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Involvement of Gαi2 in the Maintenance and Biogenesis of Epithelial Cell Tight Junctions

1998; Elsevier BV; Volume: 273; Issue: 34 Linguagem: Inglês

10.1074/jbc.273.34.21629

ISSN

1083-351X

Autores

Chandana Saha, Sanjay K. Nigám, Bradley M. Denker,

Tópico(s)

Connexins and lens biology

Resumo

Polarized epithelial cells have highly developed tight junctions (TJ) to maintain an impermeant barrier and segregate plasma membrane functions, but the mechanisms that promote TJ formation and maintain its integrity are only partially defined. Treatment of confluent monolayers of Madin-Darby canine kidney (MDCK) cells with AlF4− (activator of heterotrimeric G protein α subunits) results in a 3–4-fold increase in transepithelial resistances (TER), a reliable indicator of TJ integrity. MOCK cells transfected with activated Gα0 (Q205L) have acclerated TJ formation (Denker, B. M., Saha, C., Khawaja, S., and Nigam, S. J. (1996) J. Biol. Chem. 271, 25750–25753). Gαi2 has been localized within the tight junction, and a role for Gαi2in the formation and/or maintenance of the tight junction was studied by transfection of MDCK cells with vector without insert (PC), wild type Gαi2, or a GTPase-deficient mutant (constitutively activated), Q205Lαi2. Tryptic conformational analysis confirmed expression of a constitutively active Gαi2 in Q205Lαi2-MDCK cells, and confocal microscopy showed a similar pattern of Gαi2 localization in the three cell lines. Q205Lαi2-MDCK cells had significantly higher base-line TER values than wild type Gαi2- or PC-MDCK cells (1187 ± 150 versus 576 ± 89 (Gαi2); 377 ± 52 Ω·cm2 (PC)), and both Gαi2- and Q205Lαi2-transfected cell lines more rapidly develop TER in the Ca2+ switch, a model widely used to study the mechanisms of junctional assembly. Treatment of cells with AlF4− during the Ca2+ switch had little effect on the kinetics of TER development in Gαi2- or Q205Lαi2-MDCK cells, but PC cells reached half-maximal TER significantly sooner in the presence of AlF4− (similar times to Gαi2-transfected cells). Base-line TER values obtained after the switch were significantly higher for all three cell lines in the presence of AlF4−. These findings indicate that Gαi2 is important for both the maintenance and development of the TJ, although additional Gα subunits are likely to play a role. Polarized epithelial cells have highly developed tight junctions (TJ) to maintain an impermeant barrier and segregate plasma membrane functions, but the mechanisms that promote TJ formation and maintain its integrity are only partially defined. Treatment of confluent monolayers of Madin-Darby canine kidney (MDCK) cells with AlF4− (activator of heterotrimeric G protein α subunits) results in a 3–4-fold increase in transepithelial resistances (TER), a reliable indicator of TJ integrity. MOCK cells transfected with activated Gα0 (Q205L) have acclerated TJ formation (Denker, B. M., Saha, C., Khawaja, S., and Nigam, S. J. (1996) J. Biol. Chem. 271, 25750–25753). Gαi2 has been localized within the tight junction, and a role for Gαi2in the formation and/or maintenance of the tight junction was studied by transfection of MDCK cells with vector without insert (PC), wild type Gαi2, or a GTPase-deficient mutant (constitutively activated), Q205Lαi2. Tryptic conformational analysis confirmed expression of a constitutively active Gαi2 in Q205Lαi2-MDCK cells, and confocal microscopy showed a similar pattern of Gαi2 localization in the three cell lines. Q205Lαi2-MDCK cells had significantly higher base-line TER values than wild type Gαi2- or PC-MDCK cells (1187 ± 150 versus 576 ± 89 (Gαi2); 377 ± 52 Ω·cm2 (PC)), and both Gαi2- and Q205Lαi2-transfected cell lines more rapidly develop TER in the Ca2+ switch, a model widely used to study the mechanisms of junctional assembly. Treatment of cells with AlF4− during the Ca2+ switch had little effect on the kinetics of TER development in Gαi2- or Q205Lαi2-MDCK cells, but PC cells reached half-maximal TER significantly sooner in the presence of AlF4− (similar times to Gαi2-transfected cells). Base-line