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• W2035973222 abstract "The gap junction-forming connexin (Cx) 50 is truncated gradually during lens development. Premature cleavage of lens connexins is thought to be associated with cataract formation. We have shown previously that Cx50 is likely to be cleaved by caspase-3 like protease during chick lens development. Here, using HPLC-electrospray tandem mass spectrometry, we mapped two cleavage sites at the C terminus of Cx50 after Glu-368 and Asp-379 and identified caspase-3 and caspase-1 as the responsible proteases, respectively. The activity of caspase-1, like caspase-3, was detected in the outer cortex increased during lens development, which coincided with the accumulation of the truncated fragments of Cx50 in the core region of the lens. The truncated Cx50 fragments present in older lenses were reproduced in the younger lens after treatment with UV radiation; this cleavage could be partially blocked by caspase-1/3-specific inhibitors. Interestingly, as compared with full-length Cx50, caspase-truncated Cx50 showed a dramatic decrease in gap junction coupling and a loss of hemichannel function. Furthermore, expression of caspase-truncated Cx50 fragments increased cell viability against UV radiation as compared with full-length Cx50. Together, these results suggest that both caspase-1 and -3 are responsible for the cleavage at the C terminus of Cx50 during lens development. The reduction of gap junction coupling and closure of hemichannels formed by truncated Cx50 are likely to adaptively protect cells against elevated oxidative stress associated with lens aging. The gap junction-forming connexin (Cx) 50 is truncated gradually during lens development. Premature cleavage of lens connexins is thought to be associated with cataract formation. We have shown previously that Cx50 is likely to be cleaved by caspase-3 like protease during chick lens development. Here, using HPLC-electrospray tandem mass spectrometry, we mapped two cleavage sites at the C terminus of Cx50 after Glu-368 and Asp-379 and identified caspase-3 and caspase-1 as the responsible proteases, respectively. The activity of caspase-1, like caspase-3, was detected in the outer cortex increased during lens development, which coincided with the accumulation of the truncated fragments of Cx50 in the core region of the lens. The truncated Cx50 fragments present in older lenses were reproduced in the younger lens after treatment with UV radiation; this cleavage could be partially blocked by caspase-1/3-specific inhibitors. Interestingly, as compared with full-length Cx50, caspase-truncated Cx50 showed a dramatic decrease in gap junction coupling and a loss of hemichannel function. Furthermore, expression of caspase-truncated Cx50 fragments increased cell viability against UV radiation as compared with full-length Cx50. Together, these results suggest that both caspase-1 and -3 are responsible for the cleavage at the C terminus of Cx50 during lens development. The reduction of gap junction coupling and closure of hemichannels formed by truncated Cx50 are likely to adaptively protect cells against elevated oxidative stress associated with lens aging. The primary function of the lens, an avascular organ, is to transmit light and focus it on the retina. The lens is composed of an anterior epithelial cell layer and fiber cells that form the bulk of the organ. Epithelial cells differentiate into fiber cells, accompanied by cell elongation and organelle loss; this differentiation process continues throughout the lifespan of the organism. Lens fiber cells do not turnover throughout the lifespan of the animal and rely on a large network of intercellular gap junction communications between fiber cells and cells at the lens surface for the exchange of metabolites and ions (1Cooper K. Mathias R.T. Rae J.L. Peracchia C. Biophysics of Gap Junction Channels. CRC Press, Boca Raton, FL1991: 57-64Google Scholar). Gap junctions are clusters of transmembrane channels between two adjacent cells that allow the passage of small molecules (molecular mass = 1 kDa), such as ions, metabolites, and second massagers (2Goodenough D.A. Goliger J.A. Paul D.L. Connexins, connexons, and intercellular communication.Annu. Rev. Biochem. 