Role of nestin/14-3-3/mTOR signaling in airway smooth muscle cell proliferation and airway remodeling

doi:10.21203/rs.3.rs-2869821/v1

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Role of nestin/14-3-3/mTOR signaling in airway smooth muscle cell proliferation and airway remodeling

https://doi.org/10.21203/rs.3.rs-2869821/v1

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Airway smooth muscle (ASM) cell proliferation plays a critical role in the progression of asthma. Asthma is characterized by airway remodeling, a cardinal feature of which is aberrant ASM cell proliferation. ASM hyperplasia leads to thickening of the airway wall, which exacerbates airway hyperresponsiveness and narrowing during asthma attacks. Although Th2 cytokines are prominent drivers of asthma pathogenesis, they do not drive ASM hyperplasia in asthma. Here, we discover that nestin, a type VI intermediate filament protein, regulates human ASM cell proliferation via 14-3-3/mechanistic target of rapamycin (mTOR) signaling. More importantly, nestin is upregulated in asthmatic human ASM, an epigenetic effect dependent on ten-eleven translocation methylcytosine dioxygenase 1-mediated 5-hydroxymethylcytosine. Smooth muscle conditional nestin knockout reduces airway smooth muscle thickening, airway hyperresponsiveness, inflammation, and mucus hypersecretion in an allergen-induced murine model of asthma. Moreover, comprehensive in vivo studies demonstrate nestin drives ASM hyperplasia and airway remodeling via 14-3-3/mTOR signaling.

Biological sciences/Cell biology/Cytoskeleton/Intermediate filaments

Health sciences/Medical research/Preclinical research

Airway smooth muscle

asthma

intermediate filament protein

cytoskeletal protein

signal transduction

hyperplasia

Airway smooth muscle (ASM) cell proliferation plays a critical role in the development and homeostasis of the respiratory system as well as the progression of diseases such as asthma. Asthma is a complex pulmonary disorder that affects 300 million patients worldwide^1,2, and is characterized by airway remodeling, a cardinal feature of which is aberrant ASM cell proliferation^3–6. ASM hyperplasia leads to thickening of the airway wall, which exacerbates airway hyperresponsiveness and narrowing during asthma attacks. T helper type 2 (Th2) lung inflammation is viewed as a key mechanism for asthma progression^2,7. However, Th2 cytokines are not major drivers for ASM cell proliferation^6,8. Thus, other mechanisms must exist to regulate ASM hyperplasia in asthma.

Nestin is a class VI intermediate filament (IF) protein that was first described as a neuronal stem/progenitor cell marker during central nerve system development ⁹. Subsequently, nestin has been found in lung adenocarcinoma¹⁰, regenerative skeletal muscles¹¹, and developing vascular smooth muscle¹². Nestin expression is associated with the proliferation of cancer cells, skeletal muscle, and vascular smooth muscle^10–13. Moreover, the type III IF protein vimentin is widely expressed in a variety of cell types including smooth muscle cells^14–17. In addition to maintaining the integrity of the cells, vimentin affects cell/tissue homeostasis via a number of mechanisms including epithelial-mesenchymal transition and division ¹⁶. Despite the large body of literature during the past decade characterizing nestin, the role and mechanisms of nestin in ASM cell proliferation and airway remodeling remain largely unknown.

mTOR1 (mechanistic target of rapamycin 1) is a serine/threonine protein kinase that is implicated in the proliferation of cancer cells and vascular smooth muscle cells^18,19. mTORC1 phosphorylates p70S6 Kinase (p70S6K) and eIF4E Binding Protein (4E-BP), which promotes protein translation and lipid synthesis^18,19. p70S6K also phosphorylates ribosomal protein S6, which facilitates protein translation ²⁰. Protein translation and lipid synthesis are essential for cell proliferation. Moreover, Akt phosphorylation and activation also phosphorylate TSC2 and activate mTORC1^18,19. On the other hand, mTORC2 can phosphorylate Akt, which promotes cell survival and proliferation through the phosphorylation of several key substrates^18,19.

The 14-3-3 adapter proteins are a family of conserved regulatory molecules that are expressed in all eukaryotic cells. The 14-3-3 proteins have the ability to bind a multitude of functionally diverse signaling proteins including kinases and regulate the functions of kinases²¹. In epithelial cells, keratin 17 (an IF protein in epithelial cells) regulates mTOR signaling and the growth of keratinocytes by affecting 14-3-3²².

