bronchial asthma is a chronic disorder characterized by airway inflammation , remodeling of the airways , and their hyperresponsiveness to environmental stimuli .
it is accompanied by thickening of the bronchial walls , epithelial damage , subepithelial fibrosis , and the increased deposition of extracellular matrix proteins .
the bulk of these changes results from the phenotypic transitions of bronchial epithelial cells , smooth muscle cells , and fibroblasts [ 1 , 2 ] . in general , phenotypic shifts , such as epithelial - mesenchymal transition ( emt ) and fibroblast - myofibroblast transition ( fmt ) ,
in particular , erroneous function of myofibroblasts , representing the contractile subset of fibroblasts which have undergone fmt , was found to be crucial for the development of asthma [ 6 , 7 ] .
fmt , which is also involved in wound contraction and participates in the development of fibrotic changes in several tissues [ 810 ] , can be induced by a range of proinflammatory cytokines , in particular by factors belonging to the transforming growth factor  ( tgf- ) family [ 11 , 12 ] . on the other hand ,
an increase in the local concentration of tgf- was observed in bronchoalveolar lavage fluid sampled from asthmatic patients , and prolonged exposure to tgf- effectively induced fmt in hbfs in vitro models .  
it is generally accepted that tgf- regulates transcription of profibrotic genes , including cell adhesion molecules and extracellular matrix proteins [ 15 , 16 ] .
the canonical tgf- signaling pathway depends on the phosphorylation of r - smad proteins ( smad2 and 3 ) by activated tgf- receptor , their interaction with smad4 , and accumulation in the nuclei [ 17 , 18 ] .
tgf- signaling activity is modulated by other signaling systems , such as the wnt - dependent pathway [ 19 , 20 ] , which has also been implicated in the pathophysiology of lung cancers , asthma , and lung fibrotic disease development [ 2125 ] .
tgf- was shown to induce fmt in human lung fibroblast populations ; however , it attenuated the allergic inflammation and asthmatic symptoms in an ovalbumin - induced mouse model of asthma .
activation of wnt pathway results in the inhibition of gsk-3 ( glycogen synthase kinase 3 beta ) and subsequent cytoplasmic accumulation of -catenin .
it forms complexes with transcription factors of the lef and tcf family activating the expression of wnt - responsive genes .
a range of interactions between wnt and tgf- effectors have been identified , which conceivably account for synergic effects of tgf- and wnt signaling observed in vitro during fmt in mouse embryonic fibroblast populations .
for instance , mapk can phosphorylate both r - smads and gsk-3 [ 25 , 26 ] , while -catenin was found to interact with i - smads .
these interactions may also underline cellular context - dependent role of gsk-3 activity in asthma development .  
lithium is a nonspecific gsk-3 inhibitor widely used in the therapy of bipolar affective disorder and other psychiatric diseases .
interestingly , lithium treatment of schizoaffective disorders coincided with the remission of asthmatic symptoms , whereas the recurrence of symptomatic asthma was observed after discontinuation of lithium therapy . moreover , treatment with lithium led to a reduction in bronchial hyperresponsiveness in asthmatic patients .
we have previously shown that hbfs derived from patients with asthma displayed more pronounced fmt in response to tgf- in comparison with nonasthmatic counterparts .
this indicates susceptibility towards a tgf--induced phenotypic switch , an event crucial for the asthmatic process [ 14 , 34 ] . in the current study , we aimed to elucidate the role of lithium compounds in tgf-1-induced fmt in hbf populations and the mechanisms responsible for this process .
we demonstrate inhibition of gsk-3 activity by lithium attenuated tgf-1-induced fmt in hbf populations derived from asthmatic donors .
this effect correlated with deficient nuclear localization of -catenin and p - smad2 in tgf-1/licl - stimulated hbfs .
bronchial biopsies were obtained from 11 patients with bronchial asthma ( as ) and 11 non - asthmatic subjects ( na ) during fiberoptic bronchoscopy performed in the department of medicine ( jagiellonian university medical college ) .
primary hbf were isolated from bronchial biopsies as described previously [ 14 , 34 ] .
the first group consisted of patients with moderate to severe bronchial asthma ( as group ) with a mean age of 43.1  9.5 years ( females 64% ) , the average duration of the disease was 11.3  9.4 years , and decrease in the first second forced expiratory volume ( fev1 : 78.4  22.4% of predicted ) .
the second group comprised 11 subjects in whom diagnostic bronchoscopy ruled out any serious airway pathology , including asthma , chronic inflammatory lung disease , or cancer ( nonasthmatics ; na group ) , with mean age of 58.7  7.8 years ( females 36% ) and normal lung function tests ( fev1 : 97.3  12.8% of predicted ) .
