Makareeva Elena, Omari Shakib, Roberts-Pilgrim Anna M, Gorrell Laura, Radant Bella, Sellamani Muthulakshmi, Mertz Edward L, Khoury Basma, Kozloff Kenneth, Leikin Sergey
Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD), National Institutes of Health, Bethesda, MD, USA.
Biomedical Engineering Department, Rensselaer Polytechnic Institute, Troy, NY, USA.
Autophagy. 2025 Sep 9:1-16. doi: 10.1080/15548627.2025.2551478.
Bone synthesis should depend on autophagy because over 10% of type I procollagen (PC1) - a heterotrimer of COL1A1 and COL1A2 chains and the precursor of the main bone matrix molecule - is misfolded and rerouted from osteoblast endoplasmic reticulum (ER) to lysosomes. However, osteoblast-specific macroautophagy knockouts in mice have produced only mild bone effects. To reconcile these observations, we compared how hypomorphic expression and a conditional knockout (cKO) of - encoding a protein required for autophagosome formation - affected versus wild-type osteoblasts and . The Gly610-to-Cys substitution (G610C) in the triple helical region of the COL1A2/proα2(I) chain increases PC1 misfolding, causing its accumulation in the ER, cell stress, and osteoblast malfunction. Because autophagy reroutes misfolded PC1 from the ER to lysosomes, disruption of PC1 autophagy should significantly increase osteoblast malfunction and bone pathology in mice. Nonetheless, the present study revealed only minor effects of the cKO on osteoblast function and bone formation in the mice, like in controls. The cKO did not reduce the autophagy flux of misfolded G610C or wild-type PC1 in primary osteoblast cultures, even though the LC3 and GABARAP lipidation and therefore autophagosome formation were disrupted. Live-cell imaging in cKO osteoblasts demonstrated that PC1 was efficiently delivered to lysosomes without LC3 via ER exit site (ERES) microautophagy. Taken together, these observations indicate that LC3- and GABARAP-independent ERES microautophagy is the primary pathway of misfolded procollagen degradation in osteoblasts both in culture and .: ATG5: autophagy related 5; ATG7: autophagy related 7; Baf: bafilomycin A; BFA: brefeldin A; BGLAP/Ocn/osteocalcin: bone gamma-carboxyglutamate protein; COL1A1/proα1(I): collagen type I alpha 1 chain; COL1A2/proα2(I): collagen type I alpha 2 chain; cKO: conditional knockout; ER: endoplasmic reticulum; ERES: ER exit site; G610C mutation: COL1A2 p.Gly706Cys replacing Gly in position 610 from the start of the triple helix with Cys; GABARAP: GABA type A receptor-associated protein; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MAR: mineral apposition rate; Ob: osteoblast; Oc: osteoclast; OI: osteogenesis imperfecta; PC1: procollagen type I, a heterotrimer of two COL1A1 and one COL1A2 chains, precursor of collagen type I; PDI: protein disulfide isomerase; RB1CC1/FIP200: RB1 inducible coiled-coil 1; SP7/osterix: Sp7 transcription factor; SQSTM1/p62: sequestosome 1; WT: wild type.
骨合成应该依赖自噬，因为超过10%的I型前胶原（PC1）——一种由COL1A1和COL1A2链组成的异源三聚体，也是主要骨基质分子的前体——发生错误折叠，并从成骨细胞内质网（ER）重新定向至溶酶体。然而，小鼠中敲除成骨细胞特异性巨自噬仅产生了轻微的骨骼效应。为了协调这些观察结果，我们比较了自噬体形成所需蛋白的低表达和条件性敲除（cKO）如何影响与野生型成骨细胞和。COL1A2/proα2（I）链三螺旋区域中的甘氨酸610到半胱氨酸的取代（G610C）增加了PC1的错误折叠，导致其在内质网中积累、细胞应激以及成骨细胞功能障碍。由于自噬将错误折叠的PC1从内质网重新定向至溶酶体，PC1自噬的破坏应该会显著增加小鼠的成骨细胞功能障碍和骨病理。尽管如此，本研究仅揭示了cKO对小鼠成骨细胞功能和骨形成的轻微影响，类似于对照小鼠。cKO并未降低原代成骨细胞培养物中错误折叠的G610C或野生型PC1的自噬通量，尽管LC3和GABARAP脂化以及因此的自噬体形成受到了破坏。cKO成骨细胞的活细胞成像表明，PC1通过内质网出口位点（ERES）微自噬在没有LC3的情况下有效地被递送至溶酶体。综上所述，这些观察结果表明，不依赖LC3和GABARAP的ERES微自噬是培养和成骨细胞中错误折叠的前胶原降解的主要途径。：ATG5：自噬相关5；ATG7：自噬相关7；Baf：巴弗洛霉素A；BFA：布雷菲德菌素A；BGLAP/Ocn/骨钙素：骨γ-羧基谷氨酸蛋白；COL1A1/proα1（I）：I型胶原α1链；COL1A2/proα2（I）：I型胶原α2链；cKO：条件性敲除；ER：内质网；ERES：内质网出口位点；G610C突变：COL1A2 p.Gly706Cys，从三螺旋起始处第610位的甘氨酸被半胱氨酸取代；GABARAP：GABA A型受体相关蛋白；MAP1LC3/LC3：微管相关蛋白1轻链3；MAR：矿物质沉积率；Ob：成骨细胞；Oc：破骨细胞；OI：成骨不全；PC1：I型前胶原，由两条COL1A1和一条COL1A2链组成的异源三聚体，I型胶原的前体；PDI：蛋白二硫键异构酶；RB1CC1/FIP200：RB1诱导卷曲螺旋1；SP7/osterix：Sp7转录因子；SQSTM1/p62：聚集体蛋白1；WT：野生型
