Knockdown of Fibronectin Induces Mitochondria-Dependent Apoptosis in Rat Mesangial Cells
Di Wu*,
XiangMei Chen*,
DengFu Guo,
Quan Hong*,
Bo Fu*,
Rui Ding*,
LiFang Yu*,
Kai Hou*,
Zhe Feng*,
XiaoJie Zhang* and
JianZhong Wang*
* Department of Nephrology, Kidney Center, and Key Laboratory of the People’s Liberty Army (PLA), General Hospital of PLA, Beijing, People’s Republic of China; and Department of Medicine, University of Montreal and Research Centre, Centre Hospitalier de l’Universite de Montreal, Hotel-Dieu, Montreal, Quebec, Canada
Address correspondence to: Dr. XiangMei Chen, Department of Nephrology, Kidney Center and Key Laboratory of PLA, General Hospital of PLA, 28 Fuxing Road, Beijing 100853, China. Phone: 86-10-66935462; Fax: 86-10-68130297; E-mail: xmchen{at}public.bta.net.cn
Extracellular matrix (ECM) expansion and mesangial cell (MC)proliferation are prominent features of most types of glomerulosclerosis.A delicate balance between the ECM and MC regulates cell survival.Increasing evidence shows that a loss of ECM components cancause mitochondrial dysfunction and induce cell apoptosis. Itis proposed that directly blocking the synthesis of ECM componentscould lighten ECM accumulation and suppress cell overproliferationstatus. Fibronectin, one of the predominant adhesive glycoproteinsof the mesangial ECM, provides the survival signal for cells.Its accumulation can be observed in most types of glomerulosclerosis.In this study, angiotensin II–induced fibronectin wassuppressed by an RNA interference technique. It is interestingthat MC slowly underwent apoptosis after infection with a retrovirusthat continuously suppressed fibronectin synthesis. It was foundthat MC apoptosis occurred in a mitochondria-dependent mannermainly as a result of cytochrome c release and downstream caspase-3and -9 activation. Furthermore, it was demonstrated that fibronectinknockdown affected mitochondrial handling of Ca2+ release fromthe endoplasmic reticulum. Importantly, blocking the inositol1,4,5-triphosphate receptor with, 3,4,5-trimethoxybenzoate ordecreasing Ca2+ in the ECM with EGTA partially saved the cellsfrom apoptosis. These studies, which explored a new method forsimultaneously inhibiting MC proliferation and ECM accumulation,may represent a novel therapeutic approach to glomerulosclerosis.
Mesangial cell (MC) proliferation and extracellular matrix (ECM)overproduction are the predominant pathologic features of manyforms of glomerulonephritis (1,2). Disrupting interactions betweenthe ECM and MC, by depriving the ECM or by arginine-glycine-aspartate(RGD)-like peptide treatment (3), is the usual means of inducingcell apoptosis. However, few laboratories have tried to inhibitdirectly ECM synthesis to suppress ECM overproduction. Recently,Irwin et al. (4) provided very interesting evidence showingthat mutation of the collagen VI gene, one of the ECM proteins,causes ultrastructural alterations of the mitochondria and sarcoplasmicreticulum with the induction of spontaneous apoptosis in muscles
MC are embedded in a solid network of adhesive proteins, includingfibronectin, laminin, and vitronectin, which mediate pivotalsurvival signals to cells (5). When cells are detached fromthe ECM, they undergo apoptosis (or anoikis) (6–9). Fibronectinis one of the extracellular adhesive glycoproteins involvedin adhesion and migration for important physiologic processes.It attaches cells to matrices that contain fibrous collagens.It is considered to provide survival signals for many cell typesthrough its RGD and heparin-binding domains (10). Studies havedemonstrated that fibronectin, not collagen I, protects cellsfrom detachment-induced apoptosis. Fibronectin accumulationhas been found in most types of glomerulosclerosis (11–13).Chemical synthesis or in vitro transcription is the most convenientmethod of producing RNA interference (siRNA), but the effectis short-lived (14,15). To overcome this problem, several groupshave worked on the transcription of short hairpin RNA (shRNA)by RNA polymerase III to generate siRNA for silencing gene expression(16–18)
