Full Length ArticleGH prevents adipogenic differentiation of mesenchymal stromal stem cells derived from human trabecular bone via canonical Wnt signaling
Graphical abstract
Introduction
Mesenchymal stem cells (MSCs), present in bone and in several other tissues [1], are multi-potent cells able to differentiate into adipocytes, osteoblasts, chondrocytes, myocytes, endothelial cells, and smooth muscle cells. Among these cells, adipogenic differentiation has particular relevance in bone, as the processes of adipogenesis and osteoblastogenesis are reciprocally associated and play important roles in regulating the bone mass homeostasis, especially during aging [2]. Indeed, in both aged humans and rats, there is a decrease in bone volume while bone marrow fat increases, suggesting that during aging, there is a balance shift favoring adipogenesis. This observation was originally outlined in the bone marrow of SAMP6, a murine model of increased adipogenesis and defective osteoblastogenesis [3]. A marked accumulation of bone marrow adipocytes was also observed in senile osteoporosis [[4], [5], [6]] with increased infiltration of fat in bone marrow cavities of long bones [7]. As a consequence, osteoblastic cell number decreases resulting in an age-related decline in bone formation with potential development of bone diseases, such as osteoporosis and increased risk of bone fractures [8,9].
Bone marrow provides a microenvironment for controlling the potential of MSCs towards osteoblasts or adipocytes [10], but this control is disrupted by the aging process. Several groups found that the inhibition of adipogenesis in MSCs could support bone health [6,11]; intrinsic and extrinsic factors can regulate MSCs differentiation potential and modulate the changing balance between osteoblastogenesis and adipogenesis in aging [12].
Besides its well-known regulatory role in postnatal skeletal development [13,14], growth hormone (GH) is of particular interest in aging. The decrease in the daily secretion of GH with advancing age [15] is thought to be involved in the progressive decline of bone mineral density (BMD). In addition to low BMD, GH-deficient adults show an increase in relative adiposity and a reduction in lean body mass, which can be restored by GH treatment [16]. Administration of GH to patients with severe osteopenia results in not only an increase in bone formation markers, osteoblast number, and osteoid surface, but also in a concomitant decrease in the number and size of marrow adipocytes [17]. These observations suggest a role for GH in modulating bone marrow balance between adipogenesis and osteoblastogenesis. A suggestion supported by several recent findings showing that GH has an inhibitory effect on the differentiation of primary bovine pre-adipocytes into adipocytes [18]. Moreover, GH reduces the adipogenesis of subcutaneous adipose tissue-derived mesenchymal stem cells (AT-MSC), whereas the lack of GH signaling increases the adipogenic differentiation while it impairs osteogenic differentiation in AT-MSC from subcutaneous depot [19].
However, none of the previous studies outlined the molecular mechanisms by which GH can modulate adipogenesis in human bone marrow. Thus, we designed the following study to investigate the intracellular pathways activated by GH to regulate the fate of MSCs derived from human trabecular bone. After having shown that GH decreases adipogenesis in human MSCs cultured in adipogenic medium, we examined the expression of the adipogenesis-related and osteogenesis-related genes. Considering the large body of evidence demonstrating that Wnt/β-catenin pathway is important for both mass accrual and bone remodeling [20], we evaluated the possible involvement of the canonical Wnt signaling in GH anti-adipogenic effect and demonstrated its requirement for the fulfilment of GH action. This study provides for the first time a closer insight into the molecular mechanisms that govern bone marrow adipogenesis and osteoblastogenesis in the context of age related bone mass loss.
Section snippets
Drugs
Human recombinant growth hormone (GH), isobutyl-1-methylxanthine (IBMX), dexamethasone, and indomethacin were purchased from Sigma-Aldrich srl (Milan, Italy).
Cell culture: isolation of MSCs
Human mesenchymal stromal cells (hMSCs) were isolated from trabecular bone samples obtained from waste material of female patients during orthopedic surgery for traumatic fractures of the femoral neck requiring osteotomy. None of the patients (aged 70–81 yr) had any malignant bone diseases and all of them gave their written consent for the
Reduced lipid droplets formation in GH-treated hMSCs
First, hMSC were tested for their in vitro differentiation potential (Figs. 1 and 1S). When cultured 14 days in adipogenic medium, compared to normal medium, hMSCs significantly accumulated lipid droplets (Fig. 1A, B), and significantly (P < 0.001) increased over time the transcription of adipogenesis related genes, such as ADN and C/EBPα (Fig. 1C).
After having assessed the presence of the receptor for GH in hMSCs (Fig. 2S), we investigated whether GH could affect the adipogenic differentiation
Discussion
In the present study, we analyzed the effect of GH on the differentiation of primary hMSCs, isolated from human trabecular bone, into adipocytes. We showed that hMSCs express GH receptor and that GH activates its intracellular signaling, JAK2/STAT5 (Fig. 2S). From our results, it emerged that GH reduced adipogenic differentiation of hMSCs, associated with a decreased lipid accumulation. Our data are in agreement with previous studies that showed that the bone marrow of GH-deficient rats
Acknowledgement
The authors are grateful to Courtney L. Long, PhD, for the scientific revision and English editing of the manuscript. This study did not receive any specific grant from funding agencies in the public, commercial or non-profit sectors.
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Present address: Heisenberg-Group for Molecular Skeletal Biology (MSB-Lab) Department of Trauma, Hand and Reconstructive Surgery University Medical Center Hamburg-Eppendorf, Hamburg, Germany.