claudin-1 is a member the claudin family of proteins , which are important in tight junction formation 1 .
they play roles in major cellular functions such as growth and adhesion and are responsible for regulating the paracellular transport of molecules 2 .
claudins were first shown to be abnormally expressed in breast and ovarian cancer 3 , and have since been found to play roles in other cancers such as melanoma 4 , renal and squamous cell carcinomas 2 , to name just a few .
however , these proteins , and other tight junction proteins such as zona occludens-1 ( zo-1 ) have been shown to alter their subcellular localization during malignant progression 5 . in melanocytic lesions
, we have shown that claudin-1 expression is not only increased , but that its subcellular localization becomes dysregulated , moving away from its typical location at the cell membrane 4 .
benign lesions and less aggressive melanomas express claudin-1 in the nucleus , whereas aggressive melanomas have an abundance of claudin-1 in the cytoplasm .
we have shown that increasing claudin-1 expression in less metastatic melanoma cells caused an increase in melanoma cell motility , as well as in levels of mmp-2 activation and secretion .
increased claudin-1 expression did not correlate to increases in tight junction function , presumably due to the fact that it was expressed in the cytoplasm , rather than solely at the cell membrane 4 .
other studies have shown that protein kinase activity is important for the regulation and localization of claudin expression . it has been shown that protein kinases such as protein kinase a ( pka ) and protein kinase c ( pkc ) can phosphorylate claudins 6,7 .
phorbol ester treatment , which activates conventional pkc isoforoms , increased the expression 8 and cytoplasmic distribution of claudin-1 , and simultaneously reduced tight junction barrier integrity 7 .
conversely , activation of atypical pkc isoforms using bryostatin instead increased the expression of claudin-1 in the tight junction complex , and increased tight junction barrier integrity 9 . in melanoma , we have shown that claudin-1 expression levels are increased upon phorbol ester treatment , and decreased by inhibitors of conventional pkc isoforms , implying that , in melanoma , claudin-1 expression is dependent upon pkc isoforoms such as  ,  and  4 . in this study
, we also examine the effects of pka on claudin-1 expression and localization , and how the localization of claudin-1 can affect melanoma cell motility .
tight junction proteins have long been thought to be solely responsible for the transport of paracellular molecules .
recent data from cancer studies indicate that these proteins also play important roles in signal transduction . in part , this is facilitated by the translocation of claudins to subcellular locations other than their  normal  location at the cell membrane .
we show here that phosphorylation modifications of the tight junction protein claudin-1 cause its translocation to the cytoplasm and nucleus and that the subcellular localization of claudin-1 may dictate the metastatic capacity of melanoma cells .
our findings suggest that nuclear versus cytoplasmic expression of claudin-1 may become a valuable marker for diagnosis of malignant melanoma .
nuclear localization of claudin-1 is evident in nevi , and less metastatic melanoma cells 4 . to determine if the nuclear localization of claudin-1 was related to the increased invasive capacity of melanoma cells , we created a claudin-1 expression vector expressing claudin-1 containing a nuclear localization signal ( pdsredcldn1-nls )
however , transfection of this vector into g361 melanoma cells ( which have low levels of claudin-1 ) resulted in both nuclear and cytoplasmic expression of claudin-1 ( figure 1 a ) despite the fact that claudin-1 was attached to a nls .
this led us to ask whether a post - translational modification such as phosphorylation might be resulting in the transport of claudin-1 out of the nucleus . since we have previously implicated pkc in melanoma progression , and claudin-1 expression , we transfected pdsredcldn1-nls into g361 cells , and then treated the transfected cells with phorbol ester ( pma ) .
treatment of cells with 200 nm pma resulted in an almost complete exclusion of claudin-1 from the nucleus ( figure 1b ) .
this implies that active pkc may exist in the nucleus of melanoma cells , and the presence of active pkc isoforms in the nuclei of many cell types has been confirmed 10 - 12 .