1996; 65: 475-502Crossref PubMed Scopus (1077) Google Scholar). The structural units of gap junction channels are a family of proteins called connexins, which contains >20 members expressed in an overlapping, tissue-dependent pattern (3Meşe G. Richard G. White T.W. Gap junctions: Basic structure and function.J. Invest. Dermatol. 2007; 127: 2516-2524Abstract Full Text Full Text PDF PubMed Scopus (304) Google Scholar). Each connexin has four conserved transmembrane domains and two extracellular loops in which their cytoplasmic regions are variable, especially the C-terminal domain. Connexins expressed in most of the other tissues are highly dynamic proteins that undergo rapid turnover both in cell lines and in animal organs; their degradation has been shown to involve either the lysosome or the ubiquitin-proteasome pathway (4Musil L.S. Le A.C. VanSlyke J.K. Roberts L.M. Regulation of connexin degradation as a mechanism to increase gap junction assembly and function.J. Biol. Chem. 2000; 275: 25207-25215Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar, 5Beardslee M.A. Laing J.G. Beyer E.C. Saffitz J.E. Rapid turnover of connexin43 in the adult rat heart.Circ. Res. 1998; 83: 629-635Crossref PubMed Scopus (367) Google Scholar, 6Kjenseth A. Fykerud T. Rivedal E. Leithe E. Regulation of gap junction intercellular communication by the ubiquitin system.Cell Signal. 2010; 22: 1267-1273Crossref PubMed Scopus (65) Google Scholar, 7Laing J.G. Tadros P.N. Westphale E.M. Beyer E.C. Degradation of connexin43 gap junctions involves both the proteasome and the lysosome.Exp. Cell Res. 1997; 236: 482-492Crossref PubMed Scopus (215) Google Scholar). In addition, the C-terminal domain of connexin 50 (Cx50) 2The abbreviations used are: Cx50connexin 50LYLucifer yellowRDrhodamine dextranPIpropidium iodideCEFchicken embryonic fibroblast. contains several predicted protease consensus sequences (8Lin J.S. Fitzgerald S. Dong Y. Knight C. Donaldson P. Kistler J. Processing of the gap junction protein connexin50 in the ocular lens is accomplished by calpain.Eur. J. Cell Biol. 1997; 73: 141-149PubMed Google Scholar, 9Yin X. Gu S. Jiang J.X. The development-associated cleavage of lens connexin 45.6 by caspase-3-like protease is regulated by casein kinase II-mediated phosphorylation.J. Biol. Chem. 2001; 276: 34567-34572Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). connexin 50 Lucifer yellow rhodamine dextran propidium iodide chicken embryonic fibroblast. Three connexins, Cx43, Cx46, and Cx50, have been identified in the vertebrate lens. Cx43 is mainly expressed in anterior epithelial cells and the lens bow region. Cx43 is down-regulated and replaced by the other two lens fiber connexins, Cx50 and Cx46, during differentiation of epithelial cells to new lens fibers (10Gong X. Cheng C. Xia C. Connexins in lens development and cataractogenesis.J. Membr. Biol. 2007; 218: 9-12Crossref PubMed Scopus (75) Google Scholar). Cx50 plays an important role in maintenance of lens transparency and in normal lens development and epithelial fiber differentiation (11White T.W. Goodenough D.A. Paul D.L. Targeted ablation of connexin50 in mice results in microphthalmia and zonular pulverulent cataracts.J. Cell Biol. 1998; 143: 815-825Crossref PubMed Scopus (295) Google Scholar, 12Rong P. Wang X. Niesman I. Wu Y. Benedetti L.E. Dunia I. Levy E. Gong X. Disruption of Gja8 (α8 connexin) in mice leads to microphthalmia associated with retardation of lens growth and lens fiber maturation.Development. 