A novel function of the human oncogene Stil: Regulation of PC12 cell toxic susceptibility through the Shh pathway

The human oncogene SCL/TAL1 interrupting locus (Stil) is highly conserved in vertebrate species. Here, we report new findings of Stil in the regulation of toxic susceptibility in mammalian dopaminergic (DA)-like PC12 cells. RNAi-mediated knockdown of Stil expression did not affect the survival of proliferating PC12 cells but caused a significant amount of cell death in differentiated neurons after toxic drug treatment. In contrast, overexpression of Stil increased toxic susceptibility only in proliferating cells but produced no effect in mature neurons. Exogenetic inactivation or activation of the Sonic hedgehog (Shh) signaling transduction mimicked the effect of Stil knockdown or overexpression in regulation of PC12 cell toxic susceptibility, suggesting that Stil exerts its role through the Shh pathway. Together, the data provide evidence for novel functions of the human oncogene Stil in neural toxic susceptibility.

and Caspase-3 mediated signal transduction pathways. These findings provide evidence of differential effect of Stil expression in proliferating and differentiated cells, and reveal a novel function for Stil in neural protection.
Previous studies have demonstrated that Stil functions in the Shh pathway in DA neurons 9,10 . To investigate if the effect of Stil in regulation of PC12 cell toxic susceptibility is mediated by Shh signaling transduction, we measured PC12 cell survival in response to 6-OHDA treatment while the transduction of the Shh signals was modified by knockdown/overexpression of the Stil transcript or by inhibition/ activation of Smo receptors which function in the Shh pathway [23][24][25] . In proliferating PC12 cells, transfection with pSIREN-Stil-shRNA or pCS2+ Flh-Stil altered the level of STIL protein expression ( Fig. 2A,D). Inhibition or activation of Smo receptors (by treatment with Smo receptor antagonist cyclopamine or receptor agonist purmorphamine) not only altered Shh signaling transduction (i.e., Gli1 transcription), but also modified the expression of STIL proteins. In response to cyclopamine treatment (10 and 20 μ M), for example, the expression of GLI1 was decreased to levels that were 71.8 ± 9.8 and 11.0 ± 7.2% of the control cells, and the expression of STIL was increased to levels approximately 4-5 folds higher than controls (Fig. 2B,E). In purmorphamine (5 and 10 μ M) treated cells, the expression of Gli1 was increased by 3-4 folds as compared to control cells but the expression STIL proteins was decreased, i.e., to 90.3 ± 3.6 and 65.1 ± 9.5% of the control level (Fig. 2C,F).
In proliferating PC12 cells, inhibition of Shh signaling transduction (i.e., by cyclopamine treatment) produced no obvious effect on cell's drug susceptibility to 6-OHDA (Fig. 2G). However, when the Shh signaling in cyclopamine-treated cells was factitiously elevated, i.e., by overexpression of Stil transcripts (transfected with pCS2+ Flh-Stil plasmid), the toxic susceptibility of PC12 cells to 6-OHDA was increased. For example, in response to 6-OHDA treatment (75 μ M), the survival rate of cyclopamine-treated and plasmid-transfected cells was decreased to 64.9 ± 8.7% of the control level (Fig. 2G). Activation of Shh signaling transduction (by treatment with purmorphamine) increased the toxic drug susceptibility of proliferating PC12 cells, but this could be reversed by factitious down-regulation of Shh signaling transduction. In purmorphamine-treated cells, for example, in response to 6-OHDA treatment (75 μ M) the survival rate was decreased to 56.8 ± 10.3% of the control level (Fig. 2G). Down-regulation of Stil expression (by transfection with pSIREN-Stil-shRNA plasmid) in purmorphamine-treated cells blocked the effect of Smo receptor-initiated Shh signaling on drug toxicity, i.e., in response to 6-OHDA treatment the survival rate of the cell was increased to the control level (Fig. 2G).
In NGF-induced PC12 cells, inhibition of Shh signaling transduction (by cyclopamine) increased PC12 cell's toxic susceptibility, thereby decreasing the rate of cell survival (i.e., to 55.0 ± 10.8% of the control level) (Fig. 2H). A complete rescue in cell survival could be achieved by increasing STIL expression (by transfection with pCS2+ Flh-Stil plasmid) (Fig. 2H). Activation of the Shh pathway (by purmorphamine) produced no effect on drug susceptibility in differentiated PC12 cells (Fig. 2H). However, when the expression of Stil was factitiously inhibited (transfection with pSIREN-Stil-shRNA plasmid), the survival rate of purmorphamine-treated cells was decreased (i.e., to 60.3 ± 10.2% of the control level) (Fig. 2H). In this case, although the early signaling events in the Shh pathway were promoted, the production of STIL was inhibited by shRNA and thereby the transcription of Gli1 was inhibited.