the study was approved by the jagiellonian university 's ethics committee ( kbet/362/b/2003 ) , and informed consent was obtained from all study participants .  
hbfs were cultured in dmem with 10% fetal bovine serum ( fbs ) at 37c in a humidified atmosphere with 5% co2 and used between 5 and 15 passages . for experiments
, cells were plated at a density of 5000 cells / cm and cultured in serum - free dmem supplemented with 0.1% bovine serum albumin ( bsa ; sigma - aldrich , st .
louis , mo , usa ) . when indicated , human recombinant tgf-1 ( bd biosciences , franklin lakes , nj , usa ) , lithium chloride ( licl ; sigma - aldrich ) or their cocktail , and gsk-3 inhibitor xii , tws119 ( calbiochem , la jolla , ca , usa ) were administered 24  h after cell seeding .
lithium chloride was administered at the concentration of 10  mm , which remained in a range of serum concentrations observed in patients subjected to the chronic therapy of bipolar affective disorder [ 35 , 36 ] .
myofibroblasts were identified by immunodetection of -sma as described previously . in brief , cells growing on glass coverslips were fixed in 3.7% paraformaldehyde , permeabilised in 0.1% triton x-100 , blocked with 1% bsa , and incubated with a mouse monoclonal antibody against human -sma ( clone 1a4 , sigma - aldrich ) and alexa fluor 488 goat anti - mouse igg ( clone a11001 , sigma - aldrich ) .
visualization of specimens mounted in polyvinyl alcohol ( mowiol ; sigma - aldrich ) was performed with a leica dm ire2 microscope equipped with 40x , na-1.25 hcx plan apo objective , leica dc350fx camera , and leica fw4000 software .
a similar protocol was used for immunodetection of other cellular antigens including tgf- receptor ii ( with a mouse monoclonal antibody against tgf--rii , clone mm0056 - 4f14 , abcam , cambridge , uk ) , -catenin ( with a mouse monoclonal antibody against -catenin , sigma - aldrich ) or p - smad ( with a rabbit polyclonal antibody against smad2 , phospho s467 , abcam ) , and compatible secondary antibodies conjugated with alexa fluor 488 or alexa fluor 546 ( all from invitrogen , carlsbad , ca , usa ) . when indicated , nuclei were stained with hoechst 33342 ( sigma - aldrich ) .   for the analyses of tgf- receptor ii expression on the surface of hbf ,
confluent cell monolayers were nonenzymatically detached using the cell dissociation solution ( sigma - aldrich ) according to the manufacturer 's protocol , centrifuged , fixed and permeabilized with cytofix / cytoperm kit ( bd biosciences ) , and stained with primary mouse monoclonal anti - tgf- receptor ii antibody ( abcam ) . after washing with perm - wash buffer ( bd biosciences ) , cell suspensions were stained with secondary fitc - conjugated goat anti - mouse igg ( sigma - aldrich ) and analysed by flow cytometry ( coulter epics xl , beckman coulter , fullerton , ca ) .
results were expressed as a ratio of mean fluorescent intensity ( mfi ) of tgf--rii - specific staining to mfi of the corresponding goat - igg2a isotype control sample ( expression index ) .
hbfs cultured in 6  cm petri dishes for 7 days ( until confluency ) in control conditions or stimulated with tgf-1 ( 5  ng / ml ) , licl ( 10  mm ) and their mixture ( tgf-1/licl ) , or tws119 ( 5  m ) were washed , harvested using a cell scraper , centrifuged , and dissolved in lysis buffer ( 50  mm tris - hcl ph 7.4 , 150  mm nacl , 2% tritonx-100 , 0.02% nan3 with the addition of a proteinase inhibitor ) .
alternatively , total protein was isolated using m - per buffer ( pierce , rockford , il , usa ) with protease and phosphatase inhibitors ( sigma - aldrich ) , and the nuclear fraction was isolated using neb - b buffer ( 29  mm hepes ph 7.9 ; 0.4  m nacl ; 1  mm edta ; 1  mm egta ) . in both cases ,
cellular proteins were separated on 15% sds - polyacrylamide gels and transferred to nitrocellulose membranes .
