Patent Application: US-7968808-A

Abstract:
described are methods of increasing the barrier function of a cell - cell junction , comprising activating a pdz - gef2 protein , wherein the pdz - gef2 protein comprises an amino acid sequence encoded by a gene corresponding to the rapgef6 gene . further described are methods for selecting an activator of pdz - gef2 , comprising contacting a plurality of candidate compounds to at least two cells of a first cell type and at least two cells of a second cell type , both cell types capable of forming cell - cell junctions , measuring the maturation of cell - cell junctions before and after contacting the two cell types with the plurality of candidate compounds , and selecting a compound from the plurality of candidate compounds that is more capable of increasing the integrity of a cell - cell junction between at least two cells of the first cell type , as compared to its capability of increasing the integrity of a cell - cell junction between cells of the second type , wherein in the second cell type , the amount and / or activity of pdz - gef2 is decreased , compared to the first cell type .

Description:
293t cells were maintained in dmem ( biowhittaker ) supplemented with 10 % fetal bovine serum ( fbs ) ( cambrex ), 1 . 2 mm l - glutamine ( biowhittaker ) and antibiotics ( 100 units / ml penicillin , 100 μg / ml streptomycin ) ( biowhittaker ). a549 cells were cultured in rpmi ( biowhittaker ) supplemented with 10 % fbs and antibiotics as described herein . huvec were isolated according to jaffe et al . [ 16 , 17 ] and cultured on 1 % gelatin ( sigma ) in ebm - 2 bulletkit culture medium ( ebm - 2 supplemented with egm - 2 singlequots ( hegf , hydrocortisone , fetal bovine serum , vegf , hfgf - b , r3 - igf - 1 , ascorbic acid , ga - 1000 , heparin ) ( clonetics )). second to fifth passage cells were used . monoclonal antibodies 5d3 against epac1 have been described previously . [ 7 ] additional primary antibodies were purchased from abcam ( anti - e - cadherin hecd1 ), genetex ( anti - e - cadherin hecd1 ), invitrogen ( anti - v5 ), oncogene ( anti - alpha - tubulin ), santa cruz biotechnology ( anti - rap ), sigma ( anti - magi - 3 ), transduction laboratories ( p130cas , β - catenin , ve - cadherin : cadherin - 5 ), zymed ( anti - occludin , anti - zo - 1 ), anti - hemagglutinin ( ha ) monoclonal antibody 12ca5 . anti - mouse and anti - rabbit fluorescent secondary antibodies ( fitc and tritc ) were from molecular probes ( eugene , oreg ., us ). to produce antibodies reactive against pdz - gef2 , the first nucleotide binding domain of human pdz - gef2 corresponding to amino acids 1 - 123 was subcloned into the bacterial expression vector pgex . glutathione s - transferase ( gst ) fusion protein was produced and used to immunize rabbits . full - length pdz - gef2 was subcloned into the pb38 vector and used as a bait in a yeast - two - hybrid system with a human placenta cdna library . the screen was performed by hybrigenics as described in reference 18 . the following hemagglutinin ( ha )- tagged expression vectors were described previously : pmt2 - ha - pdz - gef2 full length and pmt2 - ha - pdz - gef2 δc ; [ 9 ] pmt2 - ha - epac1 ; [ 19 ] pmt2 - ha - v12rap . [ 20 ] the mouse magi - 3 cdna clone imagp998a228567q was obtained from the german resource center for genome research ( rzpd ) and used to clone the ww domains and the second pdz domain of magi - 3 , which correspond to the interacting part found in the yeast - two - hybrid screen ( clone 6 ). amino acids 291 - 504 corresponding to the mouse ww - ww - pdz domains of magi - 3 were generated by pcr amplification and cloned into a gateway entry vector ( invitrogen ) using the following primer set : reverse 5 ′- ggggaccactttgtacaagaaagctgggtcttaacgg cataaagtaaggttgacat - 3 ′ ( seq id no : ______ ). the magi - 3 y2h ( clone 6 ) entry clone was recombined into a v5 - pcdna3 destination vector following the manufacturer &# 39 ; s instructions . the v5 - pcdna3 - magi - 3 y2h vector was sequenced to ensure proper identity of the amino acids . 293t and a549 cells were transfected using fugene6 reagent for 48 hours ( roche diagnostics corporation ). for sirna , a549 cells and huvec were transfected using oligofectamine ( invitrogen ) for 24 to 48 hours according to the manufacturer &# 39 ; s instructions . in the case of the huvec , transfection was repeated after 24 hours . samples were analyzed 48 hours after the second round of transfection . oligos sequences ( dharmacon ): pdz - gef2 ( 285 : ggatccaacttatatagaa ) ( seq id no : ______ ); ( 289 : gacacggattgtattatta ) ( seq id no : ______ ); * epac1 ( gcaccta cgtctgcaacaa ) ( seq id no : ______ ) and ( ccatcatcctgcgagaaga ) ( seq id no : ______ ). 