dkk1 is a 29  kda secreted protein belonging to the dickkopf ( dkk ) family , which comprises four main glycoproteins in vertebrates ( dkk1 - 4 ) .
dkk1 has been identified as a potent inhibitor of the canonical wnt signaling due to its ability to bind to the wnt coreceptor lrp5/6 , thus blocking the canonical wnt/-catenin pathway .
canonical wnt pathway activation is initiated by the direct binding of the wnt glycoprotein to frizzled ( fz ) membrane receptor and to the lrp5/6 coreceptor [ 46 ] . in the absence of wnt
wnt - mediated assembly of the activated fz - lrp5/6 receptor complex is followed by the recruitment of the axin - gsk3 to the plasma membrane , resulting in the reduction of the phosphorylation and degradation of -catenin [ 9 , 10 ] . stabilized -catenin accumulates in the cytoplasm and translocates to the nucleus , where it interacts with dna - bound tcf - lef proteins and activates the transcription of target genes .
the wnt pathway is involved in many stages of invertebrate and vertebrate development and in adult tissue homeostasis [ 8 , 12 ] .
dysfunction within the wnt/-catenin signaling cascade has been associated with many human pathologies [ 8 , 13 ] , such as cancer [ 1417 ] and bone disease [ 18 , 19 ] .
lrp5-activating mutations are mainly associated with high - bone mass , while loss - of - function mutations on lrp5 are linked to bone degeneration and osteoporosis [ 20 , 21 ] . the inhibition of wnt signaling by dkk1 has been related to bone degeneration processes and reduced bone mass . in the central nervous system , dkk1 has been associated with the pathophysiology of neuronal degeneration in alzheimer disease ( ad ) [ 2326 ] .
dkk1 expression is increased in cortical neurons exposed to -amyloid peptide ( -ap ) , where its aberrant expression is responsible for hyperphosphorylation of tau in neurons challenged with -ap
. moreover , increased dkk1 expression was also observed in degenerating neurons in brain samples of ad patients .
dkk1 was shown to be neurotoxic when locally infused into brain regions where neurodegenerative processes associated with brain ischemia or ad normally take place .
moreover , dkk1 is also associated with neuronal death in cellular and animal models of excitotoxic / ischemic neuronal death , and the treatment of ischemic animals with dkk1 antisense oligonucleotides protects hippocampal neurons against ischemic damage and cultured cortical neurons against nmda toxicity . for these reasons ,
the dkk1-lrp6 interaction can be considered as a potentially interesting therapeutic intervention point and dkk1 a potential drug target for the treatment of bone and neurodegenerative disorders .
a small molecule ( nci8642 ) has been described as an inhibitor of the interaction between dkk1 and one of its receptors ( lrp5 ) , as well as an inhibitor of dkk1 activity in reducing wnt/-catenin signaling activation . in this study
, we sought to further characterise nci8642 activity , using biochemical and biophysical approaches , and to extend its characterization in relation to lrp6 , given the prominent expression in brain of lrp6 and its relevance to the neurodegenerative diseases field .
human embryonic kidney cells ( hek293 ) were obtained from the german collection of microorganisms and cell cultures . wnt3a
all cell lines were cultured in dmem supplemented with fbs and glutamax ( gibco ) .
the monoclonal antibody against -catenin was obtained from bd biosciences , the polyclonal goat anti - dkk1 antibody from r&d , while the fluorescently conjugated secondary antibodies were all from invitrogen .
the dna encoding the full - length human lrp6 sequence was cloned into the pcdna3.1/zeo(+ ) expression vector ( invitrogen ) .
the dna encoding the human dkk1 sequence was cloned into pcdna6.2/clumio - dest ( invitrogen ) .
the sequence encoding the secreted alkaline phosphatase from clontech was amplified by pcr and inserted at the c - terminus of dkk1 sequence into the pcdna6.2/clumio - dest - dkk1 plasmid .
the luciferase reporter plasmid p4tcf - luc comprises four copies of a tcf - responsive element upstream of a tata element luciferase coding sequence transcriptional unit .
all cell lines were cultured in dmem supplied with 10% fbs , 2  mm glutamax , 100  units / ml penicillin , 100  g / ml streptomycin , and the appropriate selection antibiotic ( 50 and 100  g / ml zeocine for pc12-tcfluc and hek293-lrp6 , resp .
