Hepatic fibrosis is the liver′s wound healing response to virtually all forms of chronic liver injury: toxic insult, viral infection, immunological conditions and metabolic diseases. Uncontrolled liver fibrosis eventually results in cirrhosis and associated complications, such as cancer and liver failure.1,2 Given the enormous public health implications, recent improvements in the treatment of chronic liver disease have accelerated interest in uncovering the mechanisms underlying hepatic fibrosis and its resolution.3 Currently, it is believed that activation of quiescent hepatic stellate cells (HSC) into proliferative, contractile, and fibrogenic cells in response to liver injury appears to be the dominant driving force in fibrosis.2
Phosphatase and tensin homolog deleted on chromosome ten (PTEN), a tumor suppressor phosphatase that dephosphorylates both protein and lipid substrates, inhibits the proliferation and promotes the apoptosis of tumor cells. In addition to its role in the development of both primary and malignant tumors, PTEN has also been investigated as a mediator of fibrogenesis. PTEN can arrest proliferation and drive apoptosis of lung fibroblasts both in vitro and in vivo.4,5 In a previous study, we showed that the expression of PTEN mRNA and protein were down-regulated in a rat model of hepatic fibrosis and that diminished PTEN expression was negatively correlated with the activation and proliferation of HSC in vivo.6 Currently, though, the effects of PTEN over-expression on the proliferation and apoptosis of cultured HSC remain unclear. In this study, we set out to determine the effects of PTEN over-expression, via adenoviral transduction, on the proliferation and apoptosis of HSC in vitro.
Adenovirus containing cDNA constructs encoding wild-type PTEN and green fluorescent protein (Ad-PTEN) and the empty virus control (Ad-GFP) were kindly provided by Prof. ZHU Jun-shang from the Third Military Medical University in China. Rabbit anti-PTEN polyclonal antibody, rabbit anti-Bcl-2 polyclonal antibody and rabbit anti-Bax polyclonal antibody were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, California, USA). Reverse transcription reaction system and SYBR® Green Real-Time Master Mix were bought from Tiangen Corporation (China). Terminal deoxyribonucleotidyl transferase mediated dNTP nick end labeling (TUNEL) assay kit was from Boehringer Mannheim Corporation (Germany).
The HSC-T6 cell line, which are phenotypically activated HSC, were obtained from Beijing Tumor Research Institute of the Chinese Academy of Medical Sciences. This cell line was cultured in supplemented DMEM (supplement: 8% fetal bovine serum (FBS), 100 U/ml penicillin, 100 µg/ml streptomycin, 4 mmol/L glutamine, and 1 mol/L HEPES) and grew in a 37°C, 5% CO2 humidified incubator. All experiments were conducted while cells were undergoing exponential growth. HSC were grouped as follows: (1) control group, where an equal volume of DMEM was used in place of the adenovirus; (2) Ad-GFP group, where HSC were infected with adenovirus expressing green fluorescent protein (GFP) alone; (3) Ad-PTEN group, where HSC were infected with adenovirus harboring genes for both wild type PTEN and GFP.
In vitro adenoviral gene delivery
After culturing for 24 hours, HSC were infected with Ad-PTEN or Ad-GFP at multiplicity of infection (MOI) 100. The adenoviral solution was diluted in enough DMEM (without FBS and antibiotics) to cover the monolayer of cells. After HSC were washed in DMEM (without FBS and antibiotics) twice, the adenoviral solution was added into culture dishes. After gentle agitation, the culture dishes were incubated at 37°C and 5% CO2. During the first 2 hours, dishes were agitated every 15 minutes. At 2 hours after infection, complete culture medium was replenished, and cells were incubated for 22 hours (24 hours in total). After this, culture medium was replenished and cells were then allowed to grow for times indicated below. GFP expression in HSC was observed under an inverted fluorescent microscope to confirm adenoviral infection, and flow cytometry (FCM) detection showed that 80% of cells were infected by adenovirus.
Western blotting analysis
At 72 hours after adenovirus infection, HSC were harvested, washed with phosphate-buffered saline (PBS), and then lysed with 1 ml of lysis buffer. After lysates were incubated in an ice bath for 30 minutes and centrifuged at 12 000 r/min for 15 minutes at 4°C; supernatants were collected and protein concentrations were determined using Coomassie brilliant blue staining. Western blotting analysis was subsequently performed using rabbit anti-PTEN polyclonal antibody, rabbit anti-Bcl-2 polyclonal antibody, rabbit anti-Bax polyclonal antibody or rabbit anti-β-actin polyclonal antibody. Blots were developed using enhanced chemiluminescene (ECL). The results were analyzed with Kodak 1D digital imaging software (Kodak, USA) and were reported as the optical density ratio of target protein to β-actin.
