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: airway smooth muscle, cytoskeletal protein, signal transduction, DNA methylation
Clinical Relevance
The mechanisms that drive airway smooth muscle proliferation in asthma are not entirely elucidated. In this study, we demonstrate that ten-eleven translocation methylcytosine dioxygenase 1 promotes airway smooth muscle cell proliferation via the nestin-mTOR pathway. This finding unveils a new mechanism that contributes to airway smooth muscle hyperplasia in asthma.
Asthma is characterized by airway remodeling, a cardinal feature of which is aberrant airway smooth muscle (ASM) proliferation (1–4). Abnormal ASM proliferation contributes to ASM thickening within the airway wall, which exacerbates airway obstruction during asthma attacks (3, 5, 6). Asthma is frequently viewed as a T helper cell type 2 (Th2)-dependent disease involving increased production of Th2 cytokines, including IL-4, IL-5, and IL-13 (7, 8). However, Th2 cytokines are not major drivers for ASM proliferation (4, 6). Thus, other mechanisms must exist to regulate ASM hyperplasia in asthma.
Nestin is a class VI intermediate filament protein that plays a role in regulating the proliferation of various cell types, including cancer cells (9) and ASM cells (10). Nestin regulates the activation of mTOR, which is a serine/threonine protein kinase that plays a role in the proliferation of cancer cells and smooth muscle cells (10–12). mTORC1 phosphorylates p70S6 kinase (p70S6K) and eIF4E binding protein (4E-BP), which promotes protein translation and lipid synthesis (11, 12). p70S6K also phosphorylates ribosomal protein S6, which facilitates protein translation (13). Protein translation and lipid synthesis are essential for cell proliferation. Moreover, Akt phosphorylation and activation also phosphorylate TSC2 and activate mTORC1 (11, 12). On the other hand, mTORC2 can phosphorylate Akt, which promotes cell survival and proliferation through the phosphorylation of several key substrates (11, 12).
Our recent studies have shown that nestin is upregulated in cell cultures of asthmatic human airway smooth muscle (HASM), which contributes to airway hyperresponsiveness, remodeling, and inflammation (10). Moreover, nestin knockdown (KD) inhibited mTORC activation and HASM cell proliferation upon stimulation with growth factors, which was restored by nestin rescue (10). Because these cultured HASM cells are not exposed to inflammatory mediators or medicines (e.g., β-agonists and corticosteroids) and do not interact with other structural cells (e.g., epithelium) (14), nestin upregulation in asthmatic ASM cells is likely to be an intrinsic feature. Moreover, nestin has been shown to regulate ASM cell proliferation via mTOR signaling (10).
Ten-eleven translocation methylcytosine dioxygenase (TET) is an enzyme that participates in the regulation of DNA methylation, one of the important epigenetic hallmarks that is dysregulated in certain cancers (15). DNA methylation occurs on the C-5 atom of cytosine generating 5-methylcytosine (5-mC) in the CpG islands and inhibits gene expression by interacting with methyl-binding proteins (16). TET catalyzes the hydroxylation of 5-mC to 5- hydroxymethylcytosine (5-hmC), which can be further converted to 5-fluorocytosine and 5-carboxylcytosine by the same enzymes (17). The generation of 5-hmC disinhibits the gene silencing effect of 5-mC because 5-hmC is not recognized by transcriptional repressors (18). Furthermore, it has been shown that global DNA hydroxymethylation is upregulated in HASM cells, suggesting a potential role of DNA methylation in asthma pathogenesis (19). Nevertheless, the role of TET in regulating nestin expression and mTOR activation had not previously been investigated. The objective of the present study was to determine whether TET activity and/or protein are altered in asthmatic HASM cells and whether TET plays a role in regulating nestin protein expression, mTOR activation, and the proliferation of HASM cells.
Methods
Cell Culture
HASM cells were prepared from human bronchi and adjacent tracheas obtained from the International Institute for Advanced Medicine as previously described (3, 20–25), with studies herein approved by the Albany Medical College Committee on Research Involving Human Subjects. The donor human lungs used to procure tissue and cells were not suitable for transplant and not identifiable; thus, these studies were determined not to be human subjects research. Primary cell cultures (passages 3–10) from six donors without asthma and six donors with asthma were used for the experiments (10, 26–28). Donor characteristics are included in Table E1 in the data supplement. The purity of HASM cells was determined by immunostaining for smooth muscle α-actin. Nearly 100% of these cells expressed α-actin (28).
