Neuronal calcium sensor proteins are unable to modulate NFAT activation in mammalian cells Calcium activated gene transcription through Nuclear Factor of Activated T-cells , -LRB- NFAT -RRB- proteins , is emerging as a ubiquitous mechanism for the control of important physiological processes . Of the five mammalian NFAT isoforms , transcriptional activities of NFATs 1-4 are stimulated by a calcium driven association between the ubiquitous phosphatase calcineurin and the calcium-sensing protein calmodulin . Published in vitro evidence has suggested that other members of the calmodulin super-family , in particular the neuronal calcium sensor -LRB- NCS -RRB- proteins , can similarly modulate calcineurin activity . In this study we have assessed the ability of NCS proteins to interact directly with calcineurin in vitro and report a specific if weak association between various NCS proteins and the phosphatase . In an extension to these analyses we have also examined the effects of over-expression of NCS-1 or NCS-1 mutants on calcineurin signalling in HeLa cells in experiments examining the dephosphorylation of an NFAT-GFP reporter construct as a readout of calcineurin activity . Results from these experiments indicate that NCS-1 was not able to detectably modulate calcineurin/NFAT signalling in a live mammalian cell system , findings that are consistent with the idea that calmodulin and not NCS-1 or other NCS family proteins is the physiologically relevant modulator of calcineurin activity . 1 Introduction Calcineurin -LRB- CN -RRB- -LSB- 1 -RSB- is a serine/threonine specific protein phosphatase present in all eukaryotes that is responsible for the modulation of numerous cell signalling pathways . Calcineurin interacts with the ubiquitous EF-hand containing calcium -LRB- Ca2 + -RRB- sensor calmodulin -LRB- CaM -RRB- -LSB- 2 -RSB- and this obligate association drives calcium dependent phosphatase activity , one of the primary cellular targets of which are the Nuclear Factor of Activated T-cell -LRB- NFAT -RRB- family of transcription factors . This family of gene regulatory proteins is composed of five members -LRB- NFAT1-5 -RRB- -LSB- 3,4 -RSB- , that were first identified as fundamental components of the recombinatorial immune system of vertebrates -LSB- 5 -RSB- , but which have now been implicated in a far broader range of diverse and important physiological processes . Expression of NFAT proteins is not restricted to cells of the immune system , demonstrated by their presence and effect on key functions in a selection of other cell and tissue types including brain and muscle . In a neuronal setting , NFATs have been implicated in axonal remodelling , synaptic plasticity and memory function -LSB- 6-9 -RSB- . Transcriptional events mediated by activated NFATs have been linked to aspects of cardiovascular development -LSB- 10,11 -RSB- and they have also been suggested to drive proliferative and apoptotic pathways in epithelial cells , fibroblasts , pre-adipocytes , pancreatic β-cells and osteoblasts -LSB- 12-16 -RSB- . The discovery that NFAT proteins can influence cell cycle regulation has led to the proposal that they may also be involved in certain aspects of tumorigenesis -LSB- 17 -RSB- . NFAT proteins in unstimulated cells are heavily phosphorylated on multiple serine residues by of a number of constitutively active kinases -LSB- 18 -RSB- . This modification acts to mask a nuclear localisation signal and under such circumstances NFATs remain predominantly cytosolic and transcriptionally inactive . Stimulation of cells with agonists coupled to the phospholipase-C/IP3 generating pathway , depletion of endoplasmic reticulum Ca2 + stores and subsequent sustained elevation of intracellular Ca2 + concentration -LRB- -LSB- Ca2 + -RSB- i -RRB- due to opening of plasma membrane Ca2 + release activated Ca2 + channels elicits the rapid dephosphorylation of NFAT proteins , their nuclear import and upregulation of target gene expression -LSB- 3 -RSB- . Under such circumstances , the Ca2 + signal is integrated upstream of NFAT by CN in conjunction with CaM and it is this protein complex that represents the critical regulatory mechanism for NFAT dephosphorylation and hence activation of NFAT dependent gene expression . The relationship between CN/CaM and NFATs is so deeply intertwined that nuclear translocation of NFAT proteins has been employed as a robust assay of CN activity in intact mammalian cells -LSB- 19-21 -RSB- and has even found application in large scale screens for proteins involved in calcium induced calcium release -LSB- 22 -RSB- . CaM is the primordial member of a superfamily of small EF-hand containing Ca2 + binding proteins that function to transduce spatial and temporal cellular Ca2 + signals . Other members of the CaM family include the neuronal calcium sensor -LRB- NCS -RRB- subgroup of neuronal/neuroendocrine specific proteins -LSB- 23,24 -RSB- . Although only distantly related -LRB- 21 % sequence identity between CaM and NCS-1 -RRB- published in vitro studies have identified potential overlap in target protein interactions between CaM and NCS-1 including an apparent positive interaction of both proteins with CN -LSB- 25 -RSB- . Since CaM exhibits high and ubiquitous expression these observations led to the speculation that NCS proteins may simply represent redundant or alternative modulators of classical CaM targets . Evidence from our laboratory and others has gone some way to disproving this idea with the identification of NCS specific binding proteins that , at present , have no documented interaction with CaM -LSB- 26 -RSB- . Further evidence for distinct functionality of the NCS protein family is apparent from their higher Ca2 + affinity compared to CaM , their differential cellular/tissue distributions and their non-redundancy revealed in genetic studies -LSB- 23,27 -RSB- . Together these data indicate that NCS proteins possess the requisite biochemical properties to allow them respond to unique Ca2 + signals and to bind to distinct effector proteins . The possibility exists however that overlap between CaM and NCS target specificities may be of physiological significance and in the case of CN , identification of a functionally relevant interaction with NCS proteins would be of great interest in view of recent findings implicating CN activity in aspects of neuronal function . In order to resolve some of the outstanding questions concerning potentially redundant functions of NCS proteins we have examined whether or not they are able to elicit CN activation in the physiologically relevant setting of intact mammalian cells using NFAT activation as a coupled reporter . We present in vitro biochemical data indicating that various recombinantly expressed NCS family members are able to directly bind CN although to lower levels compared with CaM . Consistent with these data we have also determined that expression of NCS-1 , mutants of this protein that have previously been demonstrated to disrupt NCS-1 function in vivo , along with other NCS family members have no effect on NFAT dephosphorylation in live HeLa cells in response to elevation of -LSB- Ca2 + -RSB- i . These data are supported by studies indicating that the nuclear translocation of NFAT on elevation of -LSB- Ca2 + -RSB- i to cell nuclei is unaffected in cells expressing NCS-1 or NCS-1 mutants . We therefore suggest that NCS proteins , although exhibiting a detectable in vitro affinity for CN , may not be likely to act as physiologically relevant modulators of CN activity or NFAT mediated gene transcription in intact cells . 