Regulation of the blood-testis barrier by coxsackievirus and adenovirus receptor

Am J Physiol Cell Physiol 33: C843 C853, 212. First pulished August 8, 212; doi:1.1152/jpcell.218.212. Regultion of the lood-testis rrier y coxsckievirus nd denovirus receptor Linlin Su, Dolores D. Mruk, nd C. Yn Cheng Center for Biomedicl Reserch, Popultion Council, New York, New York Sumitted 28 June 212; ccepted in finl form 7 August 212 Su L, Mruk DD, Cheng CY. Regultion of the lood-testis rrier y coxsckievirus nd denovirus receptor. Am J Physiol Cell Physiol 33: C843 C853, 212. First pulished August 8, 212; doi:1.1152/jpcell.218.212. The lood-testis rrier (BTB) divides the seminiferous epithelium into the sl nd the dluminl comprtment. It restricts prcellulr diffusion of molecules etween Sertoli cells, confers cell polrity, nd cretes unique microenvironment in the dluminl comprtment for spermtid development. However, it undergoes restructuring during the epithelil cycle so tht preleptotene spermtocytes differentited from type B spermtogoni residing in the sl comprtment cn trverse the BTB t stge VIII of the cycle, while the immunologicl rrier is mintined. Herein, coxsckievirus nd denovirus receptor (), tight junction (TJ) integrl memrne protein in the testis nd multiple epitheli nd endotheli, ws found to ct s regultory protein t the BTB, esides serving s structurl dhesion protein. RNAi-medited knockdown of in Sertoli cell epithelium with n estlished TJ-permeility rrier tht mimicked the BTB in vivo resulted in disruption of the TJ rrier nd n increse in endocytosis of the TJ-protein occludin. Furthermore, such n enhncement in occludin endocytosis ws ccompnied y downregultion of Thr-phosphoryltion in occludin nd n increse in the ssocition of endocytosed occludin with erly endosome ntigen-1. These findings were confirmed y overexpressing in Sertoli cells, which ws found to tighten the Sertoli cell TJ rrier, promoting BTB function. These findings support the emerging concept tht is not only structurl protein, it is involved in conferring the phosphoryltion sttus of other dhesion proteins t the BTB (e.g., occludin) possily medited vi its structurl interctions with nonreceptor protein kinses, therey modulting endocytic vesicle-medited protein trfficking. testis; spermtogenesis; seminiferous epithelil cycle; ; Sertoli cell; lood-testis rrier; tight junction; ectoplsmic speciliztion Address for reprint requests nd other correspondence: C. Y. Cheng, Mry M. Wohlford Lortory for Mle Contrceptive Reserch, Center for Biomedicl Reserch, Popultion Council, 123 York Ave., New York, NY 165 (e-mil: Y-Cheng@popcr.rockefeller.edu). COXSACKIEVIRUS AND ADENOVIRUS RECEPTOR (), 46-kD integrl memrne protein, ws first reported in 1997 s common receptor for coxsckie virus group B nd denovirus groups 2 nd 5 (2). llows virl ttchment nd entry into cells (3). Susequent studies (1 12, 61) hve shown tht is found mostly t the tight junction (TJ) in multiple tissues nd orgns including rin, hert, muscles, nd testes. Interestingly, it is highly expressed in oth Sertoli cells nd metoliclly quiescent germ cells including highly differentited hploid spermtids (36, 37, 62), nd it is lso component of the picl ectoplsmic speciliztion [picl ES, testis-specific typicl dherens junction (AJ)] esides the Sertoli cell TJ rrier (62). Since the presence of on cell surfce is crucil determining fctor for the entry of denoviruses nd coxsckieviruses (3), hs een extensively studied nd trgeted for gene therpy (47, 5). Recent studies (1, 18, 45) hve shown tht is structurl dhesion protein t the TJ in multiple cell epitheli vi homotypic interctions etween neighoring epithelil cells. lso possesses PSD-95/Dlg1/zonul occludens 1 (ZO-1)-inding (PDZ-inding)motif to fcilitte protein-protein interctions, s such, hs een shown to interct with dptor proteins ZO-1 nd -ctenin (1, 6) nd gp junction protein connexin 45 (Cx45) (31), forming protein complex. In the testis, mong more thn dozen BTB-ssocited structurl nd regultory proteins tht were exmined, ws lso found to interct with JAM-C (36), -ctenin, vinculin, nd most importntly non-receptor protein tyrosine kinse c-src sed on studies (62) using coimmunoprecipittion (Co-IP), suggesting my hve other functionl nd/or regultory roles. In fct, is importnt in trioventriculr-node conduction nd crdic function s demonstrted in studies (31) using crdicspecific knockout (-KO) mice, since ws shown to e involved in the locliztion of Cx45, ZO-1, nd -ctenin t the interclted disc in the hert nd Cx45 ws known to e involved in trioventriculr node conduction. Its tissue/orgn-specific knockout in dult mice lso led to dilted intestinl trct nd trophy of the exocrine pncres, illustrting its inctivtion led to multiple phenotypic chnges (42). my lso ct s tumor suppressor (54). Collectively, these recent findings illustrte the criticl role of in multiple cellulr functions esides serving s receptor for virl entry nd s structurl dhesion protein t the TJ nd/or AJ, possily medited vi its inding prtner(s) such s c-src. Since the findings of s puttive integrl memrne proteins in Sertoli nd germ cells in the testis (36, 62), it ws speculted tht my serve s regultory molecule to llow the trnsit of preleptotene spermtocytes t the BTB t stge VIII of the epithelil cycle (37, 61); however, the moleculr mechnism(s) underlying this event remins elusive. Herein, we report findings using techniques of gene silencing y RNAi nd trnsient overexpression tht plys regultory role t the Sertoli cell BTB vi its involvement in modulting the phosphoryltion sttus of integrl memrne proteins t the BTB (e.g., occludin), which, in turn, modultes endocytic vesicle-medited protein trfficking events t the site, therey ffecting cell dhesion sttus t the Sertoli cell-cell interfce. In short, ppers to tke prt in dynmic nd efficient regultory mechnism so tht germ cells cn e in trnsit t the BTB while the immunologicl rrier function remins intct during the epithelil cycle of spermtogenesis. MATERIALS AND METHODS Animls nd ntiodies. Mle Sprgue-Dwley rts, oth dults ( 25 3 g ody wt) nd 2-dy-old pups, were purchsed from http://www.jpcell.org 363-6143/12 Copyright 212 the Americn Physiologicl Society C843