after blocking in pbs - t ( 0.1% tween 80 in pbs containing 5% skimmed milk ) , membranes were incubated with mouse monoclonal antibodies ( all from sigma - aldrich ) , anti--sma ( 1  :  1000 ) , anti--catenin ( 1  :  2000 ) , anti--tubulin ( 1  :  1000 ) , anti - gapdh ( 1  :  10000 ) , and rabbit polyclonal antibodies anti - human retinoblastoma - associated protein 46 ( rbap46 , 1  :  2000 ) . after washing
, membranes were incubated for 1 hour with horseradish peroxidase - conjugated anti - mouse igg ( 1  :  3000 , invitrogen ) or anti - rabbit igg ( 1  :  3000 , invitrogen ) antibodies , treated with the chemiluminescent reagent super signal west pico substrate ( pierce , rockford , il ) and exposed to kodak x - omat film ( sigma - aldrich ) .
we have previously demonstrated that hbfs derived from patients with bronchial asthma display inherent features in vitro that facilitate fmt in response to prolonged incubation with tgf-1 [ 14 , 34 ] . here ,
we aimed to investigate the interference of lithium with tgf-1-induced fmt in hbf populations derived from asthmatic patients ( as , n = 8) and non - asthmatic subjects ( na , n = 8) . therefore , we first quantified -sma - positive cells in hbf populations which had undergone long - term tgf-1 stimulation .
we have found a considerably higher fraction of -sma expressing hbfs in as as compared to na populations ( figure 1(a ) ) which indicates their propensity towards a tgf-1-induced phenotypic switch .
this effect was independent of the total density of tgf- receptor as both immunofluorescence microscopy and flow cytometry analyses did not reveal any significant differences in expression levels between as and na patients ( figure 1(b ) ) .
furthermore , cytoplasmic levels of p - smad2 protein , an effector of tgf- signalling , and its nuclear localisation were increased in tgf-1-treated hbf populations , both in na and as groups .
however , we observed a slightly more pronounced nuclear accumulation of p - smad2 in tgf-1-stimulated asthmatic hbf populations ( figure 1(c ) ) .
these results suggest that mechanisms downstream of the tgf- receptor which modulate nuclear transport of smad protein(s ) determine the differences in tgf-1 reactivity between hbf derived from asthmatic and non - asthmatic patients .  
it has been found that gsk-3-dependent pathways interact with tgf- signaling in a variety of cellular systems [ 37 , 38 ] .
similarly , lithium which at millimolar concentrations displays gsk-3 inhibiting activity , has been shown to interfere with the asthmatic process [ 32 , 33 ] .
application of the experimental system based on quantification of fmt in hbf populations derived from as and na patients enabled us to elucidate the effect of cellular context on fmt - related cell reactivity to lithium ( figure 2 ) .
we have not observed any significant differences in the viability and percentage of myofibroblasts between untreated ( control ) and treated by licl alone in hbf cultures , regardless of the source of cells ( data not shown ) .
in contrast to tgf-1-treated hbf from the na group , which reacted to the administration of lithium with the induction of fmt , as hbfs treated with tgf-1/licl displayed a decrease in the fraction of myofibroblasts compared to the cells stimulated with tgf-1 alone ( 43 to 85% of -sma positive cells in the presence of tgf-1 compared to 1542% in tgf-1/licl mixture ; figure 2(b ) ) .
these data indicate that increased susceptibility of as hbfs to tgf-1-induced fmt is associated with a differential response to licl as compared to na and suggest the presence of a possible disturbance in crosstalk between signalling pathways regulated by tgf-1 and gsk-3. to check whether the attenuation of tgf-1-induced fmt in as hbf populations treated with lithium specifically results from its inhibitory effect on gsk-3 activity , we further treated the cells with tws-119 , a specific gsk-3 inhibitor .
its effect on the number of myofibroblasts in tgf-1-treated na and as hbf populations was found to be similar to that exerted by lithium ( figure 3 ) indicating the involvement of gsk-3 activity in the observed phenomena .
moreover , in na hbf populations treated with tgf-1/licl or tgf-1/tws-119 , the percentage of -sma - positive cells corresponded to -sma protein levels ( compare figure 2 to figure 4(a ) and figure 3 to figure 4(b ) ) . on the other hand ,
different results were obtained for as hbf populations . despite lower numbers of -sma - positive cells , the same amounts of -sma
this effect was not observed in tgf-1/tws-119-treated as hbf populations ( figure 4(b ) ) .
this can be explained in terms of gsk-3-unspecific induction of  supermaturation  and hypertrophy of a subset of as hbf subjected to tgf-1/licl treatment .
such a sparse subpopulation characterised by a high degree of spreading and intense -sma - specific staining was found in as hbf populations treated with the tgf-1/licl mixture ( data not shown ) .
moreover , it should be noticed that the effect of gsk-3 inhibition on fmt is much more striking when comparing the percentage of myofibroblasts to the -sma protein levels .
