ZO-1 alters the plasma membrane localization and function of Cx43 in ...

Research Article

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ZO-1 alters the plasma membrane localization and function of Cx43 in osteoblastic cells James G. Laing*, Brian C. Chou and Thomas H. Steinberg Department of Internal Medicine, Washington University School of Medicine, St Louis, MO 63110, USA *Author for correspondence (e-mail: [email protected])

Accepted 15 February 2005 Journal of Cell Science 118, 2167-2176 Published by The Company of Biologists 2005 doi:10.1242/jcs.02329

Journal of Cell Science

Summary ZO-1 is the major connexin-interacting protein in ROS 17/2.8 (ROS) osteoblastic cells. We examined the role of ZO-1 in Cx43-mediated gap junction formation and function in ROS cells that expressed the connexininteracting fragment of ZO-1 (ROS/ZO-1dn) cells. Expression of this ZO-17-444 fusion protein in ROS cells disrupted the Cx43/ZO-1 interaction and decreased dye transfer by 85%, although Cx43 was retained on the plasma membrane as assessed by surface biotinylation. Fractionation of lysates derived from ROS/ZO-1dn cells on a 5-30% sucrose flotation gradient showed that 40% of the Cx43 floated into these sucrose gradients, whereas none of the Cx43 in ROS cell lysates entered the gradients, suggesting that more Cx43 is associated with lipid rafts in

the transfected ROS cells than in lysates derived from untransfected ROS cells. In contrast to the ROS/ZO-1dn cells, ROS cells that over-expressed ZO-1 protein (ROS/ZO-1myc cells) exhibited increased gap junctional permeability and appositional membrane staining for Cx43. These data demonstrate that ZO-1 regulates Cx43mediated gap junctional communication in osteoblastic cells and alters the membrane localization of Cx43. They suggest that ZO-1-mediated delivery of Cx43 from a lipid raft domain to gap junctional plaques may be an important regulatory step in gap junction formation. Key words: Connexin, Gap junction, ZO-1, Lipid rafts

Introduction Gap junctions allow the passage of low molecular mass molecules and ions between the cytoplasm of neighboring cells, and are formed by proteins called connexins. There are 21 different connexins in mammalian species, each of which forms gap junctions with distinct physiological characteristics (Willecke et al., 2002). Osteoblasts express both Cx43 and Cx45 and changes in the relative abundance of these gap junction proteins modulate the permeability of gap junctions in bone cells and alter the expression of osteogenic proteins (Koval et al., 1995; Stains et al., 2003; Lecanda et al., 1998). Deletion of the Cx43 gene in mice leads to delayed ossification and a generalized osteoblast dysfunction (Lecanda et al., 2000). Recent studies have linked a human genetic disorder called oculodentodigital dysplasia (ODDD), which is characterized by skeletal development defects, to mutations in Cx43 (Paznekas et al., 2003). Furthermore, gap junctional communication in osteoblasts is modulated by mechanical stress and soluble factors such as parathyroid hormone, which are important regulators of bone remodeling (Civitelli et al., 1998; Ziambaras et al., 1998). These changes may involve altering the amount of Cx43 in gap junctional plaques. ROS 17/2.8 is a rat osteoblastic cell line that expresses only Cx43 on the plasma membrane. We recently demonstrated that Cx45 and Cx43 associate with ZO-1 in Cx45-transfected ROS (ROS/Cx45) cells (Laing et al., 2001). ZO-1 is a member of the family of proteins called membrane-associated guanylate kinases, and it has been studied primarily in the context of tight junctions in epithelial cells and adherens junctions in non-