C844 REGULATES BTB DYNAMICS Chrles River Lortories (Kingston, NY). The use of nimls for the studies reported herein ws pproved y the Rockefeller University Lortory Animl Cre nd Use Committee with Protocol No. 9 16 nd 12 56. Antiodies were commercilly otined except for nti-desmoglein-2, which ws prepred in our lortory s erlier descried (28) (Tle 1). Primry Sertoli cell cultures. Sertoli cells were isolted from 2-dy-old rt testes s descried previously (39). Sertoli cells were cultured in serum-free F-12/DMEM supplemented with sodium icronte, gentmicin, epiderml growth fctor, insulin, trnsferrin, nd citrcin t 35 C with 5% CO 2-95% ir (vol/vol) in humidified CO 2 incutor s descried previously (39). An incution temperture of 35 C ws used for testiculr cells, such s Sertoli cells, since in rodents nd humns testicles re locted outside the dominl cvity, enclosed in the scrotum to mintin temperture t 35 C, cooler thn the ody temperture of 37 C, necessry to mintin spermtogenesis nd fertility (21, 49, 55). It is noted tht these Sertoli cells were fully differentited nd cesed to divide (41). Furthermore, Sertoli cells isolted from 2-dy-old rts were indistinguishle from cells isolted from 9-dy-old rt testes oth morphologiclly nd functionlly (26, 33). However, the purity of Sertoli cells isolted from dult rt testes ws 85 vs. 98% from 2-dy-old rt testes ecuse of the lck of round/elongting/elongted spermtids in the ltter group, which were tightly emedded in Sertoli cells nd difficult to e removed including the use of hypotonic tretment (26, 33). Depending on the type of experiments, Sertoli cells were plted t different cell density for vrious pplictions. First, cells were plted t.45.5 1 6 cells/cm 2 on Mtrigel-coted 12-well dishes [Mtrigel diluted 1:7 in serum-free F-12/DMEM (Sigm-Aldrich, St Louis, MO)] with ech well contining 3-ml F-12/DMEM, nd cultures were hrvested t specified time points to otin enough protein in cell lystes for immunolotting or for nucleic cid extrction for RT-PCR or quntittive PCR. Second, Sertoli cells were plted t.4 1 6 cells/cm 2 on Mtrigel-coted coverslips for immunofluorescence nlysis to ssess chnges in protein distriution nd/or locliztion with ech coverslip plced in 6-well dishes, nd ech well contined 5-ml F-12/DMEM. Thus Sertoli cells were evenly spced, nd chnges in protein locliztion/distriution t the Sertoli cell-cell interfce in tretment vs. control groups could e esily detected. Third, Sertoli cells were plted t 1.2 1 6 cells/cm 2 on Mtrigel-coted icmerl units (Millicell cell culture inserts, 12-mm dimeter,.45- m pore size, effective surfce re,.6 cm 2 ; Millipore, Bedford, MA), nd units were plced in 24-well dishes with Tle 1. Antiodies used for different experiments in this study Antiody Ctlog No. Lot No. Host V Vendor.5-ml F-12/DMEM ech in the picl nd sl comprtment. Aout 48 h fter plting, cells were sujected to rief hypotonic tretment (2 mm Tris, ph 7.4 t 22, 2.5 min) to lyse residul germ cells s descried previously (14), nd cultures were rinsed twice with F-12/ DMEM to remove germ cell deris efore replced with fresh F-12/ DMEM contining the necessry supplements (39). As such, Sertoli cell cultures used for our studies hd purity of 98% with negligile contmintions of germ, Leydig, nd/or perituulr myoid cells when mrkers of these cells were ssessed y RT-PCR using specific primer pirs s descried previously (25). It is noted tht germ cells, nmely spermtogoni nd spermtocytes (note: round/elongting/elongted spermtids re sent in 2-dy-old rt testes; Ref. 9), re susceptile, ut not Sertoli cells, to this rief hypotonic tretment. This tretment ws found not to impede the TJ-permeility rrier function when the trnsepithelil electricl resistnce (TER) ws ssessed 12 24 h fter Sertoli cells were rinsed nd returned to norml culture conditions vs. Sertoli cell cultures not sujected to the hypotonic tretment. It is lso noted tht using this in vitro model, Sertoli cells ssemled functionl TJ-permeility rrier, nd ultrstructures of oth TJ, sl ES, gp junction (GJ), nd desmosome were found when exmined y electron microscopy, mimicking the BTB in vivo s descried previously (27, 28, 53). In fct, this system hs een used y investigtors to study the iology nd regultion of Sertoli cell BTB dynmics (4, 5, 15, 19, 2, 22, 4, 46). knockdown in Sertoli cells y RNAi using -specific smll interfering RNA duplexes. Sertoli cells otined from 2-dy-old rt testes were cultured lone for 3 dys to llow the estlishment of functionl TJ-permeility rrier, which ws mnifested y the presence of stle TER cross the cell epithelium. Therefter, cells were trnsfected with 1 nm nontrgeting or -specific smll interfering (si)rna duplexes y using RioJuice Trnsfection Regent (Novgen, EMD Biosciences) for 24 h ccording to the instructions of the mnufcturer. To silence, mixture of 1 nm (5=- CCUGAACAGAGGAUCGAAAtt, s137152; 5=-GAAAUGACUU- CACCGGUUAtt, s137153; 5=-CGAGUACACUUUACGAGUAtt, s137154; Amion) duplexes vs. 1 nm nontrgeting control duplexes (Amion) ws used. After trnsfection, rection mixture ws removed, wshed, nd replced with fresh F-12/DMEM, nd cells were cultured for nother 48 h efore termintion. For dul-leled immunofluorescence nlysis, 2 nm siglo Red Trnsfection Indictor (Dhrmcon/Thermo Fisher Scientific) were used to cotrnsfect with duplexes to confirm successful trnsfection. Working Dilution IB IF IP sc-1545 B288 Rit Snt Cruz Biotechnology 1:2 1:5 Occludin 71-15 2527 Rit Zymed/Invitrogen 1:4 1:5 1:4 JAM-A 36-17 37923A Rit Zymed/Invitrogen 1:25 Cludin-11 36-45 387613A Rit Zymed/Invitrogen 1:125 ZO-1 61-73 389452A Rit Zymed/Invitrogen 1:25 1:5 N-Cdherin sc-7939 H97 Rit Snt Cruz Biotechnology 1:2 -Ctenin sc-7894 G33 Rit Snt Cruz Biotechnology 1:1 -Ctenin 71-27 686848C2 Rit Zymed/Invitrogen 1:25 Desmoglein-2 Rit Cheng L Desmocollin-2 sc-66863 A18 Rit Snt Cruz Biotechnology 1:2 -Ctenin 61254 74819 Mouse BD Trnsduction Lortories 1:1 1:5 EEA-1 61457 8795 Mouse BD Trnsduction Lortories 1:2 1:1 1:4 Phospho-Ser 61-81 4487618 Rit Zymed/Invitrogen 1:5 Phospho-Thr 71-82 681682A Rit Zymed/Invitrogen 1:5 Phospho-Tyr 61-58 116746 Rit Zymed/Invitrogen 1:5 Actin sc-1616 F27 Got Snt Cruz Biotechnology 1:2 Anti-desmoglein-2 ntiody ws prepred in our lortory ginst rt desmoglein-2 recominnt protein of 17.5 kd s descried previously (28)., coxsckievirus nd denovirus receptor; ZO-1, zonul occludens 1; EEA-1, erly endosome ntigen 1; IB, immunolotting, IF, immunofluorescence nlysis; IP, immunoprecipittion.