Immunoblot Analysis
Western blotting of cell lysis was performed using experimental procedures as previously described (20, 28–31). Antibodies used were anti-nestin (Fisher Invitrogen, PIPA511887/L/N SH2420723H), anti-GAPDH (Santa Cruz Biotechnology, sc-32233/L/N K0315), anti-TET1 (EpigenTek, A-1020), anti-TET2 (EpigenTek, A1701), anti-phospho-S6K (T389) (Cell Signaling Technology, 9234S/L/N 12), anti-S6K (Santa Cruz Biotechnology, sc-8418/L/N 1219), anti-phospho-4E-BP (S65) (Cell Signaling Technology, 9451S/L/N 14), anti-4E-BP (Invitrogen, MA5-15313/L/N VF3009191), anti-phospho-S6 (Ser235/236) (Cell Signaling Technology, 4857S/L/N 2), anti-S6 (54D2) (Cell Signaling Technology, 2317S/L/N13), anti-phospho-Akt (S473) (D9E) (Cell Signaling Technology, 4060P/4060 s/L/N 16 and 19), and anti-Akt (Cell Signaling Technology, 2920S/L/N 3). The antibodies were validated by examining the molecular weight of target proteins. In addition, anti-nestin, anti-TET1, and anti-TET2 were validated by using KD cells. Finally, vendors have provided datasheets to show that antibodies were validated by positive controls. The concentrations of proteins were quantified by scanning densitometry of immunoblots (Fuji Multi Gauge software or GE IQTL software). The luminescent signals from all immunoblots were within the linear range.
Assessment of TET Activity and 5-hmC in Cells
For TET activity, nuclear extracts of HASM cells were collected by using the EpiQuick Nuclear Extraction Kit 1 (EpigenTek). The TET activity was determined by using the Epigenase 5 mC-Hydroxylase TET Activity Assay Kit (EpigenTek). The concentrations of 5-hmC in cells were assessed using the MethylFlash Global DNA Hydroxymethylation (5-hmC) ELISA Easy Kit (Colorimetric) (EpigenTek).
Assessment of Cell Proliferation
Cell proliferation was determined using experimental procedures as previously described with modification (2, 10, 24, 25, 28). Cells were treated with human PDGF-BB (Sigma-Aldrich, 10 ng/ml) in F12 medium containing 0.25% FBS for 24 hours. Cell numbers were measured using an automatic cell counter (Countess 3, Invitrogen). In addition, cell proliferation was assessed using the CellTiter 96 AQueous One Solution Cell Proliferation Assay (Promega) according to the manual of the manufacturer.
Immunofluorescence Microscopy
Cell immunofluorescence microscopy was performed using a protocol as previously described (10, 25, 32–34).
Statistical Analysis
All statistical analysis was performed using Prism 10 software (GraphPad Software). Differences between pairs of groups were analyzed using a two-tailed Student’s t test for two-group comparisons for normally distributed continuous data. A comparison among multiple groups was performed by one-way or two-way ANOVA followed by a post hoc test (Tukey’s multiple comparisons) for normally distributed continuous data. P < 0.05 was considered to be significant.
Results
Enhancement of TET Activity Is Mediated by TET1 in Asthmatic HASM Cells
To assess the potential role of TET in human asthma, we compared TET activity between nonasthmatic and asthmatic HASM cells by using the Epigenase 5 mC-Hydroxylase TET Activity Assay Kit (EpigenTek). TET activity was higher in asthmatic cells than in nonasthmatic cells (Figure 1A). There are three isoforms of TET in mammalian cells. TET1 and TET2 are expressed in most somatic cells, whereas TET3 expression is restricted to oocytes and zygotes (35). Thus, we also evaluated whether TET1 or TET2 contributes to the enhancement of TET activity in HASM cells. We transfected HASM cells with control siRNA or TET1 or TET2 siRNA. Immunoblot analysis verified the downregulation of TET1 or TET2 in cells (Figures 1B and 1C). KD of TET1, but not TET2, attenuated TET activity in both nonasthmatic and asthmatic cells (Figure 1D).
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.
The Expression of TET1 Protein Is Upregulated in Asthmatic HASM Cells
To further assess the role of TET1 in asthma, we evaluated the expression of TET proteins in nonasthmatic and asthmatic HASM cells. The protein concentrations of TET1 in asthmatic cells were higher than in nonasthmatic cells (Figure 2A). Moreover, we used immunofluorescence microscopy to evaluate the distribution and expression of TET1. TET1 was found in the nucleus. The fluorescence intensity of TET1 was higher in asthmatic cells than in nonasthmatic cells (Figure 2B).
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.