In this study, we investigated whether inhibition of ECM componentsynthesis lightens ECM accumulation and induces apoptosis inrat MC (RMC). We first confirmed that the siRNA technique iseffective for the suppression of fibronectin synthesis in RMC.Furthermore, we established a retroviral system to express shRNAto silence continuous fibronectin expression under the controlof the H1 promoter in RMC. We observed that RMC underwent apoptosisafter fibronectin expression was inhibited with shRNA and establishedthat the mitochondrial "intrinsic" pathway but not the deathreceptor "extrinsic" pathway is involved in RMC apoptosis evokedby silencing fibronectin expression. Finally, we found thatblocking the inositol 1,4,5-triphosphate (IP3) receptor anddecreasing Ca2+ concentration in the ECM partially reversedthe apoptotic process
FBS, FCS, and DMEM were supplied by Life Technologies (Burlington,ON, Canada). The retrovirus packaging cell line GP2–293T,retroviral vector pLXIN, and pVSV-G were obtained from ClontechLaboratories (Palo Alto, CA). l-Glutamine, penicillin, streptomycin,and Trizol reagent were from InVitrogen (San Diego, CA). Polybrene,leupeptin, aprotinin, antipain, PMSF, Ac-DEVD-CHO, verapamil,dantrolene, 3,4,5-trimethoxybenzoate (TMB-8), cyclosporin A(CsA), and fibronectin were procured from Sigma (St. Louis,MO). The NIH3T3 cell line came from the cell bank of the ChineseAcademy of Science. SuperFect was purchased from Qiagen (StudioCity, CA). Antibodies against caspase-3, -8, and -9 were fromAbcam Corp (Cambridge, UK). Anti–cytochrome c antibodywas provided by Santa Cruz Biotechnology (Santa Cruz, CA)
Cell Culture
RMC were obtained from Wistar rats (aged 3 mo). The cells weregrown in DMEM supplemented with 10% FBS, 100 units/ml penicillin,and 100 µg/ml streptomycin at 37°C. The retroviruspackaging cell line GP2–293T and NIH3T3 cells were grownin DMEM with 10% FBS, 10 mM HEPES, 2 mM l-glutamine, 1 mM MEMsodium pyruvate, 100 units/ml penicillin, and 100 µg/mlstreptomycin. RMC underwent three to five passages in this study
siRNA Experiments
A total of three different siRNA targeting fibronectin weretested as shown in Figure 1A. The best siRNA, FN1, targets ratfibronectin sequences (GenBank, NM_019143) located at positions1531 to 1551 relative to the start codon. FN-Con was designedto produce siRNA for control purposes with the primer set asfollows: sense, 5`-AAGGCCCTTATCGGTTCCAGACCTGTCTC-3`; and antisense,5`-AATCTGGAACCGATAAGGGCCCCTGTCTC-3`. The DNA template then wastranscripted into RNA using the Silencer siRNA constructionkit (Ambion, Austin, TX)
Figure 1. Significant inhibition of fibronectin expression in rat mesangial cells (RMC) that were transfected with RNA interference (siRNA). (A) Three different siRNA were tested to silence rat fibronectin mRNA expression in RMC. They target the sequence of rat fibronectin located at positions 1531 to 1551 (FN1), 1091 to 1111 (FN2), and 373 to 393 (FN3), respectively. (B) Confocal microscope analysis of the siRNA effect on fibronectin expression. Red fluorescence indicates fibronectin deposition. Fibronectin mRNA and protein expression were analyzed by Western blotting (C) or real-time PCR assay (D), respectively. Normal RMC (lane a as control), RMC that were transfected with FN1 siRNA with or without 10–7 M angiotensin II (Ang II) stimulation (lanes d and b), and RMC that were transfected with FN-Con siRNA with or without Ang II stimulation (lanes e and c) are indicated. (E) Fibronectin protein expression quantified in five experiments (*P < 0.05; **P < 0.01).
