PDGF induces PASMCs proliferation
To explore the effect of PDGF on PASMCs proliferation, cell viability was measured using CCK-8 at different concentrations (0,3,10,30,100 ng/ml) or different times (0, 12, 24,48,72 h). Figure 1a shows that cell viability was enhanced with increasing concentration of PDGF and 10 ng/ml PDGF triggers a 1.29-fold increase at 24 h compared control (P < 0.05). Figure 1b indicates that PDGF promoted cell proliferation time-dependently and cell viability reached 1.71-fold increase at 72 h over control (P < 0.05). The observed results reveal that PDGF induces PASMCs proliferation in a dose and time-dependent manner.
PDGF activates PI3K/Akt signaling pathway and upregulates c-MYC and TERT expressions
PI3K/Akt signaling pathway as a positive regulator mediates PDGF-induced PASMCs proliferation . To understand the mechanisms underlying PDGF-induced PASMCs proliferation, we examined the phosphorylation level of Akt, protein levels of c-MYC and TERT using western blotting. As shown in Figure 2a, b and c, 10 ng/ml PDGF significantly increased the phosphorylation level of Akt, c-MYC and TERT expressions to 1.50-fold, 1.63-fold and 1.84-fold over control, respectively (P < 0.05). These results suggest that PDGF activates PI3K/Akt signaling pathway and increases protein levels of c-MYC and TERT in PASMCs.
PI3K/Akt signaling pathway mediates PDGF-induced c-MYC and TERT expressions
It has reported that the PI3K/Akt signaling pathway promotes the transcription of TERT and cell proliferation by suppressing the function of MAX dimerization protein1in MCF-7 Cell Line . Here, to investigate whether it exists in PASMCs, cells were pre-treated with PI3K/Akt inhibitor LY294002 for 30 min and then stimulated with PDGF. We found that LY294002 significantly inhibited Akt phosphorylation to 1.11-fold over control (P < 0.05, versus PDGF-treated group Figure 3a). Moreover, the protein level of c-MYC was declined to 0.89-fold over control and TERT expression was dropped to 1.03-fold compared with control (P < 0.05, versus PDGF-treated group, Figure 3b and c). These results indicate that activation of Akt is responsible for PDGF-induced c-MYC and TERT expressions in PASMCs.
TERT expression is regulated by c-MYC
c-MYC promotes TERT expression through binding to the TERT proximal promoter in neoplastic cells. To clarify whether c-MYC regulates TERT expression in PASMCs, c-MYC was knocked down by siRNA and protein level of TERT was measured by western blotting. As shown in Figure 4a, the protein level of c-MYC was reduced to 29% after transfection of c-MYC siRNA in PASMCs for 48 h, meanwhile, control siRNA had no effect on the c-MYC protein level. Figure 4b indicates that PDGF stimulation increased TERT protein level to 1.96-fold over control (P < 0.05), while transfection of c-MYC notably reduced PDGF-induced TERT protein level to 1.14-fold compared with control (P < 0.05, versus PDGF-treated group). These results suggest the crucial role of c-MYC in PDGF-induced TERT expression.
Activation of PPARγ suppresses the effects of PDGF on Akt/c-MYC/TERT axis
To understand the effect of PPARγ on the process of PDGF-induced cell proliferation, cells were pretreated with pioglitazone (10 mM) as PPARγ agonist for 30min and then stimulated with PDGF for 24 h. As shown in Figure 5a, pioglitazone suppressed the activation of Akt induced by PDGF and the protein level of p-Akt was declined to 1.07-fold compared with control (P < 0.05, versus PDGF-treated group). Figure 5b and C indicates that cells pretreated with pioglitazone dramatically reduced the expression of c-MYC from 1.55-fold to 0.94-fold over control and TERT expression from 1.61-fold to 0.97-fold over control (P < 0.05, versus PDGF-treated group). All results suggested Activation of PPARγ inhibits PDGF-induced expression of p-Akt, c-MYC and TERT.
PDGF induces telomerase activation via Akt/c-MYC/TERT pathway
To identify whether telomerase activity in PASMCs was triggered by PDGF and was directly mediated via Akt/c-MYC/TERT pathway, cells were incubated with PDGF for 24 h. Figure 6 shows that PDGF significantly increased telomerase activation to 1.45-fold compared with control (P < 0.05). it also indicated that treatment cells with LY294002, BIBR1532, transfection of c-MYC-siRNA or pioglitazone reduced telomerase activation to 1.02-fold, 1.02-fold, 0.90-fold and 0.98-fold over control respectively (P < 0.05, versus PDGF-treated group). These results demonstrate PDGF activates telomerase through Akt/c-MYC/TERT signaling and activation of PPARγ impedes this process.
PDGF-induced telomerase activation promotes cell proliferation and migration by Akt/c-MYC/TERT pathway
According to the above data, we hypotheses that telomerase activation might mediate cell proliferation and migration by Akt/c-MYC/TERT pathway. We then examined cell viability by CCK-8 assay. As shown in Figure 7a, PDGF increased cell viability to 1.55-fold compared with control (P < 0.05), conversely BIBR1532 reversed this affect and attenuated the cell viability to 1.14-fold compared with control (P < 0.05, versus PDGF-treated group) due to the inhibition of telomerase activation. Meanwhile, pre-treatment cells with LY294002, pioglitazone or transfection of c-MYC-siRNA declined cell viability to 1.17-fold, 1.12-fold, 1.18-fold over control, respectively (P < 0.05, versus PDGF-treated group). Consistently, Figure 7b suggested that PDGF stimulation promoted cells migration, whereas BIBR1532 reversed PDGF-induced PASMCs migration and similar effects were observed in LY294002, c-MYC-siRNA and pioglitazone groups. All results prove the hypothesis that telomerase activation facilitates PASMCs proliferation and migration via Akt/c-MYC/TERT pathway