staining of the g361 cells with antibodies to phosphorylated pkc (  ,  ,  ) demonstrates that there is active pkc in melanoma cell nuclei as well ( figure 1c ) .
further , we performed immunoprecipitation studies using an antibody that binds to any protein that is a potential pkc substrate , followed by western analysis for claudin-1 . in uacc647 melanoma cells which are highly metastatic and have high levels of claudin-1 4 , claudin-1 co - immunoprecipitates with the pkc substrate antibody ( figure 1d ) . in the presence of pkc inhibitors , which we have previously demonstrated to decrease claudin-1 expression 4 ,
there is a decrease in the levels of claudin-1 precipitated by the pkc substrate antibody .
g361 cells , which have very little endogenous claudin-1 , and low levels of pkc , show no immunoprecipitation of claudin-1 with the pkc substrate antibody ( figure 1d ) .
to better characterize the phosphorylation of claudin-1 by pkc , we performed an in silico analysis of the putative pkc phosphorylation sites for claudin-1 .
we found that there are five putative pkc phosphorylation sites on the claudin-1 protein ( 212 amino acids long ) , but all of these are also sites of pka phosphorylation ( table 1 ) .
there are however , three unique sites of pka phosphorylation , at amino acid residues 65 - 69 , and 189 - 192 and 201 - 205 .
it should be noted that the aa189 - 192 site overlaps with a pkc / pka phosphorylation site ( aa188 - 191 ) . to assess whether pkc or pka was responsible for the phosphorylation and nuclear exclusion of claudin-1 , we used site - directed mutagenesis of our pcdna3.1-cldn1 vector to make mutants that either mimicked a constitutively phosphorylated state ( conversion of the phosphorylation site to an aspartic acid residue , referred to as
 d  mutants ) or mutants that are non - phosphorylable ( conversion of the phosphorylation site to an alanine residue , referred to as  a  mutants ) .
this vector , when transfected into melanoma cells shows both cytoplasmic and nuclear expression of claudin-1 , as compared to empty vector controls ( figure 2a ) .
the sites of phosphorylation close to the end of the protein sequence ( aa200 - 212 ) did not provide us with successful mutants .
mutations to alanine in pkc / pka sites did not affect the subcellular localization of claudin-1 , but alanine mutations of the two unique pka sites caused nuclear localization of claudin-1 ( figure 2b ) .
mutations to aspartic acid in both pka only as well as pkc / pka sites caused cytoplasmic redistribution of claudin-1 with complete exclusion from the nucleus ( figure 2c ) .
nuclear localization of claudin-1 upon mutation of the pka sites could be mimicked using pka inhibitors .
untreated m93 - 047 cells have high levels of claudin-1 and exhibit a diffuse , largely cytoplasmic pattern of claudin-1 ( figure 3a ) . upon treatment with pka inhibitors for 15 minutes , claudin-1 shuttles into the nucleus ( figure 3b ) .
after 1 hour of treatment with pka inhibitor , claudin-1 is still highly nuclear , although some claudin-1 has started to shuttle out of the nucleus ( figure 3c ) .
this is unlike the situation with pkc inhibitors , which cause a general downregulation of claudin-1 expression 4 .
furthermore , it is interesting to note that all of the cell lines have similar levels of phospho - pka ( figure 3d ) which explains why the transfected claudin-1 is shuttled out of the nucleus even when transfected into less metastatic g361 cells , which have low amounts of phospho - pkc 4,13 . taken together these data indicate that pka is likely contributing to the subcellular localization of claudin-1 .
we have previously shown that increasing the levels of cldn-1 increases the invasion of melanoma cells 4 . to determine
if the increase in claudin-1 needs to be cytoplasmic and not nuclear to affect the ability of melanoma cells to invade , we first performed a stable transfection of our g361 cells with the s69a ( pka , non - phosphorylatable ) claudin-1 mutants .