RMC were transfected with 0.15 µg of FN1 or FN-Con siRNAfor 48 h, then serum-starved for 24 h and stimulated with orwithout 10–7 M angiotensin II (Ang II) for 16 h. FibronectinmRNA and protein expression were studied by real-time PCR andWestern blotting, respectively, as described below. Cellularfibronectin deposition was analyzed by confocal laser scanningmicroscope (Lasersharp2000; Bio-Rad, Hercules, CA). Briefly,RMC that were transfected with siRNA FN1 or FN-Con were fixedwith 2% formaldehyde in PBS, permeabilized with 0.2% TritonX-100 in PBS that contained 1% FCS, and incubated with mouseanti-fibronectin antibody (Valbiotech, Paris, France) at 37°Cfor 2 h. After washing twice with PBS that contained 1% FCS,the cells were incubated with rhodamine-conjugated anti-mouseIg antibody at 37°C for 1 h
Plasmids for shRNA Expression
The H1 promoter was isolated by PCR from human genomic DNA withthe primers 5`-CCATGGGATCCGAACGCTGACGTC-3` and 5`-GCCTCGAGGTCGACAGATCTGTGGTCTCATACAGAACTTATAAGATTCCC-3`as described elsewhere (17). The PCR product was digested withXhoI and BamHI and subcloned into pLXIN (pLXIN/H1). Fibronectinand control shRNA then were subcloned into the pLXIN/H1 vectorwith the following oligonucleotides and termed pLXIN/H1-FN andpLXIN/H1-Con, respectively: 5`-GATCCCCCATGAAATGGTGCGGCACCttcaagagaGGTGCCGCACCATTTCATGTTTTTGGAAA-3`and 5`-TCGATTTCCAAAAACATGAAATGGTGCGGCACCtctcttgaaGGTGCCGCACCATTTCATGGGG-3`for fibronectin, and 5`-GATCCCCTCTGGAACCGATAAGGGCCttcaagagaGGCCCTTATCGGTTCCAGATTTTTGGAAA-3`and 5`-TCGATTTCCAAAAATCTGGAACCGATAAGGGCCttcaagagaGGCCCTTATCGGTTCCAGAGGG-3`for the control
Retrovirus Generation
The packaging cells were transfected by calcium phosphate precipitationwith 15 µg of pVSV-G and 15 µg of pLXIN/H1-FN orpLXIN/H1-Con. Polybrene (4 µg/ml) was added 5 min beforetransfection. The retroviruses were collected from the culturemedium 48 h after transfection, passaged through a 0.45-µmfilter, and spun at 50,000 x g for 1.5 h. The retroviruses wereresuspended overnight in TNE buffer with 0.l or 0.05 times theoriginal volume at 4°C and stored at –80°C. NIH3T3cells were used to determine the virus titers. Briefly, thecells were infected with the virus for 24 h, selected with 300µg/ml G418 for 1 wk, fixed with 4% formaldehyde, and Giemsastained. Viral titers (CFU/ml) then were quantified with positivelystained cells. We observed 60% efficiency in RMC that were infectedwith pLXIN/green fluorescence protein retrovirus, and expressionwas not changed in a single passage.
Real-Time PCR Assay
Fibronectin mRNA expression was measured by real-time PCR withthe primer set as follows: sense, 5`-TTATGACGACGGGAAGACCT-3`;and antisense, 5`-GCTGGATGGAAAGATTACTC-3`. It then was normalizedby rat GAPDH mRNA expression with the primer set as follows:sense, 5`-TGCACCACCAACTGCTTAGC-3`; and antisense, 5`-GGCATGGACTGTGGTCATGAG-3`.Real-time PCR was performed with SYBR green I (1:20,000; Qiagen)with one cycle at 95°C for 3 min followed by 40 cycles of95°C for 45 s, 61°C for 45 s, 72°C for 40 s, and80°C for 5 s
Western Blotting
Fibronectin and cytochrome c protein expression was assessedby Western blotting. Caspase-3, -8, and -9 activation was alsoquantified by Western blotting. The cells were lysed in RIPAbuffer composed of 50 mM Tris-Cl (pH 7.6), 5 mM EDTA, 150 mMNaCl, 0.5% NP-40, 0.5% Triton-X-100 containing 1 µg/mlleupeptin, aprotinin, and antipain, 1 mM sodium orthovanadate,and 0.5 mM PMSF. Protein concentration was measured by the Bradfordassay. Thirty micrograms of total protein was separated by 7.5%SDS-PAGE and then transferred to a membrane, which was blockedwith 5% skim milk, probed with a primary antibody for 2 h atroom temperature, and incubated with horseradish peroxidase–conjugatedsecondary antibody. Immunoreactive bands were visualized withthe enhanced chemiluminescent system. For studying cytochromec release from the mitochondria to the cytosol, the cells werewashed once with ice-cold PBS and lysed for 6 min on ice with100 µl of lysis buffer (250 mM sucrose, 80 mM KCl, 500µg/ml digitonin, 1 mM dithiothreitol, and 0.1 mM PMSF).The lysates were centrifuged at 12,000 x g at 4°C for 5min to obtain 50-µg supernatants (cytosolic extracts freeof mitochondria) for Western blotting
Mitochondrial Function Assay
