Downregulation of Connexin 43 Expression by High Glucose Induces Senescence in Glomerular Mesangial Cells
Xiaojie Zhang,
Xiangmei Chen,
Di Wu,
Weiping Liu,
Jianzhong Wang,
Zhe Feng,
Guangyan Cai,
Bo Fu,
Quan Hong and
Jing Du
Department of Nephrology, Kidney Center and Key Lab of the People’s Liberation Army, General Hospital of People’s Liberation Army, Beijing, People’s Republic of China
Address correspondence to: Dr. Xiangmei Chen, Department of Nephrology, Kidney Center and Key Lab of PLA, General Hospital of PLA, 28 Fuxing Road, Beijing 100853, People’s Republic of China. Phone: +86-10-66935462; Fax: +86-10-68130297; E-mail: xmchen{at}public.bta.net.cn
Received for publication July 27, 2005.
Accepted for publication March 14, 2006.
Connexin 43 (Cx43) plays an important role in cell differentiationand growth control, but whether it can be regulated by highglucose and whether it can mediate in glomerular mesangial cells(GMC) the phenotype alterations that are induced by high glucosestill remain to be explored. In this study, RNA interferenceand gene transfer techniques were used to knock down and overexpressCx43 gene in rat GMC to determine the contribution of Cx43 toGMC senescence that was induced by high glucose. The resultsshow that high glucose (30 mM) not only downregulated Cx43 mRNAand protein expression (P < 0.05) but also increased thepercentage of senescence-associated -galactosidase (SA--gal)stained cells and expression of p21cip1 and p27kip1 (P <0.05), indicating that high glucose promoted rat GMC senescence.Knocking down Cx43 gene expression significantly increased thepercentage of SA--gal stained cells and p27kip1 and p21cip1expression in GMC (P < 0.05), whereas overexpression of Cx43significantly decreased the percentage of SA--gal stained cells(P < 0.05). These results demonstrate for the first timethat downregulation of Cx43 expression by high glucose promotesthe senescence of GMC, which may be involved in the pathogenesisof diabetic nephropathy.
Hypertrophy of glomerular mesangial cells (GMC) is one of theearliest morphologic alterations in the kidney after the onsetof diabetes. Previous studies (1–3) have shown that culturedGMC that are exposed to high glucose arrest in the G1 phaseof the cell cycle, with an increased expression of the cyclin-dependentkinase (CDK) inhibitors p21cip1 and p27kip1, which are markersof cell senescence (4,5).
Blazer et al. (6) reported that hyperglycemia induced prematurereplicative senescence in human skin fibroblasts. Morocuttiet al. (7) demonstrated that there was retardation of the cellreplication rate, which was tightly coupled to larger cell volumein skin fibroblasts from patients with diabetic nephropathy.These results showed that high glucose was associated with thefibroblast aging process, although the mechanisms remained unclear.
There is some evidence (8–11) that connexin 43 (Cx43)expression and intercellular communication in retinal endothelialcells and aortic smooth muscle cells are reduced under conditionsof hyperglycemia, contributing to the development of the microangiopathyand macroangiopathy that are associated with diabetes. However,the relationship between Cx43 and cell senescence is yet unclear.Our hypothesis here is that high glucose may promote GMC senescencethrough downregulation of Cx43 expression and function.
This study was designed to investigate the effects of high glucoseon senescence of GMC, expression of Cx43, and changes of gapjunctional intercellular communication (GJIC). In addition,we aimed to determine the relationship between Cx43 and GMCsenescence under high glucose conditions. Our results indicatethat reduced Cx43 expression under high glucose acceleratesthe progression of mesangial cell senescence, a mechanism thatis associated with mesangial cell hypertrophy in diabetic nephropathy.
Materials
FBS and DMEM were supplied by Life Technologies BRL (Gaithersburg,MD). The retrovirus packaging cell line PT-67 and retroviralvector pLNCX2 were obtained from Clontech Laboratories (PaloAlto, CA). Penicillin, streptomycin, and TRIzol reagent werefrom Invitrogen (Carlsbad, CA). Polybrene, leupeptin, aprotinin,antipain, phenylmethylsulfonyl fluoride, and Ac-DEVD-CHO wereprocured from Sigma (St. Louis, MO). The NIH 3T3 cell line wasobtained from the cell bank of the Chinese Academy of Sciences.SuperFect was purchased from Qiagen (Valencia, CA). Antibodieswere procured from Sigma (Cx43 and hemagglutinin [HA]) and SantaCruz Biotechnology (Santa Cruz, CA; p27kip and p21cip).