2 Materials and methods 2.1 Recombinant proteins All recombinant GST fusion proteins used in this study were expressed and purified as previously described -LSB- 26 -RSB- . Purified recombinant calcineurin was obtained from Sigma -LRB- Poole , UK -RRB- or Biomol -LRB- Exeter , UK -RRB- . Unless otherwise stated all chemicals were of analytical grade and obtained from Sigma . 2.2 Small Scale GST protein binding assays In all binding assays , recombinant GST-fusions proteins -LRB- 1 μM -RRB- were immobilised by incubation with 40 μl of glutathione cellulose -LRB- GST cellulose -RRB- resin -LRB- 50 % bead slurry , Bioline , London , UK -RRB- that had been pre-washed in binding buffer -LRB- KCl 50 mM , HEPES 20 mM -LRB- pH 7.4 -RRB- , EGTA 5 mM , NTA 5 mM , CaCl2 4.6 mM -LRB- giving a free -LSB- Ca2 + -RSB- of 1 μM -RRB- -RRB- -LRB- + Ca2 + conditions -RRB- or binding buffer with no added CaCl2 -LRB- - Ca2 + conditions -RRB- by incubation with constant agitation for 30 min/4 °C . Recombinant calcineurin -LRB- 1 μM -RRB- or bovine brain cytosol -LRB- ∼ 1 mg , dialysed against the appropriate ± Ca2 + binding buffer -RRB- were then added to immobilised GST proteins in a 100 μl total binding reaction volume and samples incubated with constant agitation for 2 hrs at 4 °C . GST cellulose beads were pelleted by centrifugation -LRB- 5,000 rpm/1 min/4 °C -RRB- and washed with 1 ml of the appropriate binding buffer . This wash step was repeated three times and final bead pellets extracted into 50 μl SDS dissociation buffer -LRB- 125 mM HEPES pH -LRB- 6.8 -RRB- , 10 % -LRB- v/v -RRB- sucrose , 10 % -LRB- v/v -RRB- glycerol , 4 % -LRB- w/v -RRB- SDS , 1 % β-mercaptoethanol , 2 mM EDTA -RRB- and boiling for 5 min . Samples were resolved on SDS-PAGE -LRB- 12.5 % gel -RRB- and transferred to nitrocellulose filters for western blotting by transverse electrophoresis . Bound calcineurin was detected using a monoclonal antibody -LRB- 1:1000 , Sigma -RRB- followed by incubation with anti-mouse-HRP -LRB- 1:400 , Sigma -RRB- and visualisation with ECL reagents . All western blots were quantitated using ImageJ -LRB- National Institutes of Health -RRB- densitometry software . 2.3 Large scale GST protein binding assays This binding assay is essentially as described in -LSB- 26 -RSB- . Briefly , each recombinant GST-fusion protein -LRB- 5 -- 10 mg -RRB- , including free GST as control , was immobilised onto 3 ml glutathione-Sepharose 4B resin that had been pre-washed extensively with binding buffer by incubation for 2 h at 4 °C with constant agitation . Clarified bovine brain cytosolic extract was applied to the GST affinity columns and binding allowed to proceed for 16 h at 4 °C with constant agitation . Each column was washed with a minimum 50 volumes of binding buffer and specific Ca2 + - dependent binding proteins eluted using binding buffer containing no added Ca2 + -LRB- calcium-free or CF buffer -RRB- . An additional high-salt elution step with binding buffer supplemented with 1 M NaCl and no added Ca2 + -LRB- High Salt or HS buffer -RRB- was used to isolate potential Ca2 + - independent binding interactions . Eluted protein fractions were concentrated by methanol precipitation and pellets extracted into SDS dissociation buffer by boiling for 5 min . All western blots were quantitated using ImageJ -LRB- National Institutes of Health -RRB- densitometry software . 2.4 Sulfo-SBED cross-linking experiments The manufacturers protocol provided by Pierce -LRB- Rockford , IL , USA -RRB- was followed with minor modifications . All steps , except those indicated , were carried out in the dark . Bait recombinant protein of interest -LRB- ∼ 5 mg -RRB- was dialysed against binding buffer overnight at 4 °C . Immediately before use , the contents of one tube of No-Weigh Sulfo-SBED -LRB- Pierce -RRB- was dissolved in 22 μl of DMSO , this was then added to 1 ml of dialysed protein . Sulfo-SBED/protein mixes were incubated at room temperature for 30 minutes , centrifuged briefly to remove any precipitated , hydrolysed , Sulfo-SBED , and dialysed overnight against binding buffer . Bovine brain cytosol -LRB- 500 μl , ∼ 4 mg protein -RRB- dialysed against binding buffer was added to 500μl of the Sulfo-SBED/protein mix . This mixture was incubated on a rotator at room temperature for 1 hour . After this step , samples were exposed to long-wave UV illumination -LRB- 365 nm -RRB- at a distance of ∼ 5 cm from source for 15 minutes . During this step an appropriate volume of Neutravidin beads -LRB- Pierce -RRB- were washed extensively in binding buffer , 100 μl added to the bait/cytosol mixture and samples incubated on a rotator for 1 hour at room temperature . Bait/cytosol mixes were then centrifuged at 13,000 rpm for 1 minute , supernatants removed and beads washed with 1 ml of binding buffer . This step was repeated 6 times . Disulfide bond reduction was then achieved by incubating with 1 ml of 50 mM DTT for 1 hour at room temperature . Beads were subsequently centrifuged and washed extensively with binding buffer . After the final wash , 100 μl of SDS dissociation buffer was added to the beads and samples boiled for 10 minutes . Samples were separated on SDS PAGE -LRB- 12.5 % gel -RRB- and western blotted with a monoclonal anti-calcineurin antibody -LRB- 1:1000 , Sigma -RRB- followed by detection with anti-mouse-HRP -LRB- 1:400 , Sigma -RRB- and ECL reagents . 2.5 HeLa cell cultures and transfections HeLa cells were grown in Dulbecco 's modified Eagle 's medium -LRB- DMEM , Invitrogen , Paisley , UK -RRB- containing 5 % foetal bovine serum -LRB- Invitrogen -RRB- , 1 % non-essential amino acids -LRB- Invitrogen -RRB- , and 1 % penicillin/streptomycin -LRB- Invitrogen -RRB- at 37 °C in an atmosphere of 5 % CO2 and maintained at ∼ 1,000,000 cells per 75 cm2 flask . 24 hrs prior to transfection , cells were seeded onto glass cover slips on a 24-well plate at ∼ 50,000 cells per well . Transfection mixtures comprised 3 μl Fugene ™ -LRB- Roche , UK -RRB- per 1 μg plasmid DNA in a 100 μl total volume of DMEM and were incubated at room temperature for 30 minutes prior to being added drop-wise to cells . Cells were assayed 24-48 hrs post-transfection . The NFAT-GFP reporter construct was a kind gift from Professor Anjana Rao -LRB- Harvard Medical School , Boston , MA , USA -RRB- . 2.6 Ionomycin induced NFAT translocation Transfected HeLa cells were washed twice in Krebs buffer -LRB- NaCl 145 mM ; HEPES 20 mM -LRB- pH 7.4 -RRB- ; D - -LRB- + -RRB- - Glucose 10 mM ; KCl 5 mM ; CaCl2 3 mM ; MgCl2 1.3 mM ; NaH2PO4 1.2 mM -RRB- . Cells were subsequently incubated in 500 μl Krebs containing 3 μM ionomycin or 100 μM histamine for required times before either solubilisation in SDS dissociation buffer , to investigate the phosphorylation state of NFAT via Western blotting , or fixing in 4 % formaldehyde to observe the resulting sub-cellular localisation of NFAT post-treatment via confocal microscopy . For western blotting all samples were resolved on SDS-PAGE -LRB- 12.5 % gel -RRB- and proteins transferred to nitrocellulose filters by transverse electrophoresis . Filters were probed with monoclonal anti-GFP -LRB- 1:1000 , JL-8 clone , Invitrogen -RRB- followed by incubation with anti-mouse-HRP -LRB- 1:400 , Sigma -RRB- and protein visualised with ECL reagents . 