2002; 129: 167-174Crossref PubMed Google Scholar, 13Gu S. Yu X.S. Yin X. Jiang J.X. Stimulation of lens cell differentiation by gap junction protein connexin 45.6.Invest. Ophthalmol. Vis. Sci. 2003; 44: 2103-2111Crossref PubMed Scopus (44) Google Scholar, 14Sellitto C. Li L. White T.W. Connexin50 is essential for normal postnatal lens cell proliferation.Invest. Ophthal. Vis. Sci. 2004; 45: 3196-3202Crossref PubMed Scopus (61) Google Scholar). Mutations in the Cx50 gene are correlated with the formation of cataracts. Cx50-deficient mice not only develop cataracts but also exhibit microphthalmia (11White T.W. Goodenough D.A. Paul D.L. Targeted ablation of connexin50 in mice results in microphthalmia and zonular pulverulent cataracts.J. Cell Biol. 1998; 143: 815-825Crossref PubMed Scopus (295) Google Scholar, 12Rong P. Wang X. Niesman I. Wu Y. Benedetti L.E. Dunia I. Levy E. Gong X. Disruption of Gja8 (α8 connexin) in mice leads to microphthalmia associated with retardation of lens growth and lens fiber maturation.Development. 2002; 129: 167-174Crossref PubMed Google Scholar, 15Jiang J.X. Gap junctions or hemichannel-dependent and -independent roles of connexins in cataractogenesis and lens development.Curr. Mol. Med. 2010; 10: 851-863Crossref PubMed Scopus (47) Google Scholar). The cytoplasmic C-terminal domain of Cx50 is involved in gating and permeability of gap junction channels (16Xu X. Berthoud V.M. Beyer E.C. Ebihara L. Functional role of the carboxyl terminal domain of human connexin 50 in gap junctional channels.J. Membr. Biol. 2002; 186: 101-112Crossref PubMed Scopus (42) Google Scholar, 17DeRosa A.M. Mui R. Srinivas M. White T.W. Functional characterization of a naturally occurring Cx50 truncation.Invest. Ophthalmol. Vis. Sci. 2006; 47: 4474-4481Crossref PubMed Scopus (40) Google Scholar). In contrast to connexins in other organs, lens fiber connexins, like lens fibers, also do not turnover during the lifespan of the lens. Cx50 and Cx46 proteins are truncated gradually during lens development. Truncated connexin fragments accumulate in the center core regions of the lens (9Yin X. Gu S. Jiang J.X. The development-associated cleavage of lens connexin 45.6 by caspase-3-like protease is regulated by casein kinase II-mediated phosphorylation.J. Biol. Chem. 2001; 276: 34567-34572Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 18Wang Z. Schey K.L. Phosphorylation and truncation sites of bovine lens connexin 46 and connexin 50.Exp. Eye Res. 2009; 89: 898-904Crossref PubMed Scopus (36) Google Scholar). Cleavage of the C-terminal domain of Cx50 has been reported to result in a dramatic decrease of junctional conductance (17DeRosa A.M. Mui R. Srinivas M. White T.W. Functional characterization of a naturally occurring Cx50 truncation.Invest. Ophthalmol. Vis. Sci. 2006; 47: 4474-4481Crossref PubMed Scopus (40) Google Scholar). In one report, truncation at the Cx50 C terminus was found to cause a loss of pH sensitivity (16Xu X. Berthoud V.M. Beyer E.C. Ebihara L. Functional role of the carboxyl terminal domain of human connexin 50 in gap junctional channels.J. Membr. Biol. 2002; 186: 101-112Crossref PubMed Scopus (42) Google Scholar), but the results of another study indicated that pH sensitivity only was reduced partially after C-terminal truncation of Cx50 (19Stergiopoulos K. Alvarado J.L. Mastroianni M. Ek-Vitorin J.F. Taffet S.M. Delmar M. Hetero-domain interactions as a mechanism for the regulation of connexin channels.Circ. Res. 1999; 84: 1144-1155Crossref PubMed Scopus (123) Google Scholar). However, until now, the