NOX4-MPO 경로에 의한 골아세포 기능 및 골 형성의 조절 기전 연구 = Regulation of osteoblast function and bone remodeling through NOX4-MPO signaling

다국어 초록 (Multilingual Abstract)

Reactive oxygen species (ROS) play essential roles in cellular signaling and homeostasis, and recent evidence has linked excessive ROS generation to skeletal disorders and altered bone remodeling. Among ROS-producing enzymes, NADPH oxidase 4 (NOX4) is a major source of hydrogen peroxide (H₂O₂) in bone-forming cells. However, its regulatory relationship with myeloperoxidase (MPO) and osteopontin (OPN) during osteoblast differentiation and endochondral ossification remains unclear. This study aimed to elucidate how NOX4 modulates bone formation through a redox-associated mechanism involving MPO and OPN. To investigate this, NOX4-deficient (NOX4⁻/⁻) and ovariectomized (OVX) mice were analyzed to assess in vivo skeletal morphology and bone maturation. In parallel, primary calvarial osteoblasts derived from wild-type and NOX4⁻/⁻ mice were cultured with or without the irreversible MPO inhibitor 4-aminobenzoic acid hydrazide (4-ABAH). Cellular proliferation, early differentiation, and matrix mineralization were evaluated, along with mRNA and protein expression levels of NOX4, MPO, and OPN. Whole-mount skeletal staining at embryonic day 17.5 (E17.5) provided developmental validation in vivo. The NOX4⁻/⁻ group exhibited enhanced endochondral ossification compared with the control and OVX groups. Proteomic and immunohistochemical analyses showed upregulated MPO and OPN expression near the epiphyseal plate in bones from NOX4⁻/⁻ mice. In vitro, loss of NOX4 promoted osteoblast proliferation, ALP activity, and mineral deposition, whereas MPO inhibition by 4-ABAH suppressed these parameters in a dose-dependent manner. Notably, NOX4⁻/⁻ osteoblast cultures maintained higher osteogenic potential even under MPO inhibition. This study demonstrates that NOX4 deficiency promotes osteoblast differentiation, matrix maturation, and endochondral ossification through the upregulation of MPO and OPN. Our findings identify this regulatory interaction as a distinct redox-modulatory axis that is normally restrained by NOX4. Loss of NOX4 releases this restraint, thereby enhancing osteogenic progression and accelerating mineralized matrix expansion. These results establish the NOX4–MPO–OPN axis as a critical mediator of redox-dependent regulation during bone formation and highlight its potential as a therapeutic target for skeletal disorders associated with oxidative stress and impaired bone metabolism.

목차 (Table of Contents)

• List of Tables iii
• List of Figures iv
• List of Abbreviations vi
• Abstract vii
• I. Introduction 1
• II. Materials and Methods 5
• 1. Chapter 1: The Regulatory Role of NOX4 in Endochondral Ossification through the Modulation of MPO and OPN 5
• 1.1. Experimental animals (Mice) 5
• 1.2. Ovariectomy 5
• 1.3. Tissue processing 5
• 1.4. Proteome array 6
• 1.5. Staining for histomorphometry 6
• 1.6. Plasma estradiol level measurement 8
• 1.7. Immunohistochemistry 8
• 1.8. Western Blot 9
• 1.9. Statistical Analysis 10
• 2. Chapter II: NOX4–Mediated Modulation of the MPO–OPN Redox Axis in the Regulation of Osteoblast Maturation 11
• 2.1. Animals 11
• 2.2. Isolation and culture of primary calvarial osteoblasts 11
• 2.3. Osteoblast differentiation and MPO inhibition 11
• 2.4. Cell proliferation assay 12
• 2.5. MTT assay 12
• 2.6. Flow cytometric analysis of apoptosis 12
• 2.7. Alkaline phosphatase (ALP) activity and staining 13
• 2.8. Mineralization assays 14
• 2.9. Whole-mount skeletal staining 14
• 2.10. RNA Extraction and RT-qPCR 15
• 2.11. Immunoblot 15
• 2.12. Image analysis 16
• 2.13. Statistical analysis 17
• III. Results 19
• 1. Chapter 1: The Regulatory Role of NOX4 in Endochondral Ossification through the Modulation of MPO and OPN 19
• 1.1. Estradiol levels 19
• 1.2. Phenotypic and morphometric analysis of bone structure in Control, OVX, and NOX4⁻/⁻ mice 19
• 1.3. Femoral bone marrow-derived MSC proteomic profiling in experimental mice 20
• 1.4. Immunohistochemical analysis of MPO, OPN, Collagen II experession 21
• 1.5. Collagen amount and chondrogenesis in the femur 21
• 2. Chapter II: NOX4–Mediated Modulation of the MPO–OPN Redox Axis in the Regulation of Osteoblast Maturation 23
• 2.1. NOX4 deficiency accelerates ossification during embryonic skeletal development 23
• 2.2. NOX4 deficiency increases osteoblast proliferation, and this enhancement is only partially reduced by MPO inhibition 23
• 2.3. NOX4 deficiency enhances early osteoblast differentiation but remains partially resistant to MPO inhibition 25
• 2.4. NOX4 deficiency increases the mineralized area without altering nodule number, whereas MPO inhibition reduces the mineralized area across both genotypes 25
• 2.5. NOX4 deficiency leads to increased MPO and OPN expression, and both are lowered by MPO inhibition, supporting the presence of an MPO–OPN–linked regulatory module that operates under NOX4 control 26
• IV. Discussion 54
• V. References 62
• 국문요약 68
• 감사의 글 70