, 10  g / ml blasticidine for hek293-dkk1 , 0.4  mg / ml g418 for wnt3a l - cells , 20  g / ml hek293-ap - dkk1 ) . for the generation of the stable cell lines , hek293 or pc12 cells
were transfected with fugene 6 ( roche ) and subjected to antibiotic selection 24 hours after transfection .
clones were selected using immunofluorescence ( hek293-dkk1 , hek293-lrp6 ) , ap enzymatic assay on the culture medium ( hek293-ap - dkk1 ) , or measurement of luciferase activity upon stimulation with wnt3a cm ( pc12-tcf - luc ) .
for the production of dkk1 and ap - dkk1 conditioned media ( cm ) , hek293 cells stably expressing the appropriate construct were seeded into a 150-cm flasks at half confluence . the day after
media were collected 96  h after seeding , sterile filtered , and stored at 20c in aliquots .
the control conditioned optimem was obtained in the same way using the parental hek293 cells .
for the functional studies ( -catenin translocation and tcf - luc reporter assay ) , wnt3a cm was used at a 1  :  2 dilution .
each batch of dkk1 cm was functionally titrated in order to determine the volume of cm that gives about 50% inhibition of wnt signaling . from dot blot analysis ( data not shown ) ,
the concentration of dkk1 in the dkk1 cm was estimated to be about 1  g / ml . for the quantitative ap - dkk1 binding assay , ap - dkk1 cm was diluted to obtain a phosphatase activity of 20  mu / ml .
for the immunofluorescence detection of cell - bound dkk1 , lrp6 - 293 cells were seeded onto poly - d - lysine - coated coverslips 24 hours before the experiment and incubated with dkk1 or ap - dkk1 cm diluted in cell culture medium for 2 hours at 4c . when indicated , 100  m nci8642 ( or 1% dmso ) was added alongside dkk1 cm .
after washing , the cells were fixed in 3% paraformaldehyde ( 15  min , room temperature ) , blocked with pbs/0.1% bsa , and incubated with the primary goat - anti - dkk1 antibody , and fluorescent secondary antibody diluted into blocking solution .
images were collected using a zeiss lsm 510 meta confocal microscope . for the quantitative ap - dkk1 binding assay ,
the ap activity in the cell lysate was measured with attophos ap fluorescent substrate , according to the manufacturer 's instructions .
spr was performed using a biacore t100 ( ge healthcare ) . where not specified , experiments were conducted at 25c .
kinetic constants were calculated by nonlinear fitting to the association and dissociation curves according to the manufacturer 's instructions .
apparent equilibrium dissociation constants ( kd ) were then calculated as the ratio of kd / ka .
all immobilization steps were performed at a flow rate of 5  l / min using hbs - ep+ buffer ( 10  mm hepes buffer , ph 7.4 , containing 0.15  m nacl , 3  mm edta , and 0.05% ( v / v ) p20 surfactant ) .
full - length human lrp6-fc tagged ( r&d systems ) was immobilized via amine coupling on a flow cell of a cm3 sensor chip .
3000 resonance units ( rus ) were measured at the end of the immobilization procedure .
for the indirect capture of lrp6 , protein a was covalently coupled via amine immobilization on cm3 chip .
lrp6-fc capture on protein a coated surface was performed by injecting the protein for 180  sec at the concentration of 0.5  g / ml in hbs - ep+ with a flow rate of 10  l / min .
control sensorgrams , obtained on an empty flow cell where the coupling reaction had been conducted in the presence of coupling buffer alone , were always subtracted from binding responses .
this allowed subtraction of nonspecific binding , which might be generated by residual charged negative groups , which had not been neutralized by the coupling procedure .
bovine serum albumine ( bsa ) was immobilized via standard amine coupling procedures on a flow cell of a cm3 sensor chip to obtain the nonrelated control flow cell . for binding and kinetic assays ,
dkk1 ( r&d systems ) , diluted in hbs - ep+ at concentration ranging from 3 to 54  nm , was injected for 180  sec at the flow rate of 20  l / min over lrp6 either directly immobilized or captured via protein a as described before . for nci8642 binding and kinetic assays , the compound was diluted in hbs - ep+ buffer at concentration ranging from 3 to 50  m and injected over lrp6 surface for 60  sec at the flow rate of 30  l / min .