biological function of truncation of Cx50 in the lens was largely unknown. Proteolysis plays very important roles in many aspects of lens development. It has been suggested that the loss of all cytoplasmic organelles during the differentiation of epithelial cells to lens fiber cells resembles apoptosis to a certain degree (20Bassnett S. Mataic D. Chromatin degradation in differentiating fiber cells of the eye lens.J. Cell Biol. 1997; 137: 37-49Crossref PubMed Scopus (160) Google Scholar). Transcripts for each executioner caspases, caspase-1, -3, -6, and -7, have been identified in lens fibers although endogenous proteolytic activity has only been reported for caspase-3 (21Zandy A.J. Lakhani S. Zheng T. Flavell R.A. Bassnett S. Role of the executioner caspases during lens development.J. Biol. Chem. 2005; 280: 30263-30272Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). Until now, the mechanistic role of caspase activation in the lens has not been elucidated clearly. Therefore, it is of interest to understand the molecular targets of caspases activated during lens development. The eye lens is constantly exposed to UV radiation, which produces reactive oxygen species (22Bergamini C.M. Gambetti S. Dondi A. Cervellati C. Oxygen, reactive oxygen species, and tissue damage.Curr. Pharm. Des. 2004; 10: 1611-1626Crossref PubMed Scopus (491) Google Scholar). The elevated oxidative stress that results from UV radiation has also been observed in older lens (23Berthoud V.M. Beyer E.C. Oxidative stress, lens gap junctions, and cataracts.Antioxid. Redox Signal. 2009; 11: 339-353Crossref PubMed Scopus (197) Google Scholar) and is considered to be one of the major factors leading to the development of age-dependent cataracts (24Ayala M.N. Michael R. Söderberg P.G. In vivo cataract after repeated exposure to ultraviolet radiation.Exp. Eye Res. 2000; 70: 451-456Crossref PubMed Scopus (26) Google Scholar, 25Zigman S. Datiles M. Torczynski E. Sunlight and human cataracts.Invest. Ophthalmol Vis. Sci. 1979; 18: 462-467PubMed Google Scholar). We have previously shown that Cx50 is a likely target of a caspase-3-like protease (9Yin X. Gu S. Jiang J.X. The development-associated cleavage of lens connexin 45.6 by caspase-3-like protease is regulated by casein kinase II-mediated phosphorylation.J. Biol. Chem. 2001; 276: 34567-34572Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). In this study, specific natural truncation sites of Cx50 were identified, and both caspase-1 and caspase-3 were determined as the major enzymes responsible for the cleavage of Cx50 in vivo. Furthermore, similar truncations were observed in younger lenses after exposure to UV radiation, along with increased activity of caspase-1/3. Importantly, as compared with full-length Cx50, we showed that truncation of Cx50 enhanced cell resistance against UV radiation. Fertilized, incubated white Leghorn chicken eggs were purchased from Ideal Poultry (Cameron, TX) and incubated in a humidified incubator. The QuikChange site-directed mutagenesis kit was purchased from Stratagene (La Jolla, CA), and the annexin V and propidium iodide (PI) staining kit from Clontech (Mountain View, CA). The anti-Cx50 antibody against the intracellular loop domain was generated as described previously (9Yin X. Gu S. Jiang J.X. The development-associated cleavage of lens connexin 45.6 by caspase-3-like protease is regulated by casein kinase II-mediated phosphorylation.J. Biol. Chem. 2001; 276: 34567-34572Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). The polyclonal anti-caspase-1 antibody was purchased from Invitrogen, and polyclonal anti-caspase-3 antibody was from Cell Signaling (Danvers, MA). Caspase-1- and caspase-3-specific inhibitors and a Caspase Colorimetric Assay kit were purchased from EMD Chemicals (Gibbstown, NJ). Fetal bovine serum (FBS) was from Hyclone laboratories (Logan, UT). Tissue culture reagents, Lucifer yellow (LY) and rhodamine dextran (RD) were from Invitrogen. Cell proliferation reagent WST-1 was purchased from Roche Applied Science (Mannheim, Germany). All other chemicals were purchased from Sigma or Fisher Scientific (Pittsburgh, PA). Three-month-old chick lenses were collected and rinsed three times in PBS. Membrane proteins were prepared following the procedure described previously (9Yin X. Gu S. Jiang J.X. The development-associated cleavage of lens connexin 45.6 by caspase-3-like protease is regulated by casein kinase II-mediated phosphorylation.J. Biol. Chem. 2001; 276: 34567-34572Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). Briefly, chicken lenses were lysed and pelleted at 100,000 × g for 30 min at 4 °C, and the crude membrane pellet was washed two times with prechilled 20 mm NaOH. Proteins were separated by one-dimensional SDS-PAGE and stained with Coomassie Blue. Bands of interest were excised, and the proteins were digested in situ with trypsin (Promega, modified). The digests were analyzed by capillary HPLC-electrospray ionization-MS/MS on a Thermo Fisher LTQ fitted with a New Objective PicoView 550 nanospray interface. Online HPLC separation was accomplished with an Eksigent NanoLC micro-HPLC: column, PicoFritTM (New Objective; 75-μm inner diameter) packed to 11 cm with C18 adsorbent (Vydac; 218MSB5, 5 μm, 300 Å); mobile phase A, 0.5% acetic acid (HAc)/0.005% trifluoroacetic acid (TFA); mobile phase B, 90% acetonitrile/0.5% HAc/0.005% TFA; gradient, 2% to 42% B in 60 min; flow rate, 0.4 μl/min. MS conditions were as follows: electrospray ionization voltage, 2.9 kV; isolation window for MS/MS, 3; relative collision energy, 35%; scan strategy, survey scan followed by acquisition of data-dependent collision-induced dissociation spectra of the seven most intense ions in the survey scan above a set threshold. Mascot (Matrix Science) was used to search the uninterpreted collision-induced dissociation spectra searched against the Swiss-Prot database. Methionine oxidation was considered as a variable modification, with semi-trypsin as the proteolytic enzyme specificity. The Mascot search results were imported into Scaffold (Proteome Software), and a second search was then conducted within Scaffold by X! Tandem. The assignments were compiled and further processed by PeptideProphet and ProteinProphet for determination of the probabilities of peptide assignments and protein identifications. The reported peptide assignments were all ≥ the peptide 95% confidence level. Retroviral constructs and high-titer RCAS(A) retroviruses were prepared based on the protocol described previously (26Jiang J.X. Bruzzone R. Giaume C. Connexin Methods and Protocols. Humana Press Inc., Totowa, NJ2000: 159-174Google Scholar). With the wild-type RCAS(A)-Cx50 DNA construct as a template, other Cx50 mutants and truncated Cx50 were generated by using QuikChange site-directed mutagenesis kit according to the manufacturer's instructions. The primers used were as follows: Cx50(D379A), 5′-CTGAACTTGCCACCGCGGTGAGATCCCTCA-3′ (sense) and 5′-TGAGGGATCTCACCGCGGTGGCAAGTTCAG-3′ (antisense); Cx50(E368A), 5′-TGAGCGATGAAGTGGCAGGGCCTTCAGCAC-3′ (sense) and 5′-GTGCTGAAGGCCCTGCCACTTCATCGCTCA-3′ (antisense); Cx50(D365A), 5′-ACGTGGTGAGCGCTGAAGTGGAAGG-3′ (sense) and 5′-CCTTCCACTTCAGCGCTCACCACCT-3′ (antisense); Cx50(368T), 5′-GATGAAGTGGAATGACCTTCAGCACCTG-3′ (sense) and 5′-CAGGTGCTGAAGGTCATTCCACTTCATC-3′ (antisense); and Cx50(379T), 5′-CTTGCCACCGATTGAAGATCCCTCAGCAG-3′ (sense) and 5′-CTGCTGAGGGATCTTCAATCGGTGGCAAG-3′ (antisense). With the Cx50(E368A) DNA construct as a template, the Cx50(E368A,D379A) mutant was generated. The primers used were as