DNA methylation is one of the important epigenetic hallmarks that is dysregulated in certain cancers. 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 ²³. Ten-eleven translocation methylcytosine dioxygenase (TET) catalyzes the hydroxylation of 5-mC to 5- hydroxymethylcytosine (5-hmC), which can be further converted to 5-fluorocytosine (5-fC) and 5-carboxylcytosine (5-caC) by the same enzymes ²⁴. The generation of 5-hmC disinhibits the gene silencing effect of 5-mC because 5-hmC is not recognized by transcriptional repressors²⁵. The role of DNA hydroxymethylation in asthma pathogenesis is largely unknown.

In the current study, we discover that nestin regulates mTOR activation and the proliferation of ASM cells and is upregulated in ASM cell cultures derived from asthmatic lung donors. Extensive analysis of nestin-dependent signaling and function in both cell-based models and in vivo reveal nestin to be a key macromolecule to promotes ASM growth and the allergen-induced airway remodeling, acting via regulation of 14-3-3-mediated mTOR activation.

Nestin regulates ASM cell proliferation and mTOR activation

Because nestin has been implicated in the proliferation of cancer cells, skeletal muscle, and vascular smooth muscle^10–13, we evaluated the role of nestin in ASM cell proliferation induced by platelet-derived growth factor (PDGF), a mitogen important for cell proliferation and asthma pathogenesis ^6,26. We generated stable nestin knockdown (KD) human ASM cells by using lentivirus particles encoding shRNA against nestin. Nestin KD diminished the mitogen-induced cell proliferation whereas nestin rescue restored the PDGF-mediated cell proliferation (Fig. 1, A-C).

Because mTOR is associated with the proliferation of cancer cells and vascular smooth muscle cells^18,19, we initiated a comprehensive assessment of growth factor-induced mTOR activation in ASM cells. As described earlier, phosphorylation of p70S6K, 4E-BP, S6, and Akt represent readouts for mTOR activation^18,19. PDGF treatment enhanced phosphorylation of p70S6K, 4E-BP, S6, and Akt, which was time-dependent (Figure S1, A-E). Moreover, we also found that the effects of PDGF (10 or 30 min stimulation) on mTOR activation were PDGF receptor-dependent (Figure S1, F-J) (Figure S2).

To assess the role of nestin in mTOR activation, we evaluated the impact of nestin KD and rescue on mTOR activation. Nestin KD attenuated the PDGF-induced phosphorylation of p70S6K, 4E-BP, S6, and Akt; moreover, nestin rescue restored PDGF-induced mTOR activation (Fig. 1, D-H). We also noticed that nestin KD and rescue affected basal mTOR activation. This is not surprising because these cells were cultured in 0.25% growth medium to limit apoptosis. The results demonstrate that nestin regulates ASM cell proliferation and mTOR activation.

Upregulation of nestin in asthmatic ASM

Because airway remodeling is largely attributed to ASM hyperplasia and IF proteins have been implicated in the proliferation of nonmuscle cells, we questioned whether IF protein expression is altered in asthmatic ASM. ELISA analysis showed that the expression of nestin was upregulated in cultures of ASM derived from asthmatic lung donors; however, expression of another IF protein vimentin was not significantly different in control and asthmatic ASM cells (Fig. 2A). We further found upregulation of nestin expression in human asthmatic ASM tissues by using immunoblotting and immunostaining (Fig. 2, B and C). To explore whether nestin expression in human ASM could be recapitulated in animal models, we used a well-established murine model of asthma utilizing repeated exposure to house dust mite extract (HDME). HDM is a major perennial allergen source and a significant cause of allergic asthma²⁷, and mice exposed to HDME develop asthma-like characteristics^28,29. We exposed C57BL/6 mice to HDME or PBS (control) aerosol for 6 weeks using an InExpose system (Figure S3A) followed by immunoblot analysis. The expression of nestin in tracheal/bronchial tissues of HDME-treated mice was increased relative to that observed in PBS-treated mice (Fig. 2D). These results demonstrate the upregulation of nestin protein in human asthmatic ASM and in the animal model of allergic airway remodeling.

