Downregulation of Connexin 43 Expression by High Glucose
Total Page:16
File Type:pdf, Size:1020Kb
Downregulation of Connexin 43 Expression by High Glucose Reduces Gap Junction Activity in Microvascular Endothelial Cells Tsuyoshi Sato, Robert Haimovici, Richard Kao, An-Fei Li, and Sayon Roy Impairment of retinal vascular homeostasis is associated with the development and progression of diabetic retinop- athy involving gap junction intercellular communication ap junctions are membrane channels that allow (GJIC) activity. The principal gap junction protein of transfer of ions and small molecules between intercellular communication, connexin, was investigated adjacent cells. Morphological studies using im- to determine the effects of high glucose concentrations on munostaining show that gap junctions are com- the expression of endothelial-specific connexins (Cx37, G posed of a family of proteins collectively called connexins Cx40, and Cx43), connexin phosphorylation pattern, and GJIC activity. Rat microvascular endothelial (RME) cells (1). In most tissues, including retina, adjacent cells com- grown in high (30 mmol/l)-glucose medium for 9 days had municate through gap junctions involving connexins. In ,reduced Cx43 expression: Cx43 mRNA (68 ؎ 13% of the retina, connexin 43 (Cx43) is abundantly present and protein (55.6 ؎ 16% of which suggests a substantial amount of gap junctional (5 ؍ n ,0.019 ؍ control; P -levels were reduced; however, coupling in the glial syncytium (2,3). In particular, endo (5 ؍ n ,0.003 ؍ control; P Cx37 and Cx40 expression was not affected. Using alka- thelial cells and pericytes contain gap junctions and ex- line phosphatase and Western blot analyses, we identified hibit junctional transfer both in vitro (4) and in vivo (5). three forms of Cx43: a nonphosphorylated form (P0) and Morphometric analysis using electron microscopy of hu- two phosphorylated forms (P1 and P2). Expression of all three forms was decreased in cells grown in high-glucose man retinal capillaries has shown that the basement -P1, 57 ؎ 16% membrane interposed between endothelial cell and peri ;(0.04 ؍ medium: PO, 73 ؎ 15% of control (P cyte is sufficiently thin to permit cell membrane contacts ؍ and P2, 42 ؎ 22% of control (P ;(0.01 ؍ of control (P 0.006). Using immunofluorescence microscopy, we ob- between these cells (6). Junctional transfer of molecules served Cx43 localization at specific sites of contact involving endothelin-1 between microvascular endothelial (plaques) between adjacent cells. In cells grown in high- cells and pericytes has been reported to mediate endothe- glucose medium, we observed reduced plaque counts lial cell–dependent proliferation of pericytes (7). There- ؍ ؎ (63 6% of control; P 0.009) and decreased intensity of fore, changes in gap junction intercellular communication Cx43 immunofluorescence compared with cells grown in normal medium. Furthermore, using scrape load dye (GJIC) activity may disrupt homeostasis between retinal transfer (SLDT) technique, we found that these cells vascular cells, leading to alterations in the blood-retinal .(barrier (BRB ,0.01 ؍ exhibited reduced GJIC activity (60% of control; P The reduction in GJIC activity correlated with the Chronic hyperglycemia in diabetes is associated with .(5 ؍ n These results the development and progression of pathological changes .(0.9 ؍ decreased Cx43 protein levels (r indicate that high glucose concentrations inhibited GJIC in the retinal vasculature involving the breakdown of the activity by reducing Cx43 synthesis in RME cells. Impaired BRB. The endothelium forms the BRB in the retinal intercellular communication may contribute to break- capillary vessels, and its permeability is largely regulated down of homeostatic balance in diabetic microangiopathy. Diabetes 51:1565–1571, 2002 by intercellular junctions (8,9). High-glucose conditions have been shown to reduce GJIC in RPE cells, which form the outer BRB (10). In Cx43-knockout mice, characteristic junctional contacts and the close apposition between lens epithelial cells are lost, leading to disturbed cellular orga- nization and loss of structural integrity (11). These find- ings indicate that impaired GJIC among retinal vascular cells under high-glucose conditions may lead to structural failure and BRB breakdown. Vascular endothelial cells express three types of con- From the Department of Ophthalmology, Boston University School of Medi- nexin, Cx37, Cx40, and Cx43 (12), whose distribution cine, Boston, Massachusetts. within the vasculature (13) varies widely with the species Address correspondence and reprint requests to Sayon Roy, Department of Ophthalmology, Boston University School of Medicine, 715 Albany St., Bos- studied. Two forms of cellular Cx43 have been reported, ton, MA 02118. E-mail: [email protected]. the