follows: 5′-CTGAACTTGCCACCGCGGTGAGATCCCTCA-3′ (sense) and 5′-TGAGGGATCTCACCGCGGTGGCAAGTTCAG-3′ (antisense). All constructs were sequenced at the University of Texas Health Science Center, San Antonio, Institutional DNA Sequencing Facility. The high-titer retroviruses (1–5 × 108 colony forming units/ml) containing wild-type and mutants were prepared as described previously (26Jiang J.X. Bruzzone R. Giaume C. Connexin Methods and Protocols. Humana Press Inc., Totowa, NJ2000: 159-174Google Scholar). Frozen sections prepared from postnatal day 1 chick lens were washed three times with PBS for 5 min each and were then incubated with blocking solution containing 2% normal goat serum, 2% fish skin gelatin, 0.5% Triton X-100, and 1% bovine serum albumin in PBS for 1 h at room temperature. Polyclonal anti-caspase-1 (1:200 dilution) or anti-caspase-3 (1:200 dilution) antibody in blocking solution was then added, and the mixture was incubated at 4 °C overnight. Sections were washed three times with PBS for 5 min each and then incubated with FITC-conjugated goat anti-rabbit IgG (1:750) for 2 h at room temperature. After three washes for 5 min each in PBS, sections were then incubated with DAPI (1:20,000 dilution) in PBS for 5 min at room temperature. Sections were then washed with PBS for 5 min and mounted on a coverslip with mounting medium. The specimens were analyzed using by fluorescence microscopy (Olympus Optical, Tokyo, Japan). CEF cells were seeded at 3 × 105 cells in 60-mm tissue culture plates in Dulbecco's modified Eagle's medium (DMEM) plus 10% fetal calf serum, 2% chick serum, and 5% sodium pyruvate. Cells were infected on the second day with high-titer retroviruses (>1 × 108 colony-forming units/μl, 15–20 μl/plate). After reaching confluence, CEF cells were digested with 0.05% trypsin and plated into 35- or 60-mm culture plates. To prepare for lens organ cultures, intact lenses were isolated from chick embryo at day 12 and washed with PBS three times. Chick lens was incubated in M199 medium containing 10% FBS and 1% penicillin/streptomycin at 37 °C, 5% CO2. GST fusion proteins containing a portion of the C-terminal region of Cx50 (F1, residues 307–389) and its corresponding mutant Cx50F1(D379A) were expressed in Escherichia coli and purified by binding to glutathione-coupled agarose beads according to published procedures (27Jiang J.X. White T.W. Goodenough D.A. Paul D.L. Molecular cloning and functional characterization of chick lens fiber connexin 45.6.Mol. Biol. Cell. 1994; 5: 363-373Crossref PubMed Scopus (80) Google Scholar). Briefly, embryonic day 12 chick lens or CEF cells infected with recombinant retroviruses were lysed in lysis buffer (5 mm Tris, pH 8.0, 5 mm EDTA/EGTA) plus 2 mm PMSF, 10 mm N-ethylmaleimide, and 100 μm leupeptin. The crude membrane extract was pelleted at 50,000 rpm for 30 min at 4 °C (Beckman TLA 100.3 rotor). The GST fusion protein (150–200 ng) or membrane extract was subjected to enzymatic digestion with caspase-1 or caspase-3 in the absence or presence of corresponding caspase-specific inhibitors (caspase-1 inhibitor, Ac-YVAD-CHO, 0.05 μg/μl; caspase-3 inhibitor, Ac-DEVD-CHO, 0.05 μg/μl). The cleavage reaction was performed in 20 μl of reaction buffer (50 mm Tris-HCl, pH 8.0, 0.5 mm EDTA, 0.5 mm sucrose, 0.1% Triton X-100, and 5% glycerol) with or without 50 units of caspase-1 or caspase-3 for different time periods. The reaction was terminated by the addition of 5x electrophoresis sample buffer (50 mm Tris, pH 6.8, 1% SDS, 2% β-mercaptoethanol, and 35% glycerol) and boiling for 5 min. Dephosphorylation of the crude membrane extract was performed according to a published protocol (27Jiang J.X. White T.W. Goodenough D.A. Paul D.L. Molecular cloning and functional characterization of chick lens fiber