Regulation of Skeletal Muscle Mass by CXCL14 Signaling = CXCL14 신호 전달을 통한 골격 근육량 조절 기전 연구

다국어 초록 (Multilingual Abstract)

Skeletal muscle atrophy occurs when skeletal muscle homeostasis is disrupted by the imbalance of protein metabolism toward a catabolic state. Previously, we found that the expression of Cxcl14, a chemokine gene, was significantly reduced in the atrophic mouse skeletal muscle in which WNT regulator R-spondin2 was overexpressed. In this study, we investigated the potential role of Cxcl14 in muscle mass regulation utilizing both in vitro cell culture and in vivo mouse model. We found that CXCL14 protein treatment resulted in C2C12 myotube hypertrophy and was associated with activation of the AKT-S6K and inhibition of the AKT-FOXO-MuRF1/Atrogin1 pathway. Subsequently, we confirmed that CXCL14 protein treatment increased protein synthesis via S6K. Moreover, the receptors mediating CXCL14-induced hypertrophy were not CXCR4, IGF-1R, or LRP1. Consistent with the in vitro results, Cxcl14 gene overexpression in mouse skeletal muscle resulted in an increase of muscle mass, activation of AKT-S6K and inhibition of AKT-FOXO-MuRF1/Atrogin1. Although Cxcl14 overexpression did not regulate fiber-type determination, Cxcl14 increased muscle mass across all fiber types in vivo. RNA sequencing of Cxcl14-overexpressing mouse TA muscle showed the upregulation of genes associated with immune system, and downregulation of genes associated with muscle mass regulation. Furthermore, Cxcl14 overexpression in mice and CXCL14 treatment in vitro (C2C12-derived myotubes and primary human myotubes) reversed muscle atrophy induced by LPS and dexamethasone, along with the restoration of AKT-S6K pathway and inhibition of AKT-FOXO-MuRF1/Atrogin1 pathway. In conclusion, our data suggests a novel positive role for Cxcl14 in skeletal muscle mass regulation and raises therapeutic feasibility of CXCL14 for human skeletal muscle atrophy.

국문 초록 (Abstract)

근 위축증은 단백질 대사의 불균형에 의해 골격근의 항상성이 무너져 이화 작용 상태로 향할 때 발생한다. 이전에는 WNT 조절 인자인 R-spondin2가 과발현 된 근육 위축 마우스의 골격근에서 케모카인 유전자 인 Cxcl14의 발현이 현저하게 감소함을 발견하였다. 본 연구에서는 생체 외 세포 배양과 생체 내 마우스 모델을 활용하여 근육량 조절에서 Cxcl14의 잠재적 역할을 조사하였다. CXCL14 단백질 처리는 C2C12 근관 비대를 유발하였고, 이는 AKT-S6K의 활성화와 AKT-FOXO-MuRF1/Atrogin1 경로의 억제와 연관이 있음을 확인하였다. 그 후, CXCL14 단백질 처리가 S6K를 통해 단백질 합성을 증가시킴을 확인하였다. 또한 CXCR4, IGF-1R와 LRP1은 CXCL14 유도 근 비대를 매개하는 수용체가 아님을 확인하였다. 세포 배양 실험과 일관되게 마우스의 골격근에서 Cxcl14 유전자의 과발현은 근육량의 증가와 AKT-S6K의 활성화 및 AKT-FOXO-MuRF1/Atrogin1을 저해하는 결과를 나타냈다. Cxcl14의 과발현은 근육 섬유 유형의 결정을 조절하지 않았지만 동물 실험에서 생체 내 모든 근육 섬유 유형에 걸쳐 근육량을 증가시켰다. Cxcl14이 과발현된 마우스의 TA 근육의 전사체 염기분석은 면역계와 관련된 유전자의 증가와 근육량 조절과 관련된 유전자의 감소를 나타냈다. 더욱이 동물실험에서 Cxcl14의 과발현과 세포실험 (C2C12 유래 근관 및 인간 유래 초대 세포 근관)에서의 CXCL14 단백질의 처리는 AKT-S6K 경로의 회복과 AKT-FOXO-MuRF1/Atrogin1 경로의 저해와 함께 LPS 및 dexamethasone에 의해 유도된 근 위축증을 역전시켰다. 결론적으로 본 연구는 골격 근육의 조절에서 Cxcl14의 새로운 긍정적인 역할과 사람의 근 위축증에 대한 치료제로서의 실현가능성을 제시한다.