0.2% dmso was added to hbs - ep+ when performing the experiments with nci8642 compound , and dmso correction module of the software was used to compensate for refractive index change induced by dmso . for inhibition analysis , dkk1 ( 54  nm , diluted in hbs - ep+ buffer ph 7.4 ) was incubated with nci8642 at concentration from 5 to 20  m . after 1 hour at 25c ,
dkk1 was injected over immobilized lrp6 for 240  s at the flow rate of 10  l / min .
pc12-tcf - luc cells were seeded at the density of 100,000 cells / well into black - walled clear - bottom 96-well plates .
after 24 hours , culture medium was replaced with 50  l dkk1 cm diluted as appropriate in optimem .
after one - hour incubation ( 37c ) , the cells were stimulated by adding 50  l of wnt3a cm ( 50% of the final volume ) and supplemented with either dmso or nci8642 at the indicated final concentration ( final dmso concentration 1% ) .
after 24 hours of incubation , the luciferase activity was measured with steady lite substrate ( perkin - elmer ) , according to the manufacturer 's instructions . for each condition 3 ,
l - cells were seeded into black - walled clear - bottom 96-well plates at a density of 10,000 cells / well .
after 24 hours , culture medium was replaced with 50  l dkk1 cm diluted as appropriate in optimem .
after one - hour incubation ( 37c ) , the cells were stimulated by adding 50  l wnt3a cm supplemented with either dmso or nci8642 at the indicated final concentration ( final dmso concentration 1% ) .
after two - hour stimulation ( 37c ) , the cells were fixed in 3% paraformaldehyde ( 10  min , room temperature ) , permeabilized (
0.2% triton x-100 in pbs , 10  min , room temperature ) , and blocked with 0.1% bsa in pbs .
the cells were then incubated for 2 hours at room temperature with the primary anti--catenin antibody ( 1  :  500 ) , followed by the secondary alexafluor 555 goat - anti - mouse antibody ( 1  :  1000 ) .
the ratio of the nuclear and cytoplasmic intensity of -catenin staining was calculated for each cell and averaged within the well .
nci8642 was originally characterized as an inhibitor of the lrp5/dkk1 interaction . in order to determine
if nci8642 was also able to disrupt the binding of dkk1 to lrp6 , a recombinant hek293 cell line stably expressing lrp6 was generated ( hek293-lrp6 ) .
an additional recombinant hek293 cell line was established for the production of human recombinant dkk1 fused to the v5 tag at the c - terminus ( hek293-dkk1 ) .
when conditioned medium ( cm ) containing dkk1 was incubated on cells expressing lrp6 and the cell - bound dkk1 was detected by immunofluorescence , a strong membrane signal was observed in hek293-lrp6 cells ( figure 1(a ) , panel b ) , while it was almost absent on the parental hek293 cells ( figure 1(a ) , panel a ) .
when dkk1 cm was supplemented with 100  m nci8642 , the binding of dkk1 ( figure 1(a ) , panel d ) was reduced compared to the dmso - treated control ( figure 1(a ) , panel c ) . in order to obtain a quantitative measure of the dkk1/lrp6 interaction , a construct coding for human dkk1 fused to a secreted domain of alkaline phosphatase
was generated ( ap - dkk1 ) and used to establish a stable hek293 cell line secreting ap - dkk1 in the culture medium .
the ap tag at the c - terminus does not impair dkk1 binding to lrp6 , as shown by immunofluorescence of cell - bound ap - dkk1 on hek293-lrp6 ( figure 1(a ) , panels e and f ) .
the binding of ap - dkk1 to hek293-lrp6 , quantified by enzymatic ap assay , was reduced when ap - dkk1 was incubated in the presence of increasing concentrations of nci8642 .
the binding of dkk1 was reduced in a concentration - dependent manner compared to dmso sample ( figure 1(b ) ) .
the calculated ic50 was 14.6  m ( pic50 = 4.8 , stdev 0.2 , n = 10 independent experiments ) .
