Glucocorticoid-induced alterations of skeletal stem cell-based processes

Glucocorticoid (GC) induced osteoporosis (GIOP) and osteonecrosis remain a significant health issue with few approved therapies that can treat the bone loss and dysfunction of skeletal vasculature. Therefore, we aimed to investigate the cellular and molecular processes by which GCs affect osteogenesis and angiogenesis, as well as how treatment with parathyroid hormone (hPTH 1-34) modifies these effects in a mouse model of GIOP. GC treatment reduced bone mass through decreased bone formation by skeletal stem cells (SSCs) while also increasing osteoclast mediated resorption. Concomitantly, endothelial cells were increased in numbers but displayed distorted phenotypical features. However, hPTH treatment prevented GC induced changes in osteogenesis and angiogenesis. Transplantation studies of SSCs combined with molecular analysis by single cell RNA-sequencing and functional testing of primary human cells tied GC-induced skeletal changes to altered stem and progenitor cell differentiation dynamics. This in turn perpetuated reduced osteogenesis and vascular malformation through direct SSC-endothelial crosstalk mediated at least in part by Basigin. In accordance, the conditional heterozygous deletion of Basigin in the skeletal lineage as well as antibody-mediated blockade of Basigin during GC treatment prevented detrimental bone loss. Intriguingly, aging also increases skeletal Basigin levels. When administered to 2-year-old mice, anti-Basigin therapy reinstated bone remodeling to significantly improve bone mass independent of sex. These findings, while helping to explain the cellular and molecular basis of GC induced bone loss, provide new therapeutic vantage points for GIOP and potentially other conditions associated with bone loss. Overall design: To study the effects of glucocorticoids on bone biology, Methylprednisolone 5 mg 60-day release pellets and vehicle were (cat#SG-241, Innovative Research of America, Sarasota, FL, USA) implanted under the skin of mice near the lumbar spine at day 0, and pellets were maintained for 58 days or removed after 28 days as indicated. Human parathyroid hormone 1-34 (hPTH 1-34) was purchased from Sigma (cat#:P3796), and the treatment was administered subcutaneously at 5 µg/kg/day, 5x/week for four consecutive weeks. There were a total of four groups of mice receiving subcutaneous release pellets of: (1) Placebo (Control), (2) Methylprednisolone (GC), (3) Methylprednisolone removal (GC removal), (4) Methylprednisolone (GC) + hPTH 1-34 (GC+PTH).For renal capsule transplantation experiments cells from three-month old male GFP reporter mice (C57BL/6-Tg(CAG-EGFP)1Osb/J; JAX: 003291) were transplanted into male B6 mice (C57BL/Ka-Thy1.1-CD45.1; JAX: 000406). For single cell analysis femur bones were processed, digested, and prepared for FACS. Single cell solutions of each treatment group were then pooled (n=5 per group) and equal amounts of 1x10e6 PI-Ter119-CD45- and CD45+ cells were sorted into the same collection tube for each grouup containing FACS buffer. Cells were then processed with 10X Chromium Next GEM Single Cell 3' GEM kit (10X Genomics Inc, v3.1) according to manufacturer's instruction. Alternatively, cells were harvested from separate experiments of renal capsule grafts of groups (1), (2) and (4). To that end, renal capsule transplantations in the anaesthetized mouse a 5-mm dorsal incision was made and the kidney was exposed manually. Then, a 2-mm incision was created in the renal capsule using a needle bevel, and 5,000 FACS-purified mouse SSCs resuspended in 2 µl of Matrigel were transplanted beneath the capsule. The kidney was re-approximated manually and incisions were closed using sutures and staples. At time of surgery, mice were randomly divided into the three groups. Mice either received subcutaneous placebo or Methylprednisolone pellets as described above. One group additionally was treated with intermittent hPTH 1-34. Grafts were collected after 21 days and processed for single cell RNA-sequencing as described above.