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• Research Paper Volume 13, Issue 16 pp 20629-20650

  A systematic dissection of human primary osteoblasts in vivo at single-cell resolution

  Relevance score: 11.298982

  Yun Gong, Junxiao Yang, Xiaohua Li, Cui Zhou, Yu Chen, Zun Wang, Xiang Qiu, Ying Liu, Huixi Zhang, Jonathan Greenbaum, Liang Cheng, Yihe Hu, Jie Xie, Xuecheng Yang, Yusheng Li, Yuntong Bai, Yu-Ping Wang, Yiping Chen, Li-Jun Tan, Hui Shen, Hong-Mei Xiao, Hong-Wen Deng

  Keywords: single-cell RNA sequencing, osteoblasts, cellular heterogeneity, bone formation

  Published in Aging on August 24, 2021

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  Human osteoblasts are multifunctional bone cells, which play essential roles in bone formation, angiogenesis regulation, as well as maintenance of hematopoiesis. However, the categorization of primary osteoblast subtypes in vivo in humans has not yet been achieved. Here, we used single-cell RNA sequencing (scRNA-seq) to perform a systematic cellular taxonomy dissection of freshly isolated human osteoblasts from one 31-year-old male with osteoarthritis and osteopenia after hip replacement. Based on the gene expression patterns and cell lineage reconstruction, we identified three distinct cell clusters including preosteoblasts, mature osteoblasts, and an undetermined rare osteoblast subpopulation. This novel subtype was found to be the major source of the nuclear receptor subfamily 4 group A member 1 and 2 (NR4A1 and NR4A2) in primary osteoblasts, and the expression of NR4A1 was confirmed by immunofluorescence staining on mouse osteoblasts in vivo. Trajectory inference analysis suggested that the undetermined cluster, together with the preosteoblasts, are involved in the regulation of osteoblastogenesis and also give rise to mature osteoblasts. Investigation of the biological processes and signaling pathways enriched in each subpopulation revealed that in addition to bone formation, preosteoblasts and undetermined osteoblasts may also regulate both angiogenesis and hemopoiesis. Finally, we demonstrated that there are systematic differences between the transcriptional profiles of human and mouse osteoblasts, highlighting the necessity for studying bone physiological processes in humans rather than solely relying on mouse models. Our findings provide novel insights into the cellular heterogeneity and potential biological functions of human primary osteoblasts at the single-cell level.

• Research Paper Volume 11, Issue 10 pp 3250-3261

  A small molecular inhibitor of LRRK1 identified by homology modeling and virtual screening suppresses osteoclast function, but not osteoclast differentiation, in vitro

  Relevance score: 11.430111

  Mingjue Si, Canjun Zeng, Helen Goodluck, Sandi Shen, Subburaman Mohan, Weirong Xing

  Keywords: LRRK1, bone resorption, bone formation, osteoclast, kinase inhibitor

  Published in Aging on May 21, 2019

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  We used TGFβ activation kinase 1 as a template to build a 3D structure of the human LRRK1 kinase domain (hLRRK1 KD) and performed small molecule docking. One of the chemicals (IN04) that docked into the pocket was chosen for evaluation of biological effects on osteoclasts (OCs) in vitro. INO4 at 16 nM completely blocked ATP binding to hLRRK1 KD in an in vitro pulldown assay. In differentiation and pit assays, while the number of OCs on bone slices were comparable for OCs treated with IN04 and DMSO, IN04 treatment of OCs significantly impaired their ability to resorb bone. The area of pits on bone slices was reduced by 43% at 5 μM and 83% at 10 μM as compared to DMSO. Individual pits appeared smaller and shallower. F-actin staining revealed that DMSO-treated OCs displayed clear actin rings, and F-actin forms a peripheral sealing zone. By contrast, IN04-treated OCs showed disarranged F-actin in the cytoplasm, and F-actin failed to form a sealing zone on bone slices. IN04 treatment had no effects on OC-derived coupling factor production nor on osteoblast nodule formation. Our data indicate IN04 is a potent inhibitor of LRRK1, suppressing OC function with no effect on OC formation.

