High Glucose Down-regulates Intercellular Communication in Retinal Endothelial Cells by Enhancing Degradation of Connexin 43 by a Proteasome-dependent Mechanism

Abstract

Intercellular communication through gap junctions (GJIC) is most likely relevant to maintaining the integrity of the blood-retinal barrier. In this study, we investigated the mechanism whereby high glucose enhances degradation of connexin 43 (Cx43), thus contributing to a decrease in GJIC. The levels of Cx43 in bovine retinal endothelial cells exposed to high glucose (25 mm) decreased about 50% as compared with controls (5.5 mm glucose). Consistently, the half-life of the protein decreased from 2.3 to 1.9 h. The proteasome inhibitors MG132 and lactacystin prevented the loss of Cx43 induced by high glucose and extended Cx43 half-life. The amount of phosphorylated Cx43 increased in high glucose and after proteasome inhibition. Scrape-loading dye transfer experiments show that high glucose is associated to a decrease of 40% in GJIC. Significantly, this reduction can be reversed by proteasome inhibitors. The decrease in GJIC in cells exposed to high glucose is associated with a loss of Cx43 from the plasma membrane, as demonstrated by immunofluorescence and biotinylation of cell-surface proteins. Results indicate that increased phosphorylation of Cx43 under high glucose is the mechanism targeting Cx43 for degradation by a proteasome-dependent mechanism. Increased degradation of Cx43 and reduction of GJIC in high glucose may be of physiological importance by contributing to endothelial cell dysfunction associated with the breakdown of the blood-retinal barrier in diabetic retinopathy. Intercellular communication through gap junctions (GJIC) is most likely relevant to maintaining the integrity of the blood-retinal barrier. In this study, we investigated the mechanism whereby high glucose enhances degradation of connexin 43 (Cx43), thus contributing to a decrease in GJIC. The levels of Cx43 in bovine retinal endothelial cells exposed to high glucose (25 mm) decreased about 50% as compared with controls (5.5 mm glucose). Consistently, the half-life of the protein decreased from 2.3 to 1.9 h. The proteasome inhibitors MG132 and lactacystin prevented the loss of Cx43 induced by high glucose and extended Cx43 half-life. The amount of phosphorylated Cx43 increased in high glucose and after proteasome inhibition. Scrape-loading dye transfer experiments show that high glucose is associated to a decrease of 40% in GJIC. Significantly, this reduction can be reversed by proteasome inhibitors. The decrease in GJIC in cells exposed to high glucose is associated with a loss of Cx43 from the plasma membrane, as demonstrated by immunofluorescence and biotinylation of cell-surface proteins. Results indicate that increased phosphorylation of Cx43 under high glucose is the mechanism targeting Cx43 for degradation by a proteasome-dependent mechanism. Increased degradation of Cx43 and reduction of GJIC in high glucose may be of physiological importance by contributing to endothelial cell dysfunction associated with the breakdown of the blood-retinal barrier in diabetic retinopathy. Hyperglycemia and the consequent exposure of the intracellular milieu of the retinal capillary endothelial cells to elevated blood glucose concentrations have been implicated in the pathogenesis of vascular complications in diabetes, including the breakdown of the blood-retinal barrier (1Winegrad A.I. Diabetes. 1987; 36: 396-406Crossref PubMed Scopus (281) Google Scholar, 2King G.L. Kunisaki M. Nishio Y. Inoguchi T. Shiba T. Xia P. Diabetes. 1996; 45: S105-S108Crossref PubMed Google Scholar). The exact biochemical and molecular mechanisms that transduce chronic hyperglycemia into micro- and macrovascular complications of the retina are not clear. However, it has been suggested that several mechanisms play a role in the pathogenesis of diabetic retinopathy; those include increased nonenzymatic glycation (3Nishikawa T. Edelstein D. Du X.L. Yamagishi S. Matsumura T. Kaneda Y. Yorek M.A. Beebe D. Oates P.J. Hammes H.P. Giardino I. Brownlee M. Nature. 