REGULATES BTB DYNAMICS C845 Fig. 1. Primry nucleotide sequence of coxsckievirus nd denovirus receptor () (GenBnk ccession no.: NM_5357) tht ws used to clone the full-length rt testiculr. Rt testiculr ws cloned y PCR using primer pir designted ex- (see Tle 2) sed on the known sequence s shown herein, nnotted y the lck ox, utilizing rt Sertoli cell totl cdnas s the templte, which were reverse trnscried from Sertoli cell RNA, nd the cycling prmeters were shown in Tle 2. This rt testiculr cdna ws then used to prepre the full-length cdna construct nd its susequent ligtion into the mmmlin pci-neo vector, nd XhoI nd NotI restriction sites were dded to its 5=- nd 3=-end, respectively, y PCR using primer pir designted (Tle 2) nd nnotted y the dsh-lined ox. Numers t left represent the nucleotide sequence of NM_5357. This rt testiculr cdna clone ws verified y direct nucleotide sequencing t Genewiz (South Plinfield, NJ). Overexpression of in Sertoli cells. The full-length cdna encoding ws otined y PCR s erlier descried (64, 65) using cdnas derived from Sertoli cell totl RNA vi reverse trnscription step tht served s the templte nd -specific primer pir designted ex- (Fig. 1 nd Tle 2). The full-length cdna ws cloned into pci-neo mmmlin expression vector (Promeg) t the restriction enzyme sites etween XhoI nd NotI y using specific primers of (see Tle 2). The pci-neo mmmlin expression vector crries the humn cytomeglovirus immedite erly enhncer/promoter region tht promotes constitutive expression of the insert in Sertoli cells. The uthenticity of these clones ws confirmed y direct nucleotide sequencing (Genewiz). On dy 3 fter isoltion, Sertoli cells plted on Mtrigel-coted 12-well dishes or icmerl units t cell density of.5 1 6 or 1.2 1 6 cells/cm 2, respectively, were trnsfected with 1 or.5 g of plsmid DNA per well or insert y using Effectene Trnsfection Regent (Qigen) t rtio of 1 g DNA to 15 l trnsfection regent. Trnsfection mixture ws removed 24 h therefter nd replced with fresh F-12/DMEM. RNA nd protein lystes were extrcted from these Sertoli cell cultures 2-dy therefter (i.e., 3-dy fter trnsfection egn), s descried previously (58). The Sertoli cell-tj rrier function fter trnsient expression of vs. pcineo vector lone ws lso ssessed y TER mesurement. To ssess the trnsfection efficiency using the Mmmlin Expression Vector pci-neo in Sertoli cells, luciferse reporter plsmid (pgl3- nd prl-tk, Promeg) ws cotrnsfected into Sertoli cells with plsmid DNAs t.1 3 g nd different cell densities t.5 or 1.2 1 6 cells/cm 2 for 24-h y ssying the luciferse reporter gene ctivity s descried previously (64). With the use of this pproch, the trnsfection efficcy ws estimted to e 15 2%. Functionl ssessment of the Sertoli cell TJ-permeility rrier. The Sertoli cell TJ-permeility rrier ws quntified y the ility of the cell epithelium to restrict the flow of current (i.e., quntified s conductivity in ohm, ) tht ws sent cross the Sertoli cell epithelium when two electrodes of Millipore Millicell-ERS were plced in the corresponding picl nd sl chmer of the icmerl unit s erlier descried (16). In short, Sertoli cells cultured in F-12/DMEM were plted on Mtrigel-coted icmerl units (in triplictes) t 1.2 1 6 cells/cm 2 t time, nd TER ws recorded dily, with fresh F-12/DMEM replenished fter the TER mesurement. On dy 3, when the TJ-permeility rrier ws estlished s mnifested y stle TER cross the Sertoli cell epithelium, trnsfection ws performed using duplexes to silence or using pci-neo/ to overexpress vs. corresponding controls to ssess the effects of knockdown or its overexpression on the Sertoli cell TJ-permeility rrier function. Endocytosis ssy. Endocytosis ssy ws performed essentilly s descried previously (63, 67) nd detiled in n erlier report (24). In rief, Sertoli cells were cultured t.5 1 6 cell/cm 2 on Mtrigelcoted 6-well pltes for 3 dys. Therefter, cells were trnsfected with -specific duplexes vs. nontrgeting control duplexes for 24 h. Three dys fter trnsfection when ws knockdown y 7%, cell surfce proteins were iotinylted with.5 mg/ml sulfo-nhs-ss-iotin (Pierce, Rockford, IL; note: sulfo-nhs- SS-iotin is cell-impermele, clevle, iotinyltion regent) in PBS/CM uffer (PBS contining.9 mm CCl 2 nd.33 mm MgCl 2) t 4 C for 3 min (t this temperture, protein internliztion did not occur). Free iotin ws then quenched with 5 mm NH 4Cl in PBS/CM uffer t 4 C for 15 min. To initite endocytosis, Sertoli cell cultures were trnsferred from 4 C to CO 2 incutor in humidified tmosphere with 95% ir-5% CO 2 (vol/vol) t 35 C nd incuted for the specified time points to ssess the kinetics of iotinylted protein internliztion in Sertoli cells trnsfected with -specific duplexes vs. nontrgeting control duplexes. At termintion (i.e.,, 15, 3, 6, nd 9-min), iotins on uninternlized cell surfce proteins were stripped with 5 mm sodium 2-mercptoethnesulfonte (MESNA, reducing gent tht cleved iotin from iotinylted Sertoli cell surfce proteins; Sigm-Aldrich) in 1 mm Tris HCl, 1 mm NCl, nd 2.5 mm CCl 2, ph 8.6, t 4 C for 3 min nd quenched with 5 mg/ml iodocetmide (Sigm-Aldrich) in PBS/CM uffer t 4 C for 15 min. Cell lystes were then otined using IP lysis uffer [1 mm Tris,.15 M NCl, 1% NP-4, nd 1% glycerol (vol/vol), ph 7.4, t 22 C] supplemented with protese nd phosph- Tle 2. Primer sequences used to clone the rt Sertoli cell full-length cdna nd its insertion into pci-neo mmmlin expression vector Gene Primer Sequence Orienttion Position Length, p Tm, C Cycle No. GenBnk Accession No. Ex- 5=-CTGAGAGCGTTTACCTGC-3= Sense ( 4) ( 23) 1,288 54.3 35 NM_5357 5=-TGAGGCAGACAACAGGAT-3= Anti-sense 1,231 1,248 5=-ACCTCGAGATGGCGCTCCTACTGTG-3= Sense 1 17 1,59 8 1 1,77 56 35 NM_5357 5=- ACGCGGCCGCTTATACCACTGCAATGCCATCG-3= Anti-sense 1,38 1,59 CTCGAG, restriction site for XhoI; GCGGCCGC, restriction site for NotI; ATG, strt codon; TTA, stop codon; Tm, nneling temperture (see Fig. 1 for the primry sequence of rt testiculr ).

