progressed to chronic renal failure, even after therapy duration of more than 10 years. Hulton et al. [5, 12] showed comparable results for patients with CsA therapy, with an initial drop in GFR and a constant renal function afterwards. But the study is restricted to a short follow-up of 2 years in four patients only. In our study, a significant reincrease in GFR after therapy cessation was not found. In general, these patients showed excellent renal function in the long-term follow-up (mean 123 ml/min per 1.73 m2). In contrast, patients in Hulton et al.’s studies showed a worse GFR of 93 that reincreased back to a GFR > 100 ml/min per 1.73 m2 after CsA had been stopped.
Interestingly, in our control group of patients without CsA therapy, an initial drop in GFR was also found. Those patients showed a mean drop in GFR of about 5% within the 12 weeks of CP therapy, and GFR even dropped about 8% until the latest follow-up. Probably, the observed decrease in GFR in patients with CsA therapy cannot only be attributed to the hitherto described nephrotoxic effect of CsA.
Hulton et al. [12] mentioned the potential increase of GFR caused by the mineralocorticoid effect of concomitant steroid medication. One could speculate that the cessation of steroids might lead to a physiological drop in GFR. Additionally, a possible but not proven interpretation should be discussed: Part of the GFR reduction may be attributable to normalisation of hyperfiltration in nephrotic children during the first months after initiation of immunosuppressive therapy. In patients with relapsing nephrotic syndrome, long-term immunosuppressive drugs (e.g. cyclophosphamide, CsA, chlorambucil) are initiated after urine remission has been achieved; complete remission of serum albumin normally is not awaited.
Relapse-associated hypoproteinemia and hypoalbuminemia lead to oedema and an increase in GFR. This is explained by the decrease of the colloid osmotic pressure in the glomerular capillary. Reduced total serum protein leads to decreased colloid osmotic pressure and consequently to increased GFR [15]. Normalisation of the increased filtration extends over weeks and leads to a decrease in GFR physiologically. This phenomenon may explain the drop in GFR, even in patients without nephrotoxic treatment.
In summary, this study offers long-term follow-up of 20 patients with proven SSNS treated with CsA for a median of 5 years. Initially, the patients showed a drop in GFR but remained stable afterwards. None developed chronic renal failure. Nevertheless, the generalisation of these results are limited, as the study was retrospective, and only 5/20 patients underwent renal biopsy under long-term nephrotoxic treatment. It is questionable whether a prospective study to evaluate the outcome of long-term CsA therapy in children with MCNS is reasonable in the future. Newer, nonnephrotoxic drugs, e.g. mycophenolate mofetil (MMF) are of concern and need to be evaluated for efficacy and safety in the treatment of SDNS and FRNS. However, our analysis demonstrates that even long-term therapy with CsA for more than 5 years in children with MCNS is safe and does not impair renal function.

nephrotoxicity
minimal-change nephrotic syndrome
cyclosporine a
glomerular filtration rate
long-term outcome


The role of ATP and adenosine in the brain under normoxic and ischemic conditions

By taking advantage of some recently synthesized compounds that are able to block ecto-ATPase activity, we demonstrated that adenosine triphosphate (ATP) in the hippocampus exerts an inhibitory action independent of its degradation to adenosine. In addition, tonic activation of P2 receptors contributes to the normally recorded excitatory neurotransmission. The role of P2 receptors becomes critical during ischemia when extracellular ATP concentrations increase. Under such conditions, P2 antagonism is protective. Although ATP exerts a detrimental role under ischemia, it also exerts a trophic role in terms of cell division and differentiation. We recently reported that ATP is spontaneously released from human mesenchymal stem cells (hMSCs) in culture. Moreover, it decreases hMSC proliferation rate at early stages of culture. Increased hMSC differentiation could account for an ATP-induced decrease in cell proliferation. ATP as a homeostatic regulator might exert a different effect on cell trophism according to the rate of its efflux and receptor expression during the cell life cycle. During ischemia, adenosine formed by intracellular ATP escapes from cells through the equilibrative transporter. The protective role of adenosine A1 receptors during ischemia is well accepted. However, the use of selective A1 agonists is hampered by unwanted peripheral effects, thus attention has been focused on A2A and A3 receptors. The protective effects of A2A antagonists in brain ischemia may be largely due to reduced glutamate outflow from neurones and glial cells. Reduced activation of p38 mitogen-activated protein kinases that are involved in neuronal death through transcriptional mechanisms may also contribute to protection by A2A antagonism. Evidence that A3 receptor antagonism may be protective after ischemia is also reported.
Historical overview
The physiological roles of adenosine triphosphate (ATP) and its metabolite adenosine have been historically linked to cell metabolism since ATP is an ubiquitous intracellular energy source in a number of enzymatic processes. However, over the years both metabolites have emerged as very versatile molecules of biological systems, being implicated in a variety of cell processes, from platelet aggregation to neurotransmission. The term ‘purinergic signalling’ was first introduced in the scientific literature by Burnstock [1]. An implicit concept for sustaining the hypothesis of purinergic neurotransmission proposed in the 1970s was the existence of purinergic receptors. The first evidence in this direction suggested the existence of two different subfamilies of such ‘purinoceptors’, identified as P1 and P2 receptors, selective for adenosine and ATP respectively [2]. Four different subtypes of P1-G-protein-coupled receptors: A1, A2A, A2B and A3 are known at present [3], whereas P2 purinoceptors belong to two major families: P2X ligand-gated ion channel receptors and P2Y G-protein-coupled receptors [4]. Cloning experiments supported this classification and helped to subdivide P2 receptors into seven P2X and eight P2Y subtypes [5] (plus the recently deorphanized GPR17 receptor [6]).
Roles of ATP in neurotransmission under normoxic conditions
The first studies on the role of extracellular ATP in hippocampal neurotransmission indicated prominent inhibitory action of this purinergic nucleotide on synaptic activity [7, 8]. However, several lines of evidence led to the hypothesis that ATP-mediated inhibition of hippocampal neurotransmission was probably mediated by adenosine acting on A1 receptors. In fact, this response was theophylline-sensitive [7–9] and absent in A1 knockout mice [10]. This assumption was supported by the fact that, in the hippocampus, extracellular ATP is rapidly converted into adenosine by ecto-ATPases and ecto-nucleotidases [11] and that adenosine acting on A1 receptors exerts a well-described inhibitory role on CA1 excitatory neurotransmission [3]. In recent years, a more detailed observation of ATP-evoked effects, supported by the synthesis of new pharmacological tools, has helped to clarify the effective role of purinergic nucleotides in the hippocampus. In particular, it emerged that ATP mediates inhibitory effects by P2 receptor activation [12–14].