TER values obtained after the switch were significantly higher for all three cell lines in the presence of AlF4−. These findings indicate that Gαi2 is important for both the maintenance and development of the TJ, although additional Gα subunits are likely to play a role. Polarized epithelia have developed highly specialized membrane functions enabling vectorial transport across the cellular layer. The junctional complex of epithelial cells includes gap junctions, adherens junctions, and tight junctions. The tight junction (TJ) 1The abbreviations used are: TJtight junctionMDCKMadin-Darby canine kidneyG proteinguanine nucleotide-binding proteinZOzona occludensTERtransepithelial resistancePKCprotein kinase CNCnormal calciumPBSphosphate-buffered salineGTPγSguanosine 5′-3-O-(thio)triphosphate. is the most apical component of the junctional complex and provides two essential functions: (i) the permeability barrier to paracellular fluxes and (ii) the “fence” separating the apical and basolateral membrane domains. In developing tissues as well as cell culture models, the critical signaling events important to junction formation appear to be quite different from mechanisms that maintain junctional integrity. The TJ is composed of a complex of proteins that includes occludin, the only transmembrane protein identified so far (1Furuse M. Hirase T. Itoh M. Nagafuchi A. Yonemura S. Tsukita S. Tsukita S. J. Cell Biol. 1993; 123: 1777-1788Crossref PubMed Scopus (2143) Google Scholar). There are several peripherally attached membrane proteins found in the TJ including the zona occludens family (ZO-1, -2, and -3) (2Stevenson B.R. Goodenough D.A. J. Cell Biol. 1984; 98: 1209-1221Crossref PubMed Scopus (99) Google Scholar, 3Stevenson B.R. Silicano J.D. Mooseker M.S. Goodenough D.A. J. Cell Biol. 1986; 103: 755-766Crossref PubMed Scopus (1289) Google Scholar, 4Haskins J. Gu L. Wittchen E.S. Hibbard J. Stevenson B.R. J. Cell Biol. 1998; 141: 199-208Crossref PubMed Scopus (495) Google Scholar). ZO proteins are members of the MAGUK (membrane associatedguanylate kinase) superfamily that are often found at sites of cell-cell contact and may function to couple extracellular signaling pathways with the cytoskeleton. Other proteins found in or near the TJ include cingulin, 7H6, symplekin, unidentified phosphoproteins, and a series of signal transduction molecules (reviewed in Ref. 5Denker B.M. Nigam S.K. Am. J. Physiol. 1998; 274: F1-F9Crossref PubMed Google Scholar). tight junction Madin-Darby canine kidney guanine nucleotide-binding protein zona occludens transepithelial resistance protein kinase C normal calcium phosphate-buffered saline guanosine 5′-3-O-(thio)triphosphate. MDCK cells are a cultured epithelial cell line that has been extensively utilized for studies of epithelial polarity, targeting of proteins, and the study of intercellular junctions (6Rodriguez-Boulan E. Nelson W.J. Science. 1989; 245: 718-725Crossref PubMed Scopus (819) Google Scholar). The Ca2+ switch model of TJ formation in MDCK cells has been widely utilized to gain insights into the function of polarized epithelial cells (7Stuart R.O. Sun A. Bush K.T. Nigam S.K. J. Biol. Chem. 1996; 271: 13636-13641Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 8Stuart R.O. Nigam S.K. Semin. Nephrol. 1995; 15: 315-326PubMed Google Scholar, 9Nigam S.K. Rodriguez-Boulan E. Silver R.B. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6162-6166Crossref PubMed Scopus (73) Google Scholar, 10Balda M.S. Gonzalez-Mariscal L. Macias-Silva M. Torres-Marquez M.E. Garcia Sainz J.A. Cereijido M. J. Membr. Biol. 1991; 122: 193-202Crossref PubMed Scopus (247) Google Scholar, 11Cereijido M. Gonzalez-Mariscal L. Conreras R.G. Gallardo J.M. Garcia- Villegas R. Valdes J. J. Cell Sci. 1993; 17: 127-132Crossref Google Scholar) and recapitulates many of the critical molecular events of epithelial morphogenesis. MDCK cells cultured in low calcium (μm) lack cell-cell contact, polarity, and junctions. “Switching” to normal calcium medium (NC) triggers a series of molecular events that leads to establishment of the polarized phenotype