ABSTRACT

Senile osteoporosis (OP) is often concomitant with decreased autophagic activity. OPTN (optineurin), a macroautophagy/autophagy (hereinafter referred to as autophagy) receptor, is found to play a pivotal role in selective autophagy, coupling autophagy with bone metabolism. However, its role in osteogenesis is still mysterious. Herein, we identified Optn as a critical molecule of cell fate decision for bone marrow mesenchymal stem cells (MSCs), whose expression decreased in aged mice. Aged mice revealed osteoporotic bone loss, elevated senescence of MSCs, decreased osteogenesis, and enhanced adipogenesis, as well as optn^{–/ –} mice. Importantly, restoring Optn by transplanting wild-type MSCs to optn^{–/ –} mice or infecting optn^{–/ –} mice with Optn-containing lentivirus rescued bone loss. The introduction of a loss-of-function mutant of Optn^K193R failed to reestablish a bone-fat balance. We further identified FABP3 (fatty acid binding protein 3, muscle and heart) as a novel selective autophagy substrate of OPTN. FABP3 promoted adipogenesis and inhibited osteogenesis of MSCs. Knockdown of FABP3 alleviated bone loss in optn^{–/ –} mice and aged mice. Our study revealed that reduced OPTN expression during aging might lead to OP due to a lack of FABP3 degradation via selective autophagy. FABP3 accumulation impaired osteogenesis of MSCs, leading to the occurrence of OP. Thus, reactivating OPTN or inhibiting FABP3 would open a new avenue to treat senile OP.

Abbreviations: ADIPOQ: adiponectin, C1Q and collagen domain containing; ALPL: alkaline phosphatase, liver/bone/kidney; BGLAP/OC/osteocalcin: bone gamma carboxyglutamate protein; BFR/BS: bone formation rate/bone surface; CALCOCO2/NDP52: calcium binding and coiled-coil domain 2; CDKN1A/p21: cyclin-dependent kinase inhibitor 1A; CDKN2A/p16: cyclin dependent kinase inhibitor 2A; CDKN2B/p15: cyclin dependent kinase inhibitor 2B; CEBPA: CCAAT/enhancer binding protein (C/EBP), alpha; COL1A1: collagen, type I, alpha 1; Ct. BV/TV: cortical bone volume fraction; Ct. Th: cortical thickness; Es. Pm: endocortical perimeter; FABP4/Ap2: fatty acid binding protein 4, adipocyte; H2AX: H2A.X variant histone; HE: hematoxylin and eosin; MAP1LC3B: microtubule-associated protein 1 light chain 3 beta; MAR: mineral apposition rate; MSCs: bone marrow mesenchymal stem cells; NBR1: NBR1, autophagy cargo receptor; OP: osteoporosis; OPTN: optineurin; PDB: Paget disease of bone; PPARG: peroxisome proliferator activated receptor gamma; Ps. Pm: periosteal perimeter; qRT-PCR: quantitative real-time PCR; γH2AX: Phosphorylation of the Serine residue of H2AX; ROS: reactive oxygen species; RUNX2: runt related transcription factor 2; SA-GLB1: senescence-associated (SA)-GLB1 (galactosidase, beta 1); SP7/Osx/Osterix: Sp7 transcription factor 7; SQSTM1/p62: sequestosome 1; TAX1BP1: Tax1 (human T cell leukemia virus type I) binding protein 1; Tb. BV/TV: trabecular bone volume fraction; Tb. N: trabecular number; Tb. Sp: trabecular separation; Tb. Th: trabecular thickness; μCT: micro computed tomography.

摘要

老年性骨质疏松症 (OP) 通常伴随着自噬活性降低。OPTN（optineurin）是一种巨自噬/自噬（以下简称自噬）受体，被发现在选择性自噬中起关键作用，将自噬与骨代谢耦合。然而，它在成骨中的作用仍然是个谜。在此，我们将Optn鉴定为骨髓间充质干细胞 (MSCs) 细胞命运决定的关键分子，其在老年小鼠中的表达降低。老年小鼠和optn ^{- / -}小鼠显示骨质疏松性骨丢失、MSCs 衰老升高、成骨减少和脂肪生成增强。重要的是，通过将野生型 MSC 移植到optn来恢复Optn^{– / –}小鼠或感染optn ^{– / –}含有Optn的慢病毒的小鼠挽救了骨质流失。Optn ^K193R功能丧失突变体的未能重建骨-脂肪平衡。我们进一步将 FABP3（脂肪酸结合蛋白 3，肌肉和心脏）鉴定为 OPTN 的新型选择性自噬底物。FABP3 促进脂肪生成并抑制 MSCs 的成骨。FABP3 的敲低减轻了 optn 中的骨质流失^{– / –}老鼠和老年老鼠。我们的研究表明，衰老过程中 OPTN 表达降低可能导致 OP，因为缺乏通过选择性自噬降解 FABP3。FABP3 积累损害 MSCs 的成骨，导致 OP 的发生。因此，重新激活 OPTN 或抑制 FABP3 将为治疗老年 OP 开辟一条新途径。