  

  Small molecular inhibitor IN04 binds to the predicted active pocket of hLRRK1 kinase domain. (A) Predicted structure of hLRRK1 kinase domain with an active pocket. (B) A molecular structure of the potential LRRK1 inhibitor IN04. (C) A potential small molecular weight inhibitor docks to the active pocket of hLRRK1 KD. (D) Purified 34 kD recombinant protein of hLRRK1 expressed in E. coli stained with Coomassie blue. (E) IN04 inhibits ATP binding to the LRRK1 KD, measured by an in vitro pulldown assay.

  
  
  

  

  IN04 treatment inhibits osteoclast bone resorption function with no effect on osteoclast formation. Osteoclast precursors derived from C57BL/6J mice were seeded on bone slices (0.4 x 0.8 cm) and differentiated in the presence of DMSO or INO4 for 6–9 days. Cells were stained for TRAP (Tartrate-resistant acid phosphatase), and bone slices were stained with hematoxylin for bone resorption pits. (A) Representative images of TRAP-positive osteoclasts (upper panel) and resorption pits (lower panel) on bone slices. (B) Quantitative data of osteoclast numbers on bone slices. (C) Quantitative data of osteoclast size on bone slices. [29]. Quantitative data of pit formation on bone slices. The results are presented as percentage of pit area relative to the bone slice area. Data are presented as mean ± SEM. P < 0.05 or P < 0.01 indicates statistical significance (N=4).

  
  
  

  

  IN04 treatment impairs osteoclast function. (A) Representative images of bone resorption pits on bone slices scanned by nano-CT. Bone slices described in Figure 5, and a blank bone slice (1.5x1.5 mm) was scanned by nano-CT is shown. (B) Quantitative data of pit size in area on bone slices. (C) Representative images of vertical section of bone resorption pits on bone slices. (D) Quantitative data of pit depth on bone slices, measured by nano-CT. Data are presented as mean ± SEM. P < 0.05 or P < 0.01 indicates statistical significance (N=3).

  
  
  

  

  Dose response curve of resorptive pit formation. Osteoclast precursors derived from C57BL/6J mice were seeded on bone slices and differentiated in the presence of DMSO or the indicated concentrations of INO4 for 6–9 days followed by hematoxylin staining for bone resorption pits. The results are presented as percentage of pit formation relative to the DMSO-treated control. Results are averages of duplicates with comparable results obtained in another independent experiment (N=4). The IC₅₀ value was derived from the graph.

  
  
  

  

  IN04 treatment disrupts the cytoskeleton arrangement and F-actin ring formation of osteoclasts. Osteoclast precursors derived from C57BL/6J mice were seeded on bone slices and differentiated in the presence of DMSO or INO4 for 6–9 days. Mature osteoclasts were stained with Alexa Fluor 488-conjugated phalloidin and DAPI, and the actin ring formation and sealing zone were visualized by confocal microscopy. (A) Representative images of IN04- and DMSO-treated osteoclasts. (B) Horizontal cross sections of selected osteoclasts. Two lines in the middle of the cell represent positions of horizontal and vertical cuts, respectively. Rectangles are orthogonal views of X- and Y-sections, respectively. Arrows in red indicate sealing zones (SZ). (C) 3D rendering of a lateral view. A DMSO-treated osteoclast was invaded in a resorptive pit indicated by an arrow in green while an IN04-treated cell was on the surface of the bone slice.

  
  
  

  

  IN04 treatment does not influence osteoclast differentiation and coupling factor expression. Osteoclast precursors derived from C57BL/6J mice were cultured in 6-well plates and differentiated in the presence of DMSO or INO4 for 6–9 days followed by RNA extraction and real-time RT-PCR. Expression levels of endogenous Lrrk1 and osteoclast differentiation markers NFATc1, Acp5, and Cathepsin K, respectively (N=6). Expression levels of osteoclast coupling factors BMP6 (bone morphometric protein 6), CTHRC1 (collagen triple helix repeat containing 1), and Wnt10b (wingless-type MMTV integration site family, member 10B), respectively (N=6). Data are presented as mean ± SEM. P < 0.05 or P < 0.01 indicates statistical significance.

  
  
  

  

  IN04 treatment has no effect on nodule formation in vitro. Bone marrow stromal cells were treated with a mineralization medium containing 10 mM β-glycerophosphate, 50 μg/ml ascorbic acid, and 10% FBS in the presence of DMSO or INO4 for 24 days and stained with alizarin red. (A) Images of nodule formation. (B) Quantitative mineralization area measured by the OsteoMeasure system. Data are presented as mean ± SEM, N=3.