Cell Culture
Primary rat GMC were obtained from male Wistar rats (3 mo ofage). The cells were grown in low-glucose DMEM (5.5 mM d-glucose)that was supplemented with 15% FBS, 100 units/ml penicillin,and 100 µg/ml streptomycin at 37°C (12). Cells frompassages 3 to 5 were used in our study. For determination ofthe effects of high glucose on Cx43 expression and GJIC activity,GMC were grown in normal (5.5 mM) or high (30 mM) d-glucosemedium for 48 or 96 h. As an osmotic control, GMC were grownin normal-glucose medium that contained 24.5 mM mannitol. ThePT-67 retroviral packaging cells and NIH3T3 cells were culturedin DMEM with 10% FBS, 10 mM HEPES, 2 mM l-glutamine, 1 mM MEMsodium pyruvate, 100 units/ml penicillin, and 100 µg/mlstreptomycin.
Cells Proliferation Capacity Analyses
Cells were seeded into 96-well plates (2 x 103 cells/well).After incubation for 24, 48, 72, and 96 h, respectively, 3-(4,5-dimethylthiazol-2-yl)-2,5-(diphenyltetrazoliumbromide) (MTT) (2 mg/ml, 20 µl) was added into each welland incubated for 4 h. DMSO (100 µl) subsequently wasadded to each well to dissolve the formazan crystals, and theabsorption at 570 nm was measured.
Total Protein/Cell Number Ratio
Because the total protein/cell number ratio is a well-establishedmeasurement of cellular hypertrophy, this parameter was usedto determine whether the alteration of cell growth was accompaniedby cell hypertrophy (13). GMC were cultured for 1 to 4 d inlow- or high-glucose medium, and at the end of the treatmentperiod, cells were trypsinized and washed twice with ice-coldPBS and counted in a hemocytometer chamber. The cells then werelysed to measure the total protein content by the Bradford method.The total protein/cell number ratio expressed as µg/105cells was used as a hypertrophy index.
Senescence-Associated -Galactosidase Staining
After washing in PBS and fixation for 3 to 5 min (room temperature)in 2% formaldehyde/0.2% glutaraldehyde, the cells were incubatedovernight at 37°C (without CO2) with freshly prepared senescence-associated-galactosidase (SA--gal) stain solution (1 mg/ml X-gal, 40 mMcitric acid/sodium phosphate [pH 6.0], 5 mM potassium ferrocyanide,5 mM potassium ferricyanide, 150 mM NaCl, and 2 mM MgCl2). Cellsthen were rinsed with PBS, and 100 to 200 cells in six microscopicfields were counted to determine the percentages of SA--galstained positive cells.
Cell-Cycle Analysis
Cells (1 x 106; each sample) were treated with 0.25% trypsin,washed twice with cold PBS, and fixed with 70% alcohol in PBSfor 12 h at 4°C. The cells then were washed twice with PBSand stained for 30 to 60 min at 4°C in 100 µg/ml propidiumiodide solution (with 100 µg/ml RNase). Stained cellswere analyzed by flow cytometry (Becton Dickinson, FranklinLakes, NJ).
Real-Time PCR Assay
Cx43 mRNA expression was measured by real-time PCR with thefollowing primers: Sense 5'-TTC ATG CTG GTG GTG TCC-3' and antisense5'-TTG GCA TTC TGG TTG TCG-3' (expected product of approximately400 bp). Control glyceraldehyde-3-phosphate dehydrogenase primerswere as follows: Sense 5'-TGC ACC ACC AAC TGC TTA GC-3' andantisense 5'-GGC ATG GAC TGT GGT CAT GAG-3'. Real-time PCR wasperformed with SYBR green I (1:20,000; Qiagen), with 1 cycleat 95°C for 3 min followed by 40 cycles at 95°C for45 s, 61°C for 45 s, 72°C for 40 s, and 80°C for5 s.