as can be seen the pcdna3.1-cldn1 transfected cells have plenty of cytoplasmic claudin as compared to the stable empty vector clones ( figure 4a , b ) whereas the s69a mutants have largely nuclear expression of claudin-1 ( figure 4c ) . to test their invasive capacity ,
as compared to empty vector controls , cells transfected with the pka deactivating s69a mutation did not show any increase in invasion .
however , g361 cells transfected with the claudin-1 overexpressing vector showed a nearly 2-fold increase in invasion as compared to the empty vector control ( figure 4d ) .
these data appear to support the hypothesis that nuclear overexpression of claudin-1 does not increase the invasive capacity of melanoma cells , where cytoplasmic expression of claudin-1 does .
nuclear localization of claudin-1 is evident in nevi , and less metastatic melanoma cells 4 . to determine if the nuclear localization of claudin-1 was related to the increased invasive capacity of melanoma cells , we created a claudin-1 expression vector expressing claudin-1 containing a nuclear localization signal ( pdsredcldn1-nls )
however , transfection of this vector into g361 melanoma cells ( which have low levels of claudin-1 ) resulted in both nuclear and cytoplasmic expression of claudin-1 ( figure 1 a ) despite the fact that claudin-1 was attached to a nls .
this led us to ask whether a post - translational modification such as phosphorylation might be resulting in the transport of claudin-1 out of the nucleus . since we have previously implicated pkc in melanoma progression , and claudin-1 expression , we transfected pdsredcldn1-nls into g361 cells , and then treated the transfected cells with phorbol ester ( pma ) .
treatment of cells with 200 nm pma resulted in an almost complete exclusion of claudin-1 from the nucleus ( figure 1b ) .
this implies that active pkc may exist in the nucleus of melanoma cells , and the presence of active pkc isoforms in the nuclei of many cell types has been confirmed 10 - 12 .
staining of the g361 cells with antibodies to phosphorylated pkc (  ,  ,  ) demonstrates that there is active pkc in melanoma cell nuclei as well ( figure 1c ) .
further , we performed immunoprecipitation studies using an antibody that binds to any protein that is a potential pkc substrate , followed by western analysis for claudin-1 . in uacc647 melanoma cells which are highly metastatic and have high levels of claudin-1 4 , claudin-1 co - immunoprecipitates with the pkc substrate antibody ( figure 1d ) . in the presence of pkc inhibitors , which we have previously demonstrated to decrease claudin-1 expression 4 ,
there is a decrease in the levels of claudin-1 precipitated by the pkc substrate antibody .
g361 cells , which have very little endogenous claudin-1 , and low levels of pkc , show no immunoprecipitation of claudin-1 with the pkc substrate antibody ( figure 1d ) .
to better characterize the phosphorylation of claudin-1 by pkc , we performed an in silico analysis of the putative pkc phosphorylation sites for claudin-1 .
we found that there are five putative pkc phosphorylation sites on the claudin-1 protein ( 212 amino acids long ) , but all of these are also sites of pka phosphorylation ( table 1 ) .
there are however , three unique sites of pka phosphorylation , at amino acid residues 65 - 69 , and 189 - 192 and 201 - 205 .
it should be noted that the aa189 - 192 site overlaps with a pkc / pka phosphorylation site ( aa188 - 191 ) . to assess whether pkc or pka was responsible for the phosphorylation and nuclear exclusion of claudin-1 , we used site - directed mutagenesis of our pcdna3.1-cldn1 vector to make mutants that either mimicked a constitutively phosphorylated state ( conversion of the phosphorylation site to an aspartic acid residue , referred to as
 d  mutants ) or mutants that are non - phosphorylable ( conversion of the phosphorylation site to an alanine residue , referred to as  a  mutants ) .
this vector , when transfected into melanoma cells shows both cytoplasmic and nuclear expression of claudin-1 , as compared to empty vector controls ( figure 2a ) .
the sites of phosphorylation close to the end of the protein sequence ( aa200 - 212 ) did not provide us with successful mutants .