with characteristics of a tight transporting epithelium. Tight junction development can be followed by measuring the transepithelial resistance (TER), a rapid and reproducible assessment of tight junction integrity. Because MDCK cells are clonal and TJ development can be synchronized in the Ca2+ switch, the role of specific proteins on TJ biogenesis can be studied in this system by cDNA transfections. The critical role of calcium in the formation of intercellular junctions is well established. Extracellular calcium is required for homotypic interactions of E-cadherin and is likely to be the initial event of junctional complex formation (12Gumbiner B.M. Cell. 1996; 84: 345-357Abstract Full Text Full Text PDF PubMed Scopus (2948) Google Scholar). Regulated intracellular calcium stores are also important for tight junction biogenesis. There are local increases in intracellular calcium concentration at the points of cell-cell contact (9Nigam S.K. Rodriguez-Boulan E. Silver R.B. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6162-6166Crossref PubMed Scopus (73) Google Scholar), and chelation of intracellular calcium perturbs TER development (13Stuart R.O. Sun A. Panichas M. Hebert S.C. Brenner B.M. Nigam S.K. J. Cell. Physiol. 1994; 159: 423-433Crossref PubMed Scopus (105) Google Scholar). Thapsigargin depletes intracellular endoplasmic reticulum stores of calcium, and thapsigargin treatment of MDCK cells prior to initiation of cell-cell contact prevents TER development and the sorting of ZO-1 to the TJ (7Stuart R.O. Sun A. Bush K.T. Nigam S.K. J. Biol. Chem. 1996; 271: 13636-13641Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). The signaling events important for TJ biogenesis are complex and utilize a variety of pathways. Phosphorylation events are important as several proteins become phosphorylated in the TJ, and protein kinase C (PKC) isoforms translocate to the TJ during biogenesis. PKC inhibitors markedly inhibit the development of TER in the calcium switch, and PKC agonists stimulate ZO-1 translocation to the membrane. The importance of PKC in tight junction biogenesis, as well as regulated calcium stores, suggests important roles for heterotrimeric G proteins. The proximity of several G proteins to the TJ also suggests they may have potential roles in regulating the development and/or maintenance of the TJ. PKCζ and PKCα, have also been localized in the vicinity of the TJ (14Rosson D. O'Brien T.G. Kampherstein J.A. Szallasi Z. Bogi K. Blumberg P.M. Mullin J.M. J. Biol. Chem. 1997; 272: 14950-14953Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, 15Stuart R.O. Nigam S.K. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 6072-6076Crossref PubMed Scopus (245) Google Scholar, 16Denker B.M. Saha C. Khawaja S. Nigam S.J. J. Biol. Chem. 1996; 271: 25750-25753Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar, 17de Almeida J.B. Holtzman E.J. Peters P. Ercolani L. Ausiello D.A. Stow J.L. J. Cell Sci. 1994; 107: 507-515PubMed Google Scholar). We previously demonstrated that expressing a constitutively activated Gαo (Q205L) in MDCK cells significantly accelerated TJ biogenesis without affecting base-line resistances. Although Gαo is a member of the G protein family inhibited by pertussis toxin (∼80% similar to Gαi1–3), its receptors and effectors are distinct, and furthermore, Gαo is not detected in renal epithelia or MDCK cells (16Denker B.M. Saha C. Khawaja S. Nigam S.J. J. Biol. Chem. 1996; 271: 25750-25753Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar, 18Senkfor S.I. Johnson G.L. Berl T. J. Clin. Invest. 1993; 92: 786-790Crossref PubMed Scopus (9) Google Scholar, 19Hamilton S.E. Nathanson N.M. Biochem. Biophys. Res. Commun. 