2.7 Confocal microscopy For confocal laser scanning microscopy , transfected cells were examined with either a Leica TCS-SP-MP microscope or a Leica TCS-SP2-AOBS microscope -LRB- Leica Microsystems , Heidelberg , Germany -RRB- using a 22 μm pinhole and a 63 × water immersion objective with a 1.2 numerical aperture . EGFP was imaged using excitation at 488 nm and light collection at 500-550 nm . 3 Results 3.1 Direct binding of calcineurin to NCS proteins A previous in vitro study demonstrated that NCS-1 is capable of stimulating the phosphatase activity of CN but did not examine whether the two proteins directly associate . To confirm a direct interaction we first performed a series of in vitro binding assays utilising various recombinantly expressed and purified NCS proteins to act as baits and where either purified recombinant CN or bovine brain cytosol were employed as a source of prey protein . Initially , purified CN -LRB- 1 μM -RRB- was incubated in the presence of various GST-tagged NCS proteins -LRB- also at 1 μM -RRB- in binding buffer containing 1 μM free Ca2 + -LRB- Fig. 1a , upper panel -RRB- . CaM was included in this assay as a positive control for CN binding -LRB- Fig. 1a , upper panel , Calmodulin Sepharose -RRB- . CN was observed to bind directly to GST-NCS-1 , GST-Hippocalcin , GST-Neurocalcin-δ and GST-CaBP1 but not GST-KChIP1 , despite equal loading of the proteins -LRB- Fig. 1a , lower panel -RRB- , indicating some degree of interaction specificity . It was also apparent from this analysis that , under identical experimental conditions , seven-fold less CN was bound by GST-tagged NCS proteins than by CaM . We next examined if GST-tagged NCS proteins could efficiently interact with CN present as a component of a complex protein mixture . GST control protein , GST-NCS-1 and GST-CaBP1 -LRB- Calcium Binding Protein-1 , a calcium sensor more closely related to CaM than the NCS family proteins -LSB- 28 -RSB- -RRB- were incubated with bovine brain cytosolic protein extract in a large scale pull-down experiment -LSB- 26 -RSB- in the presence of 1 μM free Ca2 + and Ca2 + dependent binding partners eluted by application of Ca2 + free buffer containing EGTA and NTA -LRB- Fig. 1b , Calcium Free -LRB- CF -RRB- lanes -RRB- . An additional elution step using 1 M NaCl supplemented buffer was also applied to the samples to elute potential Ca2 + independent binding proteins -LRB- Fig. 1b , High Salt -LRB- HS -RRB- lanes -RRB- . Western blotting for the presence of CN in eluates confirmed a strictly Ca2 + dependent interaction with both GST-NCS-1 and GST-CaBP1 . No binding was detectable to GST control protein . The signal for CN in the bovine brain cytosolic extract was compared in the same experiment -LRB- Fig. 1b , Bovine brain cytosol -RRB- and it was clear that CN was not enriched over the brain extract in the Ca2 + free eluates . To provide an independent method to examine the interaction between NCS proteins and CN , we exploited a recently developed bi-functional cross-linking reagent -LSB- 29 -RSB- which permits the transfer of a biotin tag from a bait protein of interest to any specifically interacting prey proteins captured following a binding reaction . Samples from a GST-Neurocalcin-δ binding assay prepared via this method were probed with an anti-CN antibody and the presence of the phosphatase confirmed only in samples treated with the cross-linker -LRB- Fig. 1c , -LRB- + -RRB- Sulfo -RRB- and no CN was detectable in samples devoid of the reagent -LRB- Fig. 1c , -LRB- - -RRB- Sulfo -RRB- . Despite use of cross-linking to maintain any low affinity interactions , CN was not enriched in the eluate over brain cytosol input into each condition -LRB- Fig. 1c -LRB- + -RRB- Sulfo vs. Bovine brain cytosol -RRB- . The data presented in Fig. 1a suggests that the affinity of CN for CaM may be substantially greater than for the NCS proteins tested . In order to independently verify these observations we further examined the binding of CN from bovine brain cytosol to GST control protein and GST-NCS-1 in direct comparison to CaM -LRB- Fig. 1d -RRB- . We observed Ca2 + dependent binding of CN to CaM -LRB- Fig. 1d , Calmodulin Sepharose -LRB- + -RRB- -RRB- that was significantly enriched in the eluate compared to bovine brain cytosolic extract , in contrast , at this exposure of the western blot , binding to GST-NCS-1 was barely detectable . On longer exposure to film , NCS-1 bound CN was detectable although it should be noted that results obtained from this small scale binding assay are not directly comparable to data presented in Fig. 1b from a large scale binding assay which employed a greater amount of bovine brain cytosolic prey protein . These data are consistent with a high affinity Ca2 + dependent interaction between CaM and CN in contrast to a comparatively weak interaction between NCS-1 and CN . 3.2 Overexpression of NCS proteins has no effect on the dephosphorylation of an NFAT-GFP reporter construct in response to cytosolic Ca2 + elevation in HeLa cells Although our in vitro studies pointed to CaM as most likely the predominant interacting/activating partner for CN , we speculated that the situation in the complex physiological setting of mammalian cells might not reflect the results obtained from such a comparatively simple analysis . We therefore attempted to resolve this issue through an examination of a possible interaction between CN and NCS proteins in intact HeLa cells . For these experiments we obtained a GFP tagged NFAT reporter construct -LSB- 30 -RSB- , encoding the phosphorylated regulatory domain of the phosphatase , that undergoes a detectable dephosphorylation and nuclear translocation in response to sustained increases in -LSB- Ca2 + -RSB- i . Our rationale was that should there be a functionally relevant NCS protein/CN interaction in cells then there might be discernable alterations in dephosphorylation kinetics/nuclear translocation of the NFAT reporter in cells -LSB- 30,31 -RSB- over-expressing NCS proteins as observed for other NCS-1 specific functions -LSB- 32-35 -RSB- . In initial studies , we saw no effect on NFAT dephosphorylation or nuclear translocation of over-expression of wild-type NCS-1 , hippocalcin or neurocalcin-δ . Since NCS-1 had been identified in a previous report -LSB- 25 -RSB- as an in vitro activator of CN enzymatic activity we decided , however , to focus on this NCS family member for this part of the study . A second reason for examining NCS-1 in further detail also related to the fact that we had at our disposal several mutant NCS-1 constructs one of which has been previously characterised as a dominant inhibitor of NCS-1 function -LSB- 32 -RSB- . Such mutants , we reasoned , might assist in unmasking potential functional interactions between NCS-1 , CN and NFAT . The first of these mutants , NCS-1E120Q , has been employed in a number of studies due to its ability to interact normally with NCS-1 effectors however , as a consequence of impaired Ca2 + binding and conformational change , it can then exert a dominant inhibitory phenotype -LSB- 32-36 -RSB- . The second NCS-1 mutant we employed in this study , NCS-1G2A , is myristoylation deficient and , unlike wild type NCS-1 which is constitutively membrane associated through this lipid modification , is entirely cytosolic -LSB- 37,38 -RSB- . This protein was included in these studies due to the fact that the majority of