In a recent paper, we contributed to elucidating the role of ATP on CA1 hippocampal neurotransmission, and we reinforced the concept that ATP-mediated effects are not necessarily linked to adenosine formation in this brain region [15]. ATP is hydrolysed by ecto-NTPDase, enzymes located on the cell surface in the CNS that limit ATP, ADP and AMP spatio-temporal activity [11, 16]. There are three different known NTPDases: NTPDase1 hydrolyzes ATP and ADP equally well, NTPDase2 has a high preference for ATP, NTPDase3 is a functional intermediate, preferably hydrolyzing ATP [17]. In our study, we took advantage of some recently synthesized compounds: ARL 67156, which, at micromolar concentrations, inhibits rat NTPDase1 and 3 transiently transfected in Chinese hamster ovary cells, showing negligible activity on NTPDase2 [18]; BGO 136, a new inhibitor described as a selective NTPDase1 and 2 blocker with Ki values in the high micromolar range [19]; and the recently synthesized PV4, which strongly inhibits rat NTPDase1, 2 and 3 with Ki values in the nanomolar range [20]. By using these inhibitors that are able to block NTPDase activity without interfering with P2 receptor activation, we demonstrated that ATP exerts an inhibitory action, independent of its degradation to adenosine. As shown in Fig. 1, the application of ATP during a NTPDase activity blockade still elicits a decrease in evoked synaptic responses which is even more pronounced than that evoked by ATP alone.
Fig. 1a, bThe inhibitory effect induced by ATP on fEPSP amplitude is potentiated in the presence of different NTPDase inhibitors. a Time-course of fEPSP amplitude before, during and after the application of ATP in the absence or in the presence of the NTPDase1,2,3 inhibitor PV4. Each point in the graph represents the mean ± SE of fEPSP value measured as percent of baseline, pre-drug level. b Columns in the graph summarize the average amplitude (mean ± SE) of evoked fEPSP recorded from CA1 hippocampal region in control conditions, 5 min after superfusion of ATP alone and 5 min after ATP superfusion in the presence of different ecto-ATPases inhibitors. Note that the inhibitory effect of ATP on fEPSP amplitude is potentiated by BGO 136, PV4 and ARL 67156. *P < 0.05 one-way ANOVA, Newman-Keuls multiple comparison post-hoc test versus pre-drug value. §P < 0.05, one-way ANOVA, Newman-Keuls multiple comparison post-hoc test versus 10 μM ATP treated slices. (Modified from [15])
An excitatory effect of exogenous ATP on hippocampal neurotransmission has also been reported by different authors. This effect, which was observed after drug removal, was described to persist in in vitro preparations for a relatively prolonged period, up to 1 h [21–25]. This ‘long-lasting’ potentiation of synaptic responses was compared to electrically evoked LTP, firstly described in the hippocampus by Abrams and Kandel [26] and called ‘ATP-induced LTP’. In agreement, we observed a potentiation of the synaptic responses after drug removal when slices were superfused in the presence of the metabolically stable ATP-analogue ATPγS (Fig. 2) [15]. In addition to this ‘long-lasting’ excitatory effect evoked by the exogenous application of P2 agonists, we also demonstrated an excitatory tone exerted by endogenous ATP. Slices superfused with P2 antagonists show a small but significant reduction in synaptic transmission (Fig. 3). These data demonstrate that tonic activation of P2 receptors contributes to glutamatergic excitatory neurotransmission in the hippocampus, an observation that is in line with previous work [27].
Fig. 2Inhibitory and excitatory effects of the stable ATP analogue ATPγS. Averaged time-course (n = 4) of PS amplitude before, during and after the application of different concentrations of ATPγS. PS amplitude (mean ± SE) is measured as percent of baseline level. Upper panels represent single traces recorded in a typical experiment before, during and after ATPγS application at different concentrationsFig. 3Excitatory effects of endogenous ATP. Bars in the graphs represent the average of fEPSP amplitude in the presence of P2 antagonists: PPADS (30 μM) and MRS 2179 (10 μM). *P < 0.05, paired Student’s t-test. (Modified from [15])
Roles of ATP in ischemic conditions
The role of ATP may become critical during pathological conditions such as ischemia, when extracellular ATP concentrations increase. An enhanced outflow of radioactive ATP from hippocampal slices during in vitro ischemic-like insults was first reported by Juranyi and co-workers [28], and the first demonstration that ATP outflow increases in vivo during the induction of focal ischemia in the rat was reported by Melani and colleagues [29]. Thus, at the ischemic site, levels of extracellular nucleotides may remain elevated for long periods of time after injury. Evidence supports the idea that, under such pathological conditions, released ATP may exert an excitotoxic role by acting on its receptors, thus enhancing Ca2+ inward currents and altering synaptic activity and cellular plasticity. Non-selective antagonists of P2 receptors, suramin and PPADS, and the selective antagonists, BBG and MRS 2179 of P2X7 and P2Y1 receptors respectively, prevent the irreversible failure of neurotransmission induced by a prolonged period of OGD in hippocampal slices [15]. Moreover, they protect from development of anoxic depolarization (AD), which is a rapid and regenerative wave of depolarization that propagates in tissue and represents an unequivocal sign of sufferance [15]. Antagonists of P2 receptors have been proved protective against cell death induced by either ATP itself, hypoglycemia or glutamate exposure in primary cultures of brain-derived neurones [30–33]. Intrastriatal ATP injection in rats induced, 24 h later, a clearly lesioned area [34]. The ATP-induced damage was concentration-dependent, mimicked by ATPγS and α,β meATP (but not by ADP or adenosine) and blocked by RB2, a non-specific P2 antagonist [34]. Suramin, another non-specific P2 receptor antagonist, administered 30 min before occlusion of the middle cerebral artery, resulted in a significant decrease in infarct and oedema volume 6 h after brain injury [35]. In agreement, it was demonstrated that RB2 [36] and PPADS [37] improve neurological deficit and reduce the damage induced in rats in a model of focal ischemia in vivo. Moreover, RB2 induces the expression of P2X7 receptors on reactive microglia in the remote ipsi and contralateral cingulate and medial frontal cortex and striatum. Although a pro-apoptotic role has been attributed to the P2X7 receptor, results suggest that microglial cells expressing the P2X7 receptor can be implicated in tissue damage as well as in the defence and repairing processes in the remote ipsi and contralateral undamaged areas [36].
In considering the effect of ATP during ischemia, it must be taken into account that ATP itself is involved in control of cerebrovascular regulation [38] and that the metabolite of ATP, ADP, that interacts with P2Y1/P2Y12 receptors, is a potent platelet aggregator. However, clinical studies that checked the antiplatelet therapy of the selective P2Y12 receptor antagonist clopidogrel reported a response variability [39, 40].
Although evidence suggests that ATP exerts a detrimental role under ischemia, there are reports that it may exert a trophic role in terms of cell division and differentiation in both differentiated and undifferentiated cells [41–43], including adult neural stem cells [44]. Interesting data correlate these trophic effects with the occurrence of spontaneous Ca2+ waves that propagate among adjacent cells in a self-renewing manner. Kawano et al. [45] reported that ATP-induced initiation and propagation of intracellular Ca2+ waves in human mesenchymal stem cells (hMSCs) promote activation of transcription factors (e.g. NFAT) that are involved in cell differentiation. In the same study, they demonstrated that ATP-induced Ca2+ waves disappear in the fully differentiated adipogenic phenotype. We recently reported that ATP is spontaneously released from hMSCs during the early stages of culture (P0-P5). Moreover, we reported that ATP decreases proliferation rate (Fig. 4) and modulates specific ionic current hMSCs [46]. A high extracellular ATP concentration at earlier cell culture passages suggests an important role of ATP in regulating cell differentiation. Increased hMSC differentiation may account for an ATP-induced decrease in cell proliferation.