8 - pcpt - 2 ′ o - me - camp / 8 - pcpt - 2 ′- ome - camp ( biolog ) was used at a final concentration of 100 μm . quantification of the pdz - gef2 knockdown was performed using scion image ( scion corporation ). a549 cells transfected with dna expression vectors and / or sirna oligos were plated on coverslips . twenty - four or 48 hours later , cells were rinsed twice with ice cold pbs and fixed in a solution of 3 . 8 % formaldehyde , permeabilized in 0 . 1 % triton x - 100 and blocked with 2 % bsa in pbs at room temperature for one hour or overnight at 4 ° c . the coverslips were incubated with the indicated primary antibodies for one hour followed by 30 minutes incubation with the alexa secondary antibodies , then mounted on glass slides and dried overnight . the cells were examined using a zeiss axiokop2 microscope or by confocal microscopy . pictures were taken at 40 × magnification and were arranged and resized using adobe photoshop cs . the values are presented as the average of the number of junctions scoring immature over the total e - cadherin junction staining ± standard deviation of the mean ( se ). a549 cells transfected with scrambled or pdz - gef2 . 285 oligos were replated in growth media 24 hours before calcium switch . the dishes were then rinsed twice with pbs and serum - free and calcium - free media containing 4 mm egta was added to the cells for 30 minutes at 37 ° c . to disrupt e - cadherin - mediated cell - cell contacts . following calcium chelation , cells were lysed or incubated in media supplemented with low calcium ( 20 μm ) for 20 and 60 minutes . cells were washed in ice - cold pbs and lysed in modified ripa buffer ( 150 mm nacl , 50 mm tris - hcl ph 7 . 4 , 1 % nonidet p - 40 ) containing protease inhibitors , sodium vanadate and sodium fluoride . cell lysates were cleared by centrifugation and rotated with anti - v5 or anti - ha antibodies and protein a - agarose at 4 ° c . for two hours . precipitates were washed four times in lysis buffer , resuspended in sds sample buffer , resolved by 8 % sds - page and transferred to polyvinylidene difluoride membrane ( pvdf ; immobilon , millipore ). the membranes were blocked in 1 % bovine serum albumin ( bsa ) and 1 % non - fat milk , followed by incubation with primary antibodies overnight at 4 ° c . horseradish peroxidase - conjugated anti - mouse or anti - rabbit was used as a secondary antibody and the blot was developed using enhanced chemiluminescence . to assess endothelial permeability , we measured electrical resistance continuously across endothelial cell monolayer using the methodology known as “ electric cell - substrate impedance sensing ” ( ecis ). [ 21 ] briefly , huvec were grown to confluence on small gold electrodes and ecis was subsequently monitored for up to 15 hours . data was analyzed using the applied biophysics software . in epithelial cells , the presence of e - cadherin protein mediates the establishment of cell - cell contacts ( reviewed in reference 22 ). the presence of e - cadherin in mature cell - cell contacts can be visualized in vitro . therefore , pdz - gef2 - depleted a549 cells were replated on glass coverslips and e - cadherin staining was performed . treatment with pdz - gef2 sirnai for 48 hours effectively reduced the levels of endogenous pdz - gef2 protein to 64 % and 52 % of baseline ( fig1 , panels a and b , oligos 285 and 289 , respectively ). remarkably , pdz - gef2 depletion was associated with suppression of cell - cell contacts maturation 24 and 48 hours following seeding of the cells ( fig1 , panels a and b , respectively ). the junctions depicted are zipper - like structures , as seen in epithelial cells establishing initial cell - cell contacts . [ 23 ] we quantified the amount of these immature junctions and obtained approximately twice as much in the absence of pdz - gef2 ( graphs of fig1 , panels a and b ). a similar pattern of localization was observed for β - catenin , another marker of adherens junction integrity ( data not shown ). to exclude impairment of e - cadherin or other junctional proteins expression in absence of pdz - gef2 , we examined the levels of e - cadherin , zo - 1 and occludin in cells treated or not treated with 4 mm egta for 30 minutes and found equal amounts in scrambled and pdz - gef2 sirna - treated cells ( fig1 , panels