TET1 Regulates Nestin Expression and Human Airway Smooth Muscle Proliferation

Abstract

Asthma is characterized by aberrant airway smooth muscle (ASM) proliferation, which increases the thickness of the ASM layer within the airway wall and exacerbates airway obstruction during asthma attacks. The mechanisms that drive ASM proliferation in asthma are not entirely elucidated. Ten-eleven translocation methylcytosine dioxygenase (TET) is an enzyme that participates in the regulation of DNA methylation by catalyzing the hydroxylation of 5-methylcytosine (5-mC) to 5-hydroxymethylcytosine (5-hmC). The generation of 5-hmC disinhibits the gene silencing effect of 5-mC. In this study, TET1 activity and protein were enhanced in asthmatic human ASM cell cultures. Moreover, the concentration of 5-hmC was higher in asthmatic ASM cells than in nonasthmatic ASM cells. Knockdown (KD) of TET1, but not TET2, reduced the concentration of 5-hmC in asthmatic cells. Because the cytoskeletal protein nestin controls cell proliferation by modulating mTOR, we evaluated the effects of TET1 KD on this pathway. TET1 KD reduced nestin expression in ASM cells. In addition, TET1 inhibition alleviated the platelet-derived growth factor-induced phosphorylation of p70S6K, 4E-BP, S6, and Akt. TET1 inhibition also attenuated the proliferation of ASM cells. Taken together, these results suggest that TET1 drives ASM proliferation via the nestin-mTOR axis.

Keywords: DNA methylation; airway smooth muscle; cytoskeletal protein; signal transduction.

Figure 1.

Ten-eleven translocation methylcytosine dioxygenase (TET) activity is upregulated in asthmatic human airway smooth muscle (HASM) cells. (A) The activity of TET of nonasthmatic and asthmatic HASM cultures was evaluated using the 5 mC-Hydroxylase TET Activity Assay Kit. The t test was used for statistical analysis. (B and C) Protein expression of HASM cells treated with control (Ctrl) or TET1 or TET2 siRNA was evaluated by immunoblotting. The t test was used for statistical analysis. (D) Knockdown (KD) of TET1, but not TET2, attenuates TET activity of HASM cells. ANOVA was used for statistical analysis. Data are mean ± SEM (n = 6 donors/group). *P < 0.05. siRNA = short interfering RNA.

Figure 2.

Expression of TET1 protein is enhanced in asthmatic HASM cells. (A) Expression of TET1 in nonasthmatic and asthmatic HASM cultures was evaluated by immunoblot analysis. (B) The spatial localization of TET1 was assessed by immunofluorescence microscopy. TET1 localizes in the nucleus. Moreover, TET1 is upregulated in asthmatic HASM cells. Scale bars, 50 μm. Data are mean ± SEM (n = 6 donors/group). The t test was used for statistical analysis. *P < 0.05.

Figure 3.

5-Hydroxymethylcytosine (5-hmC) is elevated in asthmatic HASM cells and in nonasthmatic cells exposed to platelet-derived growth factor (PDGF). (A) The levels of 5-hmC are higher in asthmatic HASM cells than in nonasthmatic cells. Nonasthmatic and asthmatic HASM cells were treated with 10 ng/ml PDGF, IL-13, IL-4, IL-5, and IL-33 for 24 hours, followed by assessment of 5-hmC. Treatment with PDGF enhances 5-hmC in both nonasthmatic and asthmatic cells. (B and C) KD of TET1, but not TET2, diminishes 5-hmC levels in asthmatic cells and PDGF-treated nonasthmatic cells. One-way ANOVA was used for statistical analysis. Data are mean ± SEM (n = 6 donors/group). *P < 0.05. NS = not significant.

Figure 4.

TET1 regulates the expression of nestin and proliferation of HASM cells. (A and B) KD of TET1 diminishes nestin expression in asthmatic HASM cells (A) and PDGF (10 ng/ml, 24 h)-treated nonasthmatic cells (B). The t test was used for statistical analysis. (C and D) Control (Ctrl) and TET1 KD cells were treated with 10 ng/ml PDGF for 24 hours. Cell proliferation was determined by using cell counting and the CellTiter assay. TET1 KD inhibits the PDGF-induced proliferation of nonasthmatic cells and asthmatic cells. The values of nonasthmatic cells treated with control siRNA are used for normalization. Two-way ANOVA was used for statistical analysis. Data are mean ± SEM (n = 6 donors/group). *P < 0.05 and **P < 0.01.

Figure 5.

PDGF stimulation enhances mTOR activation, which is PDGF receptor dependent. (A–E) PDGF treatment increases the phosphorylation of p70S6K, eIF4E binding protein (4E-BP), S6, and Akt (mTOR activation readouts) in HASM cells, which is time dependent. (F–J) mTOR activation upon PDGF (10 ng/ml, 10-min) is diminished by the PDGF receptor inhibitor AG1296 (1 μm, 10-min preincubation). One-way ANOVA was used for statistical analysis. Data are mean ± SEM (n = 6 donors/group). *P < 0.05 and **P < 0.01.

Figure 6.

TET1 regulates mTOR activation in HASM cells. (A) Representative immunoblots illustrating the role of TET1 in mTOR activation. Nonasthmatic and asthmatic HASM cells were treated with 1 μm NSC-370284 (NSC) for 48 hours. They were then stimulated with PDGF (10 ng/ml, 10 min) or left unstimulated. mTOR activation was evaluated by immunoblot analysis. (B–E) Treatment with NSC attenuates the PDGF-induced phosphorylation of p70S6K, 4E-BP, S6, and Akt. Two-way ANOVA was used for statistical analysis. Data are mean ± SEM (n = 6 donors/group). *P < 0.05.

Figure 7.

Proposed model. Exposure to PDGF induces upregulation of TET1 in HASM, which enhances the production of 5-hmC. Elevated 5-hmC facilitates the expression of nestin, which activates mTOR and its substrates (p70S6K, 4E-BP and Akt) and promotes HASM hyperproliferation. The pharmacological tool NSC-370284 can inhibit the TET1-medicated process and cell proliferation.