Fig. 4a, bHuman mesenchymal stem cells in culture spontaneously release ATP that modulates cell proliferation. a Extracellular concentrations of ATP were measured in the medium containing hMSCs and in control medium not containing cells. Data are expressed as mean ± SE, n = 11, unpaired Student’s t-test: *P < 0.0001 vs medium alone. b Effect on hMSC proliferation after daily application of ATP (10 μM) and P2 antagonists, PPADS (30 μM) and MRS 2179 (10 μM). Data are expressed as percentage of proliferation. Proliferation of untreated cells was assumed as 100%. The cell number was determined after 5 days of culture by a culture counter. Each column bar represents the mean ± SE of n = 4 for each experimental condition. Paired Student’s t-test: *P < 0.05 vs respective control; one-way ANOVA, Newman-Keuls post-test: §P < 0.05 vs 10 μM ATP-treated cells. (Modified from [46])
Therefore, it can be envisaged that ATP, as an autocrine/paracrine homeostatic regulator, exerts different effects on cell trophism according to its extracellular concentrations, distinct cell populations involved, differential expression and recruitment of P2 receptors and of more or less sustained stimulation of the same receptor. Balancing these effects may be relevant in the post-ischemic brain, when a neuroregenerative process could promote tissue repair [47]. On this basis, determination of ATP extracellular concentrations at various times after ischemia induction may help to identify which types of receptors may be stimulated under ischemia and explain the role of detrimental versus trophic ATP during ischemia. ATP present in the extracellular space is rapidly metabolised by membrane-bound NTPDase. Extracellular ATP concentrations, evaluated in the brain to date, have been underestimated, since it has not been possible to selectively inhibit ecto-NTPDase. In fact, the selective inhibitor ARL 67156, tentatively used thus far to inhibit ecto-NTPDase, interferes with the ATP assay method [29].
Role of adenosine in cerebral transmission under normoxic conditions
Adenosine exerts an important tonic modulation of synaptic transmission in the brain. This tonic inhibition of synaptic transmission is evoked by stimulation of A1 receptors, as demonstrated in several brain regions, such as the hippocampus, striatum and olfactory cortex [48, 49]. The inhibitory effect of adenosine A1 receptor stimulation has a pre- and postsynaptic component. Activation of the presynaptic A1 receptors reduces Ca2+ influx through the preferential inhibition of N-type and, probably, Q-type channels [50, 51]. Inhibition of presynaptic calcium currents decreases transmitter release [52], and adenosine, by stimulation of A1 receptors, has been found to inhibit the release of virtually all classical neurotransmitters: glutamate, acetylcholine, dopamine, noradrenaline and serotonin (see in [53]). In particular, powerful suppression of glutamate release from presynaptic terminals has been described in the hippocampus [54, 55], where adenosine A1 receptor activation reduces the number of quanta released (but not the size of individual quanta nor postsynaptic glutamate receptor sensitivity) in the Schaffer collateral-commissural pathway [56]. The postsynaptic effect of A1 receptors consists of direct hyperpolarisation of neurones via activation of GIRK channels (Kir 3.2 and 3.4 channels: potassium inward rectifiers) [57, 58]. Endogenous adenosine exerts tonic inhibition of excitatory neurotransmission. The selective A1 antagonist, DPCPX, causes a 15% increase in synaptic potential amplitude in in vitro brain slices [59]. This is an expected result in a brain region where the adenosine concentration at a receptor level was calculated around 200 nM [59, 60] and A1 receptors, whose affinity for adenosine is in the low nanomolar range, are highly expressed. These data are confirmed by the fact that, in slices taken from homozygous A1 receptor knockout mice, no evidence was found for an endogenous inhibitory action by adenosine in the Schaffer collateral pathway in the CA1 region of the hippocampus or at the mossy fibre synapses in the CA3 region [61].
Opposite effects from A1-mediated synaptic inhibition are elicited by A2A receptor activation, which has been shown to mediate excitatory actions in synaptic function [62–64]. In the hippocampus in vitro, A2A receptor stimulation results in a Ca2+-dependent release of acetylcholine [65, 66]. Furthermore, the application of CGS 21680, a selective A2A receptor agonist, decreases the ability of A1 receptor agonists to inhibit excitatory neurotransmission [9, 67]. This effect suggests that A2A receptor stimulation increases synaptic transmission through A1 receptor desensitisation [68, 69]. However, there is also evidence that A2A receptors increase excitatory amino acid release. In fact, the selective stimulation of adenosine A2A receptors augments the amount of glutamate released in the hippocampus and striatum of young rats [70–72].
In spite of the excitatory role in neurotransmission brought about by A2A receptors, the net effect of adenosine is an inhibitory tonus on neurotransmission, in accordance with observations suggesting that activation of A2A receptors requires protracted stimulation to induce evident effects on synaptic transmission [59]. It is worth noticing that the role of A2A receptors in the striatum is recently gaining interest in light of their heterodimerisation with D2 dopamine receptors. The association between A2A and D2 receptors results in an antagonistic interaction that provides a rationale for evaluating A2A-selective antagonists in Parkinson’s disease, supported by epidemiological evidence indicating an inverse relationship between caffeine consumption and the risk of developing this pathology [73, 74]. It was suggested that A2A antagonists not only provide symptomatic relief but also decelerate dopaminergic neurone degeneration in patients.
Discrepancies about the role of adenosine A3 receptors in the brain are present in the literature. Activation of this receptor subtype has been associated with both excitatory and inhibitory effects, even in the same brain region. An excitatory role of A3 receptors has been supported by evidence indicating that, in the rat hippocampus, their activation attenuates LTD and allows induction of LTP elicited by a subliminal weak-burst protocol [75]. In addition, in the same brain area, A3 receptor activation through a selective adenosine A3 agonist has been shown to antagonize the adenosine A1 receptor-mediated inhibition of excitatory neurotransmission [8]. Moreover, A3 receptor stimulation always attenuates the inhibition of hippocampal slice neurotransmission, in a PKC-dependent manner, caused by presynaptic metabotropic glutamate receptors [76]. Whole-cell patch clamp recordings in CA3 hippocampal pyramidal neurones demonstrate that A3 receptor activation results in a significant potentiation of high threshold hippocampal Ca2+ currents by a PKA-dependent mechanism [77]. Finally, facilitation of the onset of epileptiform discharge has been observed in the presence of the selective A3 receptor agonist Cl-IB-MECA [78], and a reduction in such epileptic activity was observed when A3 receptors, activated by endogenously released adenosine during seizures, were blocked by the selective antagonist MRS 1191 [79].