Western Blotting
The cells were lysed in RIPA buffer that was composed of 50mM Tris-Cl (pH 7.6), 5 mM EDTA, 150 mM NaCl, 0.5% NP-40, and0.5% Triton-X-100 and contained 1 µg/ml leupeptin, aprotinin,and antipain; 1 mM sodium orthovanadate; and 0.5 mM phenylmethylsulfonylfluoride. Protein concentration was measured by the Bradfordassay. A total of 50 µg of total protein was separatedby 12% SDS-PAGE and then transferred to a membrane, which wasblocked with 5% skim milk, probed with a primary antibody overnightat 4°C, and incubated with an horseradish peroxidase–conjugatedsecondary antibody. Immunoreactive bands were visualized usingenhanced chemiluminescence.
Immunofluorescence Detection
To study the distribution and relative amounts of Cx43, we performedimmunofluorescent staining for Cx43 in GMC using a routine procedure.Briefly, rat GMC were seeded on sterile glass coverslips andgrown to confluence, fixed in 4% paraformaldehyde for 15 minat 4°C, washed in PBS, then permeabilized with 1% TritonX-100. After blocking nonspecific antibody binding with 1% BSAfor 15 min, the cells were incubated overnight at 4°C ina moist chamber with a monoclonal mouse anti-rat Cx43 antibodydiluted at 1:400 in PBS that contained 1% BSA. After washingwith PBS three times, the cells were incubated for 1 h withFITC-conjugated anti-mouse IgG (Sigma) diluted at 1:100 in PBSthat contained 1% BSA. After three PBS washes, the cells werecounterstained with propidium iodide and photographed usinga confocal microscope (Radiance 2000; Bio-Rad Laboratories)equipped with LSM510 software.
Fluorescence Recovery after Photobleaching
The degree of intercellular communication between neighboringcultured GMC was determined by fluorescence recovery after photobleaching(FRAP) (14), which has been a powerful tool to measure cell-to-celldiffusion of a fluorescent dye under a laser scanning microscope(Radiance 2000, Bio-Rad Laboratories). Briefly, the cells wereloaded for 20 min at room temperature with the membrane-permeantdye 6-carboxyfluorescein diacetate (50 µM; Molecular Probes,Eugene, OR). Then, a region of the cell is bleached brieflyusing a 488-nm argon laser spot at 90% of full power for 10s. Consequently, the cells inside the region of illuminationirreversibly lose their fluorescence, a phenomenon that is knownas photobleaching. Because cells can communicate with neighboringcells through gap junctions, the unbleached dye from adjacentcells can move into the bleached region, and fluorescence insidethe bleached zone increases and eventually reaches an equilibriumstate. Recovery of fluorescence was quantified at 5-s intervalsfor 12 min to derive recovery curves. We derived the recoveryratio to prephotobleaching and the diffusion speed of variousexperimental groups of GMC using these curves and were ableto estimate their GJIC functional status.
Small Interference RNA Experiments
The small interference RNA (siRNA) was designed to target thefollowing sequence of rat Cx43 sequence (GenBank, X06656): AATGAA GCA GAT TGA AAT CAA. The corresponding primers were usedto produce siRNA-Cx43 using the Silencer siRNA constructionkit (Ambion, Inc., Austin, TX): Sense 5'-AAT TGA TTT CAA TCTGCT TCA CCT GTC TC-3' and antisense 5'-AAT GAA GCA GAT TGA AATCAA CCT GTC TC-3'. GMC were transfected with 0.15 µg ofsiRNA-Cx43 for 48 h, then Cx43 mRNA and protein expression werestudied by real-time PCR and Western blotting, respectively.To confirm the specificity of siRNA-Cx43, we used a nonsensesequence siRNA-con as a control.
Construction of Retroviral Vectors and Transfection of GMC
cDNA encoding full-length Cx43 with an HA tag was subclonedinto the Bgl II/SalI sites of retroviral vector pLNCX2-neo (ClontechCo., Mountain View, CA) to create pLNCX2-Cx43. The packagingcell line PT-67 was transfected by Lipofectamine 2000 with 15µg of pLNCX2-Cx43 or pLNCX2-neo. The retroviruses werecollected from the culture medium 48 h after transfection, passedthrough a 0.45-µm filter, and spun at 50,000 x g for 1.5h. The retroviruses were resuspended overnight in TNE bufferwith 0.l times the original volume at 4°C and stored at–80°C. NIH3T3 cells were used to determine viral titers.Briefly, the cells were infected with the virus for 24 h, selectedwith 300 µg/ml G418 for 1 wk, fixed with 4% formaldehyde,and stained with Giemsa.