1997; 234: 1-7Crossref PubMed Scopus (16) Google Scholar). Several Gαi family members are expressed in epithelial cells, and Gαi2 has been shown to overlap with the tight junction in epithelial cell lines (16Denker B.M. Saha C. Khawaja S. Nigam S.J. J. Biol. Chem. 1996; 271: 25750-25753Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar, 17de Almeida J.B. Holtzman E.J. Peters P. Ercolani L. Ausiello D.A. Stow J.L. J. Cell Sci. 1994; 107: 507-515PubMed Google Scholar). Taken together, these observations raise the possibility that Gαi2 may be an important regulator of tight junctions. To test this hypothesis, we initially looked for effects of AlF4− (activator of Gα subunits) on tight junctions in control cell lines and then established MDCK cell lines overexpressing wild type Gαi2 and a constitutively activated Gαi2 (GTPase-deficient, Q205Lαi2). We find that AlF4− significantly increases TER in control cells and accelerates TER development during the Ca2+ switch. The effects of AlF4− can be reproduced in MDCK cells expressing activated Gαi2, indicating that this Gα subunit is critical to the development and maintenance of tight junctions. Rat Gαi2 cDNA was cloned into Bluescript (Stratagene) as described previously (20Denker B.M. Boutin P.M. Neer E.J. Biochemistry. 1995; 34: 5544-5553Crossref PubMed Scopus (37) Google Scholar) and recloned into the EcoRI andApaI sites of pcDNA3 (Invitrogen). Q205Lαi2 was provided by Dr. Gary Johnson and cloned into Bluescript using HindIII sites and then into pcDNA3 using XhoI and XbaI sites. MDCK cells were maintained in Dulbecco's modified Eagle's medium supplemented with antibiotics plus 5% fetal calf serum. Transfected cell lines were maintained in G418 (500 μg/ml; Life Technologies, Inc.) Subconfluent MDCK cells (ATCC, Manassas, VA) were transfected with 10 μg of linearized plasmid by calcium phosphate precipitation method as described previously (16Denker B.M. Saha C. Khawaja S. Nigam S.J. J. Biol. Chem. 1996; 271: 25750-25753Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). G418-resistant colonies were analyzed for increased Gαi2expression by Western blot using a rabbit polyclonal antibody directed toward the C terminus of Gαi2 (AS7; NEN Life Science Products). Control cells were obtained by transfecting pcDNA3 without insert, and all cell lines were established in parallel. Confluent PC-, Gαi2-, or Q205Lαi2 -MDCK cells were washed twice with PBS and then scraped into buffer A (50 mmTris-HCl, pH 7.5, 6 mm MgCl2, 75 mmsucrose, 1 mm dithiothreitol, 1 mm EDTA). Cells were frozen and thawed three times and triturated ten times through a 27 gauge needle. All samples were incubated at 30 °C with no added nucleotide or 100 μm GTPγS. Samples were immediately placed on ice, and trypsin was added (20 pmol ofl-1-(tosylamido)-2-phenylethyl chloromethyl ketone-treated trypsin (Sigma). All samples were incubated at 30 °C for 20 min, and digestion was terminated by the addition of SDS-polyacrylamide gel electrophoresis sample buffer followed by boiling for 5 min. Samples were then analyzed by SDS-polyacrylamide gel electrophoresis and Western blot using AS7 anti-Gαi2 rabbit polyclonal antibody (1:1,000) and ECL (Pierce) with goat anti-rabbit horseradish peroxidase (1:10,000). PC-, Gαi2-, or Q205Lαi2-MDCK cells were grown on coverslips or Transwell filters (12 mm) (Costar), rinsed with PBS, and fixed with methanol (100%, −70 °C) for 10 min. Cells were then washed with PBS and blocked as described previously (16Denker B.M. Saha C. Khawaja S. Nigam S.J. J. Biol. Chem. 1996; 271: 25750-25753Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). Samples were incubated with rabbit polyclonal Gαi2 (AS7, from NEN Life Science Products) at several dilutions and rat monoclonal to ZO-1 (undiluted supernatant; courtesy of D. Goodenough) for 1 h. Cells were washed with PBS three times at 5-min intervals and incubated with secondary antibodies (fluorescein- or Texas Red-conjugated goat anti-rabbit or anti-rat IgG; Jackson Immuno Research, West Grove, PA) at 1:100 with for 1 h. Coverslips were visualized on a Nikon Labphot-2 immunofluorescence microscope or a Bio-Rad 1024 confocal microscope using the 63× oil immersion objective. Images were processed in Adobe Photoshop (Adobe, CA) and figure compiled in Adobe Illustrator (Adobe, CA). MDCK cells were plated on 12-mm transwell filter (Costar) at confluence (∼3 × 105 cells) and allowed to attach for 24–36 h to form a tight monolayer in normal Ca2+ containing medium (NC). Cells were placed in low