the serine protease urokinase - type plasminogen activator ( upa ) receptor ( upar ) has been implicated in extracellular matrix ( ecm ) proteolysis , initiation of signal transduction , and important cell functions including migration and invasion . the active upa consists of catalytic protease domain and the upa amino terminal fragment ( upa - atf ) . upa - atf contains the kringle domain and the growth factor - like domain ( gfd ) that contains the binding sequence for the receptor . the upar protein has been shown to engage in multiple protein - protein interactions with other proteins such as vitronectin , integrins , and mrj [ 35 ] . sprouty ( spry ) proteins have been identified as inhibitors of the receptor tyrosine kinase ( rtk ) including ( epidermal growth factor receptor ) egfr . the mammalian genome contains four spry genes ( spry 14 ) encoding proteins with a conserved cysteine - rich region at the carboxyl terminus . due to its central role in ras / map kinase pathway , spry may act as a putative tumor suppressor gene , and that loss of expression or function may allow the cell to be hypersensitive to growth signals . interestingly , the tumor suppressor gene , wt1 , has been found to bind with spry1 promoter and regulate it during kidney development . prostate ductal hyperplasia and low - grade prostatic intraepithelial neoplasia ( pin ) have been observed in adult mouse with concomitant spry1 and spry2 loss of function . additionally , spry1 expression has been shown to be modulated in a variety of cancers . hspry1 expression was downregulated in human prostate carcinoma [ 11 , 12 ] and was decreased or absent in 15% of primary human prostate cancer and 42% of metastatic human prostate cancer . in addition , pcr studies showed downregulation of hspry1 expression in more than 94% of breast cancer patient samples , with respect to normal tissues . we have used previously yeast two - hybrid screening of breast cancer cdna library to identify hspry1 as a candidate protein that interacts with upar . this protein - protein interaction may influence a range of biological functions . here , we show for the first time that hspry1 may negatively regulate upar - stimulated cell migration and invasion in vitro . the goat anti - upar antibody was purchased from r&d systems ( number af807 , minneapolis , mn , usa ) . the mouse anti - human spry1 antibody ( a01 ) was purchased from abnova corporation , taiwan . bernard kwabi - addo ( department of pathology , baylor college of medicine , baylor plaza , houston , tx , usa ) . the recombinant human upa was purchased from r&d systems ( # 1310-se , minneapolis , mn , usa ) . the recombinant human upa - atf was obtained from american diagnostics ( # 146 , stamford , ct , usa ) . cells were transfected with recombinant or control vector in addition to gfp plasmid to control transfection efficiency . transfections were performed using genejuice ( # 70967 , novagen , emd bioscience , merck kgaa , darmstadt , germany ) . the human embryonic kidney hek293 cells stably transfected with upar were kindly provided by dr . the human breast cancer mda - mb-231 , the human colorectal carcinoma hct116 , and the human osteosarcoma saos-2 cell lines were obtained from the american type culture collection ( atcc ) . cells were maintained in dulbecco 's modified eagle 's medium supplemented with 10% fcs and 1% antibiotics . cells transfected with either pcdna3.1/hspry1 or pcdna3.1 empty plasmid were seeded into sterilized glass cover slips . cells were washed with pbs , fixed , and permeabilized with ice cold methanol for 10  min at 20c . cells were then washed , blocked with 1% bsa , and incubated with primary antibodies in 1% bsa , followed by rhodamine - conjugated and fitc - conjugated secondary antibodies in 1% bsa . cells were then washed , mounted , and visualized using confocal laser scanning microscopy ( olympus ix71 laser scanning microscope ) and 60x oil immersion lens . briefly , cells were seeded into culture dishes and incubated for 24  h at 37c . cells were then transfected with either pcdna3.1/hspry1 or pcdna3.1 empty plasmid and incubated to create confluent monolayer . a wound was created manually by scrapping the cell monolayer with a yellow pipette tip . the first images were taken at four different focal areas in each well , and cells were stimulated with egf , upa , or upa - atf 100  ng / ml or left without stimulation as a control . cells were incubated in the incubator , and the second images were taken at 18  h. these images were analyzed quantitatively by measuring the distance of cell migration in the wounded region . cell invasion assay is similar to cell migration assay ; however , it requires cells first to enzymatically penetrate a barrier of an ecm or basement membrane extract and then to migrate through it . here , to assess the effect of hspry1 in regulating cell invasion , we used 24-well transwell system with polycarbonate membranes