Contrary to previous results, an inhibitory action has been attributed to A3 receptors by Brand and colleagues [80], who demonstrated that, in rat cortical neurones, the selective activation of this adenosine receptor subtype is involved in inhibition of excitatory neurotransmission, suggesting a synergistic action with the inhibitory effect of adenosine brought about by A1 receptor activation. Despite results obtained by A3 receptor stimulation, evidence that a selective block of A3 receptors does not affect neurotransmission in the CA1 region of the hippocampus under normoxic conditions indicates that endogenous adenosine at physiological concentrations does not exert tonic activation of A3 receptors [8, 81]. This is in line with evidence that A3 receptor activation requires micromolar levels of extracellular adenosine, which can be reached only during pathological conditions of impairment in energy supply (for example during hypoxia or ischemia [82–85]).
Role of adenosine in ischemic conditions
Extracellular adenosine concentrations increase dramatically during ischemia [82, 86, 87]. During ischemia, following the imbalance between ATP degradation and resynthesis, the intracellular concentration of adenosine increases. Therefore, increased adenosine concentrations in the extracellular space during ischemia are likely due to the equilibrative transporter that carries adenosine out of cells. Adenosine formation can also take place at the extracellular level, through the hydrolysis of extracellular ATP operated by NTPDases and ecto-5′-nucleotidase (e5′-NTs) (see Fig. 5). Hence, these enzymes have a dual function in modulating purinergic neurotransmission: (1) they rapidly interrupt ATP-mediated signalling by degrading extracellular nucleotides and (2) they give rise to extracellular adenosine, which activates P1 receptors. However, recent evidence in vitro suggests that released ATP does not substantially contribute to the adenosine concentration in the extracellular milieu during ischemia [88].
Fig. 5Schematic drawing of intracellular and extracellular adenosine formation. In the extracellular space, adenosine and ATP act on own purinergic receptor subtypes: P1 and P2 receptors, respectively. ADP adenosine diphosphate, AMP adenosine monophosphate, ATP adenosine triphosphate, e5′-NT ecto-5′-nucleotidase, 5′-NT 5′-nucleotidase, NTDPase ecto-nucleoside triphosphate diphosphohydrolases, P1 adenosine receptor, P2 ATP receptor, SAH S-adenosylhomocysteine, T bidirectional nucleoside transporter. (Modified from [142])
Adenosine-potentiating agents, which elevate endogenous adenosine levels by either inhibiting its metabolism by adenosine deaminase or kinase [89, 90] or preventing its transport [91, 92], offer protection against ischemic neuronal damage in different in vivo ischemia models. Moreover, adenosine infusion into the ischemic striatum during transient focal ischemia proves to significantly ameliorate the neurological outcome and reduce infarct volume [93].
A temporal correlation exists between adenosine outflow and synaptic potential inhibition in rat hippocampal slices during ischemia-like conditions [84, 88]. Synaptic inhibition during ischemia is greatly dependent on adenosine, which, by stimulating A1 receptors, exerts a protective role by reducing the Ca2+ influx, thus counteracting the presynaptic release of excitatory neurotransmitters [55, 94] and, in particular, glutamate, which exerts an excitotoxic role during ischemia mainly by overstimulation of NMDA receptors [95]. By directly increasing the K+ and Cl− ion conductances, adenosine stabilises the neuronal membrane potentials, thus reducing neuronal excitability [96]. Consequent reductions in cellular metabolism and energy consumption [97] and moderate lowering of the body/brain temperature [98] are protective in ischemia.
A1 receptor agonists are shown to attenuate ischemic or excitotoxic neuronal damage in both in vitro and in vivo models of cerebral ischemia (for review, see [99, 100]). In accordance, adenosine A1 antagonists given acutely exacerbate the damage induced by ischemia in different animal models of ischemia. An unselective A1 receptor antagonist, theophylline, increased mortality [101, 102]. Unlike acute treatment, chronic administration of A1 agonists worsened survival and increased neuronal loss [103], a phenomenon thought to depend on A1 receptor desensitization.
Although data converge in demonstrating a neuroprotective effect of adenosine through A1 receptors during ischemia, the use of selective A1 agonists is hampered by unwanted peripheral effects, e.g. sedation, bradycardia, hypotension [104]. Von Lubitz and co-workers [105] have reported that post-ischemic administration of the A1 receptor agonist adenosine amine congener (ADAC), which induces fewer undesirable effects, increases survival in gerbils. Moreover, we may consider that administration of agents that elevate the local concentration of adenosine at the injury site, by inhibiting its metabolism to inosine or rephosphorylation to AMP or reuptake, may have the advantage of restricting the effect of such inhibitors to areas of injury-induced adenosine release [106].
More recently, the role of A2A receptors in ischemic neuroprotection has been studied. Gao and Phillis [107] demonstrated for the first time that the non-selective A2A receptor antagonist CGS 15943 reduces cerebral ischemic injury in the gerbil following global forebrain ischemia. Subsequently, many reports have confirmed the neuroprotective role of A2A receptor antagonists in different models of ischemia. The selective A2A receptor antagonist SCH 58261 reduced ischemic brain damage in neonatal [108] and adult [83, 109] rat models of focal cerebral ischaemia. The same antagonist, subchronically administered, was protective against both brain damage and neurological deficit in the adult rat model of focal cerebral ischemia [110, 111]. Studies in A2A receptor knockout mice supported the neuroprotective role of A2A receptor antagonists on ischemic brain damage [112]. The beneficial effects of A2A antagonists in stroke were mainly attributed to reduced glutamate outflow [83, 110, 113]. Prolonged application of the A2A selective agonist CGS 21680 significantly reduced synaptic depression brought about by OGD in the hippocampus [59] and the same agonist promotes glutamate release under normoxic and ischemic conditions [70, 71, 114, 115]. In addition to neurones, adenosine A2A receptors are located on microglia [116, 117] and astrocytes [118]. On glial cells, A2A receptors mediate inhibition of the glutamate uptake transporter, GLT-1, and stimulate glutamate outflow [119]. A2A antagonists prevent the increase in glutamate levels induced by glutamate uptake inhibitors [120]. Therefore, the protective effects of A2A antagonists in brain ischemia may be largely due to reduced glutamate outflow from neurones and glial cells.
Further support of a major role for glia in the neuroprotective effect of A2A antagonism in ischemia comes from the observation that subchronic administration of the A2A receptor antagonist SCH 58261 reduces p38 mitogen-activated protein kinase (MAPK) activation in striatal and cortical microglia 24 h after permanent focal ischemia [111]. Evidence indicates that p38 MAPK is activated in glia up to 24 h after ischemia [121, 122] and is involved, through transcriptional mechanisms, in neuronal death [123, 124]. Since SCH 58261 reduces glutamate outflow in the first hours after ischemia [83], reduced p38 MAPK activation may be due to a direct effect on glial A2A receptors or may be secondary to a reduction in the excitotoxic cascade that primes p38 activation [125]. The importance of A2A receptors under ischemia is highlighted by the observation that A2A receptor expression increases on neurones and microglia after ischemia [126]. Selective inactivation of A2A receptors on bone-marrow-derived cells (wild-type mice transplanted with A2A receptor knockout bone marrow cells) attenuates infarct volumes and ischemia-induced expression of several proinflammatory cytokines in the brain [127]. Therefore, protective effects of A2A antagonists may be attributed to inhibition of inflammation product production.