2000; 404: 787-790Crossref PubMed Scopus (3693) Google Scholar), activation of aldose reductase (4Obrosova I.G. Minchenko A.G. Vasupuram R. White L. Abatan O.I. Kumagai A.K. Frank R.N. Stevens M.J. Diabetes. 2003; 52: 864-871Crossref PubMed Scopus (178) Google Scholar), oxidative stress (3Nishikawa T. Edelstein D. Du X.L. Yamagishi S. Matsumura T. Kaneda Y. Yorek M.A. Beebe D. Oates P.J. Hammes H.P. Giardino I. Brownlee M. Nature. 2000; 404: 787-790Crossref PubMed Scopus (3693) Google Scholar), increased production of diacylglycerol, and stimulation of retinal protein kinase C (PKC) 1The abbreviations used are: PKC, protein kinase C; Cx43, connexin 43; LEC, lens epithelial cells; GJIC, intercellular communication through gap junctions; BREC, bovine retinal endothelial cells; DMEM, Dulbecco's modified Eagle's medium; TPA, 12-O-tetradecanoylphorbol 13-acetate; PBS, phosphate-buffered saline; PVDF, polyvinylidene fluoride; RT-PCR, reverse transcription-PCR; P/NP, phosphorylated/nonphosphorylated. (5Koya D. King G.L. Diabetes. 1998; 47: 859-866Crossref PubMed Scopus (1165) Google Scholar). Despite the nature of such mechanisms, early cell dysfunction related to diabetic vascular complications is often associated with abnormalities in cell-cell communication and maintenance of cell homeostasis. Gap junctions are intercellular-channels that permit the passage of small molecules such as small metabolites, ions, and second messengers (6Kumar N.M. Gilula N.B. Cell. 1996; 84: 381-388Abstract Full Text Full Text PDF PubMed Scopus (1652) Google Scholar). These channels consist of two hemi-channels, called connexons, that are located in the plasma membrane of two adjacent cells. Each connexon is composed of six subunits of the protein connexin. Gap junctions have an important role in a variety of cellular processes including homeostasis, morphogenesis, cell differentiation, and growth control (7Simon A.M. Goodenough D.A. Trends Cell. 1998; 8: 477-483Abstract Full Text Full Text PDF PubMed Scopus (347) Google Scholar). Three connexin subtypes are expressed in the endothelium of the blood vessels: Cx37, Cx40, and Cx43 (8Yeh H.I. Rothery S. Dupont E. Coppen S.R. Severs N.J. Circ. Res. 1998; 83: 1248-1263Crossref PubMed Scopus (155) Google Scholar). Endothelial cells rely on a network of gap junctions for intercellular communication (9Little T.L. Beyer E.C. Duling B.R. Am. J. Physiol. 1995; 268: H729-H739PubMed Google Scholar, 10Larson D.M. Wrobleski M.J. Sagar G.D. Westphale E.M. Beyer E.C. Am. J. Physiol. 1997; 272: C405-C415Crossref PubMed Google Scholar). Previous studies have shown that intercellular communication in endothelial cells is reduced under conditions of hyperglycemia (11Sato T. Haimovici R. Kao R. Li A.F. Roy S. Diabetes. 2002; 51: 1565-1571Crossref PubMed Scopus (131) Google Scholar). Such a decrease in intercellular communication is most likely due to a reduction of the abundance of connexin 43 (Cx43) in endothelial cells. One of the most remarkable aspects of gap junction biosynthesis is the exceptional metabolic lability of connexins. Although the half-life of the great majority of plasma membrane proteins exceeds 24 h, connexins present a rapid turnover rate, with half-lives of only 1.5–5 h (12Fallon R.F. Goodenough D.A. J. Cell Biol. 1981; 90: 521-526Crossref PubMed Scopus (225) Google Scholar, 13Beardslee M. Lerner D. Tadros P. Laing J. Beyer E. Yamada K. Kleber A. Schuessler R. Saffitz J. Circ. Res. 2000; 87: 656-662Crossref PubMed Scopus (444) Google Scholar, 14Musil L.S. Le A.C. Van Slyke J.K. Roberts L.M. J. Biol. Chem. 2000; 275: 25207-25215Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar). Until the present time, two proteolytic pathways have been implicated in connexin turnover, the lysosome (14Musil L.S. Le A.C. Van Slyke J.K. Roberts L.M. J. Biol. Chem. 2000; 275: 