cellular CN is similarly cytosolic and hence we hypothesised that the G2A mutation may exaggerate any effects of a potential NCS-1 / CN interaction . In an initial analysis , HeLa cells were co-transfected with NFAT-GFP along with control empty vector , NCS-1 or NCS-1 mutant constructs -LRB- Fig. 2a -RRB- . Cells were treated for 7 minutes with ionomycin/Ca2 + to elevate -LSB- Ca2 + -RSB- i , lysed and NFAT-GFP detected by western blotting . A band corresponding to the fully phosphorylated form of NFAT was apparent in 0 time point samples from all transfections -LRB- Fig. 2a , P , upper arrow -RRB- . Dephosphorylation of NFAT to two more rapidly migrating species after sustained -LSB- Ca2 + -RSB- i for 7 minutes was observable in all transfected samples and there was no detectable difference in the dephosphorylation pattern obtained between control transfected cells and NCS-1 or NCS-1 mutant transfected conditions -LRB- Fig. 2a , DeP , lower arrows -RRB- . These data are fully consistent with results obtained from parallel confocal imaging analyses of NFAT-GFP nuclear translocation in HeLa cells transfected and treated in an identical manner but which were then fixed for microscopy -LRB- Fig. 2b -RRB- . For all transfection conditions at time 0 of Ca2 + / ionomycin treatment NFAT-GFP was excluded from cell nuclei and was diffusely cytosolic -LRB- Fig. 2b , control , left hand panels -RRB- , compatible with the presence of the phosphorylated form of the protein -LRB- Fig. 2a -RRB- . After 7 minutes of Ca2 + / ionomycin treatment NFAT-GFP had redistributed to cell nuclei for all transfection conditions -LRB- Fig. 2b , ionomycin , right hand panels -RRB- again consistent with the rapid dephosphorylation of the protein -LRB- Fig. 2a -RRB- . We additionally verified that all NCS-1 expression constructs used in these studies over-expressed in HeLa cells to similar extents when co-expressed with NFAT-GFP -LRB- Fig. 2c -RRB- . Western blotting with an anti-NCS-1 antiserum -LSB- 39 -RSB- of total cell lystates from HeLa cells transfected identically to those used in functional assays -LRB- Figs. 2a and b -RRB- highlighted essentially equivalent levels of over-expression of NCS-1 , NCS-1G2A and NCS-1E120Q proteins . There was no detectable NCS-1 protein expression in control , pcDNA transfected , cells -LRB- Fig. 2c , pcDNA -RRB- . 3.3 Overexpression of NCS-1 , NCS-1E120Q and NCS-1G2A has no effect on the dephosphorylation of an NFAT-GFP reporter construct in response to cytosolic Ca2 + elevation in HeLa cells over an acute time course Our analyses suggested that , over a 7 minute time course , none of the NCS-1 constructs under examination were capable of affecting the dephosphorylation of NFAT-GFP in response to -LSB- Ca2 + -RSB- i elevation and , by inference , nor were they able to influence CN activity in intact mammalian cells . These whole cell data were consistent with our in vitro binding studies however we decided to complete our investigation with two further analyses , firstly examining whether or not NCS-1 might exert a far more subtle effect on the CN/NFAT pathway over an acute time course of -LSB- Ca2 + -RSB- i elevation -LRB- Fig. 3a -RRB- . In these experiments , HeLa cells were again transfected with NFAT-GFP along with control or NCS-1 expression constructs however dephosphorylation was monitored over a short 5 minute time course . For all constructs tested NFAT dephosphorylation was clearly apparent by the 5 minute time point -LRB- a 25 minute time point was also included to demonstrate complete dephosphorylation of NFAT-GFP -RRB- as determined by gel shift of the NFAT-GFP . Analysis of these earlier time points indicated there was no discernable difference in the pattern of dephosphorylation between any of the NCS-1 constructs tested and control transfected cells . In these experiments the calcium induced elevation in cytosolic free Ca2 + was artificially generated by high concentrations of ionomycin and external Ca2 + . Since NCS-1 has a high affinity for Ca2 + -LRB- 300 nM -RRB- one possibility is that it could activate CN under conditions of limited Ca2 + elevation thus increasing the overall cellular sensitivity to a Ca2 + signal . We completed , therefore , our analyses with an examination as to whether over-expression of NCS-1 or its mutants could influence NFAT dephosphorylation in response to a physiological stimulus . Histamine is a well characterised HeLa cell agonist that generates oscillatory intracellular Ca2 + signals -LSB- 40 -RSB- and activation of protein kinase C with phorbol esters has been shown to potentiate NFAT driven gene expression elicited by such Ca2 + oscillations -LSB- 41-43 -RSB- . We stimulated HeLa cells transfected identically to those in Figs. 2 and 3 with 100 μM histamine in both the presence and absence of 50 nM PMA over a three hour time course and assessed the phosphorylation state of NFAT-GFP by western blotting . No dephosphorylation of NFAT-GFP was detectable in these experiments in control vector expressing cells indicating that the stimulus was below the threshold for activation of CN/NFAT-GFP dephosphorylation . Significantly , cells expressing NCS-1 , NCS-1E120Q or NCS-1G2A also failed to exhibit dephosphorylation of NFAT-GFP indicating that over-expression of NCS-1 did not enhance the sensitivity of the CN/NFAT signalling pathway to Ca2 + signals under these conditions -LRB- data not shown -RRB- . For NCS-1E120Q to be able to exert a dominant negative effect it would have to bind to CN . In order to validate our use of the NCS-1E120Q mutant as a dominant negative construct in this study we therefore generated a recombinantly purified GST-tagged fusion construct encoding this protein which was used to confirm its ability to bind to purified CN -LRB- Fig. 3b -RRB- . GST-NCS-1E120Q retained almost wild-type CN binding activity in this assay although with less strict Ca2 + - dependency validating its use in functional assays of NFAT dephosphorylation and nuclear translocation . For GST-NCS-1 we observed a 13.7-fold increase in binding of CN in the presence of Ca2 + compared to conditions where Ca2 + was absent . In contrast , there was only a 2.2-fold increase in CN binding to GST-NCS-1E120Q in the presence of Ca2 + . 4 Discussion Diverse Ca2 + signals are transduced and decoded within cells by a variety of Ca2 + - sensing proteins . Of these Ca2 + responsive proteins the most intensively studied and best understood is calmodulin -LRB- CaM -RRB- -LSB- 2 -RSB- . CaM is the prototypical member of a super-family of evolutionarily diverse EF-hand containing Ca2 + sensors to which the NCS sub-family belongs -LSB- 23 -RSB- . Whilst the NCS proteins are only distantly related to CaM , comparative studies have identified significant in vitro overlap in their effector specificities -LSB- 25 -RSB- . It was proposed that NCS proteins might represent a set of redundant or alternative modulators of bone fide CaM target interactions -LSB- 25 -RSB- but the ubiquitous and high level expression of CaM would make this doubtful . Calcineurin -LRB- CN -RRB- is a cellular serine/threonine phosphatase critical to the activation of NFAT transcription factors and hence in the control of a variety of fundamental physiological processes -LSB- 3 -RSB- . CaM has been identified as the essential co-activator for CN function , however CN has also been identified as a potential target for NCS-1 -LSB- 25 -RSB- and as