In several studies, A2A receptor agonists have been found to be protective in the global ischemia model in the gerbil [128, 129]. Jones and co-workers [130] show that peripheral administration of the A2A receptor agonist CGS 21680 protects the hippocampus against kainate-induced excitotoxicity. However, the direct injection of CGS 21680 into the hippocampus failed to afford protection, while the A2A antagonist ZM 241385, when injected directly into the hippocampus, reduced kainate-induced neuronal damage [131]. These data suggest that the neuroprotective properties of A2A agonists are mainly due to peripherally mediated effects. Major mechanisms that may account for A2A-mediated protection include inhibition of platelet aggregation and vasodilation [89] and anti-inflammatory actions. A2A receptors on neutrophils may account for inhibition of adhesion to endothelial cells and ensuing production of free radicals [132, 133].
On this basis, adenosine and its receptors are considered targets for therapeutic implementation in the treatment of stroke. At the moment, a possible adenosynergic therapeutic strategy after ischaemia that is worth consideration is that of increasing adenosine concentrations at the ischemic sites by inhibitors of adenosine metabolism or reuptake in association with adenosine A2A antagonists. When considering the possible use of adenosine kinase inhibitors, it should be taken into account that adenosine represents only a small percentage of nucleotide content [134], therefore inhibition of its rephosphorylation to ATP by adenosine kinase inhibitors does not weigh upon the ATP content.
The few studies present in the literature concerning the role of A3 receptors in the pathophysiology of cerebral ischemia are rather contradictory. We have demonstrated that selective antagonism of A3 receptors facilitates the recovery of synaptic activity induced by ischemic preconditioning in rat hippocampal slices [81]. A harmful role of A3 receptors during in vitro OGD was confirmed by our observation that blocking the A3 adenosine receptor consistently abolishes or delays the occurrence of anoxic depolarization (AD) and significantly protects from the irreversible disruption of excitatory neurotransmission caused by a severe ischemic episode [135]. These results are in agreement with the observation that acute administration of a selective adenosine A3 agonist exacerbates the damage elicited by global ischemia in the gerbil [136]. On the contrary, it was demonstrated that chronic pre-ischemic administration of an A3 agonist protects against ischemic neuronal damage [136]. This effect may be attributed to desensitisation of A3 receptors. In fact, both human and rat A3 receptors are desensitised within a few minutes after agonist exposure [137, 138].
Contrary to the above information, Hentschel and colleagues [139] demonstrated that under hypoxic conditions, selective activation of A3 adenosine receptors brings about an inhibition of excitatory neurotransmission on cortical neurones, indicating that A3 receptors may sustain the neuroprotective action of adenosine induced by A1 receptors. Consistent with these reports, mice lacking A3 adenosine receptors show increased neurodegeneration in response to repeated episodes of moderate hypoxia [140] or an increase in cerebral infarction after transient ligation of the middle cerebral artery [141]. These opposite results regarding an excitatory or inhibitory role of A3 receptors on synaptic activity under hypoxia/ischemia may be reconciled by our recent data [135], suggesting that in a first phase of ischemia, A3 receptors play a protective synergistic role with A1 receptors. Severe ischemia would transform the A3 receptor-mediated effects from protective to injurious.
Taken together, these data suggest that the outcome of A3 receptor stimulation on synaptic transmission during hypoxic/ischemic phenomena depends on the intensity and duration of stimulation.
Concluding remarks
Purinergic signalling, e.g. adenosine and ATP, by activating specific membrane receptors (P1 and P2 respectively), is strictly correlated in orchestrating brain cell functions either under physiological normoxic or ischemic circumstances. However, the respective contribution of each single element is hard to discern from the total outcome, due to the rapid and ubiquitous enzymatic interconversion of these molecules.
In the brain, both compounds are likely to play a role in neuronal activity, and the inhibitory action of endogenous adenosine in excitatory neurotransmission (mainly due to A1 receptor activation) is well documented. Conversely our data, in line with previous observations in the literature, suggest that tonic activation of P2 receptors contributes to excitatory transmission in the hippocampus. Such an effect is modest, but it is unmasked by the application of P2 purinergic antagonists that reduce evoked synaptic responses.
An inhibitory effect elicited by exogenous ATP application has been frequently described and is mostly due to enzymatic degradation to adenosine and the subsequent activation of A1 receptors. However, our evidence demonstrates that ATP itself may inhibit hippocampal synaptic transmission since an even more pronounced effect is observed when enzymatic ATP degradation is blocked. Moreover, in our studies in the hippocampus, a potentiation of neurotransmission by the ATP analogue, ATPγS, develops a few minutes after the inhibitory effect has been washed out, and it persists for 45 min to 1 h. Within the brain, ATP is involved in the synaptic plasticity phenomenon and may be an important mediator of long-lasting effects in synaptic plasticity.
Either adenosine or ATP seems to become a particularly important signalling molecule under pathological conditions, such as ischemia, when the extracellular concentration of either compound drastically rises. It is well known that adenosine exerts important neuroprotective effects during brain ischemic insults by activating adenosine A1 receptors, which profoundly inhibit synaptic transmission and in particular the release of glutamate, known to contribute to ischemic damage. Under severe ischemia, a prolonged stimulation of both A2A and A3 receptors may be deleterious. The role of endogenously released ATP during cerebral ischemia is mainly deleterious, as found in our work and in the literature, since the application of P2 antagonists always results in a reduction in ischemic damage. The mechanisms underlying these effects are still unknown, but they can be correlated to the tonic excitatory action of endogenous ATP found in many brain regions (for example in the hippocampus and prefrontal cortex).
Purinergic signalling has an ancient phylogenetic origin. Expression of P2 receptors on cell membrane and responses to extracellular ATP are found in primitive prokaryotic species up to evolved animals and plants, suggesting an important and highly conserved role of extracellular purine nucleotides during evolution. Similarly, ontogenetic development in several species seems to involve purinergic signalling, especially during its first stages. Our observation that hMSCs, an undifferentiated line of cells able to originate a number of different cell lineages (adipocytes, chondrocytes, osteoblasts, neurones), spontaneously release ATP into the extracellular space and express functional purinergic P2 receptors, which modulate different kinds of membrane currents, and that ATP, during the early stages of culture, inhibits proliferation indicates an autocrine/paracrine mechanism of action for extracellular ATP in modulating cell functions of undifferentiated stem cells at early developmental stages. Such effects may be important in brain neurogenesis during development and in responses to neurodegenerative stimuli.