of 8.0  m pore size ( # 3422 , corning ) . briefly , membranes were coated with 20  g / ml collagen iv at 4c overnight . transfected cells ( 2  10/well ) were seeded onto the upper - side chambers in 0.2  ml of serum - free dmem medium , and 0.6  ml of the same medium containing 1% fcs was added onto the lower chamber . the cells were allowed to adhere for 1  h. then , chemotaxis was induced by the addition of egf , upa , or upa - atf ( 100  ng / ml ) to the lower chamber . media containing 10% fcs and 1% fcs were used as positive and negative controls , respectively . at the end of the incubation period ( 18  h ) , cells remaining in the upper chamber were scraped . cells that invaded through the membrane to the lower surface were giemsa stained and counted in five different fields under the light microscope . data is expressed as the mean  s.d , and , where appropriate , the student 's t - test was performed using graphpad prism v5.0 software ( graphpad software inc . , la jolla , ca , usa ) . we have previously shown the colocalization and physical interaction between upar and hspry1 in cells cultured under growth media conditions . here , we used the immunofluorescence staining to investigate the expression and subcellular distribution of upar and hspry1 upon stimulation of cells with egf , upa , or its amino terminal fragment . hek293/upar and mda - mb-231 cells both transfected with hspry1 although , in mda - mb-231 cells , upar seems to be colocalized with hspry1 in the absence of any stimulation , greater colocalization between hspry1 and upar has been observed upon stimulation with either egf , upa , or upa - atf in both hek293/upar and mda - mb-231 cells . these results suggest a physiological role for hspry1 in the regulation of upar signal transduction . hence , to investigate the role of hspry1 as inhibitor of cell migration and invasion upon stimulation with egf , upa , and upa - atf , we used the wound healing assay to demonstrate the physiological impact of hspry1 overexpression on upar - stimulated hek293/upar , mda - mb-231 , and hct-116 cell migration . as shown in figure 2 , the migration capacity of cells transfected with pcdna3.1/hspry1 compared to cells transfected with vector plasmid was significantly reduced ( p < 0.05 ) in control group ( 2% fcs treated cells ) . additionally , migration capacity of cells transfected with pcdna3.1/hspry1 and treated with egf and upa was significantly inhibited ( p < 0.01 ) when compared to pcdna3.1 transfected cells . cells treated with upa - atf showed a significant reduction ( p < 0.05 ) in the migration capacity of hek293/upar and hct-116 cells transfected with hspry1 versus control group . however , mda - mb-231 cells transfected with hspry1 showed a significant inhibition ( p < 0.01 ) in the migration capacity . we next used in vitro cell invasion assay to examine the ability of hspry1 overexpression to inhibit upar - stimulated invasion . we chose as a model the invasive breast cancer mda - mb-231 and osteosarcoma saos-2 cell lines . in cells transfected with pcdna3.1 , egf and upa both increased mda - mb-231 and saos-2 cellular invasiveness ( figure 3 ) . however , the catalytically inactive upa - atf caused a great suppression of mda - mb-231 and saos-2 cell invasiveness ( figure 3 ) . compared to vector - transfected cells , mda - mb-231 and saos-2 cells transfected with pcdna3.1/hspry1 this is reflected in the significant reduced number of invasive cells ( p < 0.01 ) expressing hspry1 upon stimulation with egf and upa ( figure 3 ) . the comparison also revealed that overexpression of hspry1 in the presence of atf caused significant inhibition of mda - mb-231 ( p < 0.001 ) and saos-2 ( p overexpression of upa - system components has been associated with aggressiveness in several types of cancer that offer attractive targets for development of new diagnostics and therapeutics . many of the biological functions of upar necessitate interactions with other proteins on the cell surface , in particular , transmembrane proteins . the high lateral mobility of upar on the cell membrane may provide the mechanism by which it associates with other transmembrane receptors . additionally , the recent crystal structure model of the soluble form of upar revealed that upa bounds to upar via a central cavity . this model left the external receptor surface free to bind and interact with other proteins . in addition to the primary ligand upa , a number of upar specific interactions have also been identified and are consistent with the varied functions regulated by upar including cell adhesion , cell migration , invasion , angiogenesis , and cancer metastasis . in fact , there is now evidence that upar can bind with the cell surface integrins [ 2123 ] , chemotactic receptors , egfr , and mrj . recently , we also reported hspry1 as a candidate protein that interacts with upar , suggesting a physiological role for hspry1 in the regulation of upar