epithelial cells. ZO-1 has at least five different domains that mediate protein-protein interactions, including three PDZ domains, an SH3 domain, and a catalytically inactive guanylate kinase (GUK) domain. These domains mediate interactions between ZO-1 and other proteins in the tight junction (ZO-2, ZO-3, occludin, JAM and claudin), or the adherens junction (α-catenin and afadin) (Gonzalez-Mariscal et al., 2000; Yokoyama et al., 2001). Recent studies indicated that ZO-1 specifically binds to actin and cross-links the tight junction protein occludin to the actin cytoskeleton in epithelial cells (Fanning et al., 1998; Fanning et al., 2002). ZO-1 also interacts with several different gap junction proteins, including Cx43 (Toyofuku et al., 1998; Toyofuku et al., 2001; Giepmans and Moolenar, 1998), Cx45 (Laing et al., 2001; Kausalya et al., 2001), Cx31.9 (Nielsen et al., 2002), Cx36 (Li et al., 2004a), Cx46, and Cx50 (Nielsen et al., 2003) and Cx47 (Li et al., 2004b). A recent NMR-based study demonstrated that the last 20 amino acids in the carboxyl terminus of Cx43 interacts with the second PDZ domain within ZO-1 (Sorgen et al., 2004). Different laboratories have used different strategies to disrupt interaction between Cx43 and ZO-1. Studies in which cells were transfected with mutant connexin polypeptides that lack the carboxyl-terminal PDZ binding domain, or in which the carboxyl terminus is blocked with an added epitope, suggest gap junctions can form without an interaction with ZO-1 (Giepmans and Moolenar, 1998; Falk and Lauf, 2001; Jordan et al., 1999; Bukauskas et al., 2000; Windoffer et al., 2000; Hunter et al., 2003). However, these gap junction

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plaques are abnormally large and a large percentage of the channels in these plaques are inactive, suggesting that ZO-1 may play a role in gap junction function (Bukauskas et al., 2000; Hunter et al., 2003). A recent study identified a frameshift mutation in Cx43 that deletes the Cx43 ZO-1 binding site and is linked to ODDD (van Steensel et al., 2004). In contrast, expression of a connexin-binding fragment derived from ZO-1 disrupts gap junction formation and diminishes gap junction permeability in neonatal rat ventricular myocytes and transfected HEK cells (Toyofuku et al., 1998), through an unelucidated mechanism. In the current study, we altered the function of ZO-1 in ROS cells by stably transfecting these cells with a connexin-binding amino-terminal fragment of ZO-1 or the full length ZO-1. We assessed the importance of the ZO-1 interaction by determining if there were any changes in connexin abundance, localization and gap junctional communication in these transfected cells.


Materials and Methods Reagents and plasmid Polyclonal Cx43 antiserum was previously characterized (Laing et al., 1997). The monoclonal Cx43 antibody (mAb 3067) was from Chemicon (Temecula, CA, USA) and the polyclonal Cx43 antibody was from Sigma (St Louis, MO, USA). Monoclonal antibodies directed against ZO-1 and N-cadherin were from Zymed (South San Francisco, CA, USA), the monoclonal Xpress tag antibody was from Invitrogen (Carlsbad, CA, USA), and the caveolin-1 monoclonal antibody against caveolin-1 was from BD-Biosciences (San Diego, CA, USA). The 9E10 anti-myc monoclonal antibody was generated from the 9E10 hybridoma (ATCC). The ZO-1 cDNA was a generous gift from the Goodenough laboratory (Harvard University Medical School, Boston, USA). The construct encoding the ZO-17-444 fusion protein was generated by excising the nucleotides 421-1753 from the mouse ZO-1cDNA with NotI and XbaI, and ligating this DNA into the pcDNA3.1 His mammalian expression vector (Invitrogen, Carlsbad, CA, USA). The resulting fusion protein contained amino acids 7-444 of the ZO-1 polypeptide and an amino-terminal 6-His and Xpress tags. This protein is called the ZO-17-444 fusion protein throughout this manuscript. The pCBZO-1myc plasmid was a generous gift from Alan Fanning and James Anderson (University of North Carolina, Chapel Hill, USA) (Fanning et al., 1998). In vitro cell free peptide binding assay The peptide-binding assay was performed as previously published (Laing et al., 2001). Briefly, radioactively labeled ZO-17-444 fusion protein was generated by in vitro transcription/translation using TNT T7 Quick coupled in vitro translation/translation system according to the manufacturer’s instructions (Promega Corp., Madison WI, USA). Carboxyl-terminal peptides from Cx43 and Cx45 were synthesized previously (Laing et al., 2001). Peptides corresponding to last 10 amino acids in the carboxyl terminus of Cx46 (amino acid residues 408-416) with an added amino-terminal cysteine residue (CRARPGDLAI) and the last ten amino acids in Cx32 (amino acid residues 274-283) (AEKSDRCAEC) were synthesized by Invitrogen, and conjugated to a Sulfolink or an Amino Link resin (Pierce Biotechnology Inc., Rockford IL, USA). These affinity resins were incubated with equal aliquots of the in vitro translated [35S]methionine-labeled ZO-17-444 fusion protein, and washed extensively with RIPA buffer (1% Triton X-100 and 0.6% SDS in PBS). The bound material was eluted by boiling for 5 minutes in 15 µl of sample buffer (2% SDS, 10% glycerol, 5% β-mercaptoethanol,