atp
adenosine
ischemia
ecto-atpase inhibitors
adenosine a2a receptors
p2 purinergic receptors


A2 isoform of mammalian translation factor eEF1A displays increased tyrosine phosphorylation and ability to interact with different signalling molecules

The eEF1A1 and eEF1A2 isoforms of translation elongation factor 1A have 98% similarity and perform the same protein synthesis function catalyzing codon-dependent binding of aminoacyl-tRNA to 80S ribosome. However, the isoforms apparently play different non-canonical roles in apoptosis and cancer development which are awaiting further investigations. We hypothesize that the difference in non-translational functions could be caused, in particular, by differential ability of the isoforms to be involved in phosphotyrosine-mediated signalling.
1
Introduction
Translation elongation factor 1A (eEF1A) is one of the core members of the elongation machinery providing high efficiency and processivity of the protein synthesis process in the eukaryotic cell (Negrutskii & El'skaya, 1998). eEF1A forms a ternary complex with aminoacyl-tRNA and GTP and delivers the correct aminoacyl-tRNA to the A site of mRNA programmed ribosome in the GTP hydrolysis-dependent mode. The GDP-bound form of eEF1A has been shown to interact with deacylated tRNA (Petrushenko et al., 1997) and can transport it to aminoacyl-tRNA synthetase as the tRNA recycling arm of a tRNA channelling cycle (Petrushenko, Shalak, Budkevich, Negruskii, & El'skaya, 2002).
It is believed that the translation function of eEF1A might be combined with its involvement in cytoskeletal network maintenance and various signalling pathways (Lamberti et al., 2004). eEF1A has been shown to interact with phospholipase C-gamma1 (PLC-gamma1) (Chang et al., 2002; Kim et al., 1999) and non-receptor tyrosine kinase Txk of the Tec family (Maruyama, Nara, Yoshikawa, & Suzuki, 2007). Importantly, Txk can form a complex with poly(ADP-ribose) polymerase 1 and eEF1A to influence interferon-gamma gene transcription in Th1 cells. Recently Lau, Castelli, Lin, and Macaulay (2006) identified eEF1A as a novel binding partner for Akt2 rho-associated kinase. In most cases it is not known whether interactions of eEF1A with protein kinase might lead to phosphorylation of eEF1A. Neither has the role of eEF1A in signal transduction pathways been identified.
In mammalian cells, two tissue- and development-specific isoforms, eEF1A1 and eEF1A2, are present. The expression of the 98% similar isoforms is mutually exclusive (Kahns et al., 1998). Interestingly, eEF1A2, which is present normally only in muscles and neurons, was recently associated with tumor development in tissues that normally express only eEF1A1 (Amiri et al., 2007; Anand et al., 2002). How eEF1A2 isoform is involved in malignant transformation is not as yet understood. Since tyrosine kinases are prominent players in cancer development, direct comparison of the ability of eEF1A1 and eEF1A2 to be involved in phosphoTyr-specific signalling processes could help to interpret cancer-related properties of eEF1A2.
In this study, we compared for the first time the interactions of eEF1A1 and eEF1A2 with SH2 and SH3 domains of various signalling molecules. Contrary to eEF1A1, eEF1A2 was able to interact with SH2 domains of Grb2, RasGAP, Shc and Shp2 as well as with SH3 domains of Crk, Fgr, Fyn and PLC-gamma1. Both eEF1A1 and eEF1A2 formed complexes with SH2 domain of PLC-gamma1. In vivo interaction between endogenous eEF1A and tyrosine phosphatase Shp2 was confirmed in HEK293 cell line and tyrosine phosphorylation of eEF1A1 and eEF1A2 was demonstrated by Western blots with anti-phosphotyrosine-specific antibodies. Importantly, the level of phosphorylation of eEF1A2 was higher than eEF1A1 when isolated from tissues, and also when overexpressed in cells. Furthermore, the level of phosphorylation has been shown to be crucial for in vitro complex formation of eEF1A2 and SH2 domain of Shp2.
2
Materials and methods
2.1
Cell lines and plasmid constructs
HEK293 cells were purchased from the American Type Culture Collection (Manassas, VA) and grown according to their instructions. To produce eEF1A1 and eEF1A2 stable cell lines pcDNA 3.1 eEF1A1 and eEF1A2 with the C-terminal His-tags were linearized by MunI and transfected in HEK293 cells using ExGene500 (Fermentas) according to manufacturer recommendations. G418 was used to select stable cell lines.
2.2
Bioinformatics
ScanSite Program was used for bioinformatic identification of possible SH2 and SH3 domains binding sites within eEF1A1 and eEF1A2. ScanSite employs the matrix of selectivity values for amino acids at each position relative to an orienting residue as determined by the oriented peptide library technique (Obenauer, Cantley, & Yaffe, 2003).
2.3
Purification of the eEF1A1 and eEF1A2 isoforms
eEF1A1 was purified from rabbit liver using a combination of the gel filtration, anion exchange, cation exchange and hydroxyapatite chromatographies as described previously (Budkevich et al., 2002). eEF1A2 was isolated from rabbit muscles using the same procedure except the gel-filtration step was omitted in some cases. To confirm the biological activity of the isolated isoforms, the GDP/[3H]GDP exchange was carried out as in (Carvalho, Carvalho, & Merrick, 1984).
2.4
Pull down assay
Purified GST-SH2 and GST-SH3 domains of various signalling molecules were kindly provided by Prof. Ivan Gout (London, UK) (Gout et al., 1993). In a pull down assay, 1.5 μg of GST, GST-SH2 or GST-SH3 domains fusion protein was incubated with glutathione-Sepharose 4B beads (Amersham Biosciences) for 2 h at 4 °C. The beads were then washed to remove unbound proteins and incubated at 4 °C for 3 h with 1.5 μg of purified eEF1A1 or eEF1A2. Non-specific interactions were removed by extensive washing and bound proteins eluted by boiling in Laemmli sample buffer. Eluted proteins were resolved by SDS-PAGE, transferred to polyvinylidene difluoride membrane, and detected by immunoblotting with specific antibodies.
2.5
Phosphatase treatment
Calf intestine alkaline phosphatase (CIAP) (Fermentas) has been used to dephosphorylate eEF1A1 and eEF1A2. The proteins were precipitated from the lysates of stable cell lines on NiNTA and extensively washed with 1× reaction buffer for CIAP (10 mM Tris–HCl (pH 7.5), 10 mM MgCl2), then 10 U of CIAP was added and reaction mix was incubated at 37 °C for 1 h. Thereafter the beads were washed two times with 1× reaction buffer and the proteins bound were eluted by boiling in Laemmli sample buffer.
For dephosphorylation of total protein fraction HEK293 cells overexpressing eEF1A1 or eEF1A2 were extracted with 1× CIAP reaction buffer. To facilitate lysis the cells were passed through a 20-gauge needle 20 times and whole cell extracts were centrifuged at 10,000 × g for 15 min at 4 °C. The supernatant was incubated with 10 U of CIAP at 37 °C for 1 h, in control samples sodium orthovanadate was added to final concentration 1 mM.
