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Anti-fibrotic Effects via Regulation of Transcription Factor Sp1 on Hepatic Stellate Cells

Background: Hepatic stellate cells (HSCs), the central cells in hepatic fibrosis, are characterized by sustaining activation, a process that consists in increased proliferation and over-expression of fibrotic genes. Transcription factor Sp1 mediates the expression of a variety of fibrotic genes expression and thereby play an important role in fibrosis. In addition, previous reports have indicated that Sp1 binding activity is greatly increased in activated HSCs. Thus, our aim was to investigate the anti-proliferative and anti-fibrotic effects of the oligonuceotide decoy of the transcription factor Sp1, ODN, a potent inhibitor of Sp1-activated transcription. Methods: We optimized Lipofectamin 2000 (LF2000):ODN DNA ratio for the transfection of hepatic stellate cells HSC-T6. Then we measure the effect of transfected ODN on HSC-T6 cells′ proliferation and fibrotic gene expression, and study the mechanism involved. Results: At a DNA concentration of 1 µM and a ratio ODN DNA:LF2000 of 1:3, HSC-T6 cells have the maximal transfection efficiency with the lowest toxicity. Transfected ODN effectively blocks Sp1 binding to the promoter regions of cell cycle regulatory proteins cyclin D1, p27KIP1 and fibrotic genes, including transforming growth factor (TGF)-β1, Platelet-derived growth factor (PDGF)-BB, α-SMA, α1 (I) collagen and tissue inhibitor of metalloproteinases-1 (TIMP-1). ODN inhibits HSC-T6 proliferation and fibrotic genes expression in vitro. Conclusion: Sp1 is a key transcription factor that mediates proliferation and fibrotic gene synthesis of HSC-T6, inhibition of Sp1 with decoy ODN may be an effective approach to prevent the progression of hepatic fibrosis.


Introduction
Hepatic fibrosis, a major medical problem worldwide, is characterized by the accumulation of excessive collagen and other extracellular matrix components, resulting in the destruction of the liver parenchyma architecture and 52 loss of function. The hepatic stellate cell (HSC) is the key cellular element in hepatic fibrosis. Upon liver injury, HSCs become active, undergoing phenotypic change from quiescent cells to myofibroblast-like cells. Activated HSCs are characterized by enhanced cellular proliferation, overproduction of extracellular matrix components and a variety of growth factors and cytokines, appearance of the characteristic activation marker smooth muscle α-actin (α-SMA), thereby playing a central role in the development and maintenance of hepatic fibrosis [1,2]. Because of its importance in the fibrotic process, there is considerable interest in establishing the molecular events that regulate HSCs function in fibrosis [3]. HSCs function at the level of gene transcription requires several key transcription factors include NF-κB, AP-1, PPAR-γ and Kruppel-like factors (KLFs).
Sp1 is a family member of Kruppel-like factors which is largely associated with GC-rich promoters [4]. In addition to its participation in regulating a great variety of housekeeping genes, there is growing evidence that Sp1 plays an important regulatory role in other diverse cellular functions including cell proliferation and fibrosis [5][6][7][8]. Sp1 regulates the expression of several genes relevant to fibrosis process, including TGF-β1, VEGF, COL1A2, and downstream targets of TGF-β1, such as PAI-1, fibronectin and MMPs [9][10][11][12][13]. In addition, previous work by other groups have shown that Sp1 DNA-binding activity is increased in various form of fibrosis include activated HSCs [14,15].
Recently, some novel anti-fibrotic approaches have been developed in an attempt to reduce the activity of specific fibrotic genes. Introduction of the oligonucleotide decoys with high affinity for their target transcription factors into specific cells leads to the selective downregulation of the specific gene expression at DNA level [16,17]. In these decoy ODNs, Sp1 decoy ODN is found to suppress the expression of VEGF, TGF-β1 and fibronectin in kidney fibroblasts and in vivo [18]. In another report, Park et al [19] have reported that Sp1 decoy inhibits the expression of TGF-β1 and its downstream target genes in vivo model of liver fibrosis, but its action in hepatic stellate cells remains to be elucidated.
In the present study, we investigated the role of Sp1 in HSC-T6 cells (an SV40-immortalized rat HSC line) using decoy ODN containing the Sp1-binding site to specifically inhibit the expression of Sp1. This investigation was performed to 1) optimize the Sp1 decoy ODN transfection conditions in HSC-T6; 2) test the hypothesis that transfection of Sp1 decoy ODN prevents HSC-T6 proliferation and fibrotic genes expression in vitro; 3) disclose its possible mechanism underlying proliferation of HSC-T6 cells.