25207-25215Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar, 15Laing J.G. Tadros P.N. Westphale E.M. Beyer E.C. Exp. Cell Res. 1997; 236: 482-492Crossref PubMed Scopus (217) Google Scholar) and the proteasome (13Beardslee M. Lerner D. Tadros P. Laing J. Beyer E. Yamada K. Kleber A. Schuessler R. Saffitz J. Circ. Res. 2000; 87: 656-662Crossref PubMed Scopus (444) Google Scholar, 14Musil L.S. Le A.C. Van Slyke J.K. Roberts L.M. J. Biol. Chem. 2000; 275: 25207-25215Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar, 15Laing J.G. Tadros P.N. Westphale E.M. Beyer E.C. Exp. Cell Res. 1997; 236: 482-492Crossref PubMed Scopus (217) Google Scholar, 16Laing J.G. Beyer E.C. J. Biol. Chem. 1995; 270: 26399-26403Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar, 17Girao H. Pereira P. Mol. Vis. 2003; 9: 24-30PubMed Google Scholar). The mechanisms and upstream events that lead to the choice of one degradation pathway over the other are largely unknown. It has, however, been shown that both pathways coexist and are active in a number of cell types and in a variety of experimental and pathological conditions (18Fuertes G. Martin De Llano J.J. Villarroya A. Rivett A.J. Knecht E. Biochem. J. 2003; 375: 75-86Crossref PubMed Scopus (139) Google Scholar, 19Qin H. Shao Q. Igdoura S.A. Alaoui-Jamali M.A. Laird D.W. J. Biol. Chem. 2003; 278: 30005-30014Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar, 20Ward W.F. Prog. Mol. Subcell. Biol. 2002; 29: 35-42Crossref PubMed Scopus (67) Google Scholar). For example, we have previously shown that in response to specific stimuli, proteasome is in the degradation of phosphorylated Cx43 in lens epithelial cells H. Pereira P. Mol. Vis. 2003; 9: 24-30PubMed Google Scholar). It has been shown that phosphorylation of Cx43 in decreased intercellular communication through gap junctions of a reduction in abundance the plasma membrane D. H. R. J. Mol. PubMed Scopus Google Scholar, E. R. Biol. 1997; PubMed Scopus Google Scholar, E.M. A.F. J. Cell Biol. 2000; PubMed Scopus Google Scholar, E. H. PubMed Scopus Google Scholar). For example, it shown that in high glucose induced the of GJIC through of Cx43 by T. Inoguchi T. H. Diabetes. 1998; 47: PubMed Scopus Google Scholar). the other a has shown that high glucose GJIC by Cx43 in endothelial cells (11Sato T. Haimovici R. Kao R. Li A.F. Roy S. Diabetes. 2002; 51: 1565-1571Crossref PubMed Scopus (131) Google Scholar). have shown that Cx43 is and that the proteasome pathway is in the degradation of Cx43 in H. Pereira P. Mol. Vis. 2003; 9: 24-30PubMed Google Scholar). we have suggested that the of phosphorylation and protein degradation by the proteasome pathway as a mechanism of intercellular communication in the H. Pereira P. Mol. Vis. 2003; 9: 24-30PubMed Google Scholar). it is thus that a mechanism may in retinal endothelial cells. the mechanism whereby hyperglycemia to decreased intercellular communication in endothelial we have that hyperglycemia may the conditions that increased degradation of Cx43, increased phosphorylation of the reduction in Cx43 half-life and a decreased abundance of Cx43 the plasma membrane thus for the reduced intercellular communication under conditions of Cell the of used to cells for from a bovine retinal endothelial cells from the and in Dulbecco's modified Eagle's and of retinal epithelial cells The to an and and with for the retinal through and and the on of the in by The in with endothelial growth and and on in glucose with to For from to the of high glucose on Cx43 and GJIC in (5.5 mm) high (25 mm) for to and and from The a that of other from from 12-O-tetradecanoylphorbol and MG132 in and exposed to and in phosphate-buffered by and cell The cells in mm mm and with mm mm mm The six for to cells and for The used to the protein by and with For studies cell on of on an and to polyvinylidene with connexin The by and the of the with are of and metabolic with cells with to with for h. The cells in with mm in the of MG132 