The decrease of cell survival of PC12 cells after 6-OHDA treatment is due to the increase of caspase-mediated cell death. This was verified by analyzing cleaved Caspase-3 protein expression, caspase activation, and by using the TUNEL assay. Western blot revealed significant increases in the expression of cleaved Caspase-3 proteins in proliferating PC12 cells in response to 6-OHDA treatment when the expression of Stil was elevated (i.e., in cells transfected with pCS2+ Flh-Stil plasmid) (Fig. 3A). Caspase activation assays also revealed an increase (1.8 ± 0.2 folds) of caspase activity in Stil-overexpression cells as compared to control cells in response to 6-OHDA treatment (Fig. 3C). In contrast, in proliferating PC12 cells knockdown of Stil expression (by transfecting the cells with pSIREN-Stil-shRNA plasmid) produced no obvious effects on Caspase-3 expression and caspase activity after 6-OHDA treatment as compared to control cells (transfected with scramble sequence plasmid) (Fig. 3A,C). The increase of caspase activity led to increased apoptotic cell death revealed by the TUNEL assay. For example, in proliferating PC12 cells, in response to 6-OHDA treatment, the amount of cell death in Stil overexpression cells was 3.1 ± 0.1 folds higher than the rate of cell death in control cells (Fig. 3E). In NGF-induced PC12 cells, in response to 6-OHDA treatment increases in cleaved Caspase-3 expression and caspase activation were seen in Stil-knockdown cells, but not in Stil-overexpression cells (Fig. 3B,D). Increases in apoptotic cell death were seen in Stil-knockdown cells when treated with 6-OHDA as revealed by the TUNEL assay (Fig. 3F).

Discussion
In vertebrates, STIL is expressed in different cell types where it regulates the process of cell proliferation. In a recent study, we examined the role of STIL in neural degeneration and regeneration in zebrafish models and mammalian PC12 cells. In zebrafish retinas, functional expression of STIL is required for DA cell regeneration after drug-induced degeneration 17,18 . In mammalian PC12 cells, the expression of STIL is required for cell proliferation but not neural differentiation 19 .
In this research, we examined the function of STIL in PC12 cell survival in response to treatment with neurotoxin 6-OHDA. RNAi-mediated knockdown of Stil mRNA expression efficiently diminished the expression of STIL proteins, whereas overexpression of the full-length human Stil sequence promoted STIL expression 19 . In addition, we manipulated the Shh signaling transduction using pharmacological  approaches. Down-regulation of Shh signaling was achieved by treatment with the steroidal alkaloid cyclopamine, which functions as an antagonist of the Smo receptor in the Shh pathway 26 . Up-regulation of Shh signaling was achieved by using a small molecule purmorphamine, which is an agonist for Shh receptor Smo 27 . Treatment with cyclopamine or purmorphamine does not induce growth arrest or trigger cell death in PC12 cells, which thereby provide a tool that allow functional analyses of the effect of STIL knockdown or overexpression in cell survival in the presence or absence of Shh signaling transduction. In proliferating PC12 cells, down-regulation of Stil expression produced no obvious effect on cells' drug susceptibility. However, increase of Stil expression resulted in increases in drug susceptibility. This is likely mediated by the Shh signaling transduction pathway. In cyclopamine-treated proliferating PC12 cells, for example, the activity of the Shh pathway was decreased. However, the same drug treatment resulted in significant increases of Stil expression (see Fig. 2B). The increase of Stil expression may counter the effect of cyclopamine treatment, and thereby increase the activity of Shh signaling transduction. Transfection of cyclopamine-treated PC12 cells with pCS2+ Flh-Stil plasmids increases the expression of Stil mRNA, which in turn, increases Shh transduction, and eventually increases the drug susceptibility of the cell (see Fig. 2G). In differentiated PC12 cells, Stil exerts its role on cell drug susceptibility in opposite ways, that is, overexpression of Stil produced no obvious effect on cells' survival in response to 6-OHDA treatment, but knockdown of Stil expression increases the rate of cell death after toxic drug insults. In differentiated mature PC12, the effect of Stil on drug susceptibility is also mediated by the Shh signaling transduction pathway.