connexin 45.6.Mol. Biol. Cell. 1994; 5: 363-373Crossref PubMed Scopus (80) Google Scholar). Briefly, 100–500 μg of protein from the chick lens membrane extract was dissolved in 1× New England Biolab buffer 3 and incubated with 7.5 units of alkaline phosphatase at 37 °C for 1.5 h. The reaction was terminated by addition of 5x sample buffer and then boiling for 5 min. The activities of caspase-1 and caspase-3 were measured according to the protocol provided in the Caspase Colorimetric Assay kit (EMD Chemicals) with minor modifications. Briefly, supernatants of lysates prepared from CEF cells or chick lenses were incubated with caspase-1-specific substrate (Ac-YAVD-pNA) or caspase-3 specific substrate (Ac-DEVD-pNA) in reaction buffer at 37 °C for 6 h. Caspase activities were determined using a microtiter plate reader to assess absorbance at 405 nm. Values were normalized based on the total protein in each sample. The dye uptake assay was performed as described previously (28Cherian P.P. Siller-Jackson A.J. Gu S. Wang X. Bonewald L.F. Sprague E. Jiang J.X. Mechanical strain opens connexin 43 hemichannels in osteocytes: A novel mechanism for the release of prostaglandin.Mol. Biol. Cell. 2005; 16: 3100-3106Crossref PubMed Scopus (390) Google Scholar) with some modifications. Briefly, CEF cells expressing exogenous Cx50 or mutants generated by retroviral infection or RCAS(A) vehicle-infected control cells were grown at low cell density to minimize physical contact among the cells. LY (457 Da) was used as a tracer to determine hemichannel activity; RD (10 kDa) was employed as a negative control. Cells were washed with Ca2+-free minimum essential medium three times and then mechanically stimulated to induce the opening of hemichannels by dropping Ca2+-free MEM from a pipette at a fixed distance. Then, cells were incubated in the presence of 0.4% LY plus 0.4% RD for 5 min. Cells were washed with Hanks' balanced salt solution buffer and fixed in 2% PFA. Dye uptake was calculated as the percentage of fluorescent cells divided by the total number of cells. Scrape-loading dye transfer was performed according to the published procedure (29el-Fouly M.H. Trosko J.E. Chang C.C. Scrape-loading and dye transfer. A rapid and simple technique to study gap junctional intercellular communication.Exp. Cell Res. 1987; 168: 422-430Crossref PubMed Scopus (643) Google Scholar) with modifications. Briefly, CEF cells expressing exogenous Cx50 and its mutants were grown to confluence to maximize physical contact. Cells were washed five times with Hanks' balanced salt solution plus 1% BSA for 3 min each and then scraped lightly with a 26.5-gauge needle in the presence of 1% LY and 1% RD dissolved in PBS. After incubation for 30 min, cells were washed with Hanks' balanced salt solution five times, 3 min each time, and then cells were fixed in 2% PFA for 30 min. Dye transfer results were measured with a fluorescence microscope in which LY and RD were detected by using fluorescein and RD filters. Acquisition conditions were kept consistent for all measurements, and no threshold adjustments were applied. The extent of dye transfer was quantified by measuring the ratio of the LY transfer distance from the scrape lines to the RD transfer distance. CEF cells expressing Cx50 and mutants or RCAS(A) vehicle control as described above were seeded at 5 × 104 cells in a 96-well plate in DMEM plus 10% fetal calf serum, 2% chick serum, 5% sodium pyruvate and were cultured at 37 °C, 5% CO2 for 24 h. Cells were then treated with 200 mJ/cm2 UV radiation (254 nm, Fisher Biotech FBUV XL-1000 Microprocessor Controlled UV Crosslinker) and then cultured in 100 μl of fresh medium for another 18 h. Live cells were stained with WST-1 reagent according to the procedure provided by the