0.1 M Tris-HCl pH 6.8) and analyzed by SDS-PAGE and fluorography. Cell culture and transfection ROS 17/2.8 is an osteosarcoma cell line that expresses Cx43 at the cell surface and forms functional Cx43 gap junctional channels (Steinberg et al., 1994). ROS cells were cultured in minimum Eagle’s medium containing 10% heat inactivated bovine calf serum containing 2 mM glutamine, 1 mM sodium pyruvate, 1% nonessential amino acids, 5 units/ml penicillin and 5 µg/ml streptomycin. ROS cells were transfected using Lipofectamine (Invitrogen, Carlsbad, CA, USA) with the plasmid encoding the ZO-17-444 fusion protein or the ZO1myc protein and stable clones were selected in G418 and isolated by limiting dilution. Stably transfected ROS cells were selected and cultured in the same culture medium, which contains 400 µg/ml G418. These transfectants are called ROS/ZO-1dn cells and ROS/ZO-1myc throughout this manuscript. Immunoprecipitation Co-immunoprecipitation studies were performed as published previously (Laing et al., 2001). Two 100-mm dishes of transfected ROS cells were washed, solubilized in immunoprecipitation (IP) buffer containing 1% Triton X-100, 0.5% CHAPS, 0.1% SDS and a 0.1% Sigma protease inhibitor cocktail in PBS, and lysates were precipitated at 4°C for 2 hours with our anti-Cx43 antibody. The antigen-antibody complexes were collected with protein A Sepharose and extensively washed with lysis buffer. Precipitated proteins were eluted by boiling in sample buffer, separated by SDS-PAGE, transferred to Immobilon-P membranes, and ZO-1 was identified by immunoblotting with 1:1000 dilution of monoclonal antibody directed against ZO-1 or the myc epitope. These blots were then probed with anti-mouse affinity purified peroxidase-conjugated secondary antibody (Jackson Immunoresearch, West Grove, PA, USA) and developed with the SuperSignal West Pico chemiluminescence system (Pierce Biotechnology Inc.). ZO-17-444 fusion protein-associated proteins were isolated from transfected ROS cells by affinity chromatography. Transfected ROS cells were lysed in lysis buffer [50 mM Tris pH 8, 5 mM MgCl2, 1% Triton X-100, 60 mM n-octyl D-glucoside and 1% Sigma protease inhibitor cocktail (v/v)]. Lysates were clarified by centrifugation and fractionated with 50 µl of a cobalt chelate affinity resin (Pierce Biotechnology Inc.), a resin that binds to 6-His modified proteins. The resins were then washed 4 times with 1 ml of lysis buffer, boiled for 5 minutes in 15 µl sample buffer and the eluted material was analyzed by immunoblotting with Cx43 antiserum. These blots were then probed with anti-rabbit affinity purified peroxidaseconjugated secondary antibody (Jackson Immunoresearch, West Grove, PA, USA) and developed with the SuperSignal West Pico chemiluminescence system (Pierce Biotechnology Inc.). Lucifer Yellow dye transfer Gap junction permeability was assessed by microinjecting Lucifer Yellow into single cells in adherent monolayers and counting the number of neighboring cells that received dye in 3 minutes using previously published techniques (Laing et al., 1994; Koval et al., 1995). Data was analyzed with the Student’s t-test. Immunoblotting Cells were scraped in PBS containing 1% SDS. The samples were sonicated and the DNA was sheared by passing the lysate through a tuberculin syringe. Protein concentrations were determined with the BCA protein assay. Approximately 10 µg of protein from each of these cells were separated by SDS-PAGE and analyzed by

ZO-1 regulates gap junction formation immunoblotting with our antibody directed against Cx43 that we characterized previously (Laing et al., 1997). Aliquots of SDS extracts were also analyzed by immunoblotting with antibody directed against ZO-1 (Zymed Laboratories) and N-cadherin (Zymed Laboratories).