C846 REGULATES BTB DYNAMICS tse inhiitor cocktils (Sigm-Aldrich). Endocytosed iotinylted proteins were pulled down with UltrLink Immoilized NeutrAvidin Plus eds (Pierce) from ech smple (note: ech smple contined 3 g protein of Sertoli cell lystes). The complexes were then wshed, nd the proteins were extrcted in SDS-smple uffer contining 2-mercptoethnol (6) t 1 C for 5 min (to cleve proteins from iotin, Mr 244.31, tht ound to NeutrAvidin Plus eds) nd sujected to SDS-PAGE nd immunolot nlysis using nti-occludin ntiody. All smples within n experimentl group were processed simultneously to void interexperimentl vritions. All endocytosis experiments reported herein were repeted t lest three times using different tches of Sertoli cells, nd ech experiment yielded similr results. Dul-leled immunofluorescence nlysis. Distriution nd/or locliztion of trget proteins in Sertoli cells following knockdown vs. its corresponding control were exmined y immunofluorescence microscopy. In rief, Sertoli cells were isolted nd cultured t density of.4 1 6 cells/cm 2 on Metrigel-coted coverslips nd plced in 6-well dishes with ech well contining 5-ml F-12-DMEM. On dy 3, cells were trnsfected with 1 nm nontrgeting or -specific duplexes with Riojuice trnsfection regent (Novgen) served s trnsfection medium for 24 h. Therefter, the trnsfection mixture ws removed nd cells were cultured for n dditionl 48 h. Sertoli cells were fixed in 4% prformldehyde in PBS (1 mm NH 2PO 4, ph 7.4, t 22 C contining.15 M NCl; vol/vol) for 1-min t room temperture (22 C). Prformldehyde-fixed cells were permeilized with.1% Triton X-1 in PBS (vol/vol). After locking with 1% norml got serum in PBS (vol/vol), cells were incuted with trget ntiody t pproprite dilution in PBS (see Tle 1). Following overnight incution, cells were incuted with secondry ntiodies conjugted to Cy3 or FITC (Invitrogen; 1:1 dilution in PBS) for 1 h. Then, cells were mounted with ProLong nti-fde regent contining DAPI for nuclei stining (Moleculr Proes, Eugene, OR), nd fluorescence microgrphs were otined using n Olympus BX61 fluorescence microscope, nd imges were cquired using Olympus MicroSuite FIVE (Version 1224) softwre pckge nd sved in TIFF formt. Dul-leled immunofluorescence imges were susequently merged for nlysis to ssess colocliztion using PhotoShop in Adoe Cretive Suite (Version 3.). RNA extrction nd RT-PCR. Totl RNAs were extrcted from Sertoli cells 3 dys fter trnsfection with duplexes using TRIzol regent (Invitrogen) ccording to the mnufcturer s instructions. Contminting genomic DNA in ech RNA smple, if ny, ws digested with RNse-free DNse I (Invitrogen) efore their use for reverse trnscription into cdnas using Moloney murine leukemi virus reverse trnscriptse (MMLV RT) regent (Invitrogen). PCR ws performed s erlier descried (56, 58) using primer pirs specific to corresponding trget genes (Tles 2 nd 3). For RT-PCR, trget gene ws complified with riosoml S16 (Tle 3), which served s n internl control for equl smple processing nd RNA loding. Immunolot nlysis. Lystes from Sertoli cell cultures were otined y treting cells in IP lysis uffer (1 mm Tris,.15 M NCl, 1% NP-4, nd 1% glycerol, ph 7.4, t 22 C) supplemented with protese nd phosphtse inhiitor cocktils (Sigm-Aldrich), sonicted, nd centrifuged t 15, g for 45 min t 4 C to otin cler superntnt. Lystes were stored t 2 C until use. Forty microgrms of Sertoli cell lyste protein from ech smple were resolved y SDS-PAGE for immunolot nlysis with trget proteins eing proed y the corresponding primry ntiodies (see Tle 1). Protein estimtion ws performed y spectrophotometry with Bio-Rd Dc (detergent comptile) protein ssy kit using BSA s stndrd nd Bio-Rd Model 68 Plte Reder. Co-IP. Co-IP ws used to monitor chnges in protein-protein interction s well s chnges in occludin phosphoryltion sttus. In rief, 2 g norml mouse or rit IgG were dded to 3 g Sertoli cell protein lyste nd incuted for 1 h efore precipitted with 1 l protein A/G grose eds (Snt Cruz) for 1 h, nd the superntnt ws otined (1, g, 5 min t 4 C) for susequent Co-IP. This preclening step ws importnt to remove nonspecific IgG-intercting proteins from cell lystes. Therefter, the superntnts were incuted with 2 g norml mouse or rit IgG to serve s negtive control, or nti-erly endosome ntigen 1 (nti-eea-1) or nti-occludin for Co-IP on Lnet MiniL Roller overnight t room temperture ( 22 1 C), to e followed y n incution with 2 l protein A/G grose eds to precipitte the immunocomplexes. Therefter, eds were wshed with IP lysis uffer nd immunocomplexes were extrcted in n SDS-PAGE smple uffer (6) t 1 C for SDS-PAGE nd immunolot nlysis s descried previously (57). Sttisticl nlysis. GB-STAT sttisticl nlysis softwre (Version 7., Dynmic Microsystems) ws used for sttisticl nlyses. Ech experiment ws repeted t lest three times, nd dt re mens SD. Sttisticl significnce ws nlyzed with Student s t-test or one-wy ANOVA coupled with two-tiled Dunnett s test. RESULTS Knockdown of y RNAi perturs Sertoli cell TJ-permeility rrier function vi chnges in the locliztion nd/or distriution of TJ proteins t the Sertoli cell BTB. Figure 1 shows the primry sequence of rt testiculr tht ws cloned from the cdnas derived from Sertoli cell RNA y PCR (Tle 2), nd the -specific duplexes were prepred sed on this rt testiculr primry sequence (see MATERIALS AND METHODS). RNAi ws used to knockdown expression y 7% y trnsfecting Sertoli cells in vitro with n estlished functionl TJ-permeility rrier tht mimicked the Sertoli cell BTB in vivo using -specific duplexes vs. the nontrgeting control duplexes (Fig. 2, A, nd, nd B, nd ). The knockdown of y 7% ws found to ssocite with downregultion of ZO-1 expression, ut not other TJ, sl ES, nd desmosome proteins exmined (Fig. 2B, nd ). This knockdown lso pertured the Sertoli cell TJ-permeility rrier function (Fig. 2C). We next investigted ny chnges in protein locliztion nd/or distriution t the Sertoli cell-cell interfce y Tle 3. Primer sequences used for RT-PCR experiments Gene Primer Sequence Orienttion Position Length, p Tm, C Cycle No. GenBnk Accession No. 5=-CGCTCCTACTGTGCTTC-3= Sense 5 21 195 54 3 NM_5357 5=-CTTTCTGGTTATCGGACGG-3= Anti-sense 181 199 OAS1A 5=-GAGTGAAGTTTGAGGTCCAGA-3= Sense 35 37 23 54.9 3 NM_138913 5=-CTCCGTGAAGCAGGTAGA-3= Anti-sense 562 579 STAT1 5=-GAGTGGAAGCGAAGACAG-3= Sense 712 729 218 54.6 3 NM_32612 5=-TGGAAGAGGACGAAGGTG-3= Anti-sense 912 929 S-16 5=-TCCGCTGCAGTCCGTTCAAGTCTT-3= Sense 15 38 385 XM_341815 5=-GCCAAACTTCTTGGTTTCGCAGCG-3= Anti-sense 376 399