the reason of it could be that not all expressed -sma is incorporated into stress fibers which are the marker of myofibroblasts .   altogether ,
these data indicate that gsk-3 participates in the attenuating effect of lithium on tgf-1-induced fmt in as hbf populations ; however , lithium may exert a gsk-3-unspecific effect on the maturation of myofibroblasts .
inhibition of gsk-3 activity results , among others , in the hypophosphorylation of -catenin and its nuclear translocation leading to the activation of gene expression .
therefore , we further focused on the effect of lithium and tws119 on -catenin levels in na and as hbf populations stimulated by tgf-1 .
interestingly , as hbfs are characterized by a notably higher amount of total -catenin ( figure 5 ) and -catenin cytoplasmic fraction ( figure 6 ) .
administration of tgf-1/licl and tgf-1/tws119 resulted in the additional increase of the -catenin level in na populations , in contrast to as cells ( figures 5(a ) and 5(b ) ) . on the other hand , inhibition of gsk-3 regardless of tgf-1 treatment resulted in the nuclear accumulation of -catenin in na hbf , whereas this effect was not observed in as hbf populations ( figures 6 and 7(b ) ) .
thus , the impaired intracellular trafficking of -catenin may account for differences in cell reactivity to tgf-1 stimulation and fmt between na and as bronchial fibroblasts . to further clarify the mechanism of the observed phenomenon
, we elucidated the effect of the licl and tgf-1/licl mixture on nuclear accumulation of p - smad2 , an event crucial for the activation of tgf- signalling activity ( figure 7 ) .
treatment with the tgf-1 and tgf-1/licl cocktail induced p - smad2 nuclear accumulation in na hbf populations but not in as hbf populations .
tgf-1-induced nuclear translocation of p - smad2 was significantly attenuated by lithium in as hbf populations ( figure 7(a ) ) .
interestingly , this effect correlated with the pattern of nuclear accumulation of -catenin ( figure 7(b ) ) .
these data suggest a role for the interference of the -catenin cytoplasmatic fraction with the function of p - smad2 during fmt in as hbf populations .
bronchial wall remodeling , a fundamental factor in the development of asthma , results from erroneous patterns of differentiation of bronchial cells , in particular fibroblasts [ 1 , 2 ] .
these changes are regulated by elevated production of proinflammatory cytokines , in particular tgf- , in regions of epithelial damage as reflected in bronchoalveolar lavage fluid from asthmatics [ 13 , 42 ] .
fmt , a crucial event during bronchial wall remodeling and development of fibrotic changes observed in asthma , is supposed to depend on a concerted action of inflammatory mechanisms and inherent features of airway wall fibroblasts facilitating their phenotypic transitions in response to il-4   and tgf- [ 14 , 34 ] .
our previous   and current observations indicate that increased local concentrations of tgf- , resulting from inflammation , and differences in the expression levels of tgf- receptors are not necessarily implicated in bronchial wall remodeling during the asthma .
in contrast , more pronounced differences in functional status of hbfs residing in healthy and asthmatic bronchi , for example , resulting from genetic patients ' background or selection of clones epigenetically predisposed to fmt , can account for the differences in attitude towards fmt observed between hbfs derived from asthmatic and non - asthmatic patients .
we show that inhibition of gsk-3 activity significantly attenuates tgf-1-induced fmt in hbf derived from asthmatic patients , whilst an opposite effect was observed in hbf isolated from non - asthmatic subjects .
thus , specific molecular mechanisms involving the interactions between smad proteins and effectors of gsk-3-dependent signaling determine fmt - related responses of hbf isolated from asthmatic patients .  
the observed augmentation of tgf-1-induced fmt by lithium in na hbf populations , along with a lack of differences in tgf- receptor expression levels between na and as hbfs , indicates the involvement of synergistic tgf-/gsk-3 signaling interplay in phenotypic shifts during differentiation of bronchial fibroblasts .
such an induction of fmt - related processes by synergic activation of tgf-/gsk-3 signaling was previously seen in mouse mesenchymal c3h10t1/2 cells , mouse embryonic fibroblasts , and pulmonary fibroblasts from individuals with chronic obstructive pulmonary disease .
the inhibition of gsk-3 is a key event during the activation of canonical wnt pathway and leads to hypophosphorylation of -catenin , its cytoplasmatic accumulation , subsequent nuclear translocation of -catenin complexes with transcription factors , and induction of wnt - responsive gene expression .