Based on the results derived from this research, we propose a model for the novel function of the human oncogene Stil in the regulation of toxic susceptibility of DA or DA-like neurons. It functions through the Shh signal transduction pathway but the underlying mechanisms are different in mature and proliferating neurons (Fig. 4). In mature PC12 cells, the expression of Stil is required for cell survival in response to toxic drug treatment. Overexpression of Stil increases Shh signaling transduction and inhibits Caspase-3 activity, and thereby prevents cell death after 6-OHDA treatment. Shh-mediated inhibition of Caspase-3 activity has been reported in various cell types [28][29][30][31] . Down-regulation of Stil expression decreases Gli1 transcription, which then decreases its inhibition to Caspase-3 and thereby increases cell death in response to toxic drug treatment. In proliferating PC12 cells, down-regulation of Stil expression decreases the rate of cell proliferation but produces no effects on cell maturation or survival in response to neurotoxins. The increase of Stil expression leads to increases in drug susceptibility. This may be regulated by different mechanisms. One possibility is that excessive Shh signaling activates anti-Shh signaling pathways, which in turn, diminishes the inhibition of Gli1to Caspase-3 32 . Another possibility is that overexpression of Gli1 activates other yet unknown biochemical pathways that either directly or indirectly increase the cell's drug sensitivity 33,34 . While the underlying mechanisms remain to be further examined, the data from this study provide evidence for the novel function of the human oncogene Stil in drug susceptibility in vertebrate neurons.
Gene transfection. Adherent cells were cultured for 3-5 days (60-70% confluency) in complete medium, trypsinized, and then passed through a 22-gauge needle in order to obtain singular cells. Approximately 5 million cells and 10 μ g DNA plasmid were used for each transfection using the Cell Line Nucleofector Kit V (Lonza). For shRNA transfections, cells were seeded for experiments after 72 hours. For overexpression transfections, cells were seeded for experiments after 24 hours. Control transfections (with scramble sequence) were performed along each experiment. In all cases, data obtained from the control experiments were normalized to 1.
Western blot. Cells were lysed in freshly prepared lysis buffer (50 mM Tris-HCl pH 7.4; 150 mM NaCl; 1% nonidet 40; 5 mM DTT; 10 μ g aprotinin; 10 μ g leupeptin; 10 mM PMSF; 10% glycerol) on ice, and centrifuged at 12,000 rpm for 15 min. The supernatant was saved as whole-cell lysate, and protein concentrations were measured using the Pierce BCA Assay Kit (Thermo Scientific). For immunoblotting, 50-100 μ g of protein was separated on a 5% stacking and 8% separating SDS-PAGE gel. Protein was transferred in Tris-glycine buffer with 20% methanol onto nitrocellulose membranes, and blocked with 5% non-fat milk in TBST, pH 7.5. After blocking, membranes were incubated overnight at 4 °C with primary antibodies (polyclonal rabbit anti-STIL, 1:200; Santa Cruz Biotechnology; polyclonal rabbit anti-ACTIN, 1:3,000, Sigma; polyclonal rabbit anti-cleaved Caspase-3, 1:3,000, Abcam) diluted in fresh blocking solution. The membranes were washed with TBST, and then incubated for 1 hour at room temperature with secondary antibodies (goat anti-rabbit IgG conjugated with HRP, 1:3000 for STIL, 1:30,000 for ACTIN) diluted in fresh blocking solution. The membranes were washed with TBST, and protein expression was detected using the Super-Signal West Pico Chemiluminescent Substrate (Thermo Scientific). Drug treatment. Cyclopamine (Sigma) was dissolved in ethanol at a concentration of 1 mM and stored in − 80 °C. Dilutions of 10 or 20 μ M were made in medium for treatment. Ethanol treatment (0.1%) was used as a vehicle control. Purmorphamine (Cayman Chemical) was dissolved in DMSO at a concentration of 1 mM and kept chilled prior to use. Dilutions of 5 or 10 μ M were made in medium for treatment. DMSO treatment (0.01%) was used as a vehicle control. Neurotoxin (6-OHDA; Sigma) was dissolved in 0.15% ascorbic acid (stock concentration: 1 mM; stored in − 80 °C) and applied to the culture medium at 0, 25, 50, 75, and 100 uM, respectively. In all cases, the duration of drug treatment was 24 hours. Drug treatment was applied to both undifferentiated proliferating cell and differentiated mature cells.
Cell survival and apoptosis assays. The MTT reduction assay was used to examine PC12 cell viability. MTT (Sigma) was dissolved in DPBS at 5.5 mg/mL. Cells were seeded in triplicate for each test. Medium was removed and replaced with 0.5 mg/mL MTT in DPBS. Cells were incubated in MTT for 1 hour at 37 °C in 5% CO 2. An equal volume of solvent (acidified isopropanol; 0.1 M HCl with 10% Triton X-100) was added to lyse the cells via trituration. Absorbance was read at 570 nm with background subtraction at 650 nm.
The TUNEL assay was performed in according to the manufactory's protocol (Life Technologies). At the end of drug treatment, cells were fixed and incubated with a methanol solution containing 0.3% H 2 O 2 and then incubated with the TUNEL reaction mixture for 1 hour at 37 °C. Using a microscopy, Scientific RepoRts | 5:16513 | DOI: 10.1038/srep16513 TUNEL-positive nuclei were counted in 10 randomly selected fields per cover slip, and converted to percentage by comparing TUNEL-positive counts with the total cell nuclei counts.