dul-leled immunofluorescence nlysis in these cultures (Fig. 2D, -f). Consistent with findings shown in Fig. 2, A nd B, knockdown of y RNAi considerly olished the expression of t the Sertoli cell-cell interfce (see white rrowheds in Fig. 2D, vs. ). This lso led to chnges in the REGULATES BTB DYNAMICS C847 locliztion nd distriution of occludin ( TJ-integrl memrne protein) nd ZO-1 ( TJ-ssocited dptor protein), with these proteins eing redistriuted, moving from ner the Sertoli cell surfce, nd into the cell cytosol (Fig. 2D, d nd f vs. Fig. 2D, c nd e). Such chnges in distriution nd/or locliztion A B p 453 M 394 298 234 22 15 TJ proteins Bsl ES proteins DS proteins Reltive protein level (ritrry unit) 1.2.8.4 D, 46 kd Occludin, 65 kd Cludin-11, 22 kd JAM-A, 36 kd ZO-1, 21 kd N-Cdherin, 127 kd α-ctenin, 12 kd β-ctenin, 92 kd Desmoglein-2, 16 kd Desmocollin-2, 15 kd γ-ctenin, 82 kd Actin, 42 kd S-16, 385 p, 195 p, 46 kd ZO-1 C TER (ohm cm 2 ) Occludin ZO-1 6 5 4 3 Trnsfection PCR, IB, IF 2 1 1 2 3 4 5 6 7 Time of Sertoli cells in culture (dy) c e d f Fig. 2. Study to ssess the effects of knockdown y RNAi on the Sertoli cell tight junction (TJ)-permeility rrier function in vitro. Sertoli cells were cultured lone on Mtrigel-coted dishes (A nd B), icmerl units (C), or coverslips (D) for 3 dys to llow the ssemly of functionl TJ permeility, possessing the ultrstructures of TJ, sl ectoplsmic speciliztion (ES), gp junction (GJ), nd desmosome (DS) s descried previously (27, 28, 53). Therefter, cells were trnsfected with either -specific duplexes vs. nontrgeting control duplexes for 24 h, nd cultures were then terminted for RT-PCR/immunolotting (IB; A), immunolotting (B), Sertoli TJ-permeility rrier ssessment (C), nd dul-leled immunofluorescence nlysis (IF; D). A: knockdown of y RNAi using -specific smll interfering (si)rna duplexes in Sertoli cells terminted 3 dys fter trnsfection ws verified y RT-PCR () nd immunolotting (). M, DNA size mrkers in sepir (p). B: following the knockdown of y 7% ( nd ), the levels of TJ [e.g., occludin, cludin-11, JAM-A, nd zonul occludens 1 (ZO-1)], sl ES (e.g., N-cdherin, -ctenin, nd -ctenin), nd desmosome proteins (e.g., desmoglein-2, desmocollin-2, nd -ctenin) t the Sertoli cell lood-testis rrier (BTB) were lso exmined y immunolotting 3 dys fter trnsfection. It ws noted tht except for the level of ZO-1, which ws downregulted y 3% following the knockdown of in Sertoli cells y 7%, the levels of other BTB proteins remined reltively unltered (). Densitometric nlyses of nd ZO-1 immunolotting dt tht were normlized ginst ctin wherein the control ws ritrrily set t 1 (). Ech r is men SD of n 3 experiments. C: knockdown of ws found to trnsiently pertur the Sertoli cell TJ rrier in vitro. PCR, immunolotting (IB), nd dul-leled immunofluorescence nlysis (IF) were performed 3 dys fter trnsfection. D: to investigte chnges in protein distriution/locliztion t the cell-cell interfce, Sertoli cells cultured on Mtrigel-coted coverslips were cotrnsfected with siglo Red ( trnsfection indictor) with either nontrgeting or -specific duplexes nd stined for (green fluorescence), occludin (green fluorescence), or ZO-1 (green fluorescence) on dy 3. Sertoli cells were processed for IF 3 dys fter trnsfection (i.e., dy 6 in culture). Loss of expression t the Sertoli cell-cell interfce ws detected in the knockdown cells (see white rrowheds in vs. ). Although RNAi hd no pprent effects on the stedy-stte level of occludin (B), its knockdown ws found to induce mis-locliztion of occludin, cusing it to move from ner the cell surfce into the cell cytosol. Anlogous to occludin, knockdown lso impired the locliztion/distriution of ZO-1 t the Sertoli cell interfce, esides downregulting ZO-1 expression. Scle rs 2 m in, which pplies to f. P.1.

C848 REGULATES BTB DYNAMICS A p 453 M 394 298 234 22 B p 453 394 298 234 22 M S-16, 385 p OAS1, 23 p S-16, 385 p STAT1, 218 p Fig. 3. Study to monitor off-trget effects during knockdown in Sertoli cells y RNAi. Two interferon-stimulted genes (ISG), nmely OAS1 (oligodenylte synthetse 1) nd STAT1 (signl trnsducer nd ctivtor of trnscription 1), were used s mrkers to ssess off-trget effects since these genes were known to e upregulted nonspecificlly when cells trnsfected y RNAi vectors for gene silencing. Neither OAS1 nor STAT1 ws upregulted in Sertoli cells following the knockdown of vs. control. M, DNA size mrkers in sepir (p). likely contriuted to destiliztion of cell dhesion t the TJ rrier, perturing the Sertoli cell TJ-permeility rrier s shown in Fig. 2C. However, it is lso possile tht such chnges contriuted to n ltertion of the prcellulr trnsport of the TJ rrier. The disruptive effects following the knockdown of in Sertoli cells y RNAi on the TJ rrier function (Fig. 2C) nd on protein distriution/locliztion t the cell-cell interfce (Fig. 2D) did not pper to e the results of off-trget effects, since the stedy-stte levels of severl BTB-ssocited proteins tht were exmined, including occludin, remined unltered, with the exception of ZO-1, which ws downregulted (Fig. 2B, nd ). Furthermore, the expression of two interferon-stimulted genes (ISG), such s oligodenylte synthetse 1 (OAS1) nd signl trnsducer nd ctivtor of trnscription 1 (STAT1) tht were known to e upregulted due to off-trget effects when cells were trnsfected y RNAi vectors for gene knockdown (1, 34, 43), were found to e unffected with S16 served s loding control in this semi-quntittive RT-PCR experiment (Fig. 3, A nd B). In short, smll regultory RNAs, such s used here, could trigger cellulr immune responses in Sertoli cells, such s y ctivting interferon-stimulted gene expression, which in turn led to nonspecific cellulr phenotypes nd side effects (e.g., TJ-rrier disruption, ltering protein distriution/locliztion) known s off-trget effects. Since neither one of the two ISGs, nmely OAS1 nd STAT1, ws upregulted in the knockdown Sertoli cells vs. control cells, these findings (Fig. 3, A nd B) coupled with immunolotting dt shown in Fig. 2B thus support our conclusion tht the chnges shown in Fig. 2, C nd D, were the results of knockdown. Attempts were mde y immunofluorescence microscopy to ssess chnges in other BTBssocited proteins t the Sertoli cell-cell interfce esides occludin nd ZO-1 following silencing, such s JAM-A nd cludin-11; however, working ntiodies were not ville. knockdown results in incresed endocytic vesicle-medited protein trfficking nd chnges in occludin phosphoryltion. Since knockdown tht led to prtil disruption of the Sertoli cell TJ-permeility rrier shown in Fig. 2C ppered to e the result of chnges in protein locliztion/distriution due to n increse in the internliztion of occludin (Fig. 2D), protein endocytosis ssy ws used to explore this possiility. It ws noted tht the knockdown of indeed significntly enhnced ut not in the control group where Sertoli cells were trnsfected with nontrgeting control duplexes, the kinetics of occludin endocytosis (Fig. 4A, nd ). It is noted tht in this endocytosis ssy of Le et l. (24), the use of cell-impermele ut thiol-clevle iotinyltion regent, sulfo- NHS-SS-iotin, only Sertoli cell surfce proteins with exposed primry mines, including occludin, were iotinylted. In the lne designted Totl, it represents totl iotinylted cell surfce proteins in the control group where Sertoli cells sujected to iotinyltion for 3 min without stripping with MESNA uffer (i.e., efore endocytosis; 3 g protein) were pulled down y NeutrAvidin Plus eds nd sujected to immunolotting to visulize occludin, which ws used to estimte the percentge of endocytosed occludin. Since t the time of iotinyltion efore the endocytosis ssy, totl cell surfce occludin ws similr in oth control nd groups; thus single totl lne from the