Optimization of transfection condition
HSC-T6 cells were trypsinized and used to seed 24-well gelatin-coated plates (10 5 cells/well), 500 µl of growth medium without antibiotics was added so that cells will be 70%-80% confluent at the time of transfection. Lipofectamine 2000 (LF2000; Invitrogen, San Diego, CA, USA) was used for all transfections. DNA (0.4-8 µg) was diluted in Opti-MEM (Invitrogen) to a final volume of 50 µl. LF2000 (1.2-24 µl) was diluted in Opti-MEM to a final volume of 50 µl. The two solutions were incubated at room temperature for 20min and then added to each well containing cells and 500ul of serum-free medium. The final concentration of decoy ODN was from 0.1 µM to 2 µM. A DNA (µg): LF2000 (µl) ratio of 1:3 was routinely used according to the optimization result, unless otherwise specified.

Microscopy and fluorescence detection
After transfection, HSC-T6 cells were washed with PBS, observed using a Bx51 fluorescence microscope (Olympus, Japan) and photographed with Olympus DP-70 digital camera (Olympus, Japan). The FITC was excited by an argon laser (488 nm) and the fluorescence was detected though a bandpass filter (510-550 nm).

Flow cytometry
After a 6h transfection period, HSC-T6 cells were washed, trypsinized, dispersed and then transferred into 500 µl PBS. Untransfected cells were included in every experiment and typically exhibited minimal autofluorescence. In this report, cellular uptake efficiencies refer to the percentage of cells that Chen/Zhou/Chen/An/Ji/Chen Cell Physiol Biochem 2012;29:51-60 captured the fluorescent ODN. The mean fluorescence intensity refers to the average of fluorescence intensity expressed by the population of cells that captured ODN.
For detection of cell cycle, HSC-T6 cells cultured 48h after transfection were washed twice with PBS and fixed in cold ethanol for 30 min, and then incubated with propidium iodide (PI) for 30 min. Thereafter, cells (1-2×10 5 ) were analyzed for each sample by flow cytometry (BD, USA). Cell death was measured using the Annexin-PE Apoptosis Detection Kit I (BD Bioscience).

Assay of cell viability and lactate dehydrogenase activity
Cell viability was determined by trypan blue exclusion. HSC-T6 cells were seeded in 96-well plates at 1×10 4 cells/well. After transfection, HSC-T6 cells were washed and harvested in PBS to prepare a single-cell suspension. 50 µl of the suspension was added to 0.95 ml 0.4% trypan blue solution. The total number of living and dead cells was counted with a haemacytometer. The activity of lactate dehydrogenase (LDH) in cultured supernatant was assayed by a LDH assay kit (Jiancheng Biology Engineering Institute, Nanjing, China) according to the manufacturer's instruction.
Cell proliferation HSC-T6 cells were seeded on 96-well plates at an initial density of 1×10 4 cells/well. At 40-60% confluence, cells were rendered quiescent by incubation for 24 h in growth medium deficient in serum. To evaluate the effect of the Sp1 decoy ODN on HSC-T6 cell proliferation, PS-Sp1 decoy ODN, M-Sp1 decoy ODN and LF2000 alone were added to the wells, cells cultured with serum-free medium were used as control. HSC-T6 Cells were then incubated at 37°C for a further 6 h and replace fresh growth medium after transfection. Cell proliferation was determined with a WST cell counting kit (Wako, Osaka, Japan) and cell counting. The PCR cycles were 95°C for 30 seconds, followed by 40 cycles of 95°C for 10 seconds and 60°C for 30 seconds. Each reaction was performed in triplicate and analyzed individually, relative to β-actin (a normalization control), calculated using the 2 -ΔΔCt method. Thereafter, data for transcript expression levels were expressed as fold difference relative to that of negative control cells.
Western blot and antibodies HSC-T6 cells from each group were centrifuged into pellets and extracted in ice-cold cell lysis buffer (Cell Signaling Technology, Inc., Danvers, Mass). After centrifugation for 10 min at 4°C, the supernatants were separated by 10% SDS-PAGE and transferred to nitrocellulose membranes. The membranes were incubated for 1h at room temperature with blocking solution (5% nonfat milk; Sigma-Aldrich Corporation, St. Louis, Mo), followed by incubation overnight at 4°C with primary antibodies. Next, the membranes were washed with 1×trisbuffered saline with Tween-20 solution and incubated with a horseradish peroxidase conjugated secondary antibody. Antibody-antigen complexes on the membranes were detected using an ECL system (Amersham Life Sciences, Buckinghamshire, UK), and the relative intensities of the protein bands were quantitated with Quantity One software (Bio-Rad).