manufacture. Briefly, 10 μl of WST-1 reagent was added to each well, and then cells were incubated for 4 h at 37 °C, 5% CO2. After vigorous shaking, a microplate reader was used to measure the absorbance of the samples at 450 nm immediately before UV treatment and 18 h after exposure. Values for cell viability were calculated as the percentage of absorbance at 18 h after UV exposure divided by the absorbance before treatment. CEF cells expressing Cx50 and mutants or RCAS(A) vehicle control were seeded at 8 × 105 cells in 60-mm dishes in DMEM plus 10% fetal calf serum, 2% chick serum, and 5% sodium pyruvate and cultured at 37 °C, 5% CO2 and grown to confluence. Cells were then treated with 200 mJ/cm2 UV radiation (254 nm, Fisher Biotech FBUV XL-1000 Microprocessor Controlled UV Cross-linker) and then cultured in 3 ml of fresh medium for another 18 h. Flow cytometry analysis was performed subsequently according to the protocol provided in the kit. Briefly, cells were trypsinized and gently washed once with serum-containing media, then rinsed with 1× binding buffer. Cells (0.1–1 × 106) were resuspended in 200 μl of 1× binding buffer. Five microliters of annexin V and 10 μl of PI were added, and then cells were incubated at room temperature for 10 min in the dark. Binding buffer was used to bring the reaction volume to 500 μl, and the cells were analyzed by flow cytometry (LSR-II, high-speed, multicolor, digital analyzer, BD Biosciences). The signal generated by annexin V was detected in the FITC signal channel, and the signal generated by PI was monitored by the detector for phycoerythrin emission. All data were analyzed using GraphPad Prism software (version 5.04, GraphPad Software, La Jolla, CA). A paired Student's t test was used for comparison between two groups, and one-way analysis of variance followed by a Student Newman Keul's test were used for comparison of more than two groups. Unless otherwise specified in the figure legends, the data are presented as the mean ± S.E. of at least three determinations. Asterisks indicate the degree of significant differences (*, p < 0.05; **, p < 0.01; ***, p < 0.001). To investigate the development-associated truncation of Cx50, membrane extracts isolated from chick lenses at embryonic days 8, 12, 15, and 18 and postnatal days 1 and 60 were immunoblotted with an anti-Cx50 antibody that recognizes the intracellular loop domain of Cx50. For optimal comparison of the truncated fragments of Cx50, we adjusted protein loading to make sure that the level of full-length Cx50 was comparable at different time points. Full-length Cx50 and its phosphorylated forms have molecular masses ∼55 kDa. A lower molecular mass fragment that migrated ∼49 kDa was detected, starting at embryonic day 8, gradually decreasing in quantity, whereas two smaller fragments ∼45 kDa appeared at embryonic day-12 becoming predominant since embryonic day 18 (Fig. 1§). These data suggest that truncation of Cx50 gradually increases concomitant with lens development. Based on the predicted sizes of these fragments, the cleavage sites are likely to be located at the intracellular C-terminal domain of Cx50. To locate the potential truncation sites of lens Cx50, mass spectrometry analyses were conducted of tryptic digests of Cx50 protein isolated from chick lenses. Based o" @default.
• W2035973222 created "2016-06-24" @default.
• W2035973222 creator A5000627709 @default.
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• W2035973222 date "2012-05-01" @default.
• W2035973222 modified "2023-09-29" @default.
• W2035973222 title "Developmental Truncations of Connexin 50 by Caspases Adaptively Regulate Gap Junctions/Hemichannels and Protect Lens Cells against Ultraviolet Radiation" @default.
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