The efficiency of pLNCX2-Cx43 infection of GMC was demonstratedby Western blotting against the HA tag and immunofluorescentdouble staining with Cx43 and HA antibodies. The latter wasdone as follows: GMC were fixed in 4% paraformaldehyde, permeabilized,and blocked, and the cells were incubated overnight at 4°Cwith a polyclonal rabbit anti-Cx43 antibody diluted at 1:400.After three PBS washes, the cells were incubated for 1 h atroom temperature with a monoclonal mouse anti-rat HA antibody(Sigma) diluted at 1:500. After three washings with PBS, thecells were incubated for 1 h with FITC-conjugated anti-rabbitIgG (Sigma) diluted at 1:100 and rhodamine-conjugated anti-mouseIgG (Sigma) separately. The cells then were viewed and photographedusing a confocal microscope. Green fluorescence indicated positiveCx43 staining, whereas red fluorescence indicated HA expression,and orange color indicated co-localization of Cx43 and HA. Theefficiency and the specificity of Cx43 overexpression on enhancementof GJIC in GMC were detected by FRAP in the presence or absenceof GJ inhibitor heptanal (0.5 mmol/L).
Statistical Analyses
All data analyses were performed with SPSS 10.0 (SPSS Inc.,Chicago, IL); data are expressed as mean ± SD. Comparisonamong groups was conducted with ANOVA. P < 0.05 was consideredsignificant.
High Glucose Accelerated Rat GMC Senescence
GMC were cultured for 96 h in normal-glucose (5.5 mM) medium,osmotic control, and high-glucose (30 mM) medium. MTT assaysdemonstrated that the cellular proliferation capacity of GMCwas inhibited by high glucose. The optical density values after72 h of incubation in high glucose were significantly lowerthan those in the other two groups (P < 0.05; Figure 1).At the same time, the levels of CDK inhibitors p27kip1 and p21cip1in GMC were significantly increased under high glucose for 48h (Figure 2). In addition, the percentage of SA--gal stainedpositive cells in GMC cultured in high glucose for 96 h (70.88± 7.19%) was significantly greater than that in normal-glucosemedium (30.55 ± 4.86%) or in osmotic control (31.37 ±5.73; P < 0.05; Figure 3), indicating that high glucose inducedsenescence in GMC.
Figure 1. Effect of high glucose on proliferation of glomerular mesangial cells (GMC). Cells were grown for specified periods of time, and cell number was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-(diphenyltetrazolium bromide) (MTT) conversion, measured spectrophotometrically (optical density [OD]). After 24 or 48 h of incubation, cellular proliferation of the high-glucose group was slightly higher than that of the normal-glucose group or the osmotic control group. However, after 72 h, cellular proliferation was permanently inhibited in the high-glucose group in which the OD value was significantly lower than those of the other two groups. Values are means ± SD (n = 6); comparison among groups was conducted with ANOVA. *P < 0.05 versus control.
Figure 2. Effect of high glucose on p27kip1 and p21cip1 expression of GMC. Cells were grown for 48 h under various glucose conditions and then were harvested for Western blot analysis. (A) Western blot of p27kip1 and p21cip1. Lanes 1 through 3, GMC in the normal-glucose group, osmotic control group, and high-glucose group, respectively, after 24 h of incubation; lanes 4 through 6, GMC of the normal-glucose group, osmotic control group, and high-glucose group, respectively, after 48 h of incubation. (B) p27kip1 and p21cip1 expression. Data are expressed as relative percentages versus actin, and groups were compared using ANOVA. *P < 0.05 versus normal-glucose group (n = 6).
Figure 3. Effect of high glucose on senescence-associated -galactosidase (SA--gal) staining of GMC. (A) GMC grown in normal glucose, osmotic control conditions, and high glucose for 96 h of incubation were stained for SA--gal, which shows blue precipitation in the cytoplasm in senescent cells. (B) The percentage of SA--gal stained positive cells. For each group, 100 to 200 cells were counted. Comparison among groups was conducted with ANOVA. *P < 0.05 versus normal-glucose group. Magnification, x200.