Alteration of 5-hmC Concentration in Asthmatic ASM Cells
Because the major function of TET is to catalyze the hydroxylation of 5-mC to 5-hmC (18, 36), we evaluated 5-hmC concentrations in nonasthmatic and asthmatic ASM cells by using the MethylFlash Global DNA Hydroxymethylation (5-hmC) ELISA Easy Kit. The concentration of 5-hmC in asthmatic ASM cells was higher than in nonasthmatic cells (Figure 3A, bar 1 vs. bar 2).
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.
Because platelet-derived growth factor (PDGF), IL-13, IL-4, IL-5, and IL-33 are associated with asthma pathogenesis (4, 25, 28, 34, 37), we treated nonasthmatic and asthmatic ASM cells 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 enhanced the 5-hmC concentration in nonasthmatic cells (Figure 3A, bar 1 versus bar 3). However, PDGF-mediated increase of 5-hmC in asthmatic cells was similar to that of nonasthmatic cells (Figure 3A, bar 3 versus bar 4). The results suggest that the PDGF’s sensitivity in terms of 5-hmC in asthmatic cells is similar to that in nonasthmatic cells. Moreover, exposure to IL-13, IL-4, IL-5, and IL-33 did not significantly affect 5-hmC concentrations in nonasthmatic and asthmatic cells (Figure 3A, bars 5–12). These results suggest that the growth factor, but not these ILs, regulates DNA methylation in HASM cells.
TET1 Catalyzes Production of 5-hmC
We assessed whether TET1 and TET2 affect 5-hmC concentration in this cell type. KD of TET1 reduced 5-hmC concentrations in asthmatic ASM cells (Figure 3B). Furthermore, KD of TET1 inhibited PDGF-induced enhancement of 5-hmC in nonasthmatic cells (Figure 3C). However, TET2 KD did not affect 5-hmC concentrations in these cells (Figures 3B and 3C).
TET1 Regulates Nestin Expression and ASM Cell Proliferation
Our recent studies have shown that nestin is upregulated in asthmatic HASM cells (10). Thus, we evaluated whether TET1 plays a role in regulating nestin expression. TET1 KD diminished the expression of nestin in asthmatic HASM cells (Figure 4A). Moreover, PDGF contributes to ASM hyperplasia in asthma, and PDGF-treated nonasthmatic HASM cells serve as an in vitro cell model for hyperproliferation (4, 25, 34, 38). Thus, we treated nonasthmatic cells with PDGF and determined the role of TET1 in nestin expression in these cells. TET1 KD also reduced nestin expression in nonasthmatic cells treated with PDGF (Figure 4B).
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.
Nestin has been shown to regulate ASM cell proliferation (10). Thus, we assessed the role of TET1 in cell proliferation by determining the effects of TET1 KD on ASM proliferation. Cell proliferation was evaluated by using cell counting and the CellTiter assay. TET1 KD alleviated the proliferation of asthmatic HASM cells as well as nonasthmatic HASM cells (Figures 4C and 4D).
PDGF Induces the Activation of mTOR in HASM Cells
Because mTOR has been implicated in cell proliferation (11, 12) and PDGF is known to induce ASM proliferation, we determined whether PDGF elicits mTOR activation in HASM cells. As described earlier, phosphorylation of p70S6K, 4E-BP, S6, and Akt represents readouts for mTOR activation (11, 12). We examined the effects of PDGF treatment on mTOR activation. PDGF treatment enhanced phosphorylation of p70S6K, 4E-BP, S6, and Akt, which was time dependent (Figures 5A–5E). Moreover, we pretreated cells with the PDGF receptor inhibitor AG1296 followed by PDGF stimulation. The effects of PDGF (10- or 30-min stimulation) on mTOR activation were PDGF receptor dependent (Figures 5F–5J and E1).
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.
mTOR Activation Is Regulated by TET1 in HASM Cells
Nestin has been shown to regulate mTOR activation in HASM cells (10). In this report, we found that TET1 orchestrated nestin protein expression in this cell type (Figures 4A and 4B). Therefore, we evaluated the role of TET1 in mTOR activation by determining the effects of the TET1 pharmacological agent NSC-370284 on phosphorylation of p70S6K, 4E-BP, S6, and Akt. NSC-370284 is a newly identified agent to downregulate TET1 expression via STAT3/5 (39). Treatment with NSC-370284 attenuated the PDGF-induced phosphorylation of p70S6K, 4E-BP, S6, and Akt in nonasthmatic cells (Figure 6). Moreover, we found that the PDGF-induced phosphorylation of p70S6K, 4E-BP, S6, and Akt was greater in asthmatic cells than in nonasthmatic cells, consistent with previous studies in which asthmatic HASM cells proliferate faster (25, 38, 40). Furthermore, treatment with NSC-370284 blunted the phosphorylation of p70S6K, 4E-BP, S6, and Akt in asthmatic cells in response to PDGF stimulation (Figure 6). We also found that treatment with NSC-370284 attenuated cell proliferation (Figure E2). Together, these results demonstrate that TET1 regulates mTOR activation in nonasthmatic and asthmatic HASM cells.