such represents a candidate dual CaM/NCS effector . A series of possibilities exist as a direct consequence of these observations . Firstly , NCS proteins could indeed simply represent redundant modulators for CaM targets and as such might be expected to elicit responses and to exhibit biochemical properties closely approximating those observed with CaM . Secondly , NCS proteins could feasibly interact with CaM targets in biochemically distinct ways to modulate their activities in specific , CaM independent , manners leading to the overall generation of unique physiological endpoints . The latter has been suggested for dual regulation of voltage-gated Ca2 + - channels by both CaBP1 and CaM -LSB- 44 -RSB- . With specific regard to possible CN/NFAT activation by NCS-1 , this possibility is intriguing in light of data identifying NFAT dependent gene expression as important to aspects of neuronal cell function . Thirdly , the possibility also exists that interactions observed between NCS proteins and CaM targets are physiologically irrelevant in cells . In this study we have attempted to resolve these issues through an investigation of firstly the biochemical characteristics of NCS protein interactions with CN , and secondly by examining the potential consequences of NCS protein function on activation of the classical CaM-CN-NFAT pathway in mammalian cells . In a series of in vitro biochemical assays we were able to show that there exists a selective and Ca2 + dependent interaction between various NCS proteins and both purified recombinant CN , and CN derived from bovine brain cytosolic protein extract . We noted during these analyses however that significantly more CN bound to immobilized CaM . These data are consistent with the findings of Schaad et al. indicating that NCS-1 is able to stimulate the phosphatase activity of CN in vitro but to a lesser extent than observed for CaM activation . In experiments utilising bovine brain cytosol as a source of prey protein the difference in binding of the NCS proteins for CN in comparison to CaM was more greatly magnified , and whilst considerable quantities of CN could be affinity purified by a calmodulin sepharose column , the amount of CN associated with NCS family members under identical experimental conditions was considerably lower . Previous work using a biotinylated protein overlay assay also found much less binding of CN to NCS-1 compared to CaM -LSB- 45 -RSB- . These data led us to conclude that the CaM/CN interaction was likely to be of a higher affinity than for interactions between CN and NCS proteins . Indeed , published biochemical parameters suggest that the in vitro Kd for CaM/CN interaction may be ≤ 0.1 nM -LSB- 46 -RSB- with that reported for NCS-1 / CN being 350 nM -LSB- 25 -RSB- . The in vitro affinity of CaM for CN is therefore three orders of magnitude greater than that determined for NCS-1 which would agree with the binding data presented in this study . Importantly , CaM is expressed at 5-200 fold higher concentrations in cells than NCS-1 -LSB- 25 -RSB- and so CN would be predicted to be bound to CaM rather than NCS-1 at elevated -LSB- Ca2 + -RSB- i levels . To assess whether NCS proteins were capable of modulating CN activity in a cellular environment we employed the dephosphorylation and nuclear translocation of a characterised NFAT-GFP reporter construct as a probe of CN phosphatase activity . Co-expression of NCS-1 did not alter the basal phosphorylation state of NFAT-GFP compared with control transfected cells nor did it exert a detectable effect on NFAT-GFP dephosphorylation/nuclear translocation after sustained elevation of -LSB- Ca2 + -RSB- i during single endpoint and acute time course analyses . Co-expression of the NCS proteins Hippocalcin , Neurocalcin-δ -LSB- 26 -RSB- , and the more closely related CaBP1 -LRB- which shares 56 % similarity with CaM -LSB- 47 -RSB- -RRB- were similarly without effect in analyses of NFAT-GFP dephosphorylation -LRB- data not shown -RRB- . From these analyses we can not formally exclude the possibility that NCS family proteins may activate a pool of cellular CN that does not signal via NFAT dephosphorylation . This seems unlikely however taking into account our biochemical data and the fact that in numerous other studies assessing diverse triggers of CN activity , concomitant activation of the NFAT pathway is always detected . In an extension to these studies we also examined the effects of two NCS-1 mutant proteins , NCS-1E120Q and NCS-1G2A , on NFAT-GFP dephosphorylation in HeLa cells . We were able to demonstrate that the E120Q mutant of NCS-1 -LSB- 32 -RSB- retained approximately wild type binding properties with respect to interaction with purified CN . However , this protein , which has been routinely observed to exert a dominant negative phenotype in various other studies of NCS-1 function -LSB- 32-35 -RSB- , was unable to generate detectable alterations in NFAT-GFP dephosphorylation . In an effort to try and understand if NCS-1 was unable to mediate an in vivo modulation of CN activity due to its restricted membrane localisation we also examined NFAT-GFP phosphorylation in the presence of co-expressed NCS-1G2A , a protein that fails to become myristoylated and as a result remains cytosolic . Since the bulk of cellular CN is also cytosolic we reasoned that expression of the G2A mutant might bias any interaction with CN to generate an observable alteration in dephosphorylation of NFAT-GFP . Our analysis of this mutant indicated that it elicited no significant change in the kinetics of NFAT-GFP dephosphorylation compared to control transfected cells . In related experiments we tested the ability of NCS-1 and NCS-1 dominant inhibitory mutants to influence NFAT dephosphorylation in response to physiological stimuli . We observed no detectable changes in NFAT dephosphorylation in the presence of over-expressed NCS-1 proteins suggesting that in addition to sustained elevation of cytosolic free Ca2 + , NCS-1 proteins are similarly unable to stimulate NFAT dephosphorylation in response to agonist derived Ca2 + signals that were below the threshold for significant activation of CN and NFAT dephosphorylation . Collectively our findings would argue that , although NCS proteins are capable of interacting with CN in vitro , they may be unable to act as functionally relevant activators of the enzyme in a cellular context . The possibility remains that there may be some level of interplay between NCS proteins and CaM within protein complexes , exemplified by observations that both CaM and CaBP1 interact with IP3-receptors -LSB- 40,48,49 -RSB- , that both NCS-1 and CaM are able to interact with Ca2 + channels -LSB- 50 -RSB- and G-protein coupled receptor kinases -LSB- 34,51,52 -RSB- and that CaM , NCS-1 and CaBP1 have all been shown to modulate the activity of the transient receptor potential channel , TRPC5 -LSB- 35 -RSB- . It is impossible to formally rule out direct modulation of CN by NCS proteins under all physiological conditions based on the data presented in this study however we have provided data to challenge the idea that CN is a cellular target of the NCS protein family and instead that CaM appears the more likely regulator of this key signalling phosphatase .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O B O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O B I I O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