when flowed over the surface of the sensor chip coated with recombinant lrp6-fc , dkk1 bound to immobilized receptor both specifically and in a concentration - dependent manner ( figure 2(a ) , kon   5.3  10  m  s , koff   1.8  10  s ) .
indirect capturing of lrp6-fc on recombinant protein a and direct lrp6-fc covalent coupling via amine groups produced similar interaction with recombinant human dkk1 ( kd of 8.4 and 3.5  10  m , resp . ) , demonstrating that the covalent coupling of the receptor to the chip surface does not influence its binding site .
the interaction constants obtained are in agreement with the literature data [ 1 , 34 , 35 ] .
the lrp6-dkk1 interaction was also verified using recombinant dkk1 cm instead of commercial recombinant dkk1 ( figure 2(b ) ) .
when we investigated whether nci8642 was able to bind to the lrp6 receptor , we observed a consistent and effective binding signal that is not present in the control flow cell .
the kinetic analysis of the interaction by injection of an nci8642 dilution series ( from 3  m to 50  m ; figure 2(c ) ) allowed the determination of a kd of 4.7  10  m for nci8642 on lrp6 .
we also analyzed the effect of nci8642 on the binding of dkk1 to lrp6 , to determine whether the compound could interfere with the interaction between dkk1 and lrp6 .
we injected dkk1 and nci8642 over lrp6 previously immobilized . when dkk1 was injected together with nci8642 after 1-hour incubation at 25c
, we observed a consistent reduction of dkk1 binding on lrp6 with 5  m and 10  m compound , and even a complete binding inhibition at the concentration of 20  m ( figures 2(d ) and 2(e)-upper panel ) . on the contrary
, sequential injections of 20  m nci8642 and 54  nm dkk1 did not inhibit the dkk1-lrp6 interaction .
nci8642 was not able to displace dkk1 when injected after it and dkk1 could still bind to lrp6 when injected after nci8642 at the concentration of 20  m ( figure 2(e)-middle and lower panels ) . to investigate the functional activity of nci8642
, we tested the compound in a tcf reporter - based assay in pc12 cells ( neural crest - derived rat pheochromocytoma cells ) .
tcf , similarly to lef-1 , is a downstream target transcription factor of the canonical wnt - signaling pathway .
a stable pc12 cell line expressing a tcf - dependent luciferase reporter was established ( pc12-tcf - luc ) .
wnt3a - mediated response was reverted by dkk1 cm in concentration - dependent manner ( figure 3(a ) ) .
for the purpose of compound testing , the dilution factor of dkk1 cm was chosen in order to have about 50% inhibition of wnt signaling ( figure 3(b ) , open symbols ) . in the presence of exogenously added dkk1
the effect was concentration - dependent and was reproducibly and significantly different from dmso treatment at 25  m and 50  m , although higher concentrations of the compound were toxic to the cells and an ic50 value could not , therefore , be calculated .
nci8642 was also tested in an assay measuring the modulation of -catenin intracellular accumulation and nuclear translocation .
the assay was performed in l - cells , an established model system for studying wnt signaling . upon treatment of l - cells with wnt3a cm , the intracellular amount of -catenin increases , with an enrichment in the nuclear compartment and a concomitant increase in the nuclear / cytoplasmic ratio of -catenin intensity . in the sample
treated with control cm , -catenin is hardly detectable , with a nuclear / cytoplasmic ratio close to one .
the wnt3a - mediated effect is reverted by dkk1 cm in a concentration - dependent manner ( figures 4(a ) and 4(b ) ) . when nci8642 was added to the cells together with dkk1 , and then the cells were stimulated with wnt3a
the measured ic50 of the compound is 12.6  m ( pic50 = 4.9 stdev 0.2 , n = 5 independent measurements ) , in good agreement with the binding data and the reporter assay ( figures 5(a ) and 5(b ) ) .
nci8642 is a small molecule that has been recently identified as a specific inhibitor of lrp5-dkk1 interaction .
given the role of the wnt signaling in many pathophysiological contexts , modulation of the wnt pathway has become a valuable target in drug discovery .
the modulation of the pathway at the level of the cell surface poses the problem of targeting a protein - protein interaction , which might be challenging .
recently , the lrp5-dkk1 interaction has been targeted with monoclonal anti - dkk1 antibodies [ 39 , 40 ] .