缩写：ADIPOQ：含有脂联素、C1Q 和胶原蛋白结构域；ALPL：碱性磷酸酶，肝/骨/肾；BGLAP/OC/骨钙素：骨γ羧基谷氨酸蛋白；BFR/BS：骨形成率/骨表面；CALCOCO2/NDP52：钙结合和卷曲螺旋结构域 2；CDKN1A/p21：细胞周期蛋白依赖性激酶抑制剂 1A；CDKN2A/p16：细胞周期蛋白依赖性激酶抑制剂 2A；CDKN2B/p15：细胞周期蛋白依赖性激酶抑制剂 2B；CEBPA：CCAAT/增强子结合蛋白 (C/EBP)，α；COL1A1：胶原蛋白，I 型，α 1；CT。BV/TV：皮质骨体积分数；CT。Th：皮质厚度；埃斯。Pm：皮质周长；FABP4/Ap2：脂肪酸结合蛋白 4，脂肪细胞；H2AX：H2A.X 变体组蛋白；HE：苏木精和伊红；MAP1LC3B：微管相关蛋白 1 轻链 3 β；MAR：矿物沉积率；MSCs：骨髓间充质干细胞；丁腈橡胶1：丁腈橡胶1，自噬货物受体；OP：骨质疏松症；OPTN：optineurin；PDB：骨佩吉特病；PPARG：过氧化物酶体增殖物激活受体γ；附言。Pm：骨膜周长；qRT-PCR：定量实时 PCR；γH2AX：H2AX 丝氨酸残基的磷酸化；ROS：活性氧；RUNX2：runt 相关转录因子 2；SA-GLB1：衰老相关 (SA)-GLB1（半乳糖苷酶，β 1）；SP7/Osx/Osterix：Sp7 转录因子 7；SQSTM1/p62：隔离体 1；TAX1BP1：Tax1（人 T 细胞白血病病毒 I 型）结合蛋白 1；结核病。BV/TV：骨小梁体积分数；结核病。N：小梁数；结核病。Sp：小梁分离；结核病。Th：小梁厚度；μCT：微型计算机断层扫描。附言。Pm：骨膜周长；qRT-PCR：定量实时 PCR；γH2AX：H2AX 丝氨酸残基的磷酸化；ROS：活性氧；RUNX2：runt 相关转录因子 2；SA-GLB1：衰老相关 (SA)-GLB1（半乳糖苷酶，β 1）；SP7/Osx/Osterix：Sp7 转录因子 7；SQSTM1/p62：隔离体 1；TAX1BP1：Tax1（人 T 细胞白血病病毒 I 型）结合蛋白 1；结核病。BV/TV：骨小梁体积分数；结核病。N：小梁数；结核病。Sp：小梁分离；结核病。Th：小梁厚度；μCT：微型计算机断层扫描。附言。Pm：骨膜周长；qRT-PCR：定量实时 PCR；γH2AX：H2AX 丝氨酸残基的磷酸化；ROS：活性氧；RUNX2：runt 相关转录因子 2；SA-GLB1：衰老相关 (SA)-GLB1（半乳糖苷酶，β 1）；SP7/Osx/Osterix：Sp7 转录因子 7；SQSTM1/p62：隔离体 1；TAX1BP1：Tax1（人 T 细胞白血病病毒 I 型）结合蛋白 1；结核病。BV/TV：骨小梁体积分数；结核病。N：小梁数；结核病。Sp：小梁分离；结核病。Th：小梁厚度；μCT：微型计算机断层扫描。Sp7 转录因子 7；SQSTM1/p62：隔离体 1；TAX1BP1：Tax1（人 T 细胞白血病病毒 I 型）结合蛋白 1；结核病。BV/TV：骨小梁体积分数；结核病。N：小梁数；结核病。Sp：小梁分离；结核病。Th：小梁厚度；μCT：微型计算机断层扫描。Sp7 转录因子 7；SQSTM1/p62：隔离体 1；TAX1BP1：Tax1（人 T 细胞白血病病毒 I 型）结合蛋白 1；结核病。BV/TV：骨小梁体积分数；结核病。N：小梁数；结核病。Sp：小梁分离；结核病。Th：小梁厚度；μCT：微型计算机断层扫描。