Alteration of 5-hmC level in asthmatic ASM cells

Because nestin is upregulated in asthmatic human ASM cell cultures that have intrinsic properties³⁰, we questioned whether an epigenetic program contributes to nestin upregulation. 5-hmC, a hallmark of epigenetics, has been implicated in gene expression and cancer progression^25,31. We therefore evaluated 5-hmC levels in control and asthmatic ASM cells. The level of 5-hmC in asthmatic ASM cells was higher than control cells (Fig. 3A). Since both growth factors and cytokines contribute to asthma pathogenesis ^6,32−35, we treated nonasthmatic ASM cells with PDGF, epidermal growth factor (EGF), interleukin (IL)-13, IL-4, IL-5, and IL-33 for 24 h. Treatment with PDGF or EGF enhanced 5-hmC level in cells. However, exposure to IL-13, IL-4, IL-5, and IL-33 did not significantly affect 5-hmC levels (Fig. 3A). TET catalyzes the hydroxylation of 5-mC to 5-hmC²⁵, we assessed whether TET1 and TET2 affect 5-hmC concentration in this cell type. KD of TET1, but not TET2, reduced 5-hmC in asthmatic cells, PDGF-treated cells, and EGF-treated cells (Fig. 3, B-D) (Figure S4, A and B). Furthermore, we found that TET1 KD diminished the expression of nestin in asthmatic, PDGF-treated, and EGF-treated cells (Fig. 3, E-G) (Figure S4, C-E). These results demonstrate that 5-hmC enhancement mediated by TET1 contributes to nestin upregulation in asthmatic ASM cells.

Role of nestin in allergen-induced airway remodeling and lung inflammation in vivo

Next, we questioned whether nestin plays a role in airway remodeling in vivo. To test this, we generated smooth muscle-conditional nestin knockout (KO) (nestin^smko) mice by crossing nestin^− lox mice with Myh11-cre mice that express Cre recombinase under control of a smooth muscle-specific myosin heavy chain promoter (Figure S3, B and C). We treated nestin^− lox and nestin^smko mice with PBS (control) or HDME aerosol for six weeks, and assessed ASM thickening, a key feature of airway remodeling. Exposure to the allergen increased the thickness of the ASM layer in the airways of nestin^− lox mice, which was reduced in nestin^smko mice (Fig. 4, A and B). Moreover, the allergen-induced fluorescent intensity of proliferating cell nuclear antigen (PCNA) was greater in the airways of nestin^− lox mice compared to that in nestin^− lox mice treated with PBS, which was reduced by conditional nestin KO (Fig. 4, A and C).

Because ASM thickening affects airway hyperresponsiveness (AHR)^5,8,36−38, we assessed the role of nestin in AHR in vivo and ASM hyperreactivity ex vivo. Exposure of HDME aerosol increased airway resistance in nestin^− lox mice, which was attenuated in nestin^smko mice (Fig. 4D). In vivo HDME treatment increased the acetylcholine-stimulated contractile response of tracheal rings isolated from nestin^− lox mice (Fig. 4E). However, HDME-sensitized ASM contraction was reduced in nestin^smko mice (Fig. 4E). These results suggest that nestin contributes to the development of AHR in allergen-induced asthma.

Allergic lung inflammation and mucus hypersecretion frequently occur with ASM thickening^32,39. Thus, we also evaluated the role of nestin in allergic lung inflammation and epithelial mucin in vivo. Nestin KO attenuated HDME-enhanced inflammatory cells and the Th2 cytokines IL-13, IL-4, and IL-5 (Fig. 4, F-H and Figure S3D). Moreover, nestin KO reduced the allergen-induced production of epithelial mucins (Fig. 4I). These effects may be attributed to the ability of ASM cells generate (and respond to) cytokines and serve an immunomodulatory role in allergic lung inflammation and mucus secretion^40,41. Taken together, these findings demonstrate that nestin contributes to the development of the allergen-induced airway remodeling, inflammation, and AHR in vivo.