our data , demonstrating a correlation between -sma expression , -catenin levels , and nuclear translocation of -catenin in the na hbf populations treated with tgf-1/licl and tgf-1/tws119 cocktails , reveals the involvement of the -catenin - dependent pathway in phenotypic transitions of bronchial fibroblasts .
these data are in line with the in vitro observations of similar interrelations in human lung   and skin fibroblast , and lung smooth muscle cell populations   and with the in vivo data on reduced bleomycin - induced pulmonary fibrosis upon -catenin silencing in the murine model .
notably , our study was focused on -sma expression as a marker of fmt ; however , wnt signaling controls the expression of a number of genes in lung cells , including fibronectin and collagen .
importantly , we have recently shown that the inhibition of gsk-3 activity by tws119 attenuates the secretion of ctgf by hbf derived from asthmatic patients .  
on the other hand , attenuation of tgf-1-induced fmt by lithium observed in hbf populations derived from asthmatic patients may indicate that inherent features facilitating fmt are accompanied by disturbed coherence of tgf-1/gsk-3 signaling pathways . actually , an induction of fmt by tgf-1 coincided with an increased abundance of -catenin in as but not na hbf populations .
in contrast , high levels of -catenin accompanied a relative decrease of -sma - positive cell numbers in tgf-1/licl - treated as hbf populations . thus , the function of -catenin during fmt may depend on the activity of gsk-3 modulated by lithium or tws119 .
the specific inhibition of gsk-3 by tws119 failed to induce the nuclear translocation of -catenin in as hbf populations .
therefore , the attenuating effect of lithium on fmt in as hbf populations may result from disturbed intracellular -catenin trafficking and its antagonistic effect on the function of downstream effectors of tgf-1 [ 29 , 5153 ] .
the phosphorylation of smad proteins and their subsequent accumulation in the nuclei are key events during the activation of tgf- signaling [ 17 , 18 ] . because we observed deficient nuclear accumulation of p - smad2 in as hbf populations treated with a tgf-1/licl cocktail , it is conceivable that cytoplasmically sequestrated -catenin may interact with smad proteins inhibiting their nuclear accumulation and tgf--specific signal transduction .
elevated levels of cytoplasmatic -catenin were previously demonstrated to inhibit p - smad2 nuclear accumulation via the inhibition of ismad phosphorylation and degradation .
therefore , further study is necessary to address the role of ismad proteins , in particular the possible involvement of their inhibitors , such as peptidyl - prolyl isomerases , in the observed differences between the lithium - evoked reactions of as and na hbfs .  
our finding of an inhibitory effect of lithium on tgf-1-induced fmt in as hbf populations provides a potential explanation for the attenuating effect of lithium on the ongoing asthmatic process , as well as coincidental remission of asthmatic symptoms and their recurrence upon discontinuation of lithium therapy in psychiatric diseases , as revealed by epidemiological studies [ 3133 ] .
however , it should be emphasized that numerous adverse effects of lithium have been reported .
nonetheless , other drugs targeting the wnt/-catenin pathway were reported to be potentially effective in treating cancer   and tissue remodeling after injury .
significant progress has recently been made in identifying such molecules and inventing new strategies of inhibition of wnt signaling in therapeutic applications .  
therefore , further research focused on the cell context - specific action of lithium or other wnt pathway activators during the asthmatic process , and the mechanisms of interference of these compounds with phenotypic transitions of bronchial fibroblasts would help to delineate borders of future clinical application in acute asthma treatment .
 bronchial asthma is a chronic disorder accompanied by phenotypic transitions of bronchial epithelial cells , smooth muscle cells , and fibroblasts . 
 human bronchial fibroblasts ( hbfs ) derived from patients with diagnosed asthma display predestination towards tgf--induced phenotypic switches . since the interference between tgf- and gsk-3 signaling contributes to pathophysiology of chronic lung diseases , we investigated the effect of lithium , a nonspecific gsk-3 inhibitor , on tgf-1-induced fibroblast to myofibroblast transition ( fmt ) in hbf and found that the inhibition of gsk-3 attenuates tgf-1-induced fmt in hbf populations derived from asthmatic but not healthy donors . 
 cytoplasmically sequestrated -catenin , abundant in tgf-1/licl - stimulated asthmatic hbfs , most likely interacts with and inhibits the nuclear accumulation and signal transduction of smad proteins . 
 these data indicate that the specific cellular context determines fmt - related responses of hbfs to factors interfering with the tgf- signaling pathway . 
 they may also provide a mechanistic explanation for epidemiological data revealing coincidental remission of asthmatic syndromes and their recurrence upon the discontinuation of lithium therapy in certain psychiatric diseases . 