control group ws shown in Fig. 4A,. Only smll frction of iotinylted occludin ws endocytosed (Fig. 4A, nd ). In short, the purpose of this ssy ws not ttempted to quntify differences etween cell surfce occludin in the knockdown vs. the control group ut to ssess ny ltertion in the kinetics of protein endocytosis. Since protein endocytosis is known to e regulted, t lest in prt, vi chnges in the protein phosphoryltion of n integrl memrne protein, such s occludin nd E-cdherin (7, 17), we next exmined ny ltertions in the phospho-ser, -Thr, nd -Tyr content in occludin etween the knockdown nd control groups. While the levels of occludin etween the control nd -silenced Sertoli cells y Co-IP were similr (Fig. 4B), consistent with dt shown in Fig. 2B, the level of phospho-throccludin ws significntly lowered, ut not phospho-ser- nor phospho-tyr-occludin, in the knockdown cells vs. control cells, illustrting there ws reduction of phospho-thr content in occludin following silencing (Fig. 4B, nd ). Knockdown of in Sertoli cells enhnces ssocition of occludin with endosomes. To further expnd nd confirm the oservtions shown in Figs. 2 nd 4 tht there ws n increse in protein endocytosis following knockdown, we exmined ny chnges in the protein expression of endocytic vesicle-ssocited proteins, such s EEA-1 (n endosome mrker; Ref. 35), or its ssocition with occludin in Sertoli cells fter knockdown. It is noted tht following knockdown, occludin ws internlized nd it ppered to ecome etter ssocited with EEA1 (Fig. 5A). While the stedy-stte level of EEA-1 in the knockdown Sertoli cells vs. control cells ws similr (Fig. 5B, nd ), sttisticlly significnt increse in the ssocition etween occludin nd EEA-1 ws detected y Co-IP (Fig. 5C, nd ), supporting findings otined y dul-leled immunofluorescence nlysis (Fig. 5A).

Overexpression of in Sertoli cells promotes TJ-permeility rrier function vi upregultion of TJ protein dptor ZO-1 nd desmosome proteins t the Sertoli cell BTB. To further vlidte dt otined y RNAi tht illustrte the regultory role of in BTB dynmics, the full-length cdna ws isolted y PCR (Fig. 1) using cdnas derived from Sertoli cell RNA s the templte, ligted to mmmlin expression vector pci-neo (Tle 2). This pci-neo/ plsmid ws used for overexpression of in Sertoli cell epithelium with n estlished TJ-permeility rrier (Fig. 6, A nd B). Consistent with findings using RNAi in which the knockdown of pertured the Sertoli TJ-rrier function, overexpression of (Fig. 6A) y 5% (Fig. 6C) led to tightening of the Sertoli cell TJ-permeility rrier (Fig. 6B). Studies y immunolotting lso illustrted tht n overexpression of led to n upregultion of ZO-1 expression, consistent with dt shown in Fig. 1 wherein its knockdown could downregulte ZO-1 expression, ut lso n upregultion of desmoglein-2 nd desmocollin-2, ut not other BTB-ssocited TJ nd sl ES proteins (Fig. 6C, nd ). A B Endocytosis of Sertoli cell surfce occludin t 35 o C Totl -ve 1536 9 15 3 6 9 Min Occludin, 65 kd Actin, 42 kd Biotinyltion IP: nti-occludin ws initilly identified in 1997 (2) s the receptor for coxsckievirus nd denovirus (3, 12). It is constituent protein nd cell dhesion molecule of the TJ in multiple epitheli nd endotheli (1, 32, 61). It is now estlished tht is n importnt TJ integrl memrne protein etween crdiomyocytes known s the interclted disc in the hert vi homotypic interctions of etween djcent cells, crucil to regulte crdic remodeling nd electricl conductnce etween tri nd ventricle (11, 42). Becuse is specific receptor for denovirus, it hs lso een extensively investigted to explore the use of for denovirus-sed gene therpy (23, 5, 68). However, recent studies hve shown tht is molecule with diversified functions; its contriution to cellulr physiology is well eyond its structurl nd cell dhesion role t the TJ nd other intercellulr junctions. For instnce, ws shown to e metsttic tumor suppressor since its expression reduces lung metsttic potentil in murine cncer cells (66). However, expression is lso needed for the efficient formtion of tumors in suset of lung cncer cells (44), nd its expression is necessry to support the estlishment of distnt metstses nd reducing poptosis in denoms in colon cncer (54). Collectively, these findings thus illustrte its involvement in tumorigenesis. Furthermore, ws recently shown to ply supporting role in regenertion of dmged myocrdium in the hert (59), in neuronl homeostsis y involving in xonl trnsport of clthrin-medited orgnelles vi endocytosis in the rin (48), nd in mediting signling function t the ventriculr interclted disc in the hert y recruiting connexin 45 ( GJ integrl memrne nd structurl protein), -ctenin, nd ZO-1 to the site for proper trioventriculr con Reltive occludin phosphoryltion (ritrry unit) Endocytosed Occludin (%) 3 2 1 1.2.8.4 15 3 6 9 Time (min) IB: Occludin, 65kD p-ser-occludin, 65kD p-thr-occludin, 65kD p-tyr-occludin, 65kD p-ser p-thr p-tyr REGULATES BTB DYNAMICS DISCUSSION C849 Fig. 4. A knockdown of in Sertoli cells with n estlished TJpermeility rrier y RNAi enhnces endocytosis of occludin nd lters the phosphoryltion sttus of occludin. A: Sertoli cells (.45 1 6 cells/cm 2 ) were cultured lone for 3 dys efore RNAi in which ws knockdown y trnsfecting cells with 1 nm -specific duplexes vs. nontrgeting control duplexes for 24 h (see Fig. 2C). Three dys therefter, endocytosis ssy ws performed. Totl iotinylted cell surfce proteins (Totl, from control group s shown herein, which ws similr in group), without stripping with 2-mercptoethnesulfonte, were used to ssess the percentge of occludin tht hve een endocytosed. Equl mount of proteins ( 3 g) were used for extrcting the iotinylted proteins y using Ultrlink Immoilized NeutrAvidin Plus eds (Pierce, including the Totl, s illustrted y the corresponding ctin lot). The -ve represents cell surfce proteins ( 3 g) without iotinyltion ut sujected to extrction with NeutrAvidin Plus eds nd immunolotting using n nti-occludin (see Tle 1). Knockdown of in Sertoli cells y 7% ws found to ccelerte the internliztion of occludin, TJ-integrl memrne protein, significntly vs. control cells (). Actin shown in served s protein loding control, representing ctin from Sertoli cell lystes efore NeutrAvdin pull-down. Composite dt of this endocytosis experiment were shown in in which ech dt point is men SD of n 3 independent experiments. Sttisticl nlysis ws performed y one-wy ANOVA. P.1. B: Sertoli cell lystes ( 3 g of protein) were first sujected to coimmunoprecipittion (Co-IP) with n nti-occludin ntiody to immunoprecipitte occludin, nd the levels of occludin in the smples etween the nontrgeting control nd the knockdown groups were similr (top pnel), consistent with findings shown in Fig. 1B, which lso illustrted equl protein loding etween the two groups. These lots were then proed with nti-phospho-ser, -Thr, or -Tyr ntiodies (see Tle 1) nd shown in the susequent pnels (). These findings were densitometriclly scnned nd normlized ginst occludin level with the level in the control ritrrily set t 1, ginst which sttisticl comprison ws performed. Ech r is men SD of n 3 4 experiments. P.5; P.1.