To examine mitochondrial function in RMC that were infectedwith pLXIN/H1-Con or pLXIN/H1-FN, with or without the additionof 10 µg of fibronectin for 48 h, we performed JC-1 assaysby confocal laser scanning microscope and flow cytometry. Forconfocal microscope, the cells were incubated with 0.1 mM JC-1fluorescence dye at 37°C for 15 min, then washed twice withcold PBS. Cell fluorescence was recorded by a confocal laserscanning microscope with a 488-nm argon laser and two fluorescencedetectors: FL1 (525-nm band-pass filter) and FL2 (590-nm band-passfilter) to detect JC-1 fluorescence of the dye monomer and liquidcrystal form, respectively. For flow cytometry experiments,the cells were harvested, resuspended in culture medium thatcontained 0.1 mM JC-1 dye, and examined 15 min later by flowcytometry (FACScan; Becton Dickinson). The data were analyzedwith CELLQuest software (BD Biosciences, Palo Alto, CA)
Apoptosis Assay
To quantify apoptosis in RMC that were infected with pLXIN/H1-Conor pLXIN/H1-FN, we tested different drugs and conducted flowcytometry experiments. Briefly, the cells were harvested, washedonce in cold PBS, and resuspended in 1 ml of Annexin V-bindingbuffer (10 mM HEPES [pH 7.4], 140 mM NaCl, and 2.5 mM CaCl2).The cells next were stained with 5 µl of Annexin V FITCand 5 µg/ml propidium iodide (PI) in 100 µl of AnnexinV-binding buffer at 4°C for 20 min, then supplemented with400 µl of the binding buffer and analyzed with a trilaserFACS Calibur flow cytometer. The data were processed with CELLQuestsoftware. This test discriminates intact cells (Annexin V–/PI–),early apoptotic cells (Annexin V+/PI–), and late apoptotic/necroticcells (Annexin V+/PI+). Different inhibitors, 2 mM TMB-8, 500ng/ml CsA, 100 µM Ac-DEVD-CHO, 50 µM dantrolene,10 µg/ml fibronectin, 2.5 mM verapamil, or 0.7 mM EGTA,were added to RMC that were infected with pLXIN/H1-FN for 48h, and apoptosis assays were performed as described above
Statistical Analyses
The results were presented as means ± SD and subjectedto analysis of unpaired t test followed by Bonferroni correction.P < 0.05 was considered statistically significant
Silencing Fibronectin Expression by RNA Interference in RMC
To identify the sequence of the siRNA that is most effectivein silencing fibronectin expression in RMC, we tested threedifferent siRNA targeting sequences located at positions 1531to 1551 (FN1), 1091 to 1111 (FN2), and 373 to 393 (FN3) of ratfibronectin, respectively. As shown in Figure 1A, we found thatthe FN1 (lane b) sequence close to the start codon of rat fibronectinwas the most effective in silencing fibronectin mRNA expressionin RMC and used it for this study. For confirming the specificityof FN1 siRNA, FN-Con siRNA served as the control. Confocal laserscanning microscope analysis revealed that fibronectin depositionwas significantly decreased in RMC that were transfected withFN1 (Figure 1B, b) compared with the control (Figure 1B, a)and RMC that were transfected with FN-Con (Figure 1B, c). FibronectinmRNA and protein expression was also significantly reduced inRMC that were transfected with FN1 (lane b) compared with thecontrol (lane a) and RMC that were transfected with FN-Con (Figure 1, C and D,lane c). These results clearly suggest that FN1siRNA is effective and specific in inhibiting fibronectin synthesisin RMC
Ang II, as a potent stimulator, has been reported to inducefibronectin expression in MC (19). To investigate whether siRNAsuppress the fibronectin expression evoked by Ang II, RMC thatwere transfected with FN1 or FN-Con siRNA were stimulated with10–7 M Ang II. As illustrated in Figure 1, C through E,lane d, Ang II failed to augment fibronectin mRNA and proteinexpression in RMC that were transfected with FN1 (lane d), comparedwith the control (lane a) and RMC that were transfected withFN-Con (lane c), whereas it significantly increased fibronectinmRNA and protein expression in RMC that were transfected withFN-Con (lane e) compared with the control (lane a) and RMC thatwere transfected with FN-Con (lane c). These data further confirmthe specificity of the FN1 sequence for inhibition of fibronectinexpression in RMC. Because synthetic siRNA expression is transient,we next established an shRNA retroviral system for long-termsilencing of fibronectin synthesis in RMC
Silencing Fibronectin Expression by the shRNA Retroviral System in RMC
