CLEC3A as a Protective Modulator of Cartilage Degradation in Osteoarthritis = 골관절염에서 연골 분해 보호 조절자로서의 CLEC3A

다국어 초록 (Multilingual Abstract)

Osteoarthritis (OA) is a chronic degenerative joint disease characterized by the disruption of extracellular matrix homeostasis. C-type lectin domain family 3 member A (Clec3a) is a chondrocyte-expressed C-type lectin protein known to promote cell adhesion to laminin-332 and fibronectin, but its involvement in OA pathogenesis has remained unclear. This study aimed to elucidate the regulation and functional significance of Clec3a in OA by integrating transcriptomic analyses with in vitro and in vivo approaches. Transcriptome profiling (microarray and RNA-seq) identified Clec3a as a gene consistently downregulated in primary mouse chondrocytes exposed to interleukin-1β (IL-1β) or other catabolic stimuli. Consistent with these findings, CLEC3A expression was markedly reduced in human OA cartilage, in chondrocytes treated with IL-1β or tumor necrosis factor-α, and in the cartilage of mice subjected to destabilization of the medial meniscus (DMM). Functional analyses demonstrated that adenoviral overexpression of Clec3a did not alter basal expression of Mmp3, Mmp13, or Adamts5, but significantly suppressed their induction under IL-1β stimulation. Furthermore, intra-articular delivery of Clec3a and cartilage-specific Clec3a transgenic mice exhibited reduced cartilage degeneration and decreased expression of MMP3 and MMP13 following DMM surgery. Collectively, these findings demonstrate that Clec3a is a gene strongly responsive to OA-associated catabolic conditions and suggest that Clec3a may function as a protective regulator that modulates catabolic enzyme expression and cartilage destruction, raising the possibility that Clec3a could serve as a potential therapeutic target for osteoarthritis.

국문 초록 (Abstract)

골관절염은 세포외기질의 항상성의 붕괴로 인해 발생하는 만성 퇴행성 관절질환이다. Clec3a는 연골세포에서 발현되는 C-type lectin 단백질로 laminin-332 및 fibronectin에 대한 세포 부착을 촉진하는 것으로 알려져 있으나, 골관절염 병인과의 연관성은 명확히 규명되지 않았다. 본 연구는 골관절염에서 Clec3a의 발현 조절 및 기능적 역할을 규명하기 위해 전사체 분석과 in vitro 및 in vivo 실험을 통합하여 수행되었다. 전사체 분석(microarray 및 RNA-seq) 결과, Clec3a는 interleukin-1β(IL-1β) 및 기타 catabolic 자극을 받은 1차 배양 마우스 연골세포에서 일관되게 하향 조절되는 유전자로 확인되었다. 이러한 결과와 일치하게, CLEC3A 발현은 사람 OA 연골, IL-1β 또는 tumor necrosis factor-α로 처리된 연골세포, 그리고 내측 반월판 불안정화(DMM) 수술을 시행한 마우스의 연골에서 모두 현저히 감소하였다. 기능적 분석에서, Clec3a의 아데노바이러스 매개 과발현은 비자극 조건에서는 Mmp3, Mmp13, Adamts5 발현에 영향을 주지 않았으나, IL-1β 자극 하에서는 이들 catabolic 유전자의 발현 유도를 유의하게 억제하였다. 또한 관절강 내 Clec3a 주입과 연골세포 특이적 Clec3a 형질전환 마우스는 DMM 수술 후 연골 파괴가 감소하고 MMP3 및 MMP13 발현이 감소하는 양상을 보였다. 종합하면, 본 연구 결과는 Clec3a가 OA 관련 catabolic 환경에 매우 민감하게 반응하는 유전자이며, catabolic 효소 발현과 연골 파괴를 조절하는 보호적 기능을 수행할 가능성을 시사한다. 이러한 점에서 Clec3a는 향후 골관절염 치료를 위한 잠재적 표적이 될 수 있다.

목차 (Table of Contents)

• Content
• LIST OF TABLES················································································ ⅰ
• LIST OF FIGURES ············································································· ⅱ
• LIST OF ABBREVIATIONS··································································· ⅲ
• ABSTRACT ························································································ 1
• I. INTRODUCTION ·············································································· 3
• Ⅱ. MATERIALS AND METHODS ·························································· 7
• 2.1. Human cartilage tissues ·····························································7
• 2.2. Induction of osteoarthritis using DMM surgery in mice ··················7
• 2.3. Cartilage-specific Clec3a transgenic (TG) mice····························8
• 2.4. Histological analysis··································································8
• 2.5. Microarray and RNA-seq analyses··············································9
• 2.6. Primary culture of articular chondrocytes ····································9
• 2.7. RT-PCR and qRT-PCR ····························································10
• 2.8. Immunofluorescence································································10
• 2.9. Statistical analysis···································································11
• Ⅲ. RESULTS······················································································ 12
• 3.1. Microarray and RNA-seq analyses identify the downregulation of Clec3a
• under catabolic stimulation ·························································· 12
• 3.2. CLEC3A expression is decreased in human OA cartilage ················ 16
• 3.3. Clec3a expression is decreased in a dose-dependent manner in mouse
• chondrocytes treated with inflammatory stimuli ····························· 19
• 3.4. Clec3a expression decreases during the progression of DMM-induced
• osteoarthritis in mice ·································································· 22
• 3.5. Clec3a overexpression suppresses Mmp3, Mmp13, and Adamts5
• expression under inflammatory conditions····································· 25
• 3.6. Intra-articular overexpression of Clec3a attenuates DMM-induced
• cartilage destruction and reduces Mmps expression ······················· 28
• 3.7. Clec3a TG mice exhibit reduced DMM-induced cartilage damage and
• suppressed Mmp expression························································ 31
• Ⅳ. DISCUSSION·················································································· 34
• References ······················································································· 38
• 국문 초록···························································································· 43