Biotinylation of cell surface proteins Confluent monolayers of cells from two 100 mm plates were incubated in PBS containing 100 µg/ml EZ-link NHS-LC-Biotin (Pierce Biotechnology Inc.) at 4°C for 1 hour, and then the excess biotin was quenched by adding 5 ml 0.1 M glycine/PBS. The cells were washed 6 times in ice cold PBS, harvested by scraping and solubilized in IP buffer. Biotinylated proteins were isolated by fractionating these lysates with Neutravidin-agarose (Pierce Biotechnology Inc.), eluted by boiling for 5 minutes in 15 µl sample buffer and analyzed by SDS-PAGE and immunoblotting with our Cx43 antibodies. The blot was stripped with Restore western blot stripping buffer (Pierce Biotechnology Inc.) and reprobed with an anti-actin antibody (1:1000 dilution). Ten micrograms of ROS cell extracts were run as a positive control for Cx43 and actin expression. Immunofluorescence microscopy Transfected ROS cells were processed for immunofluorescence microscopy as described previously (Laing et al., 2001). The cells were fixed in a 1:1 solution of methanol and acetone for 2 minutes at room temperature, permeabilized with 1% Triton X-100, and incubated in monoclonal antibody against ZO-1, Cx43, N-cadherin, the myc or the Xpress tag epitope and the appropriate secondary antibodies. We also examined Cx43 expression in the transfected ROS cells with two different polyclonal antibodies directed against Cx43 that we have used previously (Laing et al., 1997; Koval et al., 1995) or a commercially available antibody directed against the carboxylterminal 20 amino acids of Cx43 (Sigma-Aldrich, St Louis, MO, USA). In co-localization experiments the cells were stained simultaneously with the Sigma Cx43 polyclonal antibody (1:1000 dilution) and the Zymed ZO-1 monoclonal antibody (1:1000 dilution) or N-cadherin monoclonal antibody and subsequently with Cy3conjugated goat anti-mouse IgG antibodies and Alexa 488-conjugated goat anti-rabbit IgG antibodies.

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SDS-PAGE and fluorography (Fig. 1). The ZO-17-444 fusion protein bound to carboxyl-terminal peptides of Cx43, Cx45 and Cx46, but not Cx32. We transfected ROS cells with a plasmid bearing the cDNA for the ZO-17-444 polypeptide, selected stable G418-resistant transfectants and assessed the expression of the fusion protein by immunoblotting and immunofluorescence. Immunoblots with the Xpress epitope tag antibody produced a 54 kDa band in the transfected ROS cell lysates (Fig. 2A). Three of the clones (clones E, J and K) expressed large quantities of the ZO17-444 fusion protein, while one clone expressed less of the fusion protein (clone A). We then examined the expression of this fusion protein by immunofluorescence. In non-transfected cells the Xpress tag antibody produced minimal staining (Fig. 2B, left panel). In contrast, in the high expressing ROS/ZO1dn clone E, the ZO-17-444 fusion protein was seen in a diffuse

Fig. 1. The ZO-17-444 fusion protein interacts with the carboxyl termini of Cx43, Cx45 and Cx46, but not Cx32. The ZO-17-444 fusion protein was fractionated on agarose beads conjugated to the carboxyl termini of Cx43, Cx45, Cx46 or Cx32. The resins were washed and the bound material was analyzed by SDS-PAGE and fluorography. The position of molecular mass markers is given in kDa.

Sucrose gradient fractionation of ROS cell lysates Adherent cells from two 100-mm diameter plates were washed three times in cold PBS, scraped into 750 µl of MES-buffered saline (MBS, 25 mM MES pH 6.5, 150 mM NaCl) containing 1% Triton X-100, and disrupted in a tight-fitting Dounce homogenizer five times. The sample was mixed with an equal volume of 80% sucrose in MBS without Triton X-100, transferred to a 5 ml ultracentrifuge tube, and overlaid with 1.5 ml of 30% sucrose and 1.5 ml of 5% sucrose in MBS lacking Triton X-100. The samples were centrifuged for 18 hours at 200,000 g (44,000 rpm in a Sorval rotor, TH-660). Nine 500 µl fractions were collected and analyzed by SDS-PAGE and immunoblotting with antibodies directed against Cx43, ZO-1 or caveolin-1.