Several strategies to express siRNA via DNA-based plasmids withthe RNA polymerase III promoter, termed shRNA, have recentlybeen established (13–15). Although this technique providesa significant advantage over synthetic siRNA, primary MC aredifficult to transfect efficiently with plasmid DNA and showhigh variability. To overcome these problems, we establisheda retroviral system (pLXIN/H1) in which shRNA were under thecontrol of the H1 promoter (Figure 2A). For investigating theefficacy of the retroviral shRNA system, RMC were infected withpLXIN/H1-Con or pLXIN/H1-FN, and fibronectin expression wasassessed. Fibronectin deposition was significantly decreasedin RMC that were infected with pLXIN/H1-FN (Figure 2B, b) comparedwith the control (Figure 2B, a) and RMC that were infected withpLXIN/H1-Con (Figure 2B, c). Fibronectin mRNA expression wasalso markedly decreased in RMC that were infected with pLXIN/H1-FN(Figure 2C, lane b) compared with the control (lane a) and RMCthat were infected with pLXIN/H1-Con (lane c). Similar to RMCthat were transfected with siRNA FN1, Ang II failed to stimulatefibronectin expression in RMC that were infected with pLXIN/H1-FN(Figure 2C, lane d) but significantly increased it in RMC thatwere infected with pLXIN/H1-Con (lane e). These results suggestthat the retroviral shRNA system is effective and specific insilencing fibronectin expression in RMC
Figure 2. Inhibition of fibronectin expression in RMC that were infected with short hairpin RNA (shRNA) retrovirus. (A) Scheme of pLXIN/H1 construction: The hairpin sequence was inserted between the Bgl II and Sal I sites. (B) Confocal microscope analysis of the shRNA effect on fibronectin expression. Red fluorescence demonstrates fibronectin deposition. (C) Fibronectin mRNA analyzed by real-time PCR assay in five experiments (*P < 0.05; **P < 0.01).
Silencing Fibronectin Expression Triggers RMC Apoptosis
We noted that RMC that were infected with pLXIN/H1-FN becameless adherent and were detached from the culture dish afterthe second passage, compared with RMC that were infected withpLXIN/H1-Con. To determine whether the cells undergo apoptosis,we performed flow cytometry studies with double PI and AnnexinV staining. As seen in Figure 3A and Table 1, 18 and 4% of RMCthat were infected with pLXIN/H1-FN underwent early and lateapoptosis, respectively, compared with 2 and 1% of cells thatwere infected with pLXIN/H1-Con. The addition of 10 µgof fibronectin markedly reduced early and late RMC apoptosisto 3 and 1%, respectively. These findings suggest that fibronectinexpression is important in RMC apoptosis. To investigate whethermitochondrial function plays a role in RMC apoptosis inducedby pLXIN/H1-FN, we performed JC-1 assays. Indeed, the openingof mitochondrial permeability transition pore (PTP) has beendemonstrated to induce depolarization of the transmembrane potential,release of apoptotic factors, and loss of oxidative phosphorylation.As illustrated in Figure 3B, RMC that were infected with pLXIN/H1-FN(lane c) presented a significant loss of transmembrane potentialunder confocal microscope compared with the control (lane a)and RMC that were infected with pLXIN/H1-Con (lane b), indicatingmitochondrial dysfunction in RMC that were infected with pLXIN/H1-FN.The addition of 10 µg of fibronectin (lane d) reversedthe loss of transmembrane potential induced by infection withpLXIN/H1-FN. Flow cytometry revealed that 62% of mitochondrialpotential activities were lost in RMC that were infected withpLXIN/H1-FN compared with the control (Figure 3, C and D, lanea) and RMC that were infected with pLXIN/H1-Con (lane b), whereasthe addition of 10 µg of fibronectin (lane d) reversedthe loss of mitochondrial function induced by infection withpLXIN/H1-FN. These data demonstrated that mitochondrial functionis impaired by fibronectin inhibition with shRNA infection inRMC
Figure 3. Apoptosis and mitochondrial dysfunction induced by loss of fibronectin. (A) RMC that were infected with pLXIN/H1-Con or with pLXIN/H1-FN without or with the addition of fibronectin were passaged once then stained with annexin V and propidium iodide (PI), and apoptosis was analyzed by flow cytometry. The lower left quadrants show viable cells, which exclude PI and are negative for Annexin V. The upper right quadrants contain nonviable, necrotic, and late apoptotic cells, positive for FITC-Annexin V and PI uptake. The lower right quadrants represent apoptotic cells, Annexin V positive and PI negative. Transmembrane potential was assessed 96 h after RMC without infection (control, lane a), or infected with pLXIN/H1-Con (lane b), or pLXIN/H1-FN without (lane c) or with the addition of fibronectin (lane d) were passaged once, then stained with JC-1 dye, and analyzed by confocal microscope (B) or flow cytometry (C and D). Mitochondrial potential activity was quantified from four experiments (*P < 0.05).