2.6
Immunoprecipitation
HEK293 cells were treated under indicated conditions, then washed with ice-cold phosphate-buffered saline (PBS) and extracted with the lysis buffer containing 10 mM Tris–HCl (pH 7.5), 150 mM NaCl, 5 mM EDTA, 50 mM NaF, 1% Triton X-100, 1 mM sodium orthovanadate and a mixture of protease inhibitors (Roche Applied Science). Whole cell extracts were centrifuged at 10,000 × g for 15 min at 4 °C. Endogenous protein complexes were immunoprecipitated from 0.5 mg of total cell lysates with corresponding antibodies (1 μg/probe) and immobilized on protein A-Sepharose beads (Amersham Biosciences). Immune complexes were washed three times with the lysis buffer and used for immunoblotting. As a loading control Western blotting (WB) of total cell lysate with anti-β-actin has been used. The effective serum starvation (24 h in medium without serum) and stimulation (10% FBS, 30 min) has been confirmed in WB of total cell lysates with antibodies against phosphorylated residues S240/S244 in ribosomal protein S6, which is phosphorylated by S6K1/2 (effector kinases in PI3K/mTOR pathway) in response to growth factors stimulation.
2.7
Immunoblot analysis
Proteins were separated by SDS-PAGE, transferred onto polyvinylidene difluoride membrane, and incubated for 1 h with blocking solution (2% BSA, Tris-buffered saline, 0.1% Tween-40). After blocking the membranes were probed 2 h at 4 °C with anti-GST, anti-eEF1A (Upstate Biotechnology), anti-His tag (Cell Signalling), anti-Shp2 (Cell Signalling) or anti-pTyr 4G10 (Upstate Biotechnology) antibodies. After extensive washing with the solution of Tris-buffered saline and 0.1% Tween-40, the membranes were incubated with secondary horseradish peroxidase-conjugated antibody for 1 h at room temperature. The antigen–antibody complexes were detected using the ECL system (Millipore). When immunoblots had to be re-probed, the membranes were initially stripped and re-blocked prior to incubation with another type of primary antibody.
3
Results
3.1
Existence of SH2/SH3 domain binding sites in the eEF1A1 and eEF1A2 molecules are predicted by bioinformatic analysis
First we examined the possibility if the 98% similar eEF1A1 and eEF1A2 might have distinct binding motifs for signalling molecules. A low stringency Scansite (Obenauer et al., 2003) search revealed potential SH2/SH3 domain containing protein partners and predicted the existence of several phosphorylation sites for tyrosine kinases in both isoforms (Fig. 1). However, the SH3 recognition motif specific for Src kinase and adaptor protein Crk was found only in the eEF1A2 molecule. This is an important indication of the possibility of differential involvement of eEF1A2 in the formation of signalling complexes.
3.2
eEF1A1 and eEF1A2 interact in vitro with SH2/SH3 domains of different proteins
The ability of eEF1A1 and eEF1A2 to form complexes with SH2 and SH3 domains of different signalling molecules was directly analysed in pull down experiments. The preparative amounts of native eEF1A1 and eEF1A2 were purified from rabbit tissues as described in Section 2. Importantly, the rabbit and human proteins are 100% identical. SH2 domains of signalling proteins Grb2, RasGAP, PLC-gamma1, Shc, Shp2 and SH3 domains of Crk, Csk, Fgr, Fyn, Src were expressed as recombinant proteins fused with GST, purified from bacteria, immobilized on glutathione sepharose and mixed with eEF1A1 or eEF1A2. Complexes of eEF1A1 and eEF1A2 with SH2 and SH3 domains of different proteins were detected (Fig. 2A and B). Both eEF1A1 and eEF1A2 interacted with SH2 domain of PLC-gamma1, while eEF1A2 exclusively formed complexes with SH2 domains of Grb2, RasGAP, Shc, Shp2 and SH3 domains of Crk, Fgr, Fyn, PLC-gamma1.
It should be noted that principal possibility of the interaction of PLC-gamma1 and eEF1A was shown earlier. The interactions of SH2 and SH3 domains (Kim et al., 1999) or PH domain (Chang et al., 2002) of PLC-gamma1 with eEF1A were reported. Our results confirmed the interaction of eEF1A with SH2 and SH3 domains of PLC-gamma1 and were used as internal positive control of interaction with known eEF1A binding partner.
Contrary to eEF1A1, eEF1A2 was found to interact with SH2/SH3 domains of several adaptor proteins. Grb2 and Shc provide a critical link between cell surface growth factor receptors and the Ras and Myc signalling pathways via interactions with cytoplasmic Tyr kinases, G-proteins and other cellular Tyr-phosphorylated proteins. Interestingly, Shc is constitutively activated (tyrosine-phosphorylated) in most breast cancer cells where non-typical expression of eEF1A2 was reported (Frackelton et al., 2006; Tomlinson et al., 2005).
Identification of RasGAP protein as a direct partner of G-protein eEF1A2 may be interesting in two ways. On the one hand, RasGAP may play GTPase activating role for eEF1A2, because the initial rate of GTP hydrolysis by the intrinsic eEF1A2 GTPase is slightly lower when compared with eEF1A1 (Kahns et al., 1998 and our unpublished observation). On the other hand, eEF1A2 may influence RasGAP activity and consequently modulate Ras.
eEF1A2 interacts also with SH3 domain of Crk which is involved in MAPK signalling, actin cytoskeleton reorganization and insulin receptor signalling (Feller, 2001). It should be noted that eEF1A is involved in actin cytoskeleton regulation as well (Gross & Kinzy, 2005; Shiina, Gotoh, Kubomura, Iwamatsu, & Nishida, 1994).
Fgr and Fyn are Tyr kinases that participate, in particular, in the MAPK/ERK and insulin receptor signalling pathways involved in the control of initiation of translation (Alper & Bowden, 2005). The interaction of eEF1A2 with SH3 domains of Fgr and Fyn suggests eEF1A2 as a prospective substrate for these enzymes.
Thus, eEF1A2 is capable of forming complexes with SH2 and SH3 domains of several signalling molecules. The potentially increased role of eEF1A2, as compared to eEF1A1, in phosphoTyr-mediated signalling pathways lends support for the putative oncogenic role assigned to eEF1A2 earlier (Amiri et al., 2007; Lamberti et al., 2004; Lee, 2003).
3.3
eEF1A1 and eEF1A2 are in complex with Shp2 in vivo
Verification of the in vitro formed complexes was done in vivo taking as an example interaction of eEF1A with Shp2.
First, HEK 293 stable cell lines overexpressing eEF1A1-His, eEF1A2-His or GFP protein were used to confirm that endogenous Shp2 protein interacts with eEF1A2. Interestingly, specific binding of endogenous Shp2 to both isoforms of eEF1A was detected (Fig. 3A). Since only the C-terminal SH2 domain of Shp2 was used as a bait in the experiments in vitro, we suggest eEF1A1 and eEF1A2 bind to different domains of Shp2.
