Dynein과 상호작용하는 미세소관 결합 단백질 HOOK2의 천식에 대한 영향 = The impact of HOOK microtubule tethering protein 2 (HOOK2) interaction with dynein on asthma

다국어 초록 (Multilingual Abstract)

The impact of HOOK microtubule tethering protein 2 (HOOK2) interaction with dynein on asthma Min-Hyeok An Department of Interdisciplinary Program in Biomedical Science Major Graduate School of Soonchunhyang University Asan, Korea (Supervised by Professor An-Soo Jang) Purpose Airway epithelial cells constitute the primary structural and immunological barrier against inhaled allergens and environmental irritants. Disruption of epithelial polarity and barrier integrity is a central feature of asthma and is closely associated with oxidative stress, protease signaling, endoplasmic reticulum (ER) stress, and innate immune activation. These insults impair tight-junction stability, alter cytoskeletal organization, and promote airway remodeling. Microtubule based trafficking, particularly dynein-mediated retrograde transport, plays an essential role in maintaining Golgi centrosome organization and the apical delivery of polarity determinants. The HOOK protein family comprises adaptor-like microtubule-binding proteins that anchor organelles to the cytoskeleton and facilitate dynein dependent transport. Among these, HOOK2 is localized at the centrosome and Golgi apparatus, where it regulates ciliogenesis, pericentriolar organization, and polarized vesicular trafficking. Although HOOK2 has been implicated in cell polarity and centrosome positioning in other epithelial systems, its biological significance in asthma and its potential involvement in allergen induced epithelial dysfunction remain largely unknown. This study aimed to elucidate the role of the dynein adaptor HOOK2 in airway hyperresponsiveness and allergic inflammation, to define how HOOK2-dynein-PAR complex interactions contribute to epithelial polarity regulation, and to determine whether circulating HOOK2 levels reflect inflammatory activation in patients with asthma. Methods HOOK2 expression and function were evaluated across clinical, in vivo, and in vitro models. Plasma HOOK2 and dynein levels were measured in patients with stable or exacerbated asthma and healthy controls, and their associations with inflammatory markers and lung function parameters were assessed. An ovalbumin (OVA)-induced murine asthma model was used to analyze airway hyperresponsiveness, inflammatory cell infiltration, and structural remodeling. Lung tissues were examined by Western blotting, immunohistochemistry (IHC), immunofluorescence (IF), and co-immunoprecipitation (Co-IP) to assess the expression and molecular interactions among HOOK2, dynein, and PAR complex proteins (PAR3, PAR6, and aPKC). In NHBE cells, HOOK2 was silenced using siRNA, followed by stimulation with Dermatophagoides pteronyssinus 1 (Der p 1). Changes in protein expression, subcellular localization, and epithelial barrier integrity were evaluated using Western blotting, immunofluorescence, and transepithelial electrical resistance (TEER) assays. Results Plasma levels of HOOK2 and dynein were higher in patients with asthma (n = 19) than that of control subjects (n = 10). HOOK2 levels were correlated with dynein (r = 0.332, p = 0.021), total WBC (r = 0.398, p = 0.006), FEV1% (r = -0.409, p = 0.004) and FEV1/FVC (r = -0.368, p = 0.01) in patients with asthma. In OVA/OVA-sensitized/challenged mice, goblet cell hyperplasia, lung fibrosis, and the levels of Hook2, dynein, and PAR complex proteins were significantly increased compared with control mice. In NHBE cells, HOOK2, dynein, and PAR complex (PAR3, PAR6, aPKC) expression were increased at 24 hours following Der p 1 exposure. But, changed in NHBE cells treated with HOOK2 siRNA. Double immunofluorescence staining and co-immunoprecipitation confirmed enhanced colocalization and interaction between Hook2 with dynein, and PAR complex in airway epithelium under inflammatory conditions. Conclusions These findings reveal that HOOK2 is upregulated during allergic airway inflammation and functions as a dynein associated scaffold that modulates epithelial polarity and structural stability by coordinating PAR complex organization. Increased plasma HOOK2 levels further suggest its potential as a biomarker reflecting epithelial injury or activation in asthma. This study identifies HOOK2 as a previously unrecognized regulator in the pathogenesis of asthma and provides new mechanistic insight into microtubule based trafficking and polarity disruption during allergic inflammation.

목차 (Table of Contents)

• CONTENTS ⅰ
• Contents of Tables ⅲ
• Contents of Figures ⅳ
• ABSTRACT ⅴ
• Ⅰ. INTRODUCTION 1
• Ⅱ. MATERIALS AND METHODS 6
• 1. Patients 6
• 2. Animals 7
• 3. Periodic acid Schiff staining 8
• 4. Cell culture 8
• 5. Small interfering RNA (siRNA) and Der p 1 treatments 9
• 6. Transepithelial Electrical Resistance (TEER) 9
• 7. Enzyme-linked immunosorbent assays 9
• 8. Western blot 10
• 9. Immunohistochemistry 11
• 10 Immunofluorescence in vivo 11
• 11. Immunofluorescence in vitro 12
• 12. Co-Immunoprecipitation (Co-IP) 12
• 13. Statistical analysis 13
• Ⅲ. RESULTS 14
• 1. Patients with asthma characteristics 14
• 2. HOOK2 levels were associated with the clinicopathological characteristics of patients with asthma 14
• 3. OVA-induced inflammation and AHR in mice 15
• 4. Elevated Hook2 and dynein interaction with PAR complex expression in the lungs of OVA/OVA-sensitized/challenged mice 16
• 5. HOOK2 and dynein expression in NHBE Cells 16
• 6. Colocalization and Interaction of HOOK2 with dynein and PAR Complex Proteins 17
• Ⅳ. DISCUSSION 19
• REFERENCES 24
• 국 문 요 약 42
• Contents of Tables
• Table 1. Clinical characteristics in control subjects and patients with asthma 33
• Table 2. siRNAs used in this study 34
• Contents of Figures
• Figure 1. HOOK2 and dynein level in control subjects, stable, and exacerbated asthmatic patients 35
• Figure 2. Relationships of HOOK2 with FEV1%, FEV1/FVC, and total blood WBC in control subjects and patients with asthma 36
• Figure 3. Airway hyperresponsiveness (AHR) and inflammation in OVA/OVA-sensitized/challenged mice 37
• Figure 4. Protein expression and localization of Hook2, dynein, and PAR complex proteins in lung tissue 38
• Figure 5. HOOK2 expression and barrier function in NHBE cells 39
• Figure 6. Colocalization and interaction of HOOK2 with dynein and PAR complex proteins in NHBE cells 40
• Figure 7. Summary diagram of the effect of HOOK2 on asthma 41