C85 REGULATES BTB DYNAMICS duction nd crdic function (31). In short, there is mounting evidence sed on studies in multiple tissues nd/or orgns tht support the expnding physiologicl roles of in different spects of cellulr physiology. A Ctrl Occludin/ EEA1/DAPI B C 3 dys fter trnsfection IP: EEA1 Reltive protein level (ritrry unit) 1.5 1..5 Reltive EEA1-occludin interction (ritrry unit) 2. 1. EEA1 EEA1, 18 kd Actin, 42 kd IgG Norml SC ns 3 dys fter trnsfection IB: Occludin, 65 kd IgG H, 5 kd IgG L, 23 kd ns 3 dys fter trnsfection In this report, ws shown to e more thn structurl component of the TJ t the Sertoli cell BTB. First, its knockdown without ny pprent off-trget effects ws found to ssocite with downregultion of ZO-1 expression in Sertoli cell epithelium with n estlished TJ rrier tht mimicked the Sertoli cell BTB in vivo, suggesting my ply regultory role in protein expression t the BTB. Our oservtion is lso consistent with recent report (31) using crdicspecific conditionl knockout (-cko) mice, in which KO in the hert lso led to mrked decline in -ctenin nd ZO-1 expression t the interclted disc, which is n ultrstructure etween crdiomyocytes composed of TJ, AJ, nd GJ. More importntly, its knockdown ws found to ssocite with disruption of the Sertoli cell TJ-permeility rrier, nd n increse in endocytosis of TJ protein occludin t the Sertoli cell BTB. This increse in endocytic vesicle-medited protein trfficking is pprently the result of loss of phospho-thr in occludin (ut not phospho-ser or phospho- Tyr), which in turn leds to n incresed ssocition etween occludin nd EEA-1, n endosome mrker, s supported y Co-IP experiment. The mechnism y which regultes the phosphoryltion content of occludin is likely medited vi c-src, nonreceptor protein tyrosine kinse t the BTB (8), since c-src is one of the few regultory kinses tht ws shown to form protein complex with in the testis (62). For instnce, neither phosphoinositide 3-kinse nor focl dhesion kinse (FAK) ws found to structurlly ssocite with (62) even though oth phosphoinositide 3-kinse (53) nd FAK (51, 52), in prticulr p-fak-tyr 47 (3), hve een loclized to the BTB in the rt testis nd expressed stge specificlly during the epithelil cycle. In this context, it is of interest to note tht the use of Co-IP tht demonstrted interctions etween occludin nd EEA-1; this procedure my ring down other components of the endocytic vesicles (ut not the entire Fig. 5. Effects of knockdown on the endocytic vesicle-medited protein trfficking in Sertoli cell epithelium. A: cellulr locliztion of EEA-1 (red, n endosome mrker) nd occludin ( TJ-integrl memrne protein) in Sertoli cell epithelium nd their chnges following knockdown were exmined y dul-leled immunofluorescence nlysis 3 dys fter trnsfection of cells with -specific duplexes ( ) vs. nontrgeting control duplexes (Ctrl ). After knockdown, more occludin (green fluorescence) ws found to e endocytosed in Sertoli cells, consistent with findings shown in Fig. 1D. Also, occludin nd EEA-1 (red fluorescence) ppered to co-loclize more extensively in the knockdown cells (see white rrowheds which denote n increse in the colocliztion of occludin with EEA-1 fter knockdown). DAPI (lue) ws used to visulize nuclei. Scle r 2 m, which pplies to the other microgrph. B: Sertoli cells cultured for 3 dys were trnsfected with -specific duplexes vs. the nontrgeting control duplexes for 24 h. Cells were rinsed nd cultured with fresh F-12/DMEM for n dditionl 48 h nd terminted therefter nd used for immunolotting. knockdown hd no pprent effects on the stedy-stte level of EEA-1 in Sertoli cells (), nd the composite results of n 3 experiments were shown in (); ns, nonsignificntly different. C: Co-IP ws used to ssess chnges in protein-protein interctions etween occludin with EEA-1 following knockdown y using 3 g protein lyste for immunoprecipittion s shown in. Norml Sertoli cell lyste (Norml SC, 4 g protein) without Co-IP served s positive control (). Bottom: lot of IgG H nd IgG L chins, illustrting equl protein loding in this Co-IP experiment (). An increse in interction etween occludin with EEA-1 ws detected following knockdown in Sertoli cells (). Composite dt, with ech r represents men SD of n 3 experiments shown in in which the reltive ssocition of occludin with EEA-1 in control group ws ritrrily set t 1, ginst which sttisticl comprison ws performed. P.5 y ANOVA.