Real-time fluorescence quantitative PCR
At 72 hours after adenoviral infection, HSC were harvested and total RNA was isolated using the TRIzol kit. cDNA was then synthesized from 2-μg total RNA, using random primers and reverse transcriptase. PTEN-specific primers were purchased from Saibaisheng Gene Co. Ltd, China. The primer sequences are as follows: PTEN forward, 5′-ATA CCA GGA CCA GAG GAA ACC-3′ and PTEN reverse, 5′-TTG TCA TTA TCC GCA CGC TC-3′ (101-bp amplicon). Glyceraldehyde- 3-phosphate dehydrogenase (GAPDH) was used as reference gene using the following primers: GAPDH forward, 5′-TCC CTC AAG ATT GTC AGC AA-3′ and GAPDH reverse, 5′-AGA TCC ACA ACG GAT ACA TT-3′ (300-bp amplicon). Real-time fluorescence quantitative PCR was subsequently performed in an ABI7900 thermal cycler (ABI Corporation, USA). The amplified products were confirmed by agarose gel electrophoresis. Each Ct value (the cycle number when the fluorescence curve crossed baseline) represents the mean value from 3 independent experiments. Using a fold increase calculation (2–△△Ct method),7 the differences of PTEN mRNA expression among groups were compared.
Cell proliferation assay: MTT
HSC were seeded into a 96-well plate at a density of 3×104/ml × 200 µl/well. Adenoviral gene delivery was performed as described above. At 24, 48 and 72 hours after HSC were treated, MTT solution of 5 mg/ml × 20 µl/well was added into the wells respectively. Cells were allowed to grow for 4 hours and then the medium was replaced with 150-μl dimethylsulfoxide (DMSO). The absorbance (A), measured at 490 nm, was used to calculate the inhibition rate of proliferation ((Acontrol–Aexperimental)/Acontrol ×100%).
Apoptosis assay: TUNEL
After 72 hours of adenoviral infection, HSC were analyzed for apoptosis using a TUNEL assay according to the manufacturer′s protocol. The nuclei of apoptotic HSC yielded a brown-colored positive reaction. The apoptotic rate was calculated as the ratio of apoptotic cells (counting in 10 randomly selected visual fields) to total number of nucleated cells.
Apoptosis assay: FCM
At 72 hours after adenoviral gene delivery, HSC were harvested. Cells were subsequently washed with PBS twice, and fixed with 70% ice-cold ethanol at 4°C overnight. After cells were stained with propidium iodide (PI) at 4°C for 30 minutes, apoptotic rates were analyzed by FCM.
Tabulated data were expressed as mean ± standard deviation (SD), as calculated using SPSS 13.0 software. The mean variability among all groups was compared using one-way analysis of variance (ANOVA) analysis and the Tukey test. Statistical significance was assigned when P <0.05.
Expression of exogenous PTEN in HSC in vitro
Real-time fluorescence quantitative PCR was used to extrapolate the relative mRNA expression levels of PTEN in HSC. It was found that the amplification efficiency was essentially uniform and the results were reproducible. PTEN mRNA expression levels in Ad-GFP group and Ad-PTEN group were 0.99- and 1.57-fold, respectively. The expression value of the untreated control group was arbitrarily assigned an expression value of 1. Expression of PTEN mRNA was significantly higher (P <0.01) in the Ad-PTEN group than those in both the control group and the Ad-GFP group (Table 1).
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Table 1. Relative expression of PTEN mRNA in HSC at 72 hours after transduction by real-time fluorescence quantitative PCR (n=6)
Western blotting analysis recapitulated the fluorescence quantitative PCR data by showing that expression levels of PTEN protein were significantly higher (P <0.01) in the Ad-PTEN group (1.66±0.09) than those in the control group (1.10±0.07) and the Ad-GFP group (1.09±0.07). Furthermore, no significant difference was observed in expression of PTEN protein between the control group and the Ad-GFP group (P >0.05, Figure 1).
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Figure 1. Western blotting shows PTEN expression in HSC at 72 hours after adenoviral transduction. A: PTEN protein expression in HSC in each group. B: The loading control protein β-actin. C: The relative optical density values of PTEN protein in HSC in each group (n=6, *P <0.01 vs control, Ad-GFP groups).
HSC proliferation was inhibited by over-expression of PTEN
There was no significant difference in the proliferation of HSC at 24 hours after transduction (P >0.05). At 48 and 72 hours after transduction, though, a precipitous time-dependent drop in proliferation was observed in the Ad-PTEN group. Inhibition rates were 13.25% and 21.60%, respectively, when compared with the Ad-GFP group (P <0.01). The A value between control and Ad-GFP groups showed no notable difference at various time points, P >0.05 (Table 2).
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Table 2. Effect of PTEN on HSC proliferation by MTT (n=6)
HSC apoptosis was induced by over-expression of PTEN
The TUNEL assay (Figure 2) showed that the apoptotic rate of the Ad-PTEN group ((29.81±2.52)%) at 72 hours after transduction was significantly higher (P <0.01) compared with both the control group ((1.98±0.25)%) and the Ad-GFP group ((2.16±0.28)%). PI-labeled FCM (Figure 3) confirmed this observation, showing that the apoptotic rate of the Ad-PTEN group ((20.84±1.44)%) at 72 hours was markedly higher (P <0.01) compared with both the control group ((1.12±0.57)%) and the Ad-GFP group ((1.21±0.22)%).
