The skeletal cell-derived molecule sclerostin drives bone marrow adipogenesis

Abstract

The bone marrow niche is a dynamic and complex microenvironment that can both regulate, and be regulated by the bone matrix. Within the bone marrow (BM), mesenchymal stromal cell (MSC) precursors reside in a multi-potent state and retain the capacity to differentiate down osteoblastic, adipogenic, or chondrogenic lineages in response to numerous biochemical cues. These signals can be altered in various pathological states including, but not limited to, osteoporotic-induced fracture, systemic adiposity, and the presence of bone-homing cancers. Herein we provide evidence that signals from the bone matrix (osteocytes) determine marrow adiposity by regulating adipogenesis in the bone marrow. Specifically, we found that physiologically relevant levels of Sclerostin (SOST), which is a Wnt-inhibitory molecule secreted from bone matrix-embedded osteocytes, can induce adipogenesis in 3T3-L1 cells, mouse ear- and BM-derived MSCs, and human BM-derived MSCs. We demonstrate that the mechanism of SOST induction of adipogenesis is through inhibition of Wnt signaling in pre-adipocytes. We also demonstrate that a decrease of sclerostin in vivo, via both genetic and pharmaceutical methods, significantly decreases bone marrow adipose tissue (BMAT) formation. Overall, this work demonstrates a direct role for SOST in regulating fate determination of BM-adipocyte progenitors. This provides a novel mechanism for which BMAT is governed by the local bone microenvironment, which may prove relevant in the pathogenesis of certain diseases involving marrow adipose. Importantly, with anti-sclerostin therapy at the forefront of osteoporosis treatment and a greater recognition of the role of BMAT in disease, these data are likely to have important clinical implications.

Keywords: bone marrow adipose; bone marrow microenvironment; osteocyte-derived factors; sclerostin.

Figure 1

3T3-L1 Cells. (A) Growth Media- Oil Red O Images; Top- Growth Media Alone, Bottom- Growth Media + 1ng/mL SOST and quantification. Arrows indicate Oil Red O staining. (B) Quantification of Oil Red O staining from A. (C) The upregulation of adipogenic transcription factor Pparγ and (D) Cebpα in response to SOST in basal growth media. (F) 3T3-L1 adipogenesis as measured via lipid accumulation is significantly upregulated in a 50/50 mixture of adipogenic media and media conditioned by primary mouse osteocytes, which produce high levels of sclerostin (E) as measured via ELISA.

Figure 2

SOST is able to induce adipogenesis in primary stem-like progenitors. Recombinant mouse sclerostin may induce lipid accumulation in ear mesenchymal stromal cells (eMSCs) (A). 10 ng/mL SOST induced an increase in adipogenic transcription factors Pparg (B) and Cebpa (C) after 7 days of differentiation.

Figure 3

Effects of Sost on BM-MSCs from Mouse. (A) Quantification of Oil Red O in BM-MSCs and upregulation of adipogenic transcription factors with SOST treatment. (B) BM-MSCs also showed an upregulation of adipogenic transcription factor Pparγ when treated with 1 ng/mL SOST. (C, D) β-catenin responsive genes Axin2 and Smad6 (H; n.s.) were decreased in response to SOST treatment in mouse BM-MSCs (E) Conditioned media from osteocytes induced elevated lipid accumulation in BM-MSCs as quantified by Oil Red O and induced an upregulation of adipogenic transcription factors Cebpa (F) and Pparg (G; n.s.). (H) The mature adipocyte marker adiponectin was also significantly increased in BM-MSCs exposed to osteocyte CM.

Figure 4

Sclerostin knockout mice have altered bone and BMAT. (A) Bone marrow adipocytes are indicated by arrows in wild-type and (B) KO/KO animals; H&E stained sections of mouse femur. SOST-KO animals (males, 5–6 weeks of age; n=7) have higher bone parameters including trabecular bone volume/total volume (C), cortical thickness (D) and reduced cortical porosity (E) when compared to wild-type mice (n=8). SOST-KO mice have significantly lower marrow adipose tissue in both proximal (F) and distal (G) regions of the tibia as measured by osmium tetroxide μCT.

Figure 5

Anti-Sclerostin antibody reduces BMAT in vivo (A & C); (A) H&E images and (B and C) quantification; * indicates significance via ANOVA. (D) Proposed model of sclerostin effects as an MSC fate switch determinant between adipogenesis and osteogenesis.

Figure 6

Proposed model of sclerostin as an MSC fate switch determinant between adipogenesis and osteogenesis in the bone. Sclerostin, a gene that encodes the SOST protein, is expressed by osteocytes within cortical and trabecular bone. SOST protein is released from osteocytes and can regulate bone marrow progenitor cell differentiation by not only inhibiting osteogenesis, but also inducing adipogenesis. This novel role for sclerostin suggests that sclerostin-targeting therapies may not only increase osteogenesis, but also decrease bone marrow adipogenesis. In sum, our data reveal a previously undocumented role for sclerostin in development of bone marrow adipose.