Ca2+ (1–4 μm) medium (low calcium) for 1 h followed by switch to NC medium. TER was measured using a Millipore (Bedford, MA) electrical resistance system, and the results are expressed in Ω·cm2. TER was measured in stable monolayers and during Ca2+ switch in the presence and absence of aluminum fluoride (AlF4−; 3 mm NaF + 50 μmAlCl3, Sigma). Several lines of evidence have suggested the involvement of heterotrimeric G proteins in tight junction formation. Early studies with G protein modulators such as pertussis toxin, cholera toxin, AlF4−, and a variety of other agents showed variable effects on TJ biogenesis (10Balda M.S. Gonzalez-Mariscal L. Macias-Silva M. Torres-Marquez M.E. Garcia Sainz J.A. Cereijido M. J. Membr. Biol. 1991; 122: 193-202Crossref PubMed Scopus (247) Google Scholar). Several confocal studies have localized Gαi2, Gαi3, and Gα12 in the vicinity of the tight junction (16Denker B.M. Saha C. Khawaja S. Nigam S.J. J. Biol. Chem. 1996; 271: 25750-25753Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar, 17de Almeida J.B. Holtzman E.J. Peters P. Ercolani L. Ausiello D.A. Stow J.L. J. Cell Sci. 1994; 107: 507-515PubMed Google Scholar, 19Hamilton S.E. Nathanson N.M. Biochem. Biophys. Res. Commun. 1997; 234: 1-7Crossref PubMed Scopus (16) Google Scholar,21Dodane V. Kachar B. J. Membr. Biol. 1996; 149: 199-209Crossref PubMed Scopus (114) Google Scholar). Recently, we demonstrated that Gαo (a member of the Gα family inhibited by pertussis toxin) expressed in MDCK cells localizes to the subapical lateral membrane overlapping with ZO-1 in the tight junction (16Denker B.M. Saha C. Khawaja S. Nigam S.J. J. Biol. Chem. 1996; 271: 25750-25753Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar), and this was subsequently confirmed in another study (19Hamilton S.E. Nathanson N.M. Biochem. Biophys. Res. Commun. 1997; 234: 1-7Crossref PubMed Scopus (16) Google Scholar). A constitutively active mutant of Gαo (Q205L) also localizes in this region, and cells expressing Gαoshowed no differences in base-line junctional properties as determined by transepithelial resistance. However, in the Ca2+ switch, MDCK cells expressing activated Gαo(Q205Lαo-MDCK) developed tight junctions at twice the rate and reached significantly higher peak TER values than either Gαo-MDCK or PC-MDCK cells. Although Gαo is not normally expressed in epithelia, this observation raises the possibility that one of the Gα subunits normally found in this location could have a fundamental role in regulating the development and/or maintenance of the TJ. To further examine the role of G protein α subunits affecting the TJ, we studied the effects of the G protein activator AlF4− on TJ formation in wild type (not transfected; WT-MDCK) and vector (pcDNA3) transfected MDCK cells (PC-MDCK). AlF4− has no known effects on small GTP binding proteins but activates heterotrimeric Gα subunits. Crystal structures of Gαi1 obtained with GDP complexed with AlF4− reveal that the position of the γ-phosphate is occupied by AlF4−. AlF4− in this position prevents catalysis by immobilizing Gln204 and Arg178 (22Coleman D.E. Berghuis A.M. Lee E. Linder M.E. Gilman A.G. Sprang S.R. Science. 1994; 265: 1405-1412Crossref PubMed Scopus (757) Google Scholar). Fig. 1 shows that wild type MDCK cells and PC transfected cells cultured on filters develop significantly higher TER in the presence of AlF4−. Untreated steady state TER values were similar between the cell lines, and AlF4− reproducibly increased TER values 3–4-fold. This finding is consistent with activation of one or more endogenous Gα subunits that results in enhanced steady state resistances. Because AlF4− activates all Gα subunits in MDCK cells and several studies have placed Gαi2 in close proximity to the TJ, we tested the hypothesis that Gαi2 was important to this process by stably expressing Gαi2 and a constitutively activated Gαi2 (Q205Lαi2) in MDCK cells. The amount of transfected Gαi2 was determined relative to the levels of endogenous Gαi2 in PC-MDCK cells. Western blots of the three cell lines using identical amounts of total protein were analyzed (not