Role of mTOR in airway remodeling and inflammation

Next, we assessed whether mTOR plays a role in asthma pathogenesis in vivo. We exposed C57BL/6 mice to HDME aerosol or PBS for 6 weeks. For the last 2 weeks, we treated the animals with the mTOR inhibitor rapamycin or vehicle aerosol (Figure S3E). HDME aerosol induced activation of mTOR, which was reduced by rapamycin (Figure S5). Treatment with rapamycin aerosol diminished the allergen-induced ASM thickening in the airways of the animals (Fig. 5, A and B), which was associated with a reduction in PCNA staining (Fig. 5, A and C). Furthermore, rapamycin aerosol attenuated the HDME-induced enhancement of airway resistance in vivo and ASM hyperreactivity in vitro (Fig. 5, D and E). Additionally, rapamycin treatment reduced the allergen-enhanced generation of epithelial mucins, inflammatory cells, and Th2 cytokines (Fig. 5, F-I) (Figure S6, A and B). These results suggest an important role for mTOR activation in allergen-induced ASM thickening, AHR, mucus hypersecretion, and airway inflammation.

mTOR activation-induced “rescue” of asthma-like phenotype in nestin KO mice

Next, we questioned whether mTOR activation “rescues” the asthma-like phenotype in nestin KO animals. We treated nestin^smko mice with HDME aerosol or PBS for six weeks. In addition, we treated these mice with the mTOR activator MHY1485 ⁴² or vehicle for the last 2 weeks (Figure S3E). Consistent with findings presented in Fig. 4, nestin KO diminished HDME-induced ASM thickening, AHR, ASM hyperreactivity, inflammation, and mucin production; vehicle treatment did not affect the phenotype (Fig. 6). However, MHY1485 treatment restored the asthma-like phenotype of nestin KO mice (Fig. 6) (Figure S6, C and D). These findings demonstrate that mTOR is a downstream effector of nestin during the progression of allergen-induced airway remodeling, AHR, and inflammation. Of note, MHY1485 treatment did not enhance these PBS-treated pathological changes; these results suggest that mTOR activation alone is not sufficient to induce asthma-like phenotype in the animal model.

Regulation of mTOR activation by 14-3-3

Keratin 17 (an IF protein in epithelial cells) regulates the mTOR pathway and growth of keratinocytes by interacting with the adapter protein 14-3-3σ²². Thus, we questioned whether growth factors modulate the association of nestin with 14-3-3 in ASM. We found that treatment with PDGF enhanced the amount of 14-3-3γ (a major isoform in smooth muscle) ⁴³ in nestin immunoprecipitates (Fig. 7A). To determine whether 14-3-3γ plays a role in regulating mTOR, we silenced 14-3-3γ expression and assessed mTOR activation. KD of 14-3-3γ caused a reduction in basal and the PDGF-induced mTOR activation in ASM cells (Fig. 7, B-G), which was associated with reduced cell proliferation (Fig. 7, H and I).

Nestin KD reduced the expression of 14-3-3γ without affecting the expression of total p70S6K, 4E-BP, S6, and Akt (Fig. 8, A and B) (Fig. 1D). To further assess whether nestin affects mTOR activation via 14-3-3γ, we re-expressed 14-3-3γ in stable nestin KD cells. Re-expression of 14-3-3γ in nestin KD cells rescued basal and the growth factor-induced phosphorylation of p70S6K, 4E-BP, S6, and Akt (Fig. 8). These results suggest that nestin regulates mTOR by affecting 14-3-3γ.

Contribution of 14-3-3 in asthma-like phenotype

Having established 14-3-3 modulates mTOR activation and ASM cell proliferation, we assessed whether 14-3-3 is involved in more integrative asthma pathogenesis. We exposed C57/BL6 mice to HDME for 6 weeks and treated the animals with the 14-3-3 inhibitor R18 for the last 2 weeks (Figure S3E). Treatment with R18 aerosol reduced the HDME-induced ASM thickening and PCNA staining in the airways of the animals (Fig. 9, A-C). Furthermore, R18 aerosol attenuated the HDME-enhanced airway resistance in vivo and ASM hyperreactivity in vitro (Fig. 9, D and E). Moreover, R18 treatment reduced the allergen-enhanced epithelial mucins, inflammatory cells, and Th2 cytokines (Fig. 9, F-H) (Figure S7). These results suggest an important role for 14-3-3 in allergen-induced asthma-like phonotype.

Asthma is a pulmonary disorder with complex pathological mechanisms^44,45. Aberrant ASM cell proliferation contributes to progression of airway remodeling, a cardinal feature of asthma^3–6. Although Th2-high inflammation is recognized as an important mechanism in asthma pathology^2,7, Th2 cytokines are not major drivers for ASM cell proliferation^6,8. In this report, the IF protein nestin is upregulated in ASM cultures derived from asthmatic donor lungs. To the best of our knowledge, this is the first evidence implicating an IF protein in asthma pathogenesis.