these antibodies were effective in increasing bone density in vivo , both in nave normal growing female mice and in a model of postmenopausal osteoporosis .
however , the lack of blood - brain penetration of the antibodies limits their application to peripheral indications such as osteoporosis . on the contrary , a small molecule blocking dkk1 inhibition would have the advantage of being potentially useful also for the treatment of cns pathologies , such as neurodegeneration .
our study confirmed that nci8642 acts as a dkk1 inhibitor , and we extended the finding to the interaction of dkk1 to lrp6 .
in fact , the small molecule caused the displacement of dkk1 from lrp6 overexpressed in hek293 cells , as shown both by immunofluorescence and quantitative enzymatic assay .
the displacement was also demonstrated in a cell - free context by using spr technology , where nci8642 blocks the binding of dkk1 to the immobilized lrp6 .
interestingly , the compound was able to block dkk1 binding only when injected together with dkk1 , and not when injected sequentially after dkk1 , which is probably due to the high affinity of dkk1 for lrp6 compared with the micromolar affinity of nci8642 for lpr6 ( figures 2(c ) and 2(e ) ) .
spr technology also showed that nci8642 interacts with the immobilized lrp6 with a reproducible and concentration - dependent binding in the absence of dkk1 ( kd 4.7  10  m ) .
the interaction between nci8642-lrp6 is highly specific , because no significant binding was observed on two control flow cells .
when immobilized on the sensor chip , dkk1 lost the ability to bind lrp6 , suggesting that the immobilization caused a modification of dkk1 structure - function , thus the direct interaction of nci8642 to dkk1 was not measured .
nci8642 is also functionally active in the inhibition of dkk1 activity on wnt signaling mediated by lrp5/6 as demonstrated by the tcf - luc and -catenin assays in two different cellular backgrounds .
the compound ic50 measured in the tcf - luc and -catenin assays , where lrp5/6 is expressed at endogenous , physiological levels , is comparable with the one obtained in the binding assay on the overexpressed lrp6 . in the tcf - luc assay
, we observed a slight increase of the wnt3a - mediated activation of the reporter gene , even in the absence of dkk1 .
the hypothesis that it could be due to the presence of endogenous dkk1 in these cells was not confirmed , since we could not detect it by western blot or by quantitative rt - pcr ( data not shown ) .
nevertheless , this was not observed in the -catenin translocation assay ( data not shown ) , suggesting that it could be an artifact of the reporter assay , or a cell - line - specific effect .
although nci8642 has been reported as a dkk1/lrp5 inhibitor , our data suggest that it is also effective on dkk1/lrp6 interaction .
in fact , nci8642 blocked dkk1 binding on lrp6 , both in lrp6 overexpressing cells and in spr analysis .
this was also confirmed by functional studies , since only lrp6 , but not lrp5 , is expressed in pc12-tcf - luc cell line , and lrp6 mrna concentration is about 7 times that of lrp5 in l - cells , as assessed by qpcr ( data not shown ) .
our results suggest that it is possible to antagonize the dkk1-lrp6 interaction with a small molecule , and this leads to an enhancement of the wnt signaling pathway .
although nci8642 micromolar potency might be too low for its application in in vivo experiments , this compound can be considered as a reference small molecule for further drug development aiming at the inhibition of this therapeutically important protein - protein interaction .
 
 background . 
 dkk1 antagonizes canonical wnt signalling through high - affinity binding to lrp5/6 , an essential component of the wnt receptor complex responsible for mediating downstream canonical wnt signalling . 
 dkk1 overexpression is known for its pathological implications in osteoporosis , cancer , and neurodegeneration , suggesting the interaction with lrp5/6 as a potential therapeutic target . 
 results . 
 we show that the small - molecule nci8642 can efficiently displace dkk1 from lrp6 and block dkk1 inhibitory activity on canonical wnt signalling , as shown in binding and cellular assays , respectively . 
 we further characterize nci8642 binding activity on lrp6 by surface plasmon resonance ( spr ) technology . 
 conclusions . 
 this study demonstrates that the dkk1-lrp6 interaction can be the target of small molecules and unlocks the possibility of new therapeutic tools for diseases associated with dkk1 dysregulation . 
1. Background
2. Methods
3. Results
4. Discussion