REGULATES BTB DYNAMICS C851 A p 123 133 653 517 453 394 M pci-neo pci-neo/, 177 p S-16, 385 p B TER (ohm cm 2 ) 6 5 4 3 2 1 Trnsfection PCR, IB pci-neo pci-neo/ 1 2 3 4 5 6 7 Time of Sertoli cells in culture (dy) C TJ proteins Bsl ES proteins Desmosome proteins pci-neo pci-neo/, 46 kd Occludin, 65 kd Cludin-11, 22 kd JAM-A, 36 kd ZO-1, 21 kd N-Cdherin, 127 kd α-ctenin, 12 kd β-ctenin, 92 kd Desmoglein-2, 16 kd Desmocollin-2, 15 kd γ-ctenin, 82 kd Actin, 42 kd Reltive protein level (ritrry unit) Reltive protein level (ritrry unit) 2 1 2 1 ZO-1 Desmoglein-2 Desmocollin-2 pci-neo pci-neo/ Fig. 6. Effects of overexpression on the Sertoli cell TJ-permeility rrier nd the stedy-stte levels of BTB constituent proteins in Sertoli cells in vitro. To confirm findings regrding the physiologicl role of in regulting BTB function sed on the use of RNAi, Sertoli cells were trnsfected with pci-neo/ vector vs. empty pci-neo vector (control) to exmine chnges in TJ-rrier function following trnsient expression of. A: increse in the expression of in Sertoli cells trnsfected with pci-neo/ vector contining the full-length construct vs. empty vector (pci-neo) ws detected y RT-PCR. B: Sertoli cells t 1.2 1 6 cells/cm 2 were cultured lone for 3 dys to llow the estlishment of functionl TJ-permeility rrier nd were then trnsfected with pci-neo vector vs. pci-neo/ vector. Chnges in the TJ-rrier function ws monitored y quntifying TER cross the Sertoli cell epithelium. Ech dt point is men SD of n 3 replictes of representtive experiment, similr results were otined from 3 independent experiments. P.1 y one wy ANOVA followed y Dunnett s test. C: increse in expression following trnsfection of Sertoli cells with pci-neo/ vector ws further confirmed y immunolotting (). Also noted is n upregultion of ZO-1, consistent with findings shown in Fig. 2B, in which knockdown of ws found to ssocite with downregultion of ZO-1 (). An upregultion of desmosome proteins desmogelin-2 nd desmocollin-2 ws consistently detected following overexpression in Sertoli cells s shown in. Dt of immunolotting were summrized in histogrms shown in, illustrting significnt upregultion on ZO-1 (top), desmoglein-2, nd desmocollin-2 (ottom) expression following overexpression. Ech r is men SD of n 4 5 experiments in which the expression of trget gene ws normlized ginst the corresponding ctin level, nd the protein level in Sertoli cells trnsfected with pci-neo empty vector ws ritrrily set t 1, ginst which sttisticl comprison ws performed. P.5. endocytic vesicles ecuse lystes of Sertoli cells, insted of intct cells, were used), such s protein involving in recycling or protein degrdtion, which my lso e involved in perturing the Sertoli cell TJ-rrier function. However, it is unlikely tht the silencing of would enhnce endosome-medited occludin degrdtion since the stedy-stte level of protein remined unltered during the experimentl period. Collectively, these dt support the notion tht plys possile regultory role t the BTB to coordinte the proper functioning of different dhesion protein complexes t the site. In this context, it is of interest to note tht t the TJ in multiple epitheli is known to ssocite with dptor proteins ZO-1 nd -ctenin (1, 6), which my serve s linkers for tethering to the underlying ctin-sed cytoskeleton. Since ZO-1 lso structurlly intercts with the occludin t stoichiometric rtio of 1:1 (13); thus downregultion of ZO-1 cn led to loss of occludin-zo-1-sed dhesion function t the BTB, nd the free occludin not inding to ZO-1 my led to its unwnted phosphoryltion, possily vi c-src, which triggers n increse in its internliztion s shown herein. However, the precise moleculr signling event(s) nd/or mechnism(s) tht leds to downregultion of ZO-1 expression following knockdown of remins to e identified. Second, these findings were supported in studies y overexpressing in Sertoli cell epithelium, which ws found to tighten nd promote the Sertoli cell TJ-permeility rrier. Overexpression of ws lso found to upregulte ZO-1 in Sertoli cells, consistent with findings in RNAi experiments. Interestingly, overexpression of lso upregulted the expression of desmoglein-2 nd desmocollin-2, puttive

C852 REGULATES BTB DYNAMICS integrl memrne protein nd dptor, respectively, t the desmosome, which together with coexisting TJ, sl ES, nd GJ constitute the BTB (8, 29), suggesting tht, eing TJ protein, my ct s signling molecule tht regultes the expression of constituent proteins of desmosome. However, it is noted tht knockdown of filed to downregulte the expression of desmoglein-2 nd desmocollin-2 s shown herein sed on multiple experiments, suggesting tht only its overexpression cn trigger some unintended signling pthwy(s) tht leds to n upregultion of these two desmosoml proteins. Nonetheless, these findings illustrte the likely role of in coordinting other dhesion protein complexes to mintin the rrier function t the BTB. This finding is lso consistent with recent report (31) illustrting tht in crdic -cko mice the loss of lso ffects the expression of connexin 45 ( GJ protein) t the interclted disc, perturing trioventriculr conduction to coordinte crdic function. It is highly likely tht there is similr underlying moleculr mechnism(s) tht induces downregultion of ZO-1, -ctenin, nd connexin 45 in these different orgns following the knockdown or knockout of, which should e vigorously evluted in future studies. Bsed on the estlished cell dhesion properties of, nd the fct tht is found in oth Sertoli nd germ cells (36, 37, 62), it ws postulted tht during the trnsit of preleptotene spermtocytes t the BTB t stge VIII-IX of the epithelil cycle, residing in spermtocytes (SP) cn form homotypic interctions with in Sertoli cells (SC), such tht the rrier function cn e mintined in which residing in spermtocytes replces one of the two intercting s etween djcent Sertoli cells (SC/-/SC) forming trnsient SP/ -/SC rrier (61). Interestingly, it is now generlly ccepted tht to preserve the BTB integrity during the trnsit of preleptotene spermtocytes t the BTB t stge VIII-IX of the epithelil cycle, new BTB (e.g., TJ firils) is first ssemled ehind migrting preleptotene spermtocytes, which re connected in clones vi intercellulr ridges, efore the old BTB is disssemled (7, 8, 38). Thus, esides serving s homotypic intercting dhesion molecules etween spermtocytes nd Sertoli cells during the trnsit of spermtocytes cross the BTB, my lso tke prt in the ssemly of new BTB nd the disssemly of old BTB ner the sl- nd picl-region of migrting spermtocytes, respectively, vi its corresponding overexpression nd silencing t the two sites s illustrted in this report. This possiility must e crefully evluted in future studies. However, findings reported herein hve provided the frmework upon which functionl experiments cn e designed. ACKNOWLEDGMENTS This work ws supported y grnts from the Ntionl Institute of Child Helth nd Humn Development Grnts R1-HD-5634 (to C. Y. Cheng) nd U54-HD-2999 Project 5 (to C. Y. Cheng) nd Ntionl Nturl Science Foundtion of Chin Grnt 311143 (to L. Su). DISCLOSURES No conflicts of interest, finncil or otherwise, re declred y the uthor(s). AUTHOR CONTRIBUTIONS Author contriutions: L.S. nd C.Y.C. performed experiments; L.S., D.D.M., nd C.Y.C. nlyzed dt; L.S. nd C.Y.C. prepred figures; L.S., D.D.M., nd C.Y.C. pproved finl version of mnuscript; D.D.M. nd C.Y.C. conception nd design of reserch; D.D.M. nd C.Y.C. interpreted results of experiments; C.Y.C. drfted mnuscript; C.Y.C. edited nd revised mnuscript. REFERENCES 1. Buer M, Kinkl N, Meixner A, Kremmer E, Riemenschneider M, Forstl H, Gsser T, Ueffing M. Prevention of interferon-stimulted gene expression using microrna-designed hirpins. Gene Ther 16: 142 147, 29. 2. 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