목차 (Table of Contents)

• CHAPTER 1: INTRODUCTION 1
• 1.1. Skeletal muscle structure and function 1
• 1.2. Skeletal muscle niche 3
• 1.3. Skeletal muscle hypertrophy and atrophy 5
• 1.4. Current treatment options for skeletal muscle atrophy 6
• 1.5. Skeletal muscle mass regulatory signaling pathways 8
• 1.5.1. AKT-related protein synthesis and degradation pathway 9
• 1.5.2. TGF-β/BMP signaling pathway 12
• 1.5.3. NF-κB signaling pathway 12
• 1.5.4. JAK/STAT signaling pathway 13
• 1.5.5. Autophagy-lysosome pathway 14
• 1.5.6. WNT signaling pathway 15
• 1.6. Cytokines as skeletal muscle mass regulatory factors 19
• 1.6.1. Interleukins as skeletal muscle mass regulator 21
• 1.6.2. Interferon as skeletal muscle mass regulator 26
• 1.6.3. Chemokines as skeletal muscle mass regulator 27
• 1.7. Chemokine (C-X-C Motif) Ligand 14 (CXCL14) 30
• 1.7.1. CXCL14 structure 30
• 1.7.2. CXCL14 expression pattern and cellular sources 32
• 1.7.3. CXCL14 candidate receptors 33
• 1.7.4. CXCL14 function in homeostasis 35
• 1.7.5. CXCL14 function in skeletal muscle 36
• 1.7.6. Disorders associated with CXCL14 37
• OBJECTIVES AND AIMS 39
• CHAPTER 2: MATERIALS AND METHODS 40
• 2.1. Cell culture 40
• 2.2. Mice, in vivo skeletal muscle electroporation and atrophy rescue experiment 40
• 2.3. siRNA transfection 41
• 2.4. Histology and immunofluorescence 42
• 2.5. Western blot analysis 42
• 2.6. SunSET assay 43
• 2.7. Total RNA-seq 44
• 2.8. Statistical analysis 44
• CHAPTER 3: RESULTS 45
• 3.1. CXCL14 effects on skeletal muscle mass in vitro with C2C12 myotubes 45
• 3.1.1. CXCL14 induces hypertrophy in C2C12 myotubes 45
• 3.1.2. CXCL14 increases has no effect on cell differentiation, but increase cell fusion 46
• 3.1.3. CXCL14 activates AKT-S6K pathway and inhibits AKT-FOXO-Atrogin-1/MuRF-1 pathway in C2C12 myotubes 48
• 3.1.4. CXCL14 increases total protein synthesis via S6K in C2C12 myotubes 51
• 3.1.5. CXCL14 does not rely on CXCR4, IGF-1R and LRP1 as receptor on skeletal muscle 53
• 3.2. CXCL14 effects on skeletal muscle mass in vivo with mice TA muscle 55
• 3.2.1. CXCL14 increases skeletal muscle mass in vivo via activation of AKT-S6K pathway and inhibition of AKT-FOXO-Atrogin-1/MuRF-1 pathway 55
• 3.2.2. In vivo overexpression of Cxcl14 alters pathways associated with cytokine signaling and skeletal muscle mass regulation 60
• 3.2.3. In vivo overexpression of CXCL14 regulates target genes associated with NFKB1 and FOXO1 transcription factors 64
• 3.3. CXCL14 effects on skeletal muscle atrophy models in vitro and in vivo 66
• 3.3.1. CXCL14 reverses LPS-induced atrophy in C2C12 myotubes via activation of AKT-S6K and inhibition of AKT-FOXO-Atrogin-1/MuRF-1 pathway in C2C12 myotubes 66
• 3.3.2. CXCL14 protects skeletal muscle mass against LPS-induced atrophy in vivo via activation of AKT-S6K and inhibition of AKT-FOXO-Atrogin-1/MuRF-1 pathway 69
• 3.3.2. CXCL14 reverses dexamethasone-induced atrophy in C2C12 myotubes via activation of AKT-S6K and inhibition of AKT-FOXO-Atrogin-1/MuRF-1 pathway in C2C12 myotubes 71
• 3.3.3. CXCL14 protects skeletal muscle mass against dexamethasone-induced atrophy in vivo via activation of AKT-S6K and inhibition of AKT-FOXO-Atrogin-1/MuRF-1 pathway 74
• 3.3.4. CXCL14 protects skeletal muscle mass against LPS and dexamethasone-induced atrophy in primary human myotubes in vitro via activation of AKT-S6K and inhibition of AKT-FOXO-Atrogin-1/MuRF-1 pathway 76
• CHAPTER 4: DISCUSSION 79
• CHAPTER 5: CONCLUSION 90
• REFERENCES 92
• ABBREVIATIONS i
• LIST OF FIGURES iii
• DECLARATION v
• ACKNOWLEDGEMENTS vi