shown) using NIH image (Wayne Rasband, NIH). Relative to PC-MDCK cells, the level of Gαi2 in Gαi2-MDCK cells was 3.9 ± 0.4-fold (n = 7) increased, and for Q205Lαi2-MDCK the level was of Gαi2 was 1.8 ± 0.2-fold (n = 7) above PC-MDCK cells. To confirm that constitutively activated Gαi2 was expressed in these transfected MDCK cells, we utilized a tryptic cleavage analysis of Gαi2 (Fig. 2). This technique has been widely utilized as an indicator of Gα subunit conformation (23Denker B.M. Schmidt C.J. Neer E.J. J. Biol. Chem. 1992; 267: 9998-10002Abstract Full Text PDF PubMed Google Scholar, 24Denker B.M. Neer E.J. Schmidt C.J. J. Biol. Chem. 1992; 267: 6272-6277Abstract Full Text PDF PubMed Google Scholar) and is based on the observation that Gα subunits have a different cleavage pattern depending on whether they are folded into an active or inactive conformation. In the active conformation (GTP-liganded), there is only a single tryptic site accessible near the N terminus (approximately Arg21) resulting in a slightly truncated protein (39 kDa instead of 41 kDa for Gαi2). In the inactive (GDP-liganded) conformation, an additional site becomes accessible in the α2 helix or switch region (near Arg209) resulting in peptides of approximately 25 and 17 kDa. Fig. 2 demonstrates the tryptic cleavage patterns of cell homogenates from each of the transfected cell lines (PC-, Gαi2-, and Q205Lαi2-MDCK cells). In PC- and Gαi2-transfected cells, untreated Gαi2migrates at 41 kDa (first lane of each set) and is stabilized in the active conformation (39 kDa) if preincubated with the nonhydrolyzable GTP analogue, GTPγS (last lane of each set). However, in the absence of added nucleotide (middle lane of each set) the Gα subunits should be GDP bound from endogenous GTP/GDP in the cell, and Gαi2 is cleaved into 25- and 17-kDa fragments with no detectable 39-kDa peptide. The 25-kDa fragment (derived from the N terminus) is not detectable with the AS7 antibody, and the 17-kDa fragment is more labile (25Neer E.J. Lok J.M. Wolf L.G. J. Biol. Chem. 1984; 259: 14222-14229Abstract Full Text PDF PubMed Google Scholar) and consequently is not well visualized on these blots (not shown). Tryptic digestion of Q205Lαi2-transfected cells shows that in the absence of added nucleotide (middle lane), there is a fraction of Gαi2 that is tryptic-resistant, indicating persistence of Gαi2 in an “active” conformation. The amount of Gαi2 in the active conformation of Q205Lαi2-MDCK cells is a small fraction of the starting material. This is due, in part, to the observation that Gα subunits with mutations that result in an active conformation are more sensitive to proteolysis than wild type Gα subunits activated with GTPγS (20Denker B.M. Boutin P.M. Neer E.J. Biochemistry. 1995; 34: 5544-5553Crossref PubMed Scopus (37) Google Scholar). In addition, it is necessary to do these studies on whole cell lysates in the absence of protease inhibitors. The Western blots were deliberately overexposed to look for bands migrating at 39 kDa. This analysis confirms expression of constitutively activated Gαi2 in Q205Lαi2-MDCK cells. We have previously demonstrated that WT-MDCK cells express some Gαi2 in the subapical lateral membrane overlapping with ZO-1 (16Denker B.M. Saha C. Khawaja S. Nigam S.J. J. Biol. Chem. 1996; 271: 25750-25753Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). To eliminate the possibility that the transfection process affects Gαi2 localization, PC-MDCK cells were characterized by confocal microscopy. Fig.3 A shows a confocal image of PC-MDCK cells costained with antibodies to ZO-1 and Gαi2(used at 1:25 dilution). The confocal images reveal that Gαi2 partially colocalizes with ZO-1 at the level of the tight junction. There is significant intracellular staining that was also seen in WT-MDCK cells (16Denker B.M. Saha C. Khawaja S. Nigam S.J. J. Biol. Chem. 1996; 271: 25750-25753Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). To determine whether transfected Gαi2 subunits were localized in a similar manner to the endogenous Gαi2, the Gαi2 antibody (AS7) was diluted to a point where the endogenous