High Glucose Downregulated Cx43 Expression and GJIC Activity in GMC
The expression of CX43 was detected in GMC cultured in variousconcentrations of glucose by real-time PCR, Western blot analysis,and immunofluorescence staining. The results showed that themRNA and protein levels of Cx43 expression of the high-glucosegroup were significantly lower than those in cells exposed tonormal glucose (P < 0.05). In addition, immunofluorescencemicroscopy showed fewer number of Cx43 gap junction plaquesat the contact site between adjacent cells grown in high-glucosemedium (Figure 4, A through D).
Figure 4. Effect of high glucose on connexin 43 (Cx43) expression and gap junctional intercellular communication (GJIC) function of GMC. (A) Cx43 mRNA expression was detected by real-time PCR. Data are expressed as relative percentages against glyceraldehyde-3-phosphate dehydrogenase (GAPDH). *P < 0.05 versus normal glucose group (n = 6). (B) Western blot of Cx43 proteins. Lanes 1 through 3, GMC of normal-glucose group, osmotic control group, and high-glucose group, respectively, after 24 h of incubation; lanes 4 through 6, GMC of normal-glucose group, osmotic control group, and high-glucose group, respectively, after 48 h of incubation. (C) Graphic presentation of relative Cx43 protein abundances compared with actin. *P < 0.05 versus normal-glucose group (n = 6). (D) Immunofluorescence staining of Cx43 with monoclonal anti-Cx43 antibody. Note the characteristic spotted staining of Cx43 at the cell–cell contact regions. (E) Fluorescence recovery curve (in seconds) after photobleaching detected by fluorescence recovery after photobleaching (FRAP). GMC were cultured under normal-glucose medium, osmotic control, and high-glucose medium for 48 h. The data shown are from one representative experiment from a series of five with similar results. Magnification, x600.
The functional implications of decreased Cx43 expression inGMC exposed to high-glucose medium was evaluated by FRAP analysis(Figure 4E). The degree of intercellular communication was expressedwith a fluorescence recovery curve. The results indicated thatthe recovery rate after photobleaching of cells grown in highglucose (0.60 ± 0.019) was significantly lower than thoseof the other two groups (0.84 ± 0.023 and 0.82 ±0.025; P < 0.05).
Silencing Cx43 Expression Triggered Hypertrophy and Senescence in GMC
RNA interference has become available and has been proved tobe a powerful tool for studying gene function. Short 21- to23-nucleotide interfering RNA (siRNA) have been used successfullyto provide a strong and specific suppression of gene expressionin mammalian cells (15,16). In this study, 21-nucleotide interferingRNA (siRNA-Cx43) targeting rat Cx43 sequence was designed andtransfected into GMC. To investigate its efficacy, we transfectedGMC with either siRNA-Cx43 or irrelevant siRNA-con. After 48h, the Cx43 expression was assessed by real-time PCR, Westernblot, and immunocytochemistry. Our results indicated that Cx43mRNA were markedly lower in GMC transfected with siRNA-Cx43than those of control GMC and GMC transfected with siRNA-con(Figure 5). In addition, when siRNA-Cx43 was transfected, gapjunction plaques at cell–cell interfaces were less prevalentthan those in controls, which coincided with a reduction intotal Cx43 expression as revealed by Western blot (Figure 5).Meanwhile, GJIC of GMC was dramatically inhibited by siRNA-Cx43(Figure 5). All of these data demonstrated that the siRNA-Cx43was effective in downregulating Cx43 expression and functionin GMC.
Figure 5. Inhibition of Cx43 expression in GMC by small interference RNA (siRNA). (A) Cx43 mRNA expression detected by real-time PCR. GMC transfected with siRNA-Cx43 had a dramatically lower Cx43 mRNA expression than that of control and GMC transfected with siRNA-con. The graph summarizes combined real-time PCR data from at least six separate experiments in which the expression level of Cx43 mRNA was quantified and normalized to GAPDH. *P < 0.05 versus control group. (B) Western blot revealed a significant reduction of total Cx43 protein expression in GMC transfected with siRNA-Cx43 (lane 3) compared with control (lane 1) and GMC transfected with siRNA-con (lane 2). (C) GMC were transfected with siRNA-Cx43 and siRNA-con and then immunostained for Cx43. Note that siRNA-Cx43–expressing cells had a dramatically reduced number of gap junctions. (D) Fluorescence recovery curve detected by FRAP method. GJIC of GMC was inhibited dramatically by siRNA-Cx43 transfection. The data shown are from one representative experiment from a series of five with similar results. Magnification, x600.