ncs family proteins
calmodulin
calcineurin activity

nfat, nuclear factor of activated t-cells

kchip1, potassium channel interacting protein-1

page, polyacrylamide gel electrophoresis

egta, ethylene glycol-bis(2-aminoethylether)-n,n,n′n′-tetra-acetic acid


Manifestation

of

palmoplantar

pustulosis

during

or

after

infliximab

therapy

for

plaque-type

psoriasis

report

on

five

cases

Infliximab

is

a

monoclonal

antibody

directed

against

TNF-α

It

has

been

approved

for

use

in

rheumatoid

arthritis

ankylosing

spondylitis

inflammatory

bowel

disease

psoriatic

arthritis

and

plaque-type

psoriasis

In

case

reports

positive

effects

on

pustular

variants

of

psoriasis

have

also

been

reported

However

paradoxically

manifestation

of

pustular

psoriasis

and

plaque-type

psoriasis

has

been

reported

in

patients

treated

with

TNF

antagonists

including

infliximab

for

other

indications

Here

we

report

on

patients

with

chronic

plaque-type

psoriasis

who

developed

palmoplantar

pustulosis

during

or

after

discontinuation

of

infliximab

therapy

In

two

of

the

five

cases

manifestation

of

palmoplantar

pustulosis

was

not

accompanied

by

worsening

of

plaque-type

psoriasis

Possibly

site-specific

factors

or

a

differential

contribution

of

immunological

processes

modulated

by

TNF

inhibitors

to

palmoplantar

pustulosis

and

plaque-type

psoriasis

may

have

played

a

role

Introduction

Increased

expression

of

TNF-α

has

been

identified

as

an

important

pathophysiological

mechanism

in

different

types

of

chronic

inflammation

including

psoriasis

and

psoriatic

arthritis

The

knowledge

about

the

central

role

of

TNF-α

in

certain

diseases

has

successfully

been

converted

to

the

therapeutic

level

over

the

last

years

TNF

antagonizing

agents

such

as

the

anti-TNF

directed

antibodies

infliximab

and

adalimumab

and

the

fusion

protein

etanercept

have

substantially

improved

the

treatment

of

some

of

the

most

common

chronic

inflammatory

conditions

such

as

rheumatoid

arthritis

-LRB-

RA

-RRB-

With

of

the

patients

reaching

at

least

a

reduction

of

their

baseline

skin

symptoms

after

weeks

of

therapy

-LSB-

-RSB-

infliximab

is

regarded

as

one

of

the

most

potent

agents

available

at

present

for

the

treatment

of

psoriasis

vulgaris

Other

variants

of

psoriasis

such

as

pustular

psoriasis

have

not

been

formally

tested

in

larger

clinical

trials

However

in

individual

reports

positive

effects

of

infliximab

on

generalized

pustular

psoriasis

-LRB-

GPP

-RRB-

with

dramatic

improvements

in

some

cases

have

been

reported

-LSB-

-RSB-

Because

this

type

of

pustular

psoriasis

is

believed

to

correspond

to

an

extreme

activation

of

psoriatic

disease

mechanisms

the

high

bioavailability

of

infliximab

and

its

rapid

onset

of

action

following

intravenous

infusion

have

been

used

to

explain

the

surprisingly

fast

decrease

of

pustule

formation

in

patients

treated

with

the

agent

Paradoxically

on

the

other

hand

manifestation

of

pustular

psoriasis

has

been

reported

in

patients

treated

with

TNF

antagonists

including

infliximab

for

other

indications

Palmoplantar

pustulosis

-LRB-

PPP

-RRB-

has

long

been

regarded

as

a

localized

variant

of

pustular

psoriasis

although

more

recent

epidemiological

and

genetic

findings

argue

against

this

concept

-LSB-

-RSB-

Here

we

report

on

five

patients

with

chronic

plaque-type

psoriasis

who

developed

PPP

during

or

after

discontinuation

of

infliximab

therapy

Cases

and

discussion

Relevant

aspects

of

the

five

patients

with

chronic

plaque-type

psoriasis

who

developed

PPP

during

or

after

discontinuation

of

infliximab

therapy

are

presented

in

the

Table

and

Fig.