a and c ). interestingly , the pdz - gef2 knockdown phenotype ( pdz - gef2 - kd ) is similar to the one observed in rap1a - depleted cells ( data not shown ), providing evidence that pdz - gef2 is required for rap activation and further supporting a role for rap in junction establishment . expression of activated rap or epac1 rescues adherens junction maturation in pdz - gef2 - depleted cells ( pdz - gef2 - kd ) to confirm that the immature junction phenotype was caused by pdz - gef2 depletion that down - regulated rap activation , activated rap ( vl2rap ) and epac1 were re - expressed in scrambled or pdz - gef2 sirna - transfected cells and anti - e - cadherin staining was performed . expression of these constructs is known to lead to rap activation and significantly restored junction maturation to levels observed in cells transfected with the scrambled oligo and empty vector ( fig1 , panel d ). we concluded that rap activation by pdz - gef2 is critical for cell - cell contact maturation in a549 cells . we next showed the role of pdz - gef2 for adherens junction formation in endothelial cells . huvec were transfected as described and seeded on glass coverslips or on electrodes for ecis measurement of cellular resistance . experiments were performed when cells formed a confluent layer . in fig2 , panel a , ve - cadherin staining of pdz - gef2 - depleted huvec ( pdz - gef2 - kd ) clearly showed zipper - like , immature junctions , whereas scrambled sirna had no effect on junction appearance . it is known that immature junctions correlate with increased cell permeability and that the rap gef epac1 regulates this process . [ 24 - 27 ] therefore , we studied electrical resistance of huvec treated with epac1 and pdz - gef2 sirna . as previously reported , basal electrical resistance was decreased in epac1 - depleted cells ( fig2 , panel b ). the morphological phenotype was confirmed by electrical resistance functional assay . as expected , the reduced maturation of cell - cell contacts in absence of pdz - gef2 correlated with a 30 % decrease in basal electrical resistance ( fig2 , panel b ). activation of rap through epac1 by its specific agonist 8 - pcpt - 2 ′- ome - camp is known to decrease cell permeability of endothelial cells by tightening the junctions . [ 26 ] accordingly , a 30 - minute stimulation with 8 - pcpt - 2 ′- ome - camp ( 100 μm ) increased resistance in scrambled sirna cells and the decreased resistance observed in pdz - gef2 - depleted cells was completely rescued by 8 - pcpt - 2 ′- ome - camp ( fig2 , panel c ). as a control , huvec transfected with epac1 sirna were stimulated with 8 - pcpt - 2 ′- ome - camp and a minimal effect on electrical resistance was observed . these findings indicate that pdz - gef2 is required to maintain endothelial cell integrity . moreover , the impaired junction maturation was consistent with decreased electrical resistance in pdz - gef2 - depleted huvec . furthermore , activation of epac by 8 - pcpt - 2 ′- ome - camp could rescue the defect . in a yeast - two - hybrid screen using pdz - gef - 2 as a bait , we found magi - 3 as an interaction partner of pdz - gef - 2 . the magi - 3 protein comprises a pdz ( psd - 95 / disc large / zona occludens 1 ) domain and a guk ( guanylate kinase ) domain and is a member of the magi family . furthermore , ha - pdz - gef2 full - length co - immunoprecipitates with v5 - magi - 3 clone 6 when co - expressed in 293t cells ( fig3 ). to investigate whether pdz - gef - 2 is required for rap activation during junction remodeling , we introduced pdz - gef2 sirna in a549 cells and determined its effect on rap - gtp levels upon disruption of cell junctions by egta . in control sirna - treated cells , egta - induced rap1 activation ( fig4 , panel a , lane 2 ), compatible with previous results . to exclude effects of egta , we have replaced the edta for low ca 2 + ( 20 μm ). under these conditions , junctions are not formed ( data not shown ) and rap1 remains elevated ( fig4 , panel a , lanes 3 and 4 ). in pdz - gef2 - depleted cells , egta treatment was ineffective in increasing rap1 - gtp levels ( fig4 , panel a , lanes 6 - 8 ), showing that pdz - gef2 is required for rap1 activation triggered by the disruption of e - cadherin - based junctions . the contents of the entirety of each of which are incorporated by this reference ) 1 . chen x . and b . m . gumbiner , crosstalk between different adhesion molecules . curr . opin . cell biol . 2006 , 18 ( 5 ): 572 - 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