To examine the consequences of Cx43 silencing on GMC, we evaluatedthe related cell hypertrophy and senescence parameters mentionedabove. It was found that in GMC transfected with siRNA-Cx43,the total protein/cell number ratio (Table 1), the percentagesof cells at G1 phase (Table 2), the SA--gal positive stainingrate, and the expressions of CDK inhibitors p21cip1 and p27kip1were significantly higher than those in controls. In addition,the proliferation rate was significantly lower than those ofcontrol GMC or siRNA-con GMC (Figure 6). These results suggestedthat GMC that exhibited reduction in Cx43 expression underwenthypertrophy and premature senescence.
Figure 6. Senescence-related phenotypes in GMC transfected with siRNA. (A) Effect of siRNA inhibition of Cx43 on SA--gal staining of GMC. The percentage of SA--gal–positive cells after transfection with siRNA-Cx43 was higher than that of control or cells transfected with siRNA-con. (B) p27kip1 and p21cip1 protein expression detected by Western blot. Note that both p27kip1 and p21cip1 expression was higher in GMC transfected with siRNA-Cx43 (lane 3) than in GMC of control group (lane 1) and in GMC transfected with siRNA-con (lane 2). (C) Relative p27kip1 and p21cip1 protein abundances normalized to actin. The results showed that the expression levels of p27kip1 and p21cip1 proteins in cells transfected with siRNA-Cx43 were significantly elevated compared with those in the control group or siRNA-con group. *P < 0.05 versus control group (n = 6). (D) Proliferation of GMC transfected with siRNA-Cx43 was lower than that of control or cells that wee transfected with siRNA-con. *P < 0.05 versus control group (n = 6). Magnification, x200.
Cx43 Gene Overexpression Prevents GMC Hypertrophy and Premature Senescence Induced by High Glucose
Because it was difficult to transfect genes efficiently intoGMC using plasmids, we created a retroviral vector pLNCX2-Cx43,which drives HA-tagged Cx43. This construct was confirmed withBgl II and SalI digestion. The retrovirus that was producedby packaging PT-67 cells was collected and infected GMC effectively,as demonstrated by Western blot and double immunofluorescencestaining of Cx43 and HA tag (Figure 7).
Figure 7. Construction of retroviral vectors and transfection of GMC. (A) Retrovirus-mediated overexpression of Cx43 demonstrated in GMC. Proteins from cells were analyzed using a hemagglutinin (HA) antibody on Western blots. Lane 1, control GMC without transfection; lane 2, GMC transfected with pLNCX2-neo; lane 3, GMC transfected with pLNCX2-Cx43. (B) GMC transfected with pLNCX2-Cx43 were double stained with Cx43 and HA antibodies. Green fluorescence indicates positive Cx43 staining (a), red fluorescence indicates HA expression (b), and yellow color in the merged image indicates co-localization of Cx43 and HA (c). Magnification, x400.
GMC transfected with pLNCX2-Cx43 showed not only higher levelsof Cx43 mRNA and protein but also better GJIC function in highglucose for 48 h (Figure 8) than in control cells and cellstransfected by pLNCX2-neo (P < 0.05). Pretreatment of GMCwith heptanal (0.1 mM) blocked GJIC enhancement by Cx43 overexpression,which demonstrated specific activation of Cx43. Moreover, itwas found that cell hypertrophy and the senescence-related phenotypesof GMC promoted by high glucose were ameliorated in GMC thatexpressed exogenous Cx43 (Table 3, Figure 9). In GMC transfectedwith pLNCX2-Cx43 and cultured in high glucose, the percentageof cells at G1 phase (64.23 ± 2.35%) was significantlylower than that of control cells (75.95 ± 5.09%) or thatof cells transfected with pLNCX2-neo (72.09 ± 1.60%;Table 4). Cx43 gene transfection also decreased p21cip1 andp27kip1 protein levels (Figure 9) in GMC cultured in high glucose.