Table

of

duration

-LRB-

years

-RRB-

typePlaquePlaquePlaquePlaquePlaquePrior

pustular

psoriasisNoNoNoNoNoPsoriatic

arthritisYesNoNoNoYesFamiliy

history

of

psoriasisNegativePositiveNegativeUnknownNegativePrior

UV-therapyYesYesYesYesYesPrior

systemic

therapiesMethotrexateLeflunomideEtanerceptPrednisoloneFumaric

acid

estersCyclosporineFumaric

acid

estersFumaric

acid

estersCyclosporineRetinoidsMethotrexateFumaric

acid

estersCyclosporineMethotrexateInitial

response

to

infliximabExcellentcPASI

of

manifestation

of

PPPWeek

weeks

after

end

of

infliximab

treatmentWeek

worsening

of

plaque

psoriasisaYesYesNoNoYesDevelopment

of

GPP

in

addition

to

PPPYesNoNoNoNoPotential

trigger

factors

of

PPP

Infection

prior

to

PPPYesNoYesNoNo

Present

smokingNoNoYes

-LRB-

packyears

-RRB-

UnknownUnknownDiscontinuation

of

infliximabYesYesYesNoYesSystemic

therapy

with

sufficient

control

of

PV

and

PPPbAdalimumab

mg

e.o.wEtanercept

mg

BIW

plus

methotrexate

mg/week

orallyPUVA-therapy

of

palms

and

soles

-LRB-

Additional

topical

therapy

-RRB-

Etanercept

mg

BIWSystemic

therapy

that

failed

to

control

PV

or

PPPEtanercept

mg

BIWe

plus

methotrexate

mg/week

orallyEtanercept

mg

BIWPPP

Pustulosis

palmoplantaris

GPP

Generalized

pustular

psoriasis

PV

Psoriasis

vulgaris

e.o.w

Every

other

weeka

Loss

of

of

maximum

PASI

response

or

increase

of

physician

's

global

assessment

-LRB-

PGA

-RRB-

by

All

patients

received

additional

topical

therapy

with

glucocorticosteroids

and

Vitamin

D

analoguesc

Improvement

rated

by

PGA

with

almost

clear

-LRB-

PGA

-RRB-

d

Week

of

infliximab

treatmente

BIW

twice

weeklyf

PASI

Reduction

in

the

psoriasis

area

and

severity

index

-LRB-

PASI

-RRB-

by

Fig.

picture

of

pustulosis

palmoplantaris

in

patient

with

pustules

in

different

stages

of

evolution

on

a

sharply

delineated

erythematous

lesion

on

the

left

sole

-LRB-

a

-RRB-

and

yellowish

pustules

on

the

left

palm

-LRB-

b

-RRB-

Histological

examination

showing

intraepidermal

vesiculopustular

dermatitis

-LRB-

c

H.E.