Statistical analysis
Data are presented as means ± SD of at least three separate experiments. Difference between two groups was analyzed by two-tailed Student's t-test, and difference among three or more groups was analyzed by one-way ANOVA multiple comparisons. Statistical values of p±0.05 were considered to be significant.

Optimization of transfection condition
Lipoplexes condensation is the first limiting step in lipofection and determines the proportion of DNA that becomes available for uptake. In order to obtain the best delivery efficiency, we tested several LF2000:ODN ratios. Lipoplexes were formed at 1:1, 1:3, and 1:5 DNA: LF2000 ratios, using 0.5 µM of FITC-labeled decoy ODN. After transfection of HSC-T6 cells, increasing LF2000 concentrations lead to a greater proportion of cells taking up DNA (Fig. 1A, C and D), suggesting the production of greater numbers of lipoplexes. However, at the highest LF2000 concentration, DNA uptake per cell decreases. Taken together, the delivery of Sp1 decoy ODN was optimal at a DNA: LF2000 ratio of 1:3.
DNA concentration is another key step in lipofection. Insufficient concentrations of DNA will result in poor transfection efficiencies, while an excess of lipoplexes will compromise cell viability. To determine the optimal DNA concentration, HSC-T6 was transfected with increasing concentrations of FITC-labeled Sp1 decoy ODN using an optimal DNA: LF2000 ratio (1:3). As illustrated in Fig. 2A-C, DNA uptake per cell was proportional to lipoplex concentration at all concentrations tested. At DNA concentrations below 1 µM, transfection efficiency increased proportionally to DNA concentration. However, exposing HSC-T6 to concentrations above 1 µM resulted in no further increase in cellular uptake efficiency. Moreover, concentration above 1 µM of ODN was shown toxic to HSC-T6 in culture (Fig. 2D). These results suggest that a DNA concentration of 1 µM will be optimal for transfection of HSC-T6. As the transfection condition (1 µM of DNA at a DNA: LF2000 ratio of 1:3) provided maximal DNA delivery efficiency and minus toxicity for HSC-T6, this transfection condition was used in further experiments.

Sp1 decoy ODN prevents Sp1 binding activity
To investigate the effect of Sp1 decoy ODN on Sp1 DNA binding activity, EMSA was performed with nuclear extracts of HSC-T6 that had been transfected with decoy ODN. As shown in Fig. 3, compared with control HSC-T6, transfection with Sp1 decoy ODN, but not with the M-Sp1 decoy ODN, significantly attenuated the DNA binding activity. The binding was specific, as evidenced by the complete suppression of the formation of the DNA-protein complexes in the presence of an excess of unlabeled competitor ODN.
To further explore the reason for the growth inhibition, we examined the effects of Sp1 decoy ODN on cell cycle progression. As illustrated in Fig. 4C, pretreatment of HSC-T6 with Sp1 decoy ODN blocked the cell cycle in G1 phase. The G0/G1-phase fraction increased from 46.33% (untreated) to 71.97% at 48h post-transfection with 1 µM of Sp1 decoy ODN. However, no significant change in cell cycle arrest was seen in both cells treated with Sp1 M-ODN and LF2000 alone. Meanwhile, to evaluate whether the inhibition of cell growth might also have occurred due to apoptotic cell death, we also performed apoptosis assay using flow cytometry. However, there was no significant increased apoptosis in transfected cells, when compared with the control cells (Fig. 4D). Therefore, our results indicate that Sp1 decoy ODN inhibits the proliferation of HCS-T6 provoking the arrest of cells at the G1 phase of the cell cycle.
In addition, the molecular mechanism for cell cycle arrest in HSC-T6 was also investigated. At 48 h post-transfection, Sp1 decoy ODN but not Sp1 M-ODN, induced down-regulation of cyclin D1 simultaneously with up-regulation of p27 kip1 in HSC-T6 (Fig. 4E). Furthermore, we also performed quantitative real-time PCR assay to measure mRNA, and the results correlated with those of Western blot (Fig. 4F). Together, these results indicate that Sp1 decoy ODN inhibits cellular proliferation which may be associated with transcription modification of cyclin D1 and p27 kip1 in HSC-T6.

Sp1 decoy ODN inhibits the expression of fibrosis genes in HSC-T6
The effects of Sp1 decoy ODN on the expression of fibrosis genes in HSC-T6 were investigated. Introduction of the Sp1 decoy ODN into HSC-T6 significantly inhibited both mRNA and protein expression of TGF-β1, PDGF-BB, α-SMA, α1 (I) collagen and TIMP-1 compared with the control group. However, such a significant suppressive effect was not observed in HSC-T6 treated both M-Sp1 decoy ODN and LF2000 alone. These results clearly show that R-Sp1 decoy effectively inhibits fibrotic changes in HSC-T6, indicating that the transcription of these genes are at least partly regulated by Sp1 binding site in their promoter.