Results Expression of a ZO-17-444 fusion protein disrupts the interaction between Cx43 and ZO-1 in ROS cells The gap junction protein Cx43 interacts with the second PDZ domain within ZO-1. We made a fusion protein containing the Cx43 binding domain in ZO-1 (ZO-17-444) and examined the ability of 35S-labeled fusion protein to interact with carboxyl termini of Cx32, Cx43, Cx45 and Cx46 in an in vitro binding assay. The material that bound to the peptides was analyzed by

Fig. 2. Expression of the ZO-17-444 fusion protein in transfected ROS cells. (A) ROS/ZO-1dn cells were harvested and analyzed by immunoblotting with the Xpress tag antibody. A 54 kDa immunoreactive band is seen in the transfected cells. The position of molecular mass markers is given in kDa. (B) Localization of the ZO17-444 fusion protein in ROS/ZO-1dn cells. ROS and ROS/ZO-1dn cells (clone E) were fixed, permeabilized, and stained with Xpress epitope tag antibody. Scale bar: 25 µm.


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cytosolic and nuclear staining pattern by immunofluorescence microscopy (Fig. 2B, right panel), indicating that this protein was not recruited to junctional membranes. Similar staining patterns were seen in ROS/ZO-1dn clones J and K (data not shown). We then investigated whether the ZO-17-444 fusion protein associated with Cx43 in ROS/ZO-1dn cells. Cell lysates were passed over a cobalt chelate affinity resin and the bound and eluted material was analyzed by immunoblotting with a Cx43 monoclonal antibody. Cx43 was isolated on the affinity resin from the lysates derived from transfected ROS cells expressing larger quantities of the ZO-17-444 fusion protein (ROS/ZO-1dn clones E, J and K) (Fig. 3A). Co-immunoprecipitation experiments were performed to determine whether expression of ZO-17-444 disrupted the interaction between Cx43 and ZO-1. Proteins immunoprecipitated with the Cx43 antibody were analyzed by immunoblotting with a monoclonal ZO-1 antibody. ZO-1 precipitated with Cx43 in ROS cells and the low expressor clone, but was not identified in Cx43 precipitates from cells expressing large quantities of the ZO-17-444 fusion protein (Fig. 3B). Taken together the data presented in Fig. 3 indicate that expression of the ZO-17-444 fusion protein disrupted the Cx43 ZO-1 interaction in ROS cells. In the rest of this study we have focused on the ROS/ZO-1dn clones that express large amounts of the ZO-17-444 fusion protein (clones E, J and K). Expression of the ZO-17-444 fusion protein reduces gap junctional communication in ROS/ZO-1dn transfectants Gap junctional communication was assessed in ROS and ROS/ZO-1dn transfectants by microinjecting single cells with

Fig. 3. Expression of the ZO-17-444 fusion protein disrupts the Cx43ZO-1 interaction. (A) Cx43 associates with the ZO-17-444 fusion protein in transfected cells. ROS/ZO-1dn cells were solubilized in lysis buffer and lysates were fractionated on a cobalt chelate affinity resin. Bound material was analyzed by immunoblotting with Cx43 antiserum. Cx43 was detected in precipitates from high expressor ROS/ZO-1dn clones E, J, and K, but not in ROS or low expressor ROS/ZO-1dn clone A cells. (B) ROS/ZO-1dn cells were solubilized, immunoprecipitated with Cx43 antiserum, run on SDS-PAGE, and immunoblotted with an anti-ZO-1 monoclonal antibody. ZO-1 coprecipitated with Cx43 in ROS and in the low expressor ROS/ZO1dn clone A cells, but not in high expressor ROS ZO-1dn clones E, J, or K. The position of molecular mass markers is given in kDa.

the dye Lucifer Yellow and counting the number of cells that received dye within 3 minutes. Dye transfer was reduced in three independent ROS/ZO-1dn clones: Lucifer Yellow spread to an average of 20.6 cells per microinjected ROS cell, and to 2.2-3.4 cells per microinjected ROS/ZO-1dn transfectants (Fig. 4). Student’s t-test analysis of this data showed that Lucifer Yellow transfer was reduced in all of the transfected cell lines (P