mutations to alanine in pkc / pka sites did not affect the subcellular localization of claudin-1 , but alanine mutations of the two unique pka sites caused nuclear localization of claudin-1 ( figure 2b ) .
mutations to aspartic acid in both pka only as well as pkc / pka sites caused cytoplasmic redistribution of claudin-1 with complete exclusion from the nucleus ( figure 2c ) .
nuclear localization of claudin-1 upon mutation of the pka sites could be mimicked using pka inhibitors .
untreated m93 - 047 cells have high levels of claudin-1 and exhibit a diffuse , largely cytoplasmic pattern of claudin-1 ( figure 3a ) . upon treatment with pka inhibitors for 15 minutes , claudin-1 shuttles into the nucleus ( figure 3b ) .
after 1 hour of treatment with pka inhibitor , claudin-1 is still highly nuclear , although some claudin-1 has started to shuttle out of the nucleus ( figure 3c ) .
this is unlike the situation with pkc inhibitors , which cause a general downregulation of claudin-1 expression 4 .
furthermore , it is interesting to note that all of the cell lines have similar levels of phospho - pka ( figure 3d ) which explains why the transfected claudin-1 is shuttled out of the nucleus even when transfected into less metastatic g361 cells , which have low amounts of phospho - pkc 4,13 . taken together these data indicate that pka is likely contributing to the subcellular localization of claudin-1 .
we have previously shown that increasing the levels of cldn-1 increases the invasion of melanoma cells 4 . to determine
if the increase in claudin-1 needs to be cytoplasmic and not nuclear to affect the ability of melanoma cells to invade , we first performed a stable transfection of our g361 cells with the s69a ( pka , non - phosphorylatable ) claudin-1 mutants .
pooled stable clones were analyzed for the expression of claudin-1 and its subcellular localization . as can be seen the pcdna3.1-cldn1 transfected cells have plenty of cytoplasmic claudin as compared to the stable empty vector clones ( figure 4a , b ) whereas the s69a mutants have largely nuclear expression of claudin-1 ( figure 4c ) . to test their invasive capacity , stable clones were allowed to invade through a matrigel - coated invasion chamber . as compared to empty vector controls , cells transfected with the pka deactivating s69a mutation did not show any increase in invasion .
however , g361 cells transfected with the claudin-1 overexpressing vector showed a nearly 2-fold increase in invasion as compared to the empty vector control ( figure 4d ) .
these data appear to support the hypothesis that nuclear overexpression of claudin-1 does not increase the invasive capacity of melanoma cells , where cytoplasmic expression of claudin-1 does .
claudin-1 has been shown to play an important role in skin development and tumorigenesis . claudin-1
null mice die from dehydration within a few hours of birth due to the breakdown of barrier integrity in the epidermis 14 . in humans ,
patients with the pathological condition known as aec ( ankyloblepharon - ectodermal dysplasia - clefting ) also have compromised skin integrity , and this has been shown to correlate to the lack of claudin-1 and also to the loss of p63 15 .
further , we have previously shown that increasing claudin-1 expression increases melanoma metastasis 4 . in this study
we demonstrate that the nuclear localization of claudin-1 can not contribute to the migratory capacity of melanoma cells .
these data confirm our previous observations that nuclear claudin-1 is expressed largely in benign nevi , and early stage melanoma , but that highly metastatic melanoma cells tend to have increased claudin-1 in the cytoplasm and at the membrane , but very little nuclear staining 4 .
further , we demonstrate that claudin-1 subcellular localization is affected by pka , although constitutively activating both pka and pkc sites results in the cytoplasmic distribution of claudin-1 . however , the deactivation of pkc sites does not cause nuclear localization of claudin-1 . unlike pkc , which we have previously shown to decrease the levels of claudin-1 expression
, pka inhibition does not appear to affect claudin-1 levels , but does cause nuclear sequestration of claudin-1 , implying that pka is required for claudin-1 exclusion from the nucleus .
it is curious that in cases where claudin-1 is low , it is predominantly nuclear , despite abundant active pka .