for h. The in mm mm mm with protein mm mm mm by and used for the with Cx43 for h by with protein for h. The and the protein in and for by and the and from in and high glucose by the and the reverse for of Cx43, and the and the reverse for of For of Cx43 and in the that in the of the on a and with and the of cell-surface proteins of of cells with of mm and mm by the of of the the and the with mm mm and mm The cells in mm mm mm and with mm mm mm and on the cells and the for The protein of the and the of protein to of h of with the with The in of and for h The and the proteins by to and Cx43 on with in The with PBS, with in PBS, and with for to with connexin for h The in and with in bovine in For For the in after in the of from on for gap intercellular as by (14Musil L.S. Le A.C. Van Slyke J.K. Roberts L.M. J. Biol. Chem. 2000; 275: 25207-25215Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar). the from cells and The cells with bovine after a used to through the cell in the of Dulbecco's phosphate-buffered and of the with bovine and for an in the to the dye to transfer to cells. The cells and with and a with a are expressed as with a to a in the of Cx43 by a has previously been shown that hyperglycemia to a decrease in Cx43 in cell types (11Sato T. Haimovici R. Kao R. Li A.F. Roy S. Diabetes. 2002; 51: 1565-1571Crossref PubMed Scopus (131) Google Scholar, T. Inoguchi T. H. Diabetes. 1998; 47: PubMed Scopus Google Scholar, M. P. De H. J. J. Biol. PubMed Scopus Google Scholar). the mechanisms whereby high glucose to decreased Cx43 in BREC, the amount of Cx43 by The levels of Cx43 decreased about 50% in cells exposed to mm glucose for as compared with controls in mm the proteasome to be in Cx43 degradation (13Beardslee M. Lerner D. Tadros P. Laing J. Beyer E. Yamada K. Kleber A. Schuessler R. Saffitz J. Circ. Res. 2000; 87: 656-662Crossref PubMed Scopus (444) Google Scholar, 14Musil L.S. Le A.C. Van Slyke J.K. Roberts L.M. J. Biol. Chem. 2000; 275: 25207-25215Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar, 15Laing J.G. Tadros P.N. Westphale E.M. Beyer E.C. Exp. Cell Res. 1997; 236: 482-492Crossref PubMed Scopus (217) Google Scholar, 16Laing J.G. Beyer E.C. J. Biol. Chem. 1995; 270: 26399-26403Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar, 17Girao H. Pereira P. Mol. Vis. 2003; 9: 24-30PubMed Google Scholar), we the of the proteasome on Cx43 degradation in high glucose by with the proteasome inhibitors MG132 and lactacystin not shown in indicate that proteasome inhibitors to a the loss of Cx43 induced by high In the levels of Cx43 in cells exposed to high glucose to about of the levels by proteasome inhibitors Cx43 by under experiments that the levels of for Cx43 are not after exposure of to high glucose not the decreased of Cx43 under high glucose are likely to be related to an increased turnover of the the half-life of Cx43, cells with for h and for h. The in that the half-life of Cx43 in cells exposed to high glucose decreased from 2.3 h, in control to 1.9 h. the increased turnover of Cx43 in high glucose associated with increased degradation of the protein by a proteasome-dependent proteins in cells with for h and with in the of the proteasome that Cx43 degradation under high glucose in prevented by the proteasome MG132 and by the specific lactacystin not The half-life of Cx43 in cells in high glucose increased from 1.9 h to h the proteasome The that proteasome inhibitors extended Cx43 half-life the half-life of the protein in controls that proteasome has a role in degradation of Cx43 and that high glucose may the conditions that increased degradation of the protein by a proteasome-dependent mechanism. Increased of Cx43 under with Increased of the glucose shown to phosphorylation of Cx43, in may protein degradation (11Sato T. Haimovici R. Kao R. Li A.F. Roy S. Diabetes. 2002; 51: 1565-1571Crossref PubMed Scopus (131) Google Scholar, T. Inoguchi T. H. Diabetes. 