Discussion
Activation of Sp1 is an important event during fibrosis where several reports have shown it regulates a great number of fibrotic gene expression in various form of fibrosis [9][10][11][12][13]21]. Furthermore, the anti-fibrotic effects of Sp1 decoy ODN were observed in vitro and in vivo models of fibrosis, including renal [18], lung [22] and liver [19]. Nevertheless, no reports on the role of Sp1 decoy ODN in HSCs have been investigated. The major new finding of the present study is that an optimal transfection of Sp1 decoy ODN effectively inhibits the expression of the fibrotic genes and the proliferation of HSC-T6 and may play a role in the sustained activation of HSC-T6.
The use of decoy ODN for reducing the transactivity of transcription factor is an innovative and attractive strategy for gene therapy. However, the use of decoy ODN is limited by their generally low efficiency of transfection [23]. To circumvent this problem, we are therefore investigating the factors that limit the transfection of HSC-T6 with Sp1 decoy ODN, with a view to developing an efficient delivery method. During the condensation process, lower DNA: LF2000 ratios may increase the rate of decoy ODN encapsulation, generating greater numbers of lipoplexes with fewer ODNs being recruited to each lipoplex. Meanwhile, higher DNA: LF2000 ratios are likely to cause aggregation, leading to the sequestering of DNA molecules in large, untransfectable particles. In our study, an optimal DNA: LF2000 ratio of 1:3 in HSC-T6 supports this interpretation. Concentration of ODN is another important factor influencing the transfection. We observed that increasing DNA concentration above 1 µM resulted in no further increase in the proportion of cells taking up ODN, which may reflect a saturation of HSC-T6 at such concentrations. In support of this interpretation, concentration above 1 µM of ODN was shown toxic to HSC-T6 in culture. Taken together, we find that optimal transfection condition of HSC-T6 is achieved using 1 µM of DNA at a DNA: LF2000 ratio of 1:3.
Previous reports suggest a role for Sp1 in the cell cycle. It has been shown that Sp1 is increasingly phosphorylated in G1 phase of the cell cycle [7] and interacts with certain cell cycle regulatory proteins such as cyclin D1, EGFR, p27 Kip1 and p21 WAF1/cip1 [24][25][26]. Our results indicated that down-regulation of Sp1 was associated with a gradual decrease in proliferation of HSC-T6. Moreover, we did not detect significant apoptotic cell death after inhibition of Sp1 in HSC-T6, and the inhibition of cell growth can be partly attributed to the occurrence of G1 cell cycle arrest. These observations are consistent with up-regulation of p27 kip1 expression and down-regulation of cyclin D1, both of which are required for cell cycle progression in G1, suggesting that the Sp1 is involved in controlling the cell cycle in G1 phase, which is consistent with the research in epithelial cells by Grinstein et al. [27].
Among the transcription factors, Sp1 regulates the expression of a vast variety of genes, including TGF-β1, fibrotic cytokine, and a wide variety of matrix gene [12][13][14][15][16]23]. In the present study, our data showed that transfection with Sp1 decoy ODN can effectively attenuate the expression of TGF-β1, PDGF-BB and TIMP-1 in HSC-T6. It has been reported that repeated injection of R-Sp1 decoy ODN though the mouse tail vein can effectively decrease the liver fibrosis induced by CCl4 in vivo [21]. Together with the present results, it may suggest that this strategy specifically suppresses Sp1-mediated transcription of fibrotic genes in liver, and thus can be used as an effective therapy for hepatic fibrosis. In addition to these results, α-SMA, the characteristic activation marker of HSCs, was decreased after the transfection of Sp1 decoy ODN. Moreover, the expression of α1 (I) collagen, another classical markers for activated HSCs, was also down-regulated. This finding Effects of Sp1 on Hepatic Stellate Cells is supposed to be an evidence of Sp1 may partly play a role in the sustained activation of HSC-T6.
In conclusion, our data clearly demonstrate that Sp1 is a key transcription factor mediated proliferation, fibrotic genes synthesis of HSC-T6. Transfection with Sp1 decoy ODN seems to be an effective strategy to limit the progression of fibrosis [28]. However, the gene manipulation of Sp1 need to claim attention since there might be serious adverse effects relating to development, differentiation, and metabolism, especially in case targeting is insufficient [29][30][31]. To establish the Sp1-directed gene therapy for hepatic fibrosis, development of cell-specific gene modulation for HSCs will be essential.