The role and mechanism of nestin in ASM cell proliferation were not previously investigated. In this study, nestin was shown to regulate ASM cell proliferation and activation of mTOR (Fig. 1). p70S6K, 4E-BP, and S6 promote protein translation and lipid synthesis, which are essential for proliferation^18–20. Akt phosphorylates several key substrates, which enhances cell survival and proliferation^18,19. Our finding is supported by a previous study that keratin 17 (an IF protein in epithelial cells) orchestrates mTOR signaling and growth of keratinocytes²².

We initially discovered the expression of nestin is elevated in primary ASM cultures derived from asthmatic donor lungs (Fig. 2). Because these cells are not exposed to inflammatory mediators or medicines (e.g., β-agonists and corticosteroids), or interact with other structural cells (e.g., epithelium) ³⁰, nestin upregulation in asthmatic ASM cells is likely to be an intrinsic property, possibly driven by epigenetic influences, many of which have been associated with asthma ⁴⁶. Accordingly, we determined 5-hmC levels in asthmatic human ASM cells. The levels of 5-hmC are elevated in asthmatic ASM cells as well as in growth factor-treated cells, and these levels are reduced by TET1 KD (Fig. 3). Thus, this specific epigenetic influence likely contributes to the increased expression of nestin in asthmatic ASM cells. Furthermore, nestin upregulation can be recapitulated in the murine model of asthma, which provides an opportunity to interrogate the cause-and-effect relationship between nestin and asthma using the animal model.

To assess the role of nestin in asthma pathogenesis in vivo, we generated smooth muscle conditional nestin KO mice. Nestin KO diminishes the allergen-induced airway smooth muscle thickening in the animal model of asthma (Fig. 4). The results suggest that the increased expression of nestin in ASM contributes to ASM thickening in vivo. Moreover, nestin KO attenuates contractility of ASM from naïve animals, and inhibits the allergen-induced AHR in vivo and ASM hyperactivity in vitro. Airway smooth muscle hyperplasia also facilitates AHR to a variety of stimuli^5,8,36. As described earlier, Th2 cytokines do not drive ASM hyperplasia in asthma^6,8. Our initial aim was to understand the mechanisms that contribute to ASM proliferation in asthma. Having found nestin is upregulated in asthmatic human ASM and in an animal model of allergic lung inflammation/asthma, we expanded our assessment of nestin and found that nestin KO also reduces the allergen-induced airway inflammation and mucus hypersecretion in vivo. This finding is consistent with the known immunomodulatory role of ASM cells through secretion of cytokines/chemokines^40,41. Therefore, nestin may mediate AHR likely by directly affecting both hyperplasia and contractility of ASM^36,37,47, and by regulating ASM immunomodulatory functions.

Our cell-based experiments showed that nestin regulates mTOR activation and ASM cell proliferation (Fig. 1). Furthermore, inhibition of mTOR recapitulates the nestin KO phenotype of the murine asthma model (Fig. 5). To assess whether nestin drives airway remodeling via mTOR, we performed the “rescue” experiment in vivo. Pharmacological rescue of mTOR restores the asthma-like phenotype of nestin KO mice (Fig. 6). Thus, mTOR mediates nestin-induced airway remodeling and asthma-like phenotype.

Stimulation of human ASM cells with growth factors induces the association of 14-3-3 with nestin, and 14-3-3 KD reduces mTOR activation (Fig. 7). Moreover, nestin KD reduces the expression of 14-3-3 (Fig. 8). These results suggest that nestin modulates the functions of 14-3-3 by binding to the adapter protein and regulating expression of 14-3-3. Restoring 14-3-3 in nestin KD cells recovers mTOR activation in nestin KD cells (Fig. 8). These experiments suggest that 14-3-3 is a “biological bridge” between nestin and mTOR signaling. 14-3-3 proteins have been shown to interact with a variety of diverse signaling proteins and influences their activity²¹. Furthermore, the inhibition of 14-3-3 also reiterates the nestin KO phenotype of the animal asthma model (Fig. 9). Therefore, nestin upregulation in ASM may activate mTOR via 14-3-3, which subsequently induces ASM hyperplasia, AHR, airway inflammation, and mucus secretion in asthma.