functions . thus , knowing the subcellular localization of a protein can provide useful insights about its function . hence , in this study , we investigated the subcellular localization and distribution of both proteins by indirect immunofluorescence . the mammalian spry1 protein has been found predominantly in the perinuclear regions and in cytoplasmic vesicular structures of unstimulated cells . however , upon stimulation with growth factors , the protein relocates to membrane ruffles , where it shows a partial overlap with the localization of caveolin-1 . the significance of the membrane translocation of the spry proteins might control its inhibitory activity and reflect the fact that several spry - binding partners ( grb2 and raf1 ) are located at membrane ruffles . in addition , it has been found that both the tyrosine phosphorylation of spry1 and the inhibition of rtk signaling by spry1 occur at the plasma membrane , suggesting that the association with caveolin-1 might enhance spry1 function . interestingly , upar has been also found to colocalize with caveolin [ 29 , 30 ] . moreover , it has been suggested that caveolin and upar may operate within adhesion sites to organize kinase rich lipid domains in proximity to integrins , promoting efficient signal transduction . in mda - mb-231 cells , upar seems to be colocalized with hspry1 even in the absence of any stimulation . this suggests that the interaction between upar and spry1 seems not to be correlated with the stimulation of upar in the cells . this study was expanded by investigating the role of hspry as an inhibitor of signal transduction initiated by egf , upa , or upa - atf stimulation . binding of upa or upa - atf to upar at the cell surface stimulates cell migration . in addition , 1-integrins interaction with upar was found to affect the intracellular pathways that regulate cell invasion . egfr can link the signal transduction initiated by upar to erk pathway [ 33 , 34 ] , and egfr inhibitors are capable of inhibiting the signal transduction generated by upa . in addition , egfr activation is required for the invasion mediated by upar system [ 35 , 36 ] . furthermore , egf stimulates upar expression and cell invasiveness in a variety of cancer cell lines [ 37 , 38 ] . moreover , upa secretion and p56-induced cell motility are mediated by activation of egfr / erk pathways . hence , egfr appears to be a key molecule for upar - mediated tumour progression . here , the results revealed that hspry1 overexpression inhibits egf- , upa- , or upa - atf - stimulated cellular migration . in addition , hspry1 overexpression suppressed the invasive capability of cancer cells to penetrate collagen layers in response to egf , upa , or upa - atf . these data indicate that the hspry1 inhibits cell migration and invasion upon stimulation with egf , upa , or upa - atf . in conclusion , our results reveal an important role of hspry1 expression in suppression of upa system - stimulated migration and invasion in cancer cells . further , these findings may explain why hspry1 down - regulation or loss of function participates in cancer progression . additional experiments to investigate the ability of hspry1 and hax1 to modulate upar functions like metastasis and angiogenesis may be performed in the future . to assess this , in vitro and in vivo models it is yet to be investigated whether or not all three proteins interact to form a large complex or compete with one another to affect upar functions . increasing our understanding of the specific role of upar , hspry1 , and hax1 in the molecular mechanism of cancer can assist clinically in the development of new tumour markers , which may permit more accurate determination of diagnosis in patients with cancer .
the urokinase - type plasminogen activator receptor ( upar ) has been implicated in several processes in tumor progression including cell migration and invasion in addition to initiation of signal transduction . 
 since upar lacks a transmembrane domain , it uses the interaction with other proteins to modulate intracellular signal transduction . 
 we have previously identified hspry1 as a partner protein of upar , suggesting a physiological role for hspry1 in the regulation of upar signal transduction . in this study , 
 hspry1 was found to colocalize with upar upon stimulation with epidermal growth factor ( egf ) , urokinase ( upa ) , or its amino terminal fragment ( upa - atf ) , implicating a physiological role of hspry1 in regulation of upar signalling pathway . 
 moreover , hspry1 was able to inhibit upar - stimulated cell migration in hek293/upar , breast carcinoma , and colorectal carcinoma cells . 
 in addition , hspry1 was found to inhibit upar - stimulated cell invasion in breast carcinoma and osteosarcoma cell lines . 
 increasing our understanding of how hspry1 negatively regulates upar - stimulated cellular functions may determine a distinctive role for hspry1 in tumour suppression .