Figure 8. Effect of PLNCX2-Cx43 gene transfection on Cx43 expression and GJIC function in GMC. (A) Cx43 mRNA expression was detected by real-time PCR. GMC transfected with pLNCX2-Cx43 showed significantly higher levels of Cx43 mRNA than control cells or GMC transfected with pLNCX2-neo after 48 h of incubation with high glucose. *P < 0.05 versus control group (n = 6). (B) Western blots of Cx43 protein expression. Lanes 1, 3, and 5, cells were incubated in normal-glucose (5.5 mM) medium for 48 h; lanes 2, 4, and 6, cells were incubated in high-glucose (30 mM) medium for 48 h. (C) Relative Cx43 protein abundance compared with actin. GMC transfected with pLNCX2-Cx43 showed significantly higher levels of Cx43 than cells in the other two groups. *P < 0.05 versus control group (n = 6). (D) Three groups of GMC were incubated in high glucose for 48 h and then immunostained for Cx43 with a monoclonal anti-Cx43 antibody. GMC transfected with pLNCX2-Cx43 showed higher levels of staining of Cx43 at cell–cell contact regions. (E) Fluorescence recovery curve after photobleaching in GMC grown in high-glucose medium for 48 h. Note that GMC transfected with pLNCX2-CX43 showed better GJIC function than control cells or cells transfected by PLNCX2-neo. Pretreatment of GMC with the GJ inhibitor heptanal (0.5 mmol/L) for 30 min blocked the enhancement of GJIC by pLNCX2-Cx43 transfection. Magnification, 600.
Figure 9. Senescence-related phenotypes of GMC transfected with pLNCX2-Cx43. (A) Effect of Cx43 gene transfection on SA--gal staining in GMC incubated in high glucose. The percentage of SA--gal–positive cells transfected with pLNCX2-Cx43 was lower than that of control or cells transfected with pLNCX2-neo. (B) Proliferation of GMC under high glucose. Note that cell proliferation of cells transfected with pLNCX2-Cx43 was higher than that of controls or cells transfected with pLNCX2-neo. *P < 0.05 versus control group (n = 6). (C) Western blot demonstrating expression of p27kip1 and p21cip1 in cells transfected with pLNCX2-Cx43 compared with controls. Cells were incubated in normal glucose (5.5 mM) for 48 h (lanes 1, 3, and 5) or in high glucose for 48 h (lanes 2, 4, and 6). (D) Relative p27kip1 and p21cip1 protein abundances normalized to actin. *P < 0.05 versus control group cultured in high glucose (n = 6). Magnification, x200.
In this study, we examined senescence-related phenotypes andgap junction alterations of GMC exposed to high glucose concentrations.Cx43 silencing and overexpressing techniques were used to demonstratethe potential function of Cx43 in this pathologic condition.Our results address the protective role of Cx43 in regulatingGMC growth and phenotype.
It is known that most types of primary normal cells do not proliferateindefinitely. After a period of rapid proliferation, cell divisionrates decrease and ultimately cease; the cells then enter astate of senescence that is characterized by an irreversibleG1 growth arrest and functional and morphologic changes (4,17).Senescence of cells also can occur rapidly in response to variousphysiologic stresses, such as high glucose, oxidative stress(18), DNA-damaging agents, and other metabolic perturbations.
In this study, we detected -gal activity of GMC cultured invarious doses of glucose, as one of many hallmark measures ofcell replicative senescence or physiologic aging (19). The resultsindicated that the percentage of SA--gal positive stained cellscultured in high-glucose medium was significantly greater thanthat of cells in normal-glucose medium or in osmotic control.In addition, GMC cultured in high glucose were characterizedby an irreversible G1 growth arrest and also associated withthe upregulation of cell-cycle inhibitors p21cip1 and p27kip1,which were similar to the results of some other studies (2,3)in which these parameters were used to measure cell senescence.Our results demonstrated that high glucose induced senescencein GMC.