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.
Discussion
ASM proliferation plays an important role in regulating the progression of airway remodeling, a cardinal characteristic of asthma. The mechanism that drives ASM proliferation is not completely understood. In this study, TET1 activity and protein expression were enhanced in asthmatic HASM cells. KD of TET1 attenuated nestin protein expression, mTOR activation, and the proliferation of HASM cells.
DNA methylation occurs on the C-5 atom of cytosine and generates 5-mC, which is associated with gene silencing (16). TET catalyzes the hydroxylation of 5-mC to 5-hmC, which disinhibits the gene silencing effects of 5-hmC (17). In this study, TET activity was upregulated in asthmatic HASM cells, which was blunted by TET1 KD. Thus, TET1 is a major isoform that affects TET activity in ASM cells. Moreover, the expression of TET1 protein was enhanced in asthmatic HASM cells, which is consistent with analysis of the TET activity. This finding is supported by previous studies by others (19). Furthermore, the concentration of 5-hmC was higher in asthmatic HASM cells than in nonasthmatic cells. TET1 KD reduced the concentration of 5-hmC in asthmatic cells. TET1 KD also attenuated nestin expression and cell proliferation. It is known that 5-hmC facilitates gene expression (17). Thus, we propose that TET1 protein is enhanced in asthmatic HASM cells, which leads to increases in TET activity and 5-hmC. Elevated 5-hmC promotes nestin expression and cell proliferation.
PDGF expression is upregulated in patients with asthma, which contributes to ASM remodeling (4). In addition, the concentrations of IL-13, IL-4, IL-5, and IL-33 are elevated in the airways of patients with asthma (37, 41). Thus, we determined the effects of the growth factor and cytokines on 5-hmC concentrations. Exposure to PDGF, but not IL-13, IL-4, IL-5, and IL-33, increased 5-hmC concentrations. These results suggest that the growth factor is responsible for the regulation of DNA methylation in ASM cells.
Our recent studies have shown that the expression of nestin is upregulated in asthmatic HASM, which contributes to the progression of ASM proliferation (12). Because TET activity is associated with gene expression, we evaluated the role of TET in nestin expression. TET1 KD blunted the expression of nestin protein in asthmatic HASM cells. Moreover, TET1 KD reduced the PDGF-induced upregulation of nestin in nonasthmatic cells. Furthermore, TET1 KD diminished the proliferation of HASM cells. Therefore, TET1 participates in the regulation of nestin protein and the proliferation of smooth muscle cells.
Although mTOR has been implicated in cell proliferation (11, 12), knowledge regarding its activation in ASM cells is limited. In this study, we demonstrated that PDGF was able to phosphorylate p70S6K, 4E-BP, S6, and Akt (indications of mTOR activation), which was time and concentration dependent. Moreover, treatment with the PDGF receptor inhibitor attenuated the PDGF-mediated mTOR activation. These results suggest that the ligation of the PDGF receptor may trigger mTOR signaling, which subsequently promotes ASM cell proliferation. More important, we found that TET1 plays a role in regulating mTOR activation in HASM cells. Because TET1 also regulates the expression of nestin and mTOR is a target of nestin (10), it is likely that TET1 orchestrates mTOR activation via nestin in HASM cells. Our finding is supported by a previous study that keratin 17 (an intermediate filament protein in epithelial cells) orchestrates mTOR signaling and growth of keratinocytes (42). Nestin and keratin 17 are able to interact with the adapter protein 14-3-3, which influences the activity of mTOR (10, 42). In summary, we demonstrate that TET1 activity and protein are increased in asthmatic HASM, which contributes to the elevation of 5-hmC and nestin. Upregulated nestin promotes mTOR activation and the proliferation of HASM cells (Figure 7).
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.
Acknowledgments
Acknowledgment
The authors thank Yidi Wu of Albany Medical College for technical assistance.
Footnotes
Supported by National Heart, Lung, and Blood Institute grants HL-110951 (to D.D.T.) and HL-130304 (to D.D.T.).
Author Contributions: R.W. and G.L. performed experiments. D.D.T. conceived the research direction and coordinated the study. D.D.T. wrote the manuscript. All authors approved the final version of the manuscript.
This article has a data supplement, which is accessible at the Supplements tab.
Originally Published in Press as DOI: 10.1165/rcmb.2024-0139OC on June 11, 2024
Author disclosures are available with the text of this article at www.atsjournals.org.
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