1998; 47: PubMed Scopus Google Scholar, M. P. De H. J. J. Biol. PubMed Scopus Google Scholar). The of high glucose on phosphorylation of Cx43 induced by by cells with an of The decrease in the levels of Cx43 in cells exposed to high glucose in by cells with and Consistently, with TPA, an of PKC, in a decrease of about in the of Cx43 These show that high glucose may PKC, in may Cx43, to increased Results in that the phosphorylated of Cx43 are by a mechanism that the proteasome H. Pereira P. Mol. Vis. 2003; 9: 24-30PubMed Google Scholar). proteasome is the mechanism to increased degradation of phosphorylated Cx43 under high Cx43 from of cells under high glucose in the of proteasome inhibitors. by and to that with the Cx43 The of Cx43 and in a The show that the Cx43 increased in cells under high glucose of the proteasome to a in the of Cx43, as compared with control cells under the other the of Cx43 not the to cells exposed to high These that phosphorylation of Cx43 in cells exposed to high glucose may as a for degradation of the protein by a proteasome-dependent of the proteasome inhibitors on phosphorylation of Cx43 from cell of with MG132 proteins by to and with phosphorylated of to Cx43 of Cell in the of from E. The of the by the by the of Cx43 and in a are from control in glucose as by to Intercellular by a of increased Cx43 degradation in high glucose by dye transfer The intercellular communication as the by the dye after The GJIC decreased about 40% in cells exposed to high glucose not proteasome inhibitors the in GJIC, to an of about in the by the dye However, under high proteasome inhibitors not only the GJIC to control increased intercellular communication as compared with controls under that high glucose has a in GJIC by a mechanism that increased degradation of Cx43 in a proteasome-dependent of BREC, in high glucose with the in an of GJIC of about the other and as with in a of GJIC. of GJIC the of of Cx43 the communication is associated with the abundance of gap junction cell-cell an of intercellular communication is associated with in gap junction with the levels of Cx43 the plasma membrane to gap the decrease of GJIC in high glucose is related to in the of Cx43, with Cx43 and by In control Cx43 both to the plasma membrane, to cell-cell and In cells exposed to high is a decrease in the abundance of Cx43 the plasma membrane The amount of Cx43 the plasma membrane as the the of adjacent cells The of the proteasome on Cx43 in high glucose by cells with the proteasome Results in show that proteasome reversed the induced by high with the amount of Cx43, the plasma membrane, to that in control cells. These indicate that of GJIC in high glucose is related to the amount of Cx43 to gap junctions the plasma the of Cx43 from plasma membrane through a proteasome-dependent mechanism and in an increased degradation of Cx43 in retinal capillary endothelial cells. The amount of Cx43 the plasma membrane by biotinylation of cell-surface proteins. The amount of Cx43 decreased in cells exposed to high as compared with control cells However, with MG132 in a in the amount of Cx43 in of exposed to high glucose a in hyperglycemia the conditions that targeting of membrane Cx43 to degradation by a proteasome-dependent mechanism. The in this study, with that phosphorylation of Cx43 is the targeting Cx43 for degradation by a proteasome-dependent mechanism. increased degradation of Cx43 in a decrease in the amount of Cx43 to gap junctions the plasma membrane, to a reduction in intercellular communication in endothelial cells. Previous studies demonstrated that high glucose a reduction of GJIC T. Inoguchi T. H. Diabetes. 1998; 47: PubMed Scopus Google Scholar, M. P. De H. J. J. Biol. PubMed Scopus Google Scholar, T. T. H. Biochem. Res. 1995; PubMed Scopus Google Scholar, P. Vis. 1997; Google Scholar). (11Sato T. Haimovici R. Kao R. Li A.F. Roy S. Diabetes. 