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Figure 2. Apoptosis of HSC at 72 hours after adenoviral infection as detected by TUNEL (original magnification ×200). A: Control group. B: Ad-GFP group. C: Ad-PTEN group. D: Apoptotic rates of HSC detected by TUNEL (n=6, *P <0.01 vs control, Ad-GFP groups).
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Figure 3. Apoptosis of HSC at 72 hours after adenovirus infection as detected by PI-labeled FCM. A: Control group. B: Ad-GFP group. C: Ad-PTEN group. D: Apoptotic rates of HSC detected by PI-labeled FCM (n=6, *P <0.01 vs control, Ad-GFP groups).
Over-expression of PTEN down-regulated Bcl-2 and up-regulated Bax in HSC
Western blotting analysis showed that the expression of Bcl-2 was significantly decreased (P <0.01) at 72 hours in the Ad-PTEN group compared with the control group and Ad-GFP group (1.16±0.03 vs 1.37±0.06, 1.34±0.08, respectively). In contrast, the expression of Bax in HSC at 72 hours in the Ad-PTEN group was markedly higher (P <0.01) compared with the control group and Ad-GFP group (1.50±0.05 vs 1.28±0.06 and 1.26±0.08, respectively). Moreover, there were no significant differences (P >0.05) in either Bcl-2 or Bax expression between the control group and Ad-GFP group (Figure 4).
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Figure 4. A: Western blotting shows Bcl-2 and Bax expression in HSC at 72 hours after adenoviral infection in different groups. B: The relative optical density values of Bcl-2 and Bax protein in HSC in different groups (n=6, *P <0.01 vs control, Ad-GFP groups).
HSC are normally quiescent cells that store large amounts of vitamin A. During hepatic fibrogenesis, the activation of HSC is promoted by a variety of factors, such as acetaldehyde,8 cytokines, or oxygen-derived free radicals.9 Once activated, they migrate to the site of hepatic injury. There they proliferate and express various signal transduction proteins, producing pro-inflammatory cytokines and a great deal of collagen-rich extracellular matrix. At present, it is believed that the number of activated HSC is reduced mainly via apoptosis during the reparative process following liver injury.10
In this study, after HSC were made to over-express PTEN, an MTT assay was performed to determine the degree of growth inhibition of HSC in vitro. These results indicated that over-expression of PTEN inhibited HSC proliferation in a time-dependent manner. This is in accordance with our previous study, which showed that the expression of PTEN in liver tissues had a significant negative correlation with the proliferation of HSC in vivo.6
Nho and his colleagues5 found that PTEN phosphatase becomes activated during collagen matrix contraction and appears to be responsible for promoting lung fibroblast apoptosis during pulmonary fibrosis repair. Moreover, they showed that PTEN null lung fibroblasts are resistant to collagen matrix contraction-induced apoptosis. Other studies have demonstrated that PTEN inhibits the proliferation of lung fibroblasts cultured in vitro.4 Collectively, these studies showed a key role for PTEN in regulating the viability of lung fibroblasts, which plays a pivotal role in pulmonary fibrosis.
In the present study, we have showed the influence of over-expression of PTEN on HSC apoptosis in vitro. TUNEL and PI-labeled FCM results confirmed that over-expression of wild type PTEN induced apoptosis of HSC at 72 hours after transduction of PTEN gene. Interestingly, the apoptotic rate detected by TUNEL was significantly higher than that detected by PI-labeled FCM.
This perhaps can be attributed to TUNEL detection of both early and late stage of apoptosis (PI-labeled FCM is specific only to the late stages). Also, TUNEL is a more sensitive assay in general, as it is able to detect apoptotic DNA degradation fragments in relatively small cell populations (2×103 to 5×103 cells).
The genes Bcl-2 and Bax have been shown to play a central role in the process of apoptosis. Bcl-2 expression has been shown to promote survival, while Bax, a dominant negative inhibitor of Bcl-2, promotes apoptosis by forming a non-functional heterodimer with Bcl-2. As such, the ratio of Bcl-2 to Bax (Bcl-2/Bax) is used as an indicator of whether or not a cell population is apoptotic.11 The PTEN phosphatase can induce apoptosis of glioblastoma cells through negative regulation of PI3K/Akt and ERK signaling, as well as by decreasing expression of Bcl-2 and increasing expression of Bax.12 Here, we show that Bcl-2 expression decreased and Bax expression increased after PTEN transduction into HSC, resulting in a lower Bcl-2/Bax ratio.
Our results indicate that over-expression of PTEN has the potential to induce robust apoptosis in HSC, much in the same way it has been shown to do in tumor cells.13 Still, though, further investigation must be done to elucidate the precise mechanism of PTEN-mediated apoptosis in HSC. Uncovering these pathways will no doubt play a central role in the discovery of new strategies for both prevention and treatment of liver fibrosis.
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