this observation implies that the levels of claudin-1 may need to reach a certain threshold prior to being shuttled out of the nucleus .
hence , in metastatic melanoma cells that have high levels of pkc , there is increased claudin-1 expression , and when enough is expressed in the nucleus , pka causes its translocation to the cytoplasm .
it is known that the nuclear expression of other tight junction proteins such as zo-1 can inhibit proliferation 16 , but in our stable s69a transfectants , we see no difference in the rate of proliferation between the transfectants with nuclear claudin-1 and the empty vector controls ( data not shown ) .
it has been suggested that the translocation of claudin-1 into the nucleus of cells , despite the absence of a nuclear localization signal may require apc , zo-1 or zo-2 , possibly as shuttles for claudin-1 17 .
this opens up the possibility that wnt signaling is involved in claudin-1 expression and localization .
indeed , a study shows that increasing -catenin increases the expression of claudin-1 in colorectal cancer cells , and that claudin-1 expression is increased in more aggressive cancers 18 .
we have previously demonstrated the importance of the non - canonical wnt signaling pathway in melanoma 13,19,20 , and have shown that wnt5a increases the epithelial to mesenchymal transition ( emt ) in melanoma cells , leading to increased invasion 13 .
these effects all require pkc , and since we have shown that , in melanoma , claudin-1 expression is dependent upon pkc , it is likely that wnt5a may also increase claudin-1 expression
further , claudin-1 is important in mediating an emt in colon cancer cells 17 , similar to that seen with wnt5a .
all of these data taken together imply that claudin-1 is an important marker of melanoma progression , especially when its subcellular localization is considered , as we have shown that its cytoplasmic expression is critical for increased malignancy . how it interacts with other molecules
involved in melanoma malignancy , such as members of the wnt pathway , remains to be determined .
cell lines . as previously described 4 , the human melanoma cell lines uacc647 and m93 - 047 were cultured in rpmi-1640 media ( gibco - brl , bethesda , md ) supplemented with 10% fetal bovine serum ( fbs ) ( hyclone , logan , ut ) , 100 units / ml penicillin g and 100 units streptomycin ( gibco - brl ) .
g361 cells were maintained in mccoy 's 5a medium ( gibco - brl ) with 10% fbs .
site - directed mutagenesis was performed on the claudin-1 sequence in the pcdna3.1/v5-his vector using the quikchange ii site - directed mutagenesis kit ( stratagene , la jolla , ca ) to mutate the sites indicated in table 1 . for initial experiments attempting to attach cldn1 to a nuclear localization signal ,
cldn1 was then subcloned into the pdsred2-nuc vector ( clontech , mountainview , ca ) .
the cells were transfected with claudin-1 expression vectors or empty vector controls , using lipofectamine plus ( gibco - brl ) . after six hours of transfection ,
cells were treated with 500g / ml g418 , clones were selected and analyzed for cldn1 expression by confocal microscopy using a zeiss meta 510 confocal microscope ( zeiss , thornwood , ny ) . treatments . for pkc inhibition studies g 6983 ( sigma , st .
louis , mo ) was used at a concentration of 1um , and pma ( cell signaling , beverly , ma ) was used at a concentration of 200 nm as previously described 4 . for vehicle controls ,
pka inhibitor ( cell permeable , myristoylated protein kinase a inhibitor amide 14 - 22 , calbiochem , san diego , ca ) , was used at a concentration of 10m for the indicated times .
the pka activator , forskolin ( sigma ) was used at a concentration of 5 m for the indicated times .
as previously described 4 , for claudin-1 staining , cells were grown on glass slides , and allowed to reach 80% confluency .
cells were fixed using 95% methanol , washed and blocked with a fluorescent blocking buffer ( 0.2% triton - x100 , 0.2% bsa , 0.2% , casein , 5% normal goat serum , 0.2% gelatin , 0.02% sodium azide ) for 1 hour at room temperature .
incubation in claudin-1 primary antibody at 0.25g / ml ( zymed laboratories , san francisco , ca ) was performed overnight at 4c .
the cells were washed again with pbs for 30 minutes , probed with secondary antibody ( conjugated with alexa fluor-488 , invitrogen , carlsbad , ca ) , then washed again with pbs , mounted in pro - long gold ( invitrogen , carlsbad , ca ) with dapi and then examined using confocal microscopy .