In summary, we demonstrate that manipulation of nestin expression regulates mTOR activation and ASM cell proliferation. Moreover, nestin expression is increased in asthmatic ASM, which is attributed to TET1-mediated 5-hmC elevation. Smooth muscle conditional knockout of nestin inhibits the asthma-like phenotype. Mechanistically, loss-of-function studies demonstrate that nestin mediates ASM cell proliferation and asthma phenotype by regulating 14-3-3/mTOR signaling. Thus, we propose a complex model in which upregulated nestin drives ASM hyperplasia and asthma progression via the 14-3-3/mTOR pathway (Fig. 10).

Cell Culture. Human ASM cells were prepared from human bronchi and adjacent tracheas obtained from three sources- the International Institute for Advanced Medicine, Rutgers University, and Thomas Jefferson University as previously described ^5,33,47−51, 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 studies were determined to be Not Human Subjects Research. Smooth muscle cells passage 3–10 were used for the studies, as per^35,52,53. The purity of HASM was determined by immunostaining for smooth muscle α-actin. Nearly 100% of these cells expressed α-actin ³⁵. Basic characterization of these donors was described in Table S1.

For proliferation experiments, human ASM cells were treated with 10 ng/ml PDGF for 24 hrs. Cell counting was performed using a Countess™ 3 Automated Cell Counter (Invitrogen), and BrdU incorporation was determined using the procedures described previously^35,51.

Assessment of nestin and vimentin by ELISA. Proteins in cells were extracted using the buffer containing 2% Triton-X100, 2 mM EDTA, 20 mM Tris-HCl (pH7.6) and 0.2% SDS plus Halt™ Proteinase Inhibitor Single-Use Cocktail (ThermoScientific, #78425). The Nestin ELISA kit (ELH-NES-1) and vimentin ELISA kit (ELH-VIM-1) were purchased from Raybiotech (Peachtree Corners, GA ) and used to determine nestin and vimentin protein in extracts according to the manual of the manufacture. Protein concentration was evaluated by the BCA assay. Readings of ELISA and BCA were collected using a Glomax Multi-detection system (Promega). Duplicates of each sample were used for data analysis.

Immunoblot analysis and coimmunoprecipitation. Western blotting of cell lysis and coimmunoprecipitation were performed using the experimental procedures as previously described^{17,35,39,48,54}. Antibodies used were anti-nestin (1:1000, Fisher Invitrogen #PIPA511887/L/N SH2420723H and Cell Signaling #10959s/L/N 1), anti-GAPDH (1: 1000, Santa Cruz Biotechnology #sc-32233/L/N K0315), anti-vimentin (1:1000, Santa Cruz #sc-32332/L/N H1219), anti-phospho-S6K (T389)(1:500, Cell Signaling #9234S/L/N 12), anti-S6K (1:500, Santa Cruz #sc-8418/L/N 1219), anti-phospho-4E-BP (S65)(1:500, Cell Signaling #9451S/L/N 14), anti-4E-BP (1:1000, Invitrogen #MA5-15313/L/N VF3009191), anti-phospho-S6 (Ser235/236) (1:500, Cell Signaling #4857S/L/N 2), anti-S6 (54D2) (1:500, Cell Signaling#2317S/L/N 13), anti-phospho-Akt (S473)(D9E) (1:2000, Cell Signaling #4060P/4060s/L/N 16 & 19), anti-Akt (1:1000, Cell Signaling #2920S/L/N 3), anti-PCNA (Proteintech #24036-1-AP/L/N 18863), anti-14-3-3 γ (Cell Signaling #5522S/L/N 1). The antibodies were validated by examining the molecular weight of target proteins. In addition, anti-nestin and anti-14-3-3 were validated by using KD cells. Finally, vendors have provided datasheet to show that antibodies were validated by positive controls. The levels 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.

Lentiviral transduction. Stable KD cells were generated using lentiviruses encoding target shRNA as previously described. ^5,55. Briefly, lentiviruses encoding nestin shRNA (sc-36032-V) and control shRNA (sc-108080) were purchased from Santa Cruz Biotechnology. Human ASM cells were infected with control shRNA lentiviruses or target shRNA lentiviruses for 12 h followed by 3–4 day culture, and selected with puromycin to generate positive clones expressing shRNAs. The expression levels of nestin were assessed by immunoblot analysis. Nestin KD cells and cells expressing control shRNA were stable for at least five passages after initial infection. For the nestin rescue experiment, Nestin KD cells were treated with lentiviruses encoding RNAi-resistant nestin construct. Nestin rescue in the KD cells was verified by immunoblotting.

