Nestin drives allergen‐induced airway smooth muscle hyperplasia and airway remodeling

To the Editor, Asthma is a complex pulmonary disorder that affects 300 million patients worldwide, and is characterized by airway remodeling, a cardinal feature of which is aberrant airway smooth muscle (ASM) cell proliferation.1 ASM hyperplasia leads to thickening of the airway wall, which exacerbates airway hyperresponsiveness (AHR) and narrowing during asthma attacks. T helper type 2 (Th2) lung inflammation is viewed as a key mechanism for asthma progression.2 However, Th2 cytokines are not major drivers for ASM cell proliferation.1, 2 Thus, other mechanisms must exist to drive ASM hyperplasia in asthma. Nestin is a class VI intermediate filament (IF) protein that was described as a neuronal stem/progenitor cell marker,3 and was found to be associated with the proliferation of cancer cells.4 Moreover, mTOR (mechanistic target of rapamycin) is a serine/threonine protein kinase that is implicated in the proliferation of cancer cells.5 The 14-3-3 adapter proteins bind a multitude of functionally diverse signaling proteins including kinases and regulate the functions of kinases.6 Because asthma and cancer share a common property—hyperplasia, we hypothesized that the nestin/14-3-3/mTOR pathway plays a role in mediating ASM hyperplasia and airway remodeling in vivo. ELISA was used to assess nestin expression in asthmatic ASM. Nestin expression 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 (Figure 1A; Table S1). We further found upregulation of nestin expression in human asthmatic ASM tissues by using immunoblotting and immunostaining (Figure 1B,C) and in tracheal tissues of house dust mite extract (HDME)-exposed mice (Figure 1D; Figure S1A). We then generated smooth muscle conditional nestin knockout (KO) mice by crossing nestin-lox mice with Myh11-cre mice (Figure S1B,C). We exposed these mice to HDME or PBS aerosol for 6 weeks. Smooth muscle conditional nestin KO reduced the allergen-induced ASM hyperplasia, airway remodeling, and AHR (Figure 2). Allergic lung inflammation frequently occurs with ASM thickening. Thus, we also evaluated the role of nestin in allergic lung inflammation in vivo. Nestin KO attenuated HDME-enhanced inflammatory cells and the Th2 cytokines IL-13, IL-4, and IL-5 in the lungs (Figure S1D–H). Platelet-derived growth factor (PDGF) expression is upregulated in asthmatic patients, which contributes to ASM remodeling.1 In addition, mTOR is associated with cell proliferation.5 We evaluated the effects of PDGF on phosphorylation of p70S6K, 4E-BP, S6, and Akt (readouts for mTOR activation) in human ASM cells. PDGF treatment enhanced the phosphorylation of p70S6K, 4E-BP, S6, and Akt as well as cell proliferation, which was inhibited by nestin KD and restored by nestin rescue (Figure S2). Next, we assessed whether mTOR plays a role in asthma pathogenesis in vivo. We exposed C57BL/6N mice to HDME of PBS aerosol for 6 weeks. For the last 2 weeks, we treated the animals with the mTOR inhibitor rapamycin or vehicle aerosol. Treatment with rapamycin aerosol diminished the allergen-induced ASM thickening, AHR, and airway inflammation in the animals (Figure S3). We also discovered that PDGF stimulation increased the interaction of nestin with 14-3-3γ, and 14-3-3γ knockdown caused a reduction in the PDGF-induced mTOR activation and cell proliferation (Figure S4). Additionally, exposure to the 14-3-3 inhibitor R18 aerosol diminished the allergen-induced airway remodeling and AHR in mice (Figure S5). Taken together, these findings define nestin as a key player in ASM hyperplasia and airway remodeling. Nestin may bind 14-3-3, which subsequently activates mTOR, and induces ASM proliferation and airway remodeling. Furthermore, serum levels of nestin and PDGF were associated with lung functions of asthmatic patients (Figure S6), implying a translational role for these proteins in the future. Guoning Liao, Ruping Wang, Yidi Wu, and Neelam Kumari Maheshwari performed experiments. Dale D. Tang conceived the research direction and coordinated the study. Guoning Liao and Dale D. Tang wrote the manuscript. Raymond B. Penn generated ASM cultures, read and revised the manuscript. All authors approved the final version of the manuscript. The authors thank Alyssa C. Rezey, Brennan Gerlach, Olivia Gannon, Eylon Arbel, Saiyang Hu, Thomas Brown, and Asghar Pasha of Albany Medical College for technical assistance. The authors have declared that no conflict of interest exists. 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). The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions. Data S1: Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.