2002; 51: 1565-1571Crossref PubMed Scopus (131) Google Scholar) that the decreased levels of Cx43 associated with a of Cx43 in response to Endothelial cells exposed to high glucose a decrease of about in Cx43 the amount of the protein decreased that Cx43 degradation be in hyperglycemia (11Sato T. Haimovici R. Kao R. Li A.F. Roy S. Diabetes. 2002; 51: 1565-1571Crossref PubMed Scopus (131) Google Scholar). in this show that the amount of Cx43 in decreased about 50% in high the levels of Cx43 not have shown previously H. Pereira P. Mol. Vis. 2003; 9: 24-30PubMed Google Scholar) in other cell types that in to the Cx43 abundance can be by a mechanism. we that the reduction of Cx43 induced by high glucose be associated to an in the turnover of the In of this is the that the half-life of Cx43 in exposed to high glucose decreased about the lysosome is the for degradation of However, it has been that proteasome is in degradation of Cx43 in a number of cell types (13Beardslee M. Lerner D. Tadros P. Laing J. Beyer E. Yamada K. Kleber A. Schuessler R. Saffitz J. Circ. Res. 2000; 87: 656-662Crossref PubMed Scopus (444) Google Scholar, 14Musil L.S. Le A.C. Van Slyke J.K. Roberts L.M. J. Biol. Chem. 2000; 275: 25207-25215Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar, 15Laing J.G. Tadros P.N. Westphale E.M. Beyer E.C. Exp. Cell Res. 1997; 236: 482-492Crossref PubMed Scopus (217) Google Scholar, 16Laing J.G. Beyer E.C. J. Biol. Chem. 1995; 270: 26399-26403Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar, 17Girao H. Pereira P. Mol. Vis. 2003; 9: 24-30PubMed Google Scholar). in this for the time, that the proteasome is in degradation of Cx43 in blood retinal endothelial cells in high Significantly, we have shown that degradation of Cx43 by a proteasome-dependent mechanism can be by protein phosphorylation and in decreased GJIC H. Pereira P. Mol. Vis. 2003; 9: 24-30PubMed Google Scholar). The for degradation of Cx43 is such that it has been a mechanism for intercellular communication (14Musil L.S. Le A.C. Van Slyke J.K. Roberts L.M. J. Biol. Chem. 2000; 275: 25207-25215Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar, 17Girao H. Pereira P. Mol. Vis. 2003; 9: 24-30PubMed Google Scholar). It has been shown that phosphorylation of Cx43 by including E.M. A.F. J. Cell Biol. 2000; PubMed Scopus Google Scholar, A. E. K. K. T. M. King G.L. 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H. Biochem. Res. 1995; PubMed Scopus Google Scholar), and cells T. Inoguchi T. H. Diabetes. 1998; 47: PubMed Scopus Google Scholar), it has been shown that high glucose reduced the intercellular Significantly, the decrease in intercellular communication induced by high in most can be reversed by inhibitors T. Inoguchi T. H. Diabetes. 1998; 47: PubMed Scopus Google Scholar, P. Vis. 1997; Google Scholar, T. M. M. T. T. H. PubMed Scopus Google Scholar), thus that phosphorylation is the mechanism whereby hyperglycemia intercellular in this show that high glucose GJIC and that such reduction the activation of most other the intercellular communication is not reversed by we an in increased phosphorylation of Cx43 by under high glucose to of Cx43 from the plasma membrane and increased degradation of Cx43, with the consequent reduction of GJIC. that reduction of GJIC is the of the decreased abundance of Cx43 to gap junction and not the of on the of gap we biotinylation of membrane proteins and cell by that high glucose to a reduction on the abundance of Cx43 gap junctions the plasma membrane and that such reduction can be prevented by inhibitors of the the in this a in of Cx43 in hyperglycemia the for Cx43 degradation by a proteasome-dependent mechanism. that of gap junction intercellular communication vascular endothelial cells is in the breakdown of the barrier in chronic hyperglycemia (11Sato T. Haimovici R. Kao R. Li A.F. Roy S. Diabetes. 2002; 51: 1565-1571Crossref PubMed Scopus (131) Google Scholar). Increased degradation of Cx43 and reduction of GJIC may thus to the endothelial cell dysfunction associated with breakdown of the blood-retinal barrier associated with diabetic retinopathy.