Human nestin cDNA was purchased from Genomics Online Inc (pENTR223-1-human nestin cDNA, catalog number ABIN3427596, nestin cDNA NCBI Accession number GU014842). pLenti-puro mammalian expression vector was purchased from Addgene (Plasmid # 39481). We subcloned the nestin cDNA into the pLenti-puro vector through Nhe I/Spe I and EcoR V sites. To produce viruses, 293FT cells were transfected with pLenti-puro harboring the nestin cDNA plus a packaging vector pCMV and an envelope vector pVSV-G. Viruses were collected 48 h post transfection. For infection, smooth muscle cells were incubated with viruses for 12 h, then cultured in the F12 growth medium for 3 days. Positive clones were selected by puromycin.

Assessment of 5-hmC in cells. Human ASM cells were treated with 10/ng/ml PDGF, EGF, IL-4, IL-5, IL-13, and IL-33 for 24 h. The levels of 5-hmC in cells were assessed using the MethylFlash ™ Global DNA Hydroxymethylation (5-hmC ) ELISA Easy Kit (Colorimetric) (Epigentek, Farmingdale, NY).

siRNA and transfection. For TET1, TET2, and 14-3-3 KD, ASM cells were transfected with control siRNA (Santa Cruz # sc-37007), TET1 siRNA (Santa Cruz # sc-90457), TET2 siRNA (Santa Cruz # sc-88934) or 14-3-3γ siRNA (Santa Cruz #sc-29582) using the transfection protocol of the manufacture. For 14-3-3γ expression, human ASM cells were transfected with pcDNA3-HA-14-3-3γ (Addgene, Plasmid 13274) using the Fugene transfection kit (Promega).

Animals. All animal protocols were reviewed and approved by the Institutional Animal Care and Usage Committee (IACUC) of Albany Medical College. All experiments were strictly performed in accordance with approved protocols and regulations of IACUC. Animals were bred in the specific pathogen free housing of the Animal Research Facility, Albany Medical College.

To generate smooth muscle conditional nestin knockout mice (nestin^smko mice), nestin^− lox mice (genetic background, C57BL/6) were purchased from Mutant Mouse Resources and Research Center at the University of California, Davis. They were crossed with B6.Cg-Tg (Myh11-cre,-EGFP) 2Mik/J mice (from Jackson Laboratory, genetic background, C57BL/6J). Both male and female mice aging 6–8 weeks were used for the asthma models (See below).

Assessment of airway resistance. Age- and sex-matched nestin^− lox and nestin ^smko mice (6–8 weeks old) were exposed to house dust mite extract (HDME) aerosol (50 µg of d. pteronyssinus, Greer) or PBS aerosol (control) by using the InExpose system (SCIREQ, Montreal, Canada) for 5 days followed by every other day exposures weekly for 5 weeks. On Day 43, mice were anesthetized with intraperitoneal injection of ketamine/xylazine cocktail, tracheotomized, and connected to the FlexiVent system (SCIREQ, Montreal, Canada). Airway resistance was evaluated using the methods described previously^17,33.

Assessment of tracheal ring contraction. All experimental protocols were approved by the Institutional Animal Care and Usage Committee. A segment of tracheas (4–5 mm in length) was immediately removed and placed in physiological saline solution (PSS) containing 110 mM NaCl, 3.4 mM KCl, 2.4 mM CaCl₂, 0.8 mM MgSO₄, 25.8 mM NaHCO₃, 1.2 mM KH₂PO₄, and 5.6 mM glucose. The solution was aerated with 95%O₂-5%CO₂ to maintain a pH of 7.4. Two stainless steel wires were passed through the lumen of tracheal rings. One of the wires was connected to the bottom of organ baths and the other was attached to a Grass force transducer that had been connected to a computer with A/D converter (Grass). Tracheal segments were then placed in PSS at 37^oC. At the beginning of each experiment, 0.5 g passive tension was applied to tracheal rings. After 60 min equilibrium they were stimulated with 80 mM KCl repeatedly until contractile responses and passive tension reached a steady state. Contractile force in response to acetylcholine was then measured.