Gαi2 was barely detectable. Fig. 3 B shows a confocal analysis of PC-, Gαi2-, and Q205Lαi2-MDCK stained and analyzed under identical conditions using a 1:100 dilution of the Gαi2 antibody. In panel a, PC-MDCK cells only demonstrate faint intracellular staining, but in panels band c, transfected Gαi2 and Q205Lαi2 can be visualized in the subapical lateral membrane overlapping with the TJ marker, ZO-1. Again, there is intracellular staining that is similar to the endogenous Gαi2 (Fig. 3 A). Overall the pattern of transfected Gαi2 and Q205Lαi2 is very similar to that seen with the endogenous Gαi2 subunits. These results confirm that transfected Gαi2 and Q205Lαi2 partition between the lateral membrane overlapping with the TJ and intracellular compartments. This finding is similar to our prior findings with Gαo-transfected MDCK cells (16Denker B.M. Saha C. Khawaja S. Nigam S.J. J. Biol. Chem. 1996; 271: 25750-25753Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). Because transfected Gαi2 and Q205Lαi2 were localized in a manner similar to that of the endogenous Gαi2, we next determined whether Gαi2localization in the TJ had any functional consequences for the tight junction. PC-, Gαi2-, and Q205Lαi2-MDCK cells were simultaneously analyzed under steady state conditions and also by using the Ca2+ switch. TJ integrity was followed by measurement of transepithelial resistance. Fig.4 A demonstrates the base-line resistances in these cells and the pattern of TER development after the Ca2+ switch. Overexpressing Gαi2 had a small but insignificant (p = 0.07) effect on base-line resistances in comparison with PC-MDCK cells (576 ± 89versus 377 ± 52; n = 12), but Q205Lαi2-MDCK cells had significantly higher base-line TER values (1187 ± 150 Ω·cm2; p< 0.001). The base-line TER values for Gαi2- and PC-MDCK cells were similar to reported values of Gαo (16Denker B.M. Saha C. Khawaja S. Nigam S.J. J. Biol. Chem. 1996; 271: 25750-25753Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar), and several clones were analyzed with no significant differences seen among the clones. To gain insight into the mechanism of higher TER values observed in Q205Lαi2-MDCK cells, all three cell lines were simultaneously analyzed in the Ca2+ switch. The elevated TER in Q205Lαi2-MDCK cells could be achieved by differences in the kinetics of TER development. Nonlinear regression analysis of the TER data between 0–12 h for all of the cell lines (Fig. 4 A) indicates an asymptotic approach to peak TER. Although the data do not precisely fit standard kinetic models, the kinetics of TER development in these cells is similar to what has been reported in other studies (15Stuart R.O. Nigam S.K. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 6072-6076Crossref PubMed Scopus (245) Google Scholar, 26Balda M.S. Gonzalez-Mariscal L. Matter K. Cereijido M. Anderson J.M. J. Cell Biol. 1993; 123: 293-302Crossref PubMed Scopus (353) Google Scholar). The time to half-maximal TER is a useful value for discussing the effects of Gαi2expression on TER biogenesis, and these values were calculated for each cell line in the presence and absence of AlF4− (TableI). Table I shows that the time to half-maximal TER (T50) was significantly more rapid in Gαi2- and Q205Lαi2-MDCK cells (0.8 ± 0.3 and 1.2 ± 0.3 h, respectively) in comparison with PC cells (3.0 ± 0.5 h). AlF4− had no significant on Gαi2-transfected cells but significantly shortenedT50 for PC cells (1.1 ± 0.7 h, a value similar to that of Gαi2 cells). The observation that PC-MDCK cells treated with AlF4−develop TER nearly as rapidly as Q205Lαi2-MDCK cells suggests that Gαi2 may be the predominant Gα subunit critical in TER development. Fig. 4 B shows that the base-line TER values for the three cell lines on the day following the Ca2+ switch (26 h) are significantly higher in the presence of ALF4−. In contrast to AlF4− effects on the rate of TER development, all three cell lines had significantly increased base-line TER at 26 h in the presence of AlF4−. Similar effects of AlF4− were seen with the three cell lines cultured in the steady state (not shown). This