The mechanisms of cell senescence are not clear; growing evidencehas shown that Cx43 and GJIC might be involved in this process.Gap junctions are intercellular channels that permit the passageof small molecules such as small metabolites, ions, and secondmessengers. GJIC has an important role in a variety of cellularprocesses, including homeostasis, morphogenesis, cell differentiation,and growth control (20,21). Statuto et al. (22) reported a progressivedecrease of the expression of Cx43 and GJIC in replicative senescenceof cultured HEL-299 fibroblasts and termed Cx43 as a biomarkerof cell senescence. It also has been shown that the expressionand function of Cx43 in astrocytic cells and aortic endotheliumwere age related, suggesting that Cx43 and GJIC may play a rolein the process of cell aging (23–26).
Studies already have shown that Cx43 is a major connexin inGMC. There is a high density of Cx43 expression in GMC (27,28);however, little is known about its exact functions. It has beensuggested that Cx43 may have a role in connecting GMC into afunctional syncytium (29–31). Gap junction–mediatedcell–cell communication may play a role in the stringentgrowth control of GMC. There are few data, however, concerningfunctional regulation of Cx43 and GJIC in GMC under pathologiccircumstances.
This is the first report to explore the relationship betweenCx43 function and senescence of GMC in high glucose. As thefirst step, we evaluated whether high glucose affected GMC connexinexpression and GJIC activity. The results showed that Cx43 expressionand the number of Cx43 gap junctions were significantly downregulatedby high glucose, which was consistent with some previous results(8,9,11,32). Cx43 family members degrade rapidly, with half-livesof only 1.5 to 5 h (21), suggesting that the reduction of Cx43expression could be attributed to decreased transcription andtranslation of Cx43 gene in high glucose. As a result, the reducedGJIC activity in GMC that are exposed to high glucose couldimpair the transport of small molecules, such as calcium orcAMP, that are necessary for cell proliferation and maintenanceof cellular homeostasis.
To explore the exact role of Cx43 in GMC senescence that isinduced by high glucose, we used Cx43 gene knockdown and overexpressionsuccessfully to silence or enhance Cx43 expression. The resultshave shown that GMC infected with siRNA-Cx43 displayed the phenotypesof senescence, as demonstrated by SA--gal staining and increasedp27kip1 and p21cip1. In contrast, GMC transfected with the retroviruspLNCX2-Cx43 displayed both higher levels of Cx43 expressionand improved senescence-related phenotypes in high glucose.It therefore is reasonable to suggest that there may be an associationbetween decreased Cx43 expression and cell senescence in highglucose. The loss of Cx43 may result in unregulated cell proliferationand contribute, at least in part, to the cell senescence athigh glucose.
To determine the mechanism by which Cx43 regulates GMC growthand senescence, we examined potential molecules that are regulatedby Cx43. It is known that cell proliferation and senescenceare regulated at the level of the cell cycle by cell-cycle proteins,including CDK. In contrast, CDK inhibitors inactivate CDK andcause cell-cycle arrest. There is a growing body of literatureshowing that CDK inhibitors p21cip1 and p27kip1 may be criticalregulators of GMC hypertrophy (33) and cell senescence (4,34).Zheng et al. (5) found that mesangial cells isolated from kidneysof postmenopausal mice were enlarged and had elevated amountsof p27kip1 protein expression as assessed by immunochemistry.Other studies (35–37) also showed that p21cip1 and p27kip1were required for retinoblastoma protein–mediated senescence,demonstrating not only that p21cip1 and p27kip1 gene transfercould promote cell senescence but also that antisense p21cip1and p27kip1 had some effects on retarding the progress of cellhypertrophy or senescence. Our results showed that Cx43 knockdownsignificantly increased p21cip1 and p27kip1 expression in GMC,whereas Cx43 gene overexpression decreased p21cip1 and p27kip1protein levels in GMC cultured in high glucose, indicating thatCx43 could control cell proliferation and senescence progressionthrough cell-cycle regulation.
Our data have demonstrated for the first time that hyperglycemia-mediatedreduction of Cx43 expression and GJIC activity is involved inthe development of cell hypertrophy and senescence in GMC. Thismight be a potential target for treating diabetic nephropathy.
Acknowledgments
This work was supported by grants from the Creative ResearchGroup Fund of the National Foundation Committee of Natural Scienceof China (30121005), the National Natural Sciences Foundationof China (30370655 and 30300161), and the Main State Basic ResearchDevelopment Program of China.
Footnotes
Published online ahead of print. Publication date availableat www.jasn.org.
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