-tubulin ( 1:1000 ) , lamin - a ( 1:1000 ) , phospho- pka ( 1:1000 ) and pkc substrate ( 1:200 ) antibodies were obtained from cell signaling ( beverly , ma ) .
claudin-1 antibody ( 1:1000 ) was obtained from zymed laboratories ( san francisco , ca ) .
cells were grown to 80% confluency and then harvested on ice as previously described 4 .
nuclear and cytoplasmic extracts were obtained according to manufacturer 's instructions using the ne - per extraction kit ( pierce , rockford , il ) .
50g of each lysate was run out on sds - page 10% tris - glycine nu - page gels ( invitrogen ) and transferred onto 0.2 micron pvdf .
the membranes were probed with antibodies and then visualized using the ecl system ( amersham biosceinces , piscataway , nj ) . for immunoprecipitation ,
250g of lysate was incubated with protein a / g agarose ( sigma ) for 4 hours to clear the lysate .
the mixture was centrifuged gently , and the lysate was removed from the beads and placed into a new , siliconized microfuge tube .
pkc substrate antibody was added at a ratio of 1:200 , and the lysate and antibody were incubated with gentle rocking overnight at 4c .
protein a / g agarose was then added for 6h , after which time the lysate was removed , and the agarose washed in ripa three times .
sds - based sample buffer was used to remove the bound proteins from the agarose , and the entire mixture was subjected to western analysis for claudin-1 as described above .
matrigel invasion assays were performed using transwell migration chambers ( corning life sciences , lowell , ma ) as previously described 21 .
briefly , 8m filters were coated with 150l of 80g / ml reconstituted basement membrane ( matrigel , becton dickinson , franklin lakes , nj ) .
wild type or mutant claudin-1 expressing clones were serum starved for 16h , and 2 x 10 cells were seeded onto the filters . the total volume on the top of the filter was adjusted to 800l of serum free medium and 800l of medium containing 20% fetal calf serum was placed in the lower well to act as a chemoattractant .
cells were allowed to invade and adhere to the lower chamber , after which they were stained using crystal violet , and counted .
a student 's t - test was performed when required in at least 3 independent experiments .
 cytoplasmic expression of claudin-1 in metastatic melanoma cells correlates to increased migration , and increased secretion of mmp-2 in a pkc dependent manner , whereas claudin-1 nuclear expression is found in benign nevi . 
 melanoma cells were transfected with a vector expressing cldn-1 fused to a nuclear localization signal ( nls ) . despite significant nuclear localization of claudin-1 , there was still transport of claudin-1 to the cytoplasm . 
 phorbol ester treatment of cells transfected with nls - claudin-1 resulted in an exclusion of claudin-1 from the nucleus , despite the nls . to ascertain whether pkc or pka were involved in this translocation , we mutated the putative phosphorylation sites within the protein . 
 we found that mutating the pkc phosphorylation sites to mimic a non - phosphorylated state did not cause a shift of claudin-1 to the nucleus of the cells , but mutating the pka sites did . 
 mutations of either site to mimic constitutive phosphorylation resulted in cytoplasmic claudin-1 expression . 
 stable claudin-1 transfectants containing non - phosphorylatable pka sites exhibited decreased motility . 
 these data imply that subcellular localization of claudin-1 can be controlled by phosphorylation , dicating effects on metastatic capacity . 
Introduction
Significance
Results
PKC activation increases export of claudin-1 from the nucleus
PKC and PKA play distinct roles in the expression and localization of claudin-1
Nuclear claudin-1 does not increase melanoma cell motility
Discussion
Materials and Methods