raises the possibility that AlF4− activates additional Gα subunits in the steady state that enhances transepithelial resistance.Table ISummary of times to half-maximal TER for Gαi2-, Q205Lαi2-, and PC-MDCK cellsTER MaxT50Ω·cm2hGαi25250.8 ± 0.3Gαi2 + AlF4−6000.5 ± 0.4Q205Lαi29751.2 ± 0.3Q205Lαi2 + AlF4−9751.1 ± 0.5PC4503.0 ± 0.5PC + AlF4−7001.1 ± 0.7Ca+2 switch TER data from 0–12 h for each cell line was analyzed by nonlinear regression using GraphPad Prism (San Diego, CA). TER data was fit to a hyperbola TER Max×TT50+T=Yfor each transfected cell line in the absence (n = 12) and presence of AlF4−(n = 6; added in the low calcium medium and continued after the switch). TER Max is the line defined by the maximal TER obtained between 6–12 h and was held constant for calculation of the time to one-half maximal TER (T50). The differences in T50 between PC-MDCK cells and Gαi2- and Q205Lαi2-MDCK cells were significant (p < 0.02), and the difference with or without AlF4− was significant only for PC cells (p < 0.05). Open table in a new tab Ca+2 switch TER data from 0–12 h for each cell line was analyzed by nonlinear regression using GraphPad Prism (San Diego, CA). TER data was fit to a hyperbola TER Max×TT50+T=Yfor each transfected cell line in the absence (n = 12) and presence of AlF4−(n = 6; added in the low calcium medium and continued after the switch). TER Max is the line defined by the maximal TER obtained between 6–12 h and was held constant for calculation of the time to one-half maximal TER (T50). The differences in T50 between PC-MDCK cells and Gαi2- and Q205Lαi2-MDCK cells were significant (p < 0.02), and the difference with or without AlF4− was significant only for PC cells (p < 0.05). Taken together, these studies offer direct evidence that Gαi2 is a critical regulator of tight junction biogenesis and affects base-line characteristics of the tight junction. The protein composition of the TJ is complex with one integral membrane protein identified so far (occludin), several peripherally attached proteins with partially defined functions (including ZO-1, -2, and -3) and a variety of signal transduction molecules including PKC isoforms, Gα subunits, and tyrosine kinases (see Ref. 5Denker B.M. Nigam S.K. Am. J. Physiol. 1998; 274: F1-F9Crossref PubMed Google Scholar for review). How these diverse proteins function to maintain and regulate the development of tight junctions is not well understood. G proteins could be activated within the TJ through a classical seven-transmembrane receptor (although none yet identified in the TJ) or alternatively through a modulatory protein that promotes GDP release or slows GTP hydrolysis. Additional transmembrane proteins must exist within the TJ (27Saitou M. Fujimoto K. Doi Y. Itoh M. Fujimoto T. Furuse M. Takano H. Noda T. Tsukita S. J. Cell Biol. 1998; 141: 397-408Crossref PubMed Scopus (475) Google Scholar), and there are multiple examples of modulatory proteins that affect G protein function. GTPase activating proteins (RGS proteins;regulators of G protein signaling; reviewed in Ref. 28Berman D.M. Gilman A.G. J. Biol. Chem. 1998; 273: 1269-1272Abstract Full Text Full Text PDF PubMed Scopus (446) Google Scholar) interact with Gα subunits, and nucleotide exchange factors that promote GDP release have been described for many small G proteins such as Ras (29Shou C. Farnsworth C.L. Neel B.G. Feig L.A. Nature. 1992; 358: 351-354Crossref PubMed Scopus (289) Google Scholar). Although analogous proteins for Gα subunits have not yet been identified, such proteins may exist and could provide mechanisms for activation of Gα subunits in the TJ or within intracellular compartments (30Denker S.P. McCaffery J.M. Palade G.E. Insel P.A. Farquhar M.G. J. Cell Biol. 1996; 133: 1027-1040Crossref PubMed Scopus (117) Google Scholar). Our findings that Gαi2 is important for both the maintenance and development of the TJ does not exclude roles for other Gα subunits, and in fact the effects of AlF4− on the steady state TER suggests that other Gα subunits are likely to enhance this barrier.

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