A novel library screen identifies immunosuppressors that promote osteoblast differentiation

Abstract

Bone homeostasis can be compromised by an increase in osteoclast-mediated resorption and/or a decrease in osteoblast-mediated bone deposition. While many efforts have focused on treating osteoclast resorption, there has been less emphasis on identifying strategies for promoting osteoblast function. Herein, we describe a high-throughput screening assay to select for small molecules that augment bone morphogenetic protein-2 (BMP-2)-mediated osteoblast lineage commitment. After an initial screen of 5405 compounds; consisting of FDA-approved drugs, known bioactives, and compounds with novel chemical makeup, we identified 45 small molecules that promoted osteoblast commitment. Of the 45 candidates, there was a broad array of classes that included nine retinoid analogs/derivatives and four immunosuppressants, notably rapamycin and FK-506, which were chosen for further study. Treatment of osteoblast precursor cells with rapamycin or FK-506, either alone, or synergistically with BMP-2, increased levels of phospho-Smad1/5/8 protein and transcription of Runx-2, Osx and Smad-7, consistent with a role in promoting osteoblast differentiation. Only FK-506 was able to enhance osteocalcin transcripts and alizarin red staining, both late markers for differentiation. When osteoblast differentiation was suppressed with exogenous TGF-β1 treatment, rapamycin (but not FK-506) was able to rescue expression of differentiation markers, indicating distinct but overlapping activity of these compounds. Collectively, these data add to an understanding of pathways engaged in osteoblastogenesis, support a role for non-redundant immunosuppressant signaling, and provide a novel approach for the discovery of potentially therapeutic compounds that affect bone remodeling.

Autophagy controls mesenchymal stem cell properties and senescence during bone aging

“…Previous report found that rapamycin promotes osteoblast differentiation and inhibition of autophagosome formation in a mouse model showed severe osteopenia (Darcy et al., 2012; Shimada, Greer, McMahon, Bouxsein, & Schipani, 2008). Similarly, rapamycin has been proven to be able to reduce the severity of age‐related bone changes in trabecular bone of older male rats by partially activating autophagy of osteocytes (Luo, Ren, Li, Lian, & Lin, 2016), which is consistent with our findings.…”

Late-life rapamycin treatment reverses age-related heart dysfunction

“…This trend was also seen in the trabecular bone of the lumbar vertebra where a slightly higher bone volume and trabecular thickness were observed. It has been suggested that the modulation of mTOR may activate signaling events which would prevent the progression of lytic bone disease (Smink & Leutz, 2010) and can enhance the proliferation and differentiation of osteoblasts (Darcy et al , 2012). To fully determine whether rapamycin may attenuate bone aging, further studies with chronic rapamycin treatment beginning prior to any significant loss of bone due to aging (~3 months of age for C57BL/6 mice) are warranted.…”

Chimeric Allografts Induced by Short‐Term Treatment With Stem Cell–Mobilizing Agents Result in Long‐Term Kidney Transplant Survival Without Immunosuppression: A Study in Rats

“…However, CXCR4 blockade (AMD3100) alone was not successful, and the addition of low dose FK506 was essential, perhaps exerting its effect via the FK506-binding protein 12 (FKBP12) and not by calcineurin inhibition. Recently, FKBP12 has been reported to promote osteoblastic differentiation by activating the bone morphogenetic protein (BMP) receptor (32–34). Our current studies have shown that F alone increased SDF-1 in the transplant, and we have found in previous studies that cells with macrophage phenotypes are liberated from the bone marrow by FK506 (27).…”