stain

of

a

biopsy

from

the

left

plantar

arch

-RRB-

with

intraepidermal

accumulation

of

neutrophils

and

subcorneal

pustule

formation

-LRB-

d

-RRB-

To

the

best

of

our

knowledge

the

development

of

PPP

during

the

treatment

of

plaque-type

psoriasis

with

infliximab

has

not

yet

been

reported

The

occurrence

of

pustular

skin

lesions

usually

resembling

GPP

or

palmoplantar

pustular

psoriasis

has

occasionally

been

observed

in

patients

treated

with

infliximab

for

other

indications

-LSB-

-RSB-

Induction

of

pustular

skin

lesions

seems

not

to

be

limited

to

infliximab

therapy

but

has

also

been

described

in

association

with

the

use

of

the

TNF-antagonists

etanercept

and

adalimumab

including

the

use

in

one

patient

with

plaque-psoriasis

treated

with

etanercept

-LSB-

-RSB-

One

patient

with

seropositive

RA

developed

GPP

as

well

as

PPP

during

treatment

with

infliximab

-LSB-

-RSB-

This

patient

later

experienced

a

relapse

of

PPP

when

treatment

with

etanercept

was

initiated

which

also

suggests

that

a

class

effect

of

TNF-antagonists

may

play

a

role

In

two

of

the

three

cases

in

whom

an

exacerbation

of

plaque-psoriasis

occurred

parallel

to

the

manifestation

of

PPP

typical

trigger

factors

for

active

psoriasis

could

be

identified

such

as

an

infection

-LRB-

case

-RRB-

and

the

abrupt

termination

of

anti-psoriatic

treatment

-LRB-

case

-RRB-

These

two

cases

are

compatible

with

the

existence

of

common

trigger

factors

for

plaque

psoriasis

and

PPP

What

are

other

factors

that

might

contribute

to

the

development

of

PPP

during

treatment

of

psoriasis

vulgaris

While

the

exact

etiology

of

PPP

remains

to

be

established

a

history

of

smoking

is

the

most

important

known

risk

factor

for

PPP

However

only

one

out

of

the

three

patients

in

whom

a

smoking

history

had

been

obtained

was

a

smoker

at

the

time

of

onset

of

pustular

psoriasis

-LRB-

case

-RRB-

Streptococcal

infection

a

known

risk

factor

for

psoriasis

vulgaris

has

not

been

established

as

a

risk

factor

for

PPP

and

probably

plays

a

minor

role

there

However

in

the

cases

presented

here

one

patient

-LRB-

case

-RRB-

suffered

an

upper

respiratory

tract

infection

a

few

days

before

manifestation

of

PPP

while

another

patient

-LRB-

case

-RRB-

had

suffered

from

a

persistent

cold

weeks

before

manifestation

of

pustules

In

the

former

patient

the

close

temporal

relationship

between

infectious

symptoms

and

manifestation

of

PPP

may

point

to

a

possible

contribution

of

the

infection

to

triggering

PPP

and

a

modulation

of

the

immune

response

to

infliximab

appears

possible

It

is

likely

that

beyond

the

contribution

of

known

risk

factors

other

immunological

mechanisms

may

be

involved

in

the

manifestation

of

PPP

under

infliximab

therapy

Interferon

-LRB-

IFN

-RRB-

α

has

been

suggested

as

a

cytokine

mediating

the

manifestation

of

psoriasiform

lesions

in

patients

treated

with

TNF-inhibitors

as

a

consequence

of

crosstalk

of

TNF-α

and

IFN-α

TNF-α

is

known

to

suppress

the

generation

of

plasmacytoid

dendritic

cells

that

are

very

potent

producers

of

IFN-α

Appearance

of

plasmacytoid

dendritic

cells

-LRB-

and

IFN-α

-RRB-

in

ths

skin

is

considered

to

be

an

early

and

crucial

step

in

the

pathogenesis

of

psoriasis

-LRB-

reviewed

in

-LSB-

-RSB-

-RRB-

Thus

in

patients

treated

with

TNF-antagonists

the

inhibition

of

TNF-α

might

induce

an

increase

of

IFN-α

in

the

skin

favoring

the

manifestation

of

psoriasiform

dermatitis

In

fact

an

increase

of

IFN-α

signaling

has

been

shown

in

biopsy

specimens

from

psoriatic

plaques

induced

by

TNF-inhibitors

compared

with

traditional

psoriatic

plaques

-LSB-

-RSB-

The

relevance

of

IFN-α

for

PPP

and

TNF-inhibitor

induced

PPP

however

still

needs

to

be

determined

The

observation

of

an

improvement

of

pre-existing

psoriasis

plaques

parallel

to

the

first

manifestation

of

PPP

in

two

of

the

cases

described

here

supports

the

concept

that

immunological

mechanisms

and/or

local

factors

are

not

identical

in

the

pathogenesis

of

plaque-type

psoriasis

and

PPP

Differences

in

pathogenesis

between

plaque

psoriasis

and

PPP

are

also

supported

by

their

different

genetic

background

with

plaque

psoriasis

but

not

PPP

being

linked

to

the

major

susceptibility

locus

for

plaque-type

psoriasis

located

on

-LSB-

-RSB-

The

localized

nature

of

the

pustules

on

palms

and

soles

occurring

in

association

with

infliximab

therapy

in

some

patients

suggests

a

contribution

of

site-specific

factors

Possibly

eccrine

sweat

glands

that

are

numerous

in

the

palmoplantar

location

are

involved

in

these

processes

Changes

secondary

to

binding

of

infliximab

to

TNF-α

that

has

been

shown

to

be

expressed

in

eccrine

sweat

glands

have

been

suggested

as

a

possible

mechanism

-LSB-

-RSB-

Withdrawal

or

dose

reduction

of

systemic

cyclosporine

and

systemic

or

topical

glucocorticosteroids

given

for

treatment

of

psoriasis

vulgaris

have

been

associated

with

manifestation

of

GPP

but

not

with

PPP

To

the

best

of

our

knowledge

no

reports

on

manifestation

of

GPP

or

PPP

associated

with

treatment

with

methotrexate

another

drug

frequently

used

in

the

therapy

of

psoriasis

exist

However

in

the

clinical

trials

with

the

efalizumab

in

the

indication

plaque

psoriasis

development

of

PPP

has

also

been

observed

in

of

patients

during

the

first

weeks

of

treatment

with

efalizumab

compared

to

of

the

patients

in

the

placebo

groups

-LSB-

-RSB-

Whether

therapy

with

TNF-antagonists

may

induce

a

more

severe

form

of

plaque

psoriasis

or

psoriatic

arthritis

in

some

patients

treated

for

these

indications

is

difficult

to

determine

as

the

natural

course

of

both

plaque

psoriasis

and

psoriatic

arthritis

is

variable

and

a

loss

of

effect

is

not

easily

differentiated

from

an

induction

of

a

more

severe

form

of

the

respective

disease

However

as

a

manifestation

of

plaque

psoriasis

has

been

noted

in

a

number

of

patients

treated

with

TNF-antagonists

for

other

indications

without

a

personal

or

family

history

of

psoriasis

-LSB-

-RSB-

it

is

likely

that

TNF-antagonists

may

in

some

individuals

and

probably

in

conjunction

with

environmental

factors

favor

manifestation

or

worsening

of

psoriatic

skin

and/or

bone

disease

The

treatment

of

patients

with

plaque-psoriasis

developing

PPP

should

be

decided

on

an

individual

basis

In

case

plaque-psoriasis

remains

controlled

under

infliximab

therapy

addition

of

topical

therapy

may

be

sufficient

for

treatment

of

PPP

in

some

instances

-LRB-

case

-RRB-

However

in

some

cases

additional

topical

therapy

may

not

be

sufficient

and

UV-therapy

or

another

systemic

therapy

may

be

necessary

in

addition

to

or

as

replacement

of

infliximab

therapy

-LRB-

case

-RRB-

When

manifestation

of

PPP

is

accompanied

by

worsening

of

plaque-psoriasis

discontinuation

of

infliximab

therapy

is

advisable

and

initiation

of

other

systemic

antipsoriatic

agents

such

as

cyclosporine

or

a

different

TNF-antagonist

may

be

necessary

-LRB-

cases

and

-RRB-

In

summary

pustular

psoriasis

may

show

a

good

response

to

treatment

with

TNF-antagonists

such

as

infliximab

However

pustular

psoriasis

may

also

manifest

during

treatment

of

rheumatological

diseases

and

as

described

here

also

in

patients

with

plaque-type

psoriasis

under

treatment

with

infliximab

Manifestation

of

PPP

under

infliximab

is

not

necessarily

accompanied

by

worsening

of

pre-existing

plaque

psoriasis

Management

of

the

pustular

skin

lesions

has

to

be

decided

on

an

individual

basis

In

a

subgroup

of

patients

therapy

with

the

TNF-antagonist

has

to

be

discontinued

and

another

systemic

therapy

-LRB-

including

a

different

TNF-antagonist

-RRB-

will

probably

be

necessary

to

sufficiently

control

plaque-psoriasis

and

PPP

O O O B O O O B O O O B O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O B O O B O O O O O O O B O O B O O O O O O O O O O O O O O B O O O B O O O O O B O O O O O O O O O O O O B O O O O O O O B O O O O O O O O O O O O O O B O O O B O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O B O O O O O B O O O O B O O O O O O O O O O O O O O O O O O O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O B O O B O O O O O B O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O B O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O B O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O B O O O O B O O O O O O O O O O O O O O O O O O O O B O O O B O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O