nonphosphorylated and the phosphorylated high- Received for publication 28 November 2001 and accepted in revised form 18 molecular-weight form. However, the functional signifi- January 2002. AP, alkaline phosphatase; BRB, blood-retinal barrier; Cx, connexin; GJIC, cance of these forms of Cx43 in GJIC activity is unclear. gap junction intercellular communication; SLDT, scrape-loading dye transfer. The nonphosphorylated Cx43 is believed to mediate GJIC DIABETES, VOL. 51, MAY 2002 1565 GLUCOSE DOWNREGULATION OF CONNEXIN 43 activity, whereas the phosphorylated Cx43 blocks such between adjacent cells within a different and random area of 27 ϫ 27 m. activity (14). Recent studies have shown that protein Counts obtained from cells hybridized without primary antibody were used as background and subtracted from counts obtained from cells grown in normal kinase C–mediated excessive phosphorylation of Cx43 or high-glucose medium. may contribute to the reduction of GJIC activity (15) in Detection and quantitation of relative levels of Cx43 mRNA aortic endothelial cells grown in high-glucose medium RT-PCR. In each experiment, RNA from cells grown in normal medium was (16). processed in parallel with RNA from cells grown in high-glucose medium. RT Although there is considerable information on expres- reactions were performed in 20 l volume, with 1 g RNA, 200 units of reverse sion of Cx37, Cx40, and Cx43 in endothelial cells from transcriptase, 2.5 mol/l random hexamers, 1 mmol/l of each dNTPs, 5 mmol/l MgCl2, PCR buffer, and RNase inhibitor for 10 min at room temperature large vessels of bovine, rat (17,18), pig (13), and human followed by 40 min at 42°C. At the end of RT, samples were heated to 95°C for (19), little is known about connexin expression or GJIC 5 min, cooled on ice, and treated with RNase H (1 unit) for 15 min at 37°C. PCR activity in microvascular endothelial cells. In this study, was designed to measure the level of Cx43 expression relative to the we investigated the effect of high glucose concentrations expression of an endogenous internal standard gene, -actin. To prevent on GJIC activity in microvascular endothelial cells and quantitative inaccuracies deriving from competitive effects and different efficiency and ranges of amplification of the two cDNAs, the Cx43 and -actin determined whether changes in GJIC activity are associ- cDNAs generated in the same RT reaction were amplified in separate tubes ated with connexin gene expression. containing increasing volumes of the RT reaction (1, 2, and 4 l) to document amplification in the linear region for each cDNA. The primers used to amplify Cx43 (5Ј-GAATCCTGCTCCTGG-3Ј and 5Ј-GATGCTGATGATGTAG-3Ј) and RESEARCH DESIGN AND METHODS -actin (5Ј-ATGGATGACGATATCGCT-3Ј and 5Ј-ATCACAATGCCAGTGGTA- Ј Cell culture. Microvascular endothelial cells derived from rat epididymal fat 3 ) were designed from the European Molecular Biology Laboratory (EMBL)  pads ascertained positive for von Willebrand protein by immunofluorescence sequences (accession no. NM 012567 for Cx43 and J00691 for -actin). The microscopy were used in this study (20). Cells were grown in Dulbecco’s specificity of the PCR was enhanced by using the “hot start” approach (22). modified Eagle’s medium containing 10% fetal bovine serum (Sigma, St. Louis, The PCR containing the appropriate aliquot of RT material with primers, 0.2 MO) and antibiotics. To determine the effect of high glucose on Cx43 mol/l each, 2.5 U AmpliTaq DNA polymerase (Boehringer Mannheim), MgCl2 expression and GJIC activity, these cells were grown in normal (5 mmol/l) or (1.8 mmol/l), and PCR buffer in 50 l volume was performed in a DNA thermal high (30 mmol/l) D-glucose medium for 9 days to confluency. cycler (Hybaid, Middlesex, U.K.) using the following cycle conditions: dena- Gel electrophoresis and Western blot. Cells exposed to normal or high- turation for 1 min at 95°C, annealing for 1 min at 54°C, and extension for 2 min glucose medium were washed with PBS and lysed in buffer containing 10 at 72°C. PCR was performed with 25 cycles for Cx43 and 27 cycles for -actin. mmol/l Tris, pH 7.5 (Sigma), 1 mmol/l EDTA, and 0.1% Triton X-100 (Sigma). Analysis and quantitation of PCR products. PCR products were always Cellular protein content in the cell extract was measured by bicinchoninic resolved on the same gel (1.0% agarose) containing 0.05 l/ml GelStar (BMA, acid protein assay method (Pierce, Rockford, IL). After addition of an equal Rockland, ME) together with molecular weight markers (100-bp DNA ladder; volume of 2ϫ sample buffer and denaturation at 95°C for 5 min, the extracts Gibco, Grand Island, NY). The gel was photographed with Positive/Negative (containing 30 g protein) were electrophoresed at 260 V for 2 h. Molecular Instant Film (665; Polaroid, Cambridge, MA). Signal intensity was quantitated