Table 1. Loss of fibronectin triggers apoptosis of mesangial cellsa
RMC Apoptosis Induced by Silencing Fibronectin Is Mitochondria Dependent
Apoptosis consists of two classic pathways: The death receptor"extrinsic" and mitochondrial "intrinsic" pathways (20). Wefirst investigated, by Western blotting, whether the mitochondrialpathway is involved in RMC apoptosis induced by silencing fibronectinexpression in RMC that were infected with pLXIN/H1-FN, cytochromec, or caspase-9 or -3 activation by Western blotting. As illustratedin Figure 4, A and B, cytosolic cytochrome c was significantlyincreased in RMC that were infected with pLXIN/H1-FN (lane b)compared with pLXIN/H1-Con (lane a). The addition of exogenousfibronectin (lane d), treatments with CsA (lane c), PTP inhibitor,and Ac-DEVD-CHO (lane e), the caspase-3–specific inhibitor,partially decreased cytochrome c release in RMC that were infectedwith pLXIN/H1-FN. The complex sequentially cleaved procaspase-9into the active form (Figure 4, C and D; note the decrease ofprocaspase-9), and, subsequently, caspase-3 was activated (Figure 4, E and F)in RMC that were infected with pLXIN/H1-FN (laneb) compared with pLXIN/H1-Con (lane a). The addition of exogenousfibronectin (lane d), CsA (lane c), and Ac-DEVD-CHO (lane e)partially decreased activation of caspase-9 and -3. These resultsindicate that the mitochondrial pathway is important for RMCapoptosis induced by silencing fibronectin expression. For furtherconfirming these findings, RMC that were infected with pLXIN/H1-FNwere treated with CsA, fibronectin, or Ac-DEVD-CHO, and apoptosisassay was performed with double PI and Annexin V staining. Asshown in Figure 5A and Table 2, inhibition of PTP by CsA andcaspase-3 activity by Ac-DEVD-CHO abolished early RMC apoptosisto 5% compared with 16% in RMC that were infected with pLXIN/H1-FN,whereas CsA and Ac-DEVD-CHO had no effect on RMC apoptosis infectedwith pLXIN/H1-Con. These results further confirm that apoptosisinduced by silencing fibronectin is mitochondria dependent
Figure 4. Western blot analysis of cytochrome c release (A), caspase-3 (C), and caspase-9 (E). RMC were infected with pLXIN/H1-Con (lane a), with pLXIN/H1-FN (lane b), with pLXIN/H1-FN and incubated with 500 ng/ml cyclosporin A (CsA; lane c), with pLXIN/H1-FN and incubated with 10 µg/ml fibronectin (lane d), or with pLXIN/H1-FN and incubated with Ac-DEVD-CHO (lane e), and Western blotting analysis of cytochrome c, procaspase-9, and procaspase-3 expression was performed. Note that procaspase-3 cleaved into two active fragments, and procaspase-9 was decreased and cleaved into the active form. (G) Western blotting analysis of procaspase-8 expression in RMC that were infected with pLXIN/H1-Con (lane a) or with pLXIN/H1-FN (lane b). All blots were independently repeated three times, and quantification was done by laser densitometry for cytochrome c (B), caspase-3 (D), caspase-9 (F), and caspase-8 (H) (*P < 0.05).
Figure 5. (A) Effect of permeability transition pore and caspase-3 inhibitors, CsA, and Ac-DEVD-CHO on RMC apoptosis induced by silencing fibronectin expression. RMC that were infected with pLXIN/H1-Con or pLXIN/H1-FN were treated with CsA or Ac-DEVD-CHO. The cells were double stained with Annexin V and PI and analyzed by flow cytometry. (B) No effect on RMC apoptosis induced by silencing fibronectin gene expression by inhibition of caspase-8 activity. RMC that were infected with pLXIN/H1-Con or pLXIN/H1-FN were treated with Z-LETD-FMK. (C) Effect of calcium on RMC apoptosis induced by silencing fibronectin expression. RMC that were infected with pLXIN/H1-Con or pLXIN/H1-FN were treated with 3,4,5-trimethoxybenzoate (TMB-8), dantrolene, verapamil, or EGTA. TMB-8, dantrolene, verapamil, and EGTA had no influence on RMC apoptosis infected with pLXIN/H1-Con (see Table 4). The cells were double stained with Annexin V and PI, and apoptotic cells were analyzed by flow cytometry. The dot plots are representative of four independent experiments.