Also, a complex of endogenous eEF1A and Shp2 was immunoprecipitated from HEK293 cells using anti-eEF1A antibodies (Fig. 3B). The complex eEF1A/Shp2 was detected in growing cells. Interestingly, complex formation was more effective in cells synchronized by growth factors starvation and less efficient in cells stimulated with serum, indicating the regulatory nature of this interaction (Fig. 3B). Importantly, eEF1A2 was shown earlier to accumulate in G0 rather than in G1 and S phase cells (Ann et al., 1991). However, since anti-eEF1A monoclonal antibodies are specific both for eEF1A1 and eEF1A2, and the presence of both isoforms in embryonic cells is possible, it is difficult to determine precisely which isoform of eEF1A interacts with Shp2 in vivo.
3.4
eEF1A2 and Shp2 interaction is sensitive to dephosphorylation
To test if the interaction of eEF1A2 with C-terminal SH2 domain of phosphatase Shp2 is dependent on phosphorylation status of eEF1A, equal amounts of total cell lysates of the stable cell lines expressing eEF1A1 or eEF1A2 were treated with calf intestine alkaline phosphatase, or, in control samples, incubated with phosphatase inhibitor (sodium orthovanadate). Specific interaction of SH2-Shp2 and eEF1A2 was detected only in the samples not treated with CIAP indicating that phosphorylation of eEF1A2 is a necessary condition for eEF1A2/Shp2 complex formation (Fig. 3C).
3.5
eEF1A1 and eEF1A2 are phosphorylated at Tyr in vivo
The interaction of eEF1A with SH2 domains of different proteins in vitro as well as the eEF1A–Shp2 complexes formation in vivo indicate the possibility of tyrosine phosphorylation in the eEF1A molecules. To test if native eEF1A1 and eEF1A2 contain such modifications we probed the isolated eEF1A proteins with monoclonal antibodies specific to phosphorylated tyrosines (4G10). As a result, tyrosine phosphorylation of both isoforms was detected (Fig. 4A). Moreover, the phosphorylation of eEF1A1 and eEF1A2 at tyrosine residues was confirmed using the His-tagged eEF1A isoforms precipitated on Ni-NTA sepharose (Fig. 4B). The eEF1A1 and eEF1A2 samples dephosphorylated with calf intestine alcaline phosphatase were used to control specificity of 4G10 antibodies. Notably, eEF1A2 was characterized by greater phosphorylation level as compared to eEF1A1 in both experiments.
4
Discussion
Besides its function in translation, eEF1A is thought to be involved in several cellular processes, including embryogenesis, senescence, oncogenic transformation, cell proliferation and organization of cytoskeleton (reviewed in Gross & Kinzy, 2005; Lamberti et al., 2004; Lee, 2003). There are two tissue-specific isoforms of eEF1A. eEF1A1 functions in all mammalian tissues except neurons, cardiomyocytes and myotubes, where eEF1A2 is taking role and is expressed. Recently eEF1A2 was also found to express in human pancreatic islets (Ahmed, Forsberg, & Bergsten, 2005). Few attempts to differentiate the specific functions of the isoforms have been carried out so far. In vitro, eEF1A2 has slightly decreased translation functions [our unpublished observation]. Discovery of putative oncogenic role of eEF1A2 in ovarian cancer (Lee, 2003) as well as opposite apoptosis-related functions of the isoforms (Chang & Wang, 2007) indicated a possibility of distinct participation of eEF1A1 and eEF1A2 in cellular signalling processes. The mechanisms of cancer-related functions of eEF1A2 are not known. We suggested that difference of the isoforms could be related to their distinct ability to be involved into phosphoTyr-mediated signalling pathways.
To examine such a possibility we used the panel of cloned SH2 and SH3 domains of various signalling molecules. SH2 domains are known to associate with specific phosphoTyr-containing sites (Schlessinger & Lemmon, 2003). SH3 domains of adaptor proteins can support the association with a number of signalling proteins and upon cell stimulation can engage adaptor proteins with Tyr-phosphorylated cytoplasmic or membrane-attached partners (Li, 2005). As a result, we detected the interaction of eEF1A1 and eEF1A2 with SH2 domain of PLC-gamma1 indicating that both isoforms could contain phosphoTyr residues. In addition, eEF1A2 was found associated with SH2 domains of Grb2, RasGAP, Shc and Shp2 (Fig. 2A). This suggests eEF1A2 plays a more universal role in signal pathways. The finding that eEF1A2 rather than eEF1A1 can interact with SH3 domains of Crk, Fgr and Fyn supports this notion (Fig. 2B).
The association of eEF1A and Shp2 is very appealing in terms of the role Shp2 is playing in the cell-activation of Ras, with subsequent activation of PI3K and MAPK signalling pathways (Mohi & Neel, 2007). In addition, Shp2 regulates actin cytoskeleton reorganization by modulating the activity of small GTP-binding protein Rho (Burks & Agazie, 2006; Harada, Katoh, & Negishi, 2005). It is interesting that eEF1A is an actin-binding protein which can regulate actin bundling as well, but its target substrates have not been identified so far (Murray, Edmonds, Liu, & Condeelis, 1996). Since direct binding of eEF1A to Shp1 was recently found to have some regulatory consequence, namely eEF1A2-dependent increase of Shp1 phosphatase activity (Nandan, Yi, Lopez, Lai, & Reiner, 2002) the examination of possible effect of eEF1A2 on the Shp2 activity is a tempting task for future investigations.
We have observed direct interaction of eEF1A2 with SH2 domain of RasGAP protein. Here we can only speculate about possible involvement of eEF1A2 in the activation of PI3K and MAPK pathways by Ras through RasGAP inactivation (Yarwood, Bouyoucef-Cherchalli, Cullen, & Kupzig, 2006). Otherwise, RasGAP may play a role of GAP for eEF1A2. The last notion could be supported by the observation that the subunits of eEF1B, regular exchange factor for eEF1A1, were not identified as interacting partners for eEF1A2 (Mansilla et al., 2002).
The phosphorylation of native and overexpressed eEF1A1 and eEF1A2 at tyrosine residues was confirmed by WB with phosphotyrosine-specific antibodies (Fig. 4A and B). Several preps of eEF1A1 and eEF1A2 were analysed and eEF1A2 always demonstrated higher phosphorylation level than eEF1A1 (data not shown).
Recently, phosphorylation of Tyr 29 and Tyr 141 of eEF1A was found during an extensive search for all proteins containing phosphotyrosine in human cancer cells (Rush et al., 2005). However, since both identified tryptic peptides contained phosphoTyr29 or phosphoTyr141 (which are identical in eEF1A1 and eEF1A2) and the overall search was limited by the peptides containing phosphotyrosine, the unambiguous identification of the phosphorylated isoform in that case was hardly possible.
Thus, we have identified several binding partners of eEF1A2 previously unknown. Novel interaction of eEF1A with tyrosine phosphatase Shp2 has been confirmed also in vivo. Potentially augmented ability of eEF1A2 as compared to eEF1A1 to be involved in key signal transduction complexes could be linked to the putative oncogenic role of the former found recently in ovarian cancer (Anand et al., 2002). The definite role of eEF1A2 in activation of cell proliferation awaits further investigations.