Immunohistochemistry. Mouse lungs were placed in frozen tissue-embedding medium (Neg 52, Richard-Allen Scientific) and cryosectioned using Cryostats (Richard-Allen Scientific). Tissue sections were fixed for 30 min in 4% paraformaldehyde, and were then washed three times in PBS buffer followed by permeabilization with 0.2% Triton X-100 dissolved in PBS for 5 min. These tissues were incubated with α-smooth muscle actin antibody (Sigma) or proliferating cell nuclear antigen (PCNA) antibody (Thermo Scientific) followed by appropriate secondary antibody conjugated to Alexa-488 or Alex-543 (Molecular Probes/Life Technologies). The sections were also counterstained with 4', 6-diamidino-2-phenylindole to visualize the nucleus. The samples were viewed and digitally captured using a Leica microscope system (MDI 6000)^17,56. All histochemical measurements were performed by using the NIH ImageJ and LAS X software.

Analysis of airway inflammation. Lungs from sacrificed mice were lavaged three times with 1 mL sterile Hanks balanced salt solution (HBSS) containing 3 mM EDTA. Bronchoalveolar lavage fluid (BALF) was collected after centrifugation. The supernatant was removed and frozen at -80°C for cytokine/chemokine measurements (See below). The cell pellet was resuspended in HBSS, and total number of inflammatory cells in the BALF was counted by using a hemocytometer. Differential cell counts (macrophages, neutrophils, lymphocytes, and eosinophils) were performed by counting 100 cells from cytospin preparations stained with DiffQuick stain. The levels of IL-13, IL-4 and IL-5 in the BALF were determined using the ELISA kits (R&D systems) according to the manufacturer’s instructions^17,56.

Assessment of epithelial mucin. Mucus production in airway epithelium was evaluated by using the Periodic Acid- Schiff (PAS) staining kit (Newcomer Supply Inc, Middleton, WI) according to the manual of the manufacture.

Statistical analysis. All statistical analysis was performed using Prism software (GraphPad Software, San Diego, CA). Differences between pairs of groups were analyzed by a 2-tailed Student’s t-test for 2-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. Values of n refer to the number of experiments used to obtain each value. P < 0.05 was considered to be significant.

4E-BP

eIF4E Binding Protein

5-hmC

5- hydroxymethylcytosine

5-mC

5-methylcytosine

AHR

Airway hyperresponsiveness

Akt

Ak strain transforming

ASM

Airway smooth muscle

BALF

Bronchoalveolar lavage fluid

BCA

Bicinchoninic acid

EGF

Epidermal growth factor

Intermediate filament

HDME

House dust mite extract

interleukin

ILC2

Type 2 innate lymphoid cells

Knockdown

Knockout

mTOR

Mechanistic target of rapamycin

p70S6K or S6K

p70S6 Kinase

PBS

Phosphate buffered saline

PCNA

Proliferating cell nuclear antigen

PDGF

Platelet-derived growth factor

Ribosomal protein S6

TET

Ten-eleven translocation methylcytosine dioxygenase

Th2

T helper type 2

TSLP

Thymic stromal lymphopoietin

DATA AVAILABILITY

All data generated or analysed during this study are included in this published article (and its supplementary information files).

ACKNOWLEDGMENTS

The authors thank Alyssa C. Rezey, Brennan Gerlach, Olivia Gannon, Eylon Arbel, Saiyang Hu, and Thomas Brown of Albany Medical College for technical assistance.

Funding: This work was supported by NHLBI Grants HL-110951 (to Dale D. Tang), HL-130304 (to Dale D. Tang), and HL-145392 (to Raymond B. Penn and Dale D. Tang).

AUTHOR CONTRIBUTIONS

GL, RW, YW, and NM performed experiments. DT conceived the research direction and coordinated the study. GL and DT wrote the manuscript. RP generated ASM cultures, read and revised the manuscript. All authors approved the final version of the manuscript.

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Role of nestin/14-3-3/mTOR signaling in airway smooth muscle cell proliferation and airway remodeling

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Abstract

Figures

INTRODUCTION

RESULTS

Nestin regulates ASM cell proliferation and mTOR activation

Upregulation of nestin in asthmatic ASM

Alteration of 5-hmC level in asthmatic ASM cells

Role of nestin in allergen-induced airway remodeling and lung inflammation in vivo

Role of mTOR in airway remodeling and inflammation

mTOR activation-induced “rescue” of asthma-like phenotype in nestin KO mice

Regulation of mTOR activation by 14-3-3

Contribution of 14-3-3 in asthma-like phenotype

DISCUSSION

METHODS

Abbreviations

Declarations

References

Additional Declarations

Supplementary Files

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