Table 2. CsA and Ac-DEVD-CHO inhibit apoptosis in RMC that were infected with pLXIN/H1-FNa
RMC Apoptosis Induced by Silencing Fibronectin Is Extrinsic Pathway -Independent
To investigate whether the "extrinsic" pathway is involved inRMC apoptosis induced by silencing fibronectin, we studied caspase-8activation by Western blotting. No changes of procaspase-8 expressionbetween RMC that were infected with pLXIN/H1-Con (lane a) orpLXIN/H1-FN (lane b) were found (Figure 4, G and H), suggestingthat the "extrinsic" pathway might play a minor role in RMCapoptosis evoked by silencing fibronectin expression. Furthermore,we investigated whether inhibition of caspase-8 activity byits specific inhibitor Z-LETD-FMK has an effect on RMC apoptosis.Its activation inhibited by Z-LETD-FMK had no impact on RMCthat were infected with pLXIN/H1-con or pLXIN/H1-FN, as shownin Figure 5B and Table 3, further confirming that the extrinsicpathway is not involved in RMC apoptosis induced by silencingfibronectin expression
Table 3. Inhibition of caspase-8 activity has no effect on RMC apoptosis induced by silencing fibronectin gene expressiona
Ca+ Plays an Important Role in RMC Apoptosis Induced by Silencing Fibronectin
Calcium has been reported to be important in apoptosis. Ca2+is released only transiently during bouts of signaling by theopening of IP3 or ryanodine receptor (RyR) Ca2+-release channels.For further investigating the participation of Ca2+ in RMC apoptosisinduced by pLXIN/H1-FN infection, EGTA, a Ca2+ chelator andvarious Ca2+ channel antagonists, dantrolene for RyR, TMB-8for IP3, and verapamil for the plasma membrane Ca2+ channel,were tested, and cell apoptosis was analyzed as described above.All inhibitors had no influence on RMC apoptosis infected withpLXIN/H1-Con compared with the control (Table 4). As shown inFigure 5C and Table 4, however, EGTA effectively inhibited earlyand late apoptosis to 4 and 1% compared with 16 and 2% of RMCapoptosis infected with pLXIN/H1-FN, respectively. TMB-8 alsopartially reversed early apoptosis to 6%, whereas it increasedlate apoptosis to 5% compared with RMC apoptosis infected withpLXIN/H1-FN. Verapamil and dantrolene had no influence on eitherearly or late apoptosis compared with RMC apoptosis infectedwith pLXIN/H1-FN. These data suggest that Ca2+ released fromthe endoplasmic reticulum (ER) accelerated apoptosis, and decreasingthe Ca2+ concentration could also retard this parameter
Increasing evidence indicates that the mitochondria may "sense"changes of ECM components. The absence of secreted type VI collagencauses mitochondrial dysfunction and induces myofiber-proneapoptosis (4), revealing a subtle relationship between the ECMand the mitochondria. In this study, we report that loss offibronectin provokes RMC apoptosis in a mitochondria-dependentmanner. The mechanism of RMC apoptosis induced by silencingfibronectin synthesis is due, at least in part, to Ca2+ modulation
Overproliferation of MC and ECM protein synthesis causes theprogression of glomerulosclerosis. ECM accumulation disruptsthe normal architecture of glomeruli and inevitably causes glomerulardysfunction (21). Suppressing cell proliferation and ECM accumulationis the main treatment target clinically. Many factors, includinghemodynamic factors, growth factors, and cytokines, contributeto the progression of glomerulosclerosis (19,22,23). Growthand survival of normal cells depend on adequate connectionswith ECM proteins. Among ECM components, fibronectin plays animportant role in cell differentiation and tissue regeneration.We previously reported that RGD peptides of fibronectin induceMC apoptosis (3). In this study, we used siRNA and shRNA tosilence fibronectin expression and confirmed that these techniqueswere effective and specific. It is interesting that we discoveredthat RMC that were infected with pLXIN/H1-FN underwent apoptosis.It is well known that adherent cells need to anchor the ECMfor survival. Adherent cells that are completely detached fromthe ECM will immediately initiate a suicide program apoptosis,or anoikis. Fibronectin contains several domains that mediatemultiple cell functions. The heparin-binding and RGD domainsare believed to provide survival signals for adherent cells.Synthetic peptides that contain the RGD motif as the apoptosisinducer have been used to treat MC proliferation. However, theshort half-life of oligopeptides limits their application inthe clinic. We propose that the siRNA and shRNA techniques appliedin this study may be helpful tools for the treatment of MC proliferation
Apoptosis consists of two main pathways, the death receptor"extrinsic" and mitochondrial "intrinsic" pathways, which areactivated by caspase-8 and -9, respectively. In the intrinsicpathway, under various apoptotic stimuli, the mitochondria releaseapoptogenic proteins, such as cytochrome c, Smac/DIABLO, andAIF, into the cytosol. These proteins can be released throughmitochondrial PTP or via a PTP-independent mechanism. The releasedcytochrome c binds to Apaf-1, which recruits and activates caspase-9,which, in turn, activates executioners such as caspase-3, degradingkey cellular substrates proteolytically (24). We tested threerepresentative members of the intrinsic pathway, cytochromec, caspase-9, and caspase-3, and observed pronounced mitochondrialdysfunction in RMC that were infected with pLXIN/H1-FN. Themitochondrial alterations were mediated, at least in part, byPTP. Caspase-9 and -3 were also activated in RMC that were infectedwith pLXIN/H1-FN, consistent with increased cell apoptosis.Furthermore, the caspase-3–specific inhibitor Ac-DEVD-CHOreversed the apoptosis. Taken together, our results confirmedthat the mitochondrial pathway is pivotal in RMC apoptosis.In addition, exogenous fibronectin could reverse apoptosis,and the cells could grow, albeit much more slowly than normalcells. Mooney et al. (25) reported that fibronectin exertedno antiapoptotic action in serum depletion–induced RMCapoptosis. The different results obtained in our and their studiesmay be due to fibronectin knockdown and serum depletion inducingRMC apoptosis. Fibronectin expression is not downregulated byserum depletion but is significantly inhibited by shRNA retrovirusinfection. Hence, the addition of exogenous fibronectin canreverse RMC apoptosis induced by the loss of fibronectin
Irwin et al. (4) provided evidence that collagen VI deficiencycauses mitochondrial dysfunction and leads to skeletal musclecell apoptosis. Insufficiency of calcium reuptake into the sarcoplasmicreticulum also participates in the process. Other emerging evidenceindicates that ER stress and ER calcium release play a key rolein apoptosis. Boehning et al. (26) reported that in the earlystage of apoptosis, cytochrome c translocates from the mitochondriato the ER and binds to the IP3 receptor, which, in turn, enhancescalcium release from the ER. This result in calcium releasethat then spreads and triggers massive cytochrome c releasefrom the mitochondria throughout the cell. Consequently, thecaspases are activated, and the cells die of apoptosis (26).We incubated the cells with three different calcium channelblockers, dantrolene for RyR, TMB-8 for IP3, and verapamil forthe plasma membrane Ca2+ channel. Only the TMB-8 group showedan improvement of early apoptosis but increased late apoptosis(Figure 5C and Table 4). We tested three different doses ofEGTA to modulate Ca2+ concentration and found that with lowerdoses, which reduced Ca2+ to two thirds of its normal concentration,apoptosis was ameliorated. Increasing the EGTA dose to decreaseCa2+ to one third of its normal concentration significantlyaccelerated apoptosis. These results confirm the important roleof calcium and that the loss of ECM components does affect ERfunction in handling Ca2+, but calcium as an apoptosis initiatoror assister needs to be investigated
In summary, using the retroviral shRNA delivery system, we knockeddown fibronectin synthesis in RMC. The loss of fibronectin inducedMC apoptosis in a mitochondria-dependent manner. Inhibitionof fibronectin also modulated Ca2+ with ER deregulation, which,in turn, prompted apoptosis. To the best of our knowledge, thisis the first report that fibronectin knockdown induces MC apoptosis.Our results may provide a new strategy to treat glomerulosclerosis
Acknowledgments
This study was supported in part by grants from the Main StateBasic Research Development Program of China (G2000057000), theCreative Research Group Fund of the National Foundation Committeeof Natural Science of China (30121005), and the National FoundationCommittee of Natural Science of China (30370655) to X.M.C.;and the Canadian Institutes of Health Research (MT-14726) andthe Canadian Foundation for Innovation to D.F.G
We thank Ovid M. Da Silva, Editor, Research Support Office,Research Centre, CHUM, for editing this manuscript
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Received for publication June 6, 2004.
Accepted for publication December 18, 2004.
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